Handbook of Electroporation

Damijan Miklavčič
Editor
Handbook of
Electroporation
Handbook of Electroporation
Damijan Miklavčič
Editor
Handbook of
Electroporation
With 932 Figures and 125 Tables

Editor
Damijan Miklavčič
Faculty of Electrical Engineering
University of Ljubljana
Ljubljana, Slovenia
ISBN 978-3-319-32885-0 ISBN 978-3-319-32886-7 (eBook)

ISBN 978-3-319-32887-4 (print and electronic bundle)
DOI 10.1007/978-3-319-32886-7
Library of Congress Control Number: 2017946078
# Springer International Publishing AG 2017

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Foreword
In the familiar sense, a Foreword is to be a short informative introduction to a book

and its subject. Here, it is my intention to provide an escorting, in some parts slightly
more detailed, introduction to the ambitious enterprise of a Handbook of
Electroporation.
Already the list of contents is very impressive in the multitude of specific titles
under the common roof of a handbook addressing the subject electroporation.
Obviously it is the editors’ programmatic goal to establish a comprehensive com-
pendium of core knowledge on the concept of electroporation and electroporation-
based technologies both in general biomaterial science as well as in clinical
medicine.
The first book that contained the electroporation thematics appeared in 1989. This
book entitled “Electroporation and Electrofusion in Cell Biology and Biotechnol-
ogy” (eds. E. Neumann, A. E. Sowers and C. A. Jordan, Plenum) represents a
handbook-like collection of articles on various electric field phenomena and electric
field techniques commonly used in biophysics and biophysical chemistry and cell
biology. The book still is a rich source of information on the fundamentals and
technical applications of electric field effects in general biomaterial science.
In the program Handbook of Electroporation, the spectrum of specific titles
clearly reflects the appreciable extension of the original electroporation thematics.
It is finally appreciated and documented that electric field techniques are successfully
used in many other branches of bioelectric research and technology. Just a few
examples, bioelectrochemical methodologies are used in tackling biocorrosion and
biofouling in the pipes of the water distribution systems or of waste water flow or
transport of mineral oil. The electric field technologies applied in agriculture and in
particular in food processing, get increasingly into the focus of fundamental
bioelectrochemical research. All in all, the common goal of the cooperative strate-
gies is to work out perspectives to strengthen the scientific basis of these econom-
ically and socially important technologies. It is becoming increasingly visible that
the various electroporation biotechnologies are in progress towards scientific
maturity.
For a long time, the various electric field research groups were working more or
less in parallel and without common organisational roof. This situation began to
change when, in addition to the classical forum of meetings and conferences,
v
vi Foreword
communication was extended to student education and technical instruction in

interdisciplinary schools for teaching and technical instruction.
An outstanding highlight is the annual international school on “Electroporation-
Based Technologies and Treatments” in Ljubljana. Since the foundation of the
school in 2003 – in the beginning actively involved in science transfer and teaching,
now serving occasionally as an honorary consultant – I continue to appreciate the
enormous organizational efforts by the directors Damijan Miklavčič and Lluis Mir
and their extraordinary engaged coworkers. The Ljubljana School has developed to a
most effective forum for education in electric field pulse science and technology and
is highly esteemed by now all over the world.
At the latest at this stage, it appears appropriate to address the early scientific
foundations of the electroporation concept. Historically, the electric field effect
leading to uptake of DNA by mouse lyoma cells, was named in 1982 “electropora-
tion”, short for “electric pore formation”. When it was found that electric field pulses
together with alien DNA can be used to stably reprogram biological cells, familiar
electric field techniques gained enormous impetus for goal-directed cell
manipulations.
In the literature there is ongoing confusion on the nature of the electric field effect
in biomaterials; in particular, there is mixing up of cause and effect. For clarification,
the concept of electroporation inherently addresses the very structural basis of this
electric field effect. Elementary physics clearly specifies the electric field as the force
acting simultaneously on all charges. In membrane electroporation the targets of the
field force are the ionic and dipolar charges at the membrane-solution interface. Thus
the field action is essentially focused on the dipolar water molecules and the hydrated
ionic and dipolar lipid head groups.
From the beginning, the electric field effect causing electropores had been cast
into the most general thermodynamic frame of chemical mass action law and of van’t
Hoff relationships. Indeed, statistical ensemble thermodynamics can thus be used
constraintless for the analysis of electric field pulse data with the applied electric
field strength as the control variable. An essential key feature for deeper insight is the
classification of electroporation as a structural hysteresis phenomenon, manifested in
the cycle of rapid in-field electric pore formation and slower statistical post-field pore
annealing.
By definition, statistical thermodynamics is overall “physical chemical
reasoning”. Thus, the chemical thermodynamics of electroporation is not an
alternative “theory” competing with other theories. On the other hand, the
physical electroporation theories are using specific models. They are molecularly
detailed, but in essence they all must be consistent with the thermodynamic
fundamentals.
No doubt, the application of electric field pulses can lead to a variety of dramatic
alterations in functional membrane properties. Interestingly, the field effect was
originally documented in terms of transport quantities indicative for the extent of
the underlying structural changes. Most remarkable, and initially very estonishing,
has been the longevity of the induced permeability changes for small ions and even
Foreword vii
polyionic macromolecules that usually do not penetrate the unporated membrane

(electropermeabilisation). Of course, it is the long-lived structure which is respon-
sible for functional longevity.
Another important consequence of electroporation is manifested in the generation
of long-lived fusiogenicity of porated membranes. On the same structural line,
electroporation together with the concept of the hydrophilic (or inverted) pores is
the platform to rationalize the enormous acceleration of lipid flip-flop processes.
Finally, the enhancement of endocytotic and exocytotic processes is readily
rationalized by the larger mobility of the lipids in the pore wall and the larger
bending flexibility of the porous membrane.
Colloquially one may specify that electropulsation can “break the barrier func-
tion” of the lipid bilayer. In this context, the nondestructive electric field effect
causing the release of the intracellular catecholamine, ATP and Ca-ions in suspended
chromaffin granules was originally called “reversible dielectric breakdown”, in
1972. At higher field strength, chromogranin proteins are released and the organelle
suspension exhibits foaming; that indicated fragmentation of the cell membrane to
smaller vesicles.
Viewed afterwards, the nondestructive release of intracellular material may count
as the first example of reversible electroporation. In classical electrophysiology, the
destructive field effect experienced in a longer lasting voltage clamp of nerve
membranes had been called dielectric breakdown. This term was later also used to
name the membrane mechanism for the field effect of destroying plant protoplasts
and glycocalix-free bacteria. Nowadays the extraction of cell components by dis-
ruptive electroporation techniques (irreversible electroporation) has reached indus-
trial dimensions
Returning to the enterprise of a Handbook of Electroporation, the single contri-
butions to the book do not directly indicate that electroporation is indeed the
integrative concept.
A particular visible sign of successful integration, however, is the recently
founded International Society for Electroporation-Based Technologies and Treat-
ments. Beyond the formal official integration level, there is unanimous consensus in
the “electroporation community” in the appreciation of Damijan Miklavčič’s per-
sonal engagement and extraordinary skill in integrating the various research groups
working with electric field pulses in biomaterial science and technology. In my
personal opinion, Damijan has developed over the years to the spiritus rector of
electroporation. He plays a leading role in electroporation research and technical
development as well as education and technical instruction. Last but not least, the
merits in propagation and communication of the electroporation thematics predesti-
nate him especially for the responsible task of Editor-in-Chief of the enterprise
Handbook of Electroporation.
Despite the already visible success, the integrative efforts to establish electropo-
ration as the unifying (thermodynamic and technical) bioelectric concept will con-
tinue to be a great challenge for the future. No doubt, what has been achieved so far
is admirable and encouraging to go ahead.
viii Foreword
Finally, enjoy the book, it gives you a sense of the quality of people involved in
this demanding enterprise.
With privilege and pleasure I wish good luck.
Department of Physical and

Biophysical Chemistry Eberhard Neumann
Faculty of Chemistry
University of Bielefeld
Germany
Preface
Electroporation is a word most often used to describe the phenomenon of membrane

increased permeability due to cell exposure to electric field. Other words in describ-
ing this phenomenon and applications of pulsed electric fields that rely on it like PEF
(pulsed electric field) treatment, electropermeabilization, electrochemotherapy, gene
electrotransfection and electrotransformation, electrochemoembolization, etc., are
used in the literature interchangeably. In spite of the fact that we are only now
starting to understand the details of electric field effects on the membrane level and
the complexity of the cell’s and tissue responses following the electroporation, in the
past decades several therapies and treatments based on electroporation were devel-
oped and are used in medicine, food processing, biotechnology, etc. Although we are
still far from electroporation being fully exploited and used to its full potential we
can and should start to consider electroporation as a platform technology.
The knowledge which was developed in different fields of science is fragmented,
scattered through different books and journals. I believe the progress and further
development will be greatly facilitated providing a single entry to the researchers and
users of electroporation. When we started to organize the Electroporation-Based
Technologies and Treatments Postgraduate School and International Workshop
(www.ebtt.org) in Ljubljana with Lluis Mir in 2003 as co-directors, we did not
even imagine that the school will be organized ten times. The program has been
meticulously developed throughout these years by the faculty members who served
as the “program committee”: Veronique Préat, Eberhard Neumann, Justin Teissie,
and Gregor Serša; later P. Thomas Vernier and Mounir Tarek joined us; and recently
also Marie-Pierre Rols and Julie Gehl. At the beginning it was organized biannually,
and since 2011 it is organized annually.
What we were realizing over and over again when discussing program for the
next year was that the knowledge which students should get familiar with is
overwhelming and is spanning from physics, chemistry, engineering, medicine,
biology, etc., and that we are not able to offer this knowledge by ourselves, so we
were inviting more and more of our colleagues who could bring to all of us new
knowledge, and we all learned every year – us the faculty members and teachers as
well as students. The next thing that we realized was that the knowledge and
understanding of electroporation is far from being complete, that it is very dynamic
and needs a dynamic text book – not something static. And this was the motivation
ix
x Preface
which finally resulted in this Handbook of Electroporation. The list of chapters is

extensive and seems exhaustive, but I am afraid I have to admit we are short of
several chapters – some of which we already identified (both the thematic and their
respective authors) and some are yet to be identified – and I promise they will be
included in the dynamic online version of the Handbook as soon as possible and in
the 2nd and future editions of the Handbook.
The list of topics in different sections were developed in collaboration with
section editors who all are respected members of the community and have personally
contributed to the development and advances in the electroporation and
electroporation-based technologies and treatments. I have been privileged that
these great researchers (many of whom I admired already at the beginning of my
scientific carrier) were willing to work with me on this gigantic project – the
Handbook of Electroporation. Dietrich Knorr, Boris Rubinsky, Eugene Vorobiev,
P. Thomas Vernier, Javier Raso, Richard Heller, Juergen Kolb, Marie-Pierre Rols,
and Gregor Serša with whom we started our joint scientific journey 30 years ago
after he returned to Ljubljana from MD Anderson, Houston. With all of them
onboard the project was predestined to be a success, and I would cordially like to
thank each and all of them for sharing the work, ideas, and vision with me. But also
for inviting and mobilizing authors to write their chapters, reviewing them, and
sometimes bugging the authors and asking to finish their chapters on time.
The Handbook of Electroproation is a Final Dissemination Activity of the COST
TD1104 (www.electroporation.net), and it is more than appropriate to express our
appreciation to COST organization for the support in preparing it, and as this is the
final dissemination activity it also seems appropriate to acknowledge all members
and group leaders and workshop organizers who have helped to develop this
community, which resulted in establishing the International Society for
Electroporation-Based Technologies and Treatments.
Through almost three decades of research in the area of electroporation, I have
had a pleasure, joy, and privilege to work with many collaborators in the Laboratory
of Biocybernetics, which I am heading for the past 20 years, and many people and
labs from Slovenia and abroad. I have constantly enjoyed the support of the home
institution as well as granting agencies, most notably Research Agency of the
Republic of Slovenia, which allowed me to test new ideas and develop them further,
some even to products. University of Ljubljana at the same time has been a great
place where truly interdisciplinary research could be done.
On a more personal note, in all these years I have devoted to the field of
electroporation I had a constant support and understanding of my wife Atina and
my three sons, who because of my work and dedication to research had less of their
husband and father than they would like or sometimes would need.
Finally, I would like to express my sincere appreciation also to Springer Refer-
ence Editorial Team and in particular to Sarah Mathews and Audrey Wong for their
understanding and help in developing this Handbook of Electroporation.
Preface xi
Thanks, without all of you, it would not be possible. And because of you, the new
generations will have easier start and greater chance to make fewer mistakes in
research than we did.
Faculty of Electrical Engineering Damijan Miklavčič

Ljubljana, Slovenia
Acknowledgements
COST (European Cooperation in Science and Technology) is a pan-European

intergovernmental framework. Its mission is to enable breakthrough scientific and
technological developments leading to new concepts and products and thereby
contribute to strengthening Europe’s research and innovation capacities. It allows
researchers, engineers, and scholars to jointly develop their own ideas and take new
initiatives across all fields of science and technology, while promoting multi- and
interdisciplinary approaches. COST aims at fostering a better integration of less
research intensive countries to the knowledge hubs of the European Research Area.
The COST Association, an international not-for-profit association under Belgian
Law, integrates all management, governing, and administrative functions necessary
for the operation of the framework. The COST Association has currently 36 member
countries (www.cost.eu).
The Handbook of Electroporation has been prepared as a collective effort and
final dissemination activity of the COST TD1104 Action: European network for
development of electroporation-based technologies and treatments (www.electropo
ration.net), supported by COST.
COST is supported by
the EU Framework Programme
Horizon 2020
xiii
Contents
Volume 1
Part I Fundamental Mechanisms of Electroporation: Models and

Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
P. Thomas Vernier
1 Lipid Pores: Molecular and Continuum Models .............. 3
Lea Rems
2 Critical Electric Field and Transmembrane Voltage for Lipid Pore
Formation in Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Justin Teissie
3 Phospholipid Head Group Dipoles and Electropore
Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Justin Teissie
4 Membrane Permeabilization Lifetime in Experiments . . . . . . . . . . 61
Justin Teissie
5 Lipid Electropore Stabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
M. Laura Fernández and Marcelo R. Risk
6 Molecular Transmembrane Transport with Giant Unilamellar
Vesicles (GUVs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Marie-Pierre Rols
7 Lipid Electropore Lifetime in Molecular Models . . . . . . . . . . . . . . 113
Zachary A. Levine
8 Effects of Heterogeneous Membranes and Electrolytes on
Electropore Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Zachary A. Levine
9 Lipid Electropore Geometry in Molecular Models . . . . . . . . . . . . . 155
Paolo Marracino, P. Thomas Vernier, M. Liberti, and F. Apollonio
xv
xvi Contents
10 Electropermeabilization and Electrostimulation by Picosecond

Pulses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Iurii Semenov and Shu Xiao
11 Experimental Determination of Lipid Electropore Size . . . . . . . . . 187
Elena C. Gianulis and Andrei G. Pakhomov
12 Measurement of Molecular Transport After
Electropermeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Esin B. Sözer and P. Thomas Vernier
13 Electrical Conductance of Lipid Pores . . . . . . . . . . . . . . . . . . . . . . 219
Maura Casciola and Mounir Tarek
14 Electrodeformation, Electroporation, and Electrofusion of Giant
Unilamellar Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Rumiana Dimova and Karin A. Riske
Part II Basic Description of Cell Electroporation in Vitro . . . . . . . . 253

Marie-Pierre Rols
15 3D Tissue Models to Bridge the Gap Between Cell Culture and
Tissue in Assessing Electroporation . . . . . . . . . . . . . . . . . . . . . . . . 255
Laure Gibot
16 Single Cell Electrical Characterization Techniques . . . . . . . . . . . . 271
Olivier Français and Bruno Le Pioufle
17 Cell Stress Responses to Pulsed Electric Fields ............... 289
Ken-ichi Yano and Keiko Morotomi-Yano
18 Effects of Electroporation of Mammalian Cells on Cytoskeleton
and Intercellular Connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
Maja Čemažar
19 Nucleic Acid Electrotransfer in Mammalian Cells: Mechanistic
Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
Muriel Golzio and Marie-Pierre Rols
20 Different Cell Sensitivity to Pulsed Electric Field . . . . . . . . . . . . . . 337
Olga Pakhomova, Elena C. Gianulis, and Andrei G. Pakhomov
21 Electric Pulse Repetition Rate: Sensitization and
Desensitization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
Claudia Muratori, Maura Casciola, and Olga Pakhomova
22 Lightning-Triggered Electroporation as a Mechanism for
Horizontal Gene Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
Tadej Kotnik
Contents xvii
23 Gene Delivery by Electroporation In Vitro: Mechanisms ....... 387

Marie-Pierre Rols
24 Electroporation of Biofilms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403

Flavien Pillet
25 Bacterial Cell Envelopes: Composition, Architecture, and

Origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
Didier Zerbib
26 3D Culture Models to Assess Tissue Responses to

Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437
Anna A. Bulysheva and Richard Heller
27 Mechanistic Description of Membrane

Justin Teissie
28 Involvement of Reactive Oxygen Species in Membrane

Justin Teissie
29 Plant Cell Wall: Description, Role in Transport, and Effect

of Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489
Cristèle Delsart
30 Regulated and Apoptotic Cell Death After Nanosecond

Stephen J. Beebe
Part III Electroporation Phenomena on Tissue Level and

Systemic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529
Boris Rubinsky
31 Electroporation in Scars/Wound Healing and Skin Response . . . . 531

Laure Gibot and Alexander Golberg
32 Principles and Use of Magnetic Resonance Electrical Impedance

Tomography in Tissue Electroporation . . . . . . . . . . . . . . . . . . . . . 549
Eung Je Woo and Matej Kranjc
33 Current Density Imaging as Means to Follow Tissue

Igor Serša and Franci Bajd
34 Mass Transfer of Electrolytic Species During Electric

Field-Based Tumor Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
Guillermo Marshall
xviii Contents
35 Effects of Reversible and Irreversible Electroporation on

Endothelial Cells and Tissue Blood Flow . . . . . . . . . . . . . . . . . . . . 607
Bostjan Markelc, Maja Čemažar, and Gregor Serša
36 Biophysics and Metrology of Electroporation in Tissues . . . . . . . . 621
Uwe Pliquett
37 Selective Extraction of Molecules from Biomaterials by Pulsed
Electric Field Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 655
Eugène Vorobiev and Nikolai Lebovka
38 Assessment of Electroporation by Electrical Impedance
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 671
Quim Castellví, Borja Mercadal, and Antoni Ivorra
39 Blood Flow Modifying and Vascular-Disrupting Effects of
Electroporation and Electrochemotherapy . . . . . . . . . . . . . . . . . . . 691
Gregor Serša, Maja Čemažar, and Bostjan Markelc
40 Tissue Ablation by Irreversible Electroporation . . . . . . . . . . . . . . 707
Andrea Rolong, Boris Rubinsky, and Rafael V. Davalos
41 Diffusion Weighted Magnetic Resonance Imaging for Detection
of Tissue Electroporation In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . 723
Faisal Mahmood and Rasmus Hvass Hansen
42 Effect of Electroporation on Blood-Brain Barrier . . . . . . . . . . . . . 745
Shirley Sharabi and Yael Mardor
43 Medical Imaging of Electroporation . . . . . . . . . . . . . . . . . . . . . . . . 761
Eisuke Ueshima, Govindarajan Srimathveeravalli, and
Stephen B. Solomon
44 Immunological Response to Electroporation ................. 777
Bashir Akhavan Tafti and Stephen T. Kee
Volume 2
Part IV Hardware Used in Electroporation Experiments and

Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 791
Juergen F. Kolb
45 Marx-Generator Design and Development for Biomass
Martin Sack
46 Measurement and Characterization of Exposure Systems for
High-Frequency, Ultrashort Pulses . . . . . . . . . . . . . . . . . . . . . . . . . 813
Philippe Leveque, Rodney O’Connor, and Delia Arnaud-Cormos
Contents xix
47 Photoconductive Switching for Pulsed High-Voltage

Generators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837
Delia Arnaud-Cormos, Vincent Couderc, and Philippe Leveque
48 Basic Concepts of High-Voltage Pulse Generation . . . . . . . . . . . . . 859
Luís M. S. Redondo
49 Industrial Pulsed Electric Field Systems . . . . . . . . . . . . . . . . . . . . . 881
Michael A. Kempkes
50 Compact Nanosecond Pulse Generator Based on Printed Circuit
Board Transmission Lines and Solid-State Switches . . . . . . . . . . . 903
Yan Mi, Jialun Wan, and Yanyuan Zhang
51 Cliniporator: Medical Electroporation of Tumors . . . . . . . . . . . . . 927
Claudio Bertacchini
52 Development of Pulsed Antennas for Noninvasive Exposures . . . . 961
Shu Xiao
53 Beyond Electroporation Pulse Parameters: From Application
to Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 977
Matej Reberšek
Part V Modeling of Electroporation and Related Phenomena . . . 999

Damijan Miklavčič
54 Treatment Planning for Electrochemotherapy and Irreversible
Electroporation of Deep-Seated Tumors . . . . . . . . . . . . . . . . . . . . . 1001
Bor Kos
55 Different Approaches Used in Modeling of Cell Membrane
Clair Poignard, Aude Silve, and Lars H. Wegner
56 Electric Field Distribution and Electroporation Threshold . . . . . . 1043
Matej Kranjc and Damijan Miklavčič
57 Modeling of Electrochemical Reactions During Pulsed Electric
Field Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1059
Gianpiero Pataro, Giorgio Donsì, and Giovanna Ferrari
58 Modeling Transport Across the Electroporated Membrane . . . . . . 1089
Miao Yu and Hao Lin
59 Transmembrane Voltage Induced by Applied Electric Fields . . . . 1111
Tadej Kotnik
60 Mathematical Models of Pulsed Electric Field Treatment of
Plant Tissues and Simulation of Related Phenomena . . . . . . . . . . . 1129
Nikolai Lebovka and Eugène Vorobiev
xx Contents
61 Dual-Porosity Model of Liquid Extraction by Pressing from

Plant Tissue Modified by Electroporation . . . . . . . . . . . . . . . . . . . 1153
Samo Mahnič-Kalamiza
62 Computational Approach for Electrical Analysis of Biological
Tissue Using the Equivalent Circuit Model . . . . . . . . . . . . . . . . . . 1179
Airton Ramos and Daniela O. H. Suzuki
63 Mathematical Models Describing Cell Death Due to
Janja Dermol and Damijan Miklavčič
64 Modeling Transdermal Delivery by Electroporation:
The Thermodynamic Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . 1219
Sid Becker
65 Application of Numerical Simulation Techniques for Modeling
Pulsed Electric Field Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . 1237
Thies Wölken, Johannes Sailer, Francisco Daniel Maldonado-Parra,
Tobias Horneber, and Cornelia Rauh
66 Modeling Microbial Inactivation by Pulsed Electric Field . . . . . . . 1269
Micha Peleg
67 Cell Electroporation Model Using Voronoi Networks . . . . . . . . . . 1287
Qin Hu and Ravindra P. Joshi
Part VI Laboratory Techniques Used in Electroporation

Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1303
Marie-Pierre Rols, Boris Rubinsky, and Javier Raso
68 Fluorescent Indicators of Membrane Permeabilization Due to
Tina Batista Napotnik
69 Atomic Force Microscopy to Explore Electroporation Effects
on Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1325
Etienne Dague
70 Electroporation in Microfluidic Devices . . . . . . . . . . . . . . . . . . . . . 1339
Séverine Le Gac and Iris van Uitert
71 Detection of Electroporation in Microbial Cells: Techniques
and Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1359
Diego García-Gonzalo and Rafael Pagán
72 The Use of Electroporation in Developmental Biology . . . . . . . . . . 1375
Charlie Gosse, Xuan Zhao, I. Migeotte, Diana Suárez-Boomgaard,
Isabelle Hue, Séverine Degrelle, Aitana Perea-Gomez, and E. Mazari
Contents xxi
73 Different Cell Viability Assays Following Electroporation

In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1411
Saulius Šatkauskas, Baltramiejus Jakštys, Paulius Ruzgys, and
Milda Jakutavičiūt_e
74 Patch Clamp in Use of Electroporation Mechanisms Studies . . . . 1425

Lars H. Wegner
75 Parameters Affecting Cell Viability Following Electroporation

In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1449
Marie-Pierre Rols
76 Techniques to Detect Electroporation in Food Tissues . . . . . . . . . . 1467

77 Determination of Pulsed Electric Fields Effects on the Structure

of Potato Tubers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1489
Indrawati Oey, Farnaz Faridnia, Sze Ying Leong, David J. Burritt, and
Tingting Liu
Volume 3
Part VII Preclinical Development for Biomedical Application of

Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1509
Richard Heller and Gregor Serša
78 Preclinical Studies on Electrochemotherapy . . . . . . . . . . . . . . . . . . 1511

Gregor Serša, Masa Bosnjak, Maja Čemažar, and Richard Heller
79 Preclinical Studies on Irreversible Electroporation . . . . . . . . . . . . 1527

Suyashree Bhonsle, Robert N. Neal II, and Rafael V. Davalos
80 Preclinical Studies on Nanosecond Pulses . . . . . . . . . . . . . . . . . . . . 1543

Stephen J. Beebe
81 Principles of Electroporation for Gene Therapy . . . . . . . . . . . . . . 1563

Loree C. Heller
82 Tissue Engineering with Electroporation . . . . . . . . . . . . . . . . . . . . 1579

Mary Phillips Ho
83 Predictable Animal Models for Translational

Electroporation-Based Cancer Immunotherapy Studies . . . . . . . . 1601
Mariangela De Robertis, Ursa Lampreht Tratar, Maja Čemažar,
and Emanuela Signori
84 Antiangiogenic Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1623

Maja Čemažar, Tanja Dolinsek, Masa Bosnjak, and Gregor Serša
xxii Contents
85 New Drugs for Electrochemotherapy with Emphasis on

Calcium Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1637
Stine Krog Frandsen, Hanne Falk Hansen, and Julie Gehl
86 Gene Transfer to the Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1651
Anita Gothelf
87 Gene Electrotransfer for Ischemic Tissue . . . . . . . . . . . . . . . . . . . . 1665
88 Cochlear Implant Close-Field Electroporation . . . . . . . . . . . . . . . . 1679
Gary D. Housley, Cherylea J. Browne, Edward N. Crawford,
Matthias Klugmann, Nigel H. Lovell, and Jeremy L. Pinyon
89 Combined Treatment of Electrochemotherapy with
Irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1699
Simona Kranjc, Urska Kamensek, Maja Čemažar,
and Gregor Serša
90 Combined Treatment of Electrochemotherapy with
Immunomodulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1717
Vesna Todorovic and Maja Čemažar
91 Combining Electrolysis and Electroporation for Tissue
Ablation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1733
Mary Phillips Ho
92 Delivery of Cytokines Using Gene Electrotransfer . . . . . . . . . . . . . 1755
Shawna A. Shirley
Part VIII Biomedical Applications of Electroporation . . . . . . . . . . . 1769

Gregor Serša and Richard Heller
93 Electrochemotherapy and Its Clinical Applications . . . . . . . . . . . . 1771
Julie Gehl and Gregor Serša
94 Tissue Ablation Using Nanosecond Electric Pulses . . . . . . . . . . . . 1787
Richard Nuccitelli
95 International Network for Sharing Practices on
Electrochemotherapy (InspECT): An Integrative
Patients Treatment Consortium . . . . . . . . . . . . . . . . . . . . . . . . . . . 1799
Matteo Brizio, Simone Ribero, Luca Giovanni Campana,
A. James P. Clover, Julie Gehl, Christian Kunte,
Francesca De Terlizzi, and Pietro Quaglino
96 Electrochemotherapy of Cutaneous Metastases . . . . . . . . . . . . . . . 1817
Marko Snoj and Louise Wichmann Matthiessen
97 Electrochemotherapy of Basal Cell Carcinoma . . . . . . . . . . . . . . . 1831
A. James P. Clover, Johnny M. O’Mahony, and Declan Soden
Contents xxiii
98 Electrochemotherapy of Liver Tumors: Colorectal Liver

Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1843
Ibrahim Edhemovic and Erik Brecelj
99 Electrochemotherapy of Primary Liver Tumors . . . . . . . . . . . . . 1855
Blaž Trotovšek and Mihajlo Đokić
100 Electrochemotherapy of Locally Advanced Pancreatic
Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1871
Vincenza Granata, Maddalena Leongito, Roberta Fusco,
Mauro Piccirillo, Raffaele Palaia, Secondo Lastoria,
Antonella Petrillo, and Francesco Izzo
101 Minimally Invasive Treatment by Electroporation: Remaining
Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1887
Ruggero Cadossi, D’Alessio Valeria, Fusco Roberta,
and Mattia Ronchetti
102 Electrochemotherapy of Head and Neck Cancer . . . . . . . . . . . . . 1903
Marco Benazzo, Giulia Bertino, and Ales Groselj
103 Adjuvant Immunotherapy as a Tool to Boost Effectiveness
of Electrochemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1917
Urska Kamensek, Spela Kos, and Gregor Serša
104 Clinical Use of DNA Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1933
Li Liu, Matthew P. Morrow, and Mark Bagarazzi
105 Electrochemotherapy in Veterinary Oncology . . . . . . . . . . . . . . . 1953
Nataša Tozon, Youssef Tamzali, and Maja Čemažar
106 Electrochemotherapy and Gene Electrotransfer in Veterinary
Oncology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1969
Darja Pavlin, Maja Čemažar, Gregor Serša, Youssef Tamzali,
and Nataša Tozon
107 Cryosurgery and Irreversible Electroporation: The State of
the Art, Advantages, and Limitations . . . . . . . . . . . . . . . . . . . . . . 1985
Giuseppe Mucciardi, Carlo Magno, Antonino Inferrera,
and Franco Lugnani
108 Irreversible Electroporation Techniques in the Treatment of
Locally Advanced Liver and Pancreatic Cancer . . . . . . . . . . . . . 2001
Robert C. G. Martin
109 Safety of Clinical Irreversible Electroporation . . . . . . . . . . . . . . . 2017
K. R. Thomson, Helen Kavnoudias, and Robert N. Neal II
110 Irreversible Electroporation in Treatment of Prostate Cancer . . . 2037
Michael K. Stehling, Enric Guenther, and Nina Klein
xxiv Contents
111 Electrochemotherapy for Breast Cancer . . . . . . . . . . . . . . . . . . . . 2075

Luca Giovanni Campana, Sara Galuppo, Roberto Marconato,
and Louise Wichmann Matthiessen
112 Electrochemotherapy for Superficially Metastatic Melanoma . . . 2095
Luca Giovanni Campana, Matteo Sepulcri, Sara Valpione,
Luigi Corti, and Carlo Riccardo Rossi
Part IX Electroporation in Biotechnology . . . . . . . . . . . . . . . . . . . . 2113

Javier Raso
113 Effect of Pulsed Electric Fields on Food Constituents . . . . . . . . . 2115
Eldevan S. Silva, Shahin Roohinejad, Mohamed Koubaa,
Francisco J. Barba, Anet Režek Jambrak, Tomislava Vukušić,
Mauro D. Santos, Rui P. Queirós, and Jorge A. Saraiva
114 Pulsed Electric Fields in Combination with Vacuum
Impregnation for Improving Freezing Tolerance
of Vegetables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2135
Federico Gómez Galindo and Katarzyna Dymek
115 Electroporation-Based Applications in Biotechnology . . . . . . . . . 2153
Saša Haberl Meglič and Tadej Kotnik
116 Potential Application of Pulsed Electric Fields for Improving
Extraction of Plant Pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2171
Mustafa Fincan
117 Pulsed Electric Fields Bioproduction of Secondary Metabolites
in Plant Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2193
Pedro Elez-Martínez, Robert Soliva-Fortuny,
and Olga Martín-Belloso
118 Microalgal Biorefinery for Bulk and High-Value Products:
Product Extraction Within Cell Disintegration . . . . . . . . . . . . . . 2205
P.R. Postma, G.P. ‘t Lam, M.J. Barbosa, R.H. Wijffels,
M.H.M. Eppink, and Giuseppe Olivieri
119 Pulsed Electric Fields Effects on Health-Related Compounds
and Antioxidant Capacity of Tomato Juice . . . . . . . . . . . . . . . . . . 2225
Isabel Odriozola-Serrano, Robert Soliva-Fortuny,
and Olga Martín-Belloso
120 Pulsed Electric Fields for Inactivation of Endogenous Enzymes
in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2239
Wei Zhao and Ruijin Yang
Contents xxv
121 Pulsed Electric Fields-Assisted Extraction of Molecules from

Bacterial and Yeast Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2253
Saša Haberl Meglič
122 Pulsed Electric Fields for Pasteurization: Defining Processing

Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2271
Tony Z. Jin
123 Pulsed Electric Fields-Assisted Acetylation of Starch . . . . . . . . . . 2297

Zhong Han, Jing Hong, and Xin-An Zeng
124 Pulsed Electric Fields for Algal Extraction and Predator

Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2317
Michael A. Kempkes
Volume 4
Part X Electroporation in Food Science and Technology ....... 2333

Dietrich Knorr
125 Pulsed Electric Fields for Food Industry: Historical

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2335
Werner Sitzmann, Eugène Vorobiev, and Nikolai Lebovka
126 Advantages of Pulsed Electric Field Use for Treatment

of Algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2355
Marcos Teotonio Pereira, Duarte S. Rego, and Luis M. S. Redondo
127 Impact of Pulsed Electric Fields on Enzymes . . . . . . . . . . . . . . . . 2369

Mahesha M. Poojary, Shahin Roohinejad, Mohamed Koubaa,
Francisco J. Barba, Paolo Passamonti, Anet Režek Jambrak,
Indrawati Oey, and Ralf Greiner
128 Impact of Pulsed Electric Field Treatment on Must and Wine

Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2391
Lucía González-Arenzana, Javier Portu Reinares, Noelia López,
Pilar Santamaría, Teresa Garde-Cerdán, Ana Rosa Gutiérrez,
Isabel López-Alfaro, and Rosa López
129 Pulsed Electric Fields for Extraction of Secondary Metabolites

from Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2407
Nigel. P. Brunton and Elisa Luengo
130 Modification of Plant Biopolymers by Pulsed Electric Fields . . . . 2423

Xin-An Zeng and Jing Hong
xxvi Contents
131 Pulsed Electric Fields as Pretreatment for Subsequent Food

Process Operations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2439
Artur Wiktor and Dorota Witrowa-Rajchert
132 Pulsed Electric Fields Effects on Meat Tissue Quality and
Functionality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2455
Amali U. Alahakoon, Farnaz Faridnia, Phil J. Bremer,
Pat Silcock, and Indrawati Oey
133 Pulsed Electric Field Treatment for Beverage Production and
Preservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2477
Gulsun Akdemir Evrendilek
134 Pulsed Electric Field Treatment for Fruit and Vegetable
Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2495
Eduardo Puértolas, Guillermo Saldaña, and Javier Raso
135 Recovery of Primary and Secondary Plant Metabolites by
Pulsed Electric Field Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . 2517
Ankit Patras, Pintu Choudhary, and Ashish Rawson
136 Stress Induction and Response, Inactivation, and Recovery
of Vegetative Microorganisms by Pulsed Electric Fields . . . . . . . 2539
Felix Schottroff, Anna Krottenthaler, and Henry Jäger
137 Stress Response of Plants, Metabolite Production due to
Pulsed Electric Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2559
Ana Balasa
138 Application of Pulsed Electric Field Treatment for Food
Waste Recovery Operations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2573
Mahesha M. Poojary, Shahin Roohinejad, Francisco J. Barba,
Mohamed Koubaa, Eduardo Puértolas, Anet Režek Jambrak,
Ralf Greiner, and Indrawati Oey
139 Pulsed Electric Fields in Hurdle Approaches for Microbial
Inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2591
Cristina Arroyo and James G. Lyng
140 Responses of Plant Cells and Tissues to Pulsed Electric Field
Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2621
Federico Gómez Galindo
141 Application of Pulsed Electric Field for Treatment of Fish and
Seafood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2637
Guidan He, Yongguang Yin, Xiaoxia Yan, and Ying Wang
142 Pulsed Electric Fields Treatment in Food Technology:
Challenges and Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2657
Henry Jäger and Dietrich Knorr
Contents xxvii
Part XI Electroporation in Bioeconomy and Biorefinery

Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2681
Eugène Vorobiev
143 Pulsed Electric Fields and High-Voltage Electrical

Discharge-Assisted Extraction of Biocompounds from
Vine Shoots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2683
Hiba N. Rajha, Nadia Boussetta, Nicolas Louka, Richard
G. Maroun, and Eugène Vorobiev
144 Application of Pulsed Electric Energies for Oil and Polyphenol

Extraction from Sesame Cake and Sesame Seeds . . . . . . . . . . . . . 2699
Julia Ribeiro Sarkis, Nadia Boussetta, and Eugène Vorobiev
145 Pulsed Electric Energy-Assisted Extraction of Polyphenols

from Flaxseed Hulls and Cake . . . . . . . . . . . . . . . . . . . . . . . . . . . 2713
Nadia Boussetta and Eugène Vorobiev
146 Polyphenol and Protein Extraction from Rapeseed Stems and

Leaves Assisted by Pulsed Electric Fields . . . . . . . . . . . . . . . . . . . 2733
Xiaoxi Yu, Olivier Bals, Nabil Grimi, and Eugène Vorobiev
147 Selective Extraction of Biocompounds from Stevia rebaudiana

Bertoni Leaves Using Electrotechnologies . . . . . . . . . . . . . . . . . . 2751
Francisco J. Barba, Mohamed Koubaa, Nabil Grimi, and
Eugène Vorobiev
148 Pulsed Electric Fields-Assisted Extraction from Exotic Fruit

Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2763
Francisco J. Barba, Oleksii Parniakov, Mohamed Koubaa,
and Nikolai Lebovka
149 Application of Pulsed Electric Energy for Grape Waste

Biorefinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2781
150 High-Voltage Electrical Discharge-Assisted Extraction of

Phenolic Compounds from Grape Seeds . . . . . . . . . . . . . . . . . . . . 2799
Pierre Adda, Nadia Boussetta, O. Lesaint, and Eugène Vorobiev
151 Pulsed Electric Fields in Wineries: Potential Applications . . . . . . 2825

Guillermo Saldaña, Elisa Luengo, Eduardo Puértolas,
Ignacio Álvarez, and Javier Raso
152 Application of Pulsed Electric Energy for Lignocellulosic

Biorefinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2843
xxviii Contents
153 Pulsed Electric Energy-Assisted Biorefinery of Oil Crops and

Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2863
154 Pulsed Electric Fields and High-Voltage Electrical
Discharges-Assisted Extraction of Valuable Biocompounds
and Biopolymers from Rapeseed By-Products . . . . . . . . . . . . . . . 2883
Marwa Brahim, Francisco J. Barba, Nadia Boussetta,
Nicolas Brosse, and Eugène Vorobiev
155 Application of Pulsed Electric Fields for Root and Tuber
Crops Biorefinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2899
156 Pulsed Electric Fields and Electroporation Technologies in
Marine Macroalgae Biorefineries . . . . . . . . . . . . . . . . . . . . . . . . . 2923
Arthur Robin and Alexander Golberg
157 Pulsed Electric Field-Assisted Extraction of Pigments from
Chlorella vulgaris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2939
Elisa Luengo and Javier Raso
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2955
About the Editor
Damijan Miklavčič
Faculty of Electrical Engineering
Ljubljana, Slovenia
Education
1987 B.S. Electrical Engineering (University of Ljubljana)

1991 M.S. Electrical Engineering (University of Ljubljana)
1993 Ph.D. Electrical Engineering (University of Ljubljana)
Position
1993–present Faculty of Electrical Engineering, University of Ljubljana

1993–1994 Associate Researcher
1994–1997 Assistant Professor
1995–1996 Visiting Researcher at the Institut Gustave Roussy, Villejuif, France
1997–2002 Associate Professor
1998–present Head of the Laboratory of Biocybernetics
2002–present Professor
2003–2005 Vice-Dean for Research
2007–2015 Head of the Department of Biomedical Engineering
2015–present Deputy Head of the Department of Biomedical Engineering
xxix
xxx About the Editor
Awards
1986 Preseren Award for Students, University of Ljubljana

1989 International Award of the Mapfre Foundation (Spain)
1993 National Industrial Award of Krka Pharmaceuticals
1995 Award of the Republic of Slovenia for Scientific and Research Achievements
2003 Ambassador of the Republic of Slovenia in Science
2008 Vodovnik Prize for Exceptional Research
2008 Knight of the Order of Academic Palms of the French Republic
2012 IFMBE Vladimir K. Zworykin Award
2012 Rector’s Award for Innovations of the University of Ljubljana – 1st Prize
2014 Golden Plaque of the University of Ljubljana
2015 Fellow of the European Alliance for Medical and Biomedical Engineering and
Science
Bibliography
Prof. Miklavčič has authored or coauthored over 380 original articles in scientific
journals. His works have been cited over 6800 times (Source: WoS – self-citations
excluded).
Additional Activities
• Member of the Council of the Bioelectrochemical Society (2009–)

• President of the Slovenian Society for Engineering in Medicine and Biology
(2007–)
• President of the International Society for Electroporation-Based Technologies
and Treatments (2016–)
• Chairman of the Scientific Advisory Board of the Slovenian Forum on
Non-Ionizing Electromagnetic Radiation
• Associate Editor of Bioelectromagnetics (2005–2007)
• Associate Editor of IEEE Transactions on Biomedical Engineering (2002–2014)
• Associate Editor of IEEE Transactions on Nanobioscience (2004–2013)
• Member of the working group for preparation of the safety standards related to
nonionizing radiation at Slovenian National Standardization Office
• Referee for over 60 SCI-indexed journals
• Member of the Council of Science and Technology of the Republic of Slovenia
(2010–2013)
Section Editors
Part I: Fundamental Mechanisms of Electroporation: Models

and Experiments
P. Thomas Vernier Frank Reidy Research Center for Bioelectrics, Old Dominion
University, Norfolk, VA, USA
pvernier@odu.edu
Part II: Basic Description of Cell Electroporation In Vitro
Marie-Pierre Rols Institut de Pharmacologie et de Biologie Structurale, IPBS/CNRS

UMR 5089 and University of Toulouse, Toulouse, France
marie-pierre.rols@ipbs.fr
Part III: Electroporation Phenomena on Tissue Level and Systemic

Effects
Boris Rubinsky Department of Mechanical Engineering, University of California,

Berkeley, Berkeley, CA, USA
rubinsky@berkeley.edu
Part IV: Hardware Used in Electroporation Experiments

and Applications
Juergen F. Kolb Leibniz Institute for Plasma Science and Technology, Greifswald,
Germany
juergen.kolb@inp-greifswald.de
xxxi
xxxii Section Editors
Part V: Modeling of Electroporation and Related Phenomena
Damijan Miklavčič Faculty of Electrical Engineering, University of Ljubljana,

Ljubljana, Slovenia
damijan.miklavcic@fe.uni-lj.si
Part VI: Laboratory Techniques Used in Electroporation Research
Marie-Pierre Rols Institut de Pharmacologie et de Biologie Structurale, IPBS/

CNRS UMR 5089 and University of Toulouse, Toulouse, France
marie-pierre.rols@ipbs.fr

rubinsky@berkeley.edu
Javier Raso Department of Animal Production and Food Science, Tecnología de los
Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain
jraso@unizar.es
Part VII: Preclinical Development for Biomedical Application

of Electroporation
Richard Heller Frank Reidy Research Center for Bioelectrics, Old Dominion
rheller@odu.edu
Gregor Serša Department of Experimental Oncology, Institute of Oncology

Ljubljana, Ljubljana, Slovenia
gsersa@onko-i.si
Part VIII: Biomedical Applications of Electroporation
Gregor Serša Department of Experimental Oncology, Institute of Oncology Lju-

bljana, Ljubljana, Slovenia
gsersa@onko-i.si
rheller@odu.edu
Section Editors xxxiii
Part IX: Electroporation in Biotechnology
Javier Raso Department of Animal Production and Food Science, Tecnología de los
Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain
jraso@unizar.es
Part X: Electroporation in Food Science and Technology
Dietrich Knorr Department of Food Biotechnology and Food Process Engineering,

Technische Universität Berlin, Berlin, Germany
dietrich.knorr@tu-berlin.de
Part XI: Electroporation in Bioeconomy and Biorefinery

Applications
Eugène Vorobiev Université de Technologie de Compiègne, Département de Génie

des Procédés Industriels, Laboratoire Transformations Intégrées de la Matière
Renouvelable, EA 4297 - Équipe Technologies Agro-Industrielles, Centre de
Recherches de Royallieu,Compiègne, France
eugene.vorobiev@utc.fr
Contributors
Ignacio Álvarez Department of Animal Production and Food Science, Tecnología

de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza,
Spain
Pierre Adda Sorbonne Universités, Université de Technologie de Compiègne,

Laboratoire Transformations Intégrées de la Matière Renouvelable (TIMR EA
4297), Centre de Recherche de Royallieu, Compiègne Cedex, France
Grenoble Electrical Engineering Laboratory, G2Elab, CNRS, Grenoble Alpes Uni-
versity (UGA), Grenoble, France
Amali U. Alahakoon Department of Food Science, University of Otago, Dunedin,
New Zealand
F. Apollonio Department of Information Engineering, Electronics, and Telecom-

munications, Sapienza University of Rome, Rome, Italy
Delia Arnaud-Cormos University Limoges, CNRS, XLIM, UMR 7252, and

LABEX “Sigma-LIM”, Limoges, France
Cristina Arroyo School of Agriculture and Food Science, University College
Dublin, Dublin 4, Ireland
Mark Bagarazzi Inovio Pharmaceuticals, Inc., Plymouth Meeting, PA, USA

Franci Bajd Jožef Stefan Institute, Ljubljana, Slovenia
Faculty of Mathematics and Physics, University of Ljubljana, Ljubljana, Slovenia
Ana Balasa Unilever R&D Vlaardingen B.V., Vlaardingen, The Netherlands

Olivier Bals Sorbonne Universités, Université de Technologie de Compiègne,
Laboratoire Transformations Intégrées de la Matière Renouvelable (UTC/ESCOM,
EA 4297 TIMR), Centre de Recherche de Royallieu, Compiègne, France
Francisco J. Barba Department of Food Science, Faculty of Science, University of
Copenhagen, Frederiksberg C, Denmark
xxxv
xxxvi Contributors
Faculty of Pharmacy, Preventive Medicine and Public Health, Food Sciences,

Toxicology and Forensic Medicine Department, Nutrition and Food Science Area,
Universitat de València, València, Spain
M. J. Barbosa Bioprocess Engineering, AlgaePARC, Wageningen University and
Research, Wageningen, The Netherlands
Tina Batista Napotnik Faculty of Electrical Engineering, Department of Biomed-
ical Engineering, Laboratory of Biocybernetics, University of Ljubljana, Ljubljana,
Slovenia
Sid Becker Mechanical Engineering Department, University of Canterbury, Christ-
church, New Zealand
Stephen J. Beebe Frank Reidy Research Center for Bioelectrics, Old Dominion
Marco Benazzo Department of Otolaryngology Head and Neck Surgery, Univer-
sity of Pavia, IRCCS Policlinico San Matteo Foundation, Pavia, Italy
Claudio Bertacchini Igea S.p.A., Carpi, Italy
Giulia Bertino Department of Otolaryngology Head and Neck Surgery, University
of Pavia, IRCCS Policlinico San Matteo Foundation, Pavia, Italy
Suyashree Bhonsle Virginia Tech Wake Forest School of Biomedical Engineering
and Sciences, Virginia Tech, Blacksburg, VA, USA
The Bradley Department of Electrical and Computer Engineering, Virginia Tech,
Blacksburg, VA, USA
Masa Bosnjak Department of Experimental Oncology, Institute of Oncology Lju-
Nadia Boussetta Laboratoire Transformations Intégrées de la Matière
Renouvelable (UTC/ESCOM, EA 4297 TIMR), TIMR EA 4297, Centre de
recherche Royallieu, Département Génie des Procédés, Sorbonne universités,
Université de technologie de Compiègne, Compiègne cedex, France
Marwa Brahim Laboratoire Transformations Intégrées de la Matière
Renouvelable, Equipe Technologie Agro-Industrielles, Université de Technologie
de Compiègne, Compiègne, France
Faculté des Sciences et Technologies, Laboratoire d’Etudes et de Recherche sur le
Matériau Bois, Université de Lorraine, Vandœuvre-Lès-Nancy cedex, France
Erik Brecelj Department of Surgical Oncology, Institute of Oncology Ljubljana,
Ljubljana, Slovenia
Phil J. Bremer Department of Food Science, University of Otago, Dunedin, New
Zealand
Contributors xxxvii
Matteo Brizio Dermatologic Clinic, Department of Medical Sciences, University

of Turin, Turin, Italy
Nicolas Brosse Faculté des Sciences et Technologies, Laboratoire d’Etudes et de
Recherche sur le Matériau Bois, Université de Lorraine, Vandœuvre-Lès-Nancy
cedex, France
Cherylea J. Browne Translational Neuroscience Facility and Department of Phys-
iology, School of Medical Sciences, UNSW Australia, Sydney, NSW, Australia
School of Science and Health, Western Sydney University, Sydney, NSW, Australia
Nigel. P. Brunton UCD School of Agriculture and Food Science, University
College Dublin, Dublin 4, Ireland
Anna A. Bulysheva Frank Reidy Research Center for Bioelectrics, Old Dominion
David J. Burritt Department of Botany, University of Otago, Dunedin, New
Zealand
Ruggero Cadossi IGEA S.p.A., Carpi (MO), Italy
Luca Giovanni Campana Surgical Oncology Unit, Veneto Institute of Oncology
IOV-IRCCS and Department of Surgery Oncology and Gastroenterology
(DISCOG), University of Padova, Padova, Italy
Maura Casciola Frank Reidy Research Center for Bioelectrics, Old Dominion
Quim Castellví Department of Information and Communication Technologies,
Universitat Pompeu Fabra, Barcelona, Spain
Maja Čemažar Department of Experimental Oncology, Institute of Oncology
Faculty of Health Sciences, University of Primorska, Izola, Slovenia
Pintu Choudhary Food Safety and Quality Testing, Indian Institute of Food
Processing Technology, Thanjavur, TN, India
A. James P. Clover Department of Plastic Surgery, Cork University Hospital, Cork
and Cork Cancer Research Centre, Western Gateway Building, University College
Cork, Cork, Ireland
Luigi Corti Radiotherapy Unit, Veneto Institute of Oncology IOV-IRCCS, Padova,
Italy
Vincent Couderc CNRS, XLIM, UMR 7252, University of Limoges, Limoges,
France
Edward N. Crawford Translational Neuroscience Facility and Department of
Physiology, School of Medical Sciences, UNSW Australia, Sydney, NSW, Australia
xxxviii Contributors
Etienne Dague LAAS-CNRS, Université de Toulouse, CNRS, Toulouse, France

Rafael V. Davalos Department of Biomedical Engineering and Mechanics, Vir-
ginia Tech – Wake Forest University School of Biomedical Engineering and Sci-
ences, Blacksburg, VA, USA
Mariangela De Robertis Laboratory of Genetic and Clinical Pathology, University
Campus Bio-Medico of Rome, Rome, Italy
Laboratory of Molecular Pathology and Experimental Oncology, Institute of Trans-
lational Pharmacology, National Research Council (CNR), Rome, Italy
Francesca De Terlizzi IGEA Biophysics Lab, Via Parmenide, Carpi, Italy
Séverine Degrelle Physiopathologie et Pharmacotoxicologie Placentaire Humaine,
INSERM UMR-S1139, Université Paris Descartes, Sorbonne Paris Cité, Paris,
France
Cristèle Delsart Laboratoire Transformations Intégrées de la Matière
Renouvelable (UTC/ESCOM, EA 4297 TIMR), Sorbonne Universités, Université
de Technologie de Compiègne, Centre de Recherche de Royallieu, Compiègne,
France
Janja Dermol Faculty of Electrical Engineering, University of Ljubljana, Lju-
bljana, Slovenia
Rumiana Dimova Department of Theory and Bio-Systems, Max Planck Institute
of Colloids and Interfaces, Potsdam, Germany
Mihajlo Đokić Clinical Department of Abdominal Surgery, University Medical
Centre Ljubljana, Ljubljana, Slovenia
Tanja Dolinsek Department of Experimental Oncology, Institute of Oncology
Giorgio Donsì Department of Industrial Engineering, University of Salerno,
Fisciano, SA, Italy
ProdAl Scarl – University of Salerno, Fisciano, SA, Italy
Katarzyna Dymek Optifreeze AB, Lund, Sweden
Ibrahim Edhemovic Department of Surgical Oncology, Institute of Oncology
Pedro Elez-Martínez Department of Food Technology, University of Lleida-
Agrotecnio Center, Lleida, Spain
M. H. M. Eppink Bioprocess Engineering, AlgaePARC, Wageningen University
and Research, Wageningen, The Netherlands
Gulsun Akdemir Evrendilek Faculty of Engineering and Architecture, Depart-
ment of Food Engineering, Abant Izzet Baysal University, Bolu, Turkey
Contributors xxxix
Farnaz Faridnia Department of Food Science, University of Otago, Dunedin, New

Zealand
Department of Botany, University of Otago, Dunedin, New Zealand
M. Laura Fernández Consejo Nacional de Investigaciones Científicas y Técnicas,
Instituto de Física del Plasma, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Buenos Aires, Argentina
Departamento de Física, Facultad de Ciencias Exactas y Naturales, Universidad de
Buenos Aires, Buenos Aires, Argentina
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos
Aires, Argentina
Giovanna Ferrari Department of Industrial Engineering, University of Salerno,
Fisciano, SA, Italy
Mustafa Fincan Department of Food Engineering, Erciyes University, Melikgazi,
Kayseri, Turkey
Olivier Français ENS de Cachan, CNRS SATIE, Cachan, France
Stine Krog Frandsen Center for Experimental Drug and Gene Electrotransfer
(C*EDGE), Department of Oncology, Herlev and Gentofte Hospital, University of
Copenhagen, Herlev, Denmark
Roberta Fusco Department of Diagnostic Imaging, Radiant and Metabolic Ther-
apy, “Istituto Nazionale Tumori IRCCS Fondazione Pascale – IRCCS di Napoli”,
Naples, Italy
Federico Gómez Galindo Food Technology, Engineering and Nutrition, Lund
University, Lund, Sweden
Sara Galuppo Radiotherapy Unit, Veneto Institute of Oncology IOV-IRCCS,
Padova, Italy
Diego García-Gonzalo Tecnología de los Alimentos, Departamento de Producción
Animal y Ciencia de los Alimentos, Facultad de Veterinaria, Instituto
Agroalimentario de Aragón– IA2 (Universidad de Zaragoza-CITA), Zaragoza, Spain
Teresa Garde-Cerdán La Rioja Government, ICVV, Instituto de Ciencias de la
Vid y del Vino (Gobierno de La Rioja, Universidad de La Rioja, CSIC), Logroño, La
Rioja, Spain
Julie Gehl Center for Experimental Drug and Gene Electrotransfer (C*EDGE),
Department of Oncology, Herlev and Gentofte Hospital, University of Copenhagen,
Herlev, Denmark
Elena C. Gianulis Frank Reidy Research Center for Bioelectrics, Old Dominion
xl Contributors
Laure Gibot Institut de Pharmacologie et de Biologie Structurale, Université de

Toulouse, CNRS, UPS, Toulouse, France
Alexander Golberg The Porter School of Environmental Studies, Tel Aviv Uni-
versity, Tel Aviv, Israel
Muriel Golzio Institut de Pharmacologie et de Biologie Structurale, Université de

Lucía González-Arenzana La Rioja Government, ICVV, Instituto de Ciencias de

la Vid y del Vino (Gobierno de La Rioja, Universidad de La Rioja, CSIC), Logroño,
La Rioja, Spain
Charlie Gosse Laboratoire de Photonique et de Nanostructures, LPN-CNRS,
Marcoussis, France
Anita Gothelf Department of Oncology, Copenhagen University Hospital
Rigshospitalet, Copenhagen, Denmark
Vincenza Granata Department of Diagnostic Imaging, Radiant and Metabolic

Therapy, “Istituto Nazionale Tumori IRCCS Fondazione Pascale – IRCCS di
Napoli”, Naples, Italy
Ralf Greiner Department of Food Technology and Bioprocess Engineering, Max

Rubner-Institut, Federal Research Institute of Nutrition and Food, Karlsruhe,
Germany
Nabil Grimi Laboratoire Transformations Intégrées de la Matière Renouvelable
(UTC/ESCOM, EA 4297 TIMR), Centre de Recherche de Royallieu, Sorbonne
Universités, Université de Technologie de Compiègne, Compiègne Cedex, France
Ales Groselj Department of Otorhinolaryngology and Cervicofacial Surgery, Uni-
versity Medical Centre Ljubljana, Ljubljana, Slovenia
Enric Guenther University of California, Berkeley, CA, USA

Prostata-Center, Institut für Bildgebende Diagnostik, Offenbach, Germany
Ana Rosa Gutiérrez La Rioja Government, ICVV, Instituto de Ciencias de la Vid y
del Vino (Gobierno de La Rioja, Universidad de La Rioja, CSIC), Logroño, La
Rioja, Spain
Saša Haberl Meglič Faculty of Electrical Engineering, University of Ljubljana,

Ljubljana, Slovenia
Zhong Han School of Food Science and Engineering, South China University of
Technology, Guangzhou, Guangdong, China
Rasmus Hvass Hansen Department of Radiology, Herlev Hospital, University of
Contributors xli
Hanne Falk Hansen Center for Experimental Drug and Gene Electrotransfer
(C*EDGE), Department of Oncology, Herlev and Gentofte Hospital, University of
Guidan He College of Food Science and Engineering, Jilin University, Changchun,
China
Loree C. Heller Frank Reidy Research Center for Bioelectrics, Medical Diagnostic
and Translational Sciences, College of Health Sciences, Old Dominion University,
Norfolk, VA, USA
School of Medical Diagnostics and Translational Sciences, Old Dominion Univer-
sity, Norfolk, VA, USA
Mary Phillips Ho Department of Engineering, Quinnipiac University, Hamden,
CT, USA
Jing Hong School of Food Science and Engineering, South China University of
Tobias Horneber Food Biotechnology and Food Process Engineering, Technical
University of Berlin, Berlin, Germany
Gary D. Housley Translational Neuroscience Facility and Department of Physiol-
ogy, School of Medical Sciences, UNSW Australia, Sydney, NSW, Australia
Qin Hu School of Engineering and Technology, Central Michigan University, Mt.
Pleasant, MI, USA
Isabelle Hue Biologie du Développement et Reproduction, UMR 1198 INRA,
ENVA, Université Paris-Saclay, Jouy-en-Josas, France
Antonino Inferrera Unit of Urology, Dipartimento di Patologia umana dell’adulto
e dell’età evolutiva, Università degli Studi di Messina, Messina, Italy
Antoni Ivorra Department of Information and Communication Technologies,
Serra Húnter Program, Universitat Pompeu Fabra, Barcelona, Spain
Francesco Izzo Department of Surgical Oncology, Hepatobiliary Unit, “Istituto
Nazionale Tumori IRCCS Fondazione Pascale – IRCCS di Napoli”, Naples, Italy
Henry Jäger Institute of Food Technology, University of Natural Resources
and Life Sciences (BOKU), Vienna, Austria
Baltramiejus Jakštys Biophysical Research Group, Faculty of Natural Sciences,
Vytautas Magnus University, Kaunas, Lithuania
xlii Contributors
Milda Jakutavičiūtė Biophysical Research Group, Faculty of Natural Sciences,

Anet Režek Jambrak Faculty of Food Technology and Biotechnology, Depart-
ment of Food Process Engineering, University of Zagreb, Zagreb, Croatia
Tony Z. Jin U.S. Department of Agriculture, Agricultural Research Service, East-
ern Regional Research Center, Wyndmoor, PA, USA
Ravindra P. Joshi Department of Electrical and Computer Engineering, Texas
Tech University, Lubbock, TX, USA
Urska Kamensek Department of Experimental Oncology, Institute of Oncology
Helen Kavnoudias Radiology Department, Alfred Hospital, Melbourne, VIC,
Australia
Stephen T. Kee Division of Interventional Radiology, Department of Radiological
Sciences, University of California, Los Angeles (UCLA) Medical Center, Los
Angeles, CA, USA
Michael A. Kempkes Diversified Technologies, Inc., Bedford, MA, USA
Nina Klein Prostata-Center, Institut für Bildgebende Diagnostik, Offenbach,
Germany
Matthias Klugmann Translational Neuroscience Facility and Department of Phys-
iology, School of Medical Sciences, UNSW Australia, Sydney, NSW, Australia
Dietrich Knorr Department of Food Biotechnology and Food Process Engineer-
ing, Technische Universität Berlin, Berlin, Germany
Spela Kos Department of Experimental Oncology, Institute of Oncology Ljubljana,
Ljubljana, Slovenia
Bor Kos Faculty of Electrical Engineering, University of Ljubljana, Ljubljana,
Slovenia
Tadej Kotnik Faculty of Electrical Engineering, University of Ljubljana, Lju-
bljana, Slovenia
Mohamed Koubaa Laboratoire Transformations Intégrées de la Matière
Renouvelable (UTC/ESCOM, EA 4297 TIMR), Centre de Recherche de Royallieu,
Sorbonne Universités, Université de Technologie de Compiègne, Compiègne
Cedex, France
Matej Kranjc Faculty of Electrical Engineering, University of Ljubljana, Lju-
bljana, Slovenia
Simona Kranjc Department of Experimental Oncology, Institute of Oncology
Contributors xliii
Anna Krottenthaler Institute of Food Technology, University of Natural

Resources and Life Sciences (BOKU), Vienna, Austria
Christian Kunte Artemed Fachklinik München GmbH & Co., KG. München,
Germany
Noelia López CNTA, Centro Nacional de Tecnología y Seguridad Alimentaria, San
Adrián, Navarra, Spain
Rosa López La Rioja Government, ICVV, Instituto de Ciencias de la Vid y del Vino
(Gobierno de La Rioja, Universidad de La Rioja, CSIC), Logroño, La Rioja, Spain
Isabel López-Alfaro La Rioja Government, ICVV, Instituto de Ciencias de la Vid y
Rioja, Spain
Sección de Viticultura y Enología, ICVV. Finca La Grajera, Logroño, La Rioja,
Spain
Secondo Lastoria Department of Diagnostic Imaging, Radiant and Metabolic
Séverine Le Gac Applied Microfluidics for BioEngineering Research, MESA+
Institute for Nanotechnnology, MIRA Institute for Biomedical Engineering and
Technical Medicine, University of Twente, Enschede, The Netherlands
Bruno Le Pioufle ENS de Cachan, CNRS SATIE, Cachan, France
Ecole Normale Supérieure de Cachan, Cachan, France
Nikolai Lebovka Department of Physical Chemistry of Dirperse Minerals, Institute
of Biocolloidal Chemistry named after F.D. Ovcharenko, NAS of Ukraine, Kyiv,
Ukraine
Laboratoire de Transformations Intégrées de la Matière Renouvelable, EA 4297,
Centre de Recherches de Royallieu, Sorbonne Universités, Université de Techno-
logie de Compiègne, Compiègne, France
Sze Ying Leong Department of Food Science, University of Otago, Dunedin, New
Zealand
Maddalena Leongito Department of Surgical Oncology, Hepatobiliary Unit,
“Istituto Nazionale Tumori IRCCS Fondazione Pascale – IRCCS di Napoli”, Naples,
Italy
O. Lesaint Grenoble Electrical Engineering Laboratory, G2Elab, CNRS, Grenoble
Alpes University (UGA), Grenoble, France
Philippe Leveque University Limoges, CNRS, XLIM, UMR 7252, and LABEX
“Sigma-LIM”, Limoges, France
xliv Contributors
Zachary A. Levine Department of Physics, University of California Santa Barbara,

Santa Barbara, CA, USA
Department of Chemistry and Biochemistry, University of California Santa Barbara,
Santa Barbara, CA, USA
Materials Research Laboratory, University of California Santa Barbara, Santa
Barbara, CA, USA
M. Liberti Department of Information Engineering, Electronics, and Telecommu-
nications, Sapienza University of Rome, Rome, Italy
Hao Lin Department of Mechanical and Aerospace Engineering, Rutgers, The
State University of New Jersey, New Brunswick, NJ, USA
Li Liu Inovio Pharmaceuticals, Inc., Plymouth Meeting, PA, USA
Tingting Liu Department of Food Science, University of Otago, Dunedin, New
Zealand
Riddet Institute, Palmerston North, New Zealand
Nicolas Louka Centre d’Analyses et de Recherche, Unité de Recherche Technol-
ogies et Valorisation Agro-alimentaire (UR-TVA), Faculté des Sciences, Université
Saint-Joseph, Beirut, Lebanon
Nigel H. Lovell Graduate School of Biomedical Engineering, UNSW Australia,
Sydney, NSW, Australia
Elisa Luengo Institute of Food and Health, UCD School of Agriculture and Food
Science, University College Dublin, Dublin, Ireland
Franco Lugnani Kirurški sanatorij Rožna dolina, Ljubljiana, Slovenia
James G. Lyng School of Agriculture and Food Science, University College
Dublin, Dublin 4, Ireland
Carlo Magno Unit of Urology, Dipartimento di Patologia umana dell’adulto e
dell’età evolutiva, Università degli Studi di Messina, Messina, Italy
Faisal Mahmood Radiotherapy Research Unit (RRU), Center for Experimental
Drug and Gene Electrotransfer (CEDGE), Department of Oncology, Herlev Hospi-
tal, University of Copenhagen, Herlev, Denmark
Samo Mahnič-Kalamiza Laboratory of Biocybernetics, Faculty of Electrical Engi-
neering, University of Ljubljana, Ljubljana, Slovenia
Francisco Daniel Maldonado-Parra Food Biotechnology and Food Process Engi-
neering, Technical University of Berlin, Berlin, Germany
Roberto Marconato University of Padova School of Medicine, Padova, Italy
Contributors xlv
Yael Mardor Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel

The Advanced Technology Center, Sheba Medical Center, Ramat-Gan, Israel
Bostjan Markelc ÜRUK/MRC Oxford Institute for Radiation Oncology, Depart-
ment of Oncology, University of Oxford, Oxford, UK
Richard G. Maroun Centre d’Analyses et de Recherche, Unité de Recherche
Technologies et Valorisation Agro-alimentaire (UR-TVA), Faculté des Sciences,
Université Saint-Joseph, Beirut, Lebanon
Paolo Marracino Department of Information Engineering, Electronics, and Tele-
communications, Sapienza University of Rome, Rome, Italy
Guillermo Marshall Laboratorio de Sistemas Complejos, Departamento de
Computación, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos
Aires, Buenos Aires, Argentina
Instituto de Física del Plasma, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires and Consejo Nacional de Investigaciones Científicas
y Técnicas, Buenos Aires, Argentina
Olga Martín-Belloso Department of Food Technology, University of Lleida-
Robert C. G. Martin Division of Surgical Oncology, University of Louisville,
Louisville, KY, USA
Louise Wichmann Matthiessen Department of Oncology, Copenhagen University
Hospital Herlev and Gentofte, Herlev, Denmark
E. Mazari Center for International Research on MicroMechatronics, Institute of
Industrial Science, The University of Tokyo, Tokyo, Japan
Borja Mercadal Department of Information and Communication Technologies,
Yan Mi State Key Laboratory of Power Transmission Equipment and System
Security and New Technology, College of Electrical Engineering, Chongqing
University, Chongqing, China
I. Migeotte Institut de Recherche Interdisciplinaire en Biologie Humaine et
Moléculaire, Université Libre de Bruxelles, Bruxelles, Belgium
Damijan Miklavčič Faculty of Electrical Engineering, University of Ljubljana,
Ljubljana, Slovenia
Keiko Morotomi-Yano Institute of Pulsed Power Science, Kumamoto University,
Kumamoto, Japan
Matthew P. Morrow Inovio Pharmaceuticals, Inc., Plymouth Meeting, PA, USA
xlvi Contributors
Giuseppe Mucciardi Unit of Urology, Dipartimento di Patologia umana

dell’adulto e dell’età evolutiva, Università degli Studi di Messina, Messina, Italy
Claudia Muratori Frank Reidy Research Center for Bioelectrics, Old Dominion
Robert N. Neal II Radiology Department, Alfred Hospital, Melbourne, VIC,
Australia
Richard Nuccitelli Pulse Biosciences Inc., Burlingame, CA, USA
Rodney O’Connor University Limoges, CNRS, XLIM, UMR 7252, and LABEX
“Sigma-LIM”, Limoges, France
Johnny M. O’Mahony Department of Plastic Surgery, Cork University Hospital,
Cork, Ireland
Isabel Odriozola-Serrano Department of Food Technology, University of Lleida-
Indrawati Oey Department of Food Science, University of Otago, Dunedin, New
Zealand
Giuseppe Olivieri Bioprocess Engineering, AlgaePARC, Wageningen University
and Research, Wageningen, The Netherlands
Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale,
Università degli Studi di Napoli Federico II, Napoli, Italy
Rafael Pagán Tecnología de los Alimentos, Departamento de Producción Animal y
Ciencia de los Alimentos, Facultad de Veterinaria, Instituto Agroalimentario de
Aragón– IA2 (Universidad de Zaragoza-CITA), Zaragoza, Spain
Andrei G. Pakhomov Frank Reidy Research Center for Bioelectrics, Old Domin-
ion University, Norfolk, VA, USA
Olga Pakhomova Frank Reidy Research Center for Bioelectrics, Old Dominion
Raffaele Palaia Department of Surgical Oncology, Hepatobiliary Unit, “Istituto
Oleksii Parniakov Laboratoire Transformations Intégrées de la Matière
Renouvelable (UTC/ESCOM, EA 4297 TIMR), Sorbonne Universités, Centre de
Recherche de Royallieu, Université de Technologie de Compiègne, Compiègne,
France
Paolo Passamonti School of Science and Technology, Chemistry Section, Univer-
sity of Camerino, Camerino, Italy
Contributors xlvii
Gianpiero Pataro Department of Industrial Engineering, University of Salerno,

Fisciano, SA, Italy
Ankit Patras Department of Agricultural and Environmental Sciences, Tennessee
State University, Nashville, TN, USA
Darja Pavlin Veterinary Faculty, Small Animal Clinic, University of Ljubljana,
Ljubljana, Slovenia
Micha Peleg Department of Food Science, University of Massachusetts, Amherst,
MA, USA
Aitana Perea-Gomez Institut de Biologie Valrose, CNRS UMR7277, Inserm
U1091, Université Nice Sophia Antipolis, Nice, France
Marcos Teotonio Pereira EnergyPulse Systems, EPS, Lisbon, Portugal
Antonella Petrillo Department of Diagnostic Imaging, Radiant and Metabolic
Mauro Piccirillo Department of Surgical Oncology, Hepatobiliary Unit, “Istituto
Flavien Pillet CNRS; IPBS (Institut de Pharmacologie et de Biologie Structurale),
Toulouse, France
Université de Toulouse; UPS; IPBS, Toulouse, France
Jeremy L. Pinyon Translational Neuroscience Facility and Department of Physi-
ology, School of Medical Sciences, UNSW Australia, Sydney, NSW, Australia
Uwe Pliquett Institut für Bioprozess- und Analysenmesstechnik, Heilbad
Heiligenstadt, Germany
Clair Poignard Team MONC, INRIA Bordeaux-Sud-Ouest, Institut de
Mathématiques de Bordeaux, CNRS UMR 5251 and Université de Bordeaux,
Bordeaux, France
Mahesha M. Poojary School of Science and Technology, Chemistry Section,
University of Camerino, Camerino, Italy
P. R. Postma Bioprocess Engineering, AlgaePARC, Wageningen University and
Eduardo Puértolas Food Research Division, AZTI, Derio, Bizkaia, Spain
Pietro Quaglino Dermatologic Clinic, Department of Medical Sciences, University
Rui P. Queirós QOPNA, Departamento de Química, Universidade de Aveiro,
Campus Universitário de Santiago, Aveiro, Portugal
xlviii Contributors
Hiba N. Rajha Centre d’Analyses et de Recherche, Unité de Recherche Technol-

ogies et Valorisation Agro-alimentaire (UR-TVA), Faculté des Sciences, Université
Saint-Joseph, Beirut, Lebanon
EA 4297 TIMR), TIMR EA 4297, Centre de recherche Royallieu, Département
Génie des Procédés, Sorbonne universités, Université de technologie de Compiègne,
Compiègne cedex, France
Airton Ramos State University of Santa Catarina (UDESC), DEE/CCT, Joinville,
Santa Catarina, Brazil
Javier Raso Department of Animal Production and Food Science, Tecnología de
los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain
Cornelia Rauh Food Biotechnology and Food Process Engineering, Technical
Ashish Rawson Food Safety and Quality Testing, Indian Institute of Food Pro-
cessing Technology, Thanjavur, TN, India
Anet Režek Jambrak Faculty of Food Technology and Biotechnology, Depart-
ment of Food Engineering, University of Zagreb, Zagreb, Croatia
Matej Reberšek Faculty of Electrical Engineering, University of Ljubljana, Lju-
bljana, Slovenia
Luís M. S. Redondo Lisbon Engineering Superior Institute, GIAAPP/ISEL, Lis-
bon, Portugal
EnergyPulse Systems, EPS, Lisbon, Portugal
Duarte S. Rego EnergyPulse Systems, EPS, Lisbon, Portugal
Javier Portu Reinares La Rioja Government, ICVV, Instituto de Ciencias de la
Vid y del Vino (Gobierno de La Rioja, Universidad de La Rioja, CSIC), Logroño, La
Rioja, Spain
Lea Rems Department of Chemical Engineering, Delft University of Technology,
Delft, The Netherlands
Simone Ribero Dermatologic Clinic, Department of Medical Sciences, University
Marcelo R. Risk Consejo Nacional de Investigaciones Científicas y Técnicas
(CONICET), Buenos Aires, Argentina
Instituto Tecnológico de Buenos Aires (ITBA), Buenos Aires, Argentina
Karin A. Riske Departamento de Biofísica, Universidade Federal de São Paulo,
São Paulo, Brazil
Fusco Roberta IGEA S.p.A., Carpi (MO), Italy
Contributors xlix
Arthur Robin The Porter School of Environmental Studies, Tel Aviv University,
Tel Aviv, Israel
Andrea Rolong Department of Biomedical Engineering and Mechanics, Virginia
Tech – Wake Forest University School of Biomedical Engineering and Sciences,
Blacksburg, VA, USA
Marie-Pierre Rols Institut de Pharmacologie et de Biologie Structurale, IPBS/
CNRS UMR 5089 and University of Toulouse, Toulouse, France
Mattia Ronchetti IGEA S.p.A., Carpi (MO), Italy
Shahin Roohinejad Department of Food Technology and Bioprocess Engineering,
Max Rubner-Institut, Federal Research Institute of Nutrition and Food, Karlsruhe,
Germany
Carlo Riccardo Rossi Department of Surgery Oncology and Gastroenterology
(DISCOG), University of Padova, Padova, Italy
Paulius Ruzgys Biophysical Research Group, Faculty of Natural Sciences,
Esin B. Sözer Frank Reidy Research Center for Bioelectrics, Old Dominion Uni-
versity, Norfolk, VA, USA
Martin Sack Institute for Pulsed Power and Microwave Technology, Karlsruhe
Institute of Technology, Eggenstein-Leopoldshafen, Germany
Johannes Sailer Food Biotechnology and Food Process Engineering, Technical
Guillermo Saldaña Department of Animal Production and Food Science,
Tecnología de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza,
Zaragoza, Spain
Pilar Santamaría La Rioja Government, ICVV, Instituto de Ciencias de la Vid y
Rioja, Spain
Mauro D. Santos QOPNA, Departamento de Química, Universidade de Aveiro,
Jorge A. Saraiva QOPNA, Departamento de Química, Universidade de Aveiro,
Julia Ribeiro Sarkis Chemical Engineering Departament, Federal University of
Rio Grande do Sul, Porto Alegre, RS, Brazil
Saulius Šatkauskas Biophysical Research Group, Faculty of Natural Sciences,
l Contributors
Felix Schottroff Institute of Food Technology, University of Natural Resources and

Life Sciences (BOKU), Vienna, Austria
Iurii Semenov Frank Reidy Research Center for Bioelectrics, Old Dominion Uni-
versity, Norfolk, VA, USA
Matteo Sepulcri Radiotherapy Unit, Veneto Institute of Oncology IOV-IRCCS,
Padova, Italy
Gregor Serša Department of Experimental Oncology, Institute of Oncology Lju-
Igor Serša Jožef Stefan Institute, Ljubljana, Slovenia
Shirley Sharabi Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel
Shawna A. Shirley Frank Reidy Center for Bioelectrics, Norfolk, VA, USA
OncoSec Medical, San Diego, CA, USA
Emanuela Signori Laboratory of Molecular Pathology and Experimental Oncol-
ogy, Institute of Translational Pharmacology, National Research Council (CNR),
Rome, Italy
Pat Silcock Department of Food Science, University of Otago, Dunedin, New
Zealand
Eldevan S. Silva Departamento de Química, Universidade Federal de São Carlos,
São Carlos, Brazil
Aude Silve Karlsruhe Institute of Technology, Institute for Pulse Power and Micro-
wave Technology, Eggenstein-Leopoldshafen, Germany
Werner Sitzmann Solids Process Engineering and Particle Technology, Technical
University of Hamburg, Hamburg, Germany
Marko Snoj Institute of Oncology, Ljubljana, Slovenia
Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
Declan Soden Cork Cancer Research Centre, Western Gateway Building, Univer-
sity College Cork, Cork, Ireland
Robert Soliva-Fortuny Department of Food Technology, University of Lleida-
Stephen B. Solomon Interventional Radiology Service, Department of Radiology,
Memorial Sloan Kettering Cancer Center, New York, NY, USA
Govindarajan Srimathveeravalli Interventional Radiology Service, Department
of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, USA
Contributors li
Michael K. Stehling University of California, Berkeley, CA, USA

Prostata-Center, Institut für Bildgebende Diagnostik, Offenbach, Germany
Diana Suárez-Boomgaard Institut de Recherche Interdisciplinaire en Biologie
Humaine et Moléculaire, Université Libre de Bruxelles, Bruxelles, Belgium
Daniela O. H. Suzuki Institute of Biomedical Engineering, Federal University of
Santa Catarina (UFSC), IEB/EEL/CTC/UFSC, Florianopolis, Santa Catarina, Brazil
G. P. ‘t Lam Bioprocess Engineering, AlgaePARC, Wageningen University and
Bashir Akhavan Tafti Division of Interventional Radiology, Department of Radio-
logical Sciences, University of California, Los Angeles (UCLA) Medical Center,
Los Angeles, CA, USA
Youssef Tamzali Ecole Nationale Vétérinaire de Toulouse, Université de Toulouse,
Toulouse, France
Mounir Tarek Université de Lorraine, UMR 7565, Vandoeuvre les Nancy, France
CNRS, UMR 7565, Vandoeuvre les Nancy, France
Justin Teissie Institut de Pharmacologie et de Biologie Structurale, Université de
K. R. Thomson Radiology Department, Alfred Hospital, Melbourne, VIC,
Australia
Vesna Todorovic Department of Experimental Oncology, Institute of Oncology
Nataša Tozon Veterinary Faculty, Small Animal Clinic, University of Ljubljana,
Ljubljana, Slovenia
Ursa Lampreht Tratar Department of Experimental Oncology, Institute of Oncol-
ogy Ljubljana, Ljubljana, Slovenia
Blaž Trotovšek Clinical Department of Abdominal Surgery, University Medical
Centre Ljubljana, Ljubljana, Slovenia
Eisuke Ueshima Interventional Radiology Service, Department of Radiology,
Memorial Sloan Kettering Cancer Center, New York, NY, USA
Iris van Uitert Demcon bv, Enschede, The Netherlands
D’Alessio Valeria IGEA S.p.A., Carpi (MO), Italy
Sara Valpione Christie NHS, Foundation Trust, Manchester, UK
P. Thomas Vernier Frank Reidy Research Center for Bioelectrics, Old Dominion
lii Contributors
Eugéne Vorobiev Université de Technologie de Compiègne, Département de

Génie des Procédés Industriels, Laboratoire Transformations Intégrées de la Matière
Renouvelable, EA 4297 - Équipe Technologies Agro-Industrielles, Centre de
Recherches de Royallieu, Compiègne, France
Tomislava Vukušić Faculty of Food Technology and Biotechnology, Department
of Process Engineering, University of Zagreb, Zagreb, Croatia
Thies Wölken Food Biotechnology and Food Process Engineering, Technical
Jialun Wan State Key Laboratory of Power Transmission Equipment and System
Security and New Technology, College of Electrical Engineering, Chongqing Uni-
versity, Chongqing, China
Ying Wang College of Food Science and Engineering, Qingdao Agricultural
University, Qingdao, China
Lars H. Wegner Karlsruhe Institute of Technology, Institute for Pulse Power and
Microwave Technology (IHM), Eggenstein-Leopoldshafen, Germany
R. H. Wijffels Bioprocess Engineering, AlgaePARC, Wageningen University and
Faculty of Biosciences and Aquaculture, Nord University, Bodø, Norway
Artur Wiktor Department of Food Engineering and Process Management, Faculty
of Food Sciences, Warsaw University of Life Sciences (WULS-SGGW), Warsaw,
Poland
Dorota Witrowa-Rajchert Department of Food Engineering and Process Manage-
ment, Faculty of Food Sciences, Warsaw University of Life Sciences (WULS-
SGGW), Warsaw, Poland
Eung Je Woo Department of Biomedical Engineering, College of Medicine,
Kyung Hee University, Seoul, Republic of Korea
Shu Xiao Department of Electrical and Computer Engineering, Frank Reidy
Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA
Xiaoxia Yan College of Biological and Agricultural Engineering, Jilin University,
Changchun, China
Ruijin Yang State Key Laboratory of Food Science and Technology, Jiangnan
University, Wuxi, China
School of Food Science and Technology, Jiangnan University, Wuxi, China
Ken-ichi Yano Institute of Pulsed Power Science, Kumamoto University, Kuma-
moto, Japan
Yongguang Yin College of Biological and Agricultural Engineering, Jilin Univer-
sity, Changchun, China
Contributors liii
Xiaoxi Yu Sorbonne Universités, Université de Technologie de Compiègne,

EA 4297 TIMR), Centre de Recherche de Royallieu, Compiègne, France
Miao Yu Department of Mechanical and Aerospace Engineering, Rutgers, The
State University of New Jersey, New Brunswick, NJ, USA
Xin-An Zeng School of Food Science and Engineering, South China University of
Didier Zerbib LISBP, Université de Toulouse, CNRS, INRA, INSA, Toulouse,
France
IPBS, Université de Toulouse, CNRS, UPS, Toulouse, France
Yanyuan Zhang The State Grid Chongqing, Changshou Power Supply Company,
Chongqing, China
Wei Zhao State Key Laboratory of Food Science and Technology, Jiangnan Uni-
versity, Wuxi, China
Xuan Zhao Laboratoire de Photonique et de Nanostructures, LPN-CNRS,
Marcoussis, France
Part I
Fundamental Mechanisms of Electroporation:
Models and Experiments
P. Thomas Vernier
Lipid Pores: Molecular and Continuum
Models 1
Lea Rems
Abstract
Exposure of cells to pulsed electric fields has become a routine technique to
increase the permeability of cell membranes, allowing enhanced transmembrane
transport of drugs, genetic material, and other molecules. The full details of the
molecular mechanisms, which lead to the increased membrane permeability, are
not yet entirely clear. However, extensive theoretical and experimental studies on
model lipid systems demonstrated that formation of aqueous pores in the lipid
bilayer presents one of the structural alterations of the cell membrane, which are
induced under the influence of the electric field. The first theoretical arguments
supporting the pore formation hypothesis were based on simple models, which
treated the pores in terms of continuum (mesoscopic) theories. Later on, insights
from molecular dynamics (MD) simulations substantiated some of the predictions
arising from continuum models and, in addition, provided a comprehensive
molecular picture of the pore formation process. The present chapter gives a
brief overview of MD simulations and continuum modeling of lipid pores, with
specific aim to highlight their connections, agreements, and disagreements.
Establishing connections between these two modeling approaches is highly
beneficial in order to enhance the understanding of electroporation. On one
hand, MD simulations provide a direct method for seeking the molecular mech-
anisms of pore formation, and they compensate for the lack of microscopic
techniques to visualize lipid pores. On the other hand, continuum models,
which are computationally much less demanding, can often be more easily
applied to theoretically analyze complex experimental systems. MD simulations
could therefore be used to validate and improve continuum models, whereas
continuum models could serve as a bridge between MD simulations and
experiments.
L. Rems (*)
Department of Chemical Engineering, Delft University of Technology, Delft, The Netherlands
e-mail: l.rems@tudelft.nl
# Springer International Publishing AG 2017 3

D. Miklavčič (ed.), Handbook of Electroporation,
DOI 10.1007/978-3-319-32886-7_76
4 L. Rems
Keywords
Electroporation • Electric field • Transmembrane voltage • Lipid bilayer • Aque-
ous pore • Molecular dynamics simulations • Theoretical model
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Molecular Dynamics of Lipid Bilayer Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Approaches to Model the Exposure of a Lipid Bilayer to an Electric Field . . . . . . . . . . . . . . . . 6
General Molecular View of Lipid Bilayer Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Continuum Modeling of Lipid Bilayer Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Hydrophobic (HO) and Hydrophilic (HI) Pore Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Pore Creation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Dynamics of the Pore Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Ionic Conduction Through a Pore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Pore Annihilation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Introduction
The cell membrane is composed of various types of lipids (mainly phospholipids),

self-assembled into a continuous fluid lipid bilayer, which serves as a matrix for
membrane proteins that selectively control the transport of ions and molecules across
the membrane. The lipid bilayer itself is poorly permeable to hydrophilic, i.e., water-
soluble solutes due to its amphiphilic structure, yet the noncovalent interactions
between the lipid molecules allow that the bilayer becomes locally perturbed by
formation of an aqueous pore. Occurrence of pores is a natural phenomenon, which
can arise from membrane fluctuations and packing defects and is likely associated
with spontaneous lipid flip-flop and passive ion permeation through the membrane.
The penetration of water into the hydrophobic (HO) bilayer core, corresponding
local loss of lipid cohesion, and reorganization of lipid molecules nevertheless
demand a high energetic cost; thereby, spontaneous pore formation is a rare event.
However, the probability of pore formation increases significantly in the presence of
certain surfactants and pore-forming peptides or under the influence of external
forces including mechanical stress and electric field (Gurtovenko et al. 2010).
Pores induced by electric field, the main focus of this chapter, were found to
contribute to the structural alterations leading to increased cell membrane perme-
ability when exposing cells to electric pulses.
From an electrical point of view, the cell membrane acts similarly as a capacitor,
since it is characterized by almost negligible electrical conductivity and low dielec-
tric permittivity. Consequently, exposure of a cell to an electric field induces a
voltage across the cell membrane (▶ Chap. 59, “Transmembrane Voltage Induced
by Applied Electric Fields”). Experiments on cells as well as model lipid systems
have demonstrated that a sufficiently high transmembrane voltage provokes a
1 Lipid Pores: Molecular and Continuum Models 5
drastic increase in the measured electric (ionic) current flowing through the mem-
brane, which gives strong experimental evidence for formation of conductive pores
in the lipid bilayer, naming the phenomenon “electroporation.” These lipid
“electropores” are predicted to be dynamic, thermally fluctuating nanometer struc-
tures, which precludes their direct visualization by established microscopic tech-
niques (for recent advances, see Sengel and Wallace (2016)). Understanding of the
pore formation process and associated phenomena therefore highly relies on theo-
retical modeling of lipid pores.
The first theoretical arguments supporting the hypothesis that an increase in the
transmembrane voltage promotes formation of pores in the lipid bilayer were
proposed already in the late 1970s (Abidor et al. 1979). Since there were no methods
available that would allow a molecular-level insight into the behavior of a lipid
bilayer under the influence of an electric field, the models were based on indirect
experimental observations analyzed in terms of continuum (mesoscopic) theories. In
the 2000s, the advances in molecular modeling of lipid bilayers enabled investi-
gation of electroporation by means of atomistic molecular dynamics
(MD) simulations. MD is a computer simulation technique for studying the move-
ment of atoms and molecules, which provides a direct method for characterizing the
molecular mechanisms of electroporation. However, MD is computationally too
demanding to be applied for theoretically analyzing complex behavior of lipid
membranes in many experimental configurations. Consequently, continuum model-
ing still constitutes an important part of electroporation research as it enables
exploration on length scales and timescales not achievable by MD. Unfortunately,
only limited efforts have been made to relate the insights from MD to continuum
modeling of lipid pores.
The present chapter provides a brief overview of these two modeling approaches,
with specific aim to highlight their connections, agreements, and disagreements.
After introducing the concepts of modeling electroporation of lipid bilayers in MD
simulations and the general molecular view of electroporation (section “Molecular
Dynamics of Lipid Bilayer Electroporation”), the insights from MD are compared to
predictions from continuum modeling of lipid pores (section “Continuum Modeling
of Lipid Bilayer Electroporation”). Concluding remarks are summarized in section
“Conclusion.”
Molecular Dynamics of Lipid Bilayer Electroporation
MD simulation generates a trajectory of atomic positions and velocities in the

considered molecular system (in the present case a hydrated lipid bilayer, see
Fig. 1) based on integrating the classical equations of motion. The forces acting
between the individual atoms are derived from a potential energy function of
interatomic interactions, the latter being described through a given force field
consisting of the equations chosen to model the potential energy and their associated
parameters. The force fields for modeling lipid bilayers can be all-atom, where each
atom in the system is treated separately; united-atom, where CH2 groups in the lipid
6 L. Rems
total
Number density [arb. Units]

water CH2/CH3
PO4
C=O N(CH3)3
-20 0 20
z[Å]
Fig. 1 Configuration of a palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayer system from MD

simulations. (a) Particles inside the simulation box. Water molecules (O, gray; H, white) and the
phosphate (blue) and nitrogen (purple) atoms of the lipid headgroups are depicted by their van der
Waals radii, whereas the acyl chains (cyan) are represented as sticks. (b) Number density profiles
(arbitrary units) along the axis z normal to the bilayer plane, averaged over 2 ns of the MD
trajectory. The bilayer center is located at z = 0 (Reprinted with permission from Delemotte and
Tarek 2012. Copyright 2012 Springer)
acyl chains are represented as single particles; and other more coarse-grained
representations. Though MD is a very powerful tool for investigating systems on
the molecular level, it is limited by its high computational cost. In atomistic
simulations (all-atom and united-atom representation), generally a small lipid bilayer
patch comprising about 100–1,000 lipids is studied over timescales on the order of
10–100 ns, in order to obtain results in reasonable time with respect to the present
available computational resources. To reduce the effects of the small system size, the
bilayer patch together with solvating water molecules (and ions) is usually placed in
a cuboidal simulation box, which is replicated infinitely in three dimensions using
periodic boundary conditions.
Approaches to Model the Exposure of a Lipid Bilayer to an

Electric Field
To model the exposure of a lipid bilayer to an electric field in MD, two approaches
can be used (Delemotte and Tarek 2012). The first approach is to impose an external
electric field Eext in the direction normal to the bilayer by adding a force Fi = qiEext
on every i-th atom which carries a charge qi. Contrary to intuitive expectation, such
implementation, together with periodic boundary conditions, does not allow a direct
control of the electric field within the simulation system (Böckmann et al. 2008). The
water and lipid headgroup dipoles respond to the imposed Eext by changing their
average orientation, which affects the local electric field E felt by the particles in the
system (Fig. 2b–d). Because of the way how Eext is implemented, the total voltage
(i.e., electric potential difference) Ubox, which builds across the simulation box, is
4
a
c d
3
f(z) [V]
0
-20 0 20
z[Å]
b 0.2
0.1
E(z) [VÅ–1]
–0.1
V/A
0.00
0.05
0.10
0.15
0.20
–0.2
-20 0 20
z[Å]
Fig. 2 Electrostatic profiles in a POPC bilayer system in the absence and presence of an externally
imposed electric field. (a) Electric potential profiles Φ(z) along the axis z normal to the bilayer plane
when the system is subject to external electric fields Eext of 0.0 MV/m (dotted line), 600 MV/m (thin
line), and 3,000 MV/m (bold line). The electrostatic potential was derived from the Poisson’s
equation by double integration of the molecular charge density distributions. Black arrows point to
the water–bilayer interface. The difference between the electric potentials in the bulk water phase on
each side of the bilayer corresponds to the transmembrane voltage. (b) Associated electric field
profiles derived as E(z) = –@Φ(z)/@z. At rest (dotted line), the voltage across the water–bilayer
interfaces (the so-called dipole potential) gives rise locally to large electric fields, but the electric
field in the bilayer core is negligible. In the presence of an externally imposed electric field (thin and
bold line), the electric field profile changes. Note the change in the local electric field at the
water–bilayer interface and the increase in the magnitude of the electric field inside the bilayer
core. (c, d) Two-dimensional (out of plane) map of the electric field distribution at 0.0 MV/m (c) and
3,000 MV/m (d). The local electric field direction and strength are displayed as white arrows
(Reprinted with permission from Delemotte and Tarek 2012. Copyright 2012 Springer)
sensitive to the length of the simulation box Lz in the direction of the imposed
electric field Ubox LzEext. Nevertheless, most of this voltage drops across the
bilayer, which is much less polarizable than the surrounding bulk water (Fig. 2a).
Detailed analyses of electrostatic profiles demonstrated that the voltage across the
bilayer and the corresponding increase in the electric field inside the bilayer core
(Fig. 2b) arise almost exclusively due to electric field-driven reorientation of water
molecules at the bilayer interface, which also facilitates slight field-associated tilting
of the lipid headgroup dipoles (Ziegler and Vernier 2008). In simulations where the
transmembrane voltage is induced by an external electric field, the systems most
often do not contain ions; therefore, such simulations model a purely dielectric
response of the system. This can be considered as the limiting case in experimental
situations, when the applied electric pulse is too short to allow considerable redis-
tribution of ions in the bathing electrolyte solutions, more specifically, when the
8 L. Rems
pulse duration is much shorter than the charging time of the membrane. For example,
the charging time constant by which the electrophoretically driven ions in the
intracellular and extracellular solution with physiological ionic strength build a
voltage across the cell membrane is on the order of 100 ns.
The second approach to model an increase in the transmembrane voltage is to
impose a charge imbalance between the two sides of the bilayer, which mimics the
experimental conditions, when the membrane becomes fully charged by ions. The
charge imbalance can be imposed, e.g., by placing an excess number of monovalent
cations above the bilayer and a corresponding excess number of monovalent anions
below the bilayer. Since periodic boundary conditions are used on all sides of the
simulation box, the simulation setup needs to ensure that the excess ions from one
side of the bilayer do not escape to the other side by crossing the box through
periodic boundaries. This can be achieved by using two lipid bilayer patches placed
one above the other, which splits the aqueous solution into two compartments. The
disadvantage of this setup is that the system is large and computationally much more
demanding as all the equations need to be solved for two bilayers instead of one. The
overcost can be avoided by replacing the additional bilayer with a vacuum layer (see
Fig. 7a). In the charge imbalance method, the transmembrane voltage arises mostly
due to the contribution of the excess ions on each side of the bilayer, rather than the
electric field-driven reorientation of interfacial water. Nevertheless, the distribution
of the electric field inside the system is similar as when imposing an external electric
field. Since the bilayer acts as a capacitor, the transmembrane voltage (in a
nonporated bilayer) is proportional to the magnitude of the imposed charge
imbalance.
General Molecular View of Lipid Bilayer Electroporation
Regardless of the approach used to mimic the exposure of a lipid bilayer to an

electric field, the characteristic molecular picture of electroporation is the same
(Fig. 3). After the onset of imposing an external electric field or charge imbalance,
the local changes in the electric field distribution promote formation of water
defects at the water–bilayer interface, which eventually (after some lag time) grow
into an aqueous pore spanning the bilayer. The lag time required for the pore to form
is stochastic, but its average value strongly decreases with the magnitude of the
induced transmembrane voltage and the associated electric field inside the bilayer
core. In other words, the pore forms much more rapidly, if the transmembrane
voltage is higher. The sequence of events that lead to creation of a pore can be
divided into several steps. In the first step, the interfacial water molecules start to
protrude into the bilayer core as conically shaped water fingers, in which the water
dipoles are strongly aligned with the high electric field in the hydrophobic hydro-
carbon region. These oriented water molecules can easily hydrogen-bond among
themselves. The water fingers can emerge from either side of the bilayer, with some
types of bilayers expressing a preferential side of the protrusions (Ziegler and
Vernier 2008). The driving force for the water protrusions appears to be mostly
Pore Creation
Initiation Construction Maturation
Merger of H2O Groups Merger of P Groups P Connections → 10
E = Eporation
Pore Annihilation
Destabilization Degradation Deconstruction Dissolution

≥ 10 P Connections P Connections → 1 P Separation H2O Separation
E=0
Fig. 3 Steps in pore creation and annihilation in a POPC bilayer under the influence of an
externally imposed electric field. Only water (O, red; H, white) and phosphorus atoms of the lipid
headgroups (gold spheres) are shown for simplicity. After formation of a water-spanning column
(pore initiation), phospholipid headgroups in each leaflet around the water defect reorganize and
migrate into the pore (pore construction). The pore further evolves by migration of additional water
and headgroups into the pore until an arbitrarily defined mature pore structure is formed (pore
maturation). After removal of the porating electric field, the pore annihilates in the reverse sequence
of analogous events (Reprinted with permission from Levine and Vernier 2010. Copyright 2010
Springer)
determined by the local profile of the electric field gradient at the water–bilayer
interface, which is characteristic for a given lipid composition of the bilayer. Not all
water fingers ultimately result in a pore, but some water fingers further grow in
length to finally form a water-spanning column, either by crossing the bilayer
themselves or by joining with a similar water defect from the opposite side of the
bilayer. On some occasions, the water column can split again. Otherwise, formation
of the water column is in most phospholipid bilayers followed by migration of the
lipid headgroups into the pore wall, which stabilizes the pore. The pore can then
10 L. Rems
further grow in size under the influence of the electric field. When ions are present in
the aqueous solution, they electrophoretically migrate through the pore. If the
external electric field is removed, the pore annihilates in reverse sequence of
analogous events and the bilayer recovers its planar configuration.
Continuum Modeling of Lipid Bilayer Electroporation
MD simulations provided the first “visual” molecular-level evidence that the electric
field promotes formation of pores in the lipid bilayer and confirmed some of the main
hypotheses underlying the two-decade-older development of theoretical concepts of
electroporation. The main motivation in developing electroporation models is to
understand the experimental results, to predict experimental outcomes, and to use the
models for optimization of experimental protocols. For such purpose, the models
need to enable prediction of the physical quantities that are accessible by experi-
mental measurements. Results from MD often cannot be compared directly to
experiments, since the system in the simulations is representative of only a tiny
area of the lipid membrane, large enough to observe a single or at most few pores,
whereas the measurements on experimental systems (e.g., planar lipid bilayers, lipid
vesicles, single cells, or cell assemblies) are generally determined by an ensemble of
pores that are formed along the membrane. Moreover, in experimental systems, the
local electric field and the induced transmembrane voltage can vary with time and
position along the membrane, which can affect the local kinetics of pore creation and
dynamic expansion of pores. In order to model such complex behavior, it is
necessary to employ continuum theoretical approaches.
Nowadays most widely used continuum approach to describe electroporation of
lipid membranes originates from the principles of the classical nucleation theory
(Weaver and Chizmadzhev 1996). The pores are treated in terms of a time-dependent
distribution function n = n(rp, t) defined such that ndrp equals the number of pores
with radius between rp and rp + drp at time t. The time evolution of n is represented
as a diffusion process in the potential function ΔWp, which can be expressed through
the Einstein–Smoluchowski equation:

@n @ @n n @ΔW p
¼ Dp þ þS (1)
@t @r p @r p kT @r p
where Dp is the diffusion coefficient of pores in the space of pore radius, and the
source term S accounts for the rate of pore creation and annihilation. Equation 1 can
be implemented at discrete points along a numerical model of a membrane with
arbitrary geometry, representing a realistic experimental system exposed to an
electric pulse. Numerical solution of (1) yields prediction on the number and size
of the pores at a given time and position on the membrane. The entire modeling
approach is too extensive to be discussed here (see, e.g., Lattice Model of
a b 200
hydrophobic (HO) pore
400
150
Pore energy Wp [kT ]

300
hydrophilic (HI) pore 100 Um = 0.0 V
[kJ/mol]
200
50 0.3 V
HO HI 100
pore expansion 0.5 V
rp 0 0
dm 0.7 V
1.5 V 1.0 V –100
−50 rHO*
0 1 2 3
Pore radius rp [nm]
Fig. 4 Main predictions from continuum modeling of lipid pores. (a) Predicted steps in pore
evolution. The pore initially forms as hydrophobic (HO). If an HO pore reaches the radius rHO*, it
becomes energetically more favorable for the pore to transform into a hydrophilic (HI) pore. The HI
pore can then further expand in size. (b) Pore energy landscape for different values of the
transmembrane voltage Um. Pore energy was calculated based on Eqs. 2–5, using parameters
given the text and assuming γ = 20 pN. Left vertical axis expresses the pore energy in units of
the thermal energy kT, right axis in units of kJ/mol at temperature T = 298 K
Electroporation). Put simply, such modeling can be used to study and predict the
time course of the transmembrane voltage, ionic current, and molecular transport
through the electroporated membrane, i.e., typical physical quantities that can be
compared to experimental measurements.
The potential function ΔWp, which governs the dynamics of the pore population,
corresponds to the work required to form a pore in the membrane and is equivalent to
the pore free energy. Traditionally, ΔWp is expressed in terms of the pore radius and
is derived based on continuum approximations by viewing the pore as a circular hole
in a homogeneous membrane slab. The pore energy landscape ΔWp(rp) predicted
from continuum modeling is shown in Fig. 4b. The origin and interpretation of this
landscape are explained throughout the following subsections and compared to
observations from MD. Namely, the landscape has been proposed without any
molecular-level insight into electroporation and has been tested only against indirect
experimental measurements. Furthermore, continuum approximations can start to
lose their validity on the molecular scale (scale of single pores) since they inherently
neglect the finite size of atoms and often the specific interactions between individual
atoms or molecules. Unfortunately, only limited efforts have been made to quanti-
tatively compare continuum-level descriptions with results from simulations, which
relates to the fact that such comparison is often not simple and straightforward. Still,
at least a qualitative comparison can be made based on the present results from MD.
Note that in continuum modeling the pore free energy is generally separated into
three contributions – the edge energy ΔWedge, the surface energy ΔWsurf, and the
electrical energy ΔWel:
12 L. Rems
ΔW p ¼ ΔW edge þ ΔW surf þ ΔW el (2)
The edge energy and the electrical energy are explained in greater detail in section
“Hydrophobic (HO) and Hydrophilic (HI) Pore Configuration.” The surface energy
is though important when the membrane is subject to finite surface tension. Since
most of the presented MD results were obtained at conditions of practically zero
surface tension, ΔWsurf is neglected in this chapter. However, the reader is encour-
aged to see Jähnig (1996), den Otter (2009), Portet and Dimova (2010), and Casciola
et al. (2016), as well as references therein, for discussions on the surface tension and
its effect on pores in experimental and MD membrane systems.
Hydrophobic (HO) and Hydrophilic (HI) Pore Configuration
The formation of a water-spanning column as a step preceding the migration of lipid

headgroups into the pore, observed in MD, resembles the predicted transition of
pores from “hydrophobic” (HO) to “hydrophilic” (HI) (Fig. 4a) (Abidor et al. 1979).
In continuum modeling, this transition has been argued by comparing the energetic
cost for forming a pore of either of the two configurations.
Think of the following. The formation of a pore in the bilayer necessary involves
the establishment of the pore “edge.” The lipids lining the pore edge can either
remain in their planar, unperturbed state or can reorient their headgroups into the
pore wall. The first configuration, the HO pore, requires an energy penalty due to the
hydrophobic effect as the hydrophobic lipid tails are in direct contact with water. The
second configuration, the HI pore, also requires an energy penalty since the lipids
need to reorganize and bend into the pore. This energy penalty is often referred to as
the edge energy. The edge energy of an HO pore was derived from a theoretical
model describing the hydrophobic interactions between two hydrophobic surfaces
separated by a thin layer of water (Glaser et al. 1988):

I 1 r p =λw
ΔW HO ¼ 2πr p Γ hw dm (3)
edge
I 0 r p =λw
Parameter Γ hw 50 mN/m denotes the hydrocarbon–water interfacial tension, dm

5 nm the membrane thickness, and λw 1 nm a characteristic length describing how
far the hydrophobic surface perturbs the water structure, whereas Ii are the modified
Bessel functions (Fig. 4b, thick gray line).
The edge energy of an HI pore can be in the first approximation derived from the
elastic bending free energy of a lipid monolayer. For large pores, the edge energy is
proportional to the pore circumference and can be expressed as
edge ¼ 2πr p γ;
ΔW HI rp ! 1 (4)
The proportionality constant γ, termed the edge tension, ranges between ~5 and
50 pN and depends on the lipid composition of the bilayer (Portet and Dimova 2010;
West et al. 2013). For small HI pores, the edge energy is expected to increase with
decreasing pore radius due to the contribution of the 1/rp curvature of the pore, as
well as due to repulsive forces between the lipid headgroups. Formulating the exact
functional dependence of the edge energy for small HI pores though appears to be
beyond the validity of continuum approximations (Glaser et al. 1988; Wohlert
et al. 2006; West et al. 2013). To account for the predicted increase in the edge
energy, an arbitrary phenomenological term (C/rp)4 was added to (4), where
C = 9.671015 J1/4m is a fitting constant (Neu and Krassowska 1999) (Fig. 4b,
thick black line).
In the presence of transmembrane voltage Um, the energetic cost for forming both
the HO and HI pore is expected to decrease. If one assumes that this decrease can
mainly be attributed to a small change in the membrane specific capacitance, because
the lipids with low dielectric permittivity (el 2) are replaced by high permittivity
water (ew 80) inside the pore, then a simple estimate for a cylindrical pore suggests
(Abidor et al. 1979):
1 e0 ðew el Þ
ΔW el ¼ πr p 2 U m 2 (5)
2 dm
where e0 is the dielectric permittivity of the vacuum. Combining Eqs. (2–5) gives the
pore energy a landscape shown in Fig. 4b. When the electrical energy ΔWel is assumed
to be equal for HO and HI pores, the pore radius rHO*, at which the crossing between
the HO and HI pore energy occurs, remains the same regardless of the magnitude of
Um (Glaser et al. 1988). Consequently, rHO* depends only on the edge energy.
The estimates described above are rather simple and empirical. Nevertheless, they at
least provide a conceptual understanding of the HO!HI pore transition: a pore
preferentially forms in the HO configuration and remains in this configuration until it
reaches a size where the energetic cost for reorientation of the lipid headgroups becomes
comparable to the cost for maintaining the “edge” of the HO pore. Indeed, MD
simulations have demonstrated that in certain types of lipids, such as POPS
(palmitoyl-oleoyl-phosphatidylserine) with negatively charged headgroups and archaeal
lipids with headgroups formed from large sugar moieties, an HO pore can become
sufficiently large to conduct ions, and, on the timescale of the simulations, the pore does
not transform into HI configuration (Dehez et al. 2014; Polak et al. 2014). Conversely, in
bilayers where the headgroup reorientation is energetically less demanding, the forma-
tion of an HO pore is followed within few nanoseconds by its transition into HI
configuration (Levine and Vernier 2010; Bennett et al. 2014). The different behavior
of pores in bilayers with different lipid composition already clearly indicates that the
pore energy landscape can markedly (quantitatively) depend on the lipid structure.
Pore Creation
Continuum models generally assume that only HI pores are large enough to conduct
ions and are for this reason the most relevant pore configuration which can be
14 L. Rems
detected experimentally. Most continuum models therefore focus on describing the

creation of HI pores. As mentioned in section “Hydrophobic (HO) and Hydrophilic
(HI) Pore Configuration,” MD simulations demonstrate that a pore does not neces-
sarily have to transform into HI configuration to conduct ions. But to facilitate
comparison between continuum models and results from MD, all of the following
discussions refer to typical zwitterionic phospholipid bilayers, such as POPC
(palmitoyl-oleoyl-phosphatidylcholine), which exhibit a rapid HO!HI pore
transition.
According to the pore energy landscape in Fig. 4b, an HI pore can be created
provided that the system is able to overcome the energetic barrier which arises at the
crossing between the HO and HI pore energy (at rHO* 0.5 nm). The proposed
landscape is based on the following assumption (Glaser et al. 1988). If an HO pore
with radius rHO* (denoted here as HO* pore) is created, it rather passively transforms
into energetically more favorable and more stable HI pore. Hence, any intermediate
steps in the HO!HI pore transition do not require considerable energetic input, and
the energetic cost for creating a minimum-size HI pore is approximately equal to the
cost ΔWHO* for creating an HO* pore. In the absence of transmembrane voltage Um,
ΔWHO* is much higher than the thermal energy (~45 kT in Fig. 4b); therefore, HO*
pores rarely form spontaneously. By increasing Um, the probability for formation of
HO* pores increases, since Um decreases ΔWHO*. Taking into account equation (5),
the decrease in ΔWHO* with Um can be written as
2
ΔW HO ðU m Þ ¼ ΔW HO
p ðr HO , U m Þ ¼ ΔW HO,0 BU m (6)
where ΔWHO*,0 is the energetic cost for forming an HO* pore in the absence of Um
and B = 0.5e0(ew – el) πrHO*2/dm 13 kT/V2 is a proportionally constant. By
knowing ΔWHO*, it is possible to express the creation rate of HO* pores with the
Van’t Hoff–Arrhenius law:

ΔW HO ΔW HO,0 BU m 2
kHO ¼ A exp ¼ A exp þ (7)
kT kT kT
The prefactor A generally scales with the membrane area, but its exact definition
depends on the details of the theoretical framework used to describe the pore
creation rate in a given system (cf. Neu and Krassowska (1999), Vasilkoski
et al. (2006), Böckmann et al. (2008), Hänggi et al. (1990)). As HO* pores are
expected to transform into HI pores, kHO* is considered to be approximately equal to
the creation rate of HI pores.
The above theoretical assumptions and predictions are quite close to observations
from MD simulations. The latter have demonstrated that the creation sequence of an
HI pore can be divided into at least three kinetic steps (Fig. 3): pore initiation, pore
construction, and pore maturation. Pore initiation can be viewed as formation of an
HO* pore, whereas pore construction and maturation can be viewed as two inter-
mediate steps in the HO!HI pore transition. Pore initiation is indeed the rate-
limiting step, followed by rather passive pore construction and maturation within
a 15 b
Pore initiation
Pore creation
Poration time [ns]
10
0
3 4 5 6 7
Voltage [V]
Fig. 5 Dependence of the average pore creation time on the voltage across the simulation box Ubox
Um. (a) Pore creation consists of three steps: pore initiation, construction, and maturation (see
Fig. 3). Pore creation time is mostly determined by the pore initiation time (formation of a water-
spanning column), which falls exponentially with increasing voltage. Pore construction and mat-
uration follow pore initiation within ~2 ns. Pore creation time was determined in a POPC
(palmitoyl-oleoyl-phosphatidylcholine) bilayer. Error bars show standard deviation from three
independent simulations (the figure was plotted based on data published in Levine and Vernier
2010 (their Table 1)). (b) Average poration time obtained on bilayers with different lipid compo-
sition: POPC bilayer, POPE (palmitoyl-oleoyl-phosphatidylethanolamine) bilayer, and a bilayer
with asymmetric composition consisting of POPC in one and POPE in the other leaflet. The errors
bars show standard errors of the mean (Reprinted with permission from Gurtovenko and Lyulina
2014. Copyright 2014 American Chemical Society)
~2 ns. The average time tHO* required to observe pore initiation after the onset of an
external electric field falls exponentially with the voltage that develops across the
simulation box Ubox Um (Fig. 5a). Since tHO* is inversely proportional to the rate
of pore initiation, tHO* / kHO*1, this observation is consistent with an exponential
dependence on Um expected from Eq. 7. The predicted pore radius rHO* 0.5 nm at
which the HO!HI pore transition occurs is also in agreement with the simulations
(~0.3–0.5 nm (Böckmann et al. 2008; Levine and Vernier 2010)).
However, MD simulations suggest that the logarithm of tHO* does not decrease
with Um2, as would be expected from Eq. 7, but rather exhibits an approximately
linear decrease with Um, at least within the range of voltages considered (Fig. 5b). To
describe this linear relationship, it was proposed that the electric field-driven
reorientation of lipid headgroup dipoles (“Phospholipid Head Group Dipoles and
Electropore Formation”) may play a role in promoting pore creation and that log
(tHO*) falls proportionally with ΔμUm/(dmkT), where Δμ is the average change in
the dipole moment within the system (Böckmann et al. 2008). Yet, the electric field
increases the pore creation rate even in octane layers and in water–vacuum–water
systems, which does not support the idea that the lipid dipoles play a major role in
promoting pore creation. It appears that the dominant role can instead be attributed to
water molecules. Namely, MD revealed that the electric field-driven formation of a
water column across the bilayer decreases the energy of the interfacial water
molecules, which participate in the initial water protrusion into the bilayer core.
16 L. Rems
Similar slopes of the exponential fits to the “poration timevoltage” curves (Fig. 5b)
(Gurtovenko and Lyulina 2014), obtained on bilayers with different lipid composi-
tion, give some additional evidence that the lipid composition is not very important
in the mechanism by which the electric field decreases the energetic cost for pore
creation.
Nevertheless, the rate of pore creation should depend also on the energetic cost
ΔWHO*,0 for creating a pore in the absence of Um (see Eq. 7). This energetic cost
depends strongly on the lipid composition. For example, ΔWHO*,0 was estimated
from MD to be 17, 45, and 78 kJ/mol for DLPC (dilauroyl phosphatidylcholine),
DMPC (dimyristoyl phosphatidylcholine), and DPPC (dipalmitoyl phosphatidyl-
choline) bilayers, respectively (Bennett et al. 2014). The energetic cost for creating
a pore is correlated with the thickness of the bilayer, but it also depends on the
structure of the lipid headgroups and lipid tails (Hu et al. 2015). The different values
of ΔWHO*,0 in bilayers with different lipid composition are at least partially reflected
in the shift between the curves in Fig. 5b. As such, they are also reflected in the
different “threshold” electric fields and transmembrane voltages required to observe
a pore within a given time of the simulations, as well as in different “thresholds”
required to detect electroporation in experimental systems (“Critical Electric Field
and Transmembrane Voltage for Lipid Pore Formation in Experiments”). Although
theoretically there is no specific “threshold” for electroporation, the exponential
dependence of the pore creation rate on Um is manifested in the fact that electropo-
ration can be detected as a threshold-like phenomenon. Note that the “thresholds”
extracted from MD are higher than those measured in experiments, which can be
attributed to the small system size and short duration of the simulations (for
arguments see Böckmann et al. (2008), Ziegler and Vernier (2008), and Vasilkoski
et al. (2006)).
Dynamics of the Pore Size
Once an HI pore is created, the pore further tends to adapt to its energetically most
favorable configuration. From the perspective of the pore energy landscape in Fig. 4b,
the pore either tends to shrink or expand, depending on whether the derivative
@ΔWp/@rp is positive or negative, respectively. In the absence of Um, expansion of
the pore is energetically unfavorable due to the edge energy (4), for which the pore
tends to close. Within a certain range of Um, there exists a local minimum for which the
pore can remain metastable. This local minimum is predicted to shift to larger pore
radius with increasing Um. However, if Um becomes too high, the local minimum
vanishes suggesting that the pore starts to expand indefinitely.
These predictions are indeed qualitatively corroborated by results from
MD. When a pore is induced by an external electric field, the pore continuously
expands as long as the porating electric field is maintained. However, the pore
becomes stabilized, if the initial (porating) electric field is reduced to a lower
(sustaining) value. The size of such stabilized pore (“Lipid Electropore
Fig. 6 Pore stabilization in MD under conditions of constant charge imbalance. a, b Cumulative

average of the pore radius (a) and electric current through the pore (b) along the elapsed simulation
time. (c) The pore radius versus the established transmembrane voltage Um averaged over the last
60 ns of the simulation. Simulations were performed by maintaining a constant charge imbalance of
20qe (black dotted line), 32qe (red dashed line), 40qe (green dot-dashed line), and 56qe (cyan solid
line), where qe is the elementary charge (Adapted with permission from Casciola et al. 2016.
Copyright 2016 Elsevier)
Stabilization”) increases with the magnitude of the sustaining electric field. In

contrast, if the external electric field is too low, the pore collapses. In typical
simulations with charge imbalance, the situation is slightly different. Once a pore
is created, the charge imbalance gradually decreases throughout the simulation due
to the leakage of ions through the pore. When the charge imbalance (and the
resulting Um) becomes too low to sustain the pore, the pore collapses. Neverthe-
less, the simulations can also be performed by maintaining a constant charge
imbalance across the pore (Casciola et al. 2016). In such case, the pore size
similarly becomes stabilized, whereby the radius of the stabilized pore increases
with the value of the charge imbalance (Fig. 6a). Since the size of the pore is
stabilized, the ionic current through the pore and Um attain an approximately
constant value as well (Fig. 6b, c).
18 L. Rems
It should be mentioned that the radius of the stabilized pore, plotted in Fig. 6c
versus Um, cannot be quantitatively compared to the position of the local minima in
Fig. 4b for the following reasons:
(i) Equation 5 assumes that Um remains constant regardless of the change in the
pore size. In MD simulations, Um cannot be directly controlled; the pore size,
the ionic current through the pore, and the Um are all “free” to change and adapt
as the system moves toward an energetic minimum.
(ii) Even from the perspective of continuum-level modeling, Eq. 5 is only a rough
approximation for conductive pores; more appropriate derivation of the change
in the electrical energy with the pore size comes from the Maxwell stress acting
at the water–bilayer interface (Neu et al. 2003).
(iii) The pore energy landscape is expected to be quantitatively different for bilayers
with different lipid composition, as already pointed out in sections “Hydropho-
bic (HO) and Hydrophilic (HI) Pore Configuration” and “Pore Creation.”
Therefore, a quantitative comparison can only be made, if the parameters of
the continuum model are adapted to the system studied in MD.
Ionic Conduction Through a Pore
Stabilization of pores in MD simulations is particularly suited for characterizing the

equilibrium properties of the pores, including their ionic conductance. The
conductance of a pore (▶ Chap. 13, “Electrical Conductance of Lipid Pores”)
can be determined from the simulations by the ratio between the sum of the electric
currents Ii, carried by all ions of type i passing through the pore, and the transmem-
brane voltage Um:
X
Ip I
i i
Gp ¼ ¼ (8)
Um Um
In continuum modeling, conduction of ions through a pore is usually described in
terms of the coupled steady-state Nernst–Planck equations and the Poisson’s equa-
tion. The Nernst–Planck equations describe the molar flux ji of ions of type i, where
the ions are represented by ionic concentration ci (in mol/m3):
∇ji ¼ 0 (9a)
qe
ji ¼ Di ∇ci Di zi ci ∇Ψ þ vci (9b)
kT
Parameters Di and zi are the diffusion coefficient and valence of ions of type i,
respectively, v is the fluid velocity, and qe the elementary charge. The driving force
for the electrophoretic drift – the gradient in the electric potential Ψ – is given by the
Poisson’s equation:
ee e0 ∇ð∇ΨÞ ¼ ρe (10a)

X
ρe ¼ F zi ci (10b)
i
where ρe is the spatial charge distribution in the electrolyte, ee the relative dielectric
permittivity of the electrolyte, e0 the permittivity of vacuum, and F the Faraday
constant. To recover the pore conductance, ji needs to be integrated over the pore
area in order to obtain the ionic current Ii, which is then inserted into (8):
ðð
I i ¼ Fzi ji dA (11)
Apore
The comparison between the pore conductance extracted from MD simulations of

NaCl conduction through a pore in a POPC bilayer and pore conductance predicted
from a continuum model based on the Poisson–Nernst–Planck (PNP) theory
demonstrated very good agreement (Fig. 7b, c). The PNP model considered a
toroidal pore in a homogeneous membrane slab, which was a rather good approx-
imation for the shape of the pore observed in the simulations (Fig. 7a). It is important
to stress that in order to ensure a straightforward quantitative comparison between
the PNP model and results from the simulations, all relevant model parameters were
extracted directly from the MD trajectories (Rems et al. 2016).
Calculations performed with the PNP model also enabled a systematic analysis of
the mechanisms behind the difference in the pore conductance for Cl– and Na+ ions.
MD simulations, namely, showed that the ratio between the electric current carried
by Cl– and Na+ ions varies with the pore size, whereby the pore is always less
conductive to Na+ ions. The PNP model showed that lower conductance for Na+ ions
can be attributed to (i) lower bulk diffusion coefficient of Na+ compared to Cl– ions;
(ii) binding of Na+ ions to lipid headgroups inside the pore, which effectively acts as
a layer of surface charge at the electrolyte–lipid interface; and (iii) electroosmotic
flow of the fluid, which was induced through the pore in the opposite direction of the
flux of Na+ ions (Fig. 7b).
Pore Annihilation
MD simulations demonstrate that as long as Um is sufficiently high (about few

100 mV, see Fig. 6c), the pore can remain open. However, when Um is reduced to
zero, the pore annihilates (“Lipid Electropore Lifetime in Molecular Models”).
Similarly as pore formation, pore annihilation is stochastic, and generally occurs
within few nanoseconds to few hundreds of nanoseconds, depending on the lipid
composition (Levine and Vernier 2010; Bennett et al. 2014).
In continuum theoretical models, the pore annihilation kinetics is often
described simply based on empirical observations from experimental measurements.
According to experimental measurements of the decrease in membrane conductance
20 L. Rems
MD SYSTEM PNP MODEL

a
vacuum
Ψ = Um cNa = cNa,A cCl = cCl,A
rp zbox,A
dm
area of integration zbox,B
Ψ=0V cNa = cNa,B cCl = cCl,B

vacuum
b
40 5
Pore conductance Gp [nS]
MD
w/o qsurf
Selectivity ICl / INa
30 w/ qsurf 4
w/ qsurf & flow
20 3
10 2
0 1
0.5 1 1.5 2 2.5 3 0.5 1 1.5 2 2.5 3
Pore radius rp [nm] Pore radius rp [nm]
Fig. 7 Comparison of the pore conductance extracted from MD simulations and predicted by a
finite-element model based on the PNP theory. (a) Left: Slice across the central region of the system
from MD simulations. Na+ ions and Cl– ions are presented in yellow and green, respectively; the
after application of an electric pulse, most of the pores can be expected to collapse/
close within few μs (Glaser et al. 1988; Sengel and Wallace 2016), which is in
reasonable agreement with the kinetics of pore annihilation observed in
MD. However, experiments also demonstrate that the lipid bilayer does not
completely recover even up to seconds after the exposure to an electric pulse,
which is at least seven orders of magnitude longer than the time required to observe
pore annihilation in MD (Abidor et al. 1979; Glaser et al. 1988). Therefore, it cannot
be completely excluded that the electric field induces some additional effects in
realistic experimental lipid membranes, which are at present not taken into account
in MD. Complete understanding of pore annihilation and lipid bilayer recovery after
exposure to pulsed electric fields thus awaits further systematic research.
Conclusions
Continuum models of lipid pores formed under the influence of an electric field have
been supporting experimental investigations of electroporation for almost 40 years.
Considering that continuum models were developing without any direct insights into
the molecular mechanisms of how the pores form and behave in the presence of an
electric field, their agreement with results from MD simulations is quite impressive.
Nevertheless, MD now provides the possibility to enhance the understanding of
electroporation on the molecular level of the lipid bilayer and advance continuum-
level modeling, in order to enable quantitative, rather than qualitative predictions.
The main requirement for development of continuum models comes from the fact
that MD simulations are computationally too demanding to be directly applied for
studying electroporation on the scale of model lipid membranes, whole cells, or cell
assemblies. Consequently, continuum models could serve as a bridge between
insights from MD and experiments. In particular, MD offers an indispensable insight
into the kinetics of pore formation, dynamic behavior of pores, kinetics of pore
annihilation, as well as transport of small ionic or other molecular species across the
Fig. 7 (continued) lipids are in gray with phosphate atoms shown as spheres. Water is shown as
light-gray surface. The gray rectangle indicates the dimensions of the simulation box in x and
z direction. Right: Geometry of the corresponding PNP model. Lipid bilayer is shown in dark gray.
(b) Comparison of the total pore conductance (left) and the ratio of electric currents carried by Cl–
and Na+ ions through the pore, i.e., the pore selectivity (right). The results from MD simulations and
the PNP model are shown as black squares and lines, respectively. The PNP model is able to predict
the pore selectivity observed in the simulations when Na+ ions bound to the lipid headgroups are
represented in the model by a layer of surface charge qsurf = 0.27 qe/nm2 and when the electroos-
motic flow through the pore is taken into account (blue dotted line). When the electroosmotic flow is
neglected (green dashed line) or when both the electroosmotic flow and the surface charge are
neglected (red solid line), the model fails to predict the pore selectivity. (c) Comparison of the Na+
and Cl– concentration profiles in the MD system and the PNP model demonstrating good agree-
ment. The center of the pore is located at coordinate (0,0) (Adapted with permission from Rems
et al. 2016. Copyright 2016 Elsevier)
22 L. Rems
pores in membranes with different lipid composition – i.e., information which could
be implemented into more large-scale continuum models, representing realistic
experimental systems. Robust quantitative theoretical models of electroporation
can then help distinguish the contribution of lipid pores to the observed experimental
outcomes from the contributions of other structural alterations of the lipid
membrane, which could be induced by electric pulses.
Cross-References
▶ Critical Electric Field and Transmembrane Voltage for Lipid Pore Formation in
Experiments
▶ Electrical Conductance of Lipid Pores
▶ Lipid Electropore Lifetime in Molecular Models
▶ Lipid Electropore Stabilization
▶ Phospholipid Head Group Dipoles and Electropore Formation
▶ Transmembrane Voltage Induced by Applied Electric Fields
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Critical Electric Field and Transmembrane
Voltage for Lipid Pore Formation in 2
Experiments
Justin Teissie
Abstract
Electropermeabilization is the result of exposure of cells to electric field pulses.
The field pulse lasts from submicro- to several milliseconds. The field intensity
should be large enough to induce a dramatic structural local alteration of the cell
membrane organization. This results in transiently increased permeabilization of
the target cell membrane. Due to the dielectric properties of the cell membrane,
the external field induces a transmembrane voltage (TMV) which is controlled by
size distribution in a population of cells. In most experiments, permeabilization is
detected only when cell is exposed to a critical field strength. This is associated to
a critical TMV that affects a subpopulation among the treated cells. This chapter
describes the approaches to associate external field and induced TMV. The
definition of the critical TMV is discussed.
The different assays (conductance, transport) are described with the critical
consideration of their sensitivity for the definition of a threshold. The resulting
different estimations of the critical TMV are then reported for the different assays
and systems. The information provided by lipid assemblies, the simplest system,
is then described as a guide for the effects of chemical and biochemical modifi-
cations of cell membranes. But the final conclusions remain that cell membrane
electropermeabilization is indeed a complex process and its molecular character-
ization remains an intense field of investigations.
Keywords
Membrane • Transmembrane voltage • Transport • Membrane structure
J. Teissie (*)
Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS,
Toulouse, France
e-mail: Justin.teissie@ipbs.fr; justin@ipbs.fr

DOI 10.1007/978-3-319-32886-7_77
26 J. Teissie
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Electropermeabilization: a Theoretical Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
The External Field Induces a Change in the Transmembrane Voltage . . . . . . . . . . . . . . . . . . . . . . 26
The Resulting Transmembrane Voltage Induces Membrane Permeabilization
(Electropermeabilization) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Methods to Detect Permeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Conductance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Direct Assays by Electrical Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Patch Clamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Lipidic Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Does the Critical Electropermeabilization TMV Exist? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Critical Electropermeabilizing TMV on Red Blood Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Muscle Fibers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Patch Clamp Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Assay on a Cell Population . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
The Pulsing Protocol Affects the “Experimental” Critical TMV . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Fluorescence Imaging Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Electropermeabilization Is Inducing Membrane Fusogenicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
What Lipid Systems Can Tell Us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Introduction
As described all along the handbook, electropermeabilization is the result of exposure

of cells to electric field pulses. The field intensity should be large enough to induce a
dramatic structural local alteration of the cell membrane organization. This is observed
to occur only when the field strength is larger than a critical value. Due to the dielectric
properties of the cell membrane, the external field induces a transmembrane voltage
(TMV). This chapter reports the state of the art on the present knowledge on the critical
field and on the critical TMV associated to electropermeabilization. The conclusions
are that cell membrane electropermeabilization is indeed a complex process.
Electropermeabilization: a Theoretical Approach
The External Field Induces a Change in the Transmembrane Voltage
Short high electric field pulses when delivered to a cell suspension or a tissue results
in cell membrane permeabilization (electropermeabilization). Classical electro-
permeabilization results in an enhanced transport across the membrane and in a
change in its electrical conductivity. The electric field induces a transmembrane
voltage (TMV) (Rems and Miklavcic 2016).
2 Critical Electric Field and Transmembrane Voltage for Lipid Pore. . . 27
When a cell is submitted to an external electric field, the cell (where the plasma
membrane can be considered as a dielectric shell) should be considered as a spherical
capacitor, where the redistribution of electrophoretically driven charged ions in the
electrolytes surrounding the membrane, i.e., electric current, leads to an induced
transmembrane voltage (TMV). During the electric field application (electro-
pulsation) to the cell, an induced transmembrane voltage, i.e., potential difference
(ΔVi), is created which is locally associated with the dielectric properties of the
plasma membrane.

ΔViðM, Ε, tÞ ¼ fgðλÞrE cosθðMÞ 1 exp:ðt=τmÞ (1)
where M is the point on the cell that is considered, t is the time after electropulsation
is turned on, f is a factor depending on the cell geometry (for a sphere, f = 1.5), r is
the radius of the pulsed cell, E is the electric field strength, and θ(M) is the angle
between the direction of the field and the normal of the cell surface in M. g(λ) is
related to the different conductivities as (Zimmermann et al. 1974).
τm is the characteristic time constant of the membrane charging and can be
written as, (Kinosita and Tsong 1977):
τm ¼ rCmð2λe þ λiÞ=ð2λeλiÞ (2)
where Cm (0.5–1.0 μF/cm2) is the specific membrane capacitance. τm is calculated

to be in the submicrosecond time range for mammalian cells. It is strongly dependent
on the buffer composition as the internal composition is fixed by the cell metabolism.
The highest amplitude of ΔVi (M, Ε, t) on a (spherical) cell (radius r), for a field E
is obtained for θ = 0 (cos 0 = 1) or θ = π (cosπ = 1), with a long pulse duration
(T> > τm).
If the plasma membrane is considered to be a pure spherical dielectric (λm = 0),
we obtain g(λ) = 1. In these conditions and when the steady state is reached
(t> > τm) as for millisecond pulses, Eq. 1 simplifies to:
ΔViðM, EÞ ¼ 1:5rEcosθðMÞ (3)
The highest amplitude of ΔVi (M, Ε, t) is then 1.5Er.

The consequence is that it affects more dramatically the largest cell in a popula-
tion with cells with different sizes but biologically identical.
In cells of all types, there is an electrical potential difference between the inside of
the cell and the surrounding extracellular fluid. This is termed the resting transmem-
brane voltage of the cell (transmembrane voltage). When living cells are electro-
pulsed, ΔVi adds to the resting one ΔV0.
An electrical potential difference results from a net separation of electric charge by
the thin nonconductive layer, i.e., the membrane in a cell. TMVs exist across the
membranes of organelles. Electrical potentials (voltage) are measured in units of volts.
(A volt is defined in terms of energy per unit charge; that is, 1 V is equal to 1 J/C).
In a mammalian cell, its resting transmembrane voltage is about 70 mV. The
minus sign indicates that the inside of the cell is negative with respect to the
28 J. Teissie
extracellular buffer by definition, and when the ground electrode in measurements is

placed outside of the cell. ΔV0 is considered as not dependent on the cell size in a
homogeneous population.
Due to this ionic difference between the extracellular buffer and the cytoplasmic
volume, two driving forces influence the movement of an ion across a plasma
membrane.
• The concentration gradient

• The transmembrane voltage
The concentration gradient is valid for all molecules uncharged or charged

(including ions). But for ions, the flux, i.e., the movement of an ion across the
membrane, should consider the sum of the flux due to concentration gradient as well
as due to force exerted by electric field acting on charged particle, i.e., due to TMV
(electrophoresis). These two factors may act in the same direction or in opposite
directions.
The TMV is associated to a transmembrane field (V/cm).
E ¼ ΔV=d (4)
As a membrane is thin (d is about a few nm), even if the TMV is of the order of
100 mV, the associated field can be as large as 10 MV/m. If a membrane is porous,
charged molecules are submitted to an electric drift by the transmembrane field as
well as a diffusion driven by the concentration gradient.
Due to its intrinsic ion leakage, the membrane need to have active transporters to
maintain the concentration gradient. The consequence is that metabolic energy is
used. The resting TMV is maintained at the expense of this energy. If the metabolic
level of the cell is affected (starved cells for example), the gradient is not maintained
and the resting TMV is reduced.
When an external field is present, then the resulting TMV is
ΔVðEÞ ¼ ΔV0 þ ΔViðM, EÞ (5)
On Fig.1, the TMV point from negative to positive and their lengths is propor-
tional to the voltage. The large arrow on the top represents the electric field lines. On
the left, a cell is pictured with its homogeneous resting voltage, the middle is
indicative of the field-induced TMV that is highly position dependent along the
cell surface. On the right, the resulting modulation of the TMV when the field is
present, a highly complex position-dependent TMV is observed.
This gives an additive effect on one side of the cell and a subtractive one on the
opposite. The electric field effect is strongly position dependent (Fig. 1). When a
5103 V/M is delivered on a cell with a 10 μm radius, Eq. 3 predicts that the TMV can
locally be doubled.
One limit in the use of the Laplace equation to obtain the ΔV from the external
field E is that it is limited to spherical cells (Laplace equation). Biology is handling
Fig. 1 Modulation of the TMV of a spherical cell by an applied external field
irregularly shaped cells. In an approach making an analytical approach, the finite-

thickness, nonzero-conductivity membrane was replaced by a boundary condition in
which a specific surface conductivity is assigned to the interface between the cell
interior (the cytoplasm) and the exterior (Pucihar et al. 2006). The simulation was
validated on spherical cells (using the Laplace equation) and by a direct observation
of cells with irregular shapes where the field effect was monitored with a potentio-
metric fluorescent dye.
The Resulting Transmembrane Voltage Induces Membrane

Permeabilization (Electropermeabilization)
When the new membrane electric potential difference ΔVm (ΔV0 + ΔVi) locally
reaches a critical value (ΔVc) (Rems and Miklavcic 2016), a local alteration of the
membrane structure leads to membrane permeabilization (electropermeabilization).
ΔVc ¼ ΔV0 1:5rEc (6)
Ec is the lowest field strength that is supposed to trigger permeabilizing events for
a spherical cell with a radius r. As is ΔV0 = 70 mV, this is on the pole of the cell
facing one electrode (θ(M) = 0).
Electropermeabilization initial event is described as a local event on the cell
surface.
Molecular processes supporting classical electropermeabilization remain poorly
understood (Teissie et al. 2005). The membrane is called “porous.” The critical field
Ec can be experimentally approached. It is the lowest field strength that induces an
enhanced transport or an increase in membrane conductance. If ΔVc is assumed to
be the same whatever the cell, that is, considered in an homogeneous population,
then it is controlled by the metabolic state (through ΔV0). The critical field will be
affecting the largest cell in the population (through r).
30 J. Teissie
As it is an experimental detection of transport or membrane conductance, the

sensitivity of the assay plays a capital role.
Methods to Detect Permeabilization
Conductance
If at any point, ΔVm gets high enough (ΔVc) to induce a sufficient number of
conductive defects, the defects allow enhanced ionic transport through the mem-
brane and increase the membrane conductivity by several orders of magnitude
(transport, conductivity).
Direct Assays by Electrical Measurements
By measuring the voltage and the current flowing between the electrodes, one gets
access to the conductivity of the suspension. Under steady state condition, the
conductance of a diluted intact cell suspension can be considered controlled by the
conductance of the external solution. An ohmic behavior is observed when a voltage
pulse is delivered on the cell suspension (as long as the Joule heating is small).
When electropermeabilization appears, two events are present:
(i) The dielectric shell has conductive defects that increase during the pulse.
(ii) There is leak of the internal (cytoplasmic) solution.
Both events result in an increase in the cell suspension conductivity.

To obtain the critical field Ec, cell suspensions are pulsed by strong fields with
increasing strengths and the change in conductivity of the suspension is monitored
after each step (by applying low voltage AC train). The limit is indeed the sensibility
in the current detection. At the onset of permeabilization, only a very limited cap is
affected (θ(M) is close to zero). Therefore, few defects are present on the membrane
and the ionic leakage is limited (Kinosita and Tsong 1977; Pavlin et al. 2005).
A linear ramp in E with a small AC modulation can be used to detect the dynamic
change in current. When the permeabilization occurs, the amplitude of the AC
current shows fluctuations. This is described in details on planar lipid membrane
(Kramar et al. 2012). The steadily increasing voltage across the bilayer imposed by
linearly increasing current (capacitive loading) led to electropermeabilization of the
membrane when voltages above a few hundred millivolts were obtained. Small
voltage drops were detected preceding the destructive burst of the bilayer due to
“irreversible electroporation.” These voltage drops were often followed by a voltage
re-rise within tens of milliseconds. The fluctuations are associated to opening and
closing of conducting defects through the bilayer.
With a coulter counter, cells, due to their poor conductivity when intact, alter
the effective cross-section of the conductive channel (conductivity). The
electrical resistance across the channel increases, and under voltage clamp, the
electric current passing across the channel decreases. The size of the electric
current drop is a function of the size of the cell and of the leakiness of the
membrane. Therefore, when the voltage across the channel induces a field
high enough to trigger electropermeabilization, the associated current drop
is smaller. The limit is that the definition of the field distribution is poor
(Zimmermann et al. 1974).
Patch Clamp
The patch-clamp technique allows the investigation of currents through a small set or
even single ion (conducting) channels and therefore about the membrane conduc-
tance. A small pipette is brought in contact with the cell membrane and a tight seal
between the pipette and the membrane is obtained by a mild suction. A brief but
strong suction ruptured the cell membrane. The pipette has a direct access to the
cytoplasm (Ryttsen et al. 2000). The whole-cell patch-clamp technique was
employed to investigate the effect of millisecond pulsed electric fields on plant
protoplast (wall free plant cell) and DC-3F cells (Wegner et al. 2013). Cellular
membrane permeabilization was monitored by a conductance increase. It could be
established experimentally that electropermeabilization is associated to a rapid
membrane conductance change (due to the creation of conducting defects in the
cell membrane). This critical event occurs while the field is present to depolarize or
hyperpolarize the membrane to voltages beyond the critical potentials (evaluated to
be +201 mV and 231 mV, respectively, in the study).
Transport
Membrane permeabilization is associated with an enhanced transport of water-

soluble small molecules (that are with a low molecular weight, say less than 2 kD)
and ions (transport). It can be assayed by the inflow of dyes (Trypan blue, Propidium
iodide (PI), and similar) by observation under the microscope or with a flow
cytometer. Permeabilization can then be quantified by two parameters: the number
of cells in the population where the uptake can be detected and the amount of
uploaded dyes S (i.e., the distribution of fluorescence intensity in each single cell
in the population).
QðtÞ¼ Pperm Aperm ΔSðtÞ ð1 exp:ðt=TresÞÞ (7)
Q(t) is flow of S across the membrane at time t after the pulse.

Pperm is the permeability coefficient for the reporter molecule.
Aperm is the part of the cell surface that is permeabilized, proportional
to (1Ep/E).
ΔS(t) is the concentration difference of S between the bulk and the cytoplasm.
32 J. Teissie
Tres is the time constant of the membrane resealing that is controlled by the pulse
duration (resealing).
Permeabilization for S is detected only when the amount of S that is accumulated
through the flow is detectable by the assay. The critical field is the one that induces
permeabilization on the tiniest cap (A perm must be close to zero). ΔS(0) should be
large to increase Q(0), but the problem is the intrinsic leakage to S of the target cell
membrane. To keep the flow long lived, Tres must be long (long pulse duration), but
cell viability must be preserved. One problem is that there are some concerns on the
mechanisms affecting the cell with long pulses. The biomechanics of a cell should
consider it as a complex visco-elastic body, with a nonlinear response on the duration
of the stress (here the electrical forces associated to the field pulse).
A key practical information is that the inflow and the resulting accumulation (that
controls the sensitivity of detection of permeabilization) is controlled by Pperm that
is specific of the reporter molecule S.
Experimental data are obtained with fluorescent dyes either by observing the cells
under a digitized fluorescence microscope or with a flow cytometer. Another
approach is to follow the leakage of cytoplasmic compounds (endogenous such as
Adenosine triphosphate (ATP) or trapped by using loading with acetomethoxy
derivatives). Either a single cell approach is possible by following the decrease in
emission in each cell or on population by observing the increase of the signal in the
bulk phase (as in the case of ATP with the luciferin luciferase assay on a
luminometer).
Lipidic Models
A lot of studies have been developed on pure lipid assemblies: (Black (Planar) lipid
membrane (BLM), liposomes (small unilamellar vesicles (SUV), large unilamellar
vesicles (LUV), giant unilamellar vesicles (GUV), and multilamellar vesicles
(MLV)). Processes on such physical models are relevant of soft matter physics.
Stretching under the applied electrical forces is present and affects the membrane
curvature and the layer packing. Modifications of bilayer responses would only be
considered in the present chapter. Only definitions in terms of TMVs of critical
events occurring in living cell membranes will be approached.
Does the Critical Electropermeabilization TMV Exist?
Electrooptical and conductometrical relaxation methods allow a direct monitoring of

the structural events affecting lipid nanoassemblies during the electric field dis-
charge. The conclusions from such observations have given an accurate physical
insight in the molecular mechanisms of the delivery of drug-like dyes by electro-
permeabilization to cells and tissues (electropermeabilization). The degree of defect
formation as the primary field response increases continuously without a threshold
field strength, whereas secondary phenomena, such as a dramatic increase in the
membrane permeability to drug-like dyes (electropermeabilization), indicate thresh-

old field strength ranges. Membrane electropermeabilization is facilitated by an
increase in local curvature of the membrane as well as by a gradient of the ionic
strength across charged membranes, known to affect the spontaneous curvature.
Highly curved systems are affected by heterogeneities in packing on the outer layer
that appear as weaknesses facilitating the formation of defects (Neumann et al.
1999).
When unmodified planar bilayers are clamped at 150 mV, transient single defects
are observed for a long period of time (Melikov et al. 2001). Fast transitions between
different conductance levels are recorded due to opening and closing of metastable
lipid conducting defects (conductivity). The mean amplitude of conductance fluctu-
ations (approximately 500 pS) was independent of the applied voltage indicating the
local nature of the conductive defects. The distribution of defect conductance was
wide (dispersion of approximately 250 pS). Short bursts of conductance spikes
(opening and closing of defects) were followed by periods of background low
conductance. The on-off appearance of conducting defects proceeds through some
electrically invisible (silent) mismatches in the lipid matrix. Similar predefects
resulting in metastable conductive pathways are intrinsic features of cell membranes
and may be involved in different biologically relevant processes (defects).
The electropermeabilization process was directly assayed on the minimum model
of giant lipid vesicles, by using a highly sensitive fluorescence method based on
manganese ion transport (Mauroy et al. 2015). The approach at the single-lipid self-
assembly level detects divalent ion permeabilization on those cell sized giant
unilamellar lipid vesicles. Critical field and associated TMV are obtained by pulsing
with trains of increasing amplitudes. Short (0.1 ms long) pulses were delivered to
avoid electromechanical stretching of the vesicles. The critical values are observed
to vary from 10 to 150 mV. These values appear to be much lower than those
classically reported in the literature for cells and vesicles. The detection method
appears to be a decisive parameter as it is controlled by the transport of the reporter
dye. The critical TMVs are strongly dependent on the lipid composition. The
electropermeabilization process is a transient transition of the lipid self-organization
due to the dramatic loss of assembly cohesion that depends on the nature of the lipid
mixture building the bilayer.
The role of the applied membrane tension on the critical electric field strength for
membrane breakdown (rupture due to electropermeabilization) was approached by a
simple micropipette technique on lipid vesicles (Needham and Hochmuth 1989). An
increase in membrane tension is associated with an increase in the critical membrane
voltage, ΔVc (membrane tension). More cohesive membranes are more “resistant”
to the TMV effect. This is what is observed when the percentage of cholesterol in a
lipid mixture is increased. A theoretical model predicts that membrane rupture
occurs at a critical area increase. This dilatation is obtained either by the electrical
effect or through the applied membrane tension. Both stresses increase the elastic
energy up to this critical level. The model predicts a feedback between tension and
critical TMV to obtain vesicle bursting as a result of the electropermeabilization. As
a conclusion, under critical tension, the critical TMV can be zero.
34 J. Teissie
Critical Electropermeabilizing TMV on Red Blood Cells
In the 1970s, electropermeabilization studies were routinely performed on human

red blood cells (RBC, erythrocytes). Samples were obtained either from outdated
samples from the blood banks or freshly drawn from volunteers. Aging of the sample
was unfortunately never reported. This is an experimental problem as aging eryth-
rocyte undergoes alterations in their plasma membrane. Red blood cells are prepared
from whole blood by a simple centrifugation, which pellets the cells while the blood
plasma remains in the supernatant. The process is known as blood fractionation and
is a routine practice. Packed red blood cells are then kept in a saline buffer at low
temperature.
Red blood cells are fairly different from other mammalian cells (red blood cells).
A human erythrocyte has a donut shape. The diameter is approximately 6.2–8.2 μm
and a thickness at the thickest point of 2–2.5 μm and a minimum thickness in the
center of 0.8–1 μm. Therefore, RBCs are much smaller than most other human cells.
In a suspension, RBC have random orientation meaning that an homogeneous field
(achieved with parallel plate electrodes) induces a wide distribution of TMV even
when assuming that the population is highly homogeneous in size and physiology.
The red blood cell membrane complex is composed of three subunits: the
carbohydrate-rich glycocalyx, the lipid bilayer which contains many transmembrane
proteins and the membrane skeleton. Half of the membrane mass in human and most
mammalian erythrocytes are proteins. The plasma membrane is rich in proteins such
as band 3 and active transporter (such as the Na+/K+ ATPase). The proteins of the
membrane skeleton such as spectrin are responsible for the deformability, flexibility,
and durability of the red blood cell. Specific interactions with lipids such as
Phosphatidylserine (PS) are involved in the control of this flexibility. They enable
recovering the discoid shape as soon as these cells are not submitted to compressive
forces present in the capillaries. They are nonnucleated cells in their mature form.
The mature (enucleated) erythrocytes lose all other cellular organelles. Due to the
lack of mitochondria, they produce the energy carrier ATP by the glycolysis of
glucose and lactic acid fermentation. Hemoglobin is concentrated within the RBC
cytoplasm. Hypo-osmolar shocks can rupture the plasma membrane and hemolysis
occurs through hemolytic pores. This is a result of the mechanical tensions associ-
ated to the induced osmotic swelling. Permeabilization of the RBC membrane results
in an osmotic swelling, which if not prevented by osmotic agents (sucrose or larger
carbohydrates, polymers such as dextran or PEG) is followed by hemolysis
(swelling).
With human and bovine red blood cells, electric field induced so-called dielectric
breakdown of cell membranes could be detected using a Coulter Counter with a
hydrodynamic focusing orifice (Zimmermann et al. 1974). In making measurements
on red blood cells versus increasing electric field strength, the plot of the pulse
current heights versus the estimated electric field strength (obtained from the applied
voltage on the hydrodynamic flow chamber), a sharp bend in the classical linear
curve (ohmic behavior), is observed associated to what was called the dielectric
breakdown of the membranes. Solution of Laplace’s equation (assuming RBC being
spherical) for the electric field generated in the flow chamber brings a value of about
1.6 V for the critical permeabilizing TMV. This phenomenon was used to study
dielectric breakdown of red blood cells and the associated post pulse hemolysis in a
homogeneous electric field between two flat platinum electrodes. The electric field
was applied by a capacitor discharge generator (with flat parallel electrodes) on a
batch chamber. The calculated value (from the Laplace equation) of the critical TMV
in the homogeneous field is of the same order as when using the Coulter Counter.
This supports the conclusion that the hydrodynamic forces in the orifice of the
Coulter Counter do not play a role in inducing mechanical rupture of the red blood
cells. The dielectric breakdown (membrane conductance increase) TMV of human
red blood cells was measured to be at approximately 1 V (with field delivered with a
capacitive discharge generator) (Zimmermann et al. 1976). It is not affected by the
addition to the buffer of agents protecting against hemolysis (phosphate, sulfate,
sucrose, inulin, and EDTA). Indeed, hemolysis is detected for field pulses with a
larger amplitude. Electrical hemolysis is a secondary process of osmotic nature
induced by the permeability change of the membrane (dielectric breakdown/creation
of defects in the membrane) in response to an electric field pulse.
Square wave electric pulses applied to the erythrocyte suspension is a more
reliable approach where both the applied voltage and the delivered current are
recorded on line with a μs time resolution. An electrochemical coating of the
electrodes (platinum black) prevents the interfacial artifacts. Electric field induces
a transmembrane potential that, at a critical point, induces a sharp increase in the
current (conductivity). This results from a membrane structural alteration that either
opens up preexisting channels or transporters or creates defects (Kinosita and Tsong
1977) (defects). At least two steps are involved in the formation of the conducting
pathways: the initiation and the subsequent growth of the pore size. The induced
TMV is not measured but again obtained from the Laplace equation under the
simplification that a RBC is a sphere. The initiation step requires a transmembrane
potential greater than a threshold (approximately 1 V), whereas the latter process is
controlled by many factors such as the ionic strength, the field, or the pulse duration.
Leakage of ions leads to an osmotic imbalance which results in a colloidal hemolysis
of the red cells. Hemoglobin leakage and the associated craters are not mechanisms
but results from red blood cell electropermeabilization. The critical TMV is associ-
ated to the initiation step.
Muscle Fibers
A transmembrane voltage can be imposed on these giant cell plasma membrane by a

simple chemical treatment and the associated current is measured (Chen and Lee
1994) (transmembrane voltage ). Transmembrane potentials of up to 120 mV are
physiologic and well tolerated, but when the potential is more than 300 mV, large
currents are observed due to the electropermeabilization. Currents through frog
muscle cell membranes are observed under 4-ms pulses of up to 440 mV. The
relaxation of transmembrane currents under this voltage clamp approach back to
36 J. Teissie
zero after the pulse is observed by using low transmembrane potential pulses and
described in terms of membrane physical parameters. Electropermeabilization by
supra-critical clamped TMVs results in damaging the K+ channels, through an
electroconformational change in the transmembrane proteins. The membrane thresh-
old of 300 mV was detected as a result of this protein conformational change that
results in huge transmembrane current.
Patch Clamp Experiments
Threshold transmembrane potential for the induction of conductive defects in plated

NG108-15 neuroblastoma cells was obtained by treating small regions on the cell
surface (Ryttsen et al. 2000). Tiny noncontact electrodes were used to deliver highly
localized electric field on the cell membrane; 1 ms pulses were delivered with
increasing voltages. From patch-clamp measurements, the transmembrane threshold
voltage for pore formation of NG108-15 cells was determined to be 250 mV when
corrected for the voltage drop at the double layer.
A cell-attached patch clamp method was used to impose voltage waveforms of
desired amplitude to the cell membrane, on single ventricular cardiac myocytes
isolated from Sprague-Dawley rats using a collagenase and protease treatment
procedure (Tung and O’Neill 1995). Pulse waveform with amplitudes up to 1 V
(rectangular) were delivered using the patch pipette, as well as a low amplitude,
rectangular “conductance monitoring” pulse train. The onset of electro-
permeabilization was characterized as the appearance of small fluctuations or
jumps in current. The critical TMV was between 250 and 300 mV. Those transient
bursts in current do not occur instantaneously with the onset of the voItage pulse, but
rather with a variable delay that can occur over milliseconds. This reflects the
complex mechanisms supporting membrane electropermeabilization.
The patch clamp technique in the whole cell and the outside out configuration was
used to monitor current-voltage relations of protoplasts obtained from a tobacco
culture cell line (Wegner et al. 2013). Protoplasts were exposed to voltage pulses
with amplitudes up to supra-physiological values. A transition from a
low-conductance (0.1 nS/pF) to a high-conductance state (5 nS/pF) was observed
when the membrane was either hyperpolarized or depolarized beyond threshold
values of around 250 to 300 mV and +200 to +250 mV, respectively. K+ channel
activity was affected through an increase of cytosolic Ca2+ that is known to inhibit
outward-rectifying K+ channels in tobacco cells. But the high conductance is to be
associated with the formation of aqueous membrane defects at those supra-
physiological voltages. A fluorescence method confirmed the conductance experi-
ments under the patch clamp approach. Plotting voltage-sensitive dye ANNINE-6
fluorescence intensity against command voltage steps revealed saturation of the
curve at values <300 mV and >+300 mV. This saturation is indicative of the
effect of an enhanced conductance on the induced transmembrane voltage
(conductivity).
Transport
Assay on a Cell Population
Electropermeabilization is routinely detected by the transport assay (transport). Cells

are pulsed in a dye-containing buffer and permeabilization (uploading of the dye) is
investigated under increasing field conditions (at given pulse duration and number of
delivered pulses at a 1 Hz frequency). Transport resulting from electropermeabili-
zation occurs at a critical field strength Ep. Indeed experimentally this value is
difficult to estimate. When cells are pulsed with fields of larger strengths, it is
observed that more cells are detected as positive (dye is detected within the cyto-
plasm). Mathematical fittings of the electropermeabilization versus field strength can
be used such as a two-parameter sigmoidal curve (Kotnik et al. 2001),
yðxÞ ¼ 100%=ð1 þ exp:½ðxc xÞ=bÞ (8)
where x is the field pulse amplitude, y is the percentage of permeabilized cells, xc is

the x value corresponding to y = 50%, and b determines the slope of the sigmoid
curve, i.e., a fitting parameter.
For dye uptake, the intensity of fluorescence can be fitted by a three-parameter
Gaussian peak,
h i
yðxÞ ¼ ymax exp: ðxc xÞ2 =2b2 (9)
where x is again the field pulse amplitude, y is the intracellular concentration of

lucifer yellow, ymax is the maximum intracellular concentration of lucifer yellow in a
given experiment, xc is the x-value corresponding to y = ymax, and b determines the
width of the peak.
But due to the sigmoidal character of the curve, it does not provide access to the
critical field needed for electropermeabilization (i.e., conditions where the transport
is detected.)
The Pulsing Protocol Affects the “Experimental” Critical TMV
Interestingly, the critical parameter Ec is observed to decrease with an increase in the

pulse duration and the number of repetitive pulses in a train. A linear relationship is
observed between Ec(T) and the reciprocal of the pulse duration (T) or number of
pulses (N). This gives access by extrapolation to a limiting value of Ec (for an
infinite duration). As the pulse duration may affect the mechanism of electro-
permeabilization due to the nonlinear electrostretching, it may be safer to increase
the number of pulses rather a very long single pulse duration. Using a delay between
each pulse long enough to provide a significant cooling allows to avoid any Joule
heating artifact.
38 J. Teissie
Imaging electropermeabilization with the uptake of fluorescence dyes such as PI

showed that the critical Ec was different on the two cell hemisphere facing the
electrodes (imaging). This was explained by the contribution of the resting TMV
ΔV0 in the amplitude of the TMV present during the pulse delivery (see Eq. 5 and
Fig.1). The rate of transport was further affected by the electrophoretic force acting
on the charged PI.
Changing the temperature of the pulsing buffer was not shown to affect the
initiation step (i.e., the critical field needed to induce permeabilization). The mem-
brane translational and rotational dynamics are not affecting the initial event in
permeabilization (trigger of conducting defects), while they strongly affect the
expansion step of those defects and as a result the level of transport. Osmotic
pressure of the pulsing buffer has no effect on the induction step of permeabilization.
Increasing the ionic strength of the pulsing medium only affects the expansion step.
Molecules that modify membrane order (lysolecithin, ethanol), when used in
concentrations compatible with cell viability, are shown to affect only the expansion
step, but not the critical field that triggers electropermeabilization. Cell membrane
fluidity does not have significant effect on reversible electroporation (in agreement
with the effect of temperature).
From the experimental values of Ec obtained with Propidium iodide uptake,
taking into account the size distribution of the cell population, most studies used
the Laplace equation to get access to the critical TMV taking for r the mean value.
Using pulse duration of less than 1 ms within a train of 8 pulses, most published data
were associated with critical TMV in the range of 0.4 to 0.5 V (transmembrane
voltage).
Pulse electric field effects on phagocytes (human blood neutrophils and
rat peritoneal macrophages) have been investigated with an exponentially
decaying pulse (t = 90 μs, E = 1 to 7 kV/cm) (Malinin et al. 1989). The biological
response to electropermeabilization was to induce a chemiluminescent response
due to a respiratory burst in the phagocytes. When the mean diameter of the
neutrophils and macrophages is considered as 10 and 20 μm, respectively, the
Laplace equation (Eq. 3) yields the same threshold TMV, 0.75 V, for the two types
of cell.
By monitoring the enhanced influx of Ca2+, the variability of the critical TMV
inducing electropermeabilization of the plasma membrane of CHO cells (ΔVc) was
observed to be very high in a population (Towhidi et al. 2008). It ranged from 512 to
1028 mV in those irregularly shaped cells. The TMV was computed on a 3D finite-
elements model of each cell as the Laplace equation was not relevant. Cells of the
same type and exposed to the same number of pulses with the same duration are
affected by cell specific ΔVc. This cannot be explained by fluctuations in the resting
TMV ΔV0. The description of a cell provides some explanations. A cell is not a
smooth sphere. A lot of invaginations are present where the geometry and the
biomechanical properties of the membrane are far from what is expected with a
spherical shape. Enhanced local membrane curvature facilitates field-induced mem-
brane defects (membrane curvature). Packing is more heterogeneous in this part of
the membrane facilitating the creation of defects.
Fluorescence Imaging Observations
The use of a very fast fluorescence imaging microscopy connected to a pulse

generator gives access to the conductance changes (Hibino et al. 1991) (imaging).
A voltage-sensitive fluorescent dye was used as an indicator of the time-dependent
transmembrane change induced by the external field on sea urchin eggs. But when
the electric field was over a critical value, the transmembrane voltage kept locally a
constant value on a cap facing the electrodes. The size of the conductive cap is under
the control of the strength of the electric field. A high membrane conductance is
therefore present within 2 μs after the onset of the external field and affects the field-
induced TMV as predicted by Eq. 1. A steady value is observed in the cap where the
transmembrane conductance is obtained (Hibino et al. 1993). The critical field is the
one where the formation of this high conductance cap is formed.
Nanosecond pulsed electric fields pulses with a duration of 60 ns, and maximum
field strengths of approximately 100 kV/cm (100 V/cell diameter) are known to
induce membrane electropermeabilization (Frey et al. 2006). Membranes of Jurkat
cells were stained with a fast voltage-sensitive dye, ANNINE-6. A temporal resolu-
tion of 5 ns was achieved by the excitation with a ultrashort laser pulse of this dye
due to its subnanosecond voltage response time. The laser pulse was synchronized
with the applied electric field to record images at times before, during, and after
exposure. Along the pulse, the TMV increase at the anodic pole of the cell was
limited at 1.6 V after 15 ns and then decreased, indicating that electropermeabili-
zation was present (transmembrane voltage). TMV on the side facing the cathode
reached values of only 0.6 V in the same time period. The strong asymmetry in the
induced TMV difference cannot be linked to the resting TMV ΔV0. Different
conduction mechanisms seem to be present, which are associated to different critical
potentials.
Electropermeabilization Is Inducing Membrane Fusogenicity.
The field effect is not limited to permeabilization (Rems and Miklavcic 2016. When
applied on intact cell suspension, electric field pulses are known to induce membrane
permeabilization (electropermeabilization) and fusion (electrofusion) (membrane
fusion). These effects are triggered through a modulation of the membrane potential
difference. Due to the vectorial character of the electric field effects, this modulation
is position-dependent on the cell surface. Fusion results from the contact between
electropermeabilized cell membrane (Teissié and Rols 1993). When plated cells are
at confluence, delivery of pulses can induce electropermeabilization and fusion
(formation of polynucleated cells). The critical field to obtain permeabilization
(dye uptake) at given pulse duration (0.1 ms) and number of pulses is lower than
what is needed to get fusion (induction of polynucleated cells). This is the result of
the contribution of the resting TMV as shown in Eq. 5. ΔV0 facilitated the induction
of permeabilization on one side of the cell (low threshold in Ep for permeabilization)
but must be overcome by a higher field Ef on the other side to get its
40 J. Teissie
permeabilization and the resulting membrane fusion. The critical membrane poten-
tial difference which induces membrane permeabilization can be calculated from the
experimental observations of Ep and Ef and the value of ΔV0. It is observed that its
value is always about 200 mV for many different cell systems. This is much less than
assumed in most other studies on cell electropermeabilization.
What Lipid Systems Can Tell Us
A study of the voltage induction of transient transport defects in phospholipid bilayer

vesicles was obtained on Unilamellar vesicles (dipalmitoylphosphatidylcholine)
(Teissie and Tsong 1981. The 100 nm vesicles loaded with [14C]sucrose were
exposed to an intense electric field in the range of 20–40 kV/cm, with a field
decay time of 5–15 ms. A transient leakage of labeled sucrose was detected when
the field strength exceeded 30 kV/cm. When the TMV exceeded a threshold value of
200 mV, corresponding to an applied field strength of 30 kV/cm, there was a
transient permeabilization of the bilayer structure. No global and permanent dam-
ages to the vesicle bilayer were detected. These results showed the implication of a
membrane potential triggered conducting state of lipid bilayers in cell membrane
electropermeabilization. More recently, similar results were obtained on giant ves-
icles (GUV) either by a loss of contrast showing the leakage of the internal content
and the uptake of the buffer by a free exchange or by a fluorescence assay (Mauroy
et al. 2015 (lipid vesicles).
The experiments on lipid vesicles can provide a link with observations on planar
lipid membranes (BLM).
Palmitoy-oleoyl-phosphatidylcholine (POPC) bilayers were modified with incor-
poration of nonionic surfactant polyoxyethylene glycol (C12E8) surfactants
(Troiano et al. 1998). A 22% decrease of the destabilizing TMV was observed
upon addition of C12E8 to pure POPC bilayers. The addition of C12E8 at concen-
trations of 0.1, 1, and 10 μM to the bath surrounding the membranes decreased the
electropermeabilization threshold monotonically with concentration. These results
suggested that the polyoxyethylene glycol molecules play a major role in the
formation of hydrophilic defects in the bilayers above the critical TMV. The
C12E8 head groups are supporting the defect formation without the need of major
lipid head-group rearrangements. Surfactants can thus be used to manipulate the
electropermeabilization threshold of lipid bilayers and cell membranes.
The addition of the channel-forming peptide gramicidin D (gD) on the conduc-
tance and electropermeabilization thresholds of planar bilayer POPC membranes in a
ratio of 1:10,000 had no effect, but ratios of 1:500 and 1:15 significantly increased
the thresholds by 16% (p < 0.0001) and 40% (p < 0.0001), respectively (Troiano
et al. 1999). The effect of gD (1/15 ratio) on the membrane area expansivity modulus
(K) of GUVs was an increase by 110%. The effect is consistent with the prediction of
electromechanical model of electropermeabilization. The presence of membrane
proteins may affect the electropermeabilization of cell membrane by a local change
in their mechanical properties.
Conclusions
Critical electric field and transmembrane voltage for membrane defect formation is a
complex experimental problem. Very few direct evaluations of the critical TMV are
described, and most results are obtained from the effect of external field pulses.
Obtaining the TMV from the external field value is mostly through the use of
the Laplace equation considering the cell as a regular sphere (Laplace equation).
This is clearly a crude approximation. In a few cases, new numerical approaches
should give us more accurate insights on more complex geometries (Kotnik and
Miklavčič 2000).
The other experimental limit is that the detection of permeabilization is under
the control of the sensibility of the assay. This may explain why there is no
consensus between the different experiments. One open question is indeed the
physical definition of the critical TMV. The clear conclusion is that Electro-
permeabilization still needs more basic investigations (Teissie et al. 2005; Rems
and Miklavcic 2016).
Acknowledgments Research was conducted in the scope of the EBAM European Associated
Laboratory (LEA) and resulted from the networking efforts of the COST Action TD1104 (http://
www.electroporation.net).
Cross-References
▶ Different Approaches Used in Modeling of Cell Membrane Electroporation

▶ Fluorescent Indicators of Membrane Permeabilization Due to Electroporation
▶ Measurement of Molecular Transport After Electropermeabilization
▶ Mechanistic Description of Membrane Electropermeabilization
▶ Membrane Permeabilization Lifetime in Experiments
▶ Modeling Transport Across the Electroporated Membrane
▶ Parameters Affecting Cell Viability Following Electroporation In Vitro
▶ Patch Clamp in Use of Electroporation Mechanisms Studies
▶ Single Cell Electrical Characterization Techniques
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Phospholipid Head Group Dipoles and
Electropore Formation 3
Justin Teissie
Abstract
Classical electropermeabilization is the result of the delivery of electric field
pulses on cells. The field pulse lasts from submicro to several milliseconds. The
field intensity is large enough to induce a dramatic structural local alteration of the
cell membrane organization. This results in an enhanced permeabilization of the
target cell membrane. This is indeed a complex process and its molecular
characterization remains an intense field of investigations. A membrane is a
complex assembly where the polar head region is a charged system. It appears
as a target of the field effect.
A description of the lipid bilayer organization points out its role in the function
of membrane. Interfacial water appears as a key component in the structural
organization of the phospholipid head groups. A feedback between polar head
dipoles and water dipoles results in a specific organization.
Different biophysical approaches were developed to investigate the behavior
of the phospholipid head group dipoles during and after the pulse delivery. The
experiments were performed on membrane lipid models as well as on cell
membranes under reversible electropermeabilization, where the cell viability is
preserved through a resealing active process.
The external field pulse affects the mean orientation of the head group dipoles
and alters the local water content and self-organization. This is a key step in the
induction of defects in electropermeabilization and their associated properties.
Keywords
Membrane • Transmembrane voltage • Membrane structure • Interface • Polar
head group • Dipole • Interfacial water
J. Teissie (*)
Toulouse, France
e-mail: Justin.teissie@ipbs.fr; justin.teissie@ipbs.fr

DOI 10.1007/978-3-319-32886-7_78
46 J. Teissie
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
The Classical Description of Electropermeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
The External Field Induces a Transmembrane Voltage (TMV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
The Resulting TMV Induces Membrane Permeabilization (Electropermeabilization) . . . . . . 47
A Cell Membrane Is Not Just a Thin Dielectric Layer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Electropermeabilization on Lipid Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Multilayer Lipid Stacks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Planar Single Bilayer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Polar Head Group Reorientation in Pulsed Supported Lipid Multilayers . . . . . . . . . . . . . . . . . . . 52
Head Group Reorientation in “In Silico” Simulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Electropermeabilization on Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Interfacial Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Field Pulse Delivery Results in Asymmetric Event on the Cell Surface . . . . . . . . . . . . . . . . . . . . 55
Structural Events at the Cell Membrane Interphase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Introduction
Short high-field calibrated electric pulses when delivered to a cell suspension or a

tissue result in a membrane permeabilization (electropermeabilization). Classical
electropermeabilization results in an enhanced transport across the membrane and in
a change in its electrical conductivity. The general consensus is that the structural
transition in the membrane is due to the transmembrane voltage resulting for the
external field. The membrane is considered as a dielectric thin sheet. This is clearly
an oversimplification. Structural investigations of lipid bilayers pointed out the
complexity of the interfacial layer between the bulk water and the hydrocarbon
region. This chapter gives a survey of the recent knowledge on this organization.
Charge and dipole distribution in this 1 nm layer make it a target to the field effect
(forces and torques). In the second part of the chapter, the results obtained on the
field-induced effects on phospholipid head group dipoles by in silico simulations and
experiments on lipid models and on cells are presented with their consequences on
the electropermeabilization process.
The Classical Description of Electropermeabilization
The External Field Induces a Transmembrane Voltage (TMV)
When a cell is submitted to an external electric field, the cell (where the plasma
membrane can be considered as a dielectric shell) should be considered as a spherical
3 Phospholipid Head Group Dipoles and Electropore Formation 47
transmembrane voltage (TMV). During the electric field application (electro-

pulsation) on the cell, an electro-induced membrane electric potential difference
(ΔVi) is created which is locally associated with the dielectric properties of the
plasma membrane. Using a physical model based on a thin, weakly conductive shell
(the membrane conductivity λm) full of an internal conductive medium (the cyto-
plasm conductivity λi) and bathed in an external conductive medium (conductivity
λe), solution of the Laplace’s differential equation gives ΔVi as

ΔViðM, E, tÞ ¼ f gðλÞrE cos θðMÞ 1 expðt=τmÞ (1)
where M is the point on the cell that is considered, t is the time after electropulsation is
turned on, f is a factor depending on the cell geometry (for a sphere, f = 1.5), r is the
radius of the pulsed cell, E is the electric field strength, and (M) is the angle between
the direction of the field and the normal of the cell surface in M. g(λ) is related to the
different conductivities as (Zimmermann et al. 1974)
h i
gðλÞ ¼ 2λe 2λm þ λi þ ðλm λiÞðr d=r Þ3 3λmðr d=r Þ
h i (2)
= ð2λe þ λmÞð2λm þ λiÞ þ 2ðr d=r Þ3 ðλi λmÞðλm λeÞ
where d is the thickness of the membrane (a few nm) and τm is the characteristic time
constant of the membrane charging and can be written as (Kinosita and Tsong 1977)
τm ¼ rCmð2λe þ λiÞ=ð2λeλiÞ (3)

The Resulting TMV Induces Membrane Permeabilization

(Electropermeabilization)
When the new membrane electric potential difference ΔVm(ΔV0 + ΔVi) locally
reaches a critical value (ΔVc) (nowadays estimated between 0.25 and 0.6 V for
living cells) (Rems and Miklavcic 2016), a local alteration of the membrane structure
leads to membrane permeabilization. Molecular processes supporting classical
electropermeabilization remain poorly understood (Teissie et al. 2005) in spite of
more than 40 years of effort (Neumann and Rosenheck 1972). The membrane is
called “porous.” There is a general agreement that it cannot be adequately described
simply by the occurrence of reversible hydrophilic holes, so-called pores (“electro-
poration”), in the lipid bilayer.
An accurate biophysical description of a cell membrane is far from the simplify-
ing description of using a physical model based on a thin, weakly conductive shell
(the membrane conductivity λm).
48 J. Teissie
A Cell Membrane Is Not Just a Thin Dielectric Layer
Lipid bilayers have been studied for more than 50 years that it is missed by non-
experts in the field just how uncertain are structural quantities for the fully hydrated,
fluid (Lα or liquid crystalline) phase, that is relevant to biology (Nagle and Tristan
Nagle 2000). While lipid crystallography has been pursued and has provided many
relevant informations, it is important to recognize that fully hydrated lipid bilayers
are not even close to being considered as a crystalline state. This is mostly valid for
bilayers that are in the Lα phase because the hydrocarbon chains are
conformationally disordered in contrast to the all-trans chains present in lipid
crystals. Fluctuations, inherent in flexible and biologically relevant lipid bilayers,
make quantitative structure determination challenging. It makes no sense to describe
a lipid bilayer as if it had a regular crystalline structure because of the fluctuations.
Such crystalline structures simply do not exist in the biologically relevant state. The
appropriate description for the positions of atoms in the lipid molecule is that of
broad statistical distribution functions (that are non-Gaussian). Disorders present on
a long range are not inherent in the structure of the bilayer but intimately involve
bilayer undulations. Fluctuations are a central feature in membrane biology. Local
fluctuations and the induced transient mismatches support passive diffusion of
solutes and facilitate the conformational changes of transmembrane proteins. Fluc-
tuations mean transient random packing defects. They support the lateral diffusion of
membrane molecules (fast for lipids). They are locally enhanced due to the mem-
brane curvature, where peptide insertion is facilitated.
A description of the bilayer structure should take into account two regions. The
central part is the hydrocarbon chain region, but the end region is a mix between the
polar head group (carbonyls, glycerol, phosphate, and end groups such as choline)
and water molecules. There is indeed no sharp boundary between the regions as
shown by the overlap of the distribution functions. By the use of nuclear Overhauser
enhancement spectroscopy (NOESY) and spinning under the magic angle of lipid
multilayers, it was shown that lipid molecules are flexible, and the correlation times
between their different conformations are shorter than nanoseconds. Indeed, prox-
imity between polar head groups and the end of hydrocarbon chains is not a rare
event. Shifts in the position of lipid segments between neighboring molecules are
observed.
Water is strongly interacting with polar head groups. It is well accepted that at
least 12 water molecules provide the first strong hydration shell for
dioleoylphosphatidylcholine (DOPC). It is not surprising that stripping off essential
water involves strong forces that should result in drastic structural changes.
The polar region in lipid bilayers, where the polar head groups of lipids are
present, built an interphase, between the bulk of the aqueous phase and the hydro-
phobic core of the fatty acid chains. In this region, ionization of phospholipids as
well as adsorption of ions to lipid head groups generates electrostatic potentials
(Tocanne and Teissie 1990). They are added to polarization potentials associated to
the chemical structure of the polar head groups and to their hydration shell. These
complex potentials modulate the interphase concentration of protons, cations, and
anions with respect to those in the bulk aqueous phase. In the bilayer, lipids are
packed with their polar head preferentially oriented close to a perpendicular direction
to the interface normal. As already mentioned, due to fluctuations, lipids are also free
to diffuse laterally, and polar heads undergo fast vibrational and rotational motions.
This reflects the complexity of this interphase region. Strong interactions exist
between lipid polar head groups and make feasible the exchange of ions such as
protons between adjacent lipid molecules.
The dielectric constants which are measured at the surface of two phospholipid
species, PC (phosphatidylcholines) (zwitterionic) and PS (phosphatidylserine)
(charged), stand around 40–10. For these lipids, the larger dielectric constants are
found located at the interface, near the bulk water. The lower values are observed
located deeper in the lipid matrix, at the polar head/hydrocarbon core, in the region of
high probability of the ester carbonyl groups. A very steep gradient for the dielectric
constants in the interphase region is present with very local time-dependent fluctua-
tions. The dielectric constants on average decrease from 80 in the bulk phase to a value
of about 2 in the central hydrocarbon domain of the lipid bilayer. This dramatic change
takes place over a distance which appears to be less than 1 nm and is affected by the
bilayer undulations. From an electrical point of view, a membrane-bulk phase interface
cannot be considered as a sharp boundary (Nymeyer and Zhou 2008).
Of the individual potentials which comprise the potential profile of a membrane,
the least well understood is the dipole potential (Brockman 1994). In general, the
dipole potential is manifested between the hydrocarbon interior of the membrane and
the first few water layers adjacent to the lipid head groups. For a typical zwitterionic
phospholipid, like phosphatidylcholine, its measured value is approximately
280 mV in bilayer membranes, the hydrocarbon region appearing positive relative
to the bulk phase. Dipole potentials result from the ability of lipid head groups to
globally reorganize water structure at the interface. The functional group dipoles of
the terminal methyl groups of aliphatic chains, the glycerol-ester backbone, and the
hydrated polar head groups all contribute to the dipole potential. Any physical stress
affecting the organization (change in dipole orientation due to a torque induced by an
external field) results in a change in the dipole potential. Interphase water is involved
in its value, but the precise description of the microscopic nature of the interactions
from which it arises is far from being known. One should take into account that the
dipole potential is built in a thin region over which the dielectric constant of the
medium is changing from 2 to 80. Even if poorly characterized, the dipole potential
appears as an important regulator of membrane dynamical structure and associated
function. The hydration force is controlled by the dipole potentials of the mem-
branes. It is the leading factor in the close contacts in membrane-membrane and
membrane-ligand interactions. For multicomponent membranes (the case in cells),
the size and shape of lipid domains can be in a first approach considered due to the
balance between the line tension at the domain borders and the difference in dipole
density between the domains. Line tension tends to make the domains regular,
whereas dipole repulsion promotes transitions to complex shaped larger domains
(control of so-called rafts). Rotation of dipoles would affect the size and shapes of
domains!
50 J. Teissie
Cell membranes cannot be described as a lipid bilayer with embedded transmem-

brane proteins. The membrane-bound protein skeleton, a network of actin filaments
and associated proteins, covers the entire cytoplasmic surface and is closely linked to
clathrin-coated pits and caveolae, key factors in membrane traffic (Morone et al.
2006). The actin filaments are likely to form the limit of the membrane compart-
ments where membrane molecules are confined (making a “fence” preventing large
scale diffusion). The plasma membrane is a patchwork with regard to their lateral
diffusion. It is not a fluid bidimensional mosaic. A membrane in a cell is a more
complex system than a homogeneous bilayer. Membrane traffic is continuously
present. The plasma membrane is an out of equilibrium complex from a thermody-
namic point of view. It forms a complex with the extracellular matrix and the
cytoskeleton. Its transmembrane potential is dependent on the cellular energy
reserves.
As a conclusion, a cell membrane or even the “simple” single lipid bilayer is not a
thin dielectric sheet. Fluctuations in the relative position of the different partners are
affecting its organization. Definition of the “structure” should take into account all
the internal movements. The interphase with the bulk phase remains poorly under-
stood but is the region where a sharp gradient in the dielectric constant is present and
where the dipolar character of polar heads makes them sensitive to external electric
fields, but the associated torques are in opposite direction on the different water
membrane interphases.
Electropermeabilization on Lipid Models
Vesicles
Light Scattering
Unilamellar small lipid vesicles (SUVs, 30 nm in diameter), doped with the
diphenylhexatrienyl-phosphatidylcholine (beta-DPH pPC) lipids, were observed
by turbidimetry and light absorption when pulsed with short high-field rectangular
electrical field pulses (Neumann et al. 1998). The major changes of the turbidity and
absorbance dichroism result from vesicle elongation under electric Maxwell stress.
The kinetics of this electro-chemo-mechanical shape deformation are illustrative of
the very fast events of membrane stretching resulting in smoothing of thermal
undulations. Local displacements of the chromophore (beta-DPH pPC) relative to
the membrane normal are triggered by the pulsed field. Slower changes of the
turbidity and absorbance signals reflect the entrance of solvent into the membrane/
medium (change in the local refractive index), resulting from the alignment of
the bipolar lipid head groups in one of the leaflets at the pole caps of the vesicle
bilayer. One limit of this membrane system is that due to their small size, a high
curvature is present on the external layer, and packing defects are abundant on the
external layer.
Imaging
An externally applied electric field across giant unilamellar vesicles (GUVs)
(20–30 μm in diameter) leads to transient permeabilization of the membrane as
assayed by a loss of optical contrast due to a transmembrane exchange. The
distribution and lifetime of these permeabilizing defects on DOPC phospholipid
vesicles were assayed under a fluorescent microscope as well as from negatively
stained electron micrographs (after fixation) (Tekle et al. 2001). An asymmetrical
behavior was observed. During the pulsed field application, a single membrane hole
up to 7 μm in diameter (i.e., huge on the vesicle surface) was observed on the vesicle
side facing the negative electrode. Nothing was detected at the anode-facing hemi-
sphere where Ca2+ influx was nevertheless present supporting the formation of many
defects supporting its transport. A clear difference in the permeabilizing defects is
present associated with the direction of the transmembrane electric field (parallel to
the external field).
Coherent Anti-Stokes Raman Scattering (CARS) Microscopy

Spontaneous Raman spectroscopy (SRS) is a powerful probe in the analysis of the
chemical bounds of samples. Electropermeabilization-linked molecular events were
monitored by comparing the vibrational spectra of biological sample before and after
pulsed electric field through the detection of critical bandwidth. Coherent anti-
Stokes Raman scattering (CARS) microscopy acquires images of a sample at a
specific vibrational frequency. Based on the critical bandwidths previously deter-
mined with Raman spectroscopy, it is possible to follow the evolution of the
biochemical composition of the membrane and associated water molecules during
a pulsed electric field. The behavior of interfacial water molecules was observed on
lipid GUVs. Two different spectral signatures were obtained associated to two
different hydrogen bonds: one for lipid associated and the other for the water
clusters. A dramatic drop of the water-associated signal is induced by the pulsed
electropermeabilizing field. This reflects that interfacial water organization due to the
lipid polar heads is dramatically affected in the electropermeabilized state.
Multilayer Lipid Stacks
Polar Head Group Dipoles Are Sensitive to Their Local Environment

Phosphorus-31 and deuterium nuclear magnetic resonance (P31 NMR and 2H NMR)
measurements on PC, where the phosphocholine head group that was deuterated at
the two methylene segments monitored the head group dipole orientation on lipid
multilayers. The electric surface charge is the essential driving force in changing the
phospholipid head group orientation and conformation (Seelig et al. 1987). While
the P-N dipole is rotating nearly parallel to the membrane surface in the pure
phospholipid membrane, the addition of a positively charged amphiphile or the
binding of cationic molecules moves the N+ end of the dipole toward the water
phase, changing the orientation of the phosphate segment by more than 30 at the
52 J. Teissie
highest amphiphile concentration. The effect is present only on the PC head groups
with no perturbations on the hydrocarbon region and the glycerol backbone.
Hydration of the lipid stacks can be controlled. When it is changed in the range of
10–70 wt.% H2O, a distinct change in the field-induced alignment of the PC head
group is present. At low hydration, the N+ end of the PC head group dipole moves
closer to the hydrocarbon layer. A similar conformational change is obtained upon
addition of polyhydroxyl compounds.
Perturbations in the polar head environment (local electric charge, level of
hydration) result in a change in the orientation of their dipole. Such an effect affects
the dipolar potential and its associated control on the membrane functions.
Planar Single Bilayer
Bilayer lipid membranes (black lipid membranes, BLM) are a convenient model to
observe transmembrane potential effects on lipid bilayers. The transition at a critical
value of the applied electric field (transmembrane electric field) is characterized by a
sharp increase in conductance and represents electropermeabilization. Under current-
clamp conditions, the classical rupture of the membrane is avoided, and the process is
observed on a long term at the critical transmembrane voltage (Genco et al. 1993). It
depends dramatically on pH and ionic concentration with zwitterionic lipids, indicating
that the surface electrical properties of the interphase determine the characteristics of
such a transition (electropermeabilization). Asymmetric bilayers consisting of a
charged and an uncharged monolayer mimic natural membranes. The instability
inducing the increase in conductance starts in the uncharged monolayer, and the final
electropermeabilization process results from the coupling between the two monolayers.
The stability and rupture kinetics of bilayer negatively charged lipid membranes
covered with electrostatically adsorbed polyelectrolytes, polylysines (PLs) of different
molecular weights (MW), were analyzed under the short-voltage clamp conditions
(Diederich et al. 1998). Adsorption of PL on one side of the bilayer results in an
asymmetric transmembrane potential, which adds to the externally applied voltage.
High MW PL decreases the critical breakdown voltage of the membrane significantly
but also increases the delay time between the onset of the voltage pulse and conduc-
tance increase. The time course of conductance increase (defect expansion) is PL
molecular weight dependent. Interfacial adsorption of high MW PLs causes a dramatic
decrease of the velocity of defect expansion. 2D (surface) viscosity is increased by the
interphase modification and affects the electropermeabilization process.
Polar Head Group Reorientation in Pulsed Supported Lipid

Multilayers
Stacks of lipid bilayers can be formed and spread on the surface of conductive
electrodes. The polar head region can be directly observed by different spectroscopic
methods when the field is present and induce the conductive state of the assembly.
The influence of the field pulse on the molecular structure of the phosphatidylcho-
line head group was monitored by NMR of the phosphorus atom. The shape and the
position of the peak in the P31 NMR signal reflect the average configurational position
of the phosphate group from the polar head region. Multiple bilayers of dimyristoyl
phosphatidylcholine and potassium oleate were macroscopically oriented between
silver-coated slides, and a voltage bringing an electric field of up to 105 V.cm1
was delivered between the parallel slides (Stulen 1981). At electric fields above
104 V.cm1, spontaneous current fluctuations associated with the formation of the
conducting defects are observed; structural effects are transient and reversible. The
conformation of the head group is greatly affected as shown by the drastic change in
the P31 NMR signature. A broadening and a shift of the peak are obtained. This
reflects a dramatic average configurational change of the phosphate group region when
the permeabilizing pulse is present but no definitive detail can be obtained.
Polarized attenuated total reflection (ATR) experiments were designed to study the
effect of electric fields on membrane molecule structure and orientation by Fourier
transform-infrared (FTIR) spectroscopy (Le Saux et al. 2001; Miller 2002). Fully
reversible orientational changes in the lipid head groups are induced by an electric
potential difference field delivered on an oriented stack of lipid bilayers made of
dioleoylphosphatidylcholine (DOPC) or of dipalmitoyl phosphatidylcholine (DPPC).
Potentials above 600–700 V on 1-μm-thick multibilayer are needed to trigger the direct
reorientation of the dipoles of the different polar residues by the electric field. The electric
fields varied up to 5.5 106 V/cm in the hydrocarbon layer and up to 1.1 106 V/cm
in the polar head region (being considered as a homogeneous dielectric!) due to the
change in the dielectric constant between the interphase and the hydrocarbon regions. The
inhomogeneous field between the polar and the hydrocarbon layers drives the polar
groups into the hydrocarbon layer, exerting a pressure and penetrating them.
Polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS)
experiments provided IR spectra for 1,2-dimyristoyl-sn-glycero-3-phosphocholine
(DMPC) bilayer formed on or close to gold electrodes (Zawisza et al. 2003). Changing
the electrodes surface charge density induces a modulation of the electrode potential
and of the local static electric field present on the bilayer. The quantitative analysis of
the IR bands provided information on both the conformation and orientation of the
polar head region of the DMPC molecule. The hydration and conformation of the head
group of the DMPC molecule strongly depend on the electrode potential.
The different spectroscopic approaches (P31 NMR, ATR-FTIR, PM-IRRAS) all
brought the same information: the polar head region mean orientation is affected
when a permeabilizing electric field is present. As a result, the distribution of water
in the interphase region is changed.
Head Group Reorientation in “In Silico” Simulation
New insights on the molecular process are given by computer-assisted simulations

(Vernier and Ziegler 2007). Conditions for the simulation are very high field
(300 MV/m) (higher than under experimental conditions where the voltage to
54 J. Teissie
electrode width is about 0.1 MV/m) and short pulse duration (a few ns at the best)
due to the computing time limit. Using molecular dynamic simulations on one single
phospholipid bilayer, defect formation results from a dramatic change in the orga-
nization of the interfacial water molecules. The process of conductive defect forma-
tion is driven by the transition to a more energetically favorable configurations of
water dipoles at the water-lipid interfaces. The water molecular dipoles are oriented
in the external field (torque on the dipole). Interfacial water molecules are the only
players in the process, being its seed and its driving force. The field-generated torque
nevertheless tilts the mean head group dipole orientation by a few degrees away from
its equilibrium (at no field) position relative to the bilayer normal. Field-directed
rotation of the head group dipoles, in combination with water dipole reorientation
and solvation interactions at the aqueous-lipid interface, participates to the coordi-
nated ensemble leading to the defect formation.
Electropermeabilization on Cells
Interfacial Modifications
The organization of the membrane water interphase (dipolar potential) gives rise to a
set of different forces that are called hydration forces (Leikin et al. 1993). Due to
their repulsive nature, they prevent the spontaneous fusion of two cells when their
respective membranes are brought in close contact. A strong repulsion is detected
when the intermembrane space is reduced to a few nanometers. The associated
forces are described resulting from a local organization of the interfacial water. As
shown on lipid layers, water dipoles are sensitive to the local field, and as a result a
regular organization of the water takes place, bringing a collective regular assembly
of dipoles. The associated dipolar fields on the two membranes are in opposite
directions during the close approach. A strong repulsion is observed when a close
contact is attempted. When cells are electropermeabilized, a spontaneous fusion is
detected (Sowers 1986). The repulsive forces vanished during the pulse delivery, but
the ability of the membranes to coalesce remains present in the few seconds
following the pulse delivery. This is indicative that the structures supporting these
repulsive forces, i.e., the regular organization of interfacial water molecules, have
been altered under the electropermeabilization processes. This interfacial modifica-
tion needed to obtain cell membrane coalescence is restricted to the electro-
permeabilized part during and in the few seconds following the pulse delivery.
This local perturbation is present only where the critical electropermeabilization
transvoltage has been induced by the external field.
Spontaneous Raman spectroscopy (SRS) is a powerful probe in the analysis of the
chemical bounds not only on simple lipid assemblies as reported above but on cells
of samples. Cells were pulsed, and the CARS signature of the water molecules
was monitored before and after the pulse. Again a dramatic drop of the water-
associated signal is induced by the pulsed electropermeabilizing field. This reflects
that interfacial water organization due to the interphase is dramatically affected in

the electropermeabilized state, as predicted by the spontaneous fusion of electro-
permeabilized cells.
Field Pulse Delivery Results in Asymmetric Event on the Cell Surface
Asymmetry in the Critical TMV

Nanosecond pulsed electric fields (nsPEF) with a “long” duration of 60 ns and high
field strengths up to 100 kV/cm (100 V on a 10-μm cell diameter) are shown to
trigger membrane permeabilization by a process rather similar to the one in classical
electropermeabilization. A fast voltage-sensitive dye, ANNINE-6, has a sub-
nanosecond voltage response time and was used to follow the change in the
transmembrane voltage during the pulse delivery (Frey et al. 2006). A temporal
resolution of 5 ns of the fluorescence signal (i.e., of the transmembrane voltage
(TMV)) was obtained by the excitation of this dye with a tunable laser pulse. The
very short dye excitation by the flash was synchronized with the longer applied
electric field pulse to observe cell imaging (spatial resolution) at times before,
during, and after the field delivery. The TMV reached values of 1.6 V after 15 ns
at the anodic pole of the cell, several times larger than the TMV level supposed to be
required for inducing a conductive state of the membrane. The TMV then decreases
illustrative of a high local membrane conductance. TMV on the side facing the
cathode reached values of only 0.6 V in the same time period, indicating a strong
asymmetry in the field-dependent conduction mechanisms created in the membranes
of the two opposite cell hemispheres.
The difference in behavior of the caps of a cell facing the two electrodes was
present in classical electropermeabilization. The membrane conductance of sea
urchin eggs was investigated over the time range of 0.5–1 ms by directly imaging
the transmembrane potential at a submicrosecond resolution with the voltage-
sensitive fluorescent dye RH292 (Hibino et al. 1993). When a rectangular electric
pulse of a non-permeabilizing intensity was delivered on a cell, a position-dependent
fluorescence change illustrative of the TMV developed synchronously with the
pulse. Under an electric pulse of higher intensity, the rise of the induced potential
stopped at a certain level and then slowly decreased on the microsecond time scale.
Again this is the result of the creation of finite membrane conductance or permeabi-
lization of the membrane. The two opposite sides of the egg facing the positive and
negative electrodes were affected; a slight asymmetry was observed as the conduc-
tance on the positive side appeared higher. During the pulse duration, the conduc-
tance increased steadily up to ten times during the next 1 ms. Again asymmetry in the
processes is detected. This conductance increase was larger on the negative electrode
side; by 1 ms the conductance on the negative side was more than twice that on the
positive side, while it was lower at the 0.5-μs image. The defects induced by
the critical TMV are structurally different on both sides of the cell membrane facing
the electrodes.
56 J. Teissie
Asymmetry in the Transport by Diffusion

Fluorescence imaging under the microscope provides an approach of the transmem-
brane transport in cells with a good spatial resolution. Transport of divalent cation
(Ca2+) and three probes with a high affinity for DNA (ethidium bromide (EB),
propidium iodide (PI), and ethidium homodimer (EthD-1)) across electro-
permeabilized membranes of several mammalian cell lines was found to be selective
and asymmetrical (Tekle et al. 1994). The conductivity of the pulsing buffer affects
the ionization of the cell surface (charged groups are present on the external layer of
a cell membrane), and it is observed to modulate the transport. In low-salt medium
(where electrostatic forces are strong), Ca2+ and EB were mostly crossing the anode
facing cell membrane, while PI and EthD-1 predominately entered at the site facing
the cathode. In high-salt medium (low electrostatic repulsion), the entry site for Ca2+
and EB was reversed to the cathode-facing hemisphere while it remained unchanged
for PI and EthD-1. In all these experiments, the observed transport patterns result
from a diffusion-driven process where the flow was controlled by the permeability
coefficient of the molecule that was assayed. These observations support the con-
clusion that the defects induced by the critical TMV are structural different on both
sides of the cell membrane facing the electrodes.
Biphotonic Microscopy
Molecular center asymmetry is required for the creation of second-harmonic gener-
ation (SHG) signals. It makes this spectroscopy a powerful technique for visualizing
changes in interfacial layers in the plasma membrane of biological cells. Lipophilic
SHG probes such as di-4-ANEPPDHQ (Di-4) can detect minute perturbations in the
plasma membrane. Rapid changes in membrane symmetry could be detected using
SHG (Moen et al. 2014). Following pulsed permeabilizing electric field exposure, an
instantaneous drop of ~50% in SHG signal was detected only from the anodic pole of
the cell. This dramatic loss in SHG signal reflects a local dramatic perturbation in the
interfacial nature of the membrane but with a vectoriality of the effect.
Fluorescence Investigations with FM1-43

Millisecond-long permeabilizing pulses induced membrane disorganization
supporting the formation of long-lived permeant structures that were present during
membrane resealing. The insertion of FM1-43, a fluorescent dye, into the membrane
from the bulk is a fast process controlled by a decrease of lipid packing and/or an
electrostatic process as FM1-43 is positively charged. The amphiphilic fluorescent
probe FM1-43 was used to detect those post pulse. A fluorescence post-pulse
overshoot is associated with the membrane assembly changes (Escoffre et al.
2014). FM1-43 fluorescence gave a local response in the membrane as shown by
fluorescence imaging. This increased insertion reflects either an increase in the lipid
assembly fluctuations or a change in the membrane surface charge. Moreover, the
fluorescence increase facing the anode was twice that of the one facing the cathode
showing that the perturbation of the interface was not the same on both sides of
the cell.
These observations on the induced TMV and the associated transport when
electropermeabilization was triggered support the conclusion of asymmetric events
on the cell membrane following the direction of the transmembrane field. The
dynamical structure of the conducting defects is dependent on the direction of the
electrical constraints acting on the interfacial regions.
Structural Events at the Cell Membrane Interphase
31P NMR spectroscopy gives structural informations on the lipid polar head region.
Chinese hamster ovary (CHO) cells were reversibly permeabilized by submitting
them to short, high-intensity, square wave pulses (1.8 kV/cm, 0.1 ms) (Lopez et al.
1988). Due to these pulsing conditions (high field, short duration), a large cap was
electropermeabilized covering the majority of the cell surface, but the population
viability was preserved. It was taken advantage for the NMR investigation (which
needed averaging of the collected signal) from the observation that cells remained in
a permeable state without loss of viability for several hours at 4 C. This gave a good
signal-to-noise ratio in the definition of the spectroscopic signatures. A differential
method in the NMR pulse sequence was used to analyze the phospholipid head
groups and to get rid of the signals coming from other phosphorus groups. A new
anisotropic peak with respect to control cells was observed on 31P NMR spectro-
scopic analysis of the phospholipid components. This peak was only present when
the cells were permeable, and normal anisotropy was recovered after resealing.
Taking into account the fusogenicity of electropermeabilized cells, comparative
studies were performed on 5% poly(ethylene glycol) treated cells. The 31P NMR
spectra of the phospholipids displayed the same anisotropic peak as in the case of the
electropermeabilized cells. In the two cases, this anisotropic peak was located
downfield from the main peak associated with the phospholipids when organized
in bilayers. The localization of this anisotropic peak was very different from the one
of a hexagonal phase. As a conclusion it was proposed that cell membrane reversible
electropermeabilization is associated with a reorganization of the polar head group
region (new mean orientation or randomization of the orientations) leading to a
weakening of the hydration forces to account for these observations.
Conclusions
The complexity in the structural organization of membrane models (lipid bilayers)

or of cell membranes is most of the time neglected in the description of
electropermeabilization.
Biophysical analysis of lipid assemblies pointed out that the interfacial region
between the core of the membrane (hydrocarbon chains) and the bulk aqueous phase
is a complex assembly that must be called an interphase. This interphase is where a
sharp change in the dielectric constant is present. The presence of charged groups
58 J. Teissie
with opposite charges built dipoles that affect the local organization of water (dipolar
orientation). This interphase is therefore highly sensitive to the external field, which
applies a torque on the dipoles. Sophisticated spectroscopic investigations bring
converging conclusions: (i) structural transitions affect the interphase region during
the pulse delivery on lipid models as well as on cells, (ii) interfacial water is
modified, (iii) the effects remain present during resealing of cell membrane, and
(iv) the field effect is vectorial as different effects are observed on the two sides of the
cell facing the electrodes.
Cross-References
Experiments
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14:881–899
Membrane Permeabilization Lifetime in
Experiments 4
Justin Teissie
Abstract
Short high field electric pulses when delivered to a cell suspension or a tissue
results in a membrane permeabilization (electropermeabilization). Classical
electropermeabilization (pulse longer than 1 μs) results in an enhanced transport
across the membrane and in a change in its electrical conductivity. Electro-
permeabilization can be reversible if the pulsing parameters are selected in a
proper way. This reversibility is due to set of reactions called resealing. This
means that an enhanced membrane permeabilization is transiently induced while
preserving the cell viability. More drastic pulsing conditions can make this
permeability irreversible (cell death would result). Molecular mechanisms
supporting electropermeabilization of cell membrane are complex. They are not
limited to the lipid matrix (punching holes in the lipid bilayer as figured out in
“electroporation”) but involved the cellular machinery.
Keywords
Membrane • Transmembrane voltage • Transport • Membrane structure • Repair
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Resealing Observed from Electrical Conductance Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Online Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Patch Clamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Muscle Fibers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Electric Cell-Substrate Impedance Sensing (ECIS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Resealing as Observed by the Time-Dependent Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Resealing Is Observed by a Post Addition of the Reporter Dye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
J. Teissie (*)
Toulouse, France
e-mail: Justin.teissie@ipbs.fr; justin.teissie@ipbs.fr

DOI 10.1007/978-3-319-32886-7_79
62 J. Teissie
Dependence of the Resealing on the Pulsing Electrical Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Resealing Is Strongly Controlled by the Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Buffer Osmotic Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Buffer Ionic Strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Membrane Order . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Resealing Is an Active Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Introduction
Electropermeabilization of the cell membrane is a phenomenon caused by the direct

exposure of the cell to calibrated electric pulses. Permeabilization depends on pulse
duration, pulse amplitude, and number of pulses delivered and also on other exper-
imental conditions. With these parameters properly chosen, the process of permeabi-
lization is reversible, and cells return to their normal physiological state. The process
of permeabilization is divided into a short permeabilizing phase that is initiated
during the pulse, and a longer complex resealing phase that begins after the end of
the pulse (Puc et al. 2003). Membrane permeabilization is associated with an
enhanced transport of soluble small molecules (that are with a low molecular weight,
say less than 2 kD) and ions. It is a time-dependent process, and its kinetics can be
observed by monitoring the electrical conductivity change in the membrane or the
change in the transport parameters. The transport after the pulse is diffusion driven.
This means that its amplitude is dependent on the molecule that is detected. No post-
pulse transport is detected for macromolecules (siRNA, pDNA) (Paganin-Gioanni
et al. 2011). It can be assayed by the inflow of dyes (trypan blue, propidium iodide) by
observation under the microscope or with a flow cytometer. Permeabilization can
then be quantified by two parameters: the number of cells in the population where the
uptake can be detected and the amount of uploaded dyes (i.e., the distribution of
fluorescence intensity in each single cell in the population). As electrically induced
permeabilization is a change in the state of the membrane, it can also be detected by
the leakage of endogenous metabolites (such ATP by a luciferin luciferase chemilu-
minescent assay) or of a prepulse loaded dye (using the same methodologies as for
the uptake). Resealing is then measured by the post-pulse time dependence of one of
these assays.
Molecular processes supporting classical electropermeabilization remain poorly
understood (Teissie et al. 2005) in spite of more than 40 years of effort (Neumann and
Rosenheck 1972). The membrane is called “porous.” There is general agreement that
it cannot be adequately described simply by the occurrence of reversible hydrophilic
holes, so-called pores (“electroporation”), in the lipid bilayer. A membrane in a cell is
a more complex system. It is an out of equilibrium complex from a thermodynamic
4 Membrane Permeabilization Lifetime in Experiments 63
point of view. It forms a complex with the extracellular matrix and the cytoskeleton.
Its transmembrane potential is dependent on the cellular energy reserves. Therefore
the resealing of the structural defects supporting the transmembrane transport can be
reported only in an empiric way: i.e., an accurate description of its control by the
different physical and biological parameters but a speculative approach of the
molecular mechanisms.
A lot of studies have been developed on pure lipid assemblies (planar lipid
membrane, liposomes). Processes on such physical models are relevant of soft matter
physics and would not be considered in the present chapter. Only events occurring in
membranes within cells will be approached.
Resealing is assayed by different biophysical approaches. As mentioned above
they are shortly described in a first part of this chapter. They are used to analyze the
processes and their control by biophysical and physiological parameters.
Resealing Observed from Electrical Conductance Changes
Online Assay
access to the conductance of the suspension. Under steady-state condition, the
conductance of a diluted cell suspension can be considered controlled by the con-
ductance of the external solution. An ohmic behavior is observed when a voltage
pulse is delivered on the cell suspension (as long as the Joule heating is small).
i. The dielectric shell has conductive defects that increase during the pulse.
ii. There is leak of the internal (cytoplasmic) solution.
Both events result in an increase in the cell suspension conductivity, when it is

initially low. This is a very fast process that can be detected on the submicrosecond
time scale after correcting from the interfacial electrochemical reactions on the
electrodes. The kinetic is biphasic: a very sharp increase during the first microsecond
followed by a slower continuous increase.
After the pulse, the conductivity of the cell suspension is due to the contribution of
the leaked ions and the defects under resealing. If a second permeabilizing pulse is
applied, the conducting defects will again increase. If the delay between the two
pulses is short (a few microseconds), the conductivity of the suspension at the onset
of the second pulse is the same as at the end of the first one, and no resealing of the
defects will be detected. With longer delay (10 ms), almost complete resealing
occurs, and the observed initial increase in conductivity observed at the onset of
the second pulse is due to the contribution of the leaked ions. The resealing of the
dielectric defects is biphasic, a large but partial resealing is detected within a few
64 J. Teissie
milliseconds, but the remaining contribution is long lived. The ion leakage from the
cytoplasm is long lived and appears as the major contribution in the long-lived cell
suspension conductance change, when the ionic content of the pulsing buffer is low
(Kinosita and Tsong 1979; Hibino et al. 1991).
Increased permeability of a cell membrane is accompanied by increased mem-
brane conductivity; by measuring electric conductivity of a cell suspension, the mean
extent of permeabilized tissue could be monitored in real time. Total conductivity
changes and impedance measurements showed easy to detect significant changes in
conductivity due to this increased membrane conductivity, the ion efflux in
low-conductive medium and colloid-osmotic swelling in low- and high-conductive
medium. By measuring electric conductivity online during the pulses permeabi-
lization, threshold can be detected, but permeabilization levels are not obtained as
the signature of the electric changes is too complex (Pavlin et al. 2005).
Patch Clamp
The patch-clamp technique allows the investigation of currents through a small set or
even single ion (conducting) channels and therefore about the membrane conduc-
tance. The whole-cell patch-clamp technique was employed to investigate the effect
of millisecond pulsed electric fields on mammalian cells (Wegner et al. 2015).
Cellular membrane permeabilization was monitored by a conductance increase. It
could be established experimentally that electropermeabilization consists of two
clearly separate processes: a rapid membrane conductance change (due to the crea-
tion of conducting defects in the cell membrane or transient “electroporation”) that
occurs while the field is present to depolarize or hyperpolarize the membrane to
voltages beyond so-called threshold potentials (evaluated to be +201 mV and
231 mV, respectively in the study) and is reversible within 100 ms after the
pulse, and a long-term, or persistent, permeabilization covering the whole voltage
range. The latter remained after the pulse for at least 40 min at room temperature.
Muscle Fibers
A transmembrane voltage can be delivered directly on isolated muscle fibers that

are giant cells, and the associated current is measured (Bier et al. 2002). Transmem-
brane potentials of up to 120 mV are physiologic and well tolerated, but when the
potential is more than 300 mV, large currents are observed due to the electro-
permeabilization. Currents through frog muscle cell membranes are observed under
4 ms pulses of up to 440 mV. The relaxation of transmembrane currents under this
voltage clamp approaches back to zero after the pulse is observed by using low
transmembrane potential test pulses and described in terms of membrane physical
parameters.
Electric Cell-Substrate Impedance Sensing (ECIS)
An approach referred to as (ECIS) studied the behavior of adherent animal cells

grown to confluence on circular gold-film electrodes of 250 μm in diameter within a
culture dish (Stolwijk et al. 2011). A large coplanar counter electrode was used. The
impedance of the cell-covered electrode was measured by applying a low AC voltage
with designated frequencies to monitor the time-dependent normalized impedance
behavior of the cells. The gold-film electrodes were used to deliver well-defined
electropermeabilizing voltage pulses (several volts amplitude and several hundred
milliseconds duration) across the adherent cells in order to achieve reversible mem-
brane electropermeabilization. Time-resolved impedance measurements before and
immediately after the pulse pictured the kinetics of membrane resealing as well as
associated changes in cell morphology. Membrane permeabilization and resealing
were only clearly visible on rather short time scales. A fast and sharp drop of the
normalized impedance to less than 0.55 was followed by its quick recovery within a
few seconds to 0.9. Full recovery to pre-pulse relative impedance values (1 by
definition) resulted from a second process with a much slower time constant assigned
in part to the induced fluctuations in cell shape. Full cell membrane (electrical and
morphological) recovery was slow and obtained within less than 90 min.
Resealing as Observed by the Time-Dependent Transport
Transport is present across electropermeabilized membrane. After the pulse, when no

external field is present, the transport is diffusion driven and is controlled by the
gradient of concentration of the reporter molecules between the two sides of the
plasma membrane. It depends on the permeability of the electropermeabilized mem-
brane to the reporter molecule (in many cases a water soluble fluorescent dye such as
propidium iodide). At the single cell level or on a population, this can be monitored
by the post-pulse inflow, i.e., the fluorescence time-dependent change in the cell
cytoplasm.
A competition is present between the diffusion process and the resealing that
decreases the permeabilization. Deconvolution of the kinetics of the fluorescence
change gives the parameters of the resealing. A good fit is obtained under the
assumption that the resealing processes are all first order. A very fast approach
under a microscope showed that three steps were present (Pucihar et al. 2008). The
pulsing chamber was set on a microscope stage, and the fluorescence signal was
observed by a fast photomultiplier tube. Its output voltage U was proportional to the
fluorescence signal emitted by the cell. The fluorescence increase was due to the
inflow of PI.
When a permeabilizing field pulse was delivered with different pulse durations, a
long-lived propidium iodide (PI) uptake (fluorescence increase) was observed with
amplitudes that depended on the pulse duration.
66 J. Teissie
The complex kinetic of the fluorescence increase (I(t)) was deconvoluted as a

three-step process:
I ðtÞ ¼ I1ð1 expðt=T1ÞÞ þ I2ð1 expðt=T2ÞÞ þ I3ð1 expðt=T3ÞÞ (1)
The faster ones were all with time constant (T1, T2) shorter than 100 ms (for a
square waved pulse of 1 ms), and their detection was achievable only due to the
advanced technology of the study. With classical equipment, only the slow step
(T3) could be detected and was on the scale of several seconds or minutes (Teissie
et al. 2005; Prausnitz et al. 1995). Its amplitude I3 was large meaning that uploading
occurred mostly during this slow resealing step by a diffusion-driven process. This is
a key information for practical applications, a slow resealing will increase the amount
of molecules that are uploaded (or released):
QðtÞ ¼ Pperm Aperm ΔSðtÞð1 expðt=T3ÞÞ (2)
where Q(t) is the flow of S across the membrane at time t after the pulse, Pperm is the
permeability coefficient for the reporter molecule, Aperm is the part of the cell
surface that is permeabilized, and ΔS(t) is the concentration difference of S between
the bulk and the cytoplasm.
A key practical information is that the inflow and the resulting accumulation (that
controls the sensitivity of detection of permeabilization) are controlled by Pperm that
is specific of the reporter molecule.
Resealing Is Observed by a Post Addition of the Reporter Dye
The change in the transport properties of the electropermeabilized membrane can be

easily approached by adding the reporter dye at different delays after the pulse
delivery (inducing the membrane permeabilization) and counting the number of
cells where the dye uptake can be detected (i.e., that are still permeabilized) or the
amount of dye uptake:
QdelðtÞ ¼ PpermAperm ΔSðtÞð1 expðt Tadd=T3ÞÞ (3)
Qdel(t), flow of S across the membrane at time t after the pulse.

Tadd, delay for the addition after the pulse delivery.
Qdel(t) decreases with an increase in Tadd.
The first approach (relative number of cells where the transport is detected) is the
most popular, and permeabilization is assayed as the percentage of positive cells
(cells where uptake is detected). The second approach gives more informations. It can
be approached either by digitized fluorescence microscopy by single cell analysis,
but of course the number of cells that are analyzed remains limited, or by flow
cytometry. In this latter case, by a proper setting of the intensity windows, three
populations were detected: cells with no permeabilization, where no fluorescence
could be detected (population 1); cells that remained permeabilized with a significant
uptake (population 2), i.e., fluorescence signal; and cells that were irreversibly
permeabilized with a high fluorescence signal (population 3). Resealing was
observed by a shift over time of cells from population 2 to population 1. The mean
intensity in population 2 was indicative of the uptake along the delayed dye addition
and was observed to decrease upon time. The increase in population 3 was reflecting
irreversible permeabilization and the associated cell death (Teissie et al. 2005).
Of course the assay is strongly dependent on the sensibility of the detection assay.
The assay is slow (time needed to add the dye to the pulsed cell suspension) and
cannot detect the fast steps in resealing. Only the slow step (on the scale of several
seconds) can be observed. This approach confirms that the “slow” step in resealing is
a first-order process.
The number of permeabilized cells in the population is then
PðE, tÞ ¼ LðEÞ þ ½ðPðE, 0Þ LðEÞÞexpðKtÞ (4)
P(E,t) is the percentage of cells detected as fluorescent (populations 2 and 3 in the

FACS plot) with dye addition at time t after a pulse with an E amplitude that triggers
the lysis of a subpopulation L(E)(population 3); P(E,0) is the percentage of cells
detected as permeabilized just after the pulse (indeed where the membrane is leaky
for physiological reasons). K is the resealing constant (the reciprocal of T3).
Dependence of the Resealing on the Pulsing Electrical Parameters
Electropermeabilization is known to be under the control of the field strength E, on

the single pulse duration T, on the number of successive pulses N, and on the delay
between the pulses. Resealing lifetime (the reciprocal of K) is observed to be
controlled by these parameters in a specific way.
K is not dependent on the field strength (Teissie et al. 2005). The field strength
controls the number of cells that are permeabilized and the occurrence of the cell
lysis. When the reversible permeabilization is observed (correcting the observed
permeabilization P from the percentage of lysed cells L), then the kinetic time
constant is similar to the one observed under mild field conditions (where electro-
permeabilization is reversible, no cell lysis).
The resealing lifetime is observed to be linearly dependent on T as long as T is less
than 1 ms. The process is more complex when longer pulses (larger than 5 ms) are
delivered on the population. This supports the hypothesis that the biophysical
mechanisms affecting the cell long-lived electropermeabilization may be different
when long pulses (lasting several milliseconds each) are applied as compared with
the events triggered by much shorter pulses (in the microsecond range).
When T is less than 1 ms, the resealing lifetime is a linear function of the number
of successive pulses (delivered with a delay shorter than 1 s) as long as a limited
number is delivered (less than 10).
68 J. Teissie
As long as the delay between successive pulses is less than 1 s, there is no obvious
dependence of the resealing lifetime on the delay. The use of longer delays (several
seconds) is still the field of controversy on its consequence on electro-
permeabilization (memory effects?) (Demiryurek et al. 2015). This may result from
an excessive leakage of cytoplasmic molecules such as ATP and GTP or an exchange
of ions affecting the resting potential difference by altering the Nernst potential.
One main consequence of the dependence of the resealing lifetime on the pulse
duration and on the number of pulses is the associated control on the transport
induced by electropermeabilization. Transport after the pulse is diffusion driven
and follows Fick’s law. Resealing means a decrease in the density of defects
supporting the transmembrane transfer. This density (within the cell cap that is
affected by electropermeabilization) is known to be controlled by the pulse duration
and the number of successive pulses (when the delay between the pulses remains
short), during the so-called expansion step. Increasing the pulse duration brings an
increase in the density of defects and an associated increase in transport. This increase
is further enhanced along the resealing, as defects remain present during a longer time
as it was just described. Long pulses (up to 1 ms) give a higher cumulative transport
by acting on the density of defects and on their lifetime. A similar role is played by the
number of pulses (as long as they do not affect the viability).
Resealing Is Strongly Controlled by the Temperature
The post-pulse temperature is strongly affecting the resealing process (Teissie et al.
2005). Most experiments are run at room temperature (20–25 C). If the pulsed
sample is brought to its “physiological” temperature (37 C), a faster resealing is
observed, and the membrane enhanced permeability vanished in a short time. But if
the sample is kept on ice after the pulse train delivery, the membrane remains
permeabilized over long periods (up to several hours). Cells remain viable as
membrane resealing can be obtained by bringing the sample temperature back to
37 C. Adding the reporter dye after the “heat” jump does not bring any upload; the
membrane has lost its enhanced permeability. Cells are able to grow when brought in
the culture medium. This means that at low temperature where the resealing is not
working, cell membrane is permeabilized, but the cell viability remains preserved.
The main conclusion is that the resealing can be easily controlled by playing with
the post-pulse temperature of the sample.
Buffer Osmotic Pressure
The effects of pulsing buffer osmotic pressure were observed on short electric field
pulses (0.1 ms) induced permeabilization on Chinese hamster ovary cells (CHO)
growing either in monolayers or in suspension (Teissie et al. 2005). No osmotic
swelling was observed on mammalian nucleated cells under these experimental
conditions (this is not valid for red blood cells where electropermeabilization
under hypoosmotic conditions induced osmotic swelling and dramatic damages

in the membrane, so called craters). Osmotic pressure (increases hyperosmolarity)
(decreases (hypoosmolarity)) was shown to decrease (to increase) the efficiency of
the resealing phase. The observation was that the resealing for cells pulsed in isotonic
buffer is faster than those obtained in a hypertonic one. These results were tentatively
explained in terms of the effect of the osmotic pressure on the membrane organization
(interfacial forces and undulation forces). A more practical conclusion is that a larger
transport across the membrane is present under hyperosmolar conditions. It must be
emphasized the consequence of the observations that CHO size is not affected by
osmotic pressure. The applied pressure on the membrane keeps the same magnitude
in the hypoosmotic and in the hyperosmotic buffers. It is only the direction of
application of the pressure which is inverted. As a consequence, it is difficult to
explain the observations on cells by an effect on the stretching properties of the
membrane as it is done by others with pure lipid vesicles (Needham and Hochmuth
1989).
Osmotic effects are different when long pulses are delivered. As PI inflow
occurred mostly after the pulse (Teissie et al. 2005; Prausnitz et al. 1995; Pucihar
et al. 2008), the effect of the osmotic pressure during the 10-min incubation after 5 ms
pulses (long pulse) was investigated by changing the buffer immediately after pulsing
(Teissie et al. 2005). A large transport was observed. Under this experimental
condition, cell swelling along the pulse delivery was observed. The percentage of
permeabilized cells and the associated fluorescence intensity were not affected by this
treatment. The effect of osmolarity only existed during the application of the pulses
and not during post-pulse incubation (resealing step), when the inflow of molecules
of PI mostly took place. Resealing experiments were performed. Resealing curves
were observed to fit a first-order kinetic well. The rate constant, K, was found to be
constant whatever the osmolarity. This is a strong support for the conclusion that the
electropermeabilization process is different for short pulse at a high field and long
pulse at a lower field.
Buffer Ionic Strength
The effects of the ionic strength of the pulsing buffer on the electropermeabilization
of Chinese hamster ovary cells (CHO) were investigated (Teissie et al. 2005). Cells
were pulsed and kept at room temperature before addition of trypan blue (0.4% mass/
vol.) at various times after pulsation. The permeated state of electropulsed CHO cell
membranes (the permeabilized population) could be maintained for several minutes
at 21 C under electric field strengths larger than the critical threshold. The cells then
progressively recovered their original impermeable plasma membranes (decrease in
the permeabilized population P). The initial rate of the resealing process, i.e., the ratio
dP/dt (P = permeabilization, t = time), i.e., the kinetics of defects annihilation
(controlling the dye uptake) was followed at room temperature for CHO cells
electropulsed under the same electrical conditions (with different field strengths but
same pulse duration) but in isosmotic pulsing buffers of different ionic strengths.
70 J. Teissie
Similar curves were obtained for the different buffers: cells were maintained perme-
able for 5 min, and resealing was completed during the following 60 min.
The effect of buffer ionic concentration during the 5-min incubation period
following the pulses was also investigated by changing the buffer immediately
after application of pulses. The ion concentration during the post-pulse incubation
step did not affect the detected permeabilization. Permeabilization was only depen-
dent on the ionic content during pulsation.
Membrane Order
The energy barrier for the structural transition in the membrane bringing conducting
defects was checked for its entropic character controlled by the membrane order. A
systematic attempt tried to relate the intrinsic plasma membrane fluidity of three
different cell lines to their reversible electroporation (Kanduser et al. 2006). Mem-
brane resealing was related to cell membrane fluidity as determined by electron
paramagnetic resonance spectroscopy and computer characterization of membrane
domains. Nevertheless, this effect, if present, was masked with different time courses
of membrane resealing found for the different cell lines studied.
A more chemical approach was obtained by adding molecules known to affect the
membrane order. Cell electropermeabilization is detected only under more stringent
conditions when cells have been treated by ethanol. On the other hand, lysolecithin
was observed to facilitate cell electropermeabilization and to negatively affect the
resealing (Teissie et al. 2005). 0.4 M ethanol incubation did not inhibit the revers-
ibility of electropermeabilization. Nevertheless, these experimental results suggested
that, apparently, the loss of the defects (decrease in P) was faster when cells were
pretreated with ethanol.
When the membrane order is increased, the temporarily permeable structures
(defects) are apparently stabilized, and the lifetime of the permeable state is longer
when the cells are treated with lysolecithin before pulsing. Electropermeabilization is
inducing a transition in the membrane organization. Membrane order is modulating
the energy barrier needed to evoke this membrane transition which occurs when cells
are submitted to a field larger than a characteristic threshold (expansion step). Less
order would decrease the magnitude of this energy barrier and facilitate the resealing
(the decrease in P is faster as observed on ethanol treated cells); more order would
increase it (P remains high on a longer period after lysolecithin addition than with
untreated pulsed cells).
Resealing Is an Active Process
Electropermeabilization is affecting not only the plasma membrane, but other organ-
elles are also targets of the process. This can be a direct effect but only when very
strong fields are delivered. The field inside the cytoplasm is weaker than the external
one as the membrane, even when electropermeabilized, induces an electrical shield

(Esser et al. 2010; Weaver et al. 2012). But due to the enhanced transport, the
cytosolic composition is altered. This shown by the leak of secondary metabolites
and by the dramatic changes in Calcium content (increase). This results in an indirect
effect on organelles.
The second metabolite ATP is playing a key role in the resealing process. Its
cytoplasmic level should be high. Keeping CHO cells in PBS for 30 min before
pulsing prevented the resealing. But this was observed to starve the cells; the ATP
level dropped by 75% before the pulse delivery. Same effect on resealing was
obtained after a deoxyglucose treatment. Again the ATP level was only 25% of the
basal one (or less) and was further decreased due to the ATP leak induced by
electropermeabilization. This could not be repaired by adding external ATP to
balance the electroinduced leak.
Cytoskeleton
The membrane-bound protein skeleton, a network of actin filaments and associated

proteins, covers the entire plasma membrane cytoplasmic surface. It is closely linked
to clathrin-coated pits and caveolae, key factors in membrane traffic (Morone et al.
2006). The actin filaments are likely to form the limit of the membrane compartments
where membrane molecules are confined. Microtubules are filamentous intracellular
structures. Microtubules are involved in the organization of intracellular structure and
intracellular transport.
GTP has an active role in the polymerization of microtubules (Carlier et al. 1987)
which is prevented by colchicine (Howard and Hyman 2003). ATP plays a similar
role on the polymerization of actin microfilaments (Chhabra and Higgs 2007).
Addition of I mM GTP or ATP in the pulsing buffer, to maintain their concentration
in the cytoplasm at a constant level, did not result in any change in the permeabi-
lization, but resealing was strongly affected when GTP was added (Teissie et al.
2005). But, the resealing rate was greatly affected when cells were treated with
colchicine that prevented the tubulin polymerization: half of the cell population
became unpermeable to trypan blue in less than 2 min, with 6 min being the required
time in the case of control cells (pulsed under the same electrical conditions). In the
case of cytochalasin treatment that blocks polymerization and the elongation of actin
filaments, no difference was observed at the Iower concentration. But at 20 μM and
above, most cells were still dye permeable after 1 h incubation at room temperature,
but this could be associated with the drug lethal effect (Teissie et al. 2005).
Permeabilization appears as a structural transition in the membrane organization
with an energy cost for rupturing chemical interaction between the membrane
partners. This is strongly supported by the strong temperature dependence of the
resealing. This energy barrier could be greatly increased if interconnections between
membrane components and cytoskeleton proteins existed, mainly for microtubules.
72 J. Teissie
Such a change in the activation energy would affect the kinetics of resealing by
bringing a decrease of the rate constant of the repair process. This induces an increase
in the lifetime of permeabilization. This increase in stabilization of permeated
structures could be explained by the low lateral mobility of membrane proteins
which are linked to cytoskeleton but can be affected by the electrophoretic stress
along the membrane associated with the pulse delivery.
All these experimental observations strongly suggest that the cytoskeleton is
involved in the electropermeabilization phenomenon. Firstly, it appears to be altered
as a consequence of permeabilization (maybe not directly by the field that is weak in
the cytoplasm (Esser et al. 2010)) at a morphological level and secondly, it seems to
play a regulatory role in the stabilizating process of the defects supporting
By scanning electron microscopy, numerous microvilli and blebs were observed
almost immediately after application of short field pulses in a low conductivity buffer
(Escande-Géraud et al. 1988). No other membrane changes were observed. The
appearance of osmotic pressure-dependent “blebs” was indicative of local weakening
of the plasma membrane. The application of 100-μs rectangular pulses of 1.3 kV/cm
electric field to different types of cells (FBT, MEF, RAT-1, L-cells) in the physiolog-
ical medium (where a high conductivity was present) led to the formation and growth
of spherical and hemispherical protuberances of the cell membrane (Gass and
Chernomordik 1990). Most of these effects were fully reversible and disappeared
within 30 min at 37 C. The major effect which was induced during or in the very first
seconds following the pulse was the eruption of numerous microvilli (Escande-
Géraud et al. 1988). This increase in the density of villi was transient, and the same
density as before pulsation was observed after the 20 min post-field incubation at
37 C. The means of the size of the cell surface and of the number of filopodia are not
affected by the pulsation in a statistical significant way. The density of microvilli
increased by 40%. When cells were treated in low conductivity pulsing buffers where
nucleotides were added (cAMP, GTP, ATP all at 1 mM), a dramatic effect was
detected only when ATP was added. Both the length and the density of microvilli
increased significantly, but this effect is transient and disappeared when the cells
completely resealed (Rols and Teissié 1992).
Exocytosis
Exocytosis is described as a key process for repairing wounded membranes (McNeil

and Steinhardt 2003). One should consider cell membrane electropermeabilization as
a membrane wounding. Therefore resealing should be supported by exocytosis.
Along resealing of membrane electropermeabilization, lysosomal glycoproteins
were detected by immunofluorescence at the level of the plasma membrane. Their
density was related to the density of electrically triggered defects (by changing the
pulse duration at a given field strength) (Huynh et al. 2004). One consequence was
therefore Calcium that played a role in exocytosis should play a regulatory role in
resealing. It should be mentioned that a calcium transient increase is always present

along electropermeabilization due to the low resting cytoplasmic free concentration
of Calcium (Rems and Miklavcic 2016).
Conclusions
The main conclusion of all these experimental investigation of the lifetime of electro-
permeabilization (resealing) is that it is indeed a multistep process. Structures present as
long as the field is delivered vanished in a few milliseconds as soon as the field is
switched off. New structures remain present supporting a diffusion-driven transport of
small non-permeant species. These long-lived defects are controlled by the single pulse
duration. They support the major part of the uploading (or leakage from) of cells. Their
lifetime is dependent on complex mechanisms involving the cell metabolism with a
strong implication of exocytosis and the organization of the cytoskeleton. An important
role is played by the interfacial organization of the membrane.
Cross-References
▶ Cell Stress Responses to Pulsed Electric Fields

Experiments
▶ Different Cell Viability Assays Following Electroporation In Vitro
▶ Gene Delivery by Electroporation In Vitro: Mechanisms
▶ Nucleic Acid Electrotransfer in Mammalian Cells: Mechanistic Description
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Lipid Electropore Stabilization
5
M. Laura Fernández and Marcelo R. Risk
Abstract
The stabilization of pores can be studied by different approaches such as simu-
lations in silico or experimental procedures in vivo or in vitro. The energy to open
a pore in a lipid membrane can be delivered by different external stimuli. To
disrupt the membrane and initiate the pore opening, this energy has to reach a
threshold. Then, once the pore is open, the external stimulus can be modulated to
maintain the pore stable in time. This chapter first describes the basics of
electropermeabilization, a process also called electroporation, and the basics of
molecular dynamics in electropermeabilization. The chapter then describes in
detail the molecular changes that lead to the pore opening and evolution by
molecular dynamics. The chapter focuses on molecular dynamics because this
technique allows the study of pore stabilization at molecular level, the interpre-
tation of the lipid and water molecule rearrangements that are behind this
phenomenon, and the visualization of the pore at the scale of size and time, in
the order of nanometers and nanoseconds, respectively. Finally, the chapter also
describes other approaches where pores remain open or the permeabilized state
remains stable for a period of time, such as continuum modeling, experiments in
planar membranes, and experiments in cells. The objective of this selection is to
M.L. Fernández (*)

Consejo Nacional de Investigaciones Científicas y Técnicas, Instituto de Física del Plasma, Facultad
de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
Departamento de Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Buenos Aires, Argentina
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
e-mail: mlfernandez@dc.uba.ar
M.R. Risk
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
Instituto Tecnológico de Buenos Aires (ITBA), Buenos Aires, Argentina
e-mail: mrisk@itba.edu.ar

DOI 10.1007/978-3-319-32886-7_83
78 M.L. Fernández and M.R. Risk
relate the results obtained by molecular dynamics with those obtained experi-
mentally, or by other types of modeling, aiming to connect the mechanisms of
pore stabilization by molecular dynamics at different scales.
Keywords
Electropermeabilization • Electroporation • Pore stabilization • Molecular
dynamics
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Electropermeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Molecular Dynamics and Electropermeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Pore Stability by Molecular Dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Stable Pores Obtained by Electric Field Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Stable Pores Obtained by Charge Imbalance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Stable Pores Obtained by Mechanical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Pore Stability and Chemical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Pore Stability by Continuum Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Pore Stability in Planar Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Permeabilized State in Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Introduction
The plasma membrane defines the boundaries of all living cells. Cell membranes are
composed of lipid bilayers and proteins. The lipid bilayers of animal cell membranes
are mainly composed of phospholipids and sterols, such as cholesterol. Both of these
molecules are amphiphilic and have two defined parts: a hydrophilic one, consisting
of a polar head group, and a hydrophobic one, composed of two hydrocarbon tails in
phospholipids or four carbon rings and a hydrocarbon tail shorter than that of
phospholipids in cholesterol. Due to their amphiphilicity and shape, when phospho-
lipids are placed in water, they are spontaneously organized to form a bilayer with
the polar head interacting with the water molecules, and the nonpolar tails of each
layer facing each other in the inner part of the bilayer, preventing the contact of this
hydrophobic region with the water molecules. This lipid bilayer separates the inner
medium of the cell, called cytosol, from the extracellular fluid. The bilayer thus acts
as a barrier with a selective permeability to small polar molecules (Fig. 1a). Another
characteristic of cell membranes is the asymmetry in lipid composition of both
leaflets. Under physiological conditions, the membranes are flexible and can break
and spontaneously reseal. The disruption of the membrane exposes the hydrophobic
surface formed by the lipid tails, hidden in the core of the bilayer, to the water
molecules, creating an energetically unfavorable condition. This induces a rapid
rearrangement of the lipids, aimed to repair the rupture in the membrane.
5 Lipid Electropore Stabilization 79
Fig. 1 Evolution of the pore: Schematic three-dimensional representation of a bilayer in a

simulation box. The red beads are the phospholipid polar head groups, the green sticks are the
hydrocarbon lipid tails, and the light blue beads are the water molecules. The sizes of the
components are not in scale. (a) Intact bilayer before the application of the electric field;
(b) Beginning of the pore formation with a hydrophobic pore after the application of an external
electric field with an intensity equal to or greater than the minimum porating electric field (E0);
(c) Stage of expanded pore; (d) Stabilized pore at a value of electric field lower than E0. If the
electric field applied is equal to or greater than E0, the pore begins to be formed and evolves to an
expanded pore, from a to c. If the electric field applied is removed, the pore disappears, from c to a.
If the electric field applied decreases to a low sustaining value (Es) lower than E0, the pore shrinks,
from c to d. If the electric field applied increases to a value higher than the selected Es, the pore
expands, from d to c
The disruption in the continuity of the membrane can be artificially induced by

different stimuli such as electrical or mechanical stress or chemical agents. If the
intensity of the external electric field or the force applied to the membrane is high
enough to exceed the respective membrane thresholds, the bilayer starts to break
down due to the formation of artificial aqueous pores, which increase its permeabil-
ity and even allow the diffusion of ions and molecules to which the membrane is
normally impermeable. Thus, once the pore is open, water molecules, ions, metab-
olites, and other molecules can cross the membrane either from the inside to the
outside of the cell or in the opposite direction, depending on where they were
confined, following their own gradient. The molecules that are allowed to go through
the membrane can be normal cellular metabolites as exogenous molecules, such as
drugs, which can be placed outside the cell during an experimental procedure.
Electropermeabilization
Electropermeabilization, also called electroporation, is a methodology of transient

disruption of the membrane induced by pores due to the exposure of artificial bilayers
or cell membranes to an external electric field in vitro or in vivo (Tsong 1991). The
reversible or irreversible effects of this process on cell membranes are linked to the
intensity (which can be high or low), the repetition, the duration (which can range from
nanoseconds to microseconds), and the number of electric pulses applied. Artificial
bilayers, cells or tissues, can be successfully electroporated by means of several
protocols, which depend on the nature of the object of study selected. For instance,
to electroporate cells in a reversible way, a field magnitude in the order of kV/cm can
be applied in microsecond pulses, while higher field magnitudes in the order of MV/m
can be applied in nanosecond pulses (Silve et al. 2014; Pakhomova et al. 2011; Vernier
et al. 2006).
Molecular Dynamics and Electropermeabilization
The molecular events produced at atomic or molecular level that lead to pore
formation cannot be visualized experimentally. So, the molecular structures of the
induced pores during their formation, maturation, and closure can only be studied
and described by molecular dynamics simulations.
Molecular dynamics is a method that allows the modeling of a biological structure
such as the lipid bilayer at atomic level. In this method, the structure of interest (lipid
bilayers, proteins, nucleic acids, etc.) is placed in a tridimensional box where the initial
position of each atom is known. The structure in the box can be solvated by different
approaches, such as adding implicit or explicit solvents, thus giving the biomolecule,
or the biological structure, an adequate aqueous environment, including ions. Each
atom in the simulation box is related to and interacts with the others and has a defined
initial position and an assigned velocity. Then, by solving the Newton’s equation of
motion, the forces on each particle due to the interactions are calculated and the
movement of the atoms is simulated at each time step, in the order of femtoseconds.
At each time step, the new position or velocity of each atom is generated. Following
the movement of each atom at each time step, a trajectory is generated and the changes
of the whole simulated system can be evaluated along the time.
The molecular dynamics method works with force fields, which allow the calcu-
lation of the potential energy at each step. The force field is constituted by a
functional form to calculate the potential energy and all the parameters needed for
this calculation. With the functional form and the parameters, the potential energies
and the resulting forces can be calculated as a function of the position of each
interacting atom. In the simplest force field, the calculation of the energy is the sum
of terms that model the interactions.
The following methods have been developed for molecular dynamics simulations
to induce the formation of pores and study their further evolution.
External Electric Field Molecular dynamics procedures allow simulating the

application of an external electric field in the z-direction (transversal to the
membrane) as a new force in the system, exerted over all atoms with partial
charges, in the calculations of the potential energy. By means of this simulation,
the transmembrane potential rises until the membrane threshold is reached, causing
the formation of the pore (▶ Chap. 2, “Critical Electric Field and Transmembrane
Voltage for Lipid Pore Formation in Experiments”). This method allows the
comparison of results obtained by molecular dynamics with those obtained exper-
imentally, where high-intensity nanosecond pulsed electric fields are applied to a
system (Tieleman 2004; Tarek 2005).
Ion Charge Imbalance Another approach to describe the generation of electropores

is the induction of a transmembrane potential by the method of ion charge imbal-
ance, where the transmembrane potential is generated by imposing an ion charge
imbalance across the bilayer. This method simulates the effects on the transmem-
brane potential due to the charge imbalance in the membrane, which acts as a
capacitor. The results obtained from these molecular dynamics experiments have
been previously compared with those obtained experimentally (Sachs et al. 2004;
Gurtovenko and Vattulainen 2005; Delemotte et al. 2008).
Mechanical Stress Another way to induce pores in membranes, in the absence of an

electric field, is by mechanical stress. This method is based on the application of a
surface tension, which exceeds the bilayer threshold tension needed to maintain the
continuity of the membrane and initiates the process of pore opening (Leontiadou
et al. 2004).
Chemical Agents The addition of amphiphilic molecules to the simulated system

can affect the membrane stability and induce the pore formation. The presence of
these molecules can also lower the electroporation threshold (Fernández and
Reigada 2014; Polak et al. 2015).
The following sections in this chapter describe the principal methods to obtain
and maintain stable electropores.
Pore Stability by Molecular Dynamics
As mentioned above, the formation of pores in bilayers may be induced by several

methods by molecular dynamics. This chapter describes some of these methods,
including the application of an external electric field, the modification of the
transmembrane potential due to charge imbalance, and the poration process by

mechanical stress or by chemical agents, in order to analyze the effects of these
methods on the stability of pores.
Stable Pores Obtained by Electric Field Application
When an external electric field is applied perpendicularly to the membrane

(z-direction) and the intensity of the field reaches the electroporation threshold, the
pore begins to be formed.
The only way to analyze the molecular events involved in the initiation of the
formation and evolution of an induced pore in the membrane during electroporation
is through in silico simulations. The method of molecular dynamics simulation has
revealed that the application of an external electric field that reaches a threshold, the
minimum porating electric field (E0), in an intact membrane (Fig. 1a) induces and
then stabilizes water defects. These defects, which are also called fingers, are formed
in the interface between the water molecules and the hydrophobic part of the bilayer
membrane. These water molecules forming the defects are reorganized by the
alignment of their dipole moments with the applied electric field starting the pathway
to the formation of the pore. These fingers are extended across the hydrophobic core
of the bilayer membrane, connecting to another defect formed in the opposite leaflet,
finally resulting in a continuous path of water wires. These structures are known as
hydrophobic pores because the water molecules are in contact with the hydrophobic
part of the lipids, which constitutes the inner part of the bilayer (Fig. 1b). The new
contacts between the water molecules and the hydrophobic tails of the lipids are
energetically unfavorable. As mentioned previously, in a physiological situation, the
disruption of the membrane continuity is immediately resealed, but, in this situation,
the stimulus that causes the disruption continues exerting its effect over the mem-
brane, preventing the resealing and inducing more water molecules to penetrate into
the hydrophobic core, thus thickening the water wires. Immediately after, the lipids
start a structural rearrangement to surround the water wires. This rearrangement
begins with the movement of the polar lipid head group, in order to reach the inner
part of the bilayer, and therefore ensuring the complete lining of the water molecules.
This stage of the pore formation process is called hydrophilic pore. Then, this
process restores the prepore surface contact, where the water molecules are located
only around the polar lipid head groups and are not allowed to be in contact with the
hydrophobic region of the bilayer (Fig. 1c).
Now, at this point of the formation of the pore, and under the influence of the
external electric field, a new structure of an aqueous pore in the bilayer membrane is
created, where there is a sort of continuity between the lipids of the upper leaflet and
those of the lower leaflet, which allows the movement of lipids from one layer to the
other. Prior to the formation of the pore, this arrangement was nonexistent because
the migration of the lipids from one leaflet to the other, known as flip-flop process, is
a very infrequent event in normal conditions, so the external electric field stabilizes
this pore configuration. If the stimulus of the external electric field applied continues
in time, the hydrophilic pore keeps growing to form an expanded pore (Fig. 1c), but
if the stimulus is not removed, the growth of the pore ends up in the destabilization of
the lipid bilayer and thus leads to the rupture of the membrane. Finally, removal of
the field during the stage of expanded pore leads to the destabilization of the pore and
later to pore annihilation, returning the membrane to the ground state (Tieleman
2004; Tarek 2005; Levine and Vernier 2010; Tokman et al. 2013) (▶ Chap. 7, “Lipid
Electropore Lifetime in Molecular Models”).
Molecular dynamics studies show that electropores can be stabilized in two steps.
In the first step, high electric field strength is applied in the z-direction. This has been
tested, for instance, in the simulation of a bilayer formed by only one lipid, such as
palmitoyl-oleoyl-phosphatidylcholine, and of bilayers with a mixture of lipids mim-
icking the membranes of bacteria, such as Staphylococcus aureus and Escherichia
coli. In all the simulations, the pore evolution is the same as that depicted in
Fig. 1a–c. The simulations are stopped when an expanded pore is obtained
(Fig. 1c). In the second step, the structure of the expanded pore obtained in the
first step is used as the initial configuration in a second simulation, where the field
magnitude is reduced to obtain low field stabilized electropores for several nanosec-
onds, as depicted in Fig. 1d (Böckmann et al. 2008; Piggot et al. 2011).
This two-step protocol for the development and stabilization of the pore may
allow understanding the effect on a preformed expanded pore by modulating the
external electric field to several sustaining values (ES) lower than the minimum
porating electric field (E0) (Fernández et al. 2012).
The initial configuration for an expanded pore can be obtained in a first-step
simulation performed at the minimum porating electric field for a bilayer composed
of dioleoylphosphatidylcholine and cholesterol. Then, in the second step, several
simulations can be performed by applying different field values lower than the
minimum porating electric field to analyze the temporal evolution of the pore
volume. Three different ES values have been found to stabilize the pore indefinitely
at different final volumes. One of these results is shown in Fig. 1d, where the
sustaining value for the field maintains the pore open in a volume lower than that
obtained for the expanded pore.
Figure 2 represents the Es values obtained in the above-described simulation and
shows that the higher the Es, the higher the volume of the pore (Fig. 2a–c). The
whole volume of the pore is estimated dividing the inner part of the pore, located into
the hydrophobic core of the bilayer, into bins. In each bin, a cylindroid that includes
all the water molecules present in the pore is constructed. So, the whole volume of
the pore is calculated as the sum of the volumes of stacked cylindroids inside the
pore (Fig. 3 and Fig. 2d–g).
These results show the three field sustaining values and determine two
nonequilibrium conditions for the stability of the pore:
E < ES: If the field applied is less than the minimum sustaining value, it leads to a
decrease in the pore volume and later to the closure of the pore, as it occurs when the
field is removed (E = 0), thus allowing the complete resealing of the membrane
(Fig. 1 from 1c to 1a). So, fields lower than the minimum Es are not high enough to
maintain the pore open in a constant volume.
84
Fig. 2 Snapshots for the stabilized pore structures after 100 ns at (a) Es = 0.125 MV/m; (b) Es = 0.150 MV/m; and (c) Es = 0.175 MV/m. (d–f) Plots of the (x,
z) view of the cylindroids that form the stabilized pores (Figure from Fernández et al. 2012, taken with permission from Elsevier). Blue spheres are water oxygen
atoms, red spheres are the phosphorus atoms of dioleoylphosphatidylcholine, yellow spheres are the hydroxyl oxygen atoms of cholesterol, green spheres are the
first sn-1 carbon atoms of dioleoylphosphatidylcholine, and turquoise stick structures are the lipid hydrocarbon tails. All the snapshots were performed with
M.L. Fernández and M.R. Risk
Visual Molecular Dynamics (Humphrey et al. 1996)

Fig. 3 Schematic representation of (a) the simulation box, indicating the water region, the upper
and the lower polar regions of the bilayer and the internal nonpolar region of the bilayer and (b) the
cylindroids that constitute the pore (lateral and top view). (c) Snapshot of the initial pore structure.
(d) Plot of the (x, z) view of the cylindroids that form the starting pore. The blue spheres are the
water oxygen atoms, the red spheres are the phosphorus atoms of dioleoylphosphatidylcholine, the
yellow spheres are the hydroxyl oxygen atoms of cholesterol, and the green spheres are the first sn-1
carbon atoms of dioleoylphosphatidylcholine. (e) Plot of the (x, y) view of the cylindroids that
characterize the water pore. Crosses correspond to the center of the cylindroids. The cylindroids of
the nonpolar region that contribute to the pore volume are in red; the “external” cylindroids
excluded from the calculation are in blue (Figure from Fernández et al. 2012, taken with permission
from Elsevier)
E > ES: If the field applied is higher than the maximum sustaining value, it leads
to a continued growth of the pore volume.
These results show a direct association between the magnitude of ES and the
volume of the stable pore, indicating that the pore size can be controlled and
sustained in time (Fernández et al. 2012).
The method of controlling the pore size by adjusting the value of the sustaining
field can also be used to measure the ion conductance through nanopores (Ho et al.
2013). The simulations performed using this method have been developed in two
systems: one in the presence of sodium chloride and the other in the presence of
potassium chloride, showing that the high interaction of the sodium ions with the
interface between water and phospholipids induces a slower conductance than
potassium ions. This slowing effect can be compensated by increasing the field
intensity and as a consequence increasing the pore radius, since at higher pore radii,
the interaction of the sodium ions with the pore wall diminishes as the bulk water
predominates inside the pore.
Another way to modify the pore size is by periodically modulating the intensity of
the field (Kohler et al. 2015). This can be simulated by means of a time-varying
external electric field. Then, using a unipolar sinusoidal function, the variations in
the intensity of the field are associated with variations in the same direction of the
pore size. Therefore, a modulation in the expansion and shrinkage of the pore can be
achieved in a single simulation following the same frequency applied to the electric
field. This is schematically shown in Fig. 1 (Fig. 1c–d and d–c).
Stable Pores Obtained by Charge Imbalance
This method generates an artificial charge imbalance by simulating bilayers that

separate two water regions where different ion concentrations are imposed. This ion
imbalance generates a transmembrane potential able to induce pores as in simula-
tions where an external electric field is applied.
There are two proposed setups:
1. In the first one, two bilayers are modeled in the same simulation box, separated by
a water region. So, one leaflet of each bilayer is in contact with the middle water
slab, and the other faces a second aqueous region. This second water slab is
connected by the upper and lower borders of the box in the z-direction due to the
periodic boundary conditions.
2. In the second one, a single bilayer separating two water slabs is modeled, but
these aqueous regions are not connected because two additional vacuum/air slabs
are inserted in the upper and the lower borders of the box in the z-direction,
generating a water/vacuum interface that impedes the ionic movements between
both water reservoirs (Kirsch and Böckmann 2015).
In both methods, the ion diffusion through the pore dissipates the charge imbal-
ance, leading to a drop in the transmembrane potential.
Molecular dynamics simulations have shown that in a double bilayer of
dimyristoylphosphatidylcholine system with a sodium charge imbalance, a pore
begins to be formed within the first nanoseconds in one of the two bilayers. As the
ions cross the bilayer, the field decreases and the permeation of the sodium ions and
the pore radius decrease, leading to a stable pore for several nanoseconds, with a
remaining charge imbalance of only one sodium ion. If this residual charge imbal-
ance is removed while the pore is still stable, the pore remains open for a few
additional nanoseconds and then closes in less than 10 ns. This indicates that if the
transmembrane voltage is lower than a certain threshold, the pore becomes metasta-
ble and independent of the fall of the transmembrane potential. In this condition
where the transmembrane voltage is below the threshold, the closure is associated
with the pore size fluctuations, rather than with the disappearance of the charge
imbalance (Gurtovenko and Vattulainen 2005). This is comparable with the simula-
tions performed in size-controlled pores by electric field, where if the field applied is
lower than the Es threshold, the pore shrinks and finally closes even in the presence
of an external electric field (Fernández et al. 2012).
In the simulations performed to evaluate the charge imbalance effect on the
transmembrane potential and the pore induction using either the method of the
double bilayer or the vacuum method, as described in the previous example, when
the pore opens, the ion gradient dissipates. When this happens, the transmembrane
potential decreases and the pore cannot be longer sustained in time.
This drop in the transmembrane potential can be avoided by maintaining the
initial imposed charge imbalance, by the swapping method (Kutzner et al. 2011). In
this approach, if one ion crosses the membrane from its initial water reservoir to the
other, this ion and a water molecule from the initial reservoir are swapped and the
charge imbalance is restored. This method allows obtaining stable pores at different
transmembrane potentials and a pore radius that is linearly associated with the
transmembrane potential (Casciola et al. 2016).
Stable Pores Obtained by Mechanical Methods
The previous two sections of this chapter described the methods to open a pore and
maintain it open in a stable way by molecular dynamics, which is by using external
electric fields or the charge imbalance method. As previously mentioned, another
method that can be used to form pores is that based on mechanical stress. This
mechanical stress may be induced experimentally by the aspiration of the lipid
structures of a membrane, a vesicle or cells into a micropipette. This mechanical
stress produced by the aspiration, in other words, by a negative pressure, makes
possible the rupture of the membrane in the form of a pore, equivalent to the
electropores obtained by exposure to electric fields (Kirsch and Böckmann 2015).
Both the opening and closure of a pore can be considered to be due to the
interaction of two tensions: one aiming to maintain the pore closed (the so-called
line tension) and the other aiming to open the pore (the so-called surface tension).
The latter causes a stretching of the membrane. Then, the aspiration of the micropi-
pette causes a negative pressure, causing a surface tension at the level of the
membrane. The energy of formation of the pore (Epf), according to the theory of
pore formation, is defined by the following equation:
Epf = 2πRTL πR2TS
where R is the radius of the pore, TL is the line tension, and TS is the surface
tension (Barnett and Weaver 1991).
This equation of pore formation shows the energy necessary to open a pore,
which is the difference between the energy produced by the line tension and the
surface tension components, in a way that, if the line tension is maintained, an
increase in surface tension will result in a decrease in the energy needed to open or
maintain a pore open.
Fig. 4 Three scenarios where the radius R of the pore increases as the surface tension increases:
R 1 when the surface tension is small and increasing, R0 > R 1 when the surface tension reaches
the critical threshold for tension, and finally R1 when the surface tension is beyond the critical
threshold for tension
In molecular dynamics simulations of a dipalmitoylphosphatidylcholine lipid

bilayer membrane, a surface tension is applied, causing the formation of stable hydro-
philic pores (Leontiadou et al. 2004). This mechanical stress on the membrane, under
the form of a surface tension, was the result of the application of a lateral pressure, in
this way simulating a pressure produced by the aspiration of a micropipette. Molecular
dynamics simulations were performed in two different initial states: one using a bilayer
membrane with a metastable pore, and the other using an equilibrated bilayer mem-
brane without a pore. In both states, a threshold of critical surface tension was reported.
Below this threshold, the pores remain stable, reaching a minimum radius in the order
of 1 nm, at the center of a toroidal-shaped pore. If the critical threshold is exceeded, the
pore results unstable, thus expanding, and finally causing the rupture of the membrane.
Figure 4 shows three scenarios of the opening of the pore due to mechanical stress, in a
transversal plane view, where the radius R of the pore is increased as the surface tension
increases: R 1 when the surface tension is small and increasing, R0 > R 1 when the
surface tension reaches the critical threshold for tension, and finally R1 when the surface
tension is beyond the critical threshold for tension.
In another work, the study of the transport of ions across pores in the bilayer is
combined with mechanical stress to stabilize pores with different radii. The presence
of ions seems to induce a reduction of the stability of the pores, mainly because of
the increase in the line tension. As stated before, this tension maintains the pore
closed, and therefore, an extra mechanical stress is needed to stabilize the pore. This
can be attributed to the binding of sodium ions at the interface between the lipids and
water, in other words, the surface of the bilayer membrane (Leontiadou et al. 2007).
Pore Stability and Chemical Methods
The addition of amphiphilic agents to the system can affect the integrity of the
membrane due to the chemical nature of these compounds. A small amphiphilic
molecule such as dimethyl sulfoxide added to a dipalmitoylphosphatidylcholine
bilayer can alter the normal structure of the bilayer. Three dimethyl sulfoxide
concentration regimes are described to have different effects on the bilayer
(Gurtovenko and Anwar 2007):
• At a low concentration, the molecules of dimethyl sulfoxide locate in the interface

between the lipids and water molecules, inducing a lateral expansion of the
bilayer and a decrease in the bilayer thickness.
• An intermediate concentration induces a decrease in the bilayer thickness and the
dimethyl sulfoxide molecules locate in the interface as well as in the hydrophobic
core of the bilayer, favoring the intrusion of water molecules to the inner part of
the bilayer and leading to the stabilization of hydrophobic pores and then
allowing the formation of hydrophilic pores.
• A high concentration leads to the rupture of the bilayer.
A molecular dynamics simulation work performed in dioleoylphosphati-

dylcholine bilayers in the presence of different amounts of cholesterol combined
the effect of different concentrations of dimethyl sulfoxide with the presence of an
external electric field. The analysis of the results showed that the dimethyl sulfoxide
can facilitate the stability of the hydrophobic pores and diminishes the electropora-
tion threshold at very low concentrations of dimethyl sulfoxide even in the presence
of cholesterol (Fernández and Reigada 2014).
The effects of another amphiphilic molecule, the polyoxyethylene glycol, on
membranes can be analyzed by molecular dynamics and compared with experimen-
tal data. The results obtained using the method of charge imbalance to induce pores
showed that polyoxyethylene glycol seems to favor the formation and stabilization
of the water columns, even allowing the ion transport across the bilayer through
these stabilized pores. In a later stage, a few lipid molecules migrate to increase the
pore stabilization. The presence of this molecule lowers the electroporation threshold
(Polak et al. 2015). This finding is in line with the results described in previous
experiments performed in planar lipid bilayers electroporated in the presence of
polyoxyethylene glycol (Troiano et al. 1998).
Pore Stability by Continuum Modeling
An approach different from molecular dynamics to simulate the effects of

electroporation is the continuum modeling of a cell exposed to an electric field
(Son et al. 2014). Two different pulses are included in the model: a low-intensity
long pulse, in the order of microseconds, and a high-intensity nanosecond pulse.
In this model, the lifetime of the pore is a parameter in the order of seconds. The
results obtained using this approach show that the long pulses lead to populations
of pores with a wide range of sizes distributed in two major subpopulations with
radius of approximately 1–10 nm and that the short pulses produce only one
population of small size pores but in high number. This model indicates that the
differences in the size of the radii of the pores obtained for each pulse type are
related to the association of the radius with the duration of the pulse, because the
pores can only grow during the application of the pulse. This is consistent with
the results obtained in molecular dynamics simulations where, if the electric field
applied exceeds the threshold of the sustaining electric field, the pore keeps
growing. On the other hand, the model also explains the differences in the
number of pores formed by each pulse, associating the dependence of the pore
creation on the transmembrane voltage. So, in this model, high-intensity pulses
induce a greater number of pores (Son et al. 2014).
Another model simulating cells in 3-dimensions takes into account the
conducting and the permeable states separately in the calculation to simulate the
effects on the membrane and the transport due to the application of an external
electric field. These states depend, among other parameters, on poration character-
istic time, permeabilization dynamic, and membrane recovery time, all of them
inputs of the model. In this model also, the pores open when a voltage threshold is
exceeded (Leguèbe et al. 2014).
Pore Stability in Planar Membranes
Pore stability can also be studied in planar membranes. In these experiments, a

planar bilayer lipid membrane is formed, separating two water reservoirs, and then
the effects of membrane poration are analyzed under current-constant conditions. In
a work performed by Koronkiewicz et al., the constant current causes an increase in
the transmembrane potential and the following membrane poration. Once the current
applied reaches the membrane threshold, the potential increases up to 250 mV and
then keeps varying around 150 mV for several nanoseconds. These potential vari-
ations can be attributable to the generation of a pore with a fluctuating size in the
order of nanometers. In a different approach, the current is maintained for 10 ns and
then interrupted for few nanoseconds during several on-off current cycles. In this
approach, the pore keeps its radius stable for the whole experiment. If the interval of
interruption of the current is increased up to 100 s, the pore still remains stable in
size, but at higher interruption periods (300 s), the pore disappears and the continuity
of the membrane is fully restored (Koronkiewicz et al. 2002). This indicates that the
stability of the pore is sustained even in the absence of the stimulus for a period of
time, but in the end the membrane restores its initial continuity, as it occurs in
molecular dynamics pore simulations.
Permeabilized State in Cells
This section describes different electroporation protocols for pore stabilization in

experimental procedures in cells (▶ Chap. 20, “Different Cell Sensitivity to Pulsed
Electric Field”). Early works performed in unfertilized sea urchin eggs in a
low-calcium medium, similar to the calcium concentration of the intracellular
medium, showed that cells can be loaded with radiolabeled low molecular weight
metabolites for several minutes. These works indicated that, in these conditions, the
permeabilized state remains stable for this period of time (Swezey and Epel 1989).
In experiments performed by Pakhomov et al. in CHO cells preloaded with a
thallium-sensitive fluorophore and exposed to nanopulses, thallium was added at
different times after the application of the pulse. The measure of thallium uptake due
to electroporation indicates that the nanopores remain open for several minutes after
the application of the electroporating stimulus (Pakhomov et al. 2009).
Conclusions
Molecular dynamics simulations show that pores in a lipid bilayer membrane can be
obtained and stabilized either by electrical or mechanical stress, or by addition of
chemical agents, such as amphiphilic molecules.
In either electrical or mechanical stress, the simulations show that the external
stimulus has to reach a minimum electric field or mechanical stress, in order to
initiate the pore opening, and then the stimulus has to be maintained lower than a
critical value to stabilize the pore.
The addition of amphiphilic molecules to the simulated system facilitates the
stabilization of the water columns, which initiate the pore formation and decrease the
electroporation threshold.
The approaches using molecular dynamics establish that the modulation of the
external stimulus in values below the threshold modifies the size and keeps the pores
stable, while the stimulus is still present. In the case that the stimulus is ceased or the
intensity is lower than a sustaining value, the pore closure begins.
The approach to simulate the effects of an electric field on cells with the
continuum modeling is less detailed than molecular dynamics but more versatile in
terms of incorporating parameters as inputs of the model to describe the electropo-
ration process.
The experiments made on planar membranes show that the pore stability can also
be sustained in time.
The experiments of electroporation in cells show that the permeabilized state can
be detected for several minutes, thus indicating higher stability of this state when the
experiments are performed in cells.
The stability of the pores or the permeabilized state is related to the complexity of
the system and may range from nanoseconds in molecular dynamics simulations to
seconds in the continuum modeling and the planar membrane experiments under
constant current conditions, and to minutes in cell experiments with pulses of
different intensity and duration. Finally, the stability of the pores is due to the
modulation of the stimulus, for both molecular dynamics and planar membrane
experiments. In cells, the mechanisms that stabilize the permeabilized state after
electroporation remain unknown. This could be attributed to the complexity of the
plasma membrane and to the interactions of the plasma membrane with other
components either inside or outside the cell.
Acknowledgments This work was supported in part by grants from Universidad de Buenos Aires
(UBACyT GC 20620130100027BA), CONICET (PIP GI 11220110100379) and ITBA
(ITBACyT 2015).
Cross-References
Experiments
▶ Different Cell Sensitivity to Pulsed Electric Field
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Molecular Transmembrane Transport
with Giant Unilamellar Vesicles (GUVs) 6
Marie-Pierre Rols
Abstract
Giant vesicles provide a useful model system for measuring a variety of physical
properties of lipid membranes and for improving our understanding of the
electropermeabilization and electrofusion phenomena. The purpose of this chapter
is to present the ways GUV can be formed and submitted to electric pulses.
Moreover, it summarizes the presently known effects of electric fields on giant
vesicles and some of their practical applications. Subjecting GUVs to DC pulses
can destabilize the lipid bilayer, inducing different behavior such as deformation of
the vesicles, permeabilization, and motion of domains or fusion that can be directly
observed under a microscope. In addition to usual optical microscopies, a number
of approaches can be used to assayed molecular transport are described, including
micromanipulation, patch-clamp, and CARS spectroscopy. Electric pulses can have
dramatic consequences on membrane structure and function. With suitable electric
field parameters, it is possible to electropermeabilize the membrane leading to
exchange of molecules between the inside and the outside of the vesicle. If the
electropermeabilization is strong enough, membrane vesicles, tubules, and
macropores can be visualized and are in general accompanied with loss of lipid
membrane material and a subsequent GUV size decrease. GUV can therefore be
considered as useful tools for resolving the effect of electric fields on cells, even if
they present some clear limits in particular for the transport of plasmid DNA.
Keywords
Giant unilamellar vesicles • Model membranes • Electrodeformation • Electropo-
ration • Electrofusion • Tubules • Microscopy
M.-P. Rols (*)

Institut de Pharmacologie et de Biologie Structurale, IPBS/CNRS UMR 5089 and University
of Toulouse, Toulouse, France
e-mail: marie-pierre.rols@ipbs.fr

DOI 10.1007/978-3-319-32886-7_84
96 M.-P. Rols
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Giant Lipid Vesicles Formation and Electropulsation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Background on Lipid Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Electroformation of GUV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Electropulsation of GUV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Effects of Electric Fields on GUV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Different Approaches to Assayed Molecular Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Quantitative Optical Microscopy and Micromanipulation Studies . . . . . . . . . . . . . . . . . . . . . . . . . 104
Visualization and Quantification of Transmembrane Ion Transport . . . . . . . . . . . . . . . . . . . . . . . . 105
Monitoring and Quantifying the Passive Transport of Molecules Through
Patch-Clamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Imaging Molecular Transport Across Lipid Bilayers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Imaging Electropermeabilization Process by CARS Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . 106
Imaging Lipid Loss Associated to Electropermeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Introduction
Membranes are key constituents of cells, acting as barriers which hinder the free
passage of ions and hydrophilic molecules. If this impermeability is crucial for life to
develop, it can represent a hurdle in medicine and biotechnologies where molecule
delivery and/or extraction are required. Molecule transport across biological mem-
branes can be obtained by several methods. Virus bioinspired vectors are quite
efficient, but their safety has been questioned. Therefore, alternative chemical and
physical methods have been developed in parallel. Among the physical methods,
electropermeabilization is probably the most promising one mainly due to its
efficiency and safety. Indeed, cell membranes can efficiently be permeabilized by
applying electric pulses. Provided that the electric pulses are of a sufficient and well-
defined amplitude and duration, impermeant molecules can enter or leave the
cytoplasm of cells. Because of its efficiency, this method, also referred as electro-
poration, has rapidly becoming an established approach in medicine for the local
treatment of cancer (Yarmush et al. 2014) and it also holds great promise for gene
therapy. Electropermeabilization is also of high interest in biotechnology in partic-
ular for molecule extraction in food industry (Kotnik et al. 2015). If the exact
mechanisms of molecular uptake are still not fully understood for the moment, it is
known that the transport of molecules depends strongly on the size of the molecules.
Apart from pulse strength that has to be higher than a threshold, pulse duration is
another key parameter for the success of permeabilization, especially in the case of
large molecules such as plasmid DNA which are driven by electrophoresis toward
the permeabilized membrane (Rosazza et al. 2016). A precise description of the
events leading to membrane permeabilization, and its consequences on lipid orga-
nization, still is missing and would help design safer and more efficient protocols
particularly in the case of gene transfer. However, studying such phenomena at the
6 Molecular Transmembrane Transport with Giant Unilamellar Vesicles (GUVs) 97
molecular level in cells and tissues is unfortunately out of reach due to the high
complexity of cells. This is a major motivation for investigating the behavior of
simpler model systems, namely lipid vesicles.
The conception of the role of lipid molecules in biological membranes has
dramatically evolved over the past few decades. The lipid membrane was first
considered as a purely passive barrier and a substrate to membrane proteins which
carried out biological functions. It is now widely accepted that lipids play an active
role in a number of biological processes. Their physicochemical properties, con-
ferred by their structure and their amphiphilic nature, have consequences well
beyond their self-assembly and ability to form a barrier. It is now possible to form
cell-sized artificial membranes with well-controlled compositions. These objects are
called Giant Unilamellar Vesicles (GUVs) and are also referred to as giant vesicles or
giant liposomes. Their study has become increasingly popular in chemistry, biology,
and physics laboratories. Their primary interest is probably their size which, being of
the order of several micrometers, allows their direct observation via optical micros-
copy techniques. In addition, this system presents the advantage of having no
cytoskeleton. Dramatic membrane consequences can therefore be observed such as
deformation and lipid loss. During the 1980s, Angelova and Dimitrov developed an
efficient protocol for giant vesicles production known as electroformation (Angelova
and Dimitrov 1986), which was later shown to indeed produce unilamellar vesicles
(Rodriguez et al. 2005). Due to the ease of their fabrication and their rich phenom-
enology, artificial vesicles have received an ever growing interest from the scientific
community.
This chapter aims to present what is known about the various phenomena that
occur when GUVs are subjected to electric fields. Reviews have been already
published about the effects of electric fields on GUVs (Haluska et al. 2006) and
about the physics of GUVs in general (Dimova et al. 2006). The present paper is
therefore obviously not exhaustive. However, it should be of use to newcomers in
the field or to people familiar with previous reviews and who wish to know about
recent developments. It is organized in two main sections: (Yarmush et al. 2014)
description of synthetic lipid vesicles and tips to synthetize and submit them to
electric pulses, (Kotnik et al. 2015) presentation of different approaches to assayed
molecular transport on giant liposomes, along with their perspectives in biology and
medicine for molecule delivery when they exist.
Giant Lipid Vesicles Formation and Electropulsation
Background on Lipid Vesicles
Synthetic lipid vesicles provide membrane models suitable for systematic investi-
gations of the effects of electric fields on lipid bilayers such as electrodeformation,
electropermeabilization, and electrofusion. Membrane electropermeabilization has
been first assessed by conductance measurements or transfer of radioactive or
98 M.-P. Rols
fluorescent molecules. Different types of vesicles have therefore been used to study
the underlying process, but only the largest vesicles (above 10 μm) can offer the
direct visualization of the processes. First experiments have been performed on small
unilamellar vesicles (SUV) (15–30 nm) more than 30 years ago. A transient leakage
of radioactive sucrose was detected and was attributed to the formation of pores,
which allowed passage of sucrose. However, there was no direct proof of their
existence, or other related phenomena such as membrane deformations. DNA
electrotransfer into liposomes was first studied 20 years ago on large unilamellar
vesicles (LUV) (100–200 nm), suggesting endocytosis-like vesicles which shielded
the electrotransferred DNA from the internal medium. Yet, also in this case, the
liposomes could not be observed because of their small size. The results were
subsequently questioned by data obtained on giant liposomes supporting a mecha-
nism involving electropores and a direct entrance of fluorescent DNA into the
liposomes. GUVs containing fluorescent phospholipid analogs allowed the visuali-
zation of membrane associated perturbations under fluorescence microscope. As will
be later detailed in this chapter, lipid loss was observed in electropermeabilized
membranes and was associated with pores, vesicles, and tubules formation, struc-
tures that can be involved in transmembrane transport of molecules.
Electroformation of GUV
Electroformation is probably the most common method for vesicle production. As

already mentioned in the introduction, the process was first reported in 1986
(Angelova and Dimitrov 1986). Later refinements of the method have allowed for
unilamellar vesicles to be consistently produced (Angelova et al. 1999). The process
of electroformation involves different steps including (Yarmush et al. 2014) the
spreading of phospholipids on the surface of a conductive electrode, (Kotnik et al.
2015) the evaporation of the solvent to form a dry phospholipid film, (Rosazza et al.
2016) the immersing of the conductive electrode in an aqueous solution, and finally
(Angelova and Dimitrov 1986) the application of Alternative Current (AC) electric
pulses across the lipid film (Fig. 1). By this way, it is possible to easily obtained
GUV which sizes wary from a few μm to tens of μm. Electroformation devices can
be purchased from companies, but it is possible to build its one setup that is an easy
and cheap way to obtain GUV. Electroformation chamber can be made by two glass
layers covered with indium tin oxide to ensure the electrical conductivity of the
surface (Fig. 2).
The two layers are separated by an adhesive silicone joint of 1 mm width. The
connection with an AC generator is maintained by two wires, each one soldered on a
small copper strip stuck on the ITO slide. Phospholipids are diluted in chloroform, at
a mass concentration of 0.5 mg/mL. Fluorescent probe can be eventually added at
0.005 mg/mL in order to help GUVs visualization under fluorescent microscopes.
15 μL of lipid solution is deposited on the conducting sides of the glass slides. The
deposition is carried out slowly and at constant rate in a chamber held at 4 C to
slowly evaporate the chloroform and then the slides are dried under vacuum for a
Fig. 1 Electroformation of GUV. Left: Schematic representation of the process; Right: GUV
observed under a microscope
Fig. 2 Experimental
homemade setups for GUV
formation and
electropulsation. Left:
electroformation chamber;
Right: electropulsation device
couple of hours to entirely remove the remaining solvent molecules. Finally, the
slides are sealed together, and the chamber is filled with the formation medium
which is a 240 mM sucrose solution in water.
Sinusoidal voltage of 25 mV peak to peak is then applied at 8 Hz. The voltage is
increased by 100 mV steps every 5 min, up to a value of 1225 mV. It is maintained
under these conditions overnight. Next, a square wave of same amplitude at 4 Hz has
to be applied for 1 h to detach the liposomes from the slides. The lipids indeed
interact with the aqueous solution and electric field by “peeling off” the electrode
surface in layers and self-assembling into giant, sometimes multilamellar, vesicles.
One drawback of the first implementations of the electroformation method was
the requirement for low salt concentrations in the buffer. Since most proteins require
high salt concentrations in order to function properly, this made it very difficult to
encapsulate active proteins using electroformation. However, recent refinements to
the technique have overcome this limitation and produced liposomes by
electroformation using physiologically relevant salt concentrations. Bagatolli and
collaborators reported that GUVs can be prepared using a protocol based on
the electroformation method from either native membranes or organic lipid
mixtures at physiological ionic strength (Montes et al. 2007). They showed that
membrane proteins and glycosphingolipids preserve their natural orientation after
electroformation.
100 M.-P. Rols
One has to add that in the last ten years there have been many new developments
in liposome formation technology. Most notably, macroscale methods have been
transposed to the microscale while entirely new methods that are only possible with
microfluidic technologies were also developed. A recent review by van Swaay and
deMello provides a critical comparison of existing microfluidic technologies for
forming liposomes (van Swaay and deMello 2013). The properties of the generated
liposomes, including size, size distribution, lamellarity, membrane composition, and
encapsulation efficiency, form the basis for comparison. According to their qualities
and drawbacks, these different methods can be useful in different applications.
Electropulsation of GUV
Once obtained by electroformation, GUV can then be easily submitted to electric

field pulses by homemade pulsation chamber (Portet et al. 2009).
Homemade chamber can be composed as shown in Fig. 2 of a glass slide and a
coverslip. In the present device, two parallel copper strips of thickness 70 μm are
stuck on the slide at a distance of 1-cm apart. The coverslip is then stuck onto the
slide and strips with heated parafilm. The chamber is 1-cm long (between elec-
trodes), 2.6-cm wide (width of the coverslip), and 250-μm high (value estimated via
measurements with a microscope). 60 μL of pulsation buffer are introduced between
the slide and the coverslip, while taking care of filling the whole chamber to ensure
the conductivity of the medium. Next, 5 μL of GUV preparation are added. Capil-
larity phenomena prevent the solution from leaking out of the chamber.
The electrode thickness is about the size of the biggest liposomes, which repre-
sents only a quarter of the chamber height. It is not a priori possible to be certain of
the homogeneity of the field. However, solving numerically Laplace’s equation with
finite element software for such a geometry reveals that the field is almost homoge-
neous in the bottom part of the chamber between the electrodes, and that the size and
shape of the permeabilized area are not significantly different from that computed for
a geometry with much bigger electrodes.
Electropulsation can be carried out using different kind of electropulsator which
delivered square-wave electric pulses. An oscilloscope has to be used to monitor the
pulse shape and amplitude. The process of electropulsation can then be performed
directly under the microscope. For the phase contrast visualization, one can use an
inverted epifluorescence microscope equipped with a camera and phase contrast
objective. Visualization of the liposomes is possible thanks to the resuspension of the
GUV in a 260 mM glucose containing solution. For the fluorescence visualization,
one can use an inverted confocal microscope with an objective for fluorescence
imaging.
The pulse duration has to be set from a few hundreds of microseconds to several
ms, according to the effects that have to be studied. Short pulses are used to simply
permeabilize the cell membranes and allow the transfer of small molecule such as
cytotoxic drugs. Long pulses, at the ms time range of order, are commonly used for
gene transfer protocols in mammalian cells.
Fig. 3 Example of GUV submitted to electric pulses. Left: control, before EP; Right: 10 s after
delivery electric pulses at two electric field intensities E1 and E2 (E2 > E1). Scale bar is 10 μm
It is sometimes necessary to interrupt the pulse train for a few seconds to recenter
the image on the liposome of interest. Indeed, the observed vesicle does not always
stay immobile. It often experiences a translational motion toward the positive
electrode.
As shown in Fig. 3, direct observation of GUV submitted to electric pulses can be
performed under an inverted microscope, allowing an easy way to visualize on real
time the process of electropermeabilization. The different refractive indexes of the
internal and external media yield a contrast which enables the vesicles to be
visualized using a microscope, and the density difference allows the sedimentation
of the vesicles on the bottom of the chamber, thus reducing their distance from the
objective. Giant liposomes have different sizes ranging from a few μm to several tens
of μm. Such a heterogeneity is however very useful to study electropermeabilization
and in fact reflects the heterogeneity of cells present in tissues. Electropermeabi-
lization is, in this example where GUV are formed in a sucrose containing solution
and pulsed in the presence of glucose, quite easy to assess. Any decrease of phase
contrast indeed reflects molecule exchange between the interior and the exterior of
the vesicles.
It is well known, for many years, that the trigger of membrane electropermeabi-
lization is the transmembrane potential which must reach critical values around
200 mV. The application of the electric field indeed superimposes an electro-
induced transmembrane potential, Δψi, to the resting transmembrane potential
Δψo. The value of Δψi is given by the Schwan equation:
Δψi ¼ f gðλÞ r E cos ðθÞ, (1)
where f is a factor depending on the biological object shape, g(λ) a parameter

depending on the conductivities λ of the membrane, of the external medium, and
of the internal one, r is the radius of the biological object, E the electric field strength,
and θ the angle between the electric field direction and the normal to the membrane
at the considered point of the surface. Being dependent on the angle θ, the electric
field effect is not uniform along the membrane and its maximum effects are present
at the poles facing the electrodes.
102 M.-P. Rols
The basics for electropermeabilization imply that the transmembrane potential

must reach values closed to 200 mV. According to Eq. 1, electric field strength
values E higher than a threshold value Ec must be applied to induce membrane
permeabilization. In the case of two vesicles 1 and 2 with radii r1 and r2, respectively,
permeabilization should be induced for a constant Er value. This implies that if
r1 > r2, then E1 < E2.
As shown in Fig. 3, this is clearly the case. Large vesicles can be permeabilized
by electric field values which do not affect the smaller vesicles. There is a direct
linear relation between Ec and vesicles radius (Fig. 3, right). Moreover, the larger the
vesicles are, the lower the electric field threshold value is. By applying another train
of pulses at a higher electric fields strength (E2 > E2), it becomes then possible to
permeabilize GUV with lower sizes that were not previously affected by applying
the train of pulses (E = E1). Such experimental results are in complete agreement
with the above theory of electroporation and gives a piece of evidence that GUV are
a suitable model to assess electropermeabilization related process.
One however has to notice that the determination of the threshold values Ec by the
method used here, contrast decrease, is not a very sensitive one. Other works indeed
show that the detection method is a very important parameter for the precise
determination of the critical transmembrane potential. Fluorescence is more sensi-
tive. The use of quenching ions as probes for lipid bilayer permeabilization allows
for very high resolution detection because of their small size which implies the need
for a lower destabilization threshold than the one needed with larger molecules such
as fluorescent dyes or sugar (Mauroy et al. 2015).
Effects of Electric Fields on GUV
Both DC pulses and AC fields can strongly destabilize giant vesicles and influence
their behavior. As shown in Fig. 4, depending on the parameters of the applied field,
giant vesicles can deform, porate, fuse, or even exhibit domain motion in the case of
multicomponent liposomes (Portet et al. 2012).
Deformation of giant vesicles by electric fields was first extensively studied in the
early nineties showing that spherical liposomes deformed into prolate ellipsoids
oriented in the field direction, the degree of deformation increasing with the magni-
tude of the applied field. However, DC pulses usually have durations of the order of
several tens or hundreds of microseconds, or at most of some milliseconds. Imaging
at such high rates is impossible with classical video cameras, so if one wants to get
insight at what happens during an electric pulse application, one has to work with a
fast imaging setup allowing image acquisition at several thousand frames per
second. This approach was first applied by Riske and Dimova, and they indeed
found that GUVs deformed into prolate ellipsoids upon the application of electric
pulses of 50–300 μs duration and 1–3 kV/cm amplitude (Riske and Dimova 2005).
Later, the same authors worked in conditions closer to physiological ones and found
that vesicles subjected to electric pulses in salt solutions always adopted cylindrical
shapes, irrespective of their ionic content (Riske and Dimova 2006).
AC field
Electrodeformation Motion of domains
GUV
DC pulse
Electrodeformation Electrofusion
Size decrease Electroporation
Fig. 4 The influence of electric fields on giant lipid vesicles. GUV submitted to AC or DC pulses
can be deformed; DC pulses can induce permeabilization (which eventually leads to a decrease in
size and an exchange of molecules between interior and exterior of the vesicles) and fusion in the
case of GUV in contact (Adapted with permission from Portet et al. (2012))
Electric pulses may also have much more dramatic effects on lipid bilayers than
simple deformation. They can sufficiently disrupt the membrane as to allow the
uptake of nonpermeant molecules. No consensus exists on the way the membrane
reorganizes at the molecular level and this question still remains a challenge. Here
again, GUVs are convenient tools to investigate these questions. Some studies have
been performed, and have shown that giant vesicles can indeed become
permeabilized by DC electric pulses. It was shown by Tekle et al that the mechanism
of pore formation is asymmetric (Tekle et al. 2001). A macropore is formed on the
cathode-facing side of the vesicle, whereas the presence of many smaller pores on
the other hemisphere can be inferred from the size decrease of the liposome
following pulse application. This phenomenon of GUVs shrinkage during
electropulsation was then further extensively studied (Portet et al. 2009). By apply-
ing a sequence of long (5 ms) electric pulses, it was found that vesicles shrank, down
to a critical radius beyond which their size no longer changed. Three mechanisms for
the lipid loss were identified: formation of macropores on the cathode-facing side as
already reported, formation of tubular structures on the anode facing side, and
formation of small vesicles at both poles. These three features should probably not
be considered as distinct mechanisms. Macropore and tubules formation probably
reflect the same phenomena, as they were found to occur together. Small vesicles
expulsion can be understood as another way of expelling lipids, and may under
certain conditions be more energetically favorable than tubule formation.
104 M.-P. Rols
Increasing the membrane complexity by addition of different phospholipids and

cholesterol leads to phenomena that cannot be observed in single component GUVs.
Staykova et al managed to monitor the motion of liquid ordered domains in giant
vesicles made of DOPC/DPPC/Cholesterol (different molar ratios were explored)
subjected to AC electric fields of 500 V/cm amplitude in the kHz frequency range
(Staykova et al. 2008). They report that this movement had characteristic features
depending on the field parameters, and that it was caused by the inhomogeneous
surface tension induced by the field because of the chamber geometry. It was the first
time this phenomenon of induced charge electro-osmosis was observed and studied
on a lipid membrane.
Membrane fusion is a key process of life. However, membrane fusion does not
occur spontaneously because of large energetic barriers in biological membranes. To
promote membrane fusion, it is essential to overcome these barriers. Electrofusion
is a very convenient way to control, both spatially and temporally, fusion events.
It is thus possible to trigger and observe the whole fusion process to study the
underlying mechanisms. To occur, electrofusion requires two conditions:
(a) electropermeabilization and (b) contact between lipid membranes. When the
two membranes are close enough and lipid perturbation is high enough, fusion
occurs; for cells. Electropermeabilized membranes are thus fusogenic. For a better
understanding of the fundamental processes involved in membrane fusion, lipid
vesicles are often used as model systems. An AC field can be used to align vesicles in
the field direction and bring two vesicles into contact. A subsequent application of a
DC pulse induces the permeabilization of the two vesicles and if permeabilization is
induced in the contact area fusion is induced (Haluska et al. 2006).
Different Approaches to Assayed Molecular Transport
Quantitative Optical Microscopy and Micromanipulation Studies
Very few publications have been reported merging micropipet manipulation tech-
nique with fluorescence microscopy experiments, and so the combination of these
two techniques still remained to be fully exploited. The possibility to simultaneously
perform both types of experiments in the same microscope offers a powerful tool to
relate supramolecular membrane events at different lengths and time in the case of
lipid vesicles. Because their size that is well above the resolution limit of regular
light microscopes, GUVs are indeed suitable membrane models both for optical
microscopy and micromanipulation experimentation.
In their recent review, Bagatolli and Needham discuss the basic methodological
aspects of optical microscopy and micromanipulation methods to study membranes
(Bagatolli and Needham 2014). They focus on the use of fluorescence microscopy
and micropipet manipulation techniques to study composition–structure–property
materials relationships of free-standing lipid bilayer membranes. For instance, using
different fluorescent reporters, fluorescence microscopy allows strategies to study
membrane lateral structure/dynamics at the level of single vesicles of diverse
compositions. The micropipet manipulation technique allows studies on the mechan-

ical, thermal, molecular exchange and adhesive-interactive properties of composi-
tionally different membranes under controlled environmental conditions. By
utilization both techniques simultaneously on the same vesicle, they bring the ability
to characterize structure and strain responses together with the direct application of
well-defined stresses to a single membrane. Knowledge acquired by these studies
has informed several applications of lipid membranes including their use as drug
delivery systems.
Visualization and Quantification of Transmembrane Ion Transport
Transmembrane ion transporters are widely investigated as supramolecular agents

with potential for biological activity. Tests are usually performed in large unilamellar
vesicles (LUVs). However, transport must be followed through bulk properties of
the vesicle suspension, because LUVs are too small for individual study. Ion
transport can be revealed and quantified through direct observation employing
GUVs. This allows characterization of individual GUVs containing transporter
molecules, followed by studies of transport through fluorescence emission from
encapsulated indicators. In their publication, Valkenier and collaborators have
devised a new method whereby ion transport by small molecules into individual
giant unilamellar vesicles can be observed and quantified (Valkenier et al. 2015). By
directly visualizing transport into GUVs, their approach offers a high level of
certainty and integrity compared to experiments on bulk suspensions of smaller
vesicles. Instead of quantifying transport into a population of vesicles with a
distribution of sizes, they are able to analyze the transport into individual GUVs.
The method provides new levels of certainty and relevance, given that the GUVs are
similar in size to living cells. Indeed, positive results in this test provide clear
encouragement that a transporter has potential for biological activity. It has been
demonstrated using a highly active anion carrier, and should aid the development of
compounds for treating channelopathies such as cystic fibrosis.
Monitoring and Quantifying the Passive Transport of Molecules

Through Patch-Clamp
Transport of active molecules across biological membranes is a central issue for the
success of many pharmaceutical strategies. In their work, Amatore and collaborators
combined the patch-clamp principle with amperometric detection for monitoring
fluxes of redox-tagged molecular species across a suspended membrane patched
from single-wall GUV and the corresponding fluxes measurements quantified
(Messina et al. 2014). The quality of the patches and their proper sealing were
successfully characterized by electrochemical impedance spectroscopy. Cholesterol
was added to improve the patches stability This procedure appears versatile and
perfectly adequate to allow the investigation of transport and quantification of the
106 M.-P. Rols
transport properties through direct measurement of the coefficients of partition and

diffusion of the compound in the membrane, thus offering insight on such important
biological and pharmacological issues.
Imaging Molecular Transport Across Lipid Bilayers
In their publication, Li and collaborators used confocal microscopy to image the

transport of carboxylic acids with different lengths of carbon chains into a single
GUV (Li et al. 2011). Fluorescein-dextran, which acts as a pH-sensitive dye, was
encapsulated in the GUV to trace the transport of acid. The GUV was immobilized
on the surface of a microfluidic channel by biotin-avidin binding. This microchannel
allows the rapid and uniform exchange of the solution surrounding the GUV. Using a
spinning-disk confocal microscope, the entire concentration field is captured in a
very short exposure. This technique combines rapid buffer exchange in a
microfluidic device with high-speed confocal microscopy to accurately observe
rapid transmembrane transport processes. Results showed that more lipophilic
acids cross the bilayer more quickly. A finite difference model was developed to
simulate the experimental process and derive permeability. This technique promises
to be useful in understanding with greater detail how the molecular properties of
drug like molecules determine their transport behavior into cells.
Imaging Electropermeabilization Process by CARS Spectroscopy
In the high majority of studies, visualization of electropermeabilization is performed

by using fluorescent molecules (lipid analogue, fluorescent dye) and not pure lipid
systems thus including possible artefacts linked to these molecules. Fluorescence
microscopy, while extremely sensitive and widely used, indeed requires the intro-
duction of extrinsic fluorophores but often causes unwanted perturbations. In their
paper, Mauroy and collaborators performed experiments on pure lipid GUVs,
i.e., without any addition of exogenous molecules (lipid analogue, fluorescent dye)
and used two different methods, phase contrast and CARS microscopies (Mauroy
et al. 2012). Optical microscopy is unique in its ability to probe living specimens
with subcellular resolution. Strong vibrational signals can be obtained with coherent
anti-Stokes Raman scattering (CARS) microscopy, a nonlinear Raman technique.
GUVs were prepared in a medium containing sucrose and diluted in a medium
containing glucose. The difference in refractivity between the internal and the
external media was enhanced by phase-contrast optics. CARS has the advantage
of offering molecular specificity, without necessitating the application of external
labels. As reported by the group of Kinosita, GUVs were prepared in an internal
medium containing sucrose and diluted in the external medium containing glucose.
The insides of the liposomes looked darker than the outsides because the difference
in refractivity between the internal and the external media was enhanced by the
phase-contrast optics (Akashi et al. 1996). They present for the first time results of
electropermeabilization of pure lipid systems obtained by CARS microscopy. Their

results indicate that tubule formation indeed is not an artifact due to fluorescence
labeling. Similar results were obtained for both fluorescence and CARS microscopy.
The results underline the high potential for use of CARS microscopy as a tool to
study electropermeabilization processes in living systems.
Imaging Lipid Loss Associated to Electropermeabilization
In their work, Portet and collaborators studied the effect of permeabilizing electric
fields applied to two different types of giant unilamellar vesicles (Portet et al. 2009).
Experiments on vesicles show a decrease in vesicle radius, which is interpreted as
being due to lipid loss during the permeabilization process. They show that the
decrease in size can be qualitatively explained as a loss of lipid area, which is
proportional to the area of the vesicle that is permeabilized. The phenomenon of
electropermeabilization can be defined as a two-step process: (1) a physical change
induced in the membrane by the field (in the absence of molecules to be transported)
and (2) an interaction of the molecules that are to be transported with the modified
membrane. At the simplest level, combination of Steps 1 and 2 can be observed
experimentally as a transport phenomenon using marked molecules or via conduc-
tivity experiments. In their article, they demonstrate that Step 1 can be indirectly
detected via a change in the size of giant liposomes under electropulsation and an
associated direct visualization of the expulsion of lipids from the liposomes. Con-
cretely they study the effect of a series of permeabilizing pulses, well separated in
time, on the size of GUV. In the experiments, the radius of the GUV is measured after
each pulse and each GUV studied shows, on average, a decrease in its radius down to
a critical radius beyond which its size no longer changes. This decrease in size points
to the fact that, during the physical processes leading to electropermeabilization,
lipids are lost from the vesicle—thus leading to a reduction in their size. One of the
most fascinating aspects of the experiments is the wide variety of mechanisms of
lipid loss that can be observed: pore formation, vesicle formation, and tubule
formation (Fig. 5).
Three different mechanisms of lipid loss are observed when the lipids are
fluorescently marked, the term “lipid loss” implies loss of lipid from the bulk
spherical part of the vesicle; the lipid ejected appears, in most cases, to remain
attached to or close to the parent vesicle. The first and most frequent mechanism is
the formation of small vesicles at both the anode-and cathode-facing poles. Those
vesicles are mainly thrown out of the GUVs, but some of them were also driven
inside the GUVs. The second phenomenon was the creation of lipid tubules on the
exterior of the anode-facing hemisphere. DOPC molecules expelled from the mem-
brane rearranged in the form of tubular structures, whose lengths grew with the
number of applied pulses. These structures initiated from the pole facing the positive
electrode and remained attached to the vesicle. However, they then appeared to
diffuse away from the pole toward the equator (while remaining attached to the
membrane) and appeared to cover most of the anode-facing hemisphere. They also
108 M.-P. Rols
Fig. 5 The effects of electric fields on giant lipid vesicles. GUV submitted to DC pulses can be
permeabilized leading to the formation of vesicles and tubules which number and or size increase
with the number of pulses. Macropores can also be observed (Adapted with permission from Portet
et al. (2009))
saw on the cathode-facing side that tubules can grow on the interior surface of the
liposome. These structures also diffuse toward the equatorial regions, the number
and size of tubules, however, being smaller. This mechanism of tubule formation
appears to be stronger on the anode-facing hemisphere. Finally, they also noticed the
presence of pores on the cathode-facing hemisphere. This was a quite rare observa-
tion, but it is normal because acquisition times were of a few hundreds of millisec-
onds, the same order of magnitude as the lifetimes of such pores.
The eventual long-term evolution of the structures described above (after pulsa-
tion has been stopped) varied from one experiment to another. The small vesicles, in
most cases, diffused away from the liposome and the vesicle radius stayed constant.
However, the behavior of the tubular structures exhibited wide variation. Some of
the tubules broke away from the GUV and diffused away, sometimes forming
vesicles and sometimes not. Other tubules remained attached to the vesicles,
exhibiting polymerlike fluctuations. In some cases, they were reabsorbed into the
GUV membrane after a time of approximately minutes. In fact, the eventual fate of
tubules was strongly dependent on their environment, notably on whether other
vesicles came in contact with them or not. In the cases where tubules were
reabsorbed, the volume of the vesicle they were attached to increased, and the
final state of the vesicle was often nonspherical, and appeared to be under little
tension.
Such transient membrane alterations may be involved in the transport of mole-
cules across membranes and explain the different pathways molecules can enter
electropermeabilized cells. However, experimental results regarding the
electrotransfer of plasmid DNA into phosphatidylcholine GUVs indicate that a direct
entry is the predominant mechanism of electrotransfer (Portet et al. 2011). This is not
the case for cells, where DNA electrotransfer is a multi-step process including a key
step of interaction between the plasmid and the electropermeabilized membrane. A
quantitative analysis of the DNA concentration increments inside the GUVs can be
very well described by a simple theoretical model in which DNA entry is mostly
driven by electrophoresis. Such results pave the way towards a novel method for
encapsulating with high efficiency not only DNA, but any negatively charged
macromolecules into GUVs. They however show the limits of GUV as a model.
Conclusions
This chapter has present main data reporting that giant vesicles can be considered as
relevant biological models, for resolving the effect of electric fields on cells. GUV
have been shown as a powerful model, easy to form and observe under a microscope,
that fairly reproduce electropermeabilization associated phenomena such as defor-
mation, fusion, molecule transport. As pure lipid membranes, they however present
some limits people have to know about. According to the dictionary, a model is a
person or thing that serves as a subject for an artist, sculptor, writer. For sciences, it is
in only fact a simplified representation of a system or phenomenon.
Richard Feynmann, Nobel laureate in Physics, famously wrote: “what I cannot
create, I do not understand.” Biomimetic approach bridges the gap between the
simplest GUV consisting only of a spherical lipid bilayer enclosing a buffer, and
more complex GUVs made from lipid mixtures, carrying adhesion proteins and/or
filled with artificial cytoskeleton providing more advanced test cells. A multitude of
different modifications to the basic GUV are now available, each capturing the
essence of a different aspect of the cell (Fenz and Sengupta 2012). Such more
realistic GUV are of great interest to go deeper in the elucidation of the mechanisms
underlying transport of molecules in cells and tissues.
110 M.-P. Rols
Cross-References
Experiments
▶ Electrodeformation, Electroporation, and Electrofusion of Giant Unilamellar
Vesicles
▶ Experimental Determination of Lipid Electropore Size
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Lipid Electropore Lifetime in Molecular
Models 7
Zachary A. Levine
Abstract
Membrane electropermeabilization describes the electric field-mediated depolar-
ization, and subsequent breakdown of cellular membranes, and is widely used in
clinical and academic environments to deliver extracellular materials into the cell
interior. Recently, these methods have contributed to the optimization of food
sterilization and next-generation therapeutics that aim to enhance the susceptibil-
ity of tumor cells to traditional chemotherapies, signaling a significant shift in
biomedical and medical modalities. However, the relationship between macro-
scale membrane electropermeabilization and the influx of individual materials
through discrete electropores (often termed electroporation) is often unclear, at
best. Because detection and characterization of discrete electropores in experi-
ments containing cells or vesicles are difficult, if not impossible, this section will
describe discrete electroporation models based on theoretical molecular dynamics
simulations, with the intent of reconciling theoretical models with observations
from macroscopic experiments. Access to massively parallel supercomputing
resources have greatly enhanced the timescales and typical sample sizes modeled
in simulations, and these trends are expected to grow as computing hardware and
software become more integrated and affordable. Therefore, discussion will focus
primarily on all-atom molecular models rather than larger, coarse-grained models
that lack contributions from individual water molecules. Taken together, this
section will broadly cover studies that aim to codify the discrete biophysical
Z.A. Levine (*)

Department of Physics, University of California Santa Barbara, Santa Barbara, CA, USA
Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara,
CA, USA
Materials Research Laboratory, University of California Santa Barbara, Santa Barbara, CA, USA
e-mail: zlevine@chem.ucsb.edu

DOI 10.1007/978-3-319-32886-7_86
114 Z.A. Levine
processes that mediate membrane electropermeabilization, in an effort to better

understand its biological basis.
Keywords
Electroporation • Electropermeabilization • Electroevaporation • Computer sim-
ulations • Electropore life cycle • Phospholipid bilayers
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Electropore Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
Molecular Dynamics Simulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Electropore Lifetime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Introduction
The mechanical structure of cellular membranes, which compartmentalizes and

partitions intra- and extracellular materials from one another, can be transiently or
irreversibly permeabilized with the application of sufficiently high electric fields
(Hamilton and Sale 1967). Such electric fields, typically of MV/m amplitude and
nano- to microsecond duration, increase the electrical conductance of plasma mem-
branes and allow influx and efflux of small, normally impermeant molecules across
cellular barriers (Neumann et al. 1982). These changes in conductance, while small,
can be observed after only a few nanoseconds following the delivery of pulsed
electric fields (Benz and Zimmermann 1980). Similarly, small nanometer-sized
fluorescent dyes can be utilized as molecular markers to measure the amount of
membrane electropermeabilization and disruption, but how and where small mole-
cules enter membranes and what atomic factors mediate their interaction with
membranes under high electric fields is poorly understood and difficult to observe
directly with microscopy (Teissie et al. 2005). As a result, theoretical methods
including atomistic simulations are currently better suited for characterizing proper-
ties of discrete electropores at the atomic level, where Coulombic and Van der Waals
forces dominate.
Structurally, membranes can consist of hundreds of distinct lipid species, trans-
membrane proteins, cytoskeletal scaffolds, and a virtual zoo of interacting mole-
cules; however, the most dominant structural unit of cellular membranes is
phospholipids (Fig. 1). Phospholipids are amphiphilic molecules containing a dipo-
lar headgroup, two aliphatic (fatty acid-derived) tails, and a glycerol backbone
linking the polar headgroup to the tails. The two tails are attached to the C1 and
C2 glycerol carbon atoms, thereby denoting the tails as sn-1 and sn-2, respectively.
Depending on the fatty acid chain length, the length of lipid tails can vary
considerably from 12–22 carbon atoms and typically contain a handful of
unsaturated carbon atoms. Lipid names are then constructed by combining, in
7 Lipid Electropore Lifetime in Molecular Models 115
Fig. 1 Diagram of a typical phospholipid from molecular simulations
order, the sn-1, sn-2, backbone, and headgroup names and are often referred to by
acronyms containing the first letters of each group.
In this section, a summary of electroporation models in homogeneous phospho-
lipid membranes (i.e., membranes containing only one lipid type) will be presented;
however, in the next section (titled “Effects of Heterogeneous Membranes and
Electrolytes on Electropore Formation”), additional discussion will be given to
membranes containing multiple lipid types or electrolytes, which exhibit somewhat
different permeabilizing behaviors from homogeneous membranes.
The mechanical and electrical properties of simple lipid bilayers closely resemble
the properties of real cell membranes. Lipid bilayers in cellular membranes typically
span 4–5 nm in thickness and hold a resting potential of roughly 70 mV. This is due
to a number of factors, such as surface or zeta potentials which stem from differences
between bulk electrolyte and the membrane surface charge, dipolar potentials which
inhabit the lipid headgroup region, and other various transmembrane potentials
based on differences in electrolyte concentration from one side of the bilayer to
the other (Fig. 2). This implies that a single charge would only need 0.07 eV of
energy to cross a lipid bilayer; however, this is not the case. The lipid headgroup
116 Z.A. Levine
Fig. 2 Electrical properties of a phospholipid bilayer. ΔΨm, ΔΨs, and ΔΨd stand for the trans-
membrane, surface, and membrane dipole potentials, respectively, whereas Εm, Εs, and Εd stand for
the corresponding electric field values
region contains a very large 280 mV potential difference across it, although that
potential is often neutralized because of the equal and opposite headgroup dipoles
located on opposing leaflets. A single charge actually requires about 0.3 eVof energy
just to cross the headgroup region (consider a particle with charge 1e traveling 1 nm
against a ~300 MV/m electric field). However, a single charge needs only to traverse
halfway across the bilayer to make it through, because the hydrophobic lipid interior
will push the particle outward with little to no additional energy. For symmetric
bilayers one can imagine the headgroup polarization spanning, at most, 2.5 nm
(halfway across the bilayer) in which case the translocation energy approaches
1 eV, though this can vary if there are asymmetries present.
Electropore Models
Early efforts to model membrane reorganization under the influence of electric fields
began in the 1950s and were rooted in cellular experiments where conductive and
dielectric properties of the membrane were modulated as a function of the applied
electric field. During this time, researchers discovered that the electrical impedance of
membranes would significantly drop when large voltages generated from ion gradients
across the membrane were applied. Later in the 1970s, Neumann and Rosenheck
(1972) demonstrated that both membrane permeability and conductance could be
reversibly increased if the applied electric field was sufficiently short. Transient
electrical perturbations have since been termed “reversible electroporation” because
increases in membrane permeability and electrical conductance can be completely

reversible if the applied field is removed after a sufficiently short period of time. This is
in contrast to “irreversible electroporation,” which describes plasma membranes that
are permanently disrupted from prolonged exposure to electric fields, leading to
eventual cell death or necrosis.
Irreversible electropermeabilization of membranes was initially described by
Hamilton and Sale (1967) in a triple set of detailed publications that describe the
nonthermal destruction of bacteria, yeast, and erythrocytes under kV/cm electric
field amplitudes. Membrane lysis was found to be present at externally applied
transmembrane voltages of 0.30–1.15 volts. Reversible electropermeabilization of
cellular membranes, however, appeared to be more closely correlated with earlier
studies on changes in membrane conductance (Abidor et al. 1979). Because revers-
ible electroporation experiments were reversible, and generally nonlethal to cells, it
became an attractive system for further study and subsequently laid the groundwork
for modern continuum models to build upon.
Observing electroporation at the nanoscale is still not possible experimentally, but
a plethora of analytical models (DeBruin and Krassowska 1999; Gowrishankar and
Weaver 2003) and indirect experimental observations (Gabriel and Teissie 1997)
have been proposed in an attempt to explain the molecular mechanisms of electro-
poration using continuum electrodynamic equations. The simplest continuum
models interpret biomembranes as smooth elastic surfaces, where potentials at the
surface can be extracted using finite element models and Poisson’s equation. Electric
double-layer models that incorporate adsorbed interfacial electrolytes (built up at the
water-lipid interface) include the Gouy-Chapman-Stern model; however, individual
water molecules and phospholipid headgroups in these models are coarsely defined
and do not incorporate individual effects of water hydration or steric repulsion
between individual lipids.
Analytical electroporation models can be subdivided into two broad categories
that include (Tokman et al. 2013) “surface potential models,” or models which
calculate static potential profiles for lipid bilayers and combine them with stability
conditions (e.g., setting the surface tension equal to zero), and (Ziegler and Vernier
2008) “stochastic pore models,” which claim that there exists a complex distribution
of transient pores in membranes upon the application of external electric fields, and
that these distributions depend on macroscopic electrical properties such as resis-
tance and capacitance. Chizmadzhev and colleagues pioneered models of the first
type (Abidor et al. 1979; Chizmadzhev and Abidor 1980) using circuit analogs to
analyze the electrical permeation of lipid bilayers (e.g., that the pore energy is
proportional to the edge energy minus the surface energy minus some function of
membrane capacitance, resistance, and other electrical properties). Conversely,
Sugar and Neumann developed models of the second type (Sugar 1981) using
bilayer transition probabilities. Modern models use combinations of these such as
the asymptotic model of electroporation (Neu and Krassowska 1999) that incorpo-
rates both the electrical properties of the membrane and probabilistic estimates for
the number of open pores (N) per unit time (Eq. 1). Here, α and β are normalization
constants and Neq is the equilibrium number of pores. By taking the inverse
118 Z.A. Levine
relationship of Eq. 1, one can deduce the characteristic time that one might expect a
single electropore to form in a viscoelastic membrane. This yields a pore creation
time per unit pore, or dt/dN, that depends on exp.(V2), or on the inverse of the
voltage squared. This is not unexpected, as it resembles a Boltzmann factor with a
Hamiltonian containing a capacitive energy storage term, or U = ½ CV2, where C is
the magnitude of capacitance. Monte Carlo models can also be used to study lipid
bilayers, but because reversible electroporation is a transient, nonequilibrium event,
molecular dynamics (MD) simulations are much better suited to study these
phenomena.

dN 2 N
¼ αeβV 1 (1)
dt N eq
Recent observations have also indicated that short, nanosecond electric pulses
induce different types of cellular responses compared to microsecond pulses (Vernier
et al. 2003). These studies found that ultrashort nanosecond MV/m electric fields can
trigger intracellular calcium bursts and the externalization of anionic lipids from the
plasma membrane’s inner leaflet to the outer leaflet. This suggests that the electric
field duration is an important factor in determining electropore expansion and
function, in addition to highlighting how pulse frequency affects physical properties
of electropores and subsequent uptake of ions, dyes, and other cytosolic molecules.
Molecular Dynamics Simulations
The first molecular dynamics (MD) simulations of a lipid bilayer were performed
in 1982 by Herman J.C. Berendsen using two monolayers of 16 decane molecules
held together by a simple dihedral potential (Van der Ploeg and Berendsen 1982),
where he achieved 80 picoseconds of simulation time for every 10 h of computa-
tion time. From his early simulations, he was able to extract carbon-deuterium
order parameters relative to the bilayer normal using a second-order Legendre
polynomial as a geometric measurement of hydrocarbon orientation and rigidity.
Following this, Seelig et al. (1974) confirmed through simulations that lipid tail
rigidity decreased as the size of the lipid tails grew, a result which was consistent
with Berendsen’s work and with experimental observations at the time. These
studies showed, for the first time, that it was possible to use computer simulations
as a research tool to explain complex biological processes using simple, classical
physical models as a basis. However, because only small systems and short
timescales were available for computation at the time, only a limited number of
measurements were possible.
As computation became cheaper and more affordable, the timescales and system
sizes that could be easily simulated grew as well, enabling larger and more complex
studies to take place. Exploratory molecular dynamics (MD) simulations of electro-
poration (Tieleman 2004; Tarek 2005) showed that when large, MV/m electric fields
were applied in simulations of phospholipid membranes, a water column would form
across the bilayer interior, followed by the construction of a bridge of hydrophilic

lipid headgroups (and their hydrating water molecules). It has also been shown that
the pore creation time decreases as the pore-initiating electric field increases (Ziegler
and Vernier 2008); however, the detailed time course of electropore construction and
annihilation in MD simulations has only recently been studied. While several
pioneering studies have described key steps in the electroporation process, only a
handful of researchers have used simulations to systematically quantify and compare
objective stages in the life cycle of lipid electropores, thereby delineating a number
of field-dependent and field-independent steps that must occur in order for electro-
poration to commence.
In an early study by Böckmann (2008), the authors found a considerable increase
in the bulk water dipole moment surrounding a POPC membrane as a function of the
applied electric field. By visual inspection, they also measured the timescales of
membrane electroporation and extracted rates of permeabilization across 48 indepen-
dent trials. In each of the trials, all containing POPC lipids, they observed similar
intrusion of water molecules into the membrane interior, followed by a
rearrangement of lipids (Fig. 3). The extracted electroporation constants were then
logarithmically fit to simulation times, and extrapolated to experimental values,
which agreed with one another. This represents the enormous power of atomistic
simulations to characterize small membrane patches and extrapolate the results to
larger macroscale experiments. Finally, the authors constructed an electric field
normalized two-state system (corresponding to the intact bilayer and the
electroporated bilayer), where they show the same exponential distribution in pore
creation times across all 48 simulations. They also note that ~8 lipids border the
interior of the hydrophilic pore and that there are about 57 water molecules present in
the pore interior, with an effective pore area of 0.7 nm2.
Ziegler and Vernier (2008) utilized molecular dynamics (MD) simulations to
identify a minimum porating electric field for multiple homogeneous membranes,
where there was a 33% chance to form electropores over 25 ns (i.e., in one trial out of
three). They found that, in agreement with the asymptotic model of electroporation,
the pore formation time depended exponentially on the inverse square of the applied
voltage (Fig. 4). Minimum porating electric fields for homogeneous DLPC, DPPC,
POPC, and DOPC membranes were found to be on the order of 260 MV/m, 280 MV/m,
320 MV/m, and 380 MV/m, respectively, which increased as a function of lipid tail
length or bilayer thickness. No preference was observed for the nucleation site of
discrete electropores, but characteristic water pedestals intruding into the lipid
interface were observed that preceded the formation of hydrophobic pores. Further
analysis by Ziegler concluded that water was the primary contributor to electropore
formation in lipid membranes, as was hypothesized by Tieleman (2004). Large
electric field gradients present at the lipid/water interface, stemming from the
alignment of lipid headgroup dipoles during the application of an electric field, are
thought to be the initiator of these water pedestals, determining the rate of water
entry and subsequent pedestal growth. And, while externally applied electric fields
enhance the electric field gradients found at lipid interfaces, changes in the average
lipid headgroup tilt are small and do not significantly diverge on opposing sides of
120 Z.A. Levine
Fig. 3 Molecular dynamics simulation of a 512 POPC membrane shown after a permeabilizing
electric field of 500 MV/m is applied. Times shown are (a) 900 ps following exposure and (b) 50 ns
after exposure, where a smaller pore-stabilizing field was substituted (Reprinted from Bockmann
et al. (2008), Copyright (2008), with permission from Elsevier)
the membrane. Therefore, electropores can form from either the cathode or anode
end of a bilayer, with little affinity to one side or the other.
Electropore Lifetime
Capitalizing on previous work, Levine and Vernier (2010) proposed a number of

codified stages of electroporation that were consistent across multiple simulations,
allowing for the construction of an objective metric known as electropore “lifetime”
that could be used to characterize and compare individual electropores. After
applying 2–6 V across POPC and DOPC membranes (corresponding to
300–600 MV/m electric fields), the authors noted that certain processes always
preceded the formation of individual electropores. Electropore life cycles were
Fig. 4 Average time to the 25

formation of a hydrophilic DLPC
pore based on three DPPC
independent simulations is POPC
plotted against the applied 20 DOPC
external field. A hydrophilic
Time to Hydrophilic Pore (ns)

pore is a quasi-stable structure
defined as a continuous water
column surrounded by 15
solvated lipid headgroups.
Note that 1 mV/nm is
equivalent to 1 MV/m
(Adapted with permission
10
from Ziegler and Vernier
(2008). Copyright (2008)
American Chemical Society)
5
0
200 300 400 500 600
Field (mV/nm)
broadly divided into a number of pore creation steps and a number of pore annihi-
lation steps. Pore creation was comprised of three stages: initiation, construction, and
maturation (later referred to as expansion). Pore annihilation begins when the
external electric field is removed from a mature pore structure and proceeds through
destabilization, degradation, deconstruction, and dissolution (Fig. 5). The destabili-
zation and degradation steps were later renamed to settling and stabilization, while
the deconstruction and dissolution names remained the same (Levine and Vernier
2012).
Pore creation. Pore initiation begins with the application of an external electric
field and ends when the two groups of water molecules, initially separated by the
bilayer, merged to become a single group. Pore construction begins with the
formation of the membrane-spanning water column [hydrophobic pore (Abidor
et al. 1979)] that marks the merger of the water groups and ends when the phospho-
rus groups that are initially found on the two leaflets of the bilayer follow the water
into the membrane interior and merge into a single phosphorus group. A phosphorus
group is defined as a set of atoms, each separated by a maximum distance of 1.2 nm.
Because water and the charged phospholipid headgroups now bridge the membrane
interior, this structure is comparable to what has been called in several contexts a
hydrophilic pore (Abidor et al. 1979). Continued application of the porating electric
field resulted in an evolution or maturation/expansion of the hydrophilic pore. In this
study, a mature pore was defined as a hydrophilic pore where at least ten phosphorus
atoms from the initial anodic leaflet were found within 1.2 nm of phosphorus atoms
from the cathodic leaflet.
122 Z.A. Levine
Fig. 5 Phospholipid electropore life cycle. Structurally distinct steps in pore creation and annihi-
lation as observed in molecular dynamics simulations
Pore annihilation. Pore destabilization/settling, the first stage of pore annihila-

tion, is defined as the quasi-stable period after the field is removed during which the
number of anode to cathode phosphorus connections fluctuates around the mature
pore criterion (ten connections). Pore degradation/stabilization begins when the
number of anode-to-cathode phosphorus connections drops below ten and ends
when there is only one anode to cathode phosphorus connection. The pore diameter
decreases during this time to a minimum, about 0.4–0.6 nm. Pore deconstruction,
another quasi-stable period, terminates when the single phosphorus group of the
porated bilayer splits into two groups, which remain separate for the remainder of the
simulation. At the end of pore deconstruction, only the water column remains. The
disassembly of the water column is thereby referred to as pore dissolution.
Similar to previous models (Weaver and Chizmadzhev 1996; Ziegler and Vernier
2008), Levine and Vernier found that the pore creation times for POPC bilayers
decreased with increasing electric fields. This was almost entirely dependent on the
time required for pore initiation, the first stage of pore creation. Pore construction
and maturation/expansion times were only slightly dependent on the electric field
strength. Pore annihilation times, however, were significantly longer than pore
creation times, by an order of magnitude. In pore creation, once the initial
membrane-spanning water column had formed, it took less than 1 ns for the
headgroups to follow the water into the bilayer interior. When the electric field
was removed, however, tens of nanoseconds elapsed before the headgroups migrated
back to the bilayer leaflets. During pore destabilization, the period immediately
following removal of the electric field, the number of water molecules in the pore
(within 0.5 nm of the bilayer midplane) decreased substantially in about a nanosec-
ond, and the pore diameters decreased to about 0.6 nm. In a small percentage of
cases, pore annihilation continued to the next stage without delay, but more often the
pores remained intact, with the number of inter-leaflet phosphorus connections
fluctuating around ten, for a time that ranged from less than one to over 20 ns.
At the end of the destabilization stage, the number of anode-to-cathode phospho-
rus connections permanently declined from ten to one – the reverse evolution of the
developed pore to a minimum, single phosphorus group structure. The time required
for this decline, the pore degradation/stabilization time, was typically several nano-
seconds, significantly longer than pore maturation/expansion, the corresponding
pore creation stage. Destabilization/settling and degradation/stabilization times
exhibited large standard deviations, indicating the stochastic nature of these pro-
cesses. At the highest porating field, however, there was a significant reduction in the
destabilization time, suggesting that pore destabilization/settling has a field-
dependent component. Pore deconstruction (separation into two phosphorus groups)
and dissolution (expulsion of water from the membrane interior) appeared indepen-
dent of field strength. Deconstruction times were tens of nanoseconds with large
standard deviations, while the minimal hydrophilic pores were quasi-stable. Pore
dissolution was rapid, though, since the hydrophobic pores are known to be highly
unstable.
Levine and Vernier found no major differences in electropore lifetime between
homogeneous POPC and DOPC bilayers, though this is perhaps not surprising since
each system shares similar minimum porating electric fields (Ziegler and Vernier
2008). DOPC pore initiation was also strongly field-dependent, similar to POPC.
DOPC pore construction and maturation/expansion times, similar to POPC,
exhibited only a small dependence on electric field strength. Each of the DOPC
life cycles was comparable to the corresponding POPC times. However, pore
creation times in all of systems remained exponentially proportional to the inverse
square magnitude of the bilayer internal electric field (POPC results shown in Fig. 6
with two popular water models, SPC and SPC/E). Membrane internal electric fields
(and therefore the field gradients present at each of their interfaces) in DOPC and
POPC systems were of similar magnitude.
Electropore lifetimes, while useful, can also have significant deficiencies. First,
definitions based on arbitrary expansion cutoffs (e.g., as defined by an integer
number of phosphorus connections) may not capture more intrinsic interactions
that do not depend on, say, phosphorus. These definitions are not based on physical
124 Z.A. Levine
Fig. 6 Electropore creation times across POPC membranes as a function of the membrane internal
electric field. Note that Einternal*Lmembrane = Ebox*Lbox. Results are shown for two popular water
models – SPC and SPC/E
properties of pores in real systems, because the discrete structures are not well
understood on the experimental cellular level. It would be advantageous to define
a “mature” pore corresponding to quasi-stable structures that finish expanding in
both experiments and simulations, but at present no such structures have been
observed in experiments or simulations (Teissie et al. 2005).
Second, atomistic simulation times are (compared to experiments) very short and
are less than a microsecond. Although this molecular dynamics regime is well suited
for comparisons to data from experiments using nanosecond pulsed electric fields, it
is possible that longer rise times, lower electric fields, and longer pulses (microsec-
onds to milliseconds) associated with conventional electroporation technology result
in different energy landscapes for pore formation and porated membrane structures
that are significantly different from those observed in simulations.
Finally, important stages may be missing from these definitions. It is possible that
pore initiation actually includes two steps, similar to the first few steps in pore
annihilation. In the first of these two steps, the true initiating step, thermal jostling of
water dipoles and headgroups at the interface, results in the water pedestals
described previously (Ziegler and Vernier 2008). A hypothetical structure, which
has not yet been identified, may develop in a small percentage of these bumps, either
as the bump is formed or as a consequence of the continued interaction of the bump
with the electric field. This structure would likely facilitate a low energy path to the
formation of a membrane-spanning water column, which would be a clear signature
of commitment to pore formation. If there were additional missing stages, they
would need to connect the conversion of a pre-pore bump to a structure where
water is launched into the membrane interior.
Simulations can also model electroporation by maintaining charge imbalances
across the membrane, where the formation of pores can mediate the flow of ions
across the membrane, thereby reducing the transmembrane potential difference and
causing the pore to collapse (Tarek 2005). Other simulations have shown that
electroporation can result in permanent changes to the structure of membranes.
Fernandez et al. (2012) have also presented a formal mechanism for applying very
strong permeabilizing fields, followed by smaller stabilizing electric fields to main-
tain electropores in simulation for hundreds of nanoseconds, similar to the studies of
Böckmann et al. (2008). In larger membrane systems, Tieleman (2004) found that
lipid bilayers significantly deform in response to large electric fields. Others have
shown that electroporation in lipid bilayers precedes a wider phase transition of
lyotropic liquid crystals comprised of membrane and water, stabilized by the electric
field. Such studies state that the water hydration of lipids determines the convergent
geometry of the liquid-crystal phase (for instance, lamellar or inverted columnar),
giving greater insight into the idea that electric fields mediate phase transitions
between amphiphilic solutions and fusing cellular membranes.
Taking a step further, Tokman et al. (2013) compares the electropore lifetimes
between water/lipid systems (e.g., lipid membranes) and water/vacuum systems.
When large electric fields are applied to water/vacuum interfaces, a process known
as electroevaporation occurs where liberated water molecules form interfacial,
energy-minimized pedestals similar to those observed in membrane electropores
immediately before electroporation occurs. Therefore, the authors explored whether
lipids themselves are necessary for water permeabilization or rather a passive barrier
to entry that inhibits water entry into membranes. Interestingly, for similar external
electric fields, water electroevaporation occurs significantly faster than membrane
electroporation and yields similar water pedestal profiles (Fig. 7). This further
supports the idea that lipids inhibit electric field-mediated water entry in membranes
and do not cause the electric field gradients that ultimately displace interfacial water
into the membrane interior, as some have suggested (Bockmann et al. 2008).
Tokman and colleagues found that the dynamic progression of water columns in
both water-lipid-water (WLW) and water-vacuum-water (WVW) systems was strik-
ingly similar to one another. The dynamics of pore (water column) formation and the
similarity between WLW and WVW simulations were invariant across simulations
over a wide range of parameters (external electric field magnitude, vacuum gap
width, etc.). WLW and WVW simulations differ mainly in the timescales over which
the formation of the water bridge occurs, i.e., the pore initiation time (Levine and
Vernier 2010). To compare initiation times between WLW and WVW systems, the
authors selected a gap size for WVW systems that resulted in similar magnitudes of
the external and internal (in the lipid bilayer interior and the vacuum gap) electric
fields. This ensured that the interfacial water molecules were exposed to similar
electric fields in the WLW and WVW systems, permitting a fair comparison of pore
initiation times. After normalizing equivalent external and internal electric fields,
WVW systems formed water channels significantly faster than WLW systems
(Fig. 8).
With regard to the energetics of water during electroporation, Tokman demon-
strated that the pore formation process described above is driven by a collective
tendency of interfacial water dipoles to minimize their electrostatic interactions
126 Z.A. Levine
Fig. 7 Snapshots of the time evolution of water-lipid-water (WLW) and water-vacuum-water

(WVW) configurations under an external electric field of 500 MV/m. (a) WLW configuration at
times 5.8, 6.7, and 7.3 ns from the start of the simulation with both water molecules (oxygen, red;
hydrogen, gray) and lipid molecules (phosphorus, yellow; nitrogen, blue; lipid tail groups, silver)
displayed. (b) Same WLW data as in (a) but with only water molecules shown. (c) WVW
configuration at times 1.157, 1.160, and 1.194 ns (Figure adapted from Tokman et al. (2013))
Fig. 8 Average pore

initiation times for water-
lipid-water (WLW) and water-
vacuum-water (WVW)
systems calculated with three
sets of simulations for each
configuration (Figure adapted
from Tokman et al. (2013))
while adopting an orientation that minimizes the energy of the water dipole in the
external electric field. This is reflected in a steady drop in the per-molecule energies
of water in the nascent pore as the protrusion develops. This behavior is present in
both WVW and WLW simulations. To do this, Tokman defined an interface region
by identifying the average density of water molecules in each (1 angstrom thick)
slice of the simulation box, selecting the highest value. Slices with water density not
exceeding 50% of the bulk value were considered interfacial, while those with water
densities equal to or greater than 50% of the bulk value were labeled as bulk water.
Water molecules in the pore were selected visually and traced back into the bulk at
earlier timesteps so that their subsequent trajectories could be analyzed. Once the
water molecules comprising the protrusion were identified, Tokman computed the
average potential energy per water molecule and its constituent terms that consisted
of (i) the electrostatic interaction energy between water molecules in the protrusion
and all other water molecules, (ii) the van der Waals interaction between protrusion
and bulk water, and (iii) the interaction energy between the protrusion water dipoles
and the external electric field. For WLW simulations, Tokman also computed the
electrostatic and Lennard-Jones energies between the protrusion water molecules
and the lipids. At each time frame, energies are expressed per unit water in the
protrusion box, which varied over time. In addition to energies, Tokman calculated
the height (H) of water protrusions as the distance between the interface region
boundary and the furthest protrusion atom.
These measurements revealed a notable drop in the per-molecule energies of
waters in the protrusion as the height of the protrusion grows for both WLW and
WVW. Correlations between a drop in potential energy and the protrusion growth for
each simulation were quantified with Pearson correlation coefficients that revealed a
mean correlation of 0.65 for WVW systems and 0.5 for WLW systems. In both
128 Z.A. Levine
systems, protrusion growth is anticorrelated with potential energy or can be thought

of as correlated with a drop in potential energy. On average, Tokman found that
protruding water molecules in WLW simulations exhibited a 2.8 kJ/mol potential
energy drop during pedestal formation, while WVW protruding waters exhibited a
1.4 kJ/mol potential energy drop. In other words, energy minimization is quicker in
WVW systems because the energies needed to electroevaporate are smaller than the
energies needed to electropermeabilize. Tokman decomposes the potential energies
of water into the three components discussed earlier, (i), (ii), and (iii), to illustrate
which components are most sensitive to energy minimization. Drops in the potential
energies of water during electroporation appear to be dominated by minimization of
Coulomb forces. Contributions from dipole-field interactions only decrease slightly,
while changes in Lennard-Jones potentials remain largely unchanged. Interestingly,
the decrease in the dipole-electric field interaction corresponds with the alignment of
the protrusion molecules with the external field.
Finally, Tokman attempts to reconcile the minimum porating electric fields in
membranes described earlier (Ziegler and Vernier 2008) with simple physical
models of idealized dipoles in external electric fields. The authors construct two
model systems containing seven dipoles in either a (Tokman et al. 2013) random
bulk aggregate geometry or (Ziegler and Vernier 2008) an aligned geometry where
each dipole is stacked on top of one another and is oriented in the direction of an
electric field. When both systems are exposed to an external electric field, Tokman
tabulated contributions from Coulomb interactions, van der Waals interactions,
dipole-dipole coupling, and dipole-field coupling and ensured that each dipole
followed a Langevin function that orients each dipole in the direction of the field
based on the ratio of the electric field energy (μE) to that of the ambient thermal
energy (kT). Tokman found that as the external electric field increased, the energy
required to maintain the stacked configuration decreased, while the energy required
to maintain the bulk aggregate increased. At some threshold electric field, the
energies required to maintain these states were the same, eventually allowing one
system the opportunity to transition into the other system. This supports the idea that
at some critical electric field, interfacial water pedestals have sufficient energy to
transition from an aggregate morphology to a membrane-spanning water channel,
given enough time to sample each of the available water states.
Water translocation, even in the absence of external electric fields, can occur and
be measured in simulations, though inward water flux matches the outward water
flux, resulting in no net water transport. These events can capture how leaky
membranes are (Fig. 9) and can be used to better understand osmotic effects typically
encountered in experiments with live cells. The application of a porating electric
field has little effect on either the frequency or the direction of these random crossing
events. The primary determinant of the frequency (expressed experimentally as the
permeability coefficient) is the area per lipid. Systems with a smaller area per lipid
have smaller permeability coefficients. Consideration of this phenomenon can
enhance our understanding of the mechanism of water bridge construction, from
pre-pore pedestal to extension of the water chain across the bilayer. In Fig. 9, thermal
rotation of the yellow water molecule brings its hydrogens into close and repulsive
Fig. 9 Random permeation event in a phospholipid bilayer. Single H2O molecule (yellow) crosses
the phospholipid bilayer interface without an external electric field present, presumably due to
thermal jostling. (a) 0 ps, (b) 50 ps, (c) 105 ps, (d) 110 ps
proximity to the hydrogen on the soon-to-be crossing water molecule, which is

already poised on the edge of the low-permittivity hydrocarbon region of the lipid
tails that make up the membrane interior. Note that there are acyl oxygens surround-
ing this event, an important component of three-dimensional electrophysiological
contours of the site. Similar but less energetic interactions lead to the assembly of
intruding water pedestals, with their associated acyl oxygen and other lipid atom
neighbors, and to the molecule-by-molecule (sometimes plus one, minus two, etc.)
130 Z.A. Levine
growth of bilayer-spanning water columns. On a macroscale, this is statistical

mechanics. On the atomic scale in a phospholipid bilayer, the sum of these events
is a new structure, the chain of waters through the membrane interior that results in
the formation of a hydrophilic pore, a conduction pathway through a normally
impermeant barrier.
Conclusions
Each of these studies represents a small yet important piece of what constitutes
membrane electropermeabilization. Simulations of lipid bilayers suggest that
electropermeabilization is actually comprised of multiple electroporative events,
where a large number of discrete, water-filled pores that are capable of facilitating
the crossing of extra- and intracellular molecules through the cellular barrier work in
tandem. These discrete structures have similar morphologies, free energy barriers,
and constituent components that mediate how easily they form and determine their
resulting life cycles.
Modern simulations, while often idealized or sterilized, offer important insights
that can help guide experiments into new and previously unexplored territories. For
instance, by characterizing the many field-dependent and field-independent stages of
electroporation in simulations, experiments can better test how various perturbations
(such as changes in pH, electrolyte concentrations, addition of anionic or zwitter-
ionic lipids) affect pore creation and annihilation at various external voltages and
how changes to these properties affect membrane conductance. Simulations also
elucidate how multiple factors such as ion imbalances or fixed charged interfaces
(e.g., electrodes) contribute to membrane electropermeabilization on both the nano-
and macrolevels. In both of these situations, water appears to be the primary factor
that determines how readily membrane electropores form or, at water/vacuum
interfaces, how quickly electroevaporation occurs. Potential energy drops correlated
with the construction of pre-pore pedestals appear to precede electropore formation,
and simple toy models can be constructed that help explain how pore pedestals,
essentially aggregates of bulk water, transition at critical electric fields to stacked
water channels capable of spanning the length of a phospholipid membranes.
Simulations, then, offer a ground-up approach to incorporate what is believed to
be the most important contributing factors in biology and provide valuable first-order
estimates on the inner workings of nature.
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Effects of Heterogeneous Membranes and
Electrolytes on Electropore Formation 8
Zachary A. Levine
Abstract
Atomistic simulations such as molecular dynamics (MD) simulations have
revealed much about the fundamental biophysics of electroporation in homoge-
neous phospholipid bilayers; however, the structures and behaviors of live cellu-
lar membranes differ considerably from idealized zwitterionic lipid bilayers.
Biological membranes contain both neutral and charged lipid types and interact
with a large number of bulk and interfacial electrolytes that form complexes with
individual lipids, thereby modulating their local surface tensions and creating
domains and rafts regions. Even without considering the effects of transmem-
brane proteins, some of which are voltage-gated and are likely to perturb
electropore formation and annihilation, the differences between electroporation
in heterogeneous membranes, especially those containing salts, and homoge-
neous membranes described in the last section, are significant. This section will
focus on how local perturbations to membranes such as the inclusion of anionic
lipids, divalent cations such as calcium, oxidized lipids, and other additions can
significantly change the behaviors of membrane electropermeabilization. Simi-
larly, additional metrics such as calcium binding isotherms will be presented to
assess the validity of these simulations and how well they relate to experiments.
Finally, some additional studies will be discussed to deduce whether heteroge-
neous systems (more representative of live cellular membranes) form electropores
with an exponential inverse dependence on applied voltage and electric field, as is
observed for homogeneous systems.
Z.A. Levine (*)

Department of Physics, University of California Santa Barbara, Santa Barbara, CA, USA
Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara,
CA, USA
Materials Research Laboratory, University of California Santa Barbara, Santa Barbara, CA, USA
e-mail: zlevine@chem.ucsb.edu

DOI 10.1007/978-3-319-32886-7_87
134 Z.A. Levine
Keywords
Electroporation • Electropermeabilization • Heterogeneous Membranes • Com-
puter Simulations • Electropore Life Cycle • Anionic Lipids • Electrolytes
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Salt and Lipid Heterogeneity Affect Membrane Electrostatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Pore Lifetime in Heterogeneous Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
Introduction
Live cells exposed to short-, micro- to nano-second external electric fields develop a
time-dependent potential across the cellular membrane, which strongly depends on
the magnitude and rise time of the applied potential, and the size and diameter of the
cell, in addition to the unique dielectric solvent surrounding the cell. When cells are
exposed to micro- to milli-second, low-frequency (Hz to kHz) electric fields, the
energy is primarily absorbed in the external cellular membrane, which exhibits an
electrical charging constant (in standard physiological media) of tens to hundreds of
nanoseconds, depending on the cell shape and type. However, the application of
microsecond kilovolt-per-meter electric pulses on cells yields conductive pores in
the plasma membrane that allows for the migration of charged small and large
molecules across the mechanical and dielectric barrier presented by the lipid bilayer.
Electroporation technology, which operates in the low-field, long-pulse regime, is
widely used to facilitate transport of nucleic acids, pharmaceutical compounds, and
other materials into the cytoplasm of living cells (Neumann et al. 1982).
Quickly rising electric pulses (i.e., pulse periods that are shorter than the charging
time of the external membrane) and high frequency electric fields and voltages are
better able to perturb all parts of the cell. For instance, ns electric pulses and GHz
frequencies can bypass the plasma membrane entirely and reach into the membrane-
bound structures and biomolecular assemblies found deep in the cytosol and nucleus
(Schoenbach et al. 2001). Intracellular responses to these ultra short (ns), high-
amplitude (MV/m) electric pulses (which, while delivering megawatts of power,
do not produce significant thermal effects due to the small energy densities involved,
e.g. joules per gram) include calcium bursts and, in some situations, cell apoptosis
(Vernier et al. 2003). Thus, the potential for utilizing electroporation to kill cancer
cells in vivo, or at least to stunt tumor growth, depends heavily on the effects of large
calcium or other salt concentrations on electropore formation and on various apo-
ptosis- or phagocytosis-triggering molecules, such as the anionic lipid phosphati-
dylserine (PS) (Vernier et al. 2004).
While an attractive target of short, pulsed electroporation has historically been the
cell interior, PS externalization, or the translocation of PS from the inner leaflet of the
lipid bilayer to the exterior, representing the outside of a cell, can be triggered by ns,
8 Effects of Heterogeneous Membranes and Electrolytes on Electropore. . . 135
MV/m electric fields (Vernier et al. 2004). PS is typically confined to the interior of
cells and is regulated by a number of proteins and energy barriers (representing over
100 kJ/mol) that impede native externalization. These barriers include phospholipid
head groups, since externalized PS can result in the recognition, binding, and
signaling of platelet aggregation and catalysis of clot formation during blood
coagulation, thereby setting the stage for phagocytosis and mediating intramembrane
signal transduction in lymphocytes. Because PS externalization is a well-defined
event that is easily observed in simulations and is associated with nano-electropulse
treatment, it provides a good starting place for efforts to characterize the interactions
of pulsed electric fields with biological systems.
The need to establish objective criteria for the analysis of both the formation and
dissipation of membrane electropores in applied electric voltages and fields requires
careful measurement and comparisons of the fundamental molecular mechanisms
that are explicitly dependent on, and independent of, external electric field strengths.
Additionally, such criteria and metrics need to be evaluated across multiple model
systems containing a large variety of lipids, solvents, and proteins and under
multiple thermodynamic ensembles. This includes incorporating PS into existing
frameworks for electropore lifecycles and observing how electroporation affects PS
externalization. Many studies have already shown a large association between
electroporation and the interactions between interfacial water molecules and their
hydrated lipid headgroups (Tieleman 2004; Tarek 2005; Ziegler and Vernier 2008);
therefore, one might expect that small differences in lipid type (and other simulation
parameters) may result in large downstream effects on electropore creation and
annihilation dynamics, in addition to changes in cell viability.
Salt and Lipid Heterogeneity Affect Membrane Electrostatics
While experiments have long indicated that calcium and other cations affect the
conformation of polar lipid headgroups, simulations have also suggested that ions
such as sodium and calcium can induce changes deeper in the membrane interior,
near the lipid carbonyl region (Bockmann and Grubmuller 2004). More advanced
experiments utilizing IR spectroscopy have since confirmed these observations,
where the presence of heterogeneous lipid-ion complexes presents a huge pertur-
bation to idealized electropermeabilization processes in homogeneous (ion-free)
membranes. These complexes reduce the lateral mobility of lipids within mem-
branes and increase their surface tensions to the extent that electroporation time-
scales are affected. Stoichiometrically, monovalent cations such as sodium
typically bind to one (often anionic) phospholipid headgroup, while divalent
cations like calcium typically bind to two phospholipid headgroups. Alternatively,
calcium can bind up to four lipid carbonyl oxygen atoms when it more deeply
penetrates the phospholipid bilayer (Bockmann and Grubmuller 2004). Upon
binding to lipid headgroups, ions can affect both the net tilt and dipole moment
of headgroups (Sachs et al. 2004a), to the extent that the magnitude and direction
of these shifts depends on the ion’s charge and size. Additionally, the author
136 Z.A. Levine
observed ordering of water molecules well past the bilayer interface, though such
changes are difficult to confirm experimentally.
When ion concentrations are equal on both sides of a membrane, no external
transmembrane potential exists. However, when ion concentrations differ on each
side of a membrane (as can often be the case across cellular membranes), external
voltages can result in electrified membrane interfaces (Sachs et al. 2004b;
Gurtovenko and Vattulainen 2005), sometimes resulting in permeabilization of
the partitioning membrane until the ion imbalance is neutralized (Gurtovenko and
Vattulainen 2005). Molecular dynamics (MD) simulations can model these phe-
nomena by implementing double bilayer simulations, explicitly representing an
exterior and interior aqueous region containing different salt concentrations. Sachs
(Sachs et al. 2004b) measured a potential difference of 170 mV across a
dimyristoylphosphatidylcholine (DMPC) membrane with an ion imbalance of
only a couple of ions, and while this is likely below the electroporative threshold
required to permeabilize DMPC, local anisotropies in ion concentrations near the
membrane surface could produce external electric fields that far surpass that
threshold, though the timescales of these anisotropies are likely small. However,
it is important to note that while external ionic potentials may permeabilize
idealized, homogeneous membranes, cellular walls in living tissue often require
transmembrane potentials to be present for normal physiological processes to
occur, e.g., ATP synthesis, and may be resistant to electropermeabilization due to
additional compensating ions and transmembrane proteins. Simulations of hetero-
geneous membranes have also indicated that even in the absence of ions or
transmembrane proteins, small resting potentials across membranes are observed
due to asymmetries in lipid composition, often on the order of 100 mV
(Gurtovenko and Vattulainen 2007).
Further complicating this picture, studies by Nathan Baker (Lee et al. 2008) have
shown that when membranes are separated by different salts like NaCl and KCl, but
at similar ionic strengths, there still exists a nonzero net potential across the
membrane, on the order of 70 mV. The authors attribute this behavior to asym-
metric binding behaviors between Na and K+, where some ions bind to lipid
carbonyl oxygens, while others do not. This results in a net potential across the
membrane that tends to be localized near asymmetric binding sites. Similar results
were observed for alkali cations and halide anions (Vácha et al. 2009), where
multiple membrane binding sites were identified that uniquely perturbed electron
density profiles, resting electrostatic potentials, and area per lipid values. However,
the authors claim that such effects are largely underestimated in MD simulations due
to the lack of polarizable force fields.
To test how sensitive these interactions are to molecular force fields, Gurtovenko
and Vattulainen (2008) compared ion:lipid interactions between the GROMACS and
CHARMM force-fields and found that in both models the strongest interactions were
between phosphatidylcholine (PC) lipids and sodium ions, which resulted in even-
tual membrane compression. Potassium ions, which are larger than sodium ions,
exhibited relatively weak interactions with PC in the GROMACS force field;
however, it is known that in GROMACS, the size of potassium is over exaggerated.
Phosphatidylethanolamine (PE) lipids were much less affected by monovalent

cations, likely due to their superior ability to form both intra- and interlipid hydrogen
bonds. Thus, salt is expected to affect the inner and outer cell membrane leaflets
differently, where PE concentrations differ, asymmetrically affecting electropermea-
bilizion dynamics. To test this hypothesis, Gurtovenko (Gurtovenko and Lyulina
2014) applied permeabilizing electric fields to mixed PC:PE bilayers, and found that
water-filled electropores predominantly stem from the PC leaflet, and not the PE
leaflet. This resulted in hydrophilic electropores where only PC lipids lined the pore
exterior, not both of the lipids present in the membrane. This also yields asymmetric
lateral pressures in the membrane from one leaflet to the other, decreasing membrane
cohesion. Pore formation times for heterogeneous membranes were typically in
between the timescales expected for each of the corresponding homogeneous
membranes.
Similar studies by Sinn and Dimova (Sinn et al. 2006) found that when the
anionic lipid phosphatidylserine (PS) is inserted into PC large unilamellar vesicles
(LUVs), calcium binds preferentially to PS lipids over PC. However, when a salt
solution is mixed with lipid vesicles, there is initial osmotic shock. To compensate
for this, Sinn extruded their samples with NaCl solutions and titrated with
iso-osmolar CaCl2 solutions. Under physiological conditions, calcium interacted
with cellular membranes in the presence of NaCl, typically at concentrations of
15 mM. Therefore, it remained difficult to interpret the binding behaviors of calcium
to PS since Na+ may complete with Ca2+ for available lipid headgroups. It is thought
that the presence of NaCl actively screens the membrane from Ca2+, resulting in low
binding behaviors. In fact, Sinn found that increasing NaCl concentrations by a
factor of 10 reduced CaCl2 concentrations by a factor of 100. Despite these compli-
cations, the authors found that when 7 mM of CaCl2 was added to dioleoylpho-
sphatidylcholine (DOPC) LUVs (containing 20% DOPS and equilibrated with
10 mM NaCl), vesicle fluctuations were dramatically reduced, indicating a large
increase in membrane surface tension. As a result, the vesicles began to bleb, and
water began to leak out from the interior, eventually preceding membrane rupture
(Fig. 1).
In addition, Sinn and Dimova show well-behaved Langmuir binding isotherms
for calcium bound to heterogeneous PC:PS LUVs using titration of added calcium
(Fig. 2). For small calcium concentrations, nearly all of the added calcium binds to
the lipids. Interestingly, calcium binds to the vesicles far beyond stoichiometric
values, assuming that a single divalent calcium binds to two PS lipids. Binding of
calcium appears to exceed ratios where each PS headgroup is accounted for,
indicating that calcium binds to both PS and PC headgroups alike, though PS is
electrostatically preferred and appears to be saturated by calcium first. Fitting of the
Langmuir isotherm with a 1:2 binding model yields a binding constant of 650 M 1.
Considering the electrostatic enrichment within the double layer, this value is
notably similar to the value for pure DOPC vesicles. Therefore, it appears that the
presence of PS encourages additional calcium ions to bind electrostatically but that
the binding strength to heterogeneous PC:PS bilayers is comparable to homogeneous
PC bilayers.
138 Z.A. Levine
Fig. 1 A sequence of pictures of a giant DOPS:DOPC (γPS = 0.2) vesicle in 10 mM NaCl before
and after injection of iso-osmolar 7 mM CaCl2. Time is relative and set to zero before injection as
shown on each snapshot (min:s). (a) No CaCl2 is present; the vesicle strongly fluctuates. (b) CaCl2
solution has been injected and a significant decrease of fluctuations is observed. (c) The membrane
appears thicker and the vesicle interior fades indicating leakage; the vesicle volume has also
decreased. (d) The vesicle collapses and vanishes (Adapted from Sinn et al. 2006)
Follow-up simulations by Vernier and Ziegler (Vernier et al. 2009b) were

intended to better understand these results using MD simulations, since calcium
binding to anionic lipids in simulations must agree with experimental observations if
further studies on heterogeneous membrane electroporation are to be trusted. The
authors found that simulations of mixed, zwitterionic-anionic, asymmetrically
arranged DOPC:DOPS bilayers with monovalent and divalent cations are consistent
with experimental observations. Simulations found that in DOPC:DOPS mixtures,
the area-per-lipid decreased when either an anionic PS or a calcium ion was inserted
into the system, while membrane thickness increased after the addition of each.
Calcium also appeared to dehydrate the membrane interface, and a large affinity
between phospholipids and calcium was observed relative to sodium, especially at
PS carboxyl sites compared to PS and PC phosphate and carbonyl sites. Calcium
also induced the rotation of PC headgroups outward toward the solvent, representing
new features observed in simulations.
The authors also observe that while heterogeneous membranes exhibit conver-
gent area per lipid values at 50 ns, binding between calcium and PS/PC lipids
continues up to 100 ns, indicating that a reliance on experimental observations of
vesicle area as a marker of equilibrium can be misleading. For a 128-lipid DOPC:
DOPS system, calcium saturation of the bilayer occurs between 10 and 100 calcium
Fig. 2 Binding isotherms of

the titration of DOPS/DOPC
(γPS = 0.2) and DOPC LUVs
solution in 10 mM NaCl with
7 mM CaCl2. The line
indicated with “slope 1”
represents the case of 100%
binding. The concentration of
bound ion [CaB2+] was
determined using a calibration
curve before the
measurement. The solid curve
represents a fit with a 1:2
binding model (Adapted from
Sinn et al. 2006)
ions. The PC headgroup dipole angle distribution in PC:PS bilayers is bimodal, an

effect of the interaction between the anionic PS and the zwitterionic PC, and this
distribution can be further modulated by varying the number of calcium ions in the
system. Atomically local binding profiles for anionic versus zwitterionic phospho-
lipids and their combinations, with monovalent and divalent counter-ions, may have
significant consequences for membrane lipid and protein aggregations.
In homogeneous and heterogeneous membranes that were calcium-saturated, the
calcium concentrations in the phosphate or carboxyl region of the bilayer reached a
maximum, but then quickly dropped to a minimum at the interfacial water boundary.
However, calcium concentrations rose again in the bulk medium to their equilibrium
bulk concentrations (Fig. 3). The construction of a Gouy-Chapman double electric
layer can be seen in more detail in Fig. 3, which highlights the differences between
homogeneous PC membranes and heterogeneous PC:PS membranes. At the DOPC-
only interface (Fig. 3a), the phosphate-localized calcium peak, the counteracting
calcium and chloride profiles, and the exclusion of sodium from the headgroup
region are straightforward. At the aqueous boundary of the mixed DOPC:DOPS
leaflet on the other side of the bilayer (Fig. 3b), the calcium and chloride peaks are
distinct, but the calcium profile in the headgroup region traces a landscape compli-
cated by the presence of the serine carboxyl groups, and the calcium is accompanied
by a significantly greater amount of sodium than in the DOPC-only leaflet.
Vernier and Ziegler also observed the formation of 1:2:4 calcium:lipid:water
complexes that formed in mixed bilayers, especially between calcium and PS
carboxyl groups (Fig. 4). Calcium binding to lipid backbone carbonyl oxygens
and headgroup phosphate oxygens remained constant between homogeneous PC
membranes and heterogeneous PC:PS membranes; however, calcium’s affinity for
140 Z.A. Levine
Fig. 3 Number density

a Cl- Water/2
(nm 3) profiles for the
DOPC PO4
interface regions of the 15
DOPC-only (a) and DOPC: Ca+10
DOPS (b) leaflets of a DOPC: DOPS COO-2
DOPS (128:20) asymmetric 10 Cl-10
bilayer. The DOPC-only Ca2+ Na+10
leaflet contains 64 DOPC. The
DOPC:DOPS leaflet contains 5
Number Density (nm–3)

44 DOPC and 20 DOPS. In
addition to the other scaling
constants, the chloride and 0
–4 –3 –2 –1
sodium densities are each
multiplied by 10 (Adapted
from Vernier et al. 2009b) b Cl-
15 PO4-
Ca2+
10
COO- Ca2+
0
1 2 3 4
Distance From Bilayer Center (nm)
PS carboxyl groups was an order of magnitude higher than its affinity to carbonyl
and phosphate oxygens, according to radial distribution functions produced by the
authors. These results paint a picture where PS carboxyls are occupied by calcium
first, followed by binding to glycerol carbonyl oxygens, and to a lesser extent,
phosphate oxygens. Additionally, since PS lipids contain one additional binding
site compared to PC lipids (e.g., at the serine carboxyl group), PS lipids are the most
likely candidates that contribute to the reduction in membrane area per lipid, and
likely participate in raft or domain formation, due to their ability to preferentially
bind to calcium.
Pore Lifetime in Heterogeneous Membranes
Building on previous studies, Levine utilized MD simulations to study the life

cycles of electropores in the presence of anionic lipids and calcium (Levine and
Vernier 2012), and compared their results to existing continuum theories (Weaver
and Chizmadzhev 1996) and experiments (Sinn et al. 2006). The authors utilized
a similar lifecycle measurement to characterize these systems, which was
discussed in an earlier section (Lipid Electropore Lifetime in Molecular Models).
At smaller external electric fields, heterogeneous palmitoyloleoylphosphati-
dylcholine:palmitoyloleoylphosphatidylserine (POPC:POPS) bilayers had pore
Fig. 4 A representative
calcium-DOPS-water
complex from an equilibrated
DOPC: DOPS (108:20)
system. Calcium binds to
DOPS and water with a
stoichiometric ratio of 1:2:4.
Blue lines connect calcium
and atoms within 0.3 nm
(oxygens from water and the
DOPS carboxyl groups).
Violet: Ca; red: O; white: H;
teal: C; blue: N; gold: P.
(Adapted from Vernier et al.
2009b)
creation times that were, on average, slightly longer than homogeneous POPC
bilayers (Fig. 5), primarily because of an increase in the pore initiation time, the
time it takes water to bridge the membrane interior. The area per lipid – which is
strongly correlated with membrane permeability – in POPC:POPS systems was
about 0.60 nm2 compared to 0.66 nm2 for POPC bilayers. At higher applied
electric fields, the differences in pore creation time became minimal, and mixed
bilayers had pore creation times that were not significantly different from those
for homogenous bilayers. Pore initiation times for POPC:POPS were inversely
related to the externally applied electric field, and pore construction times
remained constant over all the electric fields sampled, similar to previously
reported studies on homogeneous POPC bilayers (Levine and Vernier 2010).
Pore expansion times decreased slightly as higher external electric fields were
applied.
POPC bilayer systems containing calcium ions had exhibited, at small external
electric fields, pore creation times which were about twice as long as systems without
calcium, and about one and a half times as long as those in mixed (POPC:POPS)
bilayers (Fig. 5). As with systems containing PS, when the external electric field was
increased, pore creation times of pure POPC systems with calcium and those without
calcium did not appear to be significantly different. The convergent area per lipid for
POPC bilayers with calcium was about 0.56 nm2, a value significantly smaller than
the area per lipid without calcium. Similar to the systems containing PS, the authors
142 Z.A. Levine
Fig. 5 Pore creation times for

three different porating
electric fields (400, 500,
600 MV/m) for bilayers
consisting of 128 POPC
(0PS:0Ca), 128 POPC
saturated with calcium
(0PS:100Ca), 108 POPC and
20 POPS on a single leaflet
without calcium present
(20PS:0Ca), and bilayers
containing both PS and Ca2+
(20PS:100Ca). Systems
containing 20 PS also contain
20 Na+ as counter ions, and
systems containing Ca2+
contain two chloride counter
ions for every calcium ion.
Systems that contain both PS
and Ca2+ have both sodium
and chloride counter ions
present. Ca2+ and POPS in the
bilayer increase the pore
initiation time
observed an inverse relationship between the externally applied electric field and the
pore initiation time and pore expansion time for POPC bilayer systems containing
calcium. Pore construction times remained constant at all the electric field values
reported.
POPC:POPS bilayers containing calcium had pore creation times which were
significantly longer than for pure POPC systems, with or without calcium, and were
also longer than POPC:POPS systems without calcium. This appeared true for all
electric field values the authors applied, even at very high fields where the pore
creation times were not significantly different. The average area per lipid of POPC:
POPS bilayers with calcium was about 0.55 nm2 before a field was applied,
somewhat smaller than the area per lipid of POPC bilayers with calcium present.
Similar to the other systems reported, both pore initiation times and pore expansion
Fig. 6 Pore annihilation

times after pore formation in
the same systems shown in
Fig. 5. Ca2+ and POPS in the
bilayer decrease the average
pore annihilation time
times decreased as the external electric field increased, while pore construction times
were similar for all the values of applied electric field.
With regard to pore annihilation, Levine and Vernier found that POPC:POPS
bilayers had pore annihilation times about three times smaller than for pure POPC
bilayers (Fig. 6). All of the pore annihilation stages (except stabilization) were
shortened relative to pure POPC, and this was true regardless of the values of the
electric field used to create the pore. The average pore radius at the start of the
annihilation step appeared to be about 2.3 nm for POPC:POPS systems, similar to
the average pore radius for pure PC bilayers, 2.2 nm. Pure POPC bilayer pore
annihilation times were heavily dominated by the pore settling and pore deconstruc-
tion phases. POPC:POPS bilayers exhibited very short pore settling times and
significantly reduced pore deconstruction times compared to POPC systems,
144 Z.A. Levine
reducing the overall time required to annihilate POPC:POPS electropores. An initial

decrease in pore radius occurred immediately after the field was removed in both
POPC and POPC:POPS bilayers, from about 2.3 nm to about 0.5 nm over the first
few nanoseconds. This initial reduction of the pore radius does not appear to be
correlated with the pore settling time.
Pure POPC systems containing calcium had dramatically reduced pore annihila-
tion times compared to POPC systems without calcium (Fig. 6). For pores created at
400 MV/m and 500 MV/m, POPC–Ca2+ systems exhibited virtually no pore settling,
while the remaining stages took no more than a few nanoseconds to complete. For
pores created at 600 MV/m, calcium still significantly reduced the pore annihilation
time, but the variation from simulation to simulation was large. As with POPC:POPS
and pure POPC bilayers without calcium, the pore radius was reduced in POPC
systems containing calcium to 0.5 nm after only a few nanoseconds, but with
calcium present the initial pore radius immediately after the external field is removed
is 1.7 nm, about 0.6 nm smaller than the pores in the annihilation simulations without
calcium.
POPC:POPS systems with calcium present had pore annihilation times which were
also significantly shorter than those for pure POPC bilayers and POPC:POPS systems
without calcium. POPC:POPS–Ca2+ annihilation times were comparable to those for
POPC–Ca2+ and POPC:POPS (without calcium) systems. For pores created at the
highest electric fields, POPC:POPS–Ca2+ systems exhibited the shortest pore annihi-
lation times of all the systems reported. Pore settling time for these systems was too
short to measure in about half of the trials; though the longest settling time measured
for POPC:POPS–Ca2+ was 200 ps. Stabilization was the dominant step in pore
annihilation for these systems, and the variance in pore annihilation time appeared
to be much smaller than for pure POPC systems without calcium. As with POPC–Ca2+
systems, the initial pore radius for POPC:POPS–Ca2+ systems after the porating field
was removed was about 1.8 nm. As soon as the pore radius decreased to about 0.5 nm
in the POPC:POPS–Ca2+ systems, the pores dissipated quickly, in stark contrast to the
pure POPC bilayers without calcium, where pores remained open with a radius around
0.5 nm for many tens of nanoseconds.
To assess the validity of the calcium ion models utilized with POPC:POPS
systems, Levine and Vernier constructed a binding isotherm (Fig. 7), which shows
the amount of bound calcium ions against the bulk calcium ion concentration. These
data were fit to a 1:2 Langmuir binding isotherm between calcium and phospholipid
and with binding isotherms taken from experiments with pure and mixed vesicles
(Sinn et al. 2006). For small calcium concentrations, the authors saw linear 1:1
binding, where calcium binded preferentially to PS carboxyl and PC:PS phosphoryl
oxygen atoms. From systems equilibrated for 150 ns (Vernier et al. 2009b), the
authors show a calcium binding coefficient of K = 2.56 M 1.
In summary, Levine and Vernier show that the incorporation of anionic phospho-
lipids into a zwitterionic homogeneous bilayer slightly increases the time required
for pore creation at lower external electric fields and drastically decreases the time
required for pore annihilation for electropores created at all external electric fields.
PS bilayers have a smaller area per lipid compared to PC bilayers, and a decrease in
Fig. 7 Calcium binding

curves show rough
correspondence between
experimental and simulated
results. The data can be fit by a
1:2 Langmuir binding
isotherm, consistent with the
formation of Ca:PS2
complexes
surface tension has been shown to affect pore formation (Tieleman 2004). At higher
electric fields, pore creation times for heterogeneous bilayers were similar to those
for homogeneous bilayers. As previous studies had shown, interfacial water was the
dominant component of pore formation (Tieleman 2004; Tarek 2005; Ziegler and
Vernier 2008), and one can speculate that the formation of bilayer-spanning water
bridges in the strong interfacial electric field gradients that result from the application
of large external electric fields is only weakly influenced by PS or PC interactions.
This is consistent with the stochastic pore hypothesis for electropermeabilization
(Neu and Krassowska 1999) and with previous observations (Levine and Vernier
2010), where pore creation time is exponentially correlated with the inverse magni-
tude of the applied electric field squared.
Similarly, calcium in homogeneous PC bilayers delays pore creation at lower
external electric fields and greatly reduces the time required for pore annihilation.
Heterogeneous PC:PS systems containing calcium had even longer pore creation
times at lower electric fields. The pore radii after creation was lower in both POPC
and POPC:POPS systems containing calcium compared to systems without calcium.
This may also be associated with significantly smaller area per lipid values found
both experimentally and in simulations (Bockmann and Grubmuller 2004) when
calcium is present. Even though the area per lipid was similar for POPC–Ca2+ and
POPC:POPS–Ca2+ systems, it has been shown experimentally (and in MD simula-
tions) that calcium binds with more affinity to PC:PS vesicles than to pure PC
vesicles (Sinn et al. 2006; Vernier et al. 2009b), forming Ca2+–PS complexes
involving PS carboxyl and phosphoryl oxygens. By increasing the total number of
calcium-lipid complexes, one effectively decreases the area per lipid of each system
and thus increases the surface tension of the membrane tension. Alternatively, when
146 Z.A. Levine
the external field is removed a change in surface tension would result in a change in
the pore edge tension since the two quantities are related; thus, one might expect to
see modified pore annihilation times for systems with PS and/or Ca2+ present,
compared to systems without PS and Ca2+.
POPC:POPS–Ca2+ systems exhibit very short pore annihilation times (a few
nanoseconds), similar to the timescales of pore creation. However, this is an order
of magnitude faster than what was observed for pure POPC systems, where pore
annihilation times were tens of (or, in some cases, over 100) nanoseconds in
simulations (Levine and Vernier 2010). These timescales are similar to the resealing
timescales reported in other molecular dynamics simulations (Tarek 2005); however,
a large discrepancy remains between simulated pore annihilation times and experi-
mental resealing times which occur on timescales of milliseconds to hundreds of
seconds. These long-lasting pores occur in living cell membranes, not simplified
phospholipid bilayers, suggesting that some membrane restructuring beyond lipid
nanopore formation occurs, and that the permeabilizing structures in cell membranes
have significantly stabilizing features.
Studies by Ho et al. (2013) have also highlighted how electropores that form in
the presence of electrolytes facilitate ionic current through permeabilized mem-
branes, allowing comparisons to be made between simulations and experiment.
Using the method of Fernandez et al. (2012) for stabilizing open electropores in
MD simulations, the authors characterized the electrical properties of open
electropores under controlled, steady-state conditions. They found that pores could
be sustained for long periods of time (hundreds of nanoseconds or longer) by
substituting a smaller, pore-stabilizing electric field in place of the larger, pore-
creating electric field. The diameters of these pores could be fine tuned by varying
the external field, enabling a systematic examination of the ion conductance of lipid
electropores (Fig. 8). Historically, increases in electric conductance were the first
indicators of the electric field-driven breakdown of the membrane barrier function
that has come to be known as electroporation; thus, conductance remains a useful
metric for characterizing open membrane pores.
Ho compares conductance values from chronopotentiometric studies of planar
lipid bilayers, allowing for the extraction of conductances from single electropores,
to those obtained from MD simulations of field-stabilized electropores in homoge-
neous POPC bilayers containing NaCl and KCl. The authors find that Na+ binds
more strongly to the phospholipid interface compared to K+ or Cl , with bound Na+
located deep in the interface at the acyl oxygens of the glycerol backbone. Con-
versely, K+ and Cl show little affinity for the bilayer, consistent with previous MD
studies. As mentioned previously, the maintenance of intracellular and extracellular
concentrations of these ions ([Na+] is much higher outside the cell; [K+] is much
higher inside the cell) is critical for the electrophysiology of living cells. Disruption
of intra- and extra-cellular ion gradients, normally maintained by ion channels and
pumps at considerable energy expense, by electroporation allows for diffusive
transport of ions and small molecules through the permeabilized membrane. This
effect subsequently has profound consequences for the cell, which must restore
osmotic and ionic balance or perish.
Fig. 8 A snapshot from an MD simulation that shows ionic current flowing through an open
electropore. Orange ions represent sodium while green ions represent chloride (Adapted from Ho
et al. 2013)
During the application of a porating electric field (Ep = 400 MV/m), an

electropore is formed in both NaCl-containing and KCl-containing POPC mem-
branes within 2 ns. During the application of such high electric fields, electropore
will continue to expand until finite size effects (artifacts of simulated unit-cells)
dominate the simulation and the bilayer becomes disorganized. Pores are stabilized
with a sustaining electric field Es, until pore radius reaches approximately 1 nm.
While Ho’s choice of Ep, and the target pore radius are arbitrary values, they should
not significantly influence the steady-state properties of the electropore. Sustaining
fields included Es = 30, 40, 50, 75, 100, and 120 MV/m for both NaCl and KCl
systems. For Es = 30 MV/m, the external field failed to maintain an open electropore
in the NaCl system (the pore annihilated within 10 ns in 2 independent trials). For
Es = 40 MV/m, a pore was sustained for more than 15 ns in the NaCl system, but no
significant Na+ transport was observed. In KCl systems, the electropores were
sustained for over 15 ns at Es = 30 MV/m (and also at 40, 50, 75, and
100 MV/m) in three independent trials. At Es = 120 MV/m, the bilayer became
disorganized within 100 ns in both NaCl and KCl systems, a result of finite size
effects and the periodic boundary conditions.
For stabilizing fields around 50, 75, and 100 MV/m, electropores were stabilized
for at least 200 ns in both NaCl and KCl systems. Pore radii (which were, on average,
1 nm) reached a field-stabilized value within 30 ns. The stabilized, time-averaged
pore radius for each stabilized electropore, taken from the last 50 ns of each
simulation, increased linearly with Es over the full range of sustaining electric fields.
For each stabilizing field, the pore radius for the NaCl system was 140 pm smaller
than for the KCl system, likely a result of the more extensive Na+ binding to POPC,
which shrank the area per lipid and increased electropore line tension.
148 Z.A. Levine
The mobilities of Na+ in MD simulations were less than that of K+ and Cl , a

consequence of the larger hydrodynamic radius of Na+. At infinite dilution, the
ratio between the known values of K+ molar conductivity and Na+ molar conduc-
tivity was approximately 1.5. Approximately the same ratio (1.6) was extracted
from MD simulations. At 0.15 M, the molar conductivity of ions decreased
according to Kohlrausch’s law. As a result of cation anion interactions, the ratio
between the molar conductivities in simulations increased to approximately 1.8.
The conductance of an electrically permeabilized lipid bilayer (for a given ion
species) was determined by Ho using a combination of the native ion conductance,
ion-pore interactions, and by the radius of the pore, which was fine tuned with the
sustaining field Es mentioned earlier. Increases in ion conduction scale with pore
radius, as one might expect, but Na+ conductance was significantly less than K+
conductance for a given radius. This was a consequence of stronger Na+ binding to
the interface, which resulted in a nonlinear relationship between pore radius and
Na+ conductance at small pore radii. At pore radii around 0.77 nm (the smallest
pore radius measured in the KCl system), the ratio of the cation conductances was
approximately 5.6, a reflection of the strong interaction between Na+ and the
phospholipid head groups in the pore wall (relative to K+). For larger pore radii
(e.g., 1.5 nm, the largest pore radius measured in NaCl-containing systems), the
ratio of conductivities was only 2.3. Clearly, as the pore size increased, a larger
fraction of bulk sodium ions were free to traverse the pore, not hindered by
interactions with the electropore wall. Confinement in these simulations increases
the likelihood for cation anion electrostatic interaction, which subsequently
decreases the net velocity of the ions. The snapshots in Fig. 9 capture this behavior.
Ho examined this effect by measuring electrophoretic mobilities in bulk water
systems at varying ion concentrations and found that for NaCl systems, the ion
mobility decreased by nearly half when the concentration increased from about 0.1
to 1 M. In KCl systems, the decrease in ion mobility was approximately 35%.
Ho also compares conductance values from MD simulations with those reported
from chronopotentiometry experiments for POPC electropores in KCl systems.
Although the two sets of conductance values lie within the same order of magnitude,
the pore radii in simulations was smaller than those reported in the chronopoten-
tiometry data. There is, unfortunately, no direct method for measuring electropore
radius experimentally; therefore, pore radius values from chronopotentiometric
results were extracted from the measured conductances using a simple model
where ions are assumed to travel through the pore as they would through a cylinder
of bulk water. In this model, the pore diameter can be estimated by d = 2(GpL/kπ)1/2,
where Gp is the pore conductance, L is the length of the pore, and k is the
conductivity of the electrolyte. Since the accuracy of the extracted pore diameter
depends on the applicability of this simple geometric model, Ho placed large bands
of uncertainty around the chronopotentiometric results. Additional assumptions in
the chronopotentiometric data were that the ion concentrations inside the pore are the
same as in the electrolyte, though Ho later finds that the concentration of KCl in
electropores was twice the bulk value. This makes direct comparisons to experiments
difficult, if not unreliable at this time.
Fig. 9 Ion interactions during migration through an electropore. External electric field of 75 MV/m
in the +z (upward) direction was applied in the snapshots. Na+ and Cl ions are represented as
magenta and green spheres, respectively. Phosphorus atoms and nitrogen atoms of POPC are shown
as brown spheres and blue spheres to delineate the pore. Lipids tails are represented as blue lines.
Water molecules and other atoms are omitted for clarity (Adapted from Ho et al. 2013)
Overall, simulations like those performed by Ho exhibit that the electropore

radius can be globally modulated by sustaining external fields. Ion conductance
was measured by only two factors: pore radius and the extent of ion interaction with
the phospholipid interface along the pore walls. The stronger binding behaviors of
Na+ resulted in significantly lower conductance for NaCl systems (compared to KCl
systems). This difference decreases as the pore radius was increased, and the pore
cross-section became more dominated by bulk water, reducing the probability of
pore wall interactions. The electric conductance of simulated electropores in
KCl-POPC systems was roughly comparable to the single-pore conductance
extracted from chronopotentiometric measurements, but different assumptions
underlying the values used for pore radius and conductance prevented precise
comparison between the two datasets. In addition, the choice of ion and water
models in simulations may also affect the ion conductance values extracted. Indeed,
better models are needed to enable quantitative comparisons between atomistic
simulations and experiment.
Vernier et al. (2009a) also considered the effects of oxidized lipids on electropore
formation after earlier studies found that membranes containing a number of oxi-
dized lipids became leakier and allowed a large number of water molecules to pass
through the membrane with ease. Since reversible electropermeabilization is widely
used to facilitate the introduction of genetic material and pharmaceutical agents into
150 Z.A. Levine
Fig. 10 Oxidized and unoxidized phospholipid conformations change over time. Composite
snapshots (21 images captured at 0.5 ns intervals over a 10 ns period) of PLPC and two oxidized
variants, oxPLPC (12-al) and oxPLPC (13-tc), showing their conformations in molecular dynamics
simulations of PLPC with 11% oxPLPC bilayers in a 360 mV/nm field. The spheres near the end of
the lipid tails mark the location of the introduced oxygens or C13 of PLPC. Structures of the
individual lipid molecules are shown below the corresponding composite. Teal – C, red – O,
gold – P, blue – N, gray – C-13 (Adapted from Vernier et al. 2009a)
living cells, Vernier shows (using molecular dynamics simulations) that oxidation of
membrane components enhances the susceptibility of the membrane to electroper-
meabilization. This occurs because additional oxygen atoms are present in the
membrane interior, which facilitates entry of water molecules across the membrane
(Fig. 10). Manipulation of the level of oxidative stress in cell suspensions and in
tissues may lead to more efficient permeabilization procedures in the laboratory and
in clinical applications such as electrochemotherapy and electrotransfection-
mediated gene therapy.
Oxidative stress is readily encountered in cultured cells and in live organisms
under a variety of conditions and significantly impacts cellular activities across the
metabolic spectrum. Because it directly affects the physical properties of the cellular
membrane, it is an important factor to consider in the lifecycle of electropores, which
is sensitive to the presence of oxygen atoms and other charges present in the
membrane interior. Studies documenting the peroxidation of membrane lipids after

electroporation have been reported, but the effects of pretreatment oxidative stress
have received little experimental attention. Since molecular dynamics simulations
have recently shown that incorporating oxidized lipids into phospholipid bilayers
increases the water permeability of these membranes, it may be expected that
bilayers containing oxidized lipids will also electroporate more readily (especially
since the formation of membrane-spanning water defects is one of the initial steps of
electroporation).
Molecular dynamics simulations and validating laboratory studies of model mem-
branes and cells in electric fields are consistent with the stochastic pore hypothesis for
electropermeabilization (Neu and Krassowska 1999). Additionally, studies such as
these show that it is possible to hasten pore formation at the molecular level by
oxidatively modifying the properties of the membrane in situ, thereby lowering the
energy barrier to poration. In Fig. 11, oxidized membrane patches are shown to be
Fig. 11 Quilted PLPC:

oxPLPC bilayer. The
simulated system, bounded by
the black square in panel A, is
divided (dashed lines) into
two regions of approximately
100% PLPC (light gray) and
two regions of approximately
50% oxPLPC (12-al) (dark
blue) and 50% PLPC, as
described in the text. To show
more clearly where poration
occurs after application of an
external electric field, copies
of the simulated system are
tiled to make a periodic
system. Preferential
electroporation of the PLPC
bilayer in regions of high
oxidized lipid content is
demonstrated in panel B,
which shows the system 1 ns
after applying a 360 mV/nm
field normal to the bilayer.
The bilayer is in the plane of
the page (Adapted from
Vernier et al. 2009a)
152 Z.A. Levine
preferential nucleation sites for membrane electroporations, while unoxidized patches

remain unaffected. Such a mechanism that might be used to localize where in the
membrane poration occurs. This demonstration of the increased sensitivity of oxida-
tively damaged cells to electropermeabilization has practical implications for the
laboratory and the clinic. For instance, an appropriately controlled peroxidizing
regimen, that is, one which enhances permeabilization without significantly affecting
viability, might increase the efficiency of electrotransfection protocols either indirectly
by enabling the use of lower porating voltages (higher voltages are associated with
lower cell survival rates) or directly by increasing the amount of genetic material that
enters the cells for a given series of electrical pulses. Alternatively, a reducing
environment would be expected to protect cells against electropermeabilization.
Extending this line of thinking, electrochemotherapy and direct ablation and
killing of tumor cells using electrical pulse therapy may likewise be enhanced by
procedures which promote oxidative stress in the tumor tissue before pulse delivery
– and inhibited when the environment in or around the tumor is reducing. In any
mixed population of cells, the different native sensitivities of various cell types to
permeabilizing electric fields and oxidative stress might be exploited by adjusting
electrical, physical, and chemical parameters to selectively transfect subsets of cells,
in vitro and in vivo.
Conclusions
Although simple, largely intuitive characterizations of the stages in the lipid

electropore life cycles provide a useful scheme for analysis of pore creation and
pore annihilation, a more sophisticated approach must build on this foundation to
incorporate systematic measurements of the pore radius and pore energies and must
include the atomic-scale electric field landscapes and interactions of water-oxygen
and water-hydrogen with the electron-dense acyl oxygens deep in the phospholipid
bilayer interface. Once these key components of the molecular structure of electropores
are accurately represented, the additional complexities of lipid heterogeneity, membrane
proteins, damaged or oxidized lipids, and cytoskeletal and glycocalyx attachments can
be added to the model one by one, until one approaches a useful representation of the
living cell membrane in porating electric fields.
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in pore creation and annihilation. J Membr Biol 236(1):27–36. doi:10.1007/s00232-010-9277-y
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Lipid Electropore Geometry in Molecular
Models 9
Paolo Marracino, P. Thomas Vernier, M. Liberti, and F. Apollonio
Abstract
Molecular dynamics (MD) simulations recently proved to be a useful tool for
unveiling many aspects of pore formation in lipid membranes under the influence
of external electric fields. In particular, the study of the structure and transport
properties of electropores must definitely take advantage of a rigorous character-
ization of pore geometry and its evolution in time. In order to compare size-
related properties of pores in bilayers of various compositions, generated and
maintained under different physical and chemical conditions, reference metrics
are needed.
In the present chapter three different methodologies to evaluate electropore
geometrical behavior will be presented: (i) the first developed method which
allows the analysis of the dimensions of the pore through an algorithm that uses a
Monte Carlo simulated annealing procedure to find the best route for a sphere
with variable radius to squeeze through the pore channel; (ii) a more recent one
allowing the three-dimensional modeling of the irregular shape of the pores
obtained as the quantification of its volume; and (iii) a new method based on a
statistical approach (following essential dynamics principles), able to describe
pore geometrical fluctuations in a robust and reproducible way.
The three approaches described here are not system specific, i.e., the methods
can be generalized for any kind of pore for which appropriate structural informa-
tion is available.
P. Marracino (*) • M. Liberti • F. Apollonio

Department of Information Engineering, Electronics, and Telecommunications, Sapienza
University of Rome, Rome, Italy
e-mail: marracino@die.uniroma1.it; liberti@die.uniroma1.it; apollonio@die.uniroma1.it
P.T. Vernier
Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA
e-mail: pvernier@odu.edu

DOI 10.1007/978-3-319-32886-7_88
156 P. Marracino et al.
Keywords
Electroporation • Molecular dynamics • Pore radius • Electropore geometry
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
The “Hole” Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
The “Water Cylindrical Slabs” Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
The “Statistical/Analytical” Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
Introduction
The complex mechanisms underlying electroporation processes in living cells are

known only in broad outline. The use of molecular dynamics (MD) simulations
provide atomic-scale information on energetic and dynamic contributions of
complex biomolecular structure and, specifically for membrane systems, it indi-
cates that application of a strong enough electric field across the membrane
bilayers leads to the intrusion of water defects and the formation of aqueous,
conductive pores (Tieleman 2004; Tarek 2005; Ziegler and Vernier 2008;
Casciola et al. 2014).
The capabilities of computer simulations are limited by the accuracy of the
underlying models describing atomic interactions and also by the computational
expense of adequately exploring all the relevant conformations of the membrane and
surrounding water and ions.
In order to compute forces, MD simulations require a potential energy function,
which describes the interactions between the atoms in the system. If the potential
energy of the system is known, given the coordinates of a starting structure and a set
of velocities, the force acting on each atom can be calculated. The accuracy of the
simulations is directly related to the potential energy function used to describe the
interactions between particles.
Microscopic all-atom models use detailed all-atom representations of both the
membrane and the solution, describing the system as a collection of particles that
interact via molecular mechanics force fields, using classical approximations of
quantum mechanical energies to describe the Coulombic (electrostatic), van der
Waals, and covalent (bond, angle) interactions.
In addition to the standard potential interactions, there is the possibility to include
an additive force as the one given by an external electromagnetic field. The field is an
external/exogenous perturbation which could, in principle, alter the charge distribu-
tion, the chemical state, and the energy of the biological structures. In particular, by
simulating a “virtual” experiment, it could be possible to observe the field action on
the microscopic structure in an accurate and rigorous way, providing a realistic
description of the interaction occurring at the atomic level.
9 Lipid Electropore Geometry in Molecular Models 157
The output of a MD simulation is the trajectory that is the series of molecular

configurations evolving in time, generated by solving the classical equations of
motion at each time step. According to the ergodic hypothesis, one can simulate a
membrane system with its surroundings for a period of time and get time-averaged
molecular properties that approach the experimentally measurable ensemble aver-
ages. In other words, if the MD simulation is long enough to cover the entire
configuration phase-space, the system reaches an equilibrium condition and
statistical-mechanics is applicable to calculate macroscopic properties as, for
example, the geometrical properties of a stable electropore.
Recently, molecular dynamics (MD) simulations have shown that a transverse
electric field can produce pores in phospholipid bilayers (Tieleman 2004), and MD
has been adopted as a useful method for studying electroporation. The molecular
mechanism has been unveiled (Tieleman 2004; Tarek 2005; Ziegler and Vernier
2008), at least for phospholipid bilayers, and the different stages of pore formation
have been characterized (Levine and Vernier 2010). However, despite its doubtless
utility, MD has a major drawback. The value of the applied field, E, used in the
simulations has to be high enough in order to electroporate (Ziegler and Vernier
2008; Fernández et al. 2010) to overcome the large activation energy required to
initiate pore formation in a reasonable time window (<100 ns). Under these circum-
stances the pore formation process can be easily followed, but the pore expands to
the size of the simulated bilayer patch (<100 nm2 in most all-atom MD simulations
of phospholipid bilayers), and strong finite size effects appear. This limitation is an
obstacle to the study of the properties and behavior of the long-lasting, quasi-stable
pores that are believed to exist in living, electroporated cell membranes on the basis
of experimental evidence, including ion conductance (Pakhomov et al. 2007).
Following the methodology proposed by Fernandez et al. (2012), consisting in a
two-step protocol based on the observation that the lipid nanopores formed in MD
simulations can be stabilized by reducing the applied electric field from a higher
porating value to a range of lower stabilizing values, one can obtain an equilibrium
condition for the electropore and thus characterize its geometry by means of a
method that should be robust, reliable, and reproducible.
Different approaches are available in literature. In order to determine electropore
dimensions it may be possible to adapt tools previously developed for protein
channels (Smart et al. 1996), all having a cavity running through them. This cavity
or pore is normally filled with solvent and provides the pathway for ion transloca-
tion. The method developed by Smart and colleagues takes into account the van der
Waals surface of molecules to infer the minimum cavity radius in a mechanistic
fashion, i.e., the probe sphere with radius equal to the cavity radius should pass the
cavity without any obstacle.
A second method uses the radius as the pore size metric (Ho et al. 2013). In this
method the length of the pore in the z-direction (normal to the bilayer plane) is
determined by the mean (xy) plane of the nitrogen (N) and phosphorus (P) atoms for
each leaflet of the bilayer. The region between the two P-N planes (the bilayer
interior) is divided into bins 1.5 nm thick in the z-direction, with each bin
overlapping its neighbor by 0.5 nm. For each bin, the minimum and maximum
x- and y-positions of the nitrogen and phosphorus atoms (in the pore walls) are
calculated, and the center of the bin is taken to be the average of the maximum and
minimum values. The radius for each bin is defined as the average distance between
the positions of all phosphorus and nitrogen atom in that bin and the center. The
overall pore radius is then selected as the smallest of the bin radii for each time point.
In other approaches the definition of electropore boundaries is based on water
density profiles (Leontiadou et al. 2004; Gurtovenko and Vattulainen 2007) rather
than phosphorus and nitrogen spatial occupancy. Following this option, in
Fernandez et al. (2012) the volume of the pore is calculated in a three-step procedure.
First the porated section of the membrane is divided into three regions: an upper head
group (polar) region, a lower head group (polar) region, and an intermediate
(nonpolar) region (the bilayer interior). The nonpolar region is divided along the
pore axis (z-axis) into cylindrical slices (cylindroids) whose radii are approximated
from the coordinates of the water molecules at the greatest distance from the pore
axis in the plane of the bilayer (x-axis and y-axis). The volume is then the sum of the
volumes of all the cylindroids.
Finally, Marracino and coauthors recently introduced (Marracino et al. 2016) a
different methodology for electropore characterization, based on the extraction of the
geometry from a statistical analysis of the tridimensional surface of the pore.
In the following sections the three different methodologies to infer electropore
geometrical behavior presented in Smart et al. (1996), Fernandez et al. (2012), and
Marracino et al. (2016) will be discussed, focusing on the differences among them,
the strong and the weak points, the possibility to catch the essential features of the
electropore, so that a measure of pore’s volume or surface could be given.
Materials and Methods
A lipid electropore is a complex three-dimensional, nanoscopic object that changes

quickly with time.
In this section, the outcomes of the three methodologies for the electropore
geometry will be presented in a general way, not dependent on membrane system
characteristics (number of lipids, ion concentration, presence of transmembrane
protein or cholesterol, etc.) or pore stage. The aim is to qualitatively compare the
three methodologies in extracting some useful quantities such as pore volume and
shape.
As a general approach, for a direct comparison among methods outcomes, it may
be convenient to adopt the strategy used in Fernandez et al. (2012): First, the pore is
initiated by applying a strong electric field, Ep E0. Second, when the electropore
has expanded, the electric field is lowered to a sustaining value Es E0. Moreover,
this approach is shown to be consistent in the sense that the dimensions of the
stabilized pore are exclusively determined by the final sustaining field Es and do not
depend either on the initial porating field or on the exact time at which the field is
reduced.
Fig. 1 The main steps of the “hole” method: panel A shows the spherical probe (blue) tangent to
the van der Waals surface of lipid’s head at pore entrance (panel B); as the probe enters more deeply
into the electropore the maximum sphere radius may change, according to the local constriction
(panel C). Molecular graphics images were generated with visual VMD 1.8.7 (Humphrey et al.
1996)
The “Hole” Method
Figure 1 presents the methodological approach to obtain the complete profile of

electropore radii along the z direction. Hereafter, an electropore perfectly normal to
membrane bilayer will be considered; the procedure requires the user to specify an
initial point p that lies anywhere within the pore. A probe sphere with radius R( p)
can be centered at p without overlapping any atom:
RðpÞ ¼ minNatom
i ½jxi pj vdW i (1)
where xi is the vector giving the position of atom i (up to Natom) and vdWi is the van
der Waals radius of an atom belonging to pore walls. The values used for the van der
Waals radii were taken from the force field used in the electropore simulation. Any
ion or solvent molecule found in the channel is excluded from consideration. The
Metropolis Monte Carlo simulated annealing procedure (Metropolis et al. 1953) is

used to adjust the point p to find the largest sphere whose center lies on the plane
through the original point orthogonal to the pore axis. All distance searches are
conducted in three dimensions.
Once the highest radius sphere center on a particular plane has been established, a
new search is initiated by taking a step of length Δr in the direction of the pore axis
so that one can obtain a series of spheres of maximum radius crossing the multiple
planes distant Δr from each other.
As apparent from panel A of Fig. 1, the first sphere is constructed starting from
the electropore upper termination. The maximum probe dimension is essentially
determined by the vdW radii of the lipid heads atoms. For the sake of simplicity, in
panel B of Fig. 1 only four representative vdW radii have been represented, hence the
maximum probe sphere is just tangent to the nearest van der Waals surface.
As the probe enters more deeply into the electropore, the maximum sphere radius
may change, according to the local constriction. The net result can be thought of as
producing the locus of a flexible sphere “squeezing” through the center of the ion
channel. The use of Monte Carlo simulated annealing reduces the possibility of the
routine becoming stuck in a local minimum.
Panel C of Fig. 1 gives a graphical representation of different probe spheres at
different pore heights: The complete probe radii profile is indicative of the
electropore shape.
As an example, in Fig. 2 the overall profile of a “test bed” electropore simulation
via the “hole” method was calculated, which is based on the approach proposed by
(Smart et al. 1996). The values for pore radius, extracted from a single simulation
frame, indicate a well-defined spatial interval with clear terminations (where the
probe sphere is larger) and a central region with wide radius oscillations. It is worth
Fig. 2 The complete pore

radius profile as obtained via
Hole program, for a single
simulation frame. The image
was generated with
KaleidaGraph 4.0
noting that the latter result represents the instantaneous geometrical picture of the
electropore; if an equilibrium simulation (i.e., a stable electropore) would be avail-
able, one can obtain a less noisy representation of pore geometrical structure by
averaging several simulation time frames.
Following this methodological approach, the direct evaluation of electropore
volume is obtained via the summation of several circular slices:
max
X
V¼ πRz ðpÞ2 Δr (2)
z¼min
where Rz( p) is the probe sphere radius at a given height z, min and max represent the
electropore upper and lower terminations (see Fig. 2), and Δr the height of each
circular slice. It is worth noting that Eq. 2 considers the summation of perfectly
circular cylindroids, i.e., the possible presence of elliptic or irregular sections cannot
be properly taken into account.
The “Water Cylindrical Slabs” Method
While the previous approach allows the geometrical characterization of the

electropore starting from lipid head atoms spatial occupancy, different method-
ologies have been recently developed which, in order to reconstruct the pore
shape, takes into account just water density profiles through the pore. The first
method proposing this approach is the one presented as the “water cylindrical
slabs” approach and proposed by Fernandez and coworkers (Fernández et al.
2010).
Figure 3 shows a density map as obtained by dividing the slice passing through
the pore geometrical center into 80 x 80 nodes, each indicative of the mean density
value in a microscopic 3D region of 0.15 0.15 0.3 nm3. As evident from the
figure, the emerging density profile does not exactly represent a circle, but rather an
ellipse as also shown in Marracino et al. (2016).
This kind of observation led Fernandez and coworkers to develop a methodology
that, provided proper electropore upper and lower terminations, represents the lipid
pore shape as a stack of elliptic slices with a fixed height Δz. The overall procedure is
depicted in Fig. 4: To accurately characterize the pore geometry at a given time, a
three-step algorithm is applied. First, the pore has to be centered inside the simula-
tion box. The centering algorithm looks for the center of mass of the membrane and
applies an offset in order to place the center of mass of the membrane at the center of
the simulation box. Second, once the pore has been centered, the membrane is
partitioned in three different parts: the upper polar region, the internal nonpolar
region, and the lower polar region (Fig. 4 panel A).
In order to detect the borders of the internal nonpolar region in the z direction, the
average position of the z coordinate is computed for a specific atom (depending on
the specific lipid model used within the simulation) belonging to lipid head groups
not implicated in the pore.
0.15 nm
z
y
b Water density (mol/l)

80
35
70
30
60
25
50
20
40
15
30
20 10
10 5
0 0
0 10 20 30 40 50 60 70 80
Number of nodes
Fig. 3 Density map of the electropore central section. The 80 80 elementary volumes cover the
entire simulation box on the xy plane. Water density in the very central part of pore sections only
slightly differs from the experimental bulk water density of 33.32 molecules/nm3 (Uematsu and
Franck 1980), reaching lower values while approaching pore walls. Molecular graphics image was
generated with visual VMD 1.8.7 (Humphrey et al. 1996). The density map was generated with
MATLAB R2013a
In Fig. 4, panel A and B these average z positions ZUP and ZDOWN, and the
corresponding standard deviation, SDUP and SDDOWN, are shown for the upper and
the lower leaflets, respectively. The nonpolar region is then considered confined
between [ZDOWN + 2 SDDOWN and ZUP + 2 SDUP], being SDDOWN and SDUP the
standard deviations associated to ZDOWN and ZUP, respectively.
9
a b
z
ZUP
ZUP Internal non 0.15 nm

polar region
ZDOWN Non polar region borders
ZDOWN
d 7
6
Lipid Electropore Geometry in Molecular Models
c
Most distant water molecule
y 5
b
4
0.15 nm a
x 3
y axis [nm]
z
2
y
1
x
0
0 1 2 3 4 5 6 7
x axis [nm]
Fig. 4 Schematic representation of the simulation box, indicating the water region, the upper and the lower polar regions of the bilayer and the internal nonpolar
163
region of the bilayer (panel A); in panel B the plot of the stack of cylindroids building up the pore are shown; in panel C a magnification of the lower cylindroid is
given, with the indication of the water molecules considered to define each elliptic slice (panel D, crosses correspond to the center of the cylindroids). Molecular
graphics images were generated with visual VMD 1.8.7 (Humphrey et al. 1996)
Third, the system volume occupied by water molecules in the pore is sliced in
cylindroids (cylinder with elliptical base in the x,y plane) with a height of 0.15 nm
(approximate radius of a water molecule) in the z direction. The radii of the
cylindroids are determined, taking into account the distances between the most
distant water molecules in x and y directions (Fig. 4 panel C), as far as a distance
of 5 nm.
The volume of the pore, V, is finally geometrically defined and computed as the
volume of the geometric figure combining all the cylindroids inside the nonpolar
region of the membrane. Figure 4 panel D points out the extremely different shapes
of the stacked elliptic sections of the pore.
Similarly to the previous methodology, here the direct evaluation of electropore
volume can be obtained via:
X
ZUP
V¼ ðπai bi ÞΔr (3)
i¼ZDOWN
where ai and bi are the i-th ellipse axes for a given slice i, ZDOWN and ZUP represent
the electropore upper and lower terminations, and Δr the height of each elliptic slice.
The “Statistical/Analytical” Method
This final method, recently proposed in Marracino et al. (2016), aims to refine and
improve the previous ones, by extracting the radius from an analysis of the tridi-
mensional surface of the pore. Pore geometry is irregular and variable on the
nanosecond time scale, but at the same time there is a significant and discernible
structure from which a tridimensional surface can be extracted. This yields a rigorous
characterization of the whole pore volume and also some biophysical variables of
interest such as water polarizability, water diffusivity, and ions and small molecules
transport.
Here the electropore geometry is reconstructed starting from water molecules
positional fluctuations, i.e., via a pure statistical approach. In particular, the present
methodological procedure aims to go beyond the method implemented in Fernandez
et al. (2012), where, although similar outcomes were produced, the cylindrical slices
forming the electropore were obtained via a mechanical approach.
Figure 5 shows the overall approach to describe in a quantitative and rigorous
way the geometry of the lipid electropore. First, to minimize the electropore trans-
lational motion during the simulation it is convenient to apply a centering algorithm,
slightly different to the one previously described. The recentering group is here
chosen by taking into account, in the very central region of the pore, only those lipids
whose translation along the z-axis do not exceed 0.2 nm (as absolute value) from
membrane center of mass. For the pore shown in Fig. 5, panel A the resulting
recentering group consisted in four lipids separated from each other around the
circumference of the pore by about 90 degrees.
One-sheet hyperboloid
z
y
x
z¢
b c 99.73%
95.45% 68.45%
e b
a
h y¢
–a
–b y¢
a b 95.45% 99.73% z
z 68.45%
x¢ y
y x¢
x
x
Fig. 5 General procedure to approximate the electropore by a three-dimensional one-sheet hyper-

boloid, with a graphical representation of the electropore (panel A) and the theoretical methods used
to extract the geometrical parameters from our molecular dynamics trajectory (panel B and C).
Molecular graphics images were generated with visual VMD 1.8.7 (Humphrey et al. 1996)
The approach aims to extract the geometrical parameters needed to fully describe
the pore as a one-sheet hyperboloid, the quadric surface that well approximates the
electropore 3D region (see Fig. 5, panel A). The one-sheet hyperboloid is a quadric
with equation
0 2 0 2 0 2
x y z
þ ¼1þ (4)
a b c
From panel B of Fig. 5 it is evident that the central section of the hyperboloid, i.e.,
z0 = 0 in Eq. 4, defines the ellipse with equation
0 2 0 2
x y
þ ¼1 (5)
a b
with a and b the length of the ellipse semiaxes along the x0 and y0 axes, respectively.
Note that the hyperboloid axes are in general rotated with respect to the Cartesian
reference system (x, y, z), i.e., all hyperboloid geometrical parameters are expressed
in the internal reference frame of the hyperboloid.
Planes with equation z0 = k intercept the hyperboloid, producing the ellipse

0 2 0 2 2
x y k
þ ¼1þ (6)
a b c
with semiaxes lengths
a pffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ffi
a0 ¼ c2 þ k2 (7)
c
b pffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ffi
b0 ¼ c2 þ k2 (8)
c
respectively. It can now be defined the three-dimensional pore surface as a
one-sheet hyperboloid: (i) a and b can be evaluated by the numerical charac-
terization of the central ellipse given in Eq. 5 and (ii) c can be evaluated with
the same procedure, by taking into account two k values in Eq. 6 suitable for
identifying the hyperboloid (electropore) lower and upper ellipses, and then
extracting c via Eqs. 7 and 8. The proper values for the k parameters, and hence
the length of the pore along its axis together with the position of pore termi-
nations, can be identified by considering the probability distributions of nitro-
gen atoms on the lipid head groups along the z-axis of the Cartesian reference
frame.
At this point one needs a theoretical tool to determine a, b, and c and eventually
electropore surface and volume via the hyperboloid equation. In order to extract
from MD simulations the correct values for the hyperboloid parameter, i.e., to
calculate the geometric fluctuations of the water molecules present in a certain
region, the covariance matrix method has been recently used (Marracino et al.
2013) to characterize the geometry of a complex biomolecular system (a protein)
by approximating its overall geometrical structure, at each MD frame, to the
ellipsoid given by the eigenvectors of the covariance matrix as obtained by the
distribution of the x, y, and z coordinates of the protein atoms (the geometrical
~ This method is a direct extension of the most general essential dynamics
matrix C).
(ED) analysis, which focuses on seeking those collective degrees of freedom that
best approximate the total amount of fluctuations of a dynamical system. ED is
based on a principal component analysis (PCA) of MD-generated structures
(Amadei et al. 1993). In the present case one can define bidimensional elliptic
sections obtained by the distribution of the x,y coordinates of the atoms of the
water molecules. Since water molecules occupy a volume, one can consider
cylindroids (cylinder with elliptical base in the x,y plane) with a height approxi-
mating the radius of a water molecule (0.15 nm) in the z direction. Note that the
slight variation along the z coordinate of water atoms does not significantly modify
the distributions along the x,y coordinates. Therefore, for a cylindrical slab defined
by water atoms, the elements of the 2 x 2 geometrical matrix at each time frame are
given by
XN
~ ¼1
C ðrl hriÞðrl hriÞT (9)
N l¼1
where rl is the l-th atom position vector and r is the mean atomic position vector
obtained by averaging all of the atom position vectors. Diagonalization of the
geometrical matrix provides two eigenvectors (ci, i = 1, 2) corresponding to the x,
y axes of the ellipse best fitting the atom distribution at that MD time frame
(geometrical axes) and two corresponding eigenvalues (λi, i = 1, 2) given by the
mean square fluctuations of the atomic positions along each eigenvector and pro-
viding the size of the ellipse along its axes.
In panel C of Fig. 5 the Gaussian atomic positional distribution along each
eigenvector is shown, with three possible measures for each semiaxis length. Hereafter
pffiffiffiffi
ai ¼ 2 λi (i = 1, 2) , i.e., the value for the geometrical semiaxes are taken as two
times (95%) the value for the mean square fluctuations of the water atomic positions
along each eigenvector. By knowing the semiaxes magnitudes (a1 , a2) and directions
(c1 , c2) at each MD frame, one can quantitatively describe each elliptic section given
in Eq. 5 in its internal reference system (i.e., a = a1; b = a2 and the x0 and y0 axes are
directed along c1 and c2). Finally, by applying the same procedure for different slices
spanning from one end of the pore to the other, one can obtain the instantaneous three-
dimensional surface that defines the pore.
As an example of the method’s outcomes in Fig. 6, the variation with time of the
minor and major axes of the three ellipses approximating the central section (k = 0
in Eq. 6) of the electropore, the upper ellipse (k = +1 nm), and the lower one
(k = 1 nm), respectively, are shown. These results refer to a stable electropore
simulation, similarly to what was explained in the previous section. From the figure
it is evident that, within the whole simulation run, axes lengths remain rather stable,
with mean values indicative of proper ellipses. As expected, the central section axes
are smaller than those for the two termination ellipses, confirming the general
properties of the one-sheet hyperboloid.
The volume calculation is straightforward, by using Cavalieri’s principle, from
which one can find the volume of a solid as a “stack of slices” between the
electropore terminations k:
ð þk ð þk ð ð
V¼ Aellipse ðz0 Þdz0 ¼ dx0 dy0 dz0 (10)
x0 2 y0 2 z0 2
k k ðÞ
a þ ðÞ
b 1 þ ðÞ
c
Moving to the more convenient elliptic coordinate system, it follows:

qffiffiffiffiffiffiffiffiffiffiffi
ð þk ð 2π ð 1þðz0c Þ
2
V¼ abρdρdθdz0 (11)
k 0 0
Where abρ is the Jacobian as obtained by coordinate’s transformation.

Fig. 6 In panel A the probability distribution of the nitrogen atom belonging to lipid head groups is
given: the two Gaussian-like peaks (centered in 5.5 and 10 nm along the z-axis) correspond to the
two interface regions of the bilayer. The nonzero nitrogen density in the interval between 6.5 nm
and 9.0 nm comes from the head groups forming the pore walls. The electropore terminations (ends)
can be properly chosen by taking into account k values inside this internal steady interval, as close
as possible to the upper and lower nitrogen distribution peaks (here 5% of the distribution peaks
for both ends of the pore were used); panel B, C, and D show the time behavior (for simulation
details see Marracino et al. 2016) of the minor and major axes of the three ellipses approximating
the central section of the electropore, the upper ellipse, and the lower one. The images were
generated with KaleidaGraph 4.0
Conclusions
In this chapter three possible methods to determine the lipid electropore geometry,
including a measure of its central radius, shape and, possibly, volume, were
presented.
A lipid electropore is a complex three-dimensional, nanoscopic object that

changes quickly with time. Characterization of the shape and size of this
dynamic biomolecular structure is essential for understanding how pore dimen-
sions affect intra- and extrapore (proximal and distal) water diffusivity, water
dipolar behavior, ion and small molecule transport, and membrane mechanical
properties.
The most popular algorithm extracting electropore geometry from lipid heads
positions (Smart et al. 1996), together with two different approaches defining
pore shape starting from water density profiles (Fernandez et al. 2012; Marracino
et al. 2016), were analyzed. All three methodologies are able to provide the
boundaries of the pore in three dimensions at any time point, so the volume of
the pore, the radius anywhere along the z-axis (normal to the plane of the bilayer),
and other geometric characteristics like the shape of pore sections can be
extracted.
Although some qualitative examples of the methods outcomes have been given
for a stable electropore simulation, in principle every kind of electropore can be
followed in time and in response to changes in simulation parameters (temperature,
pressure, bilayer composition, salt concentration). In fact, the validity of the three
methods is general and independent from the system; moreover, it is not strictly
necessary to have a permeabilized bilayer fully reequilibrated, as one can be
interested in following pore’s natural fluctuations in time.
Apart from the chemical species adopted to define the electropore, another
difference between the hole method and the latter two is the shape of the electropore
sections. The first one uses spherical probe large enough to not collide with pore
walls (defined by vdW radii of the lipid heads atoms), repeating the procedure for the
whole pore length, while the other two take into account more rigorously the actual
pore section shape.
Nonetheless, the main difference between Fernandez et al. (2012) and
Marracino et al. (2016) is the mechanical vs. statistical evaluation of pore elliptic
sections. In Marracino et al. (2016) the definition of the electropore geometry is
based only on water positional fluctuation. It was developed independently, but it
can be considered an improvement of Fernandez methodology, since here the
determination of the volume of the pore region or the pore radius is based on a
rigorous statistical approach. Moreover, it allows a quantitative description of
electropore boundaries (each elliptic slice accounts for the 95% of Gaussian atomic
positional distribution of water molecules along each semiaxis) that should
improve the electropore dimension estimation given by Fernandez et al. (where
radii are approximated from the coordinates of the water molecules at the greatest
distance from the pore axis in the plane of the bilayer [x-axis and y-axis], hence
implying a possible overestimation of pore radius) and Smart et al. (when the pore
walls are defined based on lipid atoms, the radius/volume of the pore extracted
would be systematically biased to lower values, since the water molecules that
hydrate the lipid head groups are not taken into account, hence implying a possible
underestimation of pore radius).
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lipid bilayers. J Phys Chem B 112:13588–13596
Electropermeabilization and
Electrostimulation by Picosecond Pulses 10
Iurii Semenov and Shu Xiao
Abstract
Biological effects of picosecond electric pulses have not been studied as widely as
those for longer pulses. The research has been restricted mainly due to the lack of
pulse generators and exposure systems. Recent advance in generators capable of
producing ultrashort electric pulses has alleviated the restriction and allowed
more biological studies in this short-pulse regime. As the picosecond pulse
duration is shorter than a plasma membrane charging time constant and a cell’s
dielectric relaxation time, the distribution of electric field across the cell is
determined by the dielectric properties of cell’s structures. The plasma membrane
in this case has the highest field, similar to the case when it is charged by longer
pulses through resistive current. For picosecond pulses, direct plasma membrane
poration requires very high electric fields. Cells that have voltage-gated channels
however can be permeabilized by single, 500 ps pulse with the electric field of
190 kV/cm. This is evidenced by a lack of Ca2+ response in Chinese hamster
ovary (CHO) cells and significant calcium uptake in murine pituitary tumor
(GH3) cells and neuroblastoma-glioma hybrid (NG108) cells, which both have
rich calcium voltage-gated channels. Because the pulse duration is much shorter
than the standard channel opening time, it can be speculated that the permeabi-
lization caused by picosecond pulses is different from standard channel opening,
which presents a unique opportunity for a deeper understanding of the biophysics
I. Semenov
e-mail: isemenov@odu.edu
S. Xiao (*)
Department of Electrical and Computer Engineering, Frank Reidy Research Center for Bioelectrics,
Old Dominion University, Norfolk, VA, USA
e-mail: sxiao@odu.edu

DOI 10.1007/978-3-319-32886-7_89
172 I. Semenov and S. Xiao
of both voltage-gated channel conduction and electroporation. Nevertheless, the

development of approaches to lower the electric field is warranted for practical
applications, such as stimulation that employs antennas as noninvasive pulse
delivery device.
Keywords
Picosecond pulses • Electrostimulation • Calcium • Membrane • Electroporation
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
Electropermeabilization and Electrostimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Picosecond Pulse Exposure System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
Plasma Membrane Depolarization and Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Calcium Stimulation by Picosecond Pulses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
PM Permeabilization by psEPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Direct Interaction of Membrane and psEPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Introduction
A plasma membrane (PM) charging time constant is defined by the capacitance of a

membrane and the resistances of the medium and the cytoplasm. For cells that have a
diameter of 10 μm, the charging time constant can be estimated as approximately
100 ns according to the Cole’s single-shell model. It is longer for larger cells and also
decreases with increasing conductivities of the cytoplasm and medium. Electrical
pulses longer than the PM charging time constant increase the transmembrane voltage
and consequently cause a high field across the membrane, making electroporation or
electrostimulation possible. Electrical pulses shorter than the PM charging time
constant allow direct interaction between the pulses and intracellular structures in
addition to the membrane charging. Initially, it was assumed that nanosecond electric
pulses (nsEPs) induce primarily intracellular effects while the plasma membrane
remains intact (Schoenbach et al. 2001). Later experimental results showed that
exposure to nsEPs can result in pores of nanometer size (nanopores) – smaller than
those produced by millisecond and microsecond pulses (Vernier et al. 2006a;
Pakhomov et al. 2007). But the notion that shorter pulses have a higher ability to
perturb the intracellular structures is generally correct. For example, the comparison of
300, 60, and 10 ns pulses for the same efficiency of PM permeabilization (evoking
similar Ca2+ influx via the PM) shows that a 10 ns pulse has a greater ability to
permeabilize endoplasmic reticulum (ER) membranes (evoking the highest Ca2+
efflux from ER when extracellular Ca2+ ions are absent) (Semenov et al. 2013). To
exclusively create intracellular effects, it seems reasonable to use shorter pulses in the
subnanosecond (picosecond) range. Yet, the capacitive properties of cytoplasm and
external medium must be taken into account (Kotnik and Miklavcic 2000;
10 Electropermeabilization and Electrostimulation by Picosecond Pulses 173
Gowrishankar and Weaver 2003; Timoshkin et al. 2006; Schoenbach et al. 2008).
Picosecond pulses (psEPs) still cause a significant voltage drop across PM because of
the difference in the permittivity of the cell’s membrane (low permittivity) to that of
cytoplasm and medium (high permittivity). Furthermore, charge migration in the
presence of a very high field also can cause membrane charging (Vernier et al.
2006); thus, a complete separation of the intracellular effects from the membrane
effects is unlikely. As the two most well-known and the least controversial effects of
electric pulses, i.e., electroporation and electrostimulation, arise and manifest in the
PM, the change of PM conductive properties remains the primary study of the effects
of picosecond pulses.
Studies with B-16 melanoma, liver cancer, and HeLa cells showed that extremely
high electric fields (from 20 to 1000 kV/cm) were required to observe an uptake of
trypan blue and apoptosis after exposure to picosecond pulses (Schoenbach et al.
2008; Xiao et al. 2011; Camp et al. 2012; Hua et al. 2012). It was also shown that
neurons can be stimulated by picosecond pulses at a high repetition rate (Xiao et al.
2013). Even a single subnanosecond pulse (190 kV/cm) could trigger calcium influx
through voltage-gated calcium channels (Semenov et al. 2015). On the system level,
early studies in the 1990s involving subnanosecond pulses of low amplitudes as
electromagnetic pulses (EMPs) were carried out on rats to assess their possible
hazard. The results consistently showed no biological effects after the exposures.
Physiological parameters such as blood pressure and heart rate (Walters et al. 1995)
were not affected. It was later pointed out that the electric fields in these experiments
were much lower than those required to cause measurable responses (Rogers et al.
2004).
Studies of the biological effects of psEPs are particularly motivated by the
potential applications involving noninvasive delivery as such short pulses can be
radiated via antennas. Targets deep in the tissues that are inaccessible to electrodes
may be reached with focused antennas. Two types of antenna have been suggested
for psEPs delivery: (1) impulse-radiating antennas by Baum and his colleagues
(Baum 2007) and (2) dielectric biconical antennas (DiBiCA) (Petrella et al. 2016;
Xiao 2017?). To meet the high thresholds, these antennas have to be driven by pulsed
power sources that generate very high voltages (>100 kV). Exploring biological
effects of psEPs will help to address the electric fields required for the antenna
applications and provide guidance for the antenna design. From the biophysics point
of view, psEP research will answer the question of whether psEPs cause biological
effects differently than longer pulses such as nsEPs do.
Electropermeabilization and Electrostimulation
There are two well-established biological effects on cells upon exposure to electrical
pulses: (1) PM electroporation and (2) PM electrostimulation. Electroporation
implies that pores of certain sizes and duration are actually created across PM, yet
there is no concrete proof of their actual existence. What is known is that electrical
pulses result in a long-lasting increase of PM permeability, which allows for

transport of small ions and molecules from an extracellular solution to a cell interior
and/or the other way around. This phenomenon is called electropermeabilization of
the PM. Electrostimulation of PM, on the other hand, is more specific, meaning that a
sudden membrane depolarization and activation of voltage-gated channels create
pathways for specific ions to move through. Both electropermeabilization and
electrostimulation rely on the change of the transmembrane potential Vm,impulse,
due to an external pulse (Forster 2000):
ð
1:5Rcell t=τcell
V m,impulse ¼ e Eo ðξÞdξ
τcell
where τcell is the time constant for PM charging (~100 ns) that varies according to the
cell size. To reach a threshold voltage of electropermeabilization or electro-
stimulation, the duration of the pulse (t) needs to be longer than τcell, which is the
case for long pulses (micro- or millisecond). Otherwise, Eo has to be much higher in
order to reach the same threshold Vm,impulse. As the duration of psEPs is much shorter
than τcell, Eo has to be exponentially increased compared to that of a longer pulse.
This holds true only if the mechanisms of PM electropermeabilization or stimulation
remain the same for pulses with short durations.
Picosecond Pulse Exposure System
Conventional exposure systems for studying the biological effects of psEPs are
mostly based on transverse electromagnetic (TEM) cells (Ji et al. 2006), in which
biological cells are contained in a flask. The average electric field for cells is thus
rather low as the physical size of the systems is large. One can increase the electric
field by reducing the size of the exposure system and loading the wave propagation
structures directly with the medium containing cells. One can also use two parallel
electrodes immersed in the medium with the cells (Xiao et al. 2016), which allows
much higher electric fields to be delivered to cells. The problem of air breakdown
due to high voltage is eliminated in such systems.
The complete exposure setup using parallel electrodes is shown in Fig. 1. Sub-
nanosecond electrical pulses (approximately 320 ps for full width at half maximum)
were generated by a pulse generator (FPG 20-1 PM, FID, Germany). The pulses
were sent to a π network in order to absorb and minimize the downstream reflections
before they were delivered to the electrodes above a cover slip under a microscope.
Because of multiple reflections from the source to the electrodes, the pulses were
broadened to 500 ps. The electrodes can be positioned precisely within a few μm
using a micromanipulator. The cells on the cover slip positioned in the gap between
the electrodes were exposed to the field created by the electrodes. For a given input
voltage, the electric field can be varied by raising the electrodes 50, 100, and 150 μm
above the cover slip. For an input voltage from the coaxial cable (1 V), the peak
voltage across the parallel electrodes was calculated to be 0.88 V due to the reflection
Fig. 1 The picosecond pulse stimulation setup and electric field characterization. (a) The overall
schematic of the psEP stimulation system. (b) The waveform measured before the π network
attenuator (red, left) and after it (black). The voltage scales on the left and right of the box
correspond to the left and right waveforms, respectively. (c) A 320 ps Gaussian pulse that was
used as a stimulus (red; left voltage scale) for pulse shape simulations and the resulting electric field
pulse at the center of the cover slip (black; right scale). The electric pulse was broadened to 500 ps
as measured at one half of its height. (d) The electric field strength distribution at the cover slip
surface. The gap between the lowest points of two electrodes (marked by red target signs) and the
cover slip was 50 μm. The stimulated cells were situated mostly within the central contour of the
electric field (190 kV/cm) (Semenov et al. 2015)
loss when the pulse propagated from the coaxial cable to the parallel electrodes. The
electric field in the central region on the cover slip was then calculated to be 3.2, 2.0,
and 1.3 kV/m, respectively. For all three electrode heights, the electric field in the
center region is homogenous. The area covers approximately 50 70 μm for 50 μm
and 60 70 μm for both 100 and 150 μm. Cells in this region were selected for
study. For the electrode height of 50 μm that was commonly used in the experiment,
the voltage delivered to the stimulating electrodes was kept constant (6.2 kV), which
produced an electric field of 190 kV/cm at the position of cells.
Plasma Membrane Depolarization and Electroporation
The exposure system incorporating real-time measurements of membrane potential

made it possible to observe the immediate changes induced by the electric fields that
were not immediately observable with conventional TEM cell exposure systems.
Even a single picosecond pulse (500 ps, 190 kV/cm) was shown to cause lasting PM
permeabilization. Changes of PM potential in an NG108 cell upon exposure to a
single psEP are shown in Fig. 2. Two approaches were utilized to acquire experi-
mental data. Figure 2a shows the PM potential change recorded with a current clamp
approach in the zero current injection mode. Delivery of multiple (25) pulses
enhanced the effect, but variations of the frequency in a wide range (from 8 to
1,000 Hz) were shown to be ineffective. Patch clamp is a powerful tool to study
changes of a PM potential that allows for full control over the PM potential. It
provides detailed information on the PM resistance changes due to channels opening
or electroporation. However, intense psEPs may generate noise that upset the
electronics of a patch clamp amplifier and can result in artifacts, for instance, a
sharp voltage spike following the application of a single picosecond pulse (Fig. 2a).
Thus, to confirm the experimental data, a secondary approach relying on fluores-
cence emission was necessary. A voltage-sensitive dye, such as Di-8-ANEPPS, can
be loaded into the cell membrane. The excitation spectrum of Di-8-ANEPPS shifts to
the right upon a PM depolarization. Thus, alternating the excitation of fluorescence
at the wings (440 and 530 nm) with the subsequent measurement of the
corresponding emission makes it possible to measure the ratio of the fluorescence
emission (R = 440/530 nm). The change of the ratio is proportional to the change of
PM potential. The ratio is not largely affected by the concentration or bleaching of
the dye, therefore allowing calibration of the potentiometric response. Figure 2b
shows a response of Di-8-ANEPPS to the PM potential changes triggered by the
same picosecond pulse as in Fig. 2a. Similar response confirms the PM potential
Fig. 2 Typical measurement of transmembrane potential in a single NG108 cell after exposure to a
single picosecond pulse. (a) The result measured by patch clamp. (b) The result measured by
ratiometric fluorescence imaging using di-8-ANEPPS (Xiao et al. 2016)
changes triggered by the single picosecond pulse. Upon application of the single
pulse, the PM was depolarized by 15–20 mV. This effect was transient and the PM
potential recovered to the resting level within 20–30 s after exposure (not shown in
the figure).
PM potential exists due to the difference in the ionic composition of the cell’s
interior and extracellular solutes supported by the selective permeability of the
PM. The different permeability of PM to K+, Na+, and Cl ions results in charge
separation across the membrane, establishing a resting membrane potential. PM
depolarization is generally caused by the transient opening of voltage-gated channels
(mainly VG-Na+) or PM electroporation. In the latter case, the depolarization of the
PM reflects an influx of the ions from the extracellular medium through the pores.
This influx is thought to be unselective. Since Na+ has a high chemical gradient
toward the cell’s interior and is usually the most abundant ion in the extracellular
solution, electroporation results in large Na+ uptake.
Then, is the PM potential increased by a single psEP caused by opening channels
or electroporation? It may be possible to separate these two events as the opening of
channels is selective to specific ions while electroporation is not. It can be hypoth-
esized that the ion channels can be blocked prior to pulsing, while pores, if ever
formed due to pulsing, allow for the cross-membrane ion traffic. The increase in
intracellular ion concentration of Na+, for example, can be measured by a Na+
sensitive dye such as palytoxin. This dye, however, is also known for its toxicity
and therefore is not commonly used in the measurement of sodium. Other sodium
dyes have poor selectivity as they may also be sensitive to K+, so there are limited
selections of sodium indicators. Instead of examining Na+, calcium can be chosen in
order to study the transport mechanisms through PM as many indicators that have
high selectivity for calcium are available.
Calcium Stimulation by Picosecond Pulses
Calcium is an omnipresent ion that involves signaling and regulation associated with
cell division, gene transcription, muscle contraction, secretion, release of
neuromediators from nerve terminals, and many other biological processes. Electric
fields have been shown to be effective in causing an increase in Ca2+ concentration.
The quantitative measurement of Ca2+ dynamic in cytosol can be performed with the
Ca2+-sensitive fluorescent indicators such as Fura-2, a ratiometric fluorescent dye.
There is a 20,000-fold difference between the Ca2+ concentration in the external
solution and in the cytosol, which creates a large chemical gradient. If psEPs create
pores in the plasma membrane, calcium will flow into the cell. Different cells have
different channels responsible for ion passage and can be selected for study. For
example, three cell lines, NG108, CHO, GH3, were utilized to study Ca2+ transients
evoked by exposure to psEPs (Semenov et al. 2015). NG108 cells possess L, N, P,
and Q types of voltage-gated Ca2+ channels, while GH3 cells mainly have the L-type
Ca2+ channel. In contrast, CHO cells do not have any voltage-gated Ca2+ channels.
Fig. 3 (continued)
The lack of VGCCs evident in these cells makes the pores the principal or only
mechanism for Ca2+ increase after EPs exposure.
It was shown that 500 ps pulses at 190 kV/cm, up to 100 pulses per train, failed to
elicit any Ca2+ response in CHO cells. The lack of response in CHO cells suggests
that psEP stimulation did not cause electroporation of the lipid phase of the cell
membrane. GH3 cells typically display random spontaneous Ca2+ oscillations of up
to 300–500 nM in amplitude. On top of this activity, the delivery of a single psEP at
190 kV/cm caused a synchronized increase in Ca2+ concentration, either due to
VGCC opening, or electroporation, or enhancement of the spontaneous oscillations
by some other mechanism. The psEP-induced Ca2+ transients peaked at about
100 nM within 2–15 s after the stimulus, followed by a recovery to the basal level
for about 100 s (Fig. 3a). The peak amplitude of the Ca2+ response depended on the
number of applied psEPs. Increasing the number of psEPs also shortened the rising
time of Ca2+ transients and slowed their recovery (Fig. 3a–c, e). The average
amplitude of the Ca2+ transients increased only about fourfold with an
increase in the number of psEPs from 1 to 100. Preincubation of cells with an
L-type VGCC blocker, verapamil, reduced the amplitude of Ca2+ transient at least
by 95% (Fig. 3d, e) regardless of the number of pulses used.
In the case of NG108 cells, a single pulse triggered a small (6 1.1 nM) but
reproducible Ca2+ rise (Fig. 4a). Multiple pulses evoked higher responses with the
Ca2+ increase over 100-fold larger for the greatest number of pulses (100) that were
applied (Fig. 4b, c, e). Selective blockers such as ω-conotoxin (which blocks N, P,
and Q types of Ca2+ channels) and verapamil only partially inhibited these Ca2+
transients, but with a cocktail of both blockers, 85–100% inhibition was achieved
(Fig. 4d, e). Surprisingly, this presumed activation of VGCC by psEPs was insen-
sitive to the replacement of external sodium cations with N-methyl-D-glucamine
(NMDG) impermeable for PM. Thus, Ca2+ influx could not be explained as the
“natural” opening of the voltage-gated channels due to depolarization of PM driven
by psEPs. Moreover, replacement of Na+ with NMDG increased the amplitude of the
Ca2+ response. NMDG has a lower mobility in water than Na+, which reduced the
conductivity of the physiological solution from approximately 15 to 11 mS/cm. The
reduced conductivity of the solution was reported to increase the effect of nsEP on
the cells, possibly due to the transient electrodeformation (stretching) force that
assumes its maximum value if cells are suspended in low-conductivity media
(Muller et al. 2001). However, it is not known if these findings and modeling are
ä
Fig. 3 Effect of psEP stimulation in GH3 cells. (a–d) Calcium transients evoked by 1, 5, or
50 stimuli (1p, 5p, and 50p) at 190 kV/cm, 1 kHz. Shown are representative traces in individual
cells (yellow lines, 15–25 cells per plot) and their average (black). Stimuli were delivered at 60 s into
the experiment (vertical dashed lines). (d) Pretreatment with verapamil inhibited the response to
50psEP (a magnifier is used for better viewing). (e) Average peak amplitudes of the psEP-induced
Ca2+ transient as a function of the number of pulses, in the physiological solution (control) and after
preincubation with 100 μM verapamil (ver). Shown are mean values SE for 15–25 cells per group.
The effect of verapamil is significant at p b 0.01 (two-tailed t test) for all data points (Semenov et al.
2015)
Fig. 4 (continued)
also applicable to subnanosecond pulses. Another feasible explanation involves

the inhibition of Na+/Ca2+ exchanger in the absence of extracellular Na+ and the
consequent slowing of Ca2+ removal from the cytosol that makes the Ca2+ response
larger and longer. In both NG108 and GH3 cells, Ca2+ recovery to the basal level
that preceded psEP exposure took more than 100 s in average. While it could be a
manifestation of a continued opening of VGCC, a more probable mechanism
would be the slow function of pumps and other mechanisms responsible for Ca2+
buffering and removal. To test if this extended opening of channels is the case, a
series of experiments were performed in which cells were exposed to psEPs in the
absence of extracellular Ca2+. Cells were perfused with a Ca2+-containing solution
(2 mM) 10 s after exposure. In all cases, an immediate rise of intracellular Ca2+,
proportional to the number of pulses that were applied, was observed in the cells
previously exposed to psEPs. The data indicate that at least some VGCC remained
open for at least 10 s after the stimulation. Notwithstanding slower VGCC inac-
tivation in the absence of Ca2+, this opening time is unusually long even for slow-
inactivating VGCC (Snutch et al. 2005).
PM Permeabilization by psEPs
The effect of psEPs on PM resistance seems no different from that of longer

nanosecond pulses (Semenov et al. 2016). The permeabilization of PM by psEPs
can be achieved similarly to nsEPs although the electric fields have to be much
higher. PM permeabilization, evoked by psEPs exposure, can be characterized by the
current and voltage characteristics using a patch clamp approach. It was shown that
even a single psEP at 190 kV/cm caused lasting membrane permeabilization in
NG108 cells (Semenov et al. 2016). The effect was enhanced by delivering multiple
pulses but not by varying the frequency. Whole-cell conductance in the three cell
lines (NG108, CHO, and hippocampal neurons) were decreased after a burst of
25 psEPs (500 ps, 190 kV/cm, 1 kHz). The current-voltage curves that were
measured by a step protocol initiated 1 s after the psEP delivery (Fig. 5) showed a
strong increase of inward current at negative transmembrane potentials but not much
change of the outward current at positive potentials. Moreover, the I-V curves
became steeper as the membrane potential became more negative, reflecting the
voltage sensitivity of inward conductance. The rectifier features are remarkably
similar to those obtained with much longer pulses (60 and 600 ns). The inward
ä
Fig. 4 Effect of psEP stimulation in NG108 cells. (a–d) Calcium transients evoked by 1, 5, or
100 stimuli; designations are the same as in Fig. 3. Note different scales for (a) and (b) panels versus
(c) and (d) panels; high-amplitude responses of some cells were clipped. (d) Pretreatment with
verapamil and ω-conotoxin inhibited the response to 100 psEP. (e) Average peak amplitudes of the
psEP-induced Ca2+ transient as a function of the number of pulses, in the physiological solution
(control); after preincubation with 1 μM ω-conotoxin (“ω-con”) or 100 μM verapamil and 1 μM
ω-conotoxin (“ver,ω-con”); and after the replacement of the external Na+ with NMDG (“NMDG”).
Shown are mean values SE for 15–30 cells in most groups. *p b 0.01 from the control (two-tailed
t-test) (Semenov et al. 2015)
Fig. 5 Current-voltage
curves for NG108 cells
recorded prior to psEP (open
symbols) and immediately
after exposure ( filled
symbols). A burst of
25, 500 ps pulses (190 kV/cm,
1 kHz) was applied
rectification along with voltage sensitivity was regarded as the electrophysiological

hallmarks of nanopore formation that distinguished them from the linear I–V
dependence of larger membrane pores created by conventional pulses in micro- or
millisecond ranges (Pakhomov and Pakhomova 2010). Both the inward rectification
and voltage sensitivity could potentially be explained by a conical, asymmetric
shape of nanopores or by a nanopore opening at the bottom of a funnel-shaped
membrane invagination, such as a caveole, but these speculations have not been
experimentally verified.
It is well known that in a typical strength-duration (SD) relationship, the threshold
of electrical stimulation (current, charge, or specific absorption rate) depends on the
pulse duration. Stronger electrical stimulation is necessary to achieve the stimulation
threshold for shorter pulses. In the subnanosecond range, electric field (E), pulse
number (N), and pulse duration (t) are the main parameters in the SD relationship.
For example, the trypan blue uptake was scaled with Et√N for subnanosecond pulses
(Schoenbach et al. 2008). For the case when only a single pulse is utilized, the
scaling parameters can be reduced to the electric field and the pulse duration. To
estimate the required electric field for a similar Ca2+ response in the subnanosecond
pulse range, an extrapolation of experimental data for 10, 60, and 300 ns pulses taken
from Semenov et al. (2013) indicates that at least 240 kV/cm and 370 kV/cm are
required to achieve the same Ca2+ responses of 0.15 μM and 0.5 μM, respectively
(Fig. 6). This result can explain the lack of Ca2+ uptake in CHO cells that were
exposed for the much lower electric field intensity, 190 kV/cm. It seems that the
transition of nanosecond to subnanosecond pulse results in the change of the slope of
SD relationship, suggesting the distinct mechanism of pore formation in the sub-
nanosecond range. Changing the slope and therefore lowering the critical electric
field may be possible by sensitizing cells with local heat. Evidence of the reduction
in electric field were shown in Xiao et al. (2011) and Camp et al. (2012); in one
method, the heat was generated by high-frequency pulsing, and in the other external
heat was applied.
Fig. 6 Electric fields required

to evoke the same Ca2+
responses (triangle 0.15 μM
and square 0.5 μM) in CHO
cells by pulses of different
durations (10 ns, 60 ns, and
300 ns). Both curves were
obtained for the presence of
2 mM external Ca2+. The
electric fields extrapolated for
500 ps are 240 kV/cm (empty
circle) for 0.15 μM and
370 kV/cm (filled circle) for
0.5 μM (Data taken from
Semenov et al. 2013)
Direct Interaction of Membrane and psEPs
The inability of an ultrahigh electric field (190 kV/cm, 500 ps) to permeabilize the
PM of CHO cells suggests that the PM free of VG channels is electrically more
stable for subnanosecond electric fields. The presence of voltage-sensitive channels,
however, makes the PM easier to be permeabilized as shown in NG108 cells,
although the exact mechanism may differ from activation by long pulses. The time
scale as short as a fraction of 1 ns is sufficient to stimulate these channels, while the
movement of the ion channel’s voltage sensor through several discrete intermediate
conformation steps takes at least 10–100 μs (Hille 2001), which is 4–5 orders of
magnitude longer than the membrane depolarization in subnanoseconds. The
induced membrane potential exists for approximately the duration of the psEP itself,
which is too short for VGCC opening in the standard fashion. Even if a modest
charging component is present (because of the slower low-amplitude components of
the psEP), it is difficult to explain how the membrane would hold this charge long
enough to activate VGCC.
For psEPs, the pulse duration is shorter than the dielectric relaxation time of the
cytoplasm, and the electric field distribution in the cell is mainly determined by the
combined cytoplasm and medium permittivity rather than their conductivity. The
dielectric time constant of a cytoplasm τ = ε0εcp/σcp is approximately 700 ps where
εcp~78 and σcp~1 S/m are permittivity and conductivity of the cytoplasm, respec-
tively. As the duration (500 ps) of the applied pulse is shorter than the τ of the
cytoplasm, the interaction of the pulse and the cell shifts toward the dielectric phase.
The current flowing through the plasma membrane and cytoplasm of the cell upon
exposure to 500 ps EPs is largely limited to displacement current because the
duration of the pulse is so short that no conductance (diffusion of ions is too slow
to occur in this period of time) can happen during its time span. Thus, if conductive
components of the medium and the cytoplasm can be neglected, an electric displace-
ment (D = εrε0E) at the interfaces from the medium to the PM and from the PM to
the cytoplasm is continuous. The electric field in the PM is amplified by a factor of
7 (the ratio of the dielectric constants in cytoplasm/medium (78) and PM (11)). By
other estimates (Vernier 2010), the actual electric field in the PM can be at least
20 times higher. For an electric field of 190 kV/cm, the strength of the electric field in
the membrane may reach 3.8 MV/cm. In molecular dynamics (MD) studies
(a method which yields insights of how pores are created in a clean lipid bilayer),
an electric field near 5 MV/cm was found to be sufficient to create pores (Vernier
et al. 2006b; Delemotte and Tarek 2012). Note that this field was the vacuum value in
the model, and in cell suspension, it can be lowered by a factor of 80 (62.5 kV/cm). If
that is the case, the electric field (190 kV/cm) should be sufficient for PM electro-
poration. However, the MD modeling did not take into account the membrane
proteins, which might increase the stability of the lipid bilayer and increase the
threshold electric field for pore formation. It is likely that the proteins, such as those
that the VG channels consist of, may experience a conformational change and
therefore force the gates to open directly due to the ultrahigh electric fields. This
VG opening could occur before the lipid pores open. The roles of membrane proteins
remains unclear as others have speculated that at least some membrane proteins may
represent sites of increased sensitivity to electropermeabilization (Teissie et al.
2005).
Conclusions
The selective effects of psEPs on the different cells suggest that membrane VG
channels are critical in establishing membrane permeabilization when picosecond
pulses are applied. This was shown by the observations of (a) the lack of response to
psEP in CHO cells, (b) the induction of VGCC-dependent Ca2+ transients in other
cells, and (c) the effective inhibition of such transients by specific VGCC blockers,
all of which point to the opening of VGCC by psEP. Most importantly, VGCC
opening occurred without detectable pore formation and apparently was not caused
by any transmembrane potential loss due to the electroporative ion leakage. The
phenomenon of the opening of VG channels by EP several orders of magnitude
shorter than the channel opening time suggests either a nonconventional membrane
electroporation (where pores are so short-lived that usual methods of pore detection
fail), or of a nonconventional mechanism of channel opening (e.g., by a direct effect
of the applied field on the pore gate, bypassing the shift of the voltage sensor). A
confirmation of either mechanism would have a significant impact on the under-
standing of cell membrane biophysics. These conclusions are drawn based on the
results of using Ca2+ influx as an indicator of membrane pore formation, but there is
a possibility that smaller ions, like sodium, may be possible to go through a
membrane through “smaller pores” and therefore more studies are needed to under-
stand the permeabilization resulted from picosecond pulses.
While the mechanism of psEPs effects remains to be explored, the implication of

the available findings is that cells with VG channels are better targets than those with
no VG channels. Targeting nerves or muscles can be ideal options with the possi-
bility of psEPs to regulate the membrane potentials. The need in high electric fields,
however, poses a significant challenge for the pulse delivery with antennas. Devising
solutions toward lower electric fields will be beneficial for noninvasive and deep
stimulation.
Acknowledgments This work was supported by an R21 grant (1R21EB016912-01A1) from

NIBIB and NIH and an AFOSR MURI grant “Nanoelectropulse-induced electromechanical sig-
naling and control of biological systems,” administered through Old Dominion University.
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1516-7
Experimental Determination of Lipid
Electropore Size 11
Elena C. Gianulis and Andrei G. Pakhomov
Abstract
Electroporation is a process by which nanometer-sized pores are formed in the
plasma membrane by high-power electric pulses (EP). Electroporation has many
applications, including the delivery of genes and drugs into cells, as well as tumor
ablation. Understanding the nature of pores, including size and resealing proper-
ties, under various EP exposure conditions is essential for various applications
of biomedicine and biotechnology. To better understand the properties of
electropores, researchers have employed a variety of experimental approaches
to determine electropore size. First, and perhaps the most widely used, is the
fluorescent detection of membrane-impermeable markers, such as YO-PRO-1
(YP) and propidium (Pr) iodide, into the cell following EP treatment. Similarly,
researchers can track the flow of small inorganic ions, such as thallium (Tl+),
using fluorescent detection techniques. Because Tl+ is much smaller than either
YP or Pr, comparing their relative uptake can provide a measure of the proportion
of small versus large electropores in the membrane. A third, and arguably more
accurate, approach involves the manipulation of cell volume changes following
EP exposure. This approach allows one to estimate the upper limit of electropore
size based on the molecular dimensions of solutes which block cell swelling.
Finally, the pore selectivity and pore size can be analyzed by patch clamp.
Combined, these approaches allow one to experimentally determine electropore
size under different EP treatments.
Keywords
Propidium iodide • YO-PRO-1 • Colloid-osmotic cell swelling • Patch clamp •
Thallium uptake
E.C. Gianulis (*) • A.G. Pakhomov

e-mail: egianulis@odu.edu; egianulis@gmail.com; 2andrei@pakhomov.net; apakhomo@odu.edu

DOI 10.1007/978-3-319-32886-7_114
188 E.C. Gianulis and A.G. Pakhomov
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
Electropore Definition and Major Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Fluorescent Dye Uptake Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
Membrane-Impermeable Dye Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
Fluorescent Detection of Tl+ Uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
Manipulation of Colloid-Osmotic Swelling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
Use of Patch Clamp to Estimate Pore Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Introduction
Upon exposure to high-power electric pulses (EP), the barrier function of the cell
membrane is impaired, referred to as electropermeabilization, allowing the passage
of normally impermeable solutes. It is widely accepted that electropermeabilization
results from the formation of long-lived hydrophilic pores in the lipid bilayer (see
section “Electropore Definition and Major Limitations”) (Neumann et al. 1989).
However, the exact nature of the pores formed – their structure, size, and resealing
properties – remains poorly understood.
Gaining information on the size and number of pores created by a given EP
exposure is important for determining the permeabilization efficiency, physiological
effects, and eventual cell survival following treatment. This information is essential
for many applications of electropermeabilization, such as targeted delivery of drugs
or genes, where it is important to know the upper size limit of molecules that can be
delivered into a cell. It is generally believed that the size and number of pores formed
depends directly on the duration and amplitude of electric pulses delivered. Contin-
ued exposure of a cell to an applied voltage leads to pore expansion. Hence, one can
expect that longer duration pulses (milli- and microsecond duration; msEP and μsEP,
respectively) open larger electropores than those formed by short, nanosecond
duration electric pulses (nsEP). Several studies have estimated a wide range of
sizes of pores formed by msEP and μsEP, from roughly 1 to 100 nm in diameter
(Neumann et al. 1989; Saulis 1999), while those formed by nsEP are thought to be
smaller than 1.5 nm (Bowman et al. 2010). With that in mind, it was previously
demonstrated that increasing the number of nsEP enhances permeabilization by
increasing the number, and not the size, of electropores (Pakhomov et al. 2015).
Thus, understanding the nature of pores formed under various EP exposure condi-
tions has important implications in many biomedical and biotechnology
applications.
There are several approaches to experimentally determine electropore size,
including the use of membrane-impermeable fluorescent dye molecules, tracking
the flow of ions across the membrane by either fluorescent detection or patch clamp,
and manipulation of cell volume changes. In this chapter, the various approaches
11 Experimental Determination of Lipid Electropore Size 189
used to experimentally determine electropore size will be outlined and discussed in

detail.
Electropore Definition and Major Limitations
The formation of lipid pores in cells exposed to EP is the most widely accepted but
not the only possible explanation of how the permeability of the membrane is
increased. It was not until 2016 when the existence of electropores as discrete
physical entities was proven beyond a doubt by direct observations of transport
through individual pores by total internal reflection fluorescence (TIRF) microscopy
(Sengel and Wallace 2016; Szabo and Wallace 2016). Nonetheless, the alternative
explanations include (a) the transition to a leakier (more disorganized) membrane
state without a discrete pore formation (possibly due to a chemical modification of
lipids) and (b) the involvement of other membrane constituents, such as opening or
damage of endogenous ion channels. While the exact mechanisms involved in
electropermeabilization of living cells are not fully understood, viewing it as the
formation of discrete lipid pores of a finite size is a useful concept to explain and
quantify diverse effects of different EP.
Electropores are labile formations: their diameter can change with time, can be
affected by media and by the membrane potential, and is subject to thermal fluctu-
ations. Electropore populations may be heterogeneous in pore size, and any exper-
imental estimations of pore diameter are valid only for a certain subpopulation.
Furthermore, such subpopulations are not necessarily representative of the entire
electropore population. For example, the pore size estimation based on the ratio of
propidium (Pr) and YO-PRO-1 (YP) uptake (section “Fluorescent Dye Uptake
Techniques”) will miss entirely any possible presence of a subpopulation of smaller
pores impermeable to either marker.
Different experimental methods of pore size estimation target different
electropore populations, and therefore may expectedly produce different numbers.
For example, the presence of a small subpopulation of large, Pr-permeable pores
may have no effect on the average pore size determined by the blockage of colloid-
osmotic swelling (Nesin et al. 2011).
Pore diameter estimates based on the size of permeable solutes typically disregard
the effect of hydration on the solute size. The hydrated size of marker dye molecules
and hydration energy are not precisely known and are too difficult to take into
account. It is also not known how much dehydration can (or needs to) take place
when marker molecules or ions penetrate through electropores.
With many caveats associated with the estimation of the absolute electropore
diameter, most studies focus on the relative pore size. Methods described below can
reliably and straightforwardly be employed to check if changing the pulse duration,
number, amplitude, or altering other treatment conditions affect the electropore size.
A change in the ratio of the uptake of two different size markers, such as a Pr/YP
ratio or a Pr/Tl+ ratio, even when measured in arbitrary fluorescence units, is

considered indicative of a change in the pore size. For the given examples, an
increase in the ratio (e.g., in case of stronger or longer EP) indicates that larger
electropores are formed.
The limitations of experimental methods of pore size determination should
always be carefully taken into account, especially when the experimental data are
compared to theoretical model predictions or are used as part of these models.
Fluorescent Dye Uptake Techniques
When an applied electric field reaches a critical voltage, the barrier function of the
cell membrane becomes impaired, allowing the passage of otherwise impermeable
molecules. The integrity of the membrane can be measured by monitoring the
passage of fluorescent membrane-impermeable molecules. Depending on the size
of the electropore(s) formed in the membrane, small ions (such as Tl+) and/or large
molecules (such as Pr or YP) may be able to pass through the membrane (Fig. 1). The
following sections will discuss the use of fluorescent markers to determine
electropore size.
Membrane-Impermeable Dye Molecules
The uptake of fluorescent dye molecules, such as Pr and YP, is among the most
commonly employed methods for detecting electropores. These membrane-
impermeable dye molecules bind nucleic acids inside the cell. When included in
the extracellular solution, they are essentially nonfluorescent and do not change the
fluorescence of cells. However, upon permeabilization of the plasma membrane, the
molecules can diffuse into the cell, whereupon they bind nucleic acids and show
Fig. 1 Relative molecular

dimensions of propidium,
YO-PRO-1, and Thallium
cations. Molecular views in
two perpendicular planes were
generated using Jmol open-
source Java viewer for
chemical structures in 3D
(http://www.jmol.org/). Gray
areas show van der Waals
surface of impenetrable
molecular volume (Creighton
1993). The scale is defined by
vertical and horizontal rulers
(Figure modified from
(Bowman et al. 2010) with
permission from the journal)
bright fluorescence. Thus, permeabilization of the cell membrane is measured as an

increase in cell fluorescence.
Notably, propidium iodide (PI) dissociates in water solutions into the propidium
cation (Pr) and the iodide anion. It is actually the Pr cation that passes through the
membrane, not the entire PI molecule, and causes a change in fluorescence. Hence, it
is the size of the Pr cation, which is approximately 1.5 nm (Fig. 1) (Bowman et al.
2010) that limits the passage of the dye molecule into the cell. Several studies have
used the terms “PI uptake” or “PI fluorescence,” which become misleading when
talking about the size of electropores. For the purposes of this discussion, the more
accurate term “propidium uptake,” or Pr uptake, will be used here. In contrast,
YO-PRO-1 is added to solution as an iodide salt – YO-PRO-1 iodide – which also
dissociates; so referring to YO-PRO-1 uptake is correct terminology.
The Pr uptake assay is a simple, reliable, and highly sensitive method for
detecting electropermeabilized cells. This is because, upon binding nucleic acids
within the cell, Pr fluorescence increases by 20- to 30-fold. In addition to detecting
electropermeabilized and dead or dying cells caused by μsEP and msEP (“conven-
tional electroporation”), Pr uptake can also be used to determine the optimal
exposure conditions that allow the passage of different sized macromolecules into
the cell, which is critical for targeted drug therapy. By comparing its uptake to that of
larger macromolecules, one can estimate pore size under different EP treatments. For
example, Pr uptake can be readily detected upon exposure to 100 μs EP (Rols and
Teissie 1998; Deng et al. 2003; He et al. 2007), while the cell membrane remains
essentially impermeable to macromolecules ranging in size from 10 to 456 kDa.
Thus, the electropores formed by such μsEP are at least as large as the Pr cation but
not large enough to allow the passage of much larger macromolecules. By increasing
the EF amplitude or the pulse duration into the millisecond range (to 15 ms), the cells
become permeable to both Pr as well as to each of the large macromolecules. Hence,
fine tuning of the EP exposure parameters, including adjusting the pulse amplitude
and/or duration, allows for controlled penetration of macromolecules, information
that is essential for the targeted delivery of xenomolecules of various sizes (such as
drugs, proteins, or plasmids). Using Pr uptake as a reference, one can identify the
presence of relatively small Pr-permeable pores versus larger pores (permeable to
both Pr and large macromolecules) in the cell membrane under different EP
treatments.
In contrast to conventional electroporation using msEP and μsEP, early studies
investigating the effects of nanosecond-duration electric pulses (nsEP) reported a
lack of an effect on the cell plasma membrane (Beebe et al. 2003; Deng et al. 2003;
Vernier et al. 2003; Tekle et al. 2005). These researchers observed diverse intracel-
lular effects in cells exposed to nsEP ranging from 10 to 300 ns, including Ca2+
mobilization and the induction of apoptosis, in the absence of any detectable Pr
uptake or with only delayed (secondary) Pr uptake. A conclusion was made that
nsEP can bypass the plasma membrane, leaving it undisturbed, and target intracel-
lular structures directly. However, later studies observed immediate YP uptake into
the cells following nsEP exposure indicating permeabilization of the plasma mem-
brane (Vernier et al. 2006; Batista Napotnik et al. 2012; Pakhomova et al. 2014).
Because YP is slightly smaller than the Pr cation (Fig. 1), it is likely the small size of
the electropores formed by nsEP that limits the passage of the Pr cation into the cell.
However, electropores are not rigid structures and experience thermal size fluctua-
tions (see section “Electropore Definition and Major Limitations”), and sufficiently
intense nsEP exposures can trigger detectable Pr uptake (Batista Napotnik et al.
2012; Pakhomov et al. 2015).
Fluorescent Detection of Tl+ Uptake
Given the dimensions of both YP and Pr, it is still possible that detection of
electropores smaller than ~1 nm in diameter may be missed when using these
fluorescent dye molecules. They can be revealed by the uptake of small ions, such
as Tl+ (Bowman et al. 2010). For detection of Tl+ uptake, cells are preloaded with a
nonfluorescent, Tl+-specific indicator dye and placed in a Tl+-containing bath
solution. In the presence of Tl+, the dye fluorescence increases substantially. Because
there is no Tl+ present in living cells in any appreciable amount and the intact
membrane is impermeable to Tl+, an increase in fluorescence indicates the passage of
Tl+ across the permeabilized plasma membrane into the cell. Hence, fluorescent
detection of Tl+ uptake is a sensitive technique for assaying electroporation. At the
same time, though Tl+ cannot penetrate an intact cell membrane, it is permeant to
voltage-gated K+ channels. Because of this, it is necessary to include K+ channel
blockers, such as tetraethylammonium, in the bath solution to eliminate any Tl+ entry
through channels. Alternatively, one may use cells which do not express endogenous
voltage-gated K+ channels, such as Chinese hamster ovary (CHO) cells; however,
possible Tl+ entry through non-selective cation channels cannot be fully excluded.
Tl+ is much smaller than both YP and Pr, with an approximate Van der Waals
diameter of 0.39 nm (Fig. 1). Tl+ is able to pass through small electropores, which
restrict the passage of both YP and Pr. For example, a single 600 ns pulse of 11 kV/cm
caused an immediate increase in Tl+-dependent fluorescence in CHO cells, while there
was no change in Pr fluorescence (Fig. 2) (Bowman et al. 2010). This indicates that the
electropores formed were too small to allow the passage of Pr, whereas Tl+ could enter
the cell. Only after 10, 600 ns pulses of 11 kV/cm were delivered was there a
detectable and delayed increase in Pr fluorescence. Hence, the detection of Tl+ uptake
before any detectable Pr uptake indicates the presence of small electropores in the
plasma membrane, whose diameter are estimated to be at least ~0.4 nm (to allow the
passage of Tl+) but less than ~1 nm (restricting the flow of YP and Pr).
The above described methods, namely, fluorescent detection of dye molecules
and small ion uptake, are only indicative of electropore size and do not provide
accurate measurements. These methods are most useful for comparison purposes to
characterize electropore size under different EP exposure conditions. The relative
permeability of small ions versus larger dye molecules can provide information on
the presence of very small versus larger electropores, respectively.
In some situations, one can quantitatively estimate the pore diameter based on the
relative permeability of two solutes of known molecular dimensions. When the
Fig. 2 Concurrent detection of Tl+ and Pr uptake triggered by nsEP exposures. Representa-
tive images of Tl+-sensitive FluxOR™ dye emission (top), Pr emission (middle), and DIC (bottom)
of CHO cells. Selected images shown are from before (0s) or at indicated times following nsEP
exposures. The timing and parameters for nsEP exposure are given in the top. 1p and 10p
correspond to one pulse and a train of 10 pulses, respectively. Note fast and profound uptake of
Tl+ after a single pulse but only a weak and delayed uptake of Pr after 10 pulses. Scale bars = 10 μm
(Figure modified from (Bowman et al. 2010) with permission from the journal)
concentration of both test solutes is known, both extra- and intracellularly, and
assuming the size of the electropore formed is stable with each nsEP delivered, the
pore diameter can be extracted from the permeability ratio of the two test solutes
under the excluded volume theory (Dyachok et al. 2010). This is done using the
following equation:
2
PX dpore δx
¼ 2 (1)
PY dpore δy
where PX/PY is the relative permeability of two solutes, X and Y, with diameter δX
and δY, respectively, and dpore is the diameter of the pore. This approach was
employed by Pakhomov et al. (2015) to estimate pore size by comparing the
uptake of YP and Pr. For these ions, the geometric means of the three sides of
the smallest rectangular solid that holds a space-filling model of the ion can be
substituted for δ. Hence, in this scenario, δX = 1.19 nm for YP and δY = 1.26 nm
for Pr, with a permeability ratio (PX/PY) of YP to Pr equal to 2. Using these
numbers, and assuming no significant interaction between YP or Pr with the
walls of the electropore, dpore was calculated as 1.4 nm following a 60 nsEP
exposure. The electropore diameter calculated using this approach corroborated
the predictions from molecular and mathematical models (Levine and Vernier
2010; Son et al. 2014), indicating that nsEP produce electropores with diameters
of ~1 nm.
Manipulation of Colloid-Osmotic Swelling
Though the differential uptake of fluorescent dyes is useful in estimating electropore

size, these methods are limited by dye selection and detector sensitivity. It is possible
that a dye may enter a permeabilized cell, but due to the limited sensitivity of the
detector, the signal will not be recognized. A potentially more accurate approach to
electropore size measurement is based on manipulation of cell volume changes
following EP exposure.
In a physiological solution, cell membrane permeabilization by EP leads to cell
swelling. The EP-induced swelling results from Donnan-type colloid-osmotic pres-
sure (Saulis 1999; Nesin et al. 2011; Sozer et al. 2016). In a simplified scenario, we
can consider biological cells containing both “small” and “large” solutes, which
contribute, respectively, X and Y mOsm/kg to the total intracellular osmolality Zi,
where Zi = X + Y. A typical physiological saline has an osmolality equal to that
inside the cell (Zo) but only contains “small” solutes. Once the membrane is
electroporated, it becomes permeable to small solutes only, while the large ones
are trapped inside the cell. Small solutes will tend to come to a concentration balance
inside and outside the cell. The leakage of small solutes (X) from the cell will not
appreciably change the external osmolality because the cell volume is small. In
contrast, the inflow of small solutes into the cell will increase their intracellular
concentration. If this process is allowed to proceed without water uptake, the
incoming solutes will bring the intracellular osmolality to Z + Y, where Z will be
due to the entering small solutes and Y due to large solutes still trapped inside the
cell. In practice, water inflow and cell swelling will offset the formation of this large
osmolality gradient. As long as the plasma membrane remains permeabilized, the
osmotic force will persist and water will continue to enter the cell. Hence, cell
swelling will continue until electropores are closed or the cell is lysed (a process
referred to as colloid-osmotic lysis).
This mechanism of colloid-osmotic cell swelling can be blocked or even reversed
into shrinking by replacing small solutes in the extracellular solution with an
isoosmotic amount of a larger, pore-impermeable solute. In doing so, small solutes
will flow out of an electropermeabilized cell but will not be replaced by the entry of
extracellular solutes. Hence the intracellular osmolality will be reduced, and osmot-
ically obliged water will flow out of the cell.
Using this approach to manipulate cell volume changes, one can readily estimate
electropore size by testing the impact of differently sized extracellular solutes, such
as polyethylene glycols (PEGs) or neutral sugars (Fig. 3) on cell volume change after
electroporation. Once the size of a test solute prohibits its passage through
electropores, EP-induced cell swelling will be replaced with shrinking. Hence, the
pore size can be “calibrated” based on the molecular dimensions of the test solutes.
Notably, some intermediate-sized solutes may be marginally permeable but will be
slow to diffuse into the cell. As a result, the cell may exhibit a biphasic effect – cell
shrinking followed by cell swelling. Nonetheless, using various solutes of different
sizes in the extracellular solution, one can estimate electropore size under different
EP treatments.
Fig. 3 Minimum molecular

cross-sections of different
sugars and their molecular
structures. Gray areas in the
cross-section views (left
column) limit the
impenetrable molecular
volume. The numbers
following the names of the
sugars correspond to the
geometric mean of the three
orthogonal molecular
dimensions; two values are
given in case of differently
sized isomers (Figure from
(Nesin et al. 2011) with
permission from the journal)
Most studies which utilized the manipulation of colloid-osmotic cell swelling to

estimate pore size focused on “bulk” measurements of cell volume, such as light
scattering by a cell suspension or the eventual lysis of permeabilized cells (Kinosita
and Tsong 1977a, b; Saulis 1999, 2005; Saulis and Saule 2012). They compared the
permeability of cells to small ions versus larger molecules, such as ascorbic acid,
mannitol, and sucrose, following exposure to pulses from 20 μs to 2 ms in duration.
Among the different studies, there was general agreement that under a given EP
exposure, the permeability of cells to molecules of different sizes decreases as the
molecular size increases until a critical size is reached when permeability is blocked.
Increasing the intensity of the EP treatment could increase the permeability to larger
test solutes, indicating an increase in the size and/or number of electropores in the
plasma membrane. Only when the pulse duration was 2 ms was there no difference
between the permeability of differently sized solutes. Hence, electropore size
depends, at least in part, on the duration of the EP. Collectively, these studies
reported that electropores formed by shorter EP (from 20 μs to 100 μs) were no
larger than approximately 1.6 nm in diameter, while those formed by longer EP
(2 ms) were larger.
Though these studies provided important estimations of electropore size and
lifetime, the findings were somewhat ambiguous. Cell volume estimates based on
light scattering methods could be affected by cell blebbing and cytoplasm granula-
tion, as is often seen in EP-exposed cells. Studies that relied on eventual lysis of
electroporated cells to measure cell volume could conveniently be done in erythro-
cytes, because of the presence of hemoglobin, but not in other cell types which lack a
colored pigment. Additionally, this method assumes the lack of pore size changes or
resealing during the 10–20 h prior to data measurement, and hence, bears no
information on the time dynamics of volume changes.
An alternate approach utilizes microscopy to directly measure cell volume
changes immediately following EP treatment (Nesin et al. 2011; Sozer et al.
2016). These studies used PEGs and sugars ranging from 0.7 to 1.9 nm in size to
estimate pore diameter following exposure to 5-, 60-, or 600-ns EP. Cell volume
measurements were made before, during, and after nsEP treatment. The method was
remarkably sensitive, readily distinguishing the effects of adonitol and mannitol,
which have the same cross-section and only differ by one carbon alcohol group
(Fig. 3). Both studies agree that the diameter of electropores formed by such nsEP
was <0.9 nm, based on the size of the sucrose molecule, or <1.24 nm, based on the
size of PEG 400. Notably, there was a small fraction of Pr entry following an
exposure to 600 nsEP, but it had little effect on cell volume. Even with an nsEP
exposure up to 2000 pulses, cell swelling could be blocked by PEG 1000 in the
external solution. Hence, after such an extreme exposure, pore diameter is still
smaller than the PEG 1000 molecule, about 1.9 nm in diameter. These results are
in agreement with the previous estimations of electropore size and are arguably the
most accurate assessment of electropore size to date.
Use of Patch Clamp to Estimate Pore Size
Patch clamp is widely used to compare the permeability of endogenous channels to

different ions and can similarly be employed to study the permeability of
electropores. To be permeable to an ion, a pore needs to be larger than the Stoke’s
diameter of the ion, which puts the lower estimate on the pore size. If the pore size
fluctuates (e.g., because of thermal noise), the pore permeability will be proportional
to the probability of the pore expansion beyond the minimum diameter needed to
admit the ion. Likewise, if a pore is impermeable to a certain ion, this could be the
upper estimate of the pore size. In real life, however, there is a horde of factors other
than the pore size affecting ion permeability. These factors, as well as a variety of
strategies to measure and interpret the permeability data, are extensively discussed in
specialized literature (Adams et al. 1980; Begenisich 1992; Hille 2001; Owsianik
et al. 2006) and are beyond the scope of this chapter. Here one of the applicable
approaches will only be briefly discussed.
In a whole-cell patch clamp mode, the cytosol is dialyzed and replaced with the
pipette filling solution. If the pipette and the extracellular solutions contain different
concentrations of a certain ion, a concentration driving force will be created towards
achieving the same concentration on both sides of the cell membrane. This driving
force can be counterbalanced by applying a potential across the membrane. For
given ion concentrations, this equilibrium potential is given by the Nernst equation:
RT ½ion outside cell RT ½ion outside cell

E¼ ln ¼ 2:3026 log10 (2)
zF ½ion inside cell zF ½ion inside cell
where R is the ideal gas constant, T is the temperature in kelvins, z is the ion charge,
and F is the Faraday constant. The equilibrium potential can be measured experi-
mentally in voltage clamp mode as a reversal potential (the intercept of a
current–voltage curve with abscissa).
If the intracellular and extracellular solutions can be composed to contain a single
permeable ion X outside the cell and a single permeable ion Y inside it (so-called
biionic conditions), the reversal potential Vrev will be determined by the permeability
ratio of these two ions as:

RT Px ½ X o
V rev ¼ ln (3)
zF Py ½ Y i
or

Px ½Y i ðzV rev FÞ
¼ exp (4)
PY ½Xo RT
where Px and Py are the ions’ permeabilities and [X]o and [Y]i are their concentrations
outside and inside the cell, respectively.
However, finding counterions which do not pass through electropores but are
suitable for formulating patch clamp solutions is a problem. Instead, the intra- and
extracellular solutions can be formulated to have the same ions but at different
concentrations.
For example, a pipette solution can be formulated with 150 mM of KCl, whereas
the extracellular solution will contain 30 mM of KCl. The osmolarity will be
balanced by adding approximately 240 mM of sucrose to the extracellular solution;
it is not charged and will not affect the reversal potential. The impact of charged
solutes which are present in relatively small concentrations (such as pH buffers)
usually can be neglected.
Using these solutions, current–voltage (I-V) data for a whole cell configuration
will be collected before and after the application of EP. The subtraction of the “pre-
EP” from the “post-EP” current values will be the current carried through the
electropores. Plotting this current against the applied transmembrane voltage will
generate an I-V curve for electropore current, the intercept of which with the abscissa
will be the electropore Vrev.
In the given example, if electropores are completely impermeable to Cl-, the Vrev
will be equal to the calculated equilibrium potential for K+, which is 41 mV.
Similarly, if the electropores are completely impermeable to K+, the Vrev will be
+41 mV (which is the equilibrium potential for Cl). An experimentally determined
Vrev from –41 mV to 0 mV will mean a higher permeability to K+ than to Cl; the
reversal potential of zero will mean that the ions are equally permeable, and any
value from 0 to +41 mV indicates a higher permeability to Cl. The exact perme-
ability ratio can be determined by a modified Goldmann-Hodzkin-Katz equation
(Begenisich 1992; Hille 2001). In the given example, a reversal potential of 25 mV
corresponds to a fivefold greater permeability of electropores to K+ than to Cl.
Once the relative permeability is established, measurements can be repeated for
different ions with the same counterion, e.g., with NaCl, CzCl, LiCl, tetraethy-
lammonium chloride, etc.; different anions can be compared in the same fashion.
Next, the permeabilities of different ions can be expressed relative to a single ion
chosen as a reference, and their permeabilities can be compared with the ion size
(Pakhomov and Pakhomova 2010). For detailed methods of the permeability data
analyses with respect to the pore size, readers are advised to see publications
specifically focused on this topic (Begenisich 1992; Hille 2001; Owsianik et al.
2006).
Patch clamp is a fast and convenient way to distinguish the smaller population of
electropores (“nanopores”). While larger pores respond to voltage steps simply by a
proportional increase of current, resulting in a linear I-V curve (as one would
reasonably expect from a wide opening in a membrane), nanopores are distinguished
by complex behaviors. The inward rectification can be regarded as a hallmark of
nanopore formation; other nanopore-specific features include current sensitivity, ion
selectivity, and minutes-long lifetime (Pakhomov and Pakhomova 2010). These
behaviors are similar to those of certain endogenous ion channels, and separation
of the lipid nanopore currents from ion channel currents (if certain channels are
activated by EP) remains a scientific challenge to be solved.
Conclusions
This chapter outlined the various approaches for experimentally determining

electropore size. Among the most commonly employed methods is the fluorescent
detection of membrane-impermeable dyes or small ions across the permeabilized
membrane. The relative permeability of small versus large solutes can indicate the
proportion of small versus large electropores, respectively, under a given EP treat-
ment. However, this approach does not provide an accurate measurement of
electropore size. Another approach takes advantage of the colloid-osmotic swelling
which occurs following EP treatment. By replacing small ions in the extracellular
solution with large molecules, cell swelling is blocked and replaced with shrinking.
One can “titrate” electropore size by using differently sized sugars to block cell
swelling. Because of the extreme sensitivity of this method, it is arguably the most
accurate approach for estimating electropore size. Finally, one can distinguish the
smallest electropores, termed “nanopores,” from larger electropores using the patch
clamp technique. Using the same approach, small differences in nanopore size can
be estimated by comparing the relative permeabilities of different ions in the extra-
and intracellular solutions.
Though there are many caveats associated with the estimation of electropore
diameter that must be considered, including the potential impact of hydration of
solutes and electropore size fluctuations, most studies focus on the relative pore size.
Hence, the methods described in this chapter can be used to reliably and straight-
forwardly determine how modifying EP treatments affect electropore size. This
information is valuable both for a general understanding of cell biology, as well as
in many biomedical applications.
Cross-References
▶ Lipid Electropore Geometry in Molecular Models

▶ Lipid Pores: Molecular and Continuum Models
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Measurement of Molecular Transport After
Electropermeabilization 12
Esin B. Sözer and P. Thomas Vernier
Abstract
Measurements of molecular transport after electropermeabilization of biological
membranes have been reported since the early days of electroporation research.
Monitoring molecular transport not only can help reveal underlying mechanisms
but also is critical for applications that aim to deliver molecules into cells via
electric field exposures. However, questions remain open in part because of the size
of the multi-dimensional experimental parameter spectrum that spans a wide range
of electrical and biological factors. Quantitative measurements that address these
questions are essential in guiding development of more accurate and predictive
electropermeabilization models and in optimizing parameter sets for specific appli-
cations. This chapter introduces the main experimental considerations for quanti-
tative measurements of molecular transport after electropermeabilization. A brief
discussion of the basic theoretical equations governing molecular transport is
followed by discussions of electrical exposure systems, measurement methods,
calibration of fluorescence measurements to absolute quantities, and some exam-
ples. The chapter provides perspective on the wide range of experimental design
decisions to be made and emphasizes the need for careful, quantitative, molecular
transport measurements in order to test, improve, and guide models, experiments,
and applications.
Keywords
Electroporation • Electropermeabilization • Molecular transport • Fluorescent
dyes • Quantification of molecular transport
E.B. Sözer (*) • P.T. Vernier

e-mail: esozer@odu.edu

DOI 10.1007/978-3-319-32886-7_115
202 E.B. Sözer and P.T. Vernier
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
Molecular Transport Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Experimental Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Electrical Exposure Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
Fluorescence Methods for Transport Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
Non-fluorescence Methods for Transport Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Molecular Transport Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
Introduction
Molecular transport is one of the first consequences of exposing cells to intense

electric fields, which induce an increase in the transmembrane potential that results
in permeabilization and subsequent passage of normally impermeant molecules
through the cell membrane. Decades of investigative effort have revealed many
components of electropermeabilization that include but are not limited to molecular
transport, such as increased conductance (Pakhomov et al. 2007), osmotic imbalance
(Kinosita and Tsong 1977), lipid oxidation and ATP and K+ leakage (Rols and
Teissie 1990; Saulis and Praneviciute 2007).
Measurements of molecular transport under different conditions help to define
electropermeabilization events and also to guide electroporation technologies for the
delivery of molecules into cells, like electrochemotherapy and gene electrotransfer. In
order to optimize these existing applications and facilitate new ones and to improve
models of electropermeabilization, it is critical to design and perform experiments that
measure quantitative molecular transport after electropermeabilization.
Examples of fundamental questions addressed by these measurements are:
(1) What changes occur in membrane structure and composition that allow or assist
in transport as a result of electropermeabilization?
(2) What is the amount of transport that follows electropermeabilization?
(3) Where on the membrane surface does transport occur?
(4) What is the time course of transport after electropermeabilization?
These questions are typically investigated under varying parameters of interest:
a. Electrical: pulse magnitude, duration, repetition rate, duty cycle, pattern, polarity,
and the number of pulses delivered
b. Biological and environmental: membrane composition, cell type, pulsing
medium composition, temperature, pH, etc.
Since the parameter set is multi-dimensional and large, the current understanding of
these questions is incomplete. Many investigators have addressed the simpler case of
12 Measurement of Molecular Transport After Electropermeabilization 203
small-molecule transport as a first step. Although there is no strict, established size

threshold for referring to a molecule as “small,” for the purposes of this chapter, “small”
refers to a molecular cross section no larger than 2 nm. The exposure conditions
required for the transport of small molecules, and the transport events themselves, are
simpler and more direct than those associated with the transport of larger molecules.
Transport of DNA plasmids, for example, is mediated by complex mechanisms, details
of which are far from understood (Golzio et al. 2002), and it may be that the
mechanisms of transport even for similar small molecules are quite different.
Experiments monitoring molecular transport after electropermeabilization consist
of at least the three parts shown in the block diagram in Fig. 1:
(1) Electrical exposure system, composed of a pulse generator and an applicator

with electrodes
(2) Biological target and the transport molecule
(3) Measurement instrumentation, typically a fluorescence measurement system
Quantitative analysis of raw molecular transport data is based on a careful

calibration of the measurement system, which correlates the response of the instru-
ments to input from samples containing precisely known quantities of the molecular
species under study.
This chapter covers fundamental considerations of measurements of molecular
transport after electropermeabilization, specifically focusing on measurements in
which the amount of material transported is quantified. After a brief review of
theoretical considerations of molecular transport, experimental considerations that
relate to the components of the block diagram shown in Fig. 1 will be discussed,
including electrical exposure systems, with a focus on the most common treatment
chamber designs; fluorescence and non-fluorescence measurement methods; quan-
tification of fluorescence measurements utilizing a fluorescence calibration; and
some examples of quantitative molecular transport measurements showing investi-
gative efforts utilizing a variety of pulse exposures, biological targets, and transport
molecules.
Molecular Transport Theory
Electrically, the cell membrane can be treated as a low permittivity dielectric shell
separating the cytosol and the extracellular medium – an insulator surrounded by
conductive solutions. Living cells typically maintain a transmembrane voltage called
the resting potential, a result of a small excess of negative charge in the cytoplasm.
This charge imbalance is maintained by active and passive constituents of the
membrane such as Na+–K+ exchangers and K+ leak channels.
When the cell is exposed to an electric field of sufficient magnitude and duration,
a porating potential will be induced across the membrane. The magnitude of this
induced membrane potential can be calculated using the Schwan equation, a
Fig. 1 Block diagram of electropermeabilization experiments for molecular transport

measurements
simplified, approximate solution to Poisson’s equation for a dielectric shell in

conductive media (▶ Chap. 59, “Transmembrane Voltage Induced by Applied Elec-
tric Fields” by Kotnik).
For a spherical cell, the induced transmembrane potential, Δψm, at a given
position on the membrane, is
3
Δψ m ¼ Er cos θð1 expðt=τm ÞÞ (1)
2
where E is the electric field in the region where the cell is located, r is the cell radius,
θ is the position angle measured from the center of the cell with respect to the
direction of the field, and τm is the membrane charging time constant, which is
dependent on the conductivities of membrane, extracellular medium, and cytoplasm
and can be defined by the following expression:
rem
τm ¼ σi σe (2)
2d þ rσ m
σ i þ 2σ e
where σ i, σ e, and σ m are the conductivities of cytoplasm, external medium, and
membrane respectively, em is the dielectric permittivity of the membrane, and d is the
membrane thickness.
The Schwan equation can help to conceptualize and to explain many experimen-
tal findings. It must be used very carefully, however, since its validity is based on
assumptions like negligible membrane conductivity that no longer hold after the
onset of membrane permeabilization (▶ Chap. 59, “Transmembrane Voltage
Induced by Applied Electric Fields” by Kotnik).
The porating transmembrane potential (▶ Chap. 2, “Critical Electric Field and
Transmembrane Voltage for Lipid Pore Formation in Experiments” by Teissie), the
value above which the membrane becomes electrically conductive and permeable,
has been estimated for lipid vesicles and cells to range from 200 mV to 1 V.
Once the membrane is permeabilized, transport of molecules across the mem-
brane is initiated. Two main physical mechanisms in effect immediately are diffusive
transport, which causes the particles (both neutral and charged) to migrate from
higher to lower concentrations, and the electrophoretic transport, which affects the
charged (but not neutral) particles due to a gradient in the electric potential.
Fick’s law describes aqueous diffusion in terms of a diffusion coefficient and a

concentration gradient (Hille 2001):
@cs
Js ¼ Ds (3)
@x
where cs is the local concentration of the solute S in mol/m3, Js is the molar flux
density of S in mol/m2s, and Ds is the diffusion coefficient of S.
Diffusion down a concentration gradient is a result of the random motion of
molecules. In one dimension the mean-squared displacement is
x2 ¼ 2Dt (4)
where x is the displacement, D is the diffusion coefficient, and t is the time.

A description of the diffusion of charged particles, however, must account not
only for concentration but also for electric potential gradients. Many fluorescent dye
molecules used in electropermeabilization experiments measuring molecular trans-
port are charged at physiological pH. For example, propidium and YO-PRO-1 are
cations with a +2 charge, and Lucifer yellow and calcein are anions with charge 2
and 4, respectively.
The flow of charged particles traveling across both concentration and potential
gradients is described by the Nernst-Planck equation for electrodiffusion:

d cs Fzs cs d ψ
Js ¼ Ds þ (5)
dx RT d x
where Js is the molar flux density carried by charged species S, zs is the valence of S,
ψ is the local potential in the membrane, T is absolute temperature, cs is the
concentration of S, F is the faraday constant, and R is the gas constant.
The Nernst-Planck equation defines total ionic transport as the sum of two terms:
electrophoretic, due to external electric field, and diffusive, due to the concentration
gradient. In cases where the duration of the molecular transport is orders of magni-
tude longer than the duration of the pulse and the post-permeabilization transmem-
brane potential is considered to be zero, transport will be dominated by the diffusive
component, and this is the perspective that governs most of the studies of small-
molecule transport in the literature.
The preceding analysis, however useful conceptually, fails to explain the sub-
stantial differences in experimentally observed electroporative transport of mole-
cules of similar size (Bartoletti et al. 1989; Canatella et al. 2001). One limitation of
the simple diffusion model arises from the likelihood that pore or permeabilizing
structure cross sections are similar to the sizes of the molecules being transported,
and in these cases the free diffusion assumption must be modified to account for
interactions of the molecules with lipids and other constituents of the membrane.
For example, the pore-mediated diffusive transport equation in Smith and Weaver
(2011) incorporates a hindrance factor for the mechanical interaction of the molecule
with the pore walls and a partitioning factor for the energetic cost of moving a
charged solute into the low-dielectric environment of the membrane interior. Total
diffusive flux for a solute s through pore p, Js,p is expressed as

J s, p pore1 ¼ H K J s (6)
where Js is the unhindered diffusive flux due to a concentration gradient and H and
K are the hindrance and partitioning factors with values between 0 and 1 that account
for some solute-pore interactions.
This model has been used to predict the number of molecules transported after
particular permeabilization events (Son et al. 2014) (Fig. 2). In order to validate and
improve these models, we need experimental studies that report absolute quantities
of transport.
Molecular dynamics models of transport through electric field-induced pores are
based on the physics of the interactions of individual atoms and molecules (▶ Chap. 13,
“Electrical Conductance of Lipid Pores” by Casciola and Tarek). These studies provide
insights into molecular mechanisms of transport after electropermeabilization, and when
combined with experimental observations, molecular simulations can contribute to
further improvements of models of electropermeabilized cell dynamics.
Experimental Methods
Measurement of molecular transport after electropermeabilization requires detection

of the transfer of the molecule of interest either from the extracellular medium to the
cytoplasm of the cells or in the opposite direction. Usually this means monitoring the
a log10(γcal) b log10(γcal) c log10(γcal) d log10(γcal)

t = 10 ns t = 1 ms t = 105 ms t = 20 ms
+ + + +
3 5 7
ncal= 2 ncal= 4.3⫻10 ncal= 4.3⫻10 ncal= 1.3⫻10
– – – –
–10 –9 –8 –7 –6 –5 –4 –3 –10 –9 –8 –7 –6 –5 –4 –3 –10 –9 –8 –7 –6 –5 –4 –3 –10 –9 –8 –7 –6 –5 –4 –3
e log10(γpro) f log10(γpro) g log10(γpro) h log10(γpro)

t = 10 ns t = 1 ms t = 105 ms t = 20 ms
+ + + +
5 7 8
npro= 0 ncal= 3.3⫻10 npro= 3.4⫻10 npro= 7.9⫻10
– – – –
–10 –9 –8 –7 –6 –5 –4 –3 –10 –9 –8 –7 –6 –5 –4 –3 –10 –9 –8 –7 –6 –5 –4 –3 –10 –9 –8 –7 –6 –5 –4 –3
Fig. 2 From Son et al. (2014): Calcein and propidium concentrations at different time points:
t = 10 ns (a, e), t = 1 ms (b, f), t = 105 ms (c, g), and t = 20 s (d, h) for a 40 kV/cm, 10 ns pulse. The
total intracellular amounts of each solute are displayed inside the cell. Red “+” and black “”
indicate the anodic and cathodic sides of the system, respectively
intracellular concentration of a normally impermeant compound before, during, and

after the electric field exposure.
Intracellular concentration changes can be detected by fluorescence methods if
the transport molecule is fluorescent or becomes fluorescent upon crossing the
membrane. Non-fluorescence methods like measurements of cell viability (when
the transported molecule is toxic) or chemical analysis of intracellular or extracel-
lular fractions are also used like in K+ release experiments (Saulis and Praneviciute
2007).
Fluorescence methods rely on the fluorescence mechanism of the dye. Calibration
of fluorescence intensity as a function of dye concentration is relatively simple for
fluorochromes that are innately strongly fluorescent. For dyes that experience an
enhancement of fluorescence upon transport due to interaction with specific mole-
cules, calibration is more complicated. For either case, the fluorescence intensity for
known quantities of fluorescing molecules must be determined in solutions that
replicate the transport environment. Considerations for such calibration procedures
will be discussed later in the chapter.
Some studies of molecular transport at multiple time points rely on microscopic
imaging of only a few cells. Several microscopy methods are utilized, with varying
capacities in detection sensitivity and in resolution, both temporal and spatial.
Microscopic imaging experiments that provide molecular transport kinetics only
for a small number of cells may be limited by sample size in how well they represent
larger populations. Also, the results of some “quantitative” transport studies are
reported in only arbitrary or relative units, which make comparisons to other
experimental work difficult and validation of computational models impossible.
Another group of studies of molecular transport relies on flow cytometry mea-
surements of large populations of cells at fixed time points. Although the time
resolution is limited, flow cytometry measurements can provide quantitative single-
time-point data (Gift and Weaver 1995; Canatella et al. 2001).
The electrical threshold for permeabilization for different molecules can be very
different, which means that the same electrical exposure will result in different
amounts of transport. The total transport is dependent on many variables: concen-
tration gradient, size of the transported molecule, pulse parameters (amplitude,
number, duration, repetition rate, pattern), the conductivity and osmolality of the
extracellular medium, temperature, and cell type.
Even though electrical parameters in theory should be easy to control, practical
aspects of delivery methods and designs can create variability among experiments.
Variability also arises from the complexity of biological responses and from the
natural variations in any set of biological samples.
Biological variability in electropermeabilization can be avoided by using artificial
membranes and lipid vesicles (▶ Chap. 6, “Molecular Transmembrane Transport
with Giant Unilamellar Vesicles (GUVs)” by Rols). Experiments with these systems
have shown that electrical exposure induces permeability, in some cases with pores
visible with optical microscopy (▶ Chap. 6, “Molecular Transmembrane Transport
with Giant Unilamellar Vesicles (GUVs)” by Rols). These observations contribute to
the understanding of membrane-specific electrical and biological conditions for
permeabilizing transport in the absence of complicating biological processes. There

are significant differences in results with electropermeabilized artificial membranes
and vesicles, and living cells, for example, the lifetime of induced permeability.
Artificial membranes remain permeable for only milliseconds after the electrical
stimulus, while in cells the permeabilized state can be sustained for many minutes
(▶ Chap. 4, “Membrane Permeabilization Lifetime in Experiments” by Teissie).
This is one of the indications that biological responses are a big contributor to
transport after electropermeabilization.
Results of transport experiments should be evaluated considering the whole cell
environment. For example, exposure buffers under certain conditions have little or
no effect on transport (Bowman et al. 2010), while under other conditions, the buffer
used can have dramatic effects on sensitivity to electric field exposure, specifically
when the osmolality or the conductivity is varied. Knowledge of the effects of buffer
composition for electropermeabilization experiments is far from well established,
and caution should be used when comparing results from experiments using different
pulsing buffers. Similarly, joule heating which is also dependent on pulsing medium
conductivity, is possible with long-duration pulses and even with short pulses if they
are of sufficient intensity and if enough pulses are delivered. Temperature increases
up to 5 C were reported after 100, 10 ns, 4.4 MV/m pulses delivered at 100 Hz
(Moreau et al. 2015). In addition to biological responses that can vary depending on
temperature, molecular transport is itself a function of temperature, so this variable
must be taken into account.
Table 1 summarizes some of the experimental parameter sets of studies in the
literature that report quantitative molecular transport of small molecules after
electropermeabilization. The purpose is to give a general idea of how wide the
electrical and biological parameter spectra are among these reports. This table
excludes information reported in these papers that were not quantified but reported
qualitatively. It also excludes some very informative molecular transport papers that
report transport in arbitrary or relative units.
Electrical Exposure Systems
Electrical exposure systems are composed of a pulse generator, connecting cables

and control units, and electrodes that deliver the pulse to the biological target. Pulse
generators (▶ Chap. 48, “Basic Concepts of High-Voltage Pulse Generation” by
Redondo) will not be discussed in this chapter, but considerations for choosing the
right system for pulse delivery will be briefly reviewed.
One common treatment chamber is the electroporation cuvette, a simple, com-
mercially available assembly that permits exposing a large number of cells at once.
Cuvettes have some disadvantages, however, such as a requirement for cells in
suspension (which can be circumvented by using a conductive substrate for attached
cells), an operational limit on the earliest possible observations of the cells after
pulse exposure (they must be transferred from the cuvette to the observing platform),
Table 1 Experimental parameter sets from some reports on quantitative molecular transport after
electropermeabilization
Transport Pulse width, Pulse
Source Cell type molecule amplitude number
Mir et al. (1988) NIH 3 T3-A mouse Lucifer yellow 100 μs, 8
embryo fibroblast 0–200 kV/m
Bartoletti et al. Intact yeast: FITC-dextran, 50 μs, 800 kV/m 1
(1989) Saccharomyces propidium
cerevisiae
Rols and Teissie CHO, Chinese hamster ATP 100 μs, 160 kV/ 10
(1990) ovary cells m
Prausnitz et al. Erythrocyte ghost Calcein, 1–2 msa, 1
(1993, 1994) membranes dextran 0–800 kV/m
Gift and Weaver Intact yeast: Calcein 300 μsa, 1
(1995) Saccharomyces 0–500 kV/m
cerevisiae
Canatella et al. DU 145 prostate cancer Calcein 50 μs- 20 msa, 1–10
(2001) cells 0–300 kV/m
Saulis (2005) Erythrocytes Ascorbic acid 20 μsa, 1
200–600 kV/m
Kennedy et al. HL 60 human Propidium 40 μs, 1
(2008) promyelocytic leukemia 200–350 kV/m
Pakhomov et al. CHO Chinese hamster YO-PRO-1, 60 ns, 1.3 MV/m 1–100
(2015) ovarian cells propidium
a
The exposure is an exponentially decaying pulse with the given decaying time constant
and the variabilities associated with the handling of the cells between exposure and
measurement.
Another popular option is rod/point wire electrode systems. These are custom
systems that require careful assembly. An electrostatic field distribution is calculated
to estimate the electric field between the electrodes for a given pulse exposure
(▶ Chap. 46, “Measurement and Characterization of Exposure Systems for High-
Frequency, Ultrashort Pulses” by Leveque et al.). This method is an attractive option
for real-time observations of transport because of the flexibility in the placement of
the electrodes, which can even be removed from the system if necessary during the
measurements. A disadvantage is that these systems typically expose only a few cells
at a time. Figure 3 shows an electroporation cuvette and a custom-made wire
electrode system.
Pulse exposure chambers also can be custom designed for mounting on a
microscope stage so that measurements can be made before, during, and after
pulse delivery. They typically consist of metal electrodes mounted on a transparent
substrate with a fixed inter-electrode spacing that can be filled with cell suspension
or on which the cells can be plated before experiments.
For delivery of electric pulses with rise times less than 1 ns, electrical character-
ization of the delivery system is critical, since frequency dependence of impedance
Fig. 3 (a) An electroporation cuvette. (b) Custom-made tungsten parallel wire electrodes
of most delivery systems may cause mismatches at high frequencies and a distortion
of the electric field delivered to the biological target.
Fluorescence Methods for Transport Measurements
Changes in the intracellular concentration of transported molecules can be detected

by changes in fluorescence when the molecule of interest is either naturally fluores-
cent or becomes fluorescent after interaction with molecules that are exclusively
present on one side of the membrane (▶ Chap. 68, “Fluorescent Indicators of
Membrane Permeabilization Due to Electroporation” by Tina Batista Napotnik).
Some specific examples of fluorescent dyes used for molecular transport experi-
ments are (i) propidium and YO-PRO-1, which fluoresce much more strongly when
bound to polynucleotides (Bartoletti et al. 1989; Vernier et al. 2006; Kennedy et al.
2008; Pakhomov et al. 2015); (ii) calcein (Prausnitz et al. 1993; Gift and Weaver
1995) and Lucifer yellow (Mir et al. 1988), which are fluorescent without binding to
other molecules; and (iii) dyes with fluorescence that is dependent on the concen-
tration of ions that are normally present in the cytoplasm at very low concentrations,
such as Fura-2/AM for Ca2+ (Semenov et al. 2015) or FluxOR for Tl+ (Bowman
et al. 2010). Figure 4 shows uptake of YO-PRO-1 into Jurkat cells after 100, 4 ns
pulses (Vernier et al. 2006).
ATP release is another consequence of electropermeabilization (Rols and Teissie
1990). Release of ATP can be quantified using the luciferin-luciferase assay.
Transport molecules that can be used to detect the smallest possible changes in
permeability of cell membranes are desirable, especially for studies of minimally
permeabilizing pulse exposures of nanosecond or less duration. YO-PRO-1 and Tl+
are sensitive permeability indicators that can be utilized with fluorescence methods,
while cell swelling can be used as a sensitive non-fluorescence method (Vernier et al.
2006; Bowman et al. 2010; Romeo et al. 2013). It is essential to note that the
Fig. 4 From Vernier et al. (2006): YO-PRO-1 uptake after 100, 4 ns, 8 MV/m pulses with 5 μM
YO-PRO-1
detection of permeability is a function of not only the extent of permeabilization of

the membrane to a given molecule but also the sensitivity of the measurement
system. For fluorescence methods, the latter involves detector sensitivity at the
emission wavelength, laser intensity at the excitation wavelength, quantum yield
of the fluorescent molecule, and also considerations of molecular mechanisms that
can affect the linearity of the relationship between fluorescence intensity and amount
of transport, e.g., bleaching and quenching, and kinetics of fluorochrome binding to
polynucleotides or other target molecules. The manipulation of detector sensitivity
can drastically change the minimum detectable transport (Pakhomov et al. 2015).
Assessments of molecular transport data should take into account the detection limits
of the system used for each set of measurements.
To monitor uptake kinetics with fluorescence microscopy, cells are placed in a
treatment chamber on the microscope stage, and a record of fluorescence intensity is
captured, beginning before the pulse exposure and continuing for the time set by the
protocol. Control groups with unexposed cells are recorded similarly. In every
experiment, effects like bleaching and quenching that affect fluorescence intensity
independent of the number of fluorescing molecules must be measured carefully and
appropriate corrections applied when necessary.
For flow cytometry, measurements of permeabilization cells are exposed to the
electric field in a cuvette and then transferred from the pulsing environment to the
flow cytometer, which introduces several handling steps and a potentially variable
time between pulse exposure and observation and significant sources of error
associated with handling steps.
Measurement of large molecule transport can be carried out using small-molecule
fluorescent tags, covalently attached to the large molecule under study. Plasmid
DNA, for example, can be tracked in this way.
Quantification of Fluorescence Measurements in Absolute Quantities

Quantification is important for validation of models and comparisons of independent
experiments. But truly quantified measurements of molecular transport across living
cell membranes are challenging. Fewer than 15 published reports offer quantified
measurements of the absolute amount of molecules transported in or out of the cell
after electropermeabilization.
Quantitative transport measurements using fluorescence microscopy requires cali-
bration – a systematic, reproducible correlation of the fluorescence intensity detected
from a sample with the number of molecules per unit volume (concentration) in the
sample. Calibration can be achieved in several ways. The most direct way, and the
simplest in concept, is to produce calibration curves of fluorescence intensity versus
known concentrations of the molecules generating the fluorescence. This method is
specific to the instrumentation used and to the specific settings of the illumination
power and detector gain. For calibration of the fluorescence intensity of a fluorescent
indicator of electropermeabilization, one must reproduce the environment where the
emission will occur (the interior of the cell), add known quantities of the dye to this
environment, and measure the fluorescence intensity for each known concentration of
the dye. If other parameters are not limiting, the intensity should be directly related to
the concentration. For these measurements, a confocal microscope is essential, since
the fluorescence intensity that is collected comes from a fixed volume.
Calibrated fluorescence measurements of concentration of a target species are
also possible with ratiometric fluorescence methods, where shifts in the fluorescence
excitation and emission maxima are correlated with the concentration of the target
species. As a result, the change in concentration can be measured using the change in
ratio of fluorescence intensities at two different wavelengths. Calcium ion concen-
trations, for example, can be relatively easily obtained from ratiometric fluorescence
measurements using the dye Fura-2.
Some examples of fluorescent intensity calibration from the literature are given
below. Mir et al. (1988) calibrated the fluorescence intensity of Lucifer yellow by
adding known amounts of dye to a sonicated cell homogenate, a medium similar to
the cytosol. Lucifer yellow is natively fluorescent and does not require binding to
any other substance, and quantitative measurements of dye concentration can be
obtained from confocal microscopes or spectrofluorometers.
Bartoletti et al. (1989) used fluorescein-tagged beads for quantitative flow
cytometry measurements. Two calibrations are required for this method: (1) correla-
tion of the fluorescence intensity of the beads with the number of attached fluores-
cein molecules and (2) correlation of the fluorescence of FITC-dextran suspensions
with the calibrated fluorescein fluorescence obtained in the first step.
The fluorescence of dyes like propidium and YO-PRO-1 is greatly enhanced
when these molecules bind to nucleic acids. Calibration curves are obtained by
measuring the fluorescence of known concentrations of the dye in a medium that
contains an excess of polynucleotide binding sites (Bartoletti et al. 1989; Kennedy
et al. 2008; Pakhomov et al. 2015). Figure 5 shows one such calibration (Pakhomov
et al. 2015), where fluorescence intensity changes linearly with dye concentration.
Protocols for quantitative measurements of transport using fluorescent small
molecules must include corrections for cellular volume changes that occur during
the observations. Since concentration is the number of molecules per unit volume, a
change in intracellular concentration can be due either to transport or to a change in
cell volume. Cells can swell or shrink post-exposure as a result of permeabilization-
induced osmotic imbalance; the change in volume of each cell must be taken into
account in calculations of intracellular concentration of the fluorescent target
species.
Non-fluorescence Methods for Transport Measurements
Non-fluorescence methods of quantified measurement of transport are less common,

but they contribute to the understanding of post-permeabilization transport processes
that cannot be monitored quantitatively by fluorescence methods.
Cell swelling, a consequence of the osmotic imbalance that follows membrane
permeabilization (Kinosita and Tsong 1977), has been proposed as a sensitive
indicator of electropermeabilization (Romeo et al. 2013). Although the electro-
porated membrane is permeable to inorganic ions and small molecules, large intra-
cellular solutes remain impermeant, which results in water influx as the system
approaches a Gibbs-Donnan equilibrium.
Swelling can be blocked or reversed by adding to the extracellular medium large
molecular weight impermeant solutes to counterbalance the intracellular osmotic
pressure. This method has been used to estimate pore size (▶ Chap. 11, “Experi-
mental Determination of Lipid Electropore Size” by Gianulis, E., Pakhomov, A.). If
solute molecules can pass through pores in the membrane, they cannot block osmotic
swelling by offsetting the impermeant intracellular solute concentration, because
their intracellular and extracellular concentrations will become equal. By this logic,
Fig. 5 From Pakhomov et al. (2015), published with permission. Calibration of the emission of
propidium (left panel) and YO-PRO-1 (right panel) in CHO cell lysates. Plots show dye fluores-
cence intensities in arbitrary units changing linearly with dye concentration for most of the range
measured. For a discussion of possible reasons for the nonlinearities, refer to the report itself
extracellular solutes that block swelling must be larger than the pores considered to
be responsible for the membrane permeability.
Saulis and colleagues have reported quantitative measurements of ascorbic acid
transport into erythrocytes using electron paramagnetic resonance (Saulis 2005), and
they have measured the electroporative release of K+ with potassium-selective elec-
trodes (Saulis and Praneviciute 2007). Quantitative chemical methods for measuring
intracellular concentrations of transported species require isolation of cytosol contents
and are in general more prone to error than the direct optical methods mentioned earlier.
Molecular Transport Results
Early quantitative studies of molecular transport after electropermeabilization were

designed to determine whether electroporation can deliver or extract known quan-
tities of molecules into cells in a controlled fashion. For example, one study used a
luminescence assay to measure ATP released from electropermeabilized cells (Rols

and Teissie 1990). Fluorescence flow cytometry enabled measurements of influx and
efflux of molecules of various sizes in large populations of cells, revealing a general
inverse correlation of transport with molecular size and significant cell-to-cell
variability. These findings supported the view that electroporation creates small
openings or pores in the cell membrane that allow or block transport based on the
size of the molecule (Bartoletti et al. 1989; Prausnitz et al. 1993; Gift and Weaver
1995; Canatella et al. 2001).
Molecular transport measurements were also used to study the kinetics of
post-exposure loss and subsequent restoration of the membrane barrier function
(▶ Chap. 4, “Membrane Permeabilization Lifetime in Experiments” by Teissie).
Influx of the normally impermeant fluorescent dye propidium was investigated
with sensitive, high time-resolution fluorescence microscopy (Pucihar et al. 2008).
The results showed transport beginning within 60 μs after the initiation of a porating
electric pulse. Permeabilization lifetime has been monitored by adding impermeant
substances at different times after electroporating pulse exposure (Saulis 2005;
Pakhomov et al. 2007).
Quantitative material transport data for different impermeant species can also be
correlated with electroporation models and experimental methods like those based
on colloidal osmotic swelling (▶ Chap. 11, “Experimental Determination of Lipid
Electropore Size” by Gianulis, E., Pakhomov, A.) to provide information about the
size distribution of the permeabilizing structures formed during and after pulsed
electric field exposure.
Biologically effective transport of plasmid DNA into cells involves more than just
crossing the plasma membrane. To achieve more than transient expression, the genetic
material must then move through the cytoplasm into the nucleus and integrate into the
chromosomal DNA. The efficiency of the procedure depends on pulse parameters and
exposure conditions that are presently largely empirically determined and beyond the
scope of this chapter, but some key steps of the transport mechanisms that are only a
part of this complex process have been established. Plasmid DNA, a polyanion,
migrates in the pulsed electric field and interacts with the permeabilized membrane,
forming localized aggregates, which become inaccessible to nucleases in the extracel-
lular medium. These aggregates remain associated with the membrane for several
minutes after pulse exposure. After 30 min, they can be found in the cytoplasm, and
after 24 h they are in the nucleus (Golzio et al. 2002). Interactions of these large
molecules with the membrane for many minutes are another indicator of involvement
of biological processes in post-electropermeabilization transport.
The quantitative electroporation transport data in the research literature is derived
from experiments covering a wide range of exposure conditions and pulse parame-
ters, including million-fold differences in pulse durations. On the one hand, the
transport described in most of these reports is qualitatively consistent: an electric
field amplitude threshold that is a function of pulse duration, cell size, medium
composition, etc. is observed; beyond this threshold higher fields result in greater
molecular uptake. Larger molecules require longer pulse durations, and longer
exposures lead to more transport. On the other hand, it is reasonable to expect that
there will be differences in transport for bioelectrical interactions that last a few
nanoseconds versus those that last for milliseconds (Gowrishankar and Weaver
2006; Semenov et al. 2015).
Conclusions
Quantitative measurements of molecular transport after electropermeabilization are

essential for the development of rigorous, physics-based models of electro-
permeabilization and for optimizing applications of electropermeabilization.
Experimental design for these quantitative studies involves decisions on param-
eters, conditions, and procedures that are interrelated and subject to technological
constraints. These include electrical exposure delivery, fluorescence and
non-fluorescence measurement methods, and procedures for calibration of fluores-
cent measurements.
Analysis of experimental data for presentation of quantitative results must take
into account the specific biological and environmental conditions under which the
observations were made, including the electrical field at the target, buffers and
growth media, temperature, and physical handling of electropermeabilized biologi-
cal material. This is a challenging multidisciplinary undertaking that will benefit
from new perspectives and approaches in addition to what was briefly presented in
this chapter.
Cross-References
▶ Basic Concepts of High-Voltage Pulse Generation

Experiments
▶ Measurement and Characterization of Exposure Systems for High-Frequency,
Ultrashort Pulses
▶ Molecular Transmembrane Transport with Giant Unilamellar Vesicles (GUVs)
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Electrical Conductance of Lipid Pores
13
Maura Casciola and Mounir Tarek
Abstract
Electroporation (EP), also known as electropermeabilization, is a widely used
technology employed primarily for intracellular delivery of drugs and genes,
likewise for tissue and tumor destruction.
The majority of the studies on EP hypothesize that the boost in permeability of
cell membranes, following the application of pulsed electric fields, to low- and
non-permeant molecules results from the formation of long lasting lipid defects,
termed pores. However, the exact nature (morphology, size, number, etc.) of these
pores is still unknown. Determining the pore size and their quantity under certain
condition is crucial to understand, and eventually improve, the permeabilization
efficiency.
The main limit to the direct experimental observation of “lipid pores” is their
nanometer range size, not appreciable by today’s conventional techniques. A way
to indirectly relate experimental observations to the size and numbers of pores,
relying on simplified analytical formulas, is measuring the change in electrical
conductance after the electropulsation.
The investigation of EP with atomic detail has been possible, thanks to
molecular dynamics (MD) simulations. In the past 10 years, extensive MD
studies have been carried out to model the effect of PEFs on lipid bilayers, site
M. Casciola (*)
e-mail: mcasciol@odu.edu; casciolamaura@gmail.com
M. Tarek
Université de Lorraine, UMR 7565, Vandoeuvre les Nancy, France
CNRS, UMR 7565, Vandoeuvre les Nancy, France
e-mail: mounir.tarek@univ-lorraine.fr

DOI 10.1007/978-3-319-32886-7_116
220 M. Casciola and M. Tarek
of pore formation in cell membranes. Thanks to this method, the evaluation of

structural (size) and electrical (conductance) properties of pores formed under
conditions comparable to experiments is possible, providing information hardly
accessible by direct observations.
This chapter provides an overview of MD studies on the electrical conduc-
tance of lipid pores and how they relate to experiments.
Keywords
Electroporation • Lipid bilayer • Pore size • Electrical conductance
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
MD Modeling of Membrane Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
Lipid Pore Stabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Pore Radius and Electrical Conductance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Introduction
Typical signs indicative of changes in the integrity of the cell membrane following
the application of pulsed electric fields (PEFs) are evident increases in the membrane
ionic conductance, in the molecular uptake and in the release of cytosolic contents
from the cell (Mir et al. 1988). Indeed, the application of PEFs induces the formation
of hydrophilic pores in the lipid bilayer of the plasma cell membrane, facilitating the
transport of otherwise low-permeant molecules.
Several electroporation (EP)-based technologies exploit the increase in permeabi-
lization of the plasma membrane to deliver drugs or genetic material into cells
(Breton and Mir 2012). Pore formation is likely not the only mechanism ascribable
to the boost in cell permeabilization; however, it is believed to play a significant role
in the transport of molecules across cell membranes.
The transport rate of a given molecule is, therefore, affected by the nature of “lipid
pores.” To evaluate, and eventually improve, the efficacy of the aforesaid applica-
tions is essential to quantify the size, the conductive properties, and the selectivity of
lipid pores formed under EP.
The great deal of experimental work on cells and membranes models devoted to
the structural characterization of lipid pores did not provide a clear and consistent
description of the pore size distribution (Pakhomov et al. 2015).
Indirect experimental evidences (Sengel and Wallace 2016) and in silico inves-
tigations (Tieleman 2004; Tarek 2005; Levine and Vernier 2010; Casciola and Tarek
2016) confirm the electropore presence; however their direct observation by con-
ventional techniques is limited by their nanometer range size and by the short
dynamics of their formation (ns). Such restriction implies that with today’s
13 Electrical Conductance of Lipid Pores 221
experimental techniques, it is possible to evaluate the lipid pore size exclusively by

indirect relation of the pore radius with measures of membrane conductance or of the
uptake of fluorescent dyes of different dimensions that do not pass through intact
membranes. However, indirect approaches suffer of specific shortcomings.
When the pore size is established by the selective uptake of smaller dye molecules
and ions (e.g., thallium ions (Bowman et al. 2010), YO-PRO, propidium (Pakhomov
et al. 2015), sugars, polyethylene glycols (Nesin et al. 2011), and bleomycin (Silve
et al. 2012)), the upper limit of the radius is set by the biggest molecule that can be
uptake and detected. Using this approach, estimates were obtained mainly on cells
exposed to nsPEFs of duration up to 600 ns. These investigations suggest that the
population of pores created under these conditions is composed by a high number of
small stable pores with a lifetime on the order of minutes, characterized by an
effective pore diameter not exceeding 1–1.5 nm.
The shortcoming related with such measurements is attributable to possible
interactions between the molecules and the bilayer interface. If any “long” lasting
interaction occurs, although the transport of the molecule through the pore takes
place, the time needed to the given molecule to dissociate and to be detected in the
cytosol may be not negligible. Sugars, despite are neutral molecules, are known to
change membrane properties due to strong interactions with lipids at the head group
level as result of hydrogen bonding. On the other hand, dyes are charged molecules
and can thus affect the pore morphology through electrostatic interactions and
potentially produce changes in the pore size.
As a consequence, this methodology might not be fully representative of the lipid
pore properties as directly induced by electric pulses.
Another approach for the evaluation of the pore size relies on measurements of
membrane conductance deduced by the current/voltage relationship.
Most of such results were obtained by chronopotentiometry, i.e., applying a
constant current to planar lipid bilayers (Kalinowski et al. 1998). Under this condi-
tion, the pore diameter varies from 3.8 to 12 nm, depending on the current intensity.
Other studies based on conductance measurements suggested that the pore diameter
can range from few to ~100 nm, depending on the experimental conditions. The
latter were carried out on dense cell suspensions during exposures to trains of 100 μs
pulses (Pavlin et al. 2005) and on single cells using patch clamp techniques (Krassen
et al. 2007).
With respect to monitoring the transmembrane transport of molecules, conduc-
tance measurements provide a more sensitive and less perturbing method to charac-
terize the properties of lipid pores. However, the main limit of using conductance
measurements to estimate the pore size is that continuum models must be used to
obtain the pore radius from the experimental data. Therefore, an accurate and
quantitative characterization is possible only if the theoretical model is valid and
reliable. The most common analytical models employed derive from the continuum
Poisson–Nernst–Planck theory, which intrinsically impose simplified assumptions
about the pore geometry and the effective conductivity of the electrolyte inside the
pore (Rems et al. 2016). This model relies on parameters that are in general arbitrary
or approximate, leading to possibly inaccurate predictions.
Molecular dynamic (MD) simulations are an extremely powerful tool of accom-

paniment and support to continuum models and experimental observations, provid-
ing inaccessible atomic insights that allow to shed light on molecular mechanisms of
the EP process.
MD simulation protocols, indeed, were devised to study several aspects of EP
(see Casciola and Tarek 2016 for a recent review), e.g., the mechanisms of pore
formation including the role of the interfacial water, of different lipids and complex
bilayer composition, as well as the pore characterization in terms of size and
conductance and the transport small molecules.
MD simulations give access to a direct and independent characterization of the
pore conductance, pore size, and pore geometry, which is impossible to achieve with
current experimental techniques and that can be used to validate and, eventually,
improve continuum models.
This chapter describes in section “MD Modeling of Membrane Electroporation”
on how the presence of an electric field is accounted in MD simulations.
Section “Lipid Pore Stabilization” is devoted to the protocols implemented to
stabilize lipid pores created by the methods presented in section “MD Modeling of
Membrane Electroporation” Finally, section “Pore Radius and Electric Conduc-
tance” treats what is known from MD simulation on the size and electrical conduc-
tance of stabilized lipid electropores.
MD Modeling of Membrane Electroporation
As biological membranes are characterized by very complex structures, being com-

posed by hundreds of different lipid species and proteins, most MD studies focused
their attention on lipid bilayers in their biologically relevant liquid–crystalline state.
From both a theoretical and an experimental perspective, zwitterionic phosphatidyl-
choline (PC) lipid bilayers constitute the best characterized systems. Despite their
simplicity, bilayers built from PC lipids represent remarkable test systems to gain
additional insight into the physical properties of membranes (Fig. 1).
MD simulations allow to perform in silico experiments under conditions similar
to EP experiments, modeling the presence of applied electric fields. In general, the
application of such electric fields to cells results in an increase of their transmem-
brane (TM) voltage, Um (Tieleman 2004; Tarek 2005). When the latter exceeds a
specific threshold, an intense local electric field is generated at the membrane level
leading to pore formation.
Electric field protocol. An external electric field is introduced in the direction
perpendicular to the bilayer z into the system as an additional force acting on all the
charged atoms. In such a scheme, the reorientation of water molecules located at the
solution/bilayer interface induces a TM voltage equal to Um ¼ Lz Eapp, with Lz the
length of the simulation box along z (Tieleman 2004; Tarek 2005).
Charge imbalance method. In the charge imbalance method, a TM voltage is
generated by submitting lipid bilayers to ionic salt concentration gradients
Fig. 1 Two solution baths are separated by a lipid bilayer and, in the charge imbalance scheme, a
vacuum slab. Left panel, electric field applied protocol: all the i atoms of the system are subject to a
force F = qiEapp proportional to the electric field applied and to the carried charge i. Right panel,
charge imbalance protocol: a net difference of charge is imposed by displacing a given amount of
ions from one bath to the other. The vacuum prevents the exchange of ions through the 3D periodic
boundary conditions and the collapse of the charge imbalance. The POPC head groups are shown as
cyan and white beads; the tails are shown as purple sticks; sodium and chloride ions are colored in
yellow and gray (right panel); water is transparent
considering two solution baths separated either by two bilayers (Gurtovenko and
Vattulainen 2005) (the double bilayer scheme) or by a bilayer and a vacuum slab
(Delemotte and Tarek 2012) (the single bilayer scheme). In this case the TM voltage
is Um = Qs/Csp with Qs being the charge imbalance per unit area and Csp the specific
capacitance of the bilayer (Delemotte and Tarek 2012).
Regardless of the method used, both charge imbalance and electric field provide
converged results concerning several properties of the membrane which are practi-
cally indistinguishable from each other (Melcr et al. 2016) and that lipid bilayer
electroporation takes place within identical time scales (namely, few nanoseconds),
provided that the TM voltages produced are the same (Vernier et al. 2006; Delemotte
and Tarek 2012).
Lipid Pore Stabilization
When dealing with the characteristics of electropores (e.g., size, conductance,

transport of molecules), one would expect the pore to be in this energetically
favorable state that could correspond to a stable configuration.
The two protocols presented, however, suffer from a major restriction: the
impossibility of maintaining a steady pore. In the external electric field protocol,
when the field is maintained at the poration value, the pore tends to expand above the
dimension of the simulation cell box leading to the breakdown of the bilayer, while
in the charge imbalance protocol, after EP occurs, the initially imposed difference of
charges decreases significantly within several tens/hundreds ps (depending on the
system size) due to an exchange of ions through the pores in the bilayer. The
decrease of the charge imbalance results in a TM voltage drop, which ultimately
leads to the pore collapse or resealing.
To overcome this limitation, both protocols were modified. In the case of the first
protocol presented, the pore is created by applying an intense electric field that is then
reduced to lower values resulting in stable, size-controlled electropores (Fernández
et al. 2012). The size of the pore is a function of the value of the stabilizing E field, i.e.,
the steady-state pore size depends neither on the initial porating E field nor on the exact
time the field was reduced, provided that a hydrophilic pore has been formed and that
the field is reduced before finite size effects appear. The sustaining electric field,
however, should not exceed too “high” or “low” values to avoid instability of the
pore, i.e., either its breakdown or collapse. The values of the TM voltages are system
dependent, i.e., they are strictly related to the composition of the bilayer.
To maintain the initial charge imbalance constant, the “swapping” protocol can be
used (Casciola et al. 2016). Briefly, the protocol works the following way: the
program estimates the number of ions in the two solution baths, and if this number
differs from the initial setup, an ion of one solution bath is exchanged by a water
molecule of the other solution bath. In this way, the difference of charges is
maintained during the simulation despite the pore opening and results in a constant
TM voltage across the lipid bilayer that stabilizes the pore radius to a value
dependent on the chosen charge imbalance (Fig. 2).
Pore Radius and Electrical Conductance
From MD simulations, the temporal evolution of the structure of lipid pores can be
monitored, and its radius and electrical conductance can be calculated directly and
independently.
Pore radius evaluation. The pore radius is often calculated through algorithms
based on the following concept: the center of mass of the membrane is evaluated and
positioned at the center of the simulation box. Then, the region of interest where to
calculate the pore radius is individuated measuring the average position of the z
coordinates of a given atom used as reference, i.e., generally the phosphorus atoms
of the lipid head groups; see Fig. 3a. Once the region of interest is set, the simulation
box is divided in slices (bins), usually 0.1–0.3 nm thick, along the perpendicular to
the bilayer (z axis) as shown in Fig. 3b. For each bin the radius is computed either
taking into account the distances between the most distant water molecules in the
plane parallel to the bilayer (x and y directions) or the distances between the most
distant lipid phosphorous and nitrogen atoms in plane parallel to the bilayer (x and y
Fig. 2 (a) Charge imbalance scheme; (b) water intrusion, the charge imbalance applied produces a
TM voltage that if it is high enough induces water percolation through the lipid core of the bilayer;
(c) the pore becomes conductive and ions can be exchanged between the two baths. Each time an
ion of one species crosses the pore from reservoir (a–b), another ion of the same species in B is
swapped with a water molecule in (a), such that the charge imbalance is maintained; (d) a constant
charge imbalance allows for a constant TM voltage and radius. The POPC head groups are shown as
cyan and white beads; the tails are either shown as purple sticks or omitted for simplicity; sodium
and chloride ions are colored in yellow and gray; water is transparent
directions) (Fernández et al. 2012; Ho et al. 2013) or as the largest possible sphere
which can fit the reference atoms, e.g., phosphorous and nitrogen of the lipid head
groups, without overlapping with van der Waals radii of any atom species previously
set (see Fig. 3c) (Casciola et al. 2016).
This procedure is repeated with a given time interval such that eventual pore
radius fluctuations are accounted.
Pore electrialc conductance evaluation. The lipid pore conductance, G, is defined as
the ionic current I flowing through the pore over the TM voltage applied, Um, G = I/Um.
The ionic current can be estimated counting the number of ions crossing the pore
at given intervals of time while the TM voltage as the difference between the
electrostatic potential measured at the lower and the upper baths.
The electrostatic potential Uz along the normal to the bilayer can be calculated by
solving Poisson’s equation (1), i.e., derived as double integral of ρz, the volume
charge density distribution, being ε0 the vacuum permittivity:
ð ðz
1
U z ¼ U ðzÞ U 0 ¼ ρz ðz00 Þdz00 dz0 (1)
e0
0
Fig. 3 (a) Scheme of a porated lipid bilayer. The region of interest where the algorithm calculates
the pore radius is indicated by dashed balk lines; (b) bins at a given step of the pore radius analysis
represented in solid blue boxes; (c) other possible definition of pore radius, as the largest possible
sphere which can fit the reference atoms without overlapping with van der Waals radii of any atom
species previously set. The POPC head groups are shown as green beads; the tails are shown as gray
sticks; water is in white and red beads
As reference, U0 was set to zero in the lower bulk solution. Practically, the electro-
static potential can be calculated before or after the pore formation by dividing the
system into slices along the z axis, summing the charges in each slice and integrating
the resulting charge distribution for N configurations spread out over simulation run.
A first attempt to link experimental evidences of pore conductance and radius
estimation using linear rising current technique combined with MD simulations was
carried out by Kramar et al. (2012). Their findings suggest that the opening and
closing of a single pore of conductance in the 100-nS scale would be possible for a
pore diameter of 5 nm.
More systematic MD investigations, conducted with either the electric field
(Ho et al. 2013) or the charge imbalance (Casciola et al. 2016) protocols, allowed
to quantitatively characterize the lipid pore radius and electrical conductance under
conditions similar to experiments.
Continuum models of EP (Neu and Krassowska 1999) predict the creation of
stable pores at moderate TM voltages. Once the energetic barrier for pore formation
is overcome, the formed hydrophilic pore stabilizes at a radius corresponding to the
local minimum in the pore-free energy landscape. The position of this minimum with
respect to the pore radius is also expected to shift with increasing TM voltage,
resulting in larger stable pores. If the TM voltage exceeds a certain value, however,
the minimum is expected to disappear leading to the irreversible expansion of the
pore and the ultimate rupture of the bilayer.
In general agreement with this hypothesis, MD results show that in the TM
voltage range, considered pores remain stable for tens of nanoseconds, allowing
for their geometrical and electrical characterization.
In Ho et al. (2013) all simulation schemes are bilayers composed of 128 POPC
lipids (64 per leaflet) in a 0.15 M solution of either NaCl or KCl, corresponding to an
initial system box size of approximately 7 7 10 nm.
In the porating external field Ep 400 MV/m, an electropore appears in both NaCl
and KCl POPC systems within 2 ns. In this high electric field, the pore expands until
finite size effects dominate the simulation and the bilayer becomes disorganized. To
stabilize the pore, the field is reduced to a lower sustaining value Es when the pore
Fig. 4 Pore radius versus time from selected conductance simulations. At t = 0 the external
porating field is reduced to the values in the legend. Systems containing NaCl in (a) and KCl in
(b) (Reproduced with permission from Ho et al. (2013), Copyright (2013) American Chemical
Society)
radius reaches approximately 1 nm. The latter value is arbitrary since the initial
configuration does not influence the steady-state properties of the electropore, as
reported in Fernández et al. (2012).
For Es of 50, 75, and 100 MV/m, electropores are stabilized in three independent
simulations for at least 200 ns in both NaCl and KCl systems. Figure 4 shows the
evolution of the pore size after the external field is reduced to Es. The radius (initially
about 1 nm in each case) reaches a field-stabilized value within 30 ns. The stabilized,
time-averaged pore radius for each Es is calculated from the last 50 ns of each
simulation.
Lower and higher values of Es lead either to the bilayer destabilization or to the
pore closure.
Table 1 reports the values of the pore radius, cation, and anion molar conductance
for the system containing NaCl and the one containing KCl at the different Es. In the
range of the electric field applied, the radius varies from 0.68 0.03 to
1.50 0.03 nm in the NaCl and from 0.77 0.04 to 1.56 0.05 nm in the KCl
system. For a given value of Es, the pore radius for the NaCl system is 140 pm
smaller than for the KCl system, probably a result of the more extensive Na+ binding
to POPC, which decreases the area per lipid and increases the pore line tension.
The conductance of an electroporated lipid bilayer for a given ion species is
determined by the native ion conductance, by the ion–pore interactions, by the TM
voltage induced and by the radius of the pore, which can be tuned with the sustaining
field Es. The ion conductance increases with pore radius, as one would expect
(Table 1), but Na+ conductance is significantly less than K+ conductance for a
given radius. This is a consequence of stronger Na+ binding to the interface, which
results also in the nonlinear relation between pore radius and Na+ conductance at
small values for the pore radius.
At the smallest pore radius measured in the KCl system (0.77 nm), the ratio of the
cation conductance, GK/GNa, is approximately 5.6, a reflection of the stronger
Table 1 Lipid pore properties in POPC bilayers containing 0.15 M of either NaCl or KCl
ES (MV/m) R (nm) GCATION (nS/M) GANION (nS/M)
NaCl 50 0.68 0.03 0.95 0.47 6.43 0.85
75 1.06 0.06 3.76 1.15 23.17 3.39
100 1.50 0.03 9.43 0.82 37.31 2.28
KCl 50 0.77 0.04 5.28 0.28 4.37 0.56
75 1.20 0.02 14.92 0.57 15.71 0.40
100 1.56 0.05 22.87 1.00 23.64 0.28
interaction between Na+ and the phospholipid head groups in the pore wall relative
to K+. At the largest pore radius measured in the NaCl system (1.5 nm), GK/GNa is
only 2.3. As the pore size increases, a larger and larger fraction of the sodium
ions traversing the pore sees only bulk like water and is not slowed down by pore
wall binding; thus the Na+ conductance increases relative to the K+ conductance and
GK/GNa decreases. This ratio approaches 1.8, the value in bulk water simulations, as
the pore radius increases further. K+ and Cl interact very little with the pore wall;
their conductance in lipid electropores is similar to those in bulk water.
In Casciola et al. (2016) authors applied constant charge imbalances (ΔQ) to a
porated lipid bilayer composed of 1,024 POPC molecules surrounded by 0.9 M NaCl
solution, with a total size of the simulation box of 17.8 17.8 19 nm3. The four
different ΔQ considered were 20qe, 32qe, 40qe, and 56qe, corresponding to the TM
voltages of 420, 510, 520, and 630 mV, respectively, estimated after the bilayer
electroporation.
To study the convergence of the pore radius and the ionic current in time, their
cumulative averages were estimated: after 20 ns of simulation, both the radius and
the current reach a plateau (see Casciola et al. 2016 for data) indicating stability of
the lipid pores. The average pore radius shown in Fig. 5a is almost a linear function
of the TM voltage applied; it varies from 1 to 2.5 nm in the range of charge
imbalances studied.
The ionic current was defined as the slope of the ionic flux; it reaches a plateau
after less than 10 ns. A higher TM voltage induces both an increase of the pore radius
and of the ionic current flowing through the lipid pore. Moreover, pores with larger
size allow the passage of a higher number of ions; hence the growth of the current is
both due to the increase in the TM voltage by directly increasing the electrophoretic
drift of ions, as well as the increase in the pore size. For TM voltages from 420 to
630 mV and pore radius values from 1 to 2.5 nm, the current is in the range of tens of
nA ranging from 2.69 to 18.58 nA.
In experiments aimed to investigate the nature of lipid pores by studying current/
voltage relations, the radius is indirectly deduced from the conductance measured. For
this reason, the conductance of the pore calculated from MD simulations is usually
reported as a function of the pore radius. Figure 5b shows how the conductance, which
ranges from 6.4 to 29.5 nS, is proportional to the pore radius in almost linear fashion.
Lipid pores are selective to different ion species, i.e., pores in POPC lipids are more
selective to sodium ions than to potassium and chloride ions. The pore selectivity is
defined as the ratio of anionic to cationic current. This selectivity arises from the nature
Fig. 5 (a) Minimum pore radius R over the TM voltage Um and (b) ionic conductance G as a
function of the minimum pore radius R, corresponding to ΔQ of 20qe (black triangle), 32qe (red
diamond), 40qe (green square), and 56qe (cyan circle). For each MD run, the average pore radius
and the standard deviation were estimated in the plateau, i.e., from 20 to 80 ns (Reproduced with
permission from Casciola et al. (2016), Copyright (2016) Elsevier B.V.)
of the lipid molecules constituting the pore. In POPC bilayers, the negatively charged
phosphate groups that form the walls of the pore attract sodium ions, which hinders
their passage across the bilayer but also makes the pore interior electrostatically
unfavorable for cations. The Na+ cations bind more deeply and for a longer time
than their Cl anionic counterparts; as a consequence the ratio of the Cl over the Na+
current decreases as the pore expands. Indeed, the pore expansion results in a higher
fraction of those sodium ions that pass the pore without any hindrance as they do not
interact with the pore walls. K+ cations show a similar behavior, yet less pronounced.
However, as the pore radius increases, further ions are expected to interact less with the
pore wall, and their conductance in lipid electropores is similar to those in bulk water.
In this condition, the selectivity is limited by the ratio of the electrophoretic mobility of
the different ion species in bulk water.
The pore selectivity to Cl ions partially arises also due to electroosmotic flux of
water through the pore, which enhances the current of Cl ions and at the same time
reduces the current of Na+ ions. Indeed, a net flux of water is observed in the
direction of the flux of Cl ions (i.e., opposite to the direction of the electric potential
gradient), which can be attributed to the interactions of water with electrophoreti-
cally driven charged ions.
When the same TM voltage was maintained on lipid patches of the size
(8.8 8.8 nm2, 256 lipids) usually employed in MD simulations to save computer
time, the pore radius obtained was twice smaller and the conductance almost three
times smaller than that reported for larger systems (17.8 17.8 nm2, 1,024 lipids),
likely a consequence of the constrains imposed by the finite size of the simulation box.
For comparing one set of molecular simulations of ion conductance to another, it
is essential to take into account the concentration of the ions. A common way to do
this is to express the conductance as molar conductance nS/M. Thus, comparing the
value of conductance obtained with the two protocols in Ho et al. (2013) for a
128 POPC bilayer in a salt solution of 0.1 M NaCl and for TM voltages ranging from
0.56 to 1.12 V, the pore radius is 0.68–1.50 nm and the conductance 5.48–27.88 nS/
M, while in the 1,024 POPC bilayer in a salt solution of 0.9 M NaCl when the
voltage spans from 0.42 to 0.63 V, the pore radius is 1.01–2.45 nm and its conduc-
tance 7.11–32.77 nS/M (Casciola et al. 2016).
Despite the value of conductance is in the same range, the differences may be
attributable to:
• The different force fields used, OPLS in Ho et al. (2013) and CHARMM36 in
Casciola et al. (2016).
• The different ionic concentration, interactions among the ions also affect the
conductance.
• Different simulation box, finite size effects can affect the pore conductance.
Experimental data reported from chronopotentiometry studies for POPC

electropores in KCl systems focused on single-pore conductance (Kalinowski
et al. 1998; Koronkiewicz and Kalinowski 2004). When comparing MD with the
latter, although the two sets of conductance values lie within the same order of
magnitude, the pore radii in the simulations are smaller than those reported in the
published chronopotentiometry data.
However, as discussed in the Introduction, the pore radius values of the chronopo-
tentiometric results were extracted from the measured conductance using simplified
analytical formulas. The ions are assumed to travel through the pore as they would
through a cylinder of bulk water. In this model, the pore diameter is given by d = 2
(GpL/kπ)1/2, where Gp is the pore conductance, L is the length of the pore, and k is the
conductivity of the electrolyte. Since the accuracy of the extracted pore diameter
depends on the applicability of this simple geometric model, we must place large
bands of uncertainty around the chronopotentiometric results. An additional assumption
embedded in the chronopotentiometric data is that the ion concentration inside the pore
is the same as it is in the electrolyte. However, the MD results show that concentrations
of the confined KCl in a conducting electropore are about twice the bulk value.
Rems et al. (2016) developed an approach that compares the pore conductance as
predicted by a theoretical model based on the continuum Poisson–Nernst–Planck
theory to the pore conductance obtained with MD by directly extracting all relevant
model parameters from the MD system. The authors demonstrate that a correct
description of the pore conductance is important when extracting the pore size
from experimental measurements as well as when using continuum electroporation
models and MD provides, nowadays, the most reliable tool for this scope.
Conclusions
This chapter reports on atomic characterizations of lipid pore electrical conductance.

Two MD protocols are available to induce stable pore formation in lipid bilayers in
condition similar to experiments, namely, the electric field and the charge imbalance
method. These are perceived as the most appropriate approaches to directly deter-
mine the transport of ions and selected molecules across bilayers exposed to electric
pulses, which remains a challenge for experimental methods.
The pore formed with these two protocols can be stabilized to different radii for
tens of ns when the TM voltages are lower than the EP threshold. Both the pore radii
and the pore conductance vary almost linearly with the applied voltage. Fore pores
from few to tens of nm radius, the electrical conductance is in the range of tens of
nS/M.
Moreover, lipid pores created in POPC bilayers are more selective to cations than
to anions. This selectivity originates from the structure of the lipid molecules
building the pore wall and hence may be dependent on the nature of the lipid
molecule and on its force field parameterization. Negatively charged phosphate
moieties of the head groups bind sodium ions that therefore are hindered in crossing
the pore; in addition, the presence of sodium ions in the pore wall makes the pore
interior electrostatically unfavorable for other sodium ions.
This finding indicates that the transport through electropores is sensitive to the
type of solutes, showing a different affinity for different charged species.
The use of continuum models to describe and extract information from experi-
mental observations is necessary to provide further insights to the EP phenomena.
However, theoretical models must be supported and validated by further evidences
to be quantitative. An intermediate and complementary step between the latter and
experiments can be provided by MD simulations. Studies in this direction would
allow for more quantitative and accurate predictions of the EP theory.
Cross-References
Experiments
▶ Effects of Heterogeneous Membranes and Electrolytes on Electropore Formation
▶ Lipid Electropore Stabilization
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3/4/001
Electrodeformation, Electroporation, and
Electrofusion of Giant Unilamellar Vesicles 14
Rumiana Dimova and Karin A. Riske
Abstract
This chapter summarizes a spectrum of phenomena observed on model mem-
branes exposed to electric fields. The considered model membrane system is giant
unilamellar vesicles with sizes in the range of tens of microns. Because of their
large size, the response of the membrane to electric fields can be directly
visualized under the microscope. The membrane behavior is exemplified by
several types of responses: First, the vesicles undergo morphological changes
and adopt prolate, oblate, or spherocylindrical shapes. In general, the vesicle
morphology depends on the conductivity conditions of the immersion and encap-
sulated solutions, and in the case of alternating fields – on the field frequency.
Second, after switching the electric field off, these shapes can relax back to a
sphere. The relaxation times depend on the initial membrane tension and on the
reached transmembrane potential. Third, the vesicles can undergo topological
changes such as formation of pores and, in the case of vesicles in contact, fusion.
All these processes depend on the material characteristics of the membrane such
as mechanical (bending rigidity and stretching elasticity), rheological (membrane
shear surface viscosity), and electrical (capacitance) properties of the lipid bilayer.
This chapter gives an overview of these properties and their dependence on the
membrane phase state, and presents approaches for directly assessing them using
giant unilamellar vesicles.
R. Dimova (*)
Department of Theory and Bio-Systems, Max Planck Institute of Colloids and Interfaces,
Potsdam, Germany
e-mail: dimova@mpikg.mpg.de
K.A. Riske
Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, Brazil
e-mail: kariske@unifesp.br

DOI 10.1007/978-3-319-32886-7_199
236 R. Dimova and K.A. Riske
Keywords
Giant unilamellar vesicles • Model membranes • Membrane mechanical
properties • Vesicle deformation • Poration • Membrane fusion
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
Giant Unilamellar Vesicles as Model Systems to Study Membrane Properties . . . . . . . . . . . . . . . 237
Preparation and Observation of Giant Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
Bilayer Phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Mechanical and Rheological Properties of Bilayers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Electrical Properties of Bilayers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Experiments with GUVs in Electric Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
Electrodeformation of GUVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
Vesicle Morphologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
Characteristic Relaxation Times . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
Electroporation of GUVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Electrofusion of GUVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Introduction
The effects of electric fields on cells have long been a subject of interest and
exploration. The lipid bilayer of the plasma cellular membrane acts as an electric
insulator so that a variety of electrical phenomena take place when cells are exposed
to electric fields, of either alternating (AC) or direct current (DC). AC fields, for
instance, are known to induce cell deformation, reorientation, and movement, in
some cases leading to cell alignment. Strong fields, usually applied as single or
consecutive DC pulses, can lead to the phenomenon of electroporation, also referred
to as electropermeabilization or electrical breakdown, whereby (transient) pores
open across the cellular membrane. Electroporation has obvious applications when
the target is to insert exogenous molecules into the intracellular medium, as is the
case, for instance, in gene therapy (▶ Chaps. 23, “Gene Delivery by Electroporation
In Vitro: Mechanisms,” ▶ 6, “Molecular Transmembrane Transport with Giant
Unilamellar Vesicles (GUVs),” and ▶ 19, “Nucleic Acid Electrotransfer in Mam-
malian Cells: Mechanistic Description”). When cells in contact are electroporated
they can fuse and mix their intracellular compartments and plasma membrane
(a process known as hybridization), which also finds a large range of applications.
To better understand the mechanistic details underlying electrical phenomena in
cells, model systems, mainly lipid vesicles, have been widely employed. Lipid
vesicles are handy tools to explore all sorts of membrane-related biological phe-
nomena, especially those in which the lipid phase of the biological membrane
plays a key role. That is certainly the case when regarding the effects of electric
fields on cells. Electric field-induced vesicle shape deformation, movement,
14 Electrodeformation, Electroporation, and Electrofusion of Giant. . . 237
permeabilization, and fusion have been investigated for several decades already,
and a large body of information has been collected on this topic. It has become
obvious that the response of the lipid vesicles to electric fields is defined by the
membrane material properties, such as softness, fluidity, ability to be stretched/
compressed, electrical capacitance, and surface viscosity. Of special relevance to
vesicle studies are giant unilamellar vesicles (GUVs) , cell-sized objects which can
be individually and directly observed with an optical microscope. GUVs have been
widely used in this research area, with the clear size advantage over their counter-
parts, the conventional large or small unilamellar vesicles with sizes between tens
and hundreds of nanometers.
The study of GUVs in the presence of electric fields has two main purposes. On
the one hand, the mechanistic details of cell deformation, permeabilization, and
fusion can be understood and used in the development of new protocols for bio-
technological and biomedical applications. On the other hand, electric fields can be
used to manipulate GUVs for the purpose of helping the investigation of membrane
phenomena and/or to impose a well-controlled external perturbation with the aim of
extracting material properties of lipid bilayers of different compositions and in
different conditions of biological relevance.
The chapter is organized as follows. First, the use of GUVs as biomimetic model
for deducing membrane properties are introduced, focusing on their response to
electric pulses. Then, three different field-induced effects are discussed in detail:
vesicle deformation, poration, and fusion.
Giant Unilamellar Vesicles as Model Systems to Study Membrane

Properties
Lipid vesicles, also called liposomes, have often been used as mimetic models for the
complex biological membrane because they are easy to prepare and handle, and their
lipid composition can be precisely controlled and varied. Depending on the prepa-
ration method, lipid vesicles can be unilamellar (one bilayer encapsulating internal
volume) or multilamellar (made of several bilayers) and of definite size, ranging
from tens of nanometers to tens of micrometers. This chapter will focus on the largest
ones, called giant unilamellar vesicles or GUVs which are cell-sized objects of
typical diameter around 10–50 μm. They can be easily visualized under an optical
microscope. GUVs have several advantages over the conventional small liposomes
(usually with diameters of the order of 100 nm). Giant vesicles exhibit nearly zero
membrane curvature, thus better mimicking the conditions of the plasma membrane,
and can be individually manipulated and observed. In the last decades, GUVs have
become a well-established platform on which membrane properties can be quantified
and many membrane-related phenomena, such cell adhesion, curvature sensing or
generation, membrane fusion, phase separation and domain formation, and effects of
anchored or inserted molecules, can be studied. Additionally, membrane proteins can
also be reconstituted in GUVs, thus allowing the investigation of protein functions
and mobility.
Fig. 1 Top: GUVs observed with phase contrast mode without (a) and with (b) sucrose/glucose
in/out asymmetry. Bottom: GUVs observed with fluorescence-based microscopy whereby either the
membrane is labeled and the vesicle is observed with epifluorescence (c) or both; the membrane and
the solution encapsulated in the vesicle are labeled (in green and red, respectively) and observed
with confocal microscopy (d). The dimeters of the GUVs are about 20–60 μm
Preparation and Observation of Giant Vesicles
GUVs are commonly prepared with the conventional electroformation method,

although several other protocols have been described that allow formation of
GUVs with a variety of lipids and growing media; for an overview of several
commonly used methods consult (Walde et al. 2010). To observe GUVs with optical
microscopy, transmission-light and fluorescence-based techniques are usually
employed. Among the transmission-light techniques, the most commonly used are
phase contrast, differential interference contrast (DIC), reflection interference con-
trast microscopy (RICM), and wide-field microscopy. Fluorescence-based micros-
copy includes e.g., epifluorescence, confocal microscopy, and total internal
reflection fluorescence (TIRF) microscopy. In this case, to allow observation, fluo-
rescent dyes have to be added, either to the membrane (typically ~0.5 mol%
fluorescent molecules or fluorescently labeled lipids) or to the aqueous solution
(water soluble probes added either internally or externally to the vesicles). Figure 1
shows representative images of GUVs observed with phase contrast,
epifluorescence, and confocal microscopy. The vesicles are very often prepared in a
solution containing sucrose (with concentration typically in the range 0.1–0.2 M)
and dispersed in an isoosmolar solution with glucose. This creates a sugar asymme-
try that enhances the optical contrast in phase contrast mode (see Fig. 1a, b) and
stabilizes the vesicles onto the bottom of the observation chamber due to gravity. The
asymmetry of sugars and of water soluble fluorescent dyes between the inner and
outer medium can be used to report on membrane permeability, since this asymmetry
between the inner and outer vesicle compartments is only maintained as long as the
membrane remains impermeable to sucrose/glucose and to the fluorescent molecule.
In the latter case, probes of different sizes can be used so that the pore size can be
estimated.
Bilayer Phases
Depending on the constituent lipids and the temperature, membranes can be found in
different phases or even exhibit lateral phase separation. Single-component bilayers
are either in a gel or fluid phase, depending on the temperature. At room temperature,
unsaturated lipids are often in a fluid phase, also called liquid-disordered phase,
characterized by large lateral mobility and chain disorder. Long and saturated chains
at room temperature are usually in a gel, or solid, phase, with chains that are tightly
packed and ordered and with virtually no lateral mobility. Binary mixtures of lipids
with cholesterol can be found in a liquid-ordered phase, in which the hydrocarbon
chains are ordered but the lipids still exhibit large lateral mobility. Ternary and more
complex mixtures can exhibit lateral phase coexistence. The biological membrane is
mainly in the fluid phase, since most of its phospholipids have unsaturated chains but
submicron domains resembling the liquid-ordered phase are postulated to transiently
form and to perform important tasks, as signaling and endocytosis (Simons and
Ikonen 1997). These nanodomains, the so-called membrane rafts, are rich in
sphingomyelin (a long saturated lipid), cholesterol, and certain membrane proteins.
Depending on composition and temperature, GUVs can be in the liquid-disordered,
liquid-ordered, and gel phase or exhibit coexistence phases (typically, up to three
phases can be simultaneously visualized). To visualize phase coexistence, fluores-
cently labeled lipids with known preference for one of the phases are used so that
micron-sized domains can be distinguished with fluorescence microscopy. This
chapter will predominantly discuss the properties of fluid membranes but some
information on other phases will be included as well.
Mechanical and Rheological Properties of Bilayers
Simplistically, lipid bilayers may be viewed as a single ~5-nm thick film, which may
be bent, compressed/stretched, and sheared (see Fig. 2 for a schematic illustration).
Fluid membranes are very soft and flexible, because their bending elasticity modu-
lus, κ, is only on the order of several kBT, where kBT is the thermal energy (Dimova
Fig. 2 Modes of bilayer

membrane deformations: the
response of the membrane to
out-of-plane deformations is
defined by the bending
rigidity; response to lateral
stretching is characterized by
the stretching elasticity
modulus; above certain
threshold (lysis tension), the
membrane can rupture, pore
formation being one example
of this process; lateral in-plane
displacement is characterized
by the shear surface viscosity
2014). Thus, fluid membranes exhibit thermal shape fluctuations that can be directly
observed on tensionless GUVs under the microscope. When tension is applied to the
membrane and gradually increased, one first pulls out the bilayer fluctuations and
then eventually stretches the membrane on the molecular level, by changing the area
per lipid molecule. The stretching elasticity modulus of lipid bilayers, Ka, is on the
order of that of a rubber sheet with the same thickness, i.e., approximately
200–300 mN/m (Rawicz et al. 2000). Upon stretching, the lipid bilayer can sustain
tensions up to about several mN/m (corresponding to about 5% area increase). At
certain critical tension, also known as the lysis tension, σlys, the membrane ruptures
and pores open to relieve the stress. The energy penalty to form curved pore rims
(hydrophilic pores) depends on the line (or edge) tension, γ, which is usually high for
phospholipids, so that pores tend to eventually close and restore a bilayer structure.
The resistance to shear in fluid membranes is negligible because they are thin. But
the membrane and the surrounding fluid impose a hydrodynamic drag to the motion
of an inclusion in the membrane, characterized by the membrane surface viscosity,
ηs. Another important parameter to quantify membrane fluidity is the lateral diffusion
coefficient, D, of lipids. In Table 1, some typical values of the constants discussed
above are given for fluid-phase membranes. Note that the presence of cholesterol in
fluid membranes will generally render a tighter and more ordered bilayer, with clear
impact on all the properties reported. For comparison, some data on membranes in
the gel phase are also given.
Table 1 Typical values of properties of membranes in different phase states

Liquid-disordered Liquid-ordered Gel
Bending rigidity, κ 20 kBT 100 kBT 350 kBT
Stretching elasticity, Ka 300 mN/m 1000 mN/m
Lysis tension, σ lys 5–10 mN/m 25 mN/m
Surface viscosity, ηS ~108 N s/m ~107 N s/m Diverges
Diffusion coefficient, D ~108 cm2/s ~109 cm2/s <1011 cm2/s
Edge tension, γ 30 pN 150 pN
Electrical Properties of Bilayers
Electric fields can cause a variety of effects on GUVs, most of which are also found
in cells. The lipid bilayer can withhold a salt asymmetry and separate internal and
external solutions of different conductivities and act as an insulator up to a certain
degree. Free charges in solution move in response to the electric field and can
accumulate at the membrane boundaries. The membrane then acts as a capacitor
that charges with a characteristic time and acquires a transmembrane potential Vm,
given by:
V m ¼ 1:5 R E j cos θj ½1 expðt=tc Þ (1)
where R is the vesicle radius, θ the tilt angle between the electric field direction and
the surface normal, t is time, and tc the charging time

1 1
tc ¼ RCm þ (2)
λin 2λex
with Cm being the membrane capacitance (~1 μF/cm2) and λin and λex the conduc-
tivities of the internal and external media, respectively. For low conductivity solu-
tions (sugars only), the charging time tc ~ 500 μs, and it decreases to a few μs when
salt at millimolar concentration is present in the media.
The electric field induces a stress on the membrane, given by the Maxwell stress
tensor. This leads to vesicle deformation and to an effective electrical tension σ el which
normally compresses the bilayer. At high tension, the bilayer eventually ruptures. This
occurs above some critical transmembrane potential Vc, (▶ Chap. 2, “Critical Electric
Field and Transmembrane Voltage for Lipid Pore Formation in Experiments”) which is
reached when the membrane undergoes lysis at tension σ lys = σ o + σ el, where σ o is the
initial tension before the application of the electric field. For tensionless vesicles in the
fluid phase (σ o ~ 0), Vc ~ 1 V. However, Vc can be lower for vesicles that have some
initial membrane tension (Riske and Dimova 2005). Vesicles in the liquid-ordered and
gel phases are more resistant to electroporation, and Vc is higher. For the gel phase, Vc
was reported to be around 10 V (Knorr et al. 2010). Above the poration threshold,
electrical breakdown of the bilayer occurs and pores open to relieve the stress imposed
on the membrane.
Electric fields can be of alternating (AC) and of direct (DC) current. AC fields
applied to vesicles and cells are usually relatively moderate so that the poration
threshold is not reached. The effects caused are then vesicle deformation (Dimova
et al. 2007), lipid flow due to asymmetric field around the vesicle and dielectro-
phoresis, which can cause vesicle movement and pearl-chain formation in some field
conditions. However, stronger AC fields (> 3 kV/m) are also able to induce poration
(Harbich and Helfrich 1979). DC (usually square) pulses also induces vesicle defor-
mation but can be set strong enough so as to reach the electroporation threshold and
thus opening of pores (Riske and Dimova 2005). This has been largely employed to
render cells transiently permeable. In GUVs, macropores of few micrometers open and
can be visualized with optical microscopy (see ahead). After the pulse, these
macropores usually close and the vesicle shape relaxes to its original shape. If
GUVs in contact are porated, they can fuse. The dynamics of all these processes are
governed by the material properties of the membranes as will be discussed on the
following sections.
Experiments with GUVs in Electric Fields
Effects of electric fields on model membranes can be nicely illustrated and resolved
using GUVs (Dimova et al. 2007; Dimova et al. 2009). On the one hand, GUVs have
the dimensions of cells, so the typical field parameters are similar to those used in
applications with cells. On the other hand, they can be individually observed and
followed with video microscopy. Chambers with electrodes can be easily prepared
using Pt-wires or adhesive copper tapes or can be purchased. The distance between
the electrodes should be precisely known, so that the electric field can be calculated
from the voltage applied between them. The examples shown in this chapter were
obtained with a commercially available electrofusion chamber (Eppendorf, Ger-
many) containing two Pt-wires of about 90 μm diameter and 500 μm apart. The
lipid composition of the GUVs can be controlled and varied to allow quantification
of membrane properties as a function of their composition. The results shown here
were mainly obtained with unsaturated phosphatidylcholine (PC) lipids, either
egg-PC or palmitoyloleoylphosphatidylcholine (POPC), thus with membranes in
the fluid phase. In some cases, a fraction (up to 50 mol%) of the charged lipid
palmitoyloleoylphosphatidylglycerol (POPG) was present to study the role of sur-
face charges on the membrane properties.
Electrodeformation of GUVs
Vesicle Morphologies
Lipid vesicles are deformed by electric fields. AC fields induce ellipsoidal shape
deformation which can be either prolate or oblate depending on the conductivity
ratio (λin/λex) between the inner and outer solutions and on the field frequency, ω
(Aranda et al. 2008). In both cases, the symmetry axis of the ellipsoid shape is
Fig. 3. (a) Morphological diagram of vesicle shape deformations induced by AC fields as a

function of conductivity ratio λin/λex and field frequency ω. The symmetry axes of the prolate and
oblate shapes are shown as straight arrows, and they align parallel with the electric field direction.
(b) Some effects of AC fields. Left: vesicles with excess area stored in fluctuations are deformed and
adopt prolate shapes (for (λin/λex > 1). The extent of vesicle deformation quantified by the aspect
ratio a/b is related to the field strength and surface area and can be used to extract the bending
stiffness of the membrane. Right: Due to dielectrophoresis, GUVs in AC fields move and align in
pearl chains
aligned with the electric field (the long axis in the case of prolates and the short one
in the case of oblates). A representation of the morphological diagram of vesicle
shapes induced as a function of the conductivity ratio λin/λex and the field frequency
ω is shown in Fig. 3 (Aranda et al. 2008; Dimova et al. 2009). At very high field
frequency (ω > 107 Hz), the vesicles do not deform, because the field direction
changes faster than the ions can respond to it. At lower frequencies and for λin/λex > 1,
the vesicles adopt prolate shapes. When the external conductivity is higher than that
of the internal medium (λin/λex < 1), a prolate-oblate shape transition occurs at
ω ~ 103 Hz. The critical frequency at which the vesicles undergo the prolate-oblate
transition, together with the vesicle size and the conductivity ratio, can be used to
extract the membrane capacitance. The extent of vesicle deformation depends on the
field strength and on the vesicle excess area. Thus, AC fields of moderate strength
(typically 200 V/cm) can be used to estimate the vesicle excess area stored in
fluctuations from simple geometrical considerations of ellipsoidal shapes (prolate
shape deformations are preferred because of the smaller deformation imposed by the
chamber wall). Moreover, AC fields can be used to deduce the membrane bending
stiffness, κ, by increasing the field strength and measuring the deformation induced
in terms of the ratio between the vesicle semi-axes a/b and relating it to the imposed
tension on the membrane (Gracià et al. 2010).
Moderate field frequencies (ω ~ 100 kHz) induce vesicle alignment as in a pearl
chain due to dielectrophoresis, which can be helpful for bringing vesicles into contact
and then study vesicle adhesion and/or fusion (see section “Electrofusion of GUVs”).
DC pulses also induce vesicle deformation, but of transient nature, since the
vesicle typically relaxes back to its original shape after the end of the pulse (Riske
and Dimova 2005, 2006). Typical fields applied are square DC pulses of 0.5–5 kV/
cm with duration of 0.05–5 ms, but nanosecond pulses of even higher strength have
also been used. The examples that are discussed here were obtained with pulses of
Fig. 4. (a) Shape deformations induced by DC fields. The cartoons show ellipsoidal and spherocy-
lindrical shapes induced in the absence and presence of salt outside the vesicle, respectively. The
images below are representative of the observed shapes as a function of conductivity ratio, λin/λex,
and presence of salt (~1 mM NaCl) (Adapted from Riske and Dimova (2006), Copyright 2006, with
permission from Elsevier). (b) Top: representative sequence of an egg-PC GUV during and after
application of a DC pulse (2 kV/cm, 200 μs) observed with a high temporal resolution (λin/λex > 1,
no salt out). The macropores can be visualized in the second, third, and fourth snapshots. Bottom:
Time dependence of the aspect ratio a/b for three vesicles (nonporated, porated, and porated with
excess area) and the respective relaxation characteristic times τ1, τ2, and τ3 (pulse conditions: 1 kV/
cm, 250 μs; 3 kV/cm, 100 μs; 3 kV/cm, 200 μs, respectively) (Adapted from Riske and Dimova
(2005), Copyright 2005, with permission from Elsevier)
duration in the 50–200 μs range, and strength between 1 and 4 kV/cm. The
deformation imposed to the vesicles is similar to that of low frequency AC fields.
However, the presence of salt in the external medium imposes an additional defor-
mation, a flattening of the vesicle sides along the field, see Fig. 4 (Riske and Dimova
2006). Thus, for λin/λex < 1 an oblate-like spherocylinder is observed and for
λin/λex > 1 the vesicles assume either a prolate shape in the absence of external
salt or a prolate-like spherocylindrical shape when salt is present in the vesicle
exterior. The maximum shape deformation is usually attained toward the end of
the pulse, and the dynamics of vesicle deformation during the pulse can only be
resolved with a fast camera acquisition system, as for instance in (Riske and Dimova
2005), where 50 μs resolution was employed.
Characteristic Relaxation Times
The characteristic relaxation times for the vesicle deformation after the end of the
pulse depend on the membrane material properties and on the phenomena stimulated
by the pulse (Riske and Dimova 2005). Weak fields (below the electroporation
threshold, see Eqs. 1 and 2) induce only a mild deformation due to membrane
stretching. After the end of the pulse, the vesicle quickly relaxes back to the initial
shape with characteristic time τ1 ~ 0.1 ms by releasing the acquired membrane
tension through shear in the membrane. Thus, τ1 is related to the membrane tension σ
acquired at the end of the pulse and to the surface viscosity of the bilayer, such that
τ1 ~ ηs/σ. When DC pulses strong enough to reach the poration threshold of the
membrane are applied to spherical vesicles, the deformation induced is stronger and
macropores with diameters of few micrometers may open. These pores can be clearly
visualized with phase contrast when glucose/sucrose asymmetry is employed (sub-
micron pores may be present but cannot be visualized optically; see also ▶ Chaps. 3,
“Phospholipid Head Group Dipoles and Electropore Formation,” ▶ 4, “Membrane
Permeabilization Lifetime in Experiments,” ▶ 7, “Lipid Electropore Lifetime in
Molecular Models,” ▶ 9, “Lipid Electropore Geometry in Molecular Models,” and
▶ 11, “Experimental Determination of Lipid Electropore Size”). After the pulse, the
vesicle deformation relaxation is about two orders of magnitude slower (τ2 ~ 10 ms)
than when no macropores are present. This second characteristic time τ2 is related to
pore closing time (see more details in the following section), which is set by the pore
edge tension γ and the membrane surface viscosity ηs, such that τ2 ~ ηsr/2γ, where
r is the pore radius. When the pulses are applied to GUVs with some excess area
(seen by their shape fluctuation in the absence of applied field), a third and much
longer relaxation time can be detected, τ3 ~ 0.1–1 s, depending on the excess area
available. This slow time reflects the dynamics of volume displacement around the
deformed vesicle of radius R. It is associated with shape fluctuations and entails
contributions from the medium viscosity η (of sucrose/glucose solutions), the mem-
brane bending rigidity κ and the vesicle reduced volume (v 0.95–0.99 for deflated
quasispherical vesicles), such that τ3 ~ 4πηR3/(3κ (v–1)). These relaxation times
quantified from the time dependence of the aspect ratio a/b of prolate vesicles are
illustrated in Fig. 4.
Curiously, strong (even excessive) vesicle deformation with aspect ratios a/b up
to 10 can also be observed (Sadik et al. 2011). Several theoretical studies were
motivated by the experimental results on vesicle deformation induced by electric
fields, in which the vesicle relaxation dynamics was considered using a droplet-
based model, which yielded a way of deducing the initial tension of the vesicle and
the membrane bending rigidity.
Electroporation of GUVs
Electric fields strong enough to build a transmembrane potential above a critical

value (Vm > Vc, see Eq. 1 and section “Electrical Properties of Bilayers”) cause
opening of macropores in GUVs. Submicroscopic pores are also expected to open,
but they cannot be resolved with optical microscopy. The macropores formed have a
diameter of few micrometers and can be easily detected with phase contrast and
Fig. 5 (a) Detection of pulse-induced macropores with phase contrast (top: GUV with sucrose/
glucose asymmetry), epifluorescence (middle: GUV labeled with membrane probe), and confocal
microscopy (bottom: GUV labeled with green membrane probe and encapsulating a red aqueous
soluble dye). The diameters of the vesicles are about 40 μm. (b) Electroporation used to measure
edge tension following the approach in Portet and Dimova (2010). Top: sequence of images of a
POPC GUV during and after application of a DC pulse (3 kV/cm, 150 μs). Bottom: dynamics of
pore closure with linear fit to the slow pore closure regime which gives for the edge tension
γ = 25 pN for this particular GUV
fluorescence microscopy (see Fig. 5). The pore lifetime entails four stages
(Brochard-Wyart et al. 2000): (i) pore growth, (ii) stabilization, (iii) slow pore
closure in a quasistatic leak-out regime, and (iv) fast pore closure. The third and
longer stage is governed by the medium viscosity η and by the pore edge tension γ.
The edge tension can be easily extracted from linear fits of the dynamics of the radius
of the pore r in a vesicle of radius R according to the following equation (Brochard-
Wyart et al. 2000):
2γ
R2 lnðr Þ ¼ tþC (3)
3πη
where C is a constant and t is the time. Phospholipid bilayers in the fluid phase have
γ ~ 30 pN, and this value increases typically twice in the presence of cholesterol at
physiologically relevant concentrations (Portet and Dimova 2010). The typical
lifetime of micrometer-sized pores is of about tens of milliseconds.
In fluid vesicles, DC pulses can occasionally induce macropores that remain stably
open or even never reseal, leading to vesicle destabilization and collapse. Stable and
long-lived pores can be observed in the presence of cone-shaped molecules in the
bilayer which lead to reducing the edge tension (Karatekin et al. 2003) or in the
presence of hydrogel polymers (agarose) which can physically hinder the pore closure
(Lira et al. 2014), see Fig. 6. Interestingly, pores opened across cellular membranes
might also never reseal due to physical obstruction of intracellular material leaking out.
When DC pulses are applied to GUVs containing a large fraction of negatively
charged lipids (POPC:POPG 1:1), vesicle burst/collapse is often observed (Riske
et al. 2009). After the end of the pulse, macropores can be detected, but instead of
resealing as in the case of zwitterionic membranes, the pores often continue to open
and the whole vesicle collapses into a network of lipid tubes, as shown in Fig. 6.
Therefore, the presence of negatively charged lipids has an impact on membrane
stability, most probably by reducing the edge tension significantly.
The mechanical, rheological, and electrical properties of membranes in the gel
phase (for example, sphingomyelin or dipalmitoylphosphatidylcholine – at room
temperature) differ significantly from those of fluid lipid membranes shown above.
Some of them are summarized in Table 1. These differences introduce new features in
the response of gel-phase membranes to electric fields. Because of the high bending
rigidity, gel-phase vesicles relax much faster after electrodeformation induced by
electric pulses. The critical transmembrane potential leading to the poration of
gel-phase vesicles is several times higher compared to that of fluid membranes
(Knorr et al. 2010), one of the reasons being the larger membrane thickness, which
implies that pulses of much higher field strength are needed to porate them. The
resealing of macropores in gel-phase membranes is arrested. Upon poration, gel-phase
GUVs develop cracks which do not reseal within minutes (Knorr et al. 2010).
Electrofusion of GUVs
If electroporation is induced in the contact area between two neighboring (fluid)

GUVs, vesicle fusion may occur. Exposing the GUVs to AC fields prior to applica-
tion of porating DC pulses can bring vesicles together (see Fig. 3b), thus increasing
the likelihood of vesicle fusion. The first step in the fusing process after application
of the pulse is the formation of a fusion neck from the merge of the two pore edges in
the membranes of the adjacent GUVs. The size of this fusion neck can be followed
with fast video microscopy (Haluska et al. 2006). In some cases, several fusion necks
can form (see Fig. 7 for an example). The dynamics of neck growth involves two
processes, as can be clearly seen in Fig. 7: a fast neck opening (with a velocity of a
few cm/s) within the first millisecond, followed by a much slower increase in the
fusion neck that requires a few seconds to finally result in a single vesicle. The initial
fast process is driven by the dissipation of the high membrane tension acquired in the
fusion neck zone, and it is thus dependent on the same parameters as the fast
relaxation dynamics of nonporated vesicles (τfast~ ηs/σ ~ 0.1 ms). After the release
of the membrane tension, the expansion of the fusion neck slows down by orders of
magnitude and is then dictated by the slow mixing of the inner volumes of the two
initial vesicles and by expelling of fluid around the fusion neck which is governed by
the membrane bending stiffness κ, in a similar way as the long relaxation dynamics
τ3 of fluctuating GUVs (τslow ~ η ΔV/κ ~ 10 s).
Fig. 6 – (a) Pulse-induced bursting of a POPC:POPG 1:1 GUV. The images were obtained with
phase contrast (top) and confocal microscopy (bottom). The DC pulse applied was 3 kV/cm and
150 μs. The scale bar corresponds to 25 μm. Reproduced from (Riske et al. 2009) with permission
from The Royal Society of Chemistry. (b) A POPC GUV encapsulating agarose gel is shown before,
during, and after application of a DC pulse (3 kV/cm, 150 μs). The arrow points to the opened
macropore. The scale bar corresponds to 20 μm. (c) Cartoon showing the physical obstruction of
agarose polymer hindering pore resealing after electroporation. The inner solution (pink) leaks out
and the vesicle looses contrast (b–c are adapted from (Lira et al. 2014) Copyright 2014, with
permission from Elsevier)
From a more practical viewpoint, electrofusion of GUVs can be used to initiate

targeted mixing of either their encapsulated content or their membranes. Vesicles
encapsulating two different components or of two distinct membrane composition
(and labeled differently to distinguish them) can be brought together either by AC
fields, via micromanipulation with glass capillaries of micrometer diameter (micro-
pipettes) or using microfluidic devices. After application of a strong DC pulse,
Fig. 7. (a) Electrofusion of two egg-PC GUVs. (b) Dynamics of the size of the fusion neck. (c)
Electrofusion of two egg-PC GUVs through several fusion necks. The scale bars represent 20 μm
(Adapted from Haluska et al. (2006), Copyright 2006, National Academy of Sciences)
vesicle fusion may occur thus (i) initiating the reaction of the solutes in the two
components encapsulated in the two vesicles or (ii) mixing of the membranes of the
two vesicles. An example for using vesicle electrofusion as a microreactor is the
synthesis of quantum-dot-like CdS nanoparticles in the closed compartment of a
fused vesicle couple (Yang et al. 2009) as illustrated in Fig. 8. In this case, two
GUVs, one loaded with Na2S and the other loaded with CdCl2, are fused. The
product of the reaction, quantum-dot-like CdS nanoparticles, can be visualized under
laser excitation as a fluorescent bright spot in the fusion zone (see last image in
Fig. 8b). This method could be especially suitable for the online monitoring of
several physicochemical processes such as photosynthesis, enzyme catalysis, and
photopolymerization. On the other hand, fusing of two vesicles with known surface
area and lipid composition can be used to produce GUVs with precise
multicomponent lipid composition (Bezlyepkina et al. 2013). This is important
Fig. 8. Electrofusion of giant vesicles as a method for nanoparticle synthesis. (a) Schematic
illustration of the electrofusion protocol: Two populations of vesicles containing reactant A or B
are mixed (in A- and B-free environment). The vesicles are subjected to an AC field to align them in
the direction of the field and bring them close together. A DC pulse initiates the electrofusion of the
two vesicles and the reaction between A and B proceeds to the formation of nanoparticles
encapsulated in the fused vesicle. (b) Confocal cross-sections of giant vesicles loaded with Na2S
(red) and CdCl2 (green) undergoing electrofusion. After fusion (right), fluorescence from the
product is detected in the interior of the fused vesicle. The time after applying the pulse is indicated
on the micrographs (Reproduced from Yang et al. (2009) with permission from John Wiley and
Sons)
because GUVs grown from lipid mixtures can exhibit widely different individual
composition, depending on the individual vesicle history. For example, before
observation, a phase-separated vesicle may have budded in the region of one of
the domains and the two daughter vesicles would then have attained compositions
that are very different from that of the mother vesicle. Indeed, in samples with
compositions belonging to the region of coexistence of two fluid phases, one often
finds vesicles that are not phase-separated. Particularly strong deviations in the
vesicle composition are observed for multicomponent lipid mixtures that are not
fully miscible at the temperature of observation. To overcome this problem,
electrofusion of two vesicles made of two different fully miscible lipid mixtures
can be employed (Bezlyepkina et al. 2013).
Conclusions
Electric fields induce a variety of effects on GUVs. Moderate fields cause shape
deformation and vesicle movement/alignment. Stronger fields are able to induce
electroporation and membrane fusion if vesicles are in close contact. These
phenomena and their dynamics depend on the vesicle state (e.g., its tension and
excess area) and on the material properties of the lipid bilayer, such as membrane
bending stiffness, surface viscosity, edge tension, capacitance, and poration thresh-
old. Here it was shown that AC and DC fields can be used to manipulate GUVs, for
instance to pull out excess area stored in shape fluctuations, and to extract membrane
properties such as bending stiffness and edge tension, and how these properties
change with membrane composition. Several other applications can be envisaged for
model systems in the presence of electric fields. For instance, more complex
membranes could be used for obtaining GUVs (e.g., with complex biological lipid
mixtures, with specific membrane proteins incorporated, and with a cytoskeleton
which provides mechanical stability to the vesicle). This will allow the study of
systems that capture better the complexity of real cells.
Acknowledgment K.A.R. acknowledges the financial support of FAPESP.
Cross-References
Experiments
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Part II
Basic Description of Cell Electroporation
in Vitro
Marie-Pierre Rols
3D Tissue Models to Bridge the Gap
Between Cell Culture and Tissue in 15
Assessing Electroporation
Laure Gibot
Abstract
The vast majority of studies on drug delivery by electroporation have been
performed in vitro on cell cultures in 2D (cell monolayers or cells in suspension)
and in vivo in animal models. The results obtained are often inconsistent. Three-
dimensional tissue models of increasing complexity are starting to be used to
study mechanisms of electrotransfer of cytotoxic molecules and nucleic acids in a
tissue context in vitro. These 3D models include collagen gel, normal and/or
tumor spheroids, as well as human tissue substitutes produced by tissue engi-
neering. Three-dimensional culture systems allow researchers to address the
concept of cell organization in 3D and to take into account dynamic interactions
between distinct cell types and surrounding extracellular matrix. Thus, 3D tissue
models mimic biological tissue better than classical cell culture. Besides, this
approach is an efficient and ethical alternative to the use of laboratory animals.
Moreover, cells can be isolated directly from a patient. This offers the possibility
to adapt treatment planning to that person’s biological characteristics, leading to a
potentially more effective and better tolerated therapeutic approach. Understand-
ing and mastering the mechanisms of drug delivery by electrotransfer at the tissue
scale will enable the development of new strategies to increase transfer efficiency
while avoiding damage to surrounding tissue. Finally, this knowledge of mech-
anisms at the tissue scale will open the door of clinical applications to gene
therapy and DNA vaccination and will promote the development of innovative,
effective, and personalized therapies.
Keywords
3D • Cell sheet • Gel • In vitro • Spheroid • Tissue engineering
L. Gibot (*)
Toulouse, France
e-mail: gibot@ipbs.fr

DOI 10.1007/978-3-319-32886-7_16
256 L. Gibot
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
Electroporation Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
2D Versus 3D Cell Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
Human Versus Animal Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
Tissue Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
Spheroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
Tissue Engineering Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
Introduction
Exposure of cells to high-voltage electric pulses causes membrane electroporation, or

electropermeabilization, i.e., transiently permeabilizes the plasma membrane of cells.
This phenomenon allows the entry of therapeutic molecules into cells and tissues. The
technique is easy to implement, safe, and inexpensive and facilitates tissue targeting
since only cells located between the electrodes are affected by the electric field.
Multiple developments are made of the methodology in human clinics. Electro-
poration is a safe technique for transferring nucleic acids into different target organs
and tissues, and gene electrotransfer (GET) is a promising method in gene therapy
either for systemic secretion of proteins, for vaccination, or in relation with cancer
therapy. Electroporation also ensures targeted and massive influx of cytotoxic
molecules into electropermeabilized tumor cells. This clinical application, called
electrochemotherapy (ECT), is used in many European countries for the treatment of
skin cancer and subcutaneous tumors. By potentiating the cytotoxicity of drugs, ECT
reduces the doses injected into patients and thus limits their side effects. Irreversible
electroporation (IRE) is a novel nonthermal ablation mode based on electro-induced
irreversible effect defects on cell plasma membrane. This method offers minimally
invasive treatment for selected tumors.
Optimizing therapeutic drug or nucleic acid delivery by electroporation is a
challenging hurdle in preclinical studies. This is mainly due to differences between
animal tissue characteristics and those of human cells as well as the lack of
representativeness of conventional 2D cell culture.
Since cell physiology is quite different in 2D versus 3D environments, the
development of 3D cell culture systems is a real opportunity in biology to study
cell behavior after application of stress. Cells classically cultivated in 2D do not
provide an accurate representation of in vivo morphology, and cell responses
obtained from these cultures are misrepresented in the absence of cell-cell and
cell-matrix interactions. Conventional 2D in vitro models are often considered as
highly reductionist, jeopardizing the relevance of the preclinical efficacy data.
The mechanisms involved in gene and drug electrotransfer in tissue remain
largely unknown, particularly because of the complexity of the 3D architecture.
15 3D Tissue Models to Bridge the Gap Between Cell Culture and Tissue in. . . 257
Table 1 3D in vitro model characteristics in the perspective of electroporation studies. ECM,

extracellular matrix
Intercellular Type of Ease of Ease of
junctions ECM Coculture cells handling analyses Cost
Gel No No Yes Human/ + + ++
animal
Spheroid Yes No Yes Human/ + + +
animal
Tissue engineering Yes Yes Yes Human/ ++ ++ ++
animal
Decellularized Yes Yes Yes Human ++ ++ ++
human tissue
For example, one of the main hurdles for in vivo gene electrotransfer remains the
impaired diffusion of large molecules through tissue because of dense extracellular
matrix and cell-cell junctions.
The gold standard in term of 3D in vitro tissue model has to meet several criteria.
Cells have to be organized in the three dimensions within a native extracellular
matrix rich. This extracellular matrix should be a representative of in vivo situation,
meaning composed of various class of molecules (fibers, glycosaminoglycans,
glycoproteins) and not only one such collagen. Coculture of relevant cell types
found in the tissue of interest has to be present. Cells should be able to develop
intercellular junctions with neighboring cells. Human cell types are preferred. Its
cost and time to produce should be reasonable. Its manipulation should be user-
friendly. It should be analyzed easily both on fresh and fixed tissue, especially by
microscopy techniques in order to visualize processes at cell scale. Nowadays, all the
developed 3D models only meet partially these criteria (Table 1) meaning that more
work has to be undertaken to propose more physiological 3D models.
Compared to classical in vitro studies on monolayers or cells in suspension or
in vivo on animal models, only few articles have been published on studies using
in vitro 3D models. This chapter discusses 3D studies and underlines the great
potential of 3D in vitro tissue models to reach a better understanding and control
of drug and gene electrotransfer at tissue scale.
Electroporation Principle
When the cell is exposed to high external electric field for sufficient time for plasma
membrane charging, the cell membrane becomes permeable for ions, drugs, and
molecules as large as plasmid DNA, which are otherwise impermeable. This phys-
ical phenomenon of cell permeabilization by external pulsed electric field applica-
tion is named “electroporation.” If membrane permeability is transient, the cell
survives and the membrane recovers its selective permeability, the electroporation
is reversible; if the cell dies, the electroporation is irreversible.
The mechanisms of molecule electrotransfer were mainly deciphered thanks to
single-cell studies. Electroporation can be described as a three-step process. First,
258 L. Gibot
before electric field application, the plasma membrane acts as a physical barrier with
selective permeability that prevents the free exchange of hydrophilic molecules
between the cytoplasm and the extracellular compartment. Secondly, during electric
field application, the transmembrane potential increases, leading to the formation of
membrane defects, called pores, even if no experimental data support this hypoth-
esis. These membrane defects allow the exchange of molecules both from the outside
to inside of the cell and the opposite. Finally, after electric field application,
membrane resealing occurs within about 20 min depending on the cell type. Small
molecules such as propidium iodide or antitumor drugs have free access to the
cytoplasm of electropermeabilized cells. Their transfer to the cytoplasm occurs
both during the external application of the electric pulses and after, as long as the
plasma membrane is not resealed. On the other hand, delivery of larger and charged
molecules such as plasmid DNA requires distinct consecutive steps: migration of
DNA toward the membrane, DNA insertion into the membrane, translocation across
the membrane via endocytosis and cytoskeleton implication, DNA migration to the
nucleus, and finally transfer of DNA across the nuclear envelope for effective
transcription. DNA migration toward the membrane is dependent on DNA electro-
phoresis, thus trains of long pulses lasting several milliseconds are generally applied
for nucleic acid delivery.
Due to its selectivity both in terms of time and space, electroporation enabled a
development of multiple applications into medical and industrial processes: cancer
treatment, gene therapy, wound healing, bacterial disinfection, and electroextraction
in agro-industry. Despite their wide range of practical applications in medicine,
biotechnology, and process industry, the fundamental mechanisms of cell membrane
electroporation are not well understood at tissue scale.
Since several decades, a large number of parameters such as electric field
intensity, pulse number and duration, frequency of application, media conductivity,
temperature, pH, and surface charge have been experimentally investigated, partic-
ularly in single-cell context, and theoretically modeled by different research groups.
These theoretical developed models are capable of simulating the electroporation
phenomena for the simple cases of unique cells in suspension of in monolayer.
However, they often fail to predict the cell electroporation phenomena in complex
environment such as tissues, where cells don’t display a spherical shape and are
embedded in the complex extracellular matrix with nonhomogeneous electrical
properties. Furthermore, tissue properties such as perfusion, cell density, and
orientation can affect the electric conductivity of the tissue. While theoretical
models of cell electroporation in tissues exist, they incorporate only the information
about the cells and completely ignore the impact of other tissue components such
as extracellular matrix. At tissue scale, the challenge is therefore to decipher the
impact of physicochemical properties of extracellular matrix and cell organization in
3D on electric field distribution inside the tissue and cell membrane electroporation
process. This knowledge gap results in empirical approaches for protocol develop-
ment with unpredictable electroporation success rates in basic experiments and
clinics.
2D Versus 3D Cell Environment
Although conventional cell culture monolayers or cells in suspension have provided

important scientific and conceptual advances, it is obvious that 2D cell systems lose
the architecture of the tissue from which they originate. They also lack mechanical
and biophysical signals and intercellular communication (Pampaloni et al. 2007).
The 3D environment plays a key role in cell and tissue homeostasis. The cells
communicate dynamically and plastically with each other and with the surrounding
stroma. It is this permanent and reciprocal exchange between the cells and their
extracellular environment which maintains the architecture and the functional orga-
nization of the tissues (Fig. 1).
Thus, several major studies have shown that the dynamic and reciprocal interac-
tions between the extracellular matrix and the nucleus, mediated by the cytoskeleton,
coordinate cellular functions, architecture, and environmental tissue homeostasis
(Nelson and Bissell 2006; Zaman 2013). Thus, the concept of “dynamic reciprocity”
proposed by Dr Mina Bissell lays on the reciprocal link between the extracellular
matrix and the nucleus of a cell where the extracellular compartment provides
biological, chemical, and physical signaling cues to maintain tissue integrity. Indeed,
complex environmental interactions and forces contribute profoundly to the behav-
ior, phenotype, and evolution of both normal and tumor cells. Since the extracellular
Fig. 1 Dynamic interactions between cells and extracellular environment in a tissue context:
notion of mechanobiology. In a tissue context, there is a physical link between the extracellular matrix
and the nucleus via membrane receptors such as integrins and the cytoskeleton. Communication is
dynamic and reciprocal since the extracellular environment regulates cell function and gene expression
which modulates the architecture of the environment (Reproduced with permission from Macmillan
Publishers Ltd: Nature Reviews Molecular Cell Biology, (Wang et al. 2009), Copyright (2009))
260 L. Gibot
compartment dictates cell behavior via a myriad of controlled signals, researchers

have to develop ways to mimic these functions in vitro in order to recapitulate tissue
functions in both 3D space and over time.
In order to study delivery of drugs by electroporation in a human tissue context, it
is of utmost importance to produce in vitro biologically relevant 3D models of tissue.
These powerful tools have to reproduce biological, biochemical, and biophysical
constraints in vitro related to the 3D architecture of human tissue and thus will be
much closer to reality than cell culture monolayers or animal models.
Human Versus Animal Model
The skin is the preferred tissue for studying the consequences of therapeutic drug
electrotransfer, not only because of its accessibility and knowledge gained by
clinicians with electrochemotherapy but also because it is rich in presenting cells
and thus has immunological properties essential for the development of DNA
vaccination (Gothelf and Gehl 2010).
The choice of a study model is based on many factors such as availability, cost,
ease of operation, and especially on its anatomic/functional similarity to human
tissue. Even though porcine skin has the closest anatomy to that of humans, the cost
per animal and for housing facilities explains why rodents are conventionally used
for studies on gene and drug electrotransfer in the skin. However, murine skin differs
from human skin in its architecture and its cellular composition. In addition, a
transcriptomic study shows that only 30% of genes associated with the skin are
identical between human and mouse (Gerber et al. 2014).
The development of tissue engineering for the purposes of regenerative medicine
has led to different models of relevant human tissues.
Tissue Engineering
Tissue engineering can replace, restore, maintain, or improve the function of human
tissues through the laboratory production of biological substitutes for transplantation.
In addition to these clinical applications, tissue engineering is also an invaluable tool in
basic research. The reconstructed substitutes must provide a biological, biochemical,
and biophysical model in which cells survive, differentiate, and organize themselves
and are able to perform their biological functions (Badylak et al. 2012). A number of
approaches are based on the use of materials (scaffolds), organic or not, to provide a
3D structure yielding specific biological and mechanical properties (Fig. 2).
Gels
The extracellular matrix (ECM) is the major component of the 3D cellular environ-
ment providing mechanical and biochemical support for cells in a tissue. In order to
mimic tissue architecture more closely in vitro, some authors embedded cells in a 3D
Fig. 2 Schema of the three

fundamental components of
3D tissue models. Cell culture
methods, characteristics of the
scaffold, and relevance of
biochemical and mechanical
signals are primordial to
produce a 3D bioactive
construct
gel made with extracellular matrix components. Indeed, cells cultured in 3D matrices
meet a more physiological environment relative to classical 2D monolayers, espe-
cially concerning biomechanical stimulation. Thus, 3D matrices are thought to
provide a tissue-like environment.
Collagen type I is the most abundant protein in mammals and fulfills a variety of
mechanical functions depending on the tissue in the body. Interestingly, its mechanical
properties are generally adapted by a modification of the hierarchical structure rather
than by a different chemical composition. Collagen I, mostly of bovine and rat tail
origin, is the most widely used extracellular matrix protein for 3D cell culture in gel.
By providing in vivo-like extracellular matrix structures, it facilitates cell attachment,
growth, differentiation, migration, and tissue morphogenesis. Besides its biological
relevance, collagen I presents the advantage of being commercially available, even if
quite expansive. Collagen is very viscous and a considerable amount gets lost during
pipetting for gel preparation. All the protocols last approximately 3 h to produce a
usable gel. Generally, collagen comes in an acidic solution and needs to be neutralized
with NaOH and made isotonic with 10X PBS before mixing with cells. All the
solutions used have to be kept on ice until the cells have been added in order to
prevent collagen from gelling. Cells in suspension are then added to the collagen mix.
Once placed at 37 C, the polymerization can take place. A fast polymerization allows
optimal cell distribution in the gel. The major drawback of the gel approach is that
when seeded with cells, especially fibroblasts, they will contract because of cell
remodeling. This means that gels can be analyzed only for days after treatment.
Haberl et al. embedded CHO cells in a type I collagen gel and studied the
electroporation efficiency with propidium iodide uptake as well as the effects of
plasmid concentration and pulse duration on gene electrotransfer (GET) efficiency
(Haberl and Pavlin 2010). They confirmed that gene electrotransfer efficiency is
262 L. Gibot
Fig. 3 IRE pulse delivery in collagen I gel seeded with tumors cells. (a) Picture of the
experimental setup. (b) Geometry and mesh used in the finite element model for simulating the
electrical and thermal response of the in vitro tumors to IRE (Reprinted with permission from
(Arena et al. 2012), Copyright (2012), with permission from Elsevier)
comparable to that observed in in vivo studies and depends on plasmid-DNA

concentration and pulse duration.
Cells grown in agarose gel were recently used to underline the electrosensitization
role in cell ablation by a nanosecond pulsed electric field (Muratori et al. 2016).
Electrosensitization corresponds to a delayed increase of sensitivity to electropora-
tion for cells that have already been subjected to electroporation. Muratori
et al. showed that local ablation was obtained when trains of pulses were split into
two identical fractions. They demonstrated that 3D gels containing cells enabled cell
electrosensitization to be visualized and were a useful tool to study electro-assisted
cell ablation. Thus, electrosensitization may help normal or tumor tissue ablation by
reducing the voltage or number of pulses required or by increasing inter-electrode
distance without losing ablation efficiency.
Tumor cells grown in 3D collagen gels were shown to be a relevant model for
studies on irreversible electroporation (IRE)-based cancer therapies and help to
improve treatment planning without the use of animal models (Arena et al. 2012).
Irreversible electroporation is a nonthermal, local ablation technique that is highly
promising in cancer therapy. In order to induce cell death, series of short-duration,
high-intensity electric pulses are applied to the targeted tissue. Collagen I hydrogel-
based bioengineered tumors were used to generate data for refinement of the algo-
rithms necessary to elucidate electric field distribution in tissue, which forms the basis
of current treatment planning algorithms for in vivo applications. Indeed, electrical and
thermal fluctuations were monitored during treatment (Fig. 3). Thus, 3D cell culture
models can be used for more accurate modeling of IRE-based cancer therapies in an
in vitro experimental setting, which will help to spread the clinical use of this method.
Spheroids
While the gel strategy uses a biomaterial as a scaffold to provide a 3D structure to the
model, the spheroid approach is devoid of exogenous material. In the 1980s,
Sutherland et al. proposed a multicellular 3D model named the spheroid (Sutherland
1988). This 3D model is based on cell aggregation properties and exhibits numerous
intercellular junctions.
Several techniques can be applied to promote spheroid formation (Hirschhaeuser
et al. 2010), but two are predominantly used: the hanging drop technique or the
seeding of cells in nonadherent round-bottom 96-well plates. In the hanging drop
technique, wells are coated with agarose in order to prevent cell attachment. Then, a
small drop (usually 20 μl) of cell culture medium containing 500 cells is deposited
on the interior face of the plate lid. When the lid is placed onto the plate, the drop
hangs over the agarose-coated well. After 3 days, thanks to gravity, the cells started
to attach together and form a small spheroid that has to be transferred into the
agarose-coated well in order to grow for 2 more days in large volume of cell culture
medium. This technique presents some drawbacks: Some spheroids are lost during
the transfer phase; spheroid diameters are not homogenous between all the wells; it is
time consuming. The other classical used technique is the growth of cells in
nonadherent environment. For this purpose, some plates are conveniently commer-
cially available. Homemade nonadherent plates can be produced using a coating
with poly(2-hydroxyethyl methacrylate) (poly-HEMA). The main advantage of the
nonadherent approach is that spheroids can be produced in 96-well plates, and they
are highly homogenous in size. Usually, spheroids are used around 5 days after
seeding and can be followed and analyzed for weeks after treatments.
Interestingly, both normal and tumor cells were found to form spheroids. In large
proliferating tumor spheroids, gradients of oxygen, nutrients, catabolites, and pro-
liferation are observed, mimicking those established in vivo in avascular regions of
solid tumors (Fig. 4).
Several publications have demonstrated that a 3D spheroid model is a relevant
tool to study and optimize DNA electrotransfer (for review see (Gibot and Rols
2013)).
Canatella et al. paved the way for studies on electrotransfer of small molecules in
3D spheroid structures (Canatella et al. 2004). Using calcein, they showed that
cellular uptake within a spheroid is spatially heterogeneous after electroporation,
with a decreasing gradient from the surface to the core. Indeed, an increased
diffusion lag time is required for small molecules to homogeneously diffuse within
the spheroid. Besides, dense cell populations exhibit heterogeneous electric proper-
ties, which modulate the cell response to electric field application. Finally, depending
on their location within the spheroid, cells display a variation in size. Thus, cell
radius in the core was found to be 19% smaller than at the surface. Spheroids seem to
be a suitable model to reproduce, in vitro, the cell heterogeneity found in tissue.
Comparing cell suspensions and spheroids, Chopinet et al. demonstrated that, for the
same level of electropermeabilization, GET efficiency was 25-fold higher in cells in
suspension than in cells organized in 3D (Chopinet et al. 2012). Furthermore,
efficiently transfected cells were solely located at the spheroid surface, especially
on the side facing the negative electrode (Fig. 5; Mellor et al. 2006; Wasungu
et al. 2009), stressing that large molecule diffusion is a hurdle in GET efficiency,
as it is in an in vivo context. Indeed, cell density and contacts between cells
(intercellular junctions) seem to limit/prevent uniform DNA distribution.
264 L. Gibot
Spheroid Characteristics
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Fig. 4 Schematic representation of gradients established in a growing tumor spheroid. Dotted

line delimits the necrotic core (Reprinted with permission from (Hirschhaeuser et al. 2010),
Copyright (2010), with permission from Elsevier)
Melanoma spheroids produced in a microgravity environment have proven to be

a relevant and useful tool to study gene electrotransfer following intra-spheroid
plasmid DNA injection (Marrero and Heller 2012). Large-scale spheroids of
human keratinocytes were injected with melanoma cells before in vitro determina-
tion of optimal electric parameters that allow GFP-reporter gene as well as IL-15
gene of interest efficient electrotransfer. Interestingly, these mixed spheroids
exhibited distinct sensitivity to transfection between normal cutaneous cells and
melanoma tumor cells. Finally, this model demonstrates the predictability of cell
response to GET and may facilitate future developments of clinical trials for cancer
therapies in vivo.
Tumor spheroids in vitro were shown to effectively reproduce processes observed
in vivo with electrochemotherapy (ECT) (Gibot et al. 2013), even if some limitations
Fig. 5 GET in spheroid. Cells efficiently electrotransfected with GFP-encoding DNA plasmid
(green) are only located at the spheroid surface. (a) Transmitted light images of the spheroid. (b, c)
Fluorescent images obtained by confocal microscopy (Reprinted with permission from (Wasungu
et al. 2009), Copyright (2009), with permission from Elsevier)
are still present, such as lack of blood circulation or immune system response.
Indeed, the concomitant application of an electric field with anticancer drugs con-
ventionally used in ECT, namely bleomycin and cisplatin, results in the total
destruction of the spheroid by cell death within 5 days after treatment. This faithfully
reproduces results obtained in vivo on small animals. Furthermore, the tumor
spheroid was demonstrated to be an effective tool for in vitro screening of new
candidate drugs for ECT.
Finally, this cell model in 3D will permit adaptation of therapeutic planning to the
biological profile of each patient and therefore develop personalized medicine,
which is more effective and better tolerated. Indeed, most medical treatments are
designed for the average population of patients. Even if it is successful for some
patients, it is not for others. Personalized medicine is a promising way to disease
prevention and treatment that takes into account differences in patient’s genes,
environments, and lifestyles. The final aim is to select treatments that improve
chances of survival while reducing adverse effects.
Interestingly, mixed spheroids composed of both normal and tumor human cells
were shown to be an efficient tool to reproduce in vitro phenomena observed in vivo
by clinicians after electrochemotherapy (Gibot et al. 2016). Indeed, after ECT,
clinicians observed good tissue healing and excellent esthetic and functional recov-
ery at the tumor site, meaning that normal tissue located between the electrodes was
not affected by the treatment. Experimentally, normal cells within mixed spheroids
are spared when treated with ECT. Recently, mixed spheroids also indicated differ-
ential effects in normal and malignant cell lines with calcium electroporation
(Frandsen et al. 2015). In conclusion, coculture of different cell types within
mixed spheroids will better mimic what happens in vivo in tissue submitted to
drug electrotransfer.
266 L. Gibot
Tissue Engineering Approaches
A number of approaches such as 3D gels are based on the use of exogenous materials
as scaffolds to provide a 3D structure and specific mechanical properties to their
models. Spheroid models are based on the development of intercellular junctions,
but they lack endogenous extracellular matrix. Innovative tissue-engineered
approaches focus on the use of living cells secreting their own extracellular matrix.
This scaffoldless technique is named the self-assembly method (Athanasiou
et al. 2013). Thus, cells stimulated with ascorbic acid, also named vitamin C, can
synthesize their own endogenous extracellular matrix rich in native components
such as collagens and form a handleable cell sheet. Indeed, ascorbic acid is a cofactor
of prolyl hydroxylase and lysyl hydroxylase. These enzymes catalyze the hydrox-
ylation of proline and lysine in hydroxyproline and hydroxylysine, respectively.
These hydroxylases, localized in the endoplasmic reticulum of cells, are essential to
the intracellular assembly of the collagen triple helix. Indeed, hydroxyproline resi-
dues in the procollagen sequence ensure the tertiary structure in triple helix of mature
collagen. Over 35% of procollagen proline residues have to be hydroxylated in order
to maintain the collagen triple-helix conformation at the physiological temperature
of 37 C. Furthermore, ascorbic acid was shown to act at the level of gene expression
by increasing the transcription of collagen I gene and the stabilization of its mRNA.
It also increases the excretion rate of extracellular collagen.
During production, self-assembled tissue models display biological processes
similar to those that occur in vivo. The self-assembly approach leads to reconstructed
tissue with a morphological structure and organization close to those of in vivo
tissues.
Human dermal substitutes have been produced by the self-assembly approach and
used to understand the role of each parameter of the experimental setup on the
distinct cell types present within the skin during drug electrotransfer. These models
were efficiently electropermeabilized with application of millisecond electric pulses
(Madi et al. 2015). They are valuable tools to study GET mechanisms in human
tissue since they correctly reproduce limiting steps found in vivo such as plasmid
DNA mobility within a tissue dense in extracellular matrix (Madi et al. 2016). Thus,
3D tissue models produced by the self-assembly approach appear to be promising to
determine optimal electrical parameters for distinct cell types in a tissue.
In order to develop 3D tissue models ever closer to native human tissue, a
promising and innovative strategy is to use decellularized human tissues. Once
recellularized with human cells, they provide a valuable tool to gain ex vivo assess
to drug electrotransfer mechanisms. For example, human skin grafts colonized with
human cutaneous cells (keratinocytes and fibroblasts) were efficient ex vivo human
models for optimizing gene expression which is a challenging hurdle in preclinical
studies (Bulysheva et al. 2016). Interestingly, mechanical integrity and thickness of
the tissue allow for its manipulation with forceps and for intradermal injections of
plasmid DNA or therapeutic drugs (Fig. 6). In conclusion, this recellularized skin
graft structurally and functionally resembled native human skin in tissue histological
Fig. 6 Intradermal
injection within a
recellularized human skin
graft. Grafts are easily
manipulated. Injection of
green food coloring was
performed to visualize the
location of DNA injection
within the dermis
(Reproduced from (Bulysheva
et al. 2016))
organization, proving an effective 3D human skin model for preclinical drugs and
gene delivery studies.
Conclusions
Most studies on therapeutic molecule electrotransfer, either as part of electroche-

motherapy or of plasmid DNA vectorization, are carried out on 2D monolayer
cultures of cells, or in animal models. These approaches do not allow the putative
roles of tissue architecture (extracellular matrix, cell-cell junctions) to be deciphered
in drug electrotransfer at the tissue scale. 3D models may be carefully built up in
complexity in terms of spatial positioning of different cell types, controlling matrix
density, and composition. Such improved biomimetic models are the link between
the cells cultivated in 2D and the whole animal body. However, it has to be kept in
mind that even the more sophisticated 3D in vitro model will present some limits
compared to the human body. Until today, no fluid perfusion was achieved in in vitro
produced tissue substitute. Thus, 3D models lack blood and lymphatic circulation.
Besides, even if some immune cell types can be included in studies in 3D in vitro, no
functional immune system is today available. Finally, despite the development of
“organ-on-chip” technologies, it remains difficult to in vitro reproduce dialogues and
communication existing between different tissues and organs.
In 1959, Russell and Burch published “The Principles of Humane Experimental
Techniques,” and the basic tenet of their report is that “the humanest possible
treatment of experimental animals, far from being an obstacle, is actually a prereq-
uisite for a successful animal experiment.” Thus, they proposed the 3Rs’ ethical rule
which consist in replacement, reduction, and refinement of animal experiments.
Recently, a fourth R was added to this chart for responsibility of the experimenter.
268 L. Gibot
Consequently, as a part of the implementation of the 4Rs’ ethical rule, 3D in vitro

models are a reasonable alternative to the use of laboratory animals.
3D models can potentially be reconstructed with cells from a patient, which
would allow the therapeutic protocol to be adapted to the biological characteristics
of the patient. Thus, this approach will permit the development of efficient and
personalized medicine.
Finally, the exploration of nonviral vectorization of therapeutic drugs in a human
tissue context will not only bring many relevant answers on its fundamental mech-
anisms but also promote crucial advances for medical applications. Thus, a better
understanding and control of the electroporation mechanisms at the tissue scale will
improve the targeting and effectiveness of drug transfer, whether it is anticancer
molecules in electrochemotherapy, genetic information for DNA vaccination, or
gene therapy.
Cross-References
▶ 3D Culture Models to Assess Tissue Responses to Electroporation

▶ Electrochemotherapy and Its Clinical Applications
▶ Principles of Electroporation for Gene Therapy
▶ Tissue Ablation by Irreversible Electroporation
▶ Tissue Engineering with Electroporation
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Single Cell Electrical Characterization
Techniques 16
Olivier Français and Bruno Le Pioufle
Abstract
In this chapter the possibility to electrically characterize the electroporation of
single cells is discussed. During the electroporation process, the application of
pulsed electric field (▶ Chap. 59, “Transmembrane Voltage Induced by Applied
Electric Fields”) leads to the partial permeabilization of the cell membrane,
opening access to the cytoplasmic compartment for drug delivery, for instance.
The electric impedance of the cell is modified by such a treatment. Impedance
measurement can thus be employed to characterize the permeabilization effect, on
a given angular frequencies range, which leads to an impedance spectrum. The
impedance spectrum measurement can be achieved even at the level of single
cells. In that case, the distance between the detection microelectrodes and the cell
must be considered in order to achieve a satisfactory sensitivity. In addition, when
considering the impedance measurement of cells flowing through a microfluidic
channel, fast detection is also required, as the cell is present between the mea-
surement electrodes only for a short while. A specific strategy must then be
employed to achieve such goal. In this case, instead of screening the whole
frequencies spectrum, a few set of frequencies should be selected and mixed to
be simultaneously injected prior to synchronous detection. Another way to assess
the single cell impedance is the use of indirect methods based on the mechanical
behavior of cells that polarize under the application of a stationary or propagative
electrical field. In particular, electrorotation experiment provides the electrical
characterization of the cell, which is dependent of its level of permeabilization,
O. Français
ENS de Cachan, CNRS SATIE, Cachan, France
e-mail: olivier.francais@satie.ens-cachan.fr
B. Le Pioufle (*)
ENS de Cachan, CNRS SATIE, Cachan, France
Ecole Normale Supérieure de Cachan, Cachan, France
e-mail: bruno.lepioufle@satie.ens-cachan.fr; bruno.le-pioufle@ens-cachan.fr

DOI 10.1007/978-3-319-32886-7_15
272 O. Français and B. Le Pioufle
after the application of electrical field pulses. When such approach is used, the
vicinity of electrodes is less critical as the main requirement lays on the homo-
geneity of the rotating electrical field. Finally, the possibility to focus the elec-
troporation at the subcellular level is discussed in the chapter. Indeed, the use of
micro- or nanotechnology permits to localize spatially the electroporation on a
tiny portion of the membrane of single cells. Adherent cells can be locally
electroporated, using nanosized electrodes, subsequently used to monitor the
intracellular potential.
Keywords
Bioimpedance • Single cell • Microfluidics • Electrorotation •
Microelectroporation
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
Impedance Measurement of a Single Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Electrical Characterization Using Electrorotation Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
Microelectroporation of a Single Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
Introduction
The impedance measurement is a common way to investigate the level of electro-

poration of cells (Pavlin et al. 2005). Such measurement can be achieved even at the
level of single cells. In that case, the vicinity between the detection microelectrodes
and the cell must be considered in order to achieve a satisfactory sensitivity. Indeed
the measurement electrodes probe a volume composed of the cell and the medium,
the volume ratio between those being thus of prime importance.
The impedance spectrum is defined as the ratio between the voltage applied to a
pair of electrodes and the current collected by these electrodes, in function of the
applied angular frequency. In the case where only medium is contained between the
electrodes (distance between the electrodes D, surface of electrode S, see Fig. 1), the
impedance expresses:
1 D 1
Zm ¼ ¼ (1)
σ mS em S S iωem
þ iω
D D
where σDmS represents the conductance of the medium between the two electrodes, and
em S
D its capacitance. In this equation, em represents the permittivity of the medium, σ m
its conductivity, ω stands for the angular frequency of the voltage applied to
electrodes, and i is the complex number (i2 = 1). This impedance can also be
16 Single Cell Electrical Characterization Techniques 273
Fig. 1 Single cell immersed electrode 1

within the medium between S (surface)
two electrodes (gap D, surface external medium
S). The dielectric properties of
the different compartments of
the cell (e*memb, e*in) are
mbrane
homogenized to e*cell thanks to me
the single shell model e
R
D
cytoplasm
electrode 2
expressed in function of the complex permittivity of the medium, defined as em ¼ em

i σωm , as seen in Eq. 1.
Such spectrum is modified by the presence of a cell between the electrodes, as
represented in Fig. 1. The impedance of an elementary volume of medium
containing a single cell disposed between those two measurement electrodes (dis-
tance between the electrodes D, surface of electrode S, see Fig. 1) can be approxi-
mated using the Maxwell mixture theory (Maxwell 1881):
D 1 D 1
Z mix ¼ ¼ (2)
S σ mix þ iωemix S iωemix
where e*mix is the complex permittivity of the mixture, composed of the volume of
medium containing the single cell, that can be expressed in function of the complex
permittivity of the medium itself e*m, the volume fraction Φ of the cell within the
medium, and the Claussius-Mossotti factor fCM that characterizes the contrast
between the cell and the medium in term of complex permittivities (Jones 2005):
1 þ 2Φ f CM
emix ¼ em (3)
1 2Φ f CM
with
ecell em
f CM ¼ (4)
ecell þ 2em
and Φ the fraction volume between the cell and the volume between electrodes:
4
πR3
Φ¼3 3 (5)
D
the complex permittivity e* being defined as a function of the permittivity e and
conductivity σ of the corresponding material (inner compartment of the cell, cell
membrane, medium) as:
σ
e ¼ e i (6)
ω
σ mix and emix relate respectively to the conductivity and permittivity of the equivalent
mixture (medium containing the cell).
The complex permittivity of the cell e*cell (that appears in the expression of the
Claussius-Mossotti factor fCM and thus affects the impedance of the mixture, Eq. 2)
depends on many parameters as it relates to the geometry, permittivity, and conduc-
tivity of the cell components (membrane, cytoplasm, intracellular components).
Anyway the complex permittivity of a spherical-shaped cell can be approximated,
using the single shell model (Huang et al. 1992), when considering the cell as an
averaged inner compartment having its own dielectrical properties σ in and ein,
surrounded by an outer cytoplasmic membrane (σ memb and ememb, see Fig. 1):
2 3
R 3 ein ememb
þ2
6 Re ein þ 2ememb 7
ecell ¼ ememb 6
4 3 e e 7
5 (7)
R

in memb

Re ein þ 2ememb
where R stands as the cell radius and e as the thickness of the cell membrane, as
depicted in Fig. 1.
This chapter will describe firstly the influence of the electroporation on the
impedance spectrum characteristic of single cells within miniaturized microfluidic
devices (section “Impedance Measurement of a Single Cell”). The dependance of the
measurement resolution with the volume fraction Φ between the cell and the
surrounding medium will be assessed. Methods for the impedance measurement of
flowing cells, that can be achieved by choosing a set of excitation frequencies
simultaneous injected prior to synchronous detection, will be described and illus-
trated by some important achievement found in the literature.
Section “Electrical Characterization Using Electrorotation Experiment” will
describe an alternative method to monitor the impedance spectrum of cells during
electroporation that is less dependent on the high volume fraction Φ requirement.
This method – the electrorotation spectrum – is based on the analysis of the
mechanical angular velocity of cells exposed to a rotationary electric field.
Finally the section 3 (Microelectroporation of a Single Cells) will show that the
recent improvements of the technology permit the focusing of electrical field in
such a way that the electroporation is localized on the cell membrane
(microelectroporation).
Impedance Measurement of a Single Cell
The electrical characterization of a single cell is defined by the assessment of its

dielectrical properties, the conductivity σ and permittivity e of its components
(membrane and intracellular components). The single shell model of the cell pro-
vides a fair description of its dielectric behavior, through its complex permitivity
spectrum, as shown on Fig. 2.
Indeed, as introduced in the previous section, the complex permittivity of the cell
ecell ¼ ecell i σωcell involves a real part that relates to the dielectric polarization of the
cell (permittivity ecell), while the imaginary part relates to the losses (conductivity
σ cell).
At the lowest frequencies, the cell membrane is preponderant ( ecell ! Re ememb
when ω ! 0, from Eq. 7), while at high frequencies, the interfacial relaxation
resonance does appear (see Fig. 2). The latter corresponds to the vibration of ions
confined by the cell membrane within the inner part of the cell, which is associated to
the loss peak visible in Fig. 2.
The impedance measurement should sense both low and high frequencies char-
acteristics (the membrane dielectric characteristic Re ememb at the lowest frequencies
and the relaxation phenomena induced by the resonance due to the ions confined by
the membrane in the inner compartment at higher frequencies) and should thus
provide the dielectric characteristics of both the inner compartment and the
4
x 10 Complex permitivity of the cell
4
real part
3.5 Imaginary part
membrane like
3
behavior
Complex permitivity
2.5
2 Interfacial
relaxation
1.5
0.5
0 0 2 4 6 8 10
10 10 10 10 10 10
Angular frequency ω (Rd/s)
Fig. 2 Real and imaginary parts of the complex permittivity spectrum of a cell. The comparison
between the real and imaginary parts of the complex permittivity highlights the fact that the cell
membrane is predominant in its dielectric behavior at low frequency and the relaxation resonance
induced in the inner compartment confined by the membrane that occurs at the higher frequencies.
R = 5 μm, e = 5 nm, σ memb = 108 Sm1, σ in = 1 Sm1, e0 = 8.85 1012, ememb = 11e0,
ein = 50e0
membrane. Nevertheless, the impedance measurement is affected by the dielectric

characteristics of the surrounding medium included between sensing electrodes (see
Fig. 1).
Using the Maxwell’s mixture model, the impedance spectrum of the composite
material including the cell and the surrounding medium, i.e., the dependance of Zmix
with the electrical frequency ω, can be evaluated (Sun and Morgan 2010). The
impedance of the mixture depends on the dielectric properties of the cell e*cell, on
the dielectric properties of the medium e*m, and on the volume ratio between those.
As shown in Fig. 3, where the complex resistivity spectrum is represented, the
volume ratio Φ determines the sensitivity of the cell impedance measurement (the
complex resistivity spectrum ρ*mix(ω) is defined in Eq. 8 as the adimensional
equivalent of the mixture impedance Zmix). Indeed the miniaturization of the mea-
surement system (gap and surface of electrodes) advantageously increases the
sensitivity of the spectrum to the presence of a single cell between electrodes
(10.6% variation of the impedance, for low frequencies (ω ’ 105 Rd/s), for a
characteristic dimension D = 4 Rcell).
1 1 S
ρmix ¼ ¼ ¼ Zmix (8)
σ mix þ iωemix iωemix D
Complex resistivity, mixture model

1
D=15 μm
no cell, Cdl
0.95 D=15, Cdl
D=20 μm
D=25 μm
0.9 D=30 μm
n
no cell
tio
riza
0.85
iatu
M in
0.8
0.75
0.7 5 6 7 8
10 10 10 10
Fig. 3 Impedance spectrum of a single cell within a cubic volume of medium (electrode surface
D2, electrode distance D), using the Maxwell’s mixture theory. Plain curves: the cell is present
between electrodes that are separated with a distance D varying from 15 μm to 30 μm. Bold red
curve: reference impedance spectrum (no cell). Dashed curves: the influence of the double-layer
capacitance is shown, with or without cell. R = 5 μm, e = 5 nm, σ memb = 108 Sm1, σ in = 1
Sm1, σ m = 1.4 Sm1, e0 = 8.851012, ememb = 11e0, ein = 50e0, em = 80e0
The impedance of the double-layer Zdl ¼ iωC1 D2 , due to the contact between the
s
σm
electrode and the electrolyte, should not be neglected as the cut frequency DC s
associated to its effect is also linked to the degree of miniaturization. The double-
layer capacitance is taken into account in Fig. 3 (dot lines, for a double-layer
capacitance Cs = 0.5 Fm1, corresponding to black Platinum electrodes (Martinsen
and Grimnes 2011)). Anyway this effect becomes negligible, if high surface capac-
itance materials are chosen for the electrodes, as soon as high electrical frequencies
are considered. If this is not the case, differential measurement (as in Holmes
et al. (2009), see Fig. 5) where the impedances with and without cells are subtracted
will be implemented. Such differential measurement is broadly used as it also
permits to get rid of the temperature effect, which occurs in the two measurement
zones, and can thus be eliminated by the differentiation.
Using a measurement configuration with convenient dimensions (high minia-
turization D = 20 μm, for instance, with Rcell = 5 μm), the permeabilization of
the membrane (▶ Chap. 55, “Different Approaches Used in Modeling of Cell
Membrane Electroporation”) can be monitored with impedance measurement.
As shown in Fig. 4, the permeabilization of the membrane leads to a significant
modification of the measured impedance (11% variation of the impedance for low
frequencies (ω ’ 105 Rd/s), for a characteristic dimension D = 4 Rcell, and for an
Influence of the permeabiliation on the complex resistivity, D=20 μm

0.79
−1
10−8 Sm
n
0.78 10 Sm −1
−5
tio
abi ne
liza
10−4 Sm −1
meembra
0.77
10−3 Sm −1
perm
no cell
0.76
10−3, 45°
0.75
0.74
0.73
0.72
0.71 5 6 7 8
10 10 10 10
Fig. 4 Impedance spectrum of a cell, influence of the membrane permeabilization. The distance
between electrodes is D = 20 μm. The membrane conductivity σ membrane is the variable parameter.
Plain curves: the cell is present between electrodes, and has varying levels of σ memb, from 108
Sm1 to 103 Sm1. Bold pink curve: partial permeabilization of the cell membrane, σ memb = 103
Sm1 on the poles of the cell, with a 45 solid angle, while the rest of the membrane keeps the 108
Sm1 conductivity (this curve is obtained by a 3D finite element calculation). Bold red curve:
reference curve, no cell between the electrodes
increase of the membrane conductivity σ memb from 108 Sm1 up to 103 Sm1).
On this figure is represented the evolution of the mixture complex resistivity, when
the whole membrane is permeabilized (pink curve), or when part of it is
permeabilized (permeabilization of the poles of the cell facing the electrodes
(Fig. 1), on a 45 solid angle section, bold pink curve. This curve is achieved
thanks to finite element calculation).
The impedance measurement of single cells flowing within a small channel
equipped with electrodes was firstly performed by Hoffman and Britt (1979). The
velocity of the cell is too fast for a whole impedance spectrum recording, since all
frequencies should have to be screened while the cell is passing above the electrodes.
For that reason a set of excitation frequencies has to be chosen that might provide the
relevant information about the cell status. To do so Hoffman and Britt (1979) injected
simultaneously this set of excitation frequencies, the measured spectrum being then
demodulated using “synchronous detection” techniques. Gawad et al. (2001)
performed the same type of experiment within a micromachined fluidic device. The
influence of the medium variations, due to the temperature, for instance, as well as the
effect of the double-layer capacitance is compensated thanks to a differential measure-
ment (Morgan et al. 2006). Such a strategy was used successfully to differentiate white
blood cells (neutrophils, monocytes, and lymphocytes) from the whole blood sample,
after lysis of the erythrocytes (Holmes et al. 2009). To perform those assay, two
excitation frequencies where injected for the impedance analysis, and the results were
correlated to an optical detection within the same flowing microchannel (see Fig. 5)
This approach has also been used to characterize on the same device the imped-
ance variation of single cells flowing forward and backward within a microfluidic
channel (Bürgel et al. 2015). Such a device, which structure is shown in Fig. 6,
permitted a simultaneous electrical monitoring of the permeabilization thanks to
impedance measurement (eight frequencies were simultaneously injected) as well as
a microscopy observation of the intracellular content release and cell swelling, in real
time and at the single cell level.
Interesting possibilities to assess the cytoplasm content are investigated by
extending this impedance spectroscopy towards the high frequencies. An exam-
ple of these possibilities is provided in Haandbæk et al. (2014), where the high-
frequency range (up to 500 MHz in this study) allowed to characterize and
differentiate wild or mutant yeasts through their intracellular content. The fre-
quency range is still limited by the technology, but more precise characterization
of the dielectric properties of cells thanks to impedance spectrometry is expected
in the near future.
Electrical Characterization Using Electrorotation Experiment
As recalled previously and shown in Fig. 3 and Eq. 3, the volume fraction of the cell
Φ within the measurement zone, i.e., the level of confinement, is crucial in order to
achieve an acceptable sensitivity when performing impedance spectroscopy at the
Fig. 5 Microfluidic device for the electrical differentiation of white blood cells thanks to imped-
ance measurement. Two electrical frequencies (503 kHz and 1.7 MHz) were simultaneously applied
to contiguous pairs of electrodes for a differential measurement. One pair of electrodes probes the
cell, while the other pair of electrodes probes a volume of the same medium without cell as a
reference. The differential current measurement is then demodulated using synchronous detection
techniques. Fluorescent labeling of the respective population to detect was achieved within the same
channel, cell-by-cell in the detection region of the microchannel, using dual laser excitation
(Adapted from Holmes et al. (2009), permission from Royal Society of Chemistry Publishing
group, Ltd: Lab on Chip, copyright (2009))
single cell level. An alternative to acquire single cell impedance spectrum, which
requires a lower level in term of confinement, is to perform electrorotation experi-
ment (Trainito et al. 2015). In that case the cell is submitted to a homogeneous
propagative electric field, which drags the cell due to its polarization (Pohl 1958;
Čemažar et al. 2013). In the case of the electrorotation, the propagative electrical
field is rotational. The cell experiences a torque, which directly relates to its
dielectric contrast with the medium (the contrast being characterized by the
!
Claussius-Mossotti factor (Eq. 4)). Indeed this electrorotation torque Γdep depends
Fig. 6 On-chip
a Pt electrode
electroporation and
impedance spectroscopy. (a) Glass
The cell is injected within the
Flow provided by
syringe pump
microchannel and the
impedance is measured before
and after the electroporation Cell Cell
(8 V amplitude AC voltage
applied to platinum planar
electrodes spaced 18 μm with
a 50 kHz frequency). The cell
is then flowed forward and EIS analysis Electroporation
backward between the
measurement electrodes
(to which superposed 1 V
amplitude AC signals, in the V(t)
range 20 kHz–20 MHz, are
imposed). (b) View of the Cell Cell
microdevice, and electrical
and fluidic connections (From t
Bürgel et al. (2015),
permission from Elsevier
b
Publishing group, Ltd: Fused silica
Sensors and Actuators B, Holder PCB capillaries
copyright (2015))
Measurement
region
Electrode
Microfluidic
chip
on the imaginary part of fCM, on the squared modulus of the electric field, and on the
volume of the cell (Morgan and Green 2003):
! !
Γdep ¼ 4πR3 em ℑ ðf CM ÞE2 z (9)
!
where z is the unitary vector, normal to the plan of electrodes. Varying the electrical
field angular frequency ω, a spectrum of the rotational velocity of the cell can be
measured:
e 0 e m E2
Ωcell ¼ ℑ ðf CM Þ (10)
2η
where η stands for the dynamic viscosity of the medium.
As seen from Eq. 10, the rotational velocity spectrum is proportional to the
imaginary part of the Claussius-Mossotti factor ℑ( fCM), which is affected by the
level of permeabilization (Eq. 4). The Fig. 7a points out the dependence of this
spectrum on the cell membrane conductivity σ memb. This dependency is particularly
obvious for the first peak (the positive one) of ℑ( fCM) function, i.e., for ω < 30 106
Rd/s. Indeed for these low frequencies, the complex permittivity of the mixture is
mainly dependent on the membrane properties (ecell Re ememb , as mentioned in the
previous section).
The second peak of ℑ( fCM) (the negative one) is sensitive to the dielectric
properties of the intracellular compartment (σ in and ein), as seen in Fig. 7b.
To estimate the dielectric parameters (ememb, σ memb, ein, σ in), the fitting between
the measured experimental electrorotation spectrum and the theoretical spectrum
that depends on these dielectric parameters can be achieved using least-square
algorithms.
In the case of a single cell electrorotation experiment, a strategy consists in
(i) trapping the cell thanks to conventional dielectrophoresis forces using a set of
electrodes inducing a stationary electrical field and (ii) to superpose to this sta-
tionary field a rotational field creating an electrorotation torque (Huang
et al. 1992). From the velocity spectrum, the dielectric characteristics of the cell
e*cell and finally the dielectric characteristics of the cell components (e*memb for the
membrane and e*in for the averaged cytoplasm) can be estimated. This strategy was
applied to monitor the permeabilization level of a single cell, trapped by a four-
electrodes set, and submitted to such electrorotation torque (Trainito et al. 2015),
see Fig. 8.
Microelectroporation of a Single Cell
Thanks to the degree of miniaturization achievable with microfluidic technologies,

the permeabilization can be localized precisely on the membrane of a single cell
(concept of microelectroporation). This approach is a way to focus precisely the
access to the cytoplasm and possible drug delivery, and should improve the cell
viability, compared to the conventional methods for membrane permeabilization, as
the permeabilized zone remains small. In this case, the objective is not the cell
bioimpedance measurement but creating an access to the cytoplasm for DNA
injection, drug delivery, or intracellular potential recording. The first example
showing such an approach is described in Huang and Rubinsky (1999). In Huang
and Rubinsky (2001) the cell is trapped by a microaperture etched within a thin
silicon nitride membrane that also focuses the electrical field. The current through
the microaperture (and thus through the patched membrane) is measured during the
electroporation, which provides a way to monitor electrically the process of cell
membrane electroporation.
Another version achieved in PDMS (polydimethylsiloxane) was proposed in
Khine et al. (2005), where the cell to be electroporated was trapped at the inlet of
Imaginary part of Claussius-Mossotti factor

a 0.6
10−8 Sm −1
10 Sm −1
−5
Perme
0.4
10−4 Sm −1
memabili z
10 Sm −1
−3
b ra at
ne i
0.2 on
−0.2
−0.4 5 6 7 8 9 10
10 10 10 10 10 10
b Imaginary part of Claussius-Mossotti factor

0.5
initial
0.4
low permittivity
0.3 low conductivity
0.2
(fCM )
0.1
−0.1
−0.2
−0.3
−0.4 5 6 7 8 9 10
10 10 10 10 10 10
Fig. 7 Dependance of the single cell electrorotation spectrum with the dielectric parameters of the
cell. (a) The membrane permeabilization mainly affects the first peak of the spectrum, corresponding
to the lowest frequencies. The imaginary part of Claussius-Mossotti factor is represented here, with a
parametric variation of the membrane conductance (σ memb increasing from 108 Sm1 up to 103
Sm1). (b) The second peak of the characteristic is mostly sensitive to the dielectric properties of the
intracytoplasmic compartment σ in and ein. The initial spectrum is represented (σ in = 1 Sm1, ein =
50e0, bold blue curve) as well as the low permittivity for the inner compartment (ein = 20e0, red dots
curve) and the low conductivity for the inner compartment (σ in = 0.5 Sm1, light blue curve). For
both curves (a) and (b), σ m = 0.3 Sm1 (low conductivity medium)
b
Electrorotation spectrum - B16F10 cell
0.5
0
Rotational speed [rps]
-0.5
-1
-1.5
-2
-2.5
Fitted data BP
-3 Experimental data BP
Fitted data AP
-3.5 Experimental data AP
104 105 106 107 108

Frequency [Hz]
Fig. 8 Influence of the electroporation on the electrorotation spectrum of a single cell. (a)
Experimental protocol: the cell captured in the central area using conventional dielectrophoresis
is submitted to the electrorotation torque. The electrorotation spectrum is measured before and after
electroporation. (b) Experimental electrorotation spectra obtained before and after permeabilization
of the single cell (lymphocyte). DEP dielectrophoresis, EROT electrorotation, PEF pulsed electric
field, BP before pulses, AP after pulses (Adapted from Trainito et al. (2015), permission from John
Wiley and Sons Publishing group, Ltd: Electrophoresis, copyright (2015))
a tiny microchannel (section = few μm2, see Fig. 9). Real-time monitoring of the
electroporation, by means of the current measurement, was also achieved in a
parallelized version of the device (Khine et al. 2007).
Such concept is very similar to the patch-clamp technique (▶ Chap. 74, “Patch
Clamp in Use of Electroporation Mechanisms Studies”), firstly proposed by Neher
a V/Vo b
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
5 µm
0.2
0.1
0
1
0.9
0.8
0.7
0.6
V/Vo
0.5
0.4
0.3
0.2
0.1
0
10 20 30 40
Distance x/µm
Fig. 9 Concept of the microelectroporation. (a) The cell is trapped and immobilized in contact of a
microaperture which enhances the transmembrane voltage in the patched zone of the membrane.
V corresponds to the potential, while Vo refers to the potential difference applied to electrodes. (b)
View of the cell trapped thanks to the horizontal channel micromolded in PDMS technology
(Adapted from Khine et al. (2005), permission from Royal Society of Chemistry Publishing
group, Ltd: Lab on Chip, copyright (2004))
et al. (1978) where the intracellular potential of neurons is measured by the use of a
very thin glass pipette patching the cell membrane. The microelectroporation
approach proposed in Khine et al. (2007) brings the benefit of possible
parallelization which is a key progress for future drug screening applications.
Anyway, the patch-clamp technique was later developed in its microfluidic version,
known as the planar patch-clamp (Fertig et al. 2003), which is also compatible with
the parallelization (Brüggemann et al. 2006). Such similarity in the structure of both
types of devices (devices for microelectroporation and devices for the electrophys-
iological recording) led (Xie et al. 2012) to combine both functions with the same
measurement electrodes. Indeed this device integrates nanopillars at the tip of
recording electrodes, which are used to permeabilize the membrane very locally,
and lets the contact to establish with the intracellular compartment for transmem-
brane potential recording (Fig. 10).
Fig. 10 Concept of the nanoporation: nanopillars are designed on the tip of recording microelec-
trodes (1.5 μm tall and 150 nm diameter). (c) Once the cell is placed above the electrodes, electric
field pulses application leads to nanopore opening on the membrane, which opens transiently the
access between the recording electrode and the intracellular medium (recording in (b) versus
recording in (a)) (Adapted from Xie et al. (2012), permission from Nature Publishing group, Ltd:
Nature Nanotechnology, copyright (2012))
Conclusions
This chapter shows that the impedance measurement provides an interesting

approach to characterize single cells. In particular the cell permeabilization can be
monitored, even at the level of single cells, using therefore devoted microfluidic
devices. The degree of miniaturization of such devices is a crucial parameter, as the
volume fraction Φ directly influences the sensitivity of the measurement. The
electrodes must thus be at the vicinity of the single cell to be characterized (typically
the electrode gap should not exceed two times the cell diameter).
Under adequate conditions, where the electrodes are in a strong vicinity with the
cell, a precise monitoring of the evolution of cell electrical parameters can be
achieved during the electroporation. To do so, one should use high-specific surface
materials for the electrodes, like electroplated black platinum, as the influence of the
double layer due to the interface between the medium and the electrodes might
become critical when downscaling the device. Anyway, the influence of this double-
layer capacitance can be minimized using a differential measurement setup, probing
a volume of medium without cells as a reference. Moreover, such a differential setup
has to be considered in order to get rid of the temperature influence on the impedance
measurement. High-throughput electrical characterization of cells can be envisioned
in microfluidic devices, cells flowing one by one though the sensing area within a
microchannel. In such a configuration, a specific electronics is required for fast and
real-time analysis. Indeed the whole frequency spectrum cannot be acquired during the
short time while the cell is flowing at the vicinity of electrodes, and a set of discrete
excitation frequencies should thus be chosen to be injected simultaneously, prior to
synchronous detection. Spreading the characterization spectrum towards high angular
frequency is an important technological challenge, as it may permit to assess the
dielectric properties of the intracellular content, providing increasing accuracy on the
cell status determination. Promising works are ongoing with the high-frequency
impedance spectrometry of single cells. Such progress will allow to establish an
accurate dielectric signature of the cell as well as a marker of the cell content. In
particular, it might be a promising and complementary way to characterize the effect
of ultrashort electric field pulses [Measurement and Characterization of Exposure
System for High-Frequency/Ultrashort Pulses] (like nanopulses), that permeabilize
the intracellular lipid substructures (Schoenbach et al. 2001; Dalmay et al. 2012),
as information might be provided on the dielectric properties modifications
induced by such electrical treatment.
Single cell electrical characterization can also be achieved thanks to
electrorotation experiment, exploiting therefore the polarizability of cells immersed
within a homogeneous rotary electrical field. Indeed the polarizability of the cell, in a
given medium, and thus its mechanical response to a rotating electrical field, is
related to the Claussius-Mossotti factor ( fCM, see Eq. 4), that characterizes the
dielectric contrast between the cell and the surrounding medium, and depends on
the dielectric properties of the cell components (ememb and σ memb for the membrane,
ein and σ in for the intracellular compartment, see Eq. 7). Such an approach to estimate
the dielectric properties of the cell components does not require high volume fraction
Φ, and the required level of confinement is thus less critical. With this alternative
way to characterize single cells, it might be also advantageous to increase the
characterization spectrum high-frequency limit, as it might permit to assess the cell
interior.
Finally, the new possibilities in terms of miniaturization achievable with micro-
and nanotechnology open very promising perspectives, like the possibility to target
spatially the cell electroporation on a small portion of its membrane, and give
electrical access to the cell cytoplasm. Such electrical treatment is less invasive,
improving thus the cell viability after treatment. It shows to be a convenient way to
transiently get an access through the membrane for action potential recording. The
delivery of extracellular compounds through the membrane locally electroporated
thanks to this technique might be an interesting approach to be investigated in the
near future.
Acknowledgments This work has benefited from the financial support of the LabeX LaSIPS
(ANR-10-LABX-0040-LaSIPS) managed by the French National Research Agency under the
“Investissements d’avenir” program (nANR-11-IDEX-0003-02) and COST TD1104, www.electro
poration.net. The authors acknowledge project supports from ENS Cachan-Université Paris Saclay,
Institut d’Alembert, and CNRS.
Cross-References

Ultrashort Pulses
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Cell Stress Responses to Pulsed Electric
Fields 17
Ken-ichi Yano and Keiko Morotomi-Yano
Abstract
Cells are often exposed to unfavorable conditions and stimuli, which are collec-
tively referred to as stress. To adapt to stress, cells induce a set of reactions, called
stress responses, that lead to suppression of translation initiation. Protein synthe-
sis requires considerable amounts of energy and amino acids, and hence stress-
induced translational suppression preserves cellular resources and serves as a
mechanism for survival. This chapter is aimed at providing an overview of the
stress responses induced by pulsed electric fields (PEFs). PEFs are utilized in a
broad range of the life sciences, owing to their action on the cell membrane.
Nanosecond PEFs (nsPEFs) generate small membrane pores that permeate small
molecules, such as ions, and longer PEFs yield larger membrane pores suited for
DNA transfection and tumor chemotherapy. Irrespective of the pulse width, PEFs
are essentially deleterious to cellular homeostasis, because they compromise the
membrane integrity and perturb the balance between intra- and extracellular
molecules across the cell membrane. Recently, nsPEFs have been shown to
induce stress responses in human and mouse cells. nsPEFs elicit stress-responsive
signal transduction that comprises two protein kinases, namely, PERK and
GCN2, and their substrate eIF2α, leading to translational suppression by eIF2α
phosphorylation. nsPEFs affect another signal transduction that controls transla-
tion initiation by mTORC1-mediated 4E-BP1 phosphorylation. Collectively,
accumulating evidence supports the concept of PEF-induced stress responses
that appear to have profound effects on cellular functions such as gene expression
and cell death induction.
K.-i. Yano (*) • K. Morotomi-Yano

Institute of Pulsed Power Science, Kumamoto University, Kumamoto, Japan
e-mail: yanoken@kumamoto-u.ac.jp; morotomi@kumamoto-u.ac.jp

DOI 10.1007/978-3-319-32886-7_17
290 K.-i. Yano and K. Morotomi-Yano
Keywords
Stress response • Signal transduction • Protein kinase • Protein synthesis •
Translational suppression • Nanosecond pulsed electric fields • Nucleofection •
Electroporation
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
Overview of Stress Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
General Principles of Stress Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
Integrated Stress Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
Alternative Mechanism for Stress-Induced Translational Suppression . . . . . . . . . . . . . . . . . . . . . 294
Stress Responses Induced by Nanosecond Pulsed Electric Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Nanosecond Pulsed Electric Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Activation of eIF2α Phosphorylation by nsPEFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
Activation of Stress-Responsive Protein Kinases by nsPEFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Effects of nsPEFs on 4E-BP1 and AMPK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
Suppression of Protein Synthesis by nsPEFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
Nucleofection-Induced Stress Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Possible Effects of Electroporation on Cell Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
Introduction
Living cells often encounter physical and chemical stimuli from their external
environment and respond to them by evoking intracellular reactions that ultimately
lead to changes in cellular physiology and behavior. Cellular responses to external
stimuli generally involve the induction of intracellular signaling, which is mediated
by sequential protein phosphorylation and is transduced to downstream effector
proteins. Consequently, the external stimuli cause alterations in various cellular
activities, including gene expression, metabolism, proliferation, differentiation,
and cell death induction (Alberts et al. 2014).
Among the diverse external stimuli, those with adverse impacts on the cell are
referred to as cellular stress. Human cells have an ability to adapt to cellular stress by
inducing a distinct set of intracellular reactions that are collectively called stress
responses. The stress responses play critical roles in the maintenance of homeostasis
under adverse circumstances. Dysfunctions of the stress responses are known to be
associated with many diseases, such as inflammation and neurodegenerative dis-
eases, thereby implicating the physiological significance of stress responses
(Leprivier et al. 2015).
Pulsed electric fields (PEFs) are widely used in the life sciences, because different
effects on the cell membrane can be achieved in a pulse width-dependent manner.
PEFs on the order of ms to μs are suited for macromolecule transfer, and these
17 Cell Stress Responses to Pulsed Electric Fields 291
PEFs are widely used for DNA transfection (▶ Chap. 19, “Nucleic Acid
Electrotransfer in Mammalian Cells: Mechanistic Description”) and electroche-
motherapy (Rems and Miklavcic 2016). Nanosecond PEFs (nsPEFs) produce
small membrane pores that allow permeation of small molecules, such as ions
(Vernier et al. 2006; Pakhomov et al. 2009). PEF-induced membrane permeabi-
lization allows movement of extracellular as well as intracellular molecules across
the cell membrane and consequently perturbs cellular homeostasis. Accordingly,
PEFs are essentially deleterious for cellular physiology and could be considered as
cellular stress.
This chapter is intended to provide an overview of the stress responses induced by
PEFs. Owing to very limited information currently available on the stress responses
evoked during electroporation used for macromolecule transfer, particular emphasis
is placed on the cellular responses to nsPEFs and nucleofection. Exposure of human
cells to nsPEFs is known to elicit various intracellular reactions, and molecular
details of the nsPEF-induced stress responses have been previously demonstrated
(Morotomi-Yano et al. 2012b). Nucleofection is a sort of electroporation that is
conducted under undisclosed electrical conditions optimized for individual cell types
(Gresch et al. 2004). A previous study has shown the induction of stress-responsive
signaling by nucleofection (Anderson et al. 2013). In this chapter, first, the general
mechanisms of cellular stress responses in human cells are outlined. Second, the
nsPEF-induced stress responses are described in detail. An outline of nucleofection-
induced reactions is also given. Finally, possible physiological implications of the
PEF-induced stress responses are discussed. Although this chapter focuses on
cellular responses in human and mammalian cells, other chapters (▶ Chaps. 140,
“Responses of Plant Cells and Tissues to Pulsed Electric Field Treatments,” and
▶ 137, “Stress Response of Plants, Metabolite Production due to Pulsed Electric
Fields”) provide the outline of stress responses in plant cells.
Overview of Stress Responses
General Principles of Stress Responses
Cells can sense environmental changes and external stimuli and transmit them
through intracellular signal transduction that is mainly mediated by a cascade of
protein phosphorylation. Protein phosphorylation, a covalent modification with a
phosphate group at specific sites, can alter protein conformation, causing either an
increase or a decrease in protein activity. Activation of signal transduction results
in the phosphorylation of downstream effector proteins, leading to alterations in
various cellular functions and physiology.
Figure 1 shows a simplified model for stress responses. When cells encounter
stressed conditions, phosphorylation-mediated signal transduction is rapidly
induced, leading to attenuation of translation initiation and consequent suppression
Fig. 1 Conceptual model for

stress response. When the cell
encounters adverse
environment and stimuli, a
stress-sensing protein triggers
signal transduction that is
mediated by a cascade of
protein phosphorylation.
Stress-induced signal is
transduced to downstream
effector proteins and causes
attenuated translation
initiation and consequent
suppression of protein
synthesis. Stress-induced
signal transduction often leads
to alterations of gene
expression
of protein synthesis. Because protein synthesis consumes large amounts of energy and
materials, its suppression saves biological resources and thereby serves as a survival
mechanism. In addition to translational suppression, stress-induced signal transduction
frequently causes altered expression of a specific set of genes, which contributes to
adaptation of cells to stressed conditions. Although Fig. 1 shows a simplified concep-
tual model for stress responses, human and mammalian cells have multiple mecha-
nisms, in which many cellular proteins are involved as described below.
Integrated Stress Response
Outline of Integrated Stress Response

Cells have evolved elaborate mechanisms to sense changes in the extracellular
environment. When encountering stress, cells induce distinctive signal transduction
that attenuates an overall rate of protein synthesis by inhibiting the activity of the
eukaryotic initiation factor 2 (eIF2). The stress-induced signal transduction and
consequent translational suppression involving the eIF2 inhibition are collectively
called the integrated stress response, because cellular responses to diverse forms of
stress converge to these reactions (Fig. 2) (Leprivier et al. 2015). The integrated
stress response is evolutionarily conserved in a wide range of eukaryotes from yeasts
to humans, and translational suppression is a central event in this process. Under
normal physiological conditions, cells continuously undergo protein synthesis,
which requires substantial amounts of energy and materials. Translational
Fig. 2 Integrated stress response. Various forms of stress cause the phosphorylation of eIF2α and
consequent suppression of translation initiation, which are collectively referred to as the integrated
stress response. Human and mammalian cells have four stress-responsive protein kinases, namely,
PERK, GCN2, HRI, and PKR, each of which is activated by different external cues. The activated
stress-responsive kinases phosphorylate eIF2α, which serves as a convergence point for signal
transduction induced by various forms of stress. Phosphorylated eIF2α interferes with the assembly
of a functional translational machinery on the 50 cap structure of mRNA. Most mRNA species are
translated in a cap-dependent manner; therefore, eIF2α phosphorylation leads to the suppression of
general protein synthesis. A complex of GADD34 with PP1 dephosphorylates eIF2α and serves as a
negative feedback mechanism for the integrated stress response
suppression is considered to be beneficial for stressed cells, because it saves cellular

resources (Leprivier et al. 2015). Although the integrated stress response generally
serves as a prosurvival mechanism, its prolonged activation exerts facilitative effects
on cell death induction (Woehlbier and Hetz 2011; Koromilas 2015). Thus, the
attenuation of the stress response in a timely manner is also critical for cell survival.
Translational Suppression Mediated by eIF2α Phosphorylation

The process of protein synthesis consists of multiple steps from initiation to termina-
tion of translation, and it is primarily controlled at the step of translation initiation.
Translation initiation of most messenger RNA (mRNA) species relies on the cap
structure located at the 50 end of the mRNA. In the cap-dependent translation initiation,
initiator methionyl-tRNA, eIF2, and the 40S ribosomal subunit associate to form the
43S preinitiation complex that is recruited to the cap structure bound to the eukaryotic
initiation factor 4 (eIF4) and scans for the initiation codon in the mRNA (Baird and
Wek 2012). Phosphorylation of the α subunit of eIF2 (eIF2α) inhibits the 43S
preinitiation complex formation and consequently suppresses translation initiation
(Baird and Wek 2012; Koromilas 2015). In human cells, serine 51 of eIF2α is rapidly
phosphorylated under stress conditions (Fig. 2) (Koromilas 2015).
The phosphorylation of eIF2α is conducted by stress-responsive protein kinases.
Human and mammalian cells have four stress-responsive kinases for eIF2α phos-
phorylation, each of which is differentially activated by distinct forms of stress (Fig. 2)
(Koromilas 2015). Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is
activated by endoplasmic reticulum (ER) stress, which is caused by unfolded proteins
in the ER. General control nonderepressible 2 (GCN2) is mainly activated by amino
acid deprivation. PKR (double stranded RNA-dependent protein kinase) plays a
critical role in antiviral responses, and it is activated by double-stranded RNA,
which emerges during viral infection. Heme-regulated inhibitor (HRI) has physio-
logical roles particularly in erythroid tissues and is activated by heme deprivation.
The phosphorylation of eIF2α at serine 51 is a common consequence of the activation
of these stress-responsive kinases and is thus considered a hallmark of the induction
of the integrated stress response (Baird and Wek 2012; Koromilas 2015).
Recovery from Translational Suppression Caused by eIF2α Phosphorylation

When the stress conditions pass over, cells need to restore protein synthesis through
dephosphorylation of eIF2α, and growth arrest and DNA damage-inducible protein 34
(GADD34) is involved in this process. GADD34 associates with protein phosphatase 1
(PP1) to form a catalytically active phosphatase that specifically dephosphorylates
eIF2α and relieves the translational suppression (Novoa et al. 2001). The activity of
GADD34 in the relief of the integrated stress response is regulated at both transcrip-
tional and translational levels. GADD34 gene is expressed at low levels under normal
physiological conditions and is transcriptionally activated by a wide variety of stress.
Most mRNA species in a stressed cell are poorly translated owing to eIF2α phosphor-
ylation, but GADD34 mRNA can circumvent the eIF2α-mediated translational sup-
pression. Upstream open reading frames (uORFs) in the 50 untranslated region of
GADD34 mRNA are critical elements for translation initiation under stress conditions,
although its precise mechanism is still elusive (Lee et al. 2009). In addition to GADD34
mRNA, approximately 2.5% of the total mRNA was estimated to be preferentially
translated under stress conditions through uORF-mediated and other related mecha-
nisms (Dang Do et al. 2009; Baird and Wek 2012), which allow the synthesis of a subset
of proteins that play critical roles in the control of the integrated stress response. Based
on the transcriptional and translational regulation, GADD34 constitutes a negative
feedback mechanism for the eIF2α phosphorylation-mediated translational suppression.
Alternative Mechanism for Stress-Induced Translational Suppression
Human and mammalian cells have an additional mechanism for stress-induced trans-
lational suppression (Fig. 3), which is independent of eIF2α phosphorylation. As
described above, the cap structure is important for translation initiation for most
mRNA species. eIF4E recognizes the cap structure and recruits eIF4G and
other translation initiation factors to form an active translation initiation complex.
The inhibitory 4E-binding protein 1 (4E-BP1) competes with eIF4G for binding to
Fig. 3 mTORC1-mediated translational regulation. mTORC1 is involved in stress-induced trans-

lational regulation that is independent of the integrated stress response. On the 50 cap structure of
mRNA, eIF4E and other proteins form a complex for translation initiation. 4E-BP1 has the ability to
interfere with this protein assembly by binding to eIF4E. (a) Under normal physiological condi-
tions, hyperphosphorylation by mTORC1 sequesters 4E-BP1 from eIF4E, which ensures the
cap-dependent translation initiation. (b) Stress conditions cause a reduced mTORC1 activity that
in turn results in reduced 4E-BP1 phosphorylation, leading to the suppression of the cap-dependent
translation initiation by binding of 4E-BP1 to eIF4E
eIF4E and thereby inhibits translation initiation (Ma and Blenis 2009). In normal
physiological state, mammalian target of rapamycin complex 1 (mTORC1), a member
of the phosphatidylinositol 3-kinase-related kinase family, phosphorylates 4E-BP1 at
multiple sites to sequester 4E-BP1 from eIF4E (Fig. 3). Energy deprivation and other
stress conditions cause a marked reduction in the catalytic activity of mTORC1,
resulting in the decreased phosphorylation and increased eIF4E-binding of 4E-BP1
(Fig. 3) (Ma and Blenis 2009). The catalytic activity of mTORC1 is controlled by
several regulatory proteins, one of which is AMP-activated protein kinase (AMPK).
AMPK can sense cellular energy status and negatively regulates the mTORC1 activity
(Hardie 2011). Because protein synthesis is one of the major energy-consuming
processes in the cell, its energy-dependent control involving mTORC1, AMPK, and
4E-BP1 serves as an alternative mechanism for adaptation to the stress conditions.
As described above, many proteins function in the integrated stress response
and the alternative mechanism for stress-induced translational suppression. Table 1
provides a list of proteins involved in these cellular reactions.
Stress Responses Induced by Nanosecond Pulsed Electric Fields
Nanosecond Pulsed Electric Fields
PEFs on the order of ms to μs are commonly used for electrotransfer of macromol-

ecules into living cells, because these PEFs efficiently increase membrane perme-
ability, which can be detected by incorporation of membrane-impermeant
Table 1 List of proteins involved in stress responses

Name Function Involvement in stress responses
eIF2α Translation initiation factor Translational suppression by its phosphorylation
eIF4E Translation initiation factor Binding to 50 cap of mRNA for translation initiation
eIF4G Translation initiation factor Assembly of translation initiation complex
4E-BP1 Translation regulator Inhibition of eIF4E binding to 50 cap of mRNA
PERK Kinase Activated by ER stress and PEFs
GCN2 Kinase Activated by UV, nutrition deprivation, and PEFs
PKR Kinase Activated by virus infection
HRI Kinase Activated by heme deprivation
AMPK Kinase Activated by energy deprivation and nsPEFs
mTORC1 Kinase Suppressed by energy deprivation
GADD34 Cofactor of phosphatase Dephosphorylate eIF2α
PP1 Phosphatase Dephosphorylate eIF2α
IRE1 Kinase, nuclease Activated by ER stress
ATF6 Transcription factor Activated by ER stress
CHOP Transcription factor Downstream factor of ER stress pathway
XBP1 Transcription factor Downstream factor of ER stress pathway
GADD45 Cofactor of kinases Downstream factor of UV-induced stress pathway
fluorescent dyes, such as propidium iodide, into living cells. Compared to these
PEFs, nsPEFs have less incidence of membrane permeabilization, as judged by the
fluorescent dye incorporation. Instead, nsPEFs elicit calcium influx and cell bleb-
bing, presumably caused by ion imbalance, indicating the generation of small
membrane pores that allow permeation of ions and water (White et al. 2004; Vernier
et al. 2006; Pakhomov et al. 2009). Recent studies have documented that nsPEFs
induce various intracellular reactions that include stress responses (Morotomi-Yano
et al. 2012b).
Activation of eIF2a Phosphorylation by nsPEFs
eIF2α plays a critical role in cap-dependent translation initiation, and its activity is
negatively controlled by phosphorylation under various stress conditions (Koromilas
2015). The occurrence of eIF2α phosphorylation is regarded as a hallmark of the
integrated stress response and can be examined by Western blotting using an
antibody specific to phosphorylated eIF2α (Fig. 4). When nsPEFs are applied to
cultured cells, eIF2α phosphorylation is easily detected by Western blotting, indi-
cating the induction of the integrated stress response by nsPEFs (Morotomi-Yano
et al. 2012b). Phosphorylation of eIF2α can be detected in various cell lines,
including HeLa S3, HCT116, Jurkat, and mouse embryonic fibroblasts (MEFs). In
the case of HeLa S3, eIF2α phosphorylation occurs within 1 min after nsPEF
exposure, persists at high levels for 30 min, and gradually decreases thereafter.
Downregulation of eIF2α phosphorylation is known to involve the activation of
Fig. 4 Examples of Western blot analysis of protein phosphorylation. Phosphorylation status of a

protein of interest can be examined by Western blotting. When available, a specific antibody against
a phosphorylated protein is used for the analysis of protein phosphorylation. If a protein of interest
is heavily phosphorylated at multiple sites, such hyperphosphorylation causes a slow migration of
the protein in gel electrophoresis and yields shift band(s) in Western blotting. In this case, hyperpho-
sphorylated and unphosphorylated species are distinguishable in a single Western blot. (a) Example
of use of a phospho-specific antibody. Western blot analysis of eIF2α phosphorylation was
performed using a specific antibody against phosphorylated eIF2α (upper panel) and an antibody
reacting with eIF2α irrespective of its phosphorylation status (lower panel). eIF2α exists in both
nsPEF-exposed (shown by P) and untreated (shown by -) cells (lower panel), but phosphorylated
eIF2α is detected only in the nsPEF-exposed cells (upper panel). (b) Example of a bandshift due to
hyperphosphorylation. Western blot analysis of PERK was performed using an anti-PERK anti-
body. PERK protein is detected as a single band in untreated cells (-). nsPEFs induce hyperpho-
sphorylation of PERK that causes slow electrophoretic migration of PERK protein and yields
shifted bands in the nsPEF-exposed cells (P)
GADD34 that constitutes an active eIF2α phosphatase with the catalytic subunit of
PP1. Quantitative analysis of reverse transcription-polymerase chain reactions
(RT-PCR) demonstrated that GADD34 gene expression is markedly increased at
1 h after nsPEF exposure (Morotomi-Yano et al. 2012b), supporting the idea that
GADD34 is involved in the recovery of nsPEF-induced eIF2α phosphorylation
(Fig. 5).
Recent studies have demonstrated that nsPEFs induce different intracellular
reactions in a manner dependent on nsPEF intensity (Fig. 6). Relatively weak
nsPEFs, which do not cause obvious growth retardation or cell death, trigger the
activation of several signal transduction pathways, including mitogen-activated
protein kinase pathways (Morotomi-Yano et al. 2011b; Morotomi-Yano
et al. 2011a). Western blot analysis of eIF2α phosphorylation has shown that such
weak nsPEFs are insufficient for the induction of the integrated stress response. To
achieve eIF2α phosphorylation, modest intensities of nsPEFs are required, which
cause reduced proliferation but not gross cell death (Morotomi-Yano et al. 2012b).
Intense nsPEFs induce not only eIF2α phosphorylation but also cause cell death
(▶ Chap. 63, “Mathematical Models Describing Cell Death Due to Electroporation,”
and ▶ 30, “Regulated and Apoptotic Cell Death After Nanosecond Electroporation”)
(Morotomi-Yano et al. 2013, 2014). However, it remains unclear whether the nsPEF-
induced stress responses serve only as a prosurvival mechanism or have facilitative
effects on cell death induction, when overactivated. Further investigation is needed
to clarify this point.
Fig. 5 nsPEF-induced stress responses. Previous research has demonstrated the effects of nsPEFs
on multiple stress-responsive proteins that constitute two independent pathways leading to transla-
tional suppression. nsPEFs activate PERK (a) and GCN2 (b) that phosphorylate eIF2α. PERK and
GCN2 appear to act on the nsPEF-induced eIF2α phosphorylation in a mutually compensatory
manner. (c) nsPEFs cause reduced 4E-BP1 phosphorylation, suggesting an inhibitory effect of
nsPEFs on mTORC1. It remains unclear whether nsPEFs affects mTORC1 directly or indirectly.
(d) nsPEFs cause reduction in intracellular energy levels and AMPK activation, both of which are
known to contribute to negative regulation of the mTORC1 activity and thus may account for the
reduced 4E-BP1 phosphorylation. Increased eIF2α phosphorylation and decreased 4E-BP1 phos-
phorylation serve as two independent mechanisms for translational suppression
Fig. 6 Intensity-dependency of cellular responses to nsPEFs. nsPEFs elicit distinct cellular

responses, depending on their intensities. Relatively weak nsPEFs exhibit little effects on cell
growth and cell death but can activate several signal pathways, including various MAPK pathways,
like JNK, ERK, and p38 pathways, and AMPK pathway. nsPEFs at modest intensities induce the
stress responses that include eIF2α phosphorylation, 4E-BP1 dephosphorylation, and translational
suppression. Strong nsPEFs cause cell death, and either apoptosis or necrosis is induced in a cell
type-dependent manner
Activation of Stress-Responsive Protein Kinases by nsPEFs
Human and mammalian cells have four stress-responsive protein kinases for eIF2α
phosphorylation, each of which is activated by a distinct form of stress (Koromilas
2015). Activation of these kinases is manifested by their autophosphorylation,
which can be detected by Western blotting (Fig. 4). Analysis of phosphorylation
status of the stress-responsive kinases by Western blotting demonstrated that PERK
and GCN2 are definitely activated by nsPEFs (Fig. 5) (Morotomi-Yano
et al. 2012b). Intriguingly, PERK and GCN2 act on the PEF-induced eIF2α
phosphorylation in a mutually compensatory manner. Mouse embryonic fibroblast
cells lacking either PERK or GCN2 gene do not exhibit any detectable reduction in
the nsPEF-induced eIF2α phosphorylation, suggesting the presence of a compen-
satory mechanism for the lack of either PERK or GCN2 (Morotomi-Yano
et al. 2012b). Double knockout cells for both PERK and GCN2 genes exhibit a
significant decrease in the nsPEF-induced eIF2α phosphorylation, supporting the
idea of the functional compensation between PERK and GCN2 (Morotomi-Yano
et al. 2012b), although the molecular details of this phenomenon need to be further
investigated.
PERK and GCN2 play central roles in the stress response pathways for ER
stress and UV irradiation, respectively, and the activation of these kinases by
nsPEFs raised a possibility that nsPEFs act in ways similar to ER stress or UV
irradiation. It is well established that stress responses caused by ER stress and UV
irradiation result in expression of distinct subsets of stress-inducible genes. For
example, ER stress activates three upstream signaling proteins, namely, PERK,
activating transcription factor 6 (ATF6), and inositol-requiring protein 1 (IRE1),
which in turn lead to the induction of CCAAT-enhancer-binding protein homolo-
gous protein (CHOP) gene transcription and X-box binding protein 1 (XBP1)
mRNA splicing (Szegezdi et al. 2006). UV irradiation elicits GCN2 activation
that causes altered expression of growth arrest and DNA damage-inducible 45
(GADD45) and other genes (Takekawa and Saito 1998). In the case of HeLa S3
cells, these alterations in gene expression are detectable within 1 h after treatment
with ER stress-inducing agents or UV irradiation. To clarify whether nsPEFs exert
their effects as ER stress or not, quantitative RT-PCR analysis of CHOP and XBP1
mRNAs was performed using HeLa S3 cells. However, there were no substantial
changes in these mRNAs in nsPEF-exposed cells, indicating that nsPEFs do not
act as ER stress despite the obvious PERK activation (Morotomi-Yano
et al. 2012b). In addition, RT-PCR analysis performed on the UV-inducible
genes indicated no significant changes in these genes in the nsPEF-exposed cells
(Morotomi-Yano et al. 2012b). Taken together, these observations suggest that
nsPEFs act as a new form of cellular stress distinct from ER stress and UV
irradiation. It is noteworthy that PERK and GCN2 are located most upstream in
their signaling pathways, suggesting direct effects of nsPEFs on these kinases,
although precise mechanisms for their activation by nsPEFs need further
investigation.
Effects of nsPEFs on 4E-BP1 and AMPK
Human cells have an additional mechanism for the stress-induced translational

suppression, and 4E-BP1 is involved in this process (Ma and Blenis 2009). Most
mRNA species are translated in a cap-dependent manner, and 4E-BP1 can interfere
with the assembly of translation factors on the cap structure (Fig. 3). In a normal
physiological state, 4E-BP1 is heavily phosphorylated by mTORC1, and its inhib-
itory activity on the cap-dependent translation initiation is suppressed by hyperpho-
sphorylation. Under stress conditions, particularly during energy deprivation, the
catalytic activity of mTORC1 is decreased, leading to decreased 4E-BP1 phosphor-
ylation and consequent translational suppression (Fig. 3) (Ma and Blenis 2009).
When nsPEFs are applied to cultured cells, such as HeLa S3 and MEFs, dephos-
phorylation of 4E-BP1 is induced within 5 min and persists for more than 1 h,
suggesting the occurrence of 4E-BP1-mediated translational suppression (Fig. 5)
(Morotomi-Yano et al. 2012b). Although the effects of nsPEFs on the catalytic
activity of mTORC1 have not been studied yet, the decreased 4E-BP1 phosphory-
lation appears to reflect the decreased catalytic activity of mTORC1. Recently,
electroporation using 100 μs PEFs has been reported to induce energy deprivation
in a calcium-dependent manner, which ultimately causes necrotic cell death
(Frandsen et al. 2012). Similarly, nsPEFs also induce calcium-dependent necrotic
cell death in several cell lines, including HeLa S3, K562, and HEK293 (Morotomi-
Yano et al. 2014). The calcium-dependent energy deprivation in PEF-exposed cells
appears to account for the decrease in 4E-BP1 phosphorylation. In addition, nsPEFs
have been reported to induce AMPK activation by protein phosphorylation
(Morotomi-Yano et al. 2012a). AMPK phosphorylation is rapidly induced within
1 min, persists for 15 min, and then decreases, when nsPEFs are applied to HeLa S3
or Jurkat cells. Contribution of activated AMPK to the negative regulation of
mTORC1 has been well documented (Ma and Blenis 2009). Collectively, these
observations imply the translational suppression mediated by decreased 4E-BP1
phosphorylation in nsPEF-exposed cells (Fig. 5), although the precise relationship
among 4E-BP1, mTORC1, and AMPK in nsPEF-exposed cells remains to be
confirmed at molecular levels.
Suppression of Protein Synthesis by nsPEFs
The phosphorylation-mediated control of eIF2α and 4E-BP1 activities strongly

suggests that nsPEFs activate two independent mechanisms for translational sup-
pression (Fig. 5), which prompted further analysis of protein synthesis rates in
nsPEF-exposed cells. Because living cells contain massive amounts of proteins,
discrimination of newly synthesized proteins from preexisting ones requires a
special experimental approach, namely, metabolic labeling with radioactive
amino acids (Fig. 7). In this method, cells are incubated in culture medium
containing 35S-labeled methionine and cysteine. During incubation, cells
Fig. 7 Measurement of protein synthesis rates by using radioactive amino acids. Radioactive
amino acids are used for metabolic labeling of newly synthesized proteins to measure overall
rates of protein synthesis. After nsPEF exposure, 35S-labeled amino acids, namely, methionine and
cysteine, are added to culture medium. Cells uptake these radioactive amino acids and use them for
protein synthesis, yielding radioactive proteins. Following cell extract preparation, proteins are
precipitated in the presence of trichloroacetic acid, and radioactivity in a protein fraction is
measured by liquid scintillation counting
incorporate the radioactive amino acids and use them for protein synthesis,
resulting in the production of radioactive proteins. Following cell extract prepara-
tion, a protein fraction is separated from that of free amino acids, and the radioac-
tivity in the protein fraction is quantified.
By applying this method, the suppression of general protein synthesis in nsPEF-
exposed cells was demonstrated using HeLa S3 cells as well as MEFs (Morotomi-
Yano et al. 2012b). After nsPEF exposure, protein synthesis was quickly attenuated,
and the maximum suppression was observed at 30 min. UV irradiation is commonly
used as a positive control for stress response induction and elicits persistent trans-
lational suppression for several hours. Acute suppression and rapid recovery of
protein synthesis are characteristics of the nsPEF-induced stress response, when
compared to UV irradiation. Collectively, nsPEFs induce acute suppression of
general protein synthesis that is presumably mediated by the phosphorylation of
eIF2α and 4E-BP1.
Nucleofection-Induced Stress Responses
Nucleofection is a sort of electroporation that is conducted using a specialized

device, in which undisclosed electric parameters optimized for individual cell
types are preset (Gresch et al. 2004). Nucleofection can achieve efficient transfection
and is particularly useful for primary and nondividing cells. In nucleofection-treated
cells, phosphorylation status of the signaling molecules in the integrated stress
response was analyzed by Western blotting (Anderson et al. 2013). First, eIF2α
phosphorylation was detected in MEFs as well as primary human dendritic cells after
nucleofection. Increased eIF2α phosphorylation was rapidly induced and detectable
up to 2 h. The presence of exogenous DNA was not required for nucleofection-
induced eIF2α phosphorylation, suggesting the causal effects of electric pulses in
nucleofection. Similar to nsPEFs, PERK and GCN2 were activated by nucleofection,
and GCN2 appears to play a major role in the nucleofection-induced eIF2α
phosphorylation. Luciferase activities derived from transfected expression plasmids

were decreased after nucleofection, indicative of translational suppression. Collec-
tively, these observations suggest the occurrence of the integrated stress responses by
nucleofection and provide insightful information about cellular effects of PEFs used
for electroporation (Anderson et al. 2013).
Possible Effects of Electroporation on Cell Physiology
In contrast to particular emphasis given to nsPEFs and nucleofection in the above

sections, the last part of this chapter is dedicated to PEFs used for electroporation.
Because it has become evident that both nsPEF exposure and nucleofection elicit
stress responses, it seems reasonable to assume that regular electroporation also
induces stress responses. One of the most common applications of electroporation is
transfection of exogenous DNA, and PEFs used for DNA transfection may exert
their negative effects on transgene expression by stress-induced translational sup-
pression. Accordingly, nucleofection has been shown to cause a reduction in lucif-
erase activity expressed from transfected plasmid DNA (Anderson et al. 2013). In
case of nsPEFs, translational suppression is relatively acute and recovers within a
few hours (Morotomi-Yano et al. 2012b). These observations suggest the importance
of PEF conditions in transfection, and careful optimization of PEF conditions is
required for the maximum expression of transgene.
Another possible adverse effect of electroporation is the occurrence of cell death,
and in fact, intense PEFs can induce massive cell death caused by irreparable
damages, which is called irreversible electroporation and is used for cancer treat-
ment. Currently, it remains unclear whether the PEF-induced stress responses are
beneficial for cell survival or have a facilitatory influence on cell death. When
properly controlled, stress responses collectively serve as a potent mechanism for
cell survival, whereas persistent activation of stress responses often promotes cell
death, presumably because elimination of overstressed cells is beneficial for the body
(Woehlbier and Hetz 2011; Koromilas 2015). Thus, the PEF-induced stress
responses may have dual effects depending on the extent and duration of their
activation, and future research is required to evaluate the contribution of the stress
responses to survival and death in electroporation.
In addition to DNA transfection, electroporation is utilized for the introduction of
antitumor drugs for cancer chemotherapy, in which the PEF-induced stress responses
could have therapeutic advantages. First, tumor cells have mechanisms to counteract
the antitumor drugs by degrading or excluding them, and translational suppression
could attenuate these cellular activities and augment therapeutic efficacy of the
introduced drugs. Second, stress-induced signaling generally leads to the activation
of downstream factors, many of which negatively regulate cell growth (Hamanaka
et al. 2005; Koromilas 2015). Thus, PEFs used for electroporation may induce
stress-induced antiproliferative intracellular reactions. Finally, translational
suppression itself is reasonably expected to have inhibitory effects, because cell

growth requires synthesis of various proteins. In summary, PEFs used in cancer
chemotherapy should have several antiproliferative effects that can act in synergy
with antitumor drugs.
Conclusions
Despite their usefulness in various life science fields, PEF are essentially detrimental
for cellular homeostasis because of their ability to permeabilize the cell membrane.
Therefore, PEFs can be regarded as a sort of stress for a living cell, and, indeed, it has
become increasingly evident that PEFs elicit stress responses. Exposure of human
and mammalian cells to nsPEFs induces two distinct stress response pathways, both
of which alter phosphorylation status of translation initiation factors, leading to the
suppression of protein synthesis. In addition to nsPEFs, stress responses are also
activated by nucleofection, which is a sort of electroporation conducted by a
specialized device with pre-optimized electric parameters. Collectively, these obser-
vations clearly indicate that PEFs act as stress to induce cascades of intracellular
reactions leading to the suppression of protein synthesis.
Identification of key intracellular events of PEF-induced stress responses raises
next questions to be elucidated. To date, common cell lines, such as HeLa S3 and
MEFs, under standardized culture conditions have been utilized as an experimental
model for investigating the molecular details of PEF-induced stress responses. Such
experimental setups are useful for analysis of intracellular mechanisms but insuffi-
cient to assess biological significance of PEF-induced stress responses in the in vivo
situations. Further studies employing physiologically relevant conditions are
required to advance our understanding of possible contributing factors to
PEF-induced stress in vivo, such as an influence of microenvironment and depen-
dency of tissue and cellular contexts. Furthermore, limited information is currently
available on how and to what extent the PEF-induced stress responses affect the
outcomes of electroporation-based applications. Although PEF-induced stress
responses can be speculated to facilitate cancer chemotherapy and to exert negative
effects on transgene expression in DNA transfection, experimental validation is
required. These future efforts will provide a broader and more integrative view of
PEF-induced stress responses, which pave the way for more effective applications of
PEFs in the life sciences.
Acknowledgment This work was supported by JSPS KAKENHI Grant Number 26350540.
Cross-References

▶ Regulated and Apoptotic Cell Death After Nanosecond Electroporation
▶ Responses of Plant Cells and Tissues to Pulsed Electric Field Treatments

▶ Stress Response of Plants, Metabolite Production due to Pulsed Electric Fields
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Effects of Electroporation of Mammalian
Cells on Cytoskeleton and Intercellular 18
Connections
Maja Čemažar
Abstract
Electroporation of mammalian cells affects several cellular components, includ-
ing cytoskeleton and intercellular connections. This chapter describes the effects
of electroporation alone or in combination with cytotoxic drugs or plasmid DNA
on the components of cytoskeleton. Responses of cells to electroporation depend
on the type of cells. In Chinese hamster ovary cells, mainly microtubule filaments
were depolymerized following electroporation with microsecond pulses, with a
recovery during 60 min, while recently the effect on the actin filaments in the cell
cortex was also demonstrated. Electroporation of endothelial cells caused, besides
depolymerization of microtubules, also depolymerization of actin filaments,
while vimentin intermediate filaments were not affected. In addition, the loss of
contractility and vascular endothelial cadherin (VE-cadherin) from the adherens
cell junction was also observed. All these changes lead to increased permeability
of endothelial cell monolayer. In tumor cells, electroporation does not cause
depolymerization of actin filaments, but their rearrangement into stress fibers,
lamellipodia, and filopodia. Electroporation with nanosecond pulses also affects
actin filaments. Their depolymerization and disassembly of gap junctions were
observed following exposure of cells to nanosecond pulses, while tight junctions
were not significantly affected. The role of cytoskeletal filaments was also studied
following electrochemotherapy and plasmid DNA electrotransfer into the cells
demonstrating that cytoskeletal proteins are quicker and more severely affected
following electrochemotherapy compared to electroporation alone and that the
actin filaments play an active role in gene electrotransfer.
Collectively, electroporation greatly affects cytoskeleton and intercellular
connections in different types of cells and plays an important role in the transport
M. Čemažar (*)
Department of Experimental Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia
e-mail: mcemazar@onko-i.si

DOI 10.1007/978-3-319-32886-7_18
308 M. Čemažar
of plasmid DNA into the cells and in the vascular changes observed after
treatment with electrochemotherapy. However, studies are needed to fully eluci-
date the involvement of cytoskeleton and intercellular connection in different
types of electroporation application, which will possibly lead to improved or new
treatment options based on electroporation technology.
Keywords
Cytoskeleton • Intercellular connections • Electroporation • Nanosecond pulsed
electric fields
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
Cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Intercellular Connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
The Role of Cytoskeleton in Cell Electrofusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
The Effects of Electroporation on Cytoskeleton and Intercellular Connections . . . . . . . . . . . . . . . 312
The Effects of Electrochemotherapy and Gene Electrotransfer on Cytoskeleton
and Intercellular Connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
Introduction
Electroporation of different types of cells in vitro is a powerful tool used in many

biological applications for introduction of various molecules into the cells or into the
membranes of cells, fusion of cells, and extraction of molecules from cells. In
electroporation protocols for mammalian cells, most commonly used electric pulses
are square wave-shaped electric pulses. The duration of pulses ranges from nano-
seconds to milliseconds with electric field densities up to several kV/cm for nano-
second pulses to several tens to hundreds V/cm for micro- and millisecond pulses,
depending on the desired biological application. Besides the influence of electrical
parameters on the desired outcome of the application, a significant role plays also
other physicochemical properties, such as the composition of the electroporation
medium (buffer) and its temperature, as well as biological properties of the cells.
Among the biological properties of the cells, the size and shape of the cells, whether
they are in suspension or adherent, the density of cell suspension, and the type of the
cells (fibroblasts, erythrocytes, endothelial cells, different types of tumor cells) are
the most studied properties in the electroporation studies. Since early studies on cell
electrofusion and cell electropermeabilization for introduction of small molecules
into the cells, it is known that mammalian cells behave differently compared to lipid
vesicles. Therefore, already in these early studies, the involvement of cytoskeleton in
the permeabilization process was suggested (Blangero et al. 1989). Later on influ-
ence of cell-to-cell junctions on the behavior of cells exposed to electric pulses was
also studied (Kanthou et al. 2006). Basic experimental mechanistic studies together
18 Effects of Electroporation of Mammalian Cells on Cytoskeleton and. . . 309
with realistic finite element numerical modeling provided further insights into the
process of electroporation in cells and tissues and are important for biomedical
progress of electroporation into clinics, where it is already used in electroche-
motherapy (introduction of cytotoxic drugs into the cells of tumors for tumor
destruction), plasmid DNA vaccination protocols (introduction of plasmid DNA
encoding antigen either in the skin or muscle for development of immunity), and
gene therapy protocols (introduction of plasmid DNA encoding therapeutic protein
in different types of tissue (skin, muscle, tumor) depending on the target disease). In
this chapter, firstly the basic facts about cytoskeleton and intercellular connections in
mammalian cells are described, and the role of cytoskeleton and intercellular con-
nections in electroporation-based methods and treatments, such as cell fusion,
electrochemotherapy, and gene electrotransfer, is explained.
Cytoskeleton
Cytoskeleton is present in all types of cells: archaea, bacteria, and eukaryotes.

Initially, cytoskeleton was thought to be unique to eukaryotic cells; however, in
the past 20 years, the presence of cytoskeletal proteins with mainly cytomotive
function was also demonstrated in archaeal and bacterial cells (Wickstead and Gull
2011; Backert et al. 2013).
In eukaryotes the cytoskeleton contains three major types of protein filaments:
microtubules, intermediate filaments, and actin filaments, also called microfilaments.
The overall function of cytoskeleton is to support and maintain the shape of the cell,
by giving it the mechanical resistance to deformation. In tissues, cytoskeletal pro-
teins are associated with extracellular matrix and also with neighboring cells, with
the help of accessory proteins, thus supporting the entire tissue or organ. The
cytoskeletal proteins are also involved in many other functions of cells, such as
migration, intracellular transport, signaling, endocytosis, segregation of chromo-
somes during nuclear division, and cytokinesis, the final division of the cell’s
cytoplasm. The cytoskeletal proteins are thus very dynamic structures that are
capable of rapid growth and disassembly, depending on the type of the cell and the
requirements of the cell. In addition, not all types of the cytoskeletal protein are
present in all cell types, for example, the certain types of intermediate filaments
(Fig. 1) (Wickstead and Gull 2011; Backert et al. 2013).
The microtubules are hollow cylinders of outer diameter of 23 nm that are a
polymer composed of a dimer of α- and β-tubulin. They originate from the nuclear
organization center called centrosome that is located near the cell nucleus and is
composed of γ-tubulin. From this centrosome, the microtubules grow outward, and
the growing end of the microtubules is the plus end and is oriented toward the site of
polarity, for example, to the leading edge of migratory endothelial cell. The energy
for the growth of microtubules comes for guanosine triphosphate (GTP), and due to
the quick hydrolization of GTP, when bound to β-tubulin, the microtubules can
rapidly switch between growing and shrinking, the process known as dynamic
instability, which is important for the migration of cells.
310 M. Čemažar
Fig. 1 Cytoskeleton and intercellular connections in cells. Blue – microtubules extending from the
centrosome toward the cell membrane. Green – intermediate filaments are involved in the desmo-
somes (green) connecting two neighboring cells and hemidesmosomes (light blue) that are
connecting cells to the extracellular matrix. Red – actin filaments are a component of adherens
junction and also form a cell cortex, a filamentous network under the cell membrane. Yellow – gap
junctions enable communication between cells. Black dots in the apical site of the cells – tight
junctions, which are preventing the movement of the proteins embedded into the cell membrane
from apical to basal site of the cells. The schematic representation was prepared by Dr. Urska
Kamensek
Another constituent of cytoskeletal proteins are intermediate filaments, which are

a class of different types of proteins with average diameter of 10 nm. Intermediate
filaments are composed of different types of proteins depending on the type of the
cell. Lamins are the only intermediate filaments that are located in the nucleus of all
nucleated cells and form the nuclear lamina, which supports the nuclear envelope
and helps in disassembly and assembly of the nuclear envelope during the mitosis. In
epithelial cells, the intermediate filaments, keratins or cytokeratins, are present and
are extended from the nuclear envelope toward the plasma membrane, where they
are part of desmosomes and hemidesmosomes. Vimentin is an intermediate filament
present in cells of mesenchymal origin, such as fibroblasts, endothelial cells, and
leukocytes. Its main role is to maintain cell integrity. Other types of intermediate
filaments are neurofilaments, found abundantly in axons, and desmins that are
present in sarcomeres of the muscle cells.
Actin filaments or microfilaments are the smallest among cytoskeletal proteins,
with the diameter of 7 nm. They are composed of G-actin, a globular actin, which is a
monomer that forms F-actin, a filamentous actin. Actin filaments are very flexible,
and their role in the cell is dependent on the accessory proteins that are bound to actin
filaments. They are involved in muscle contraction, together with myosin in muscle
cells. In non-muscle cells, they are the main constituents of cell cortex, they are
involved in signal transduction, they form stress fibers, and they are part of adherent
cells in cell-to-cell junction and focal adhesion in cell-to-extracellular matrix adhe-
sion (Fig. 1) (Wickstead and Gull 2011; Backert et al. 2013).
Intercellular Connections
Cell-to-cell junctions, also called intercellular bridge, are protein structures that exist
between the cells in all multicellular organisms and provide contact between the
cells. They are especially abundant in epithelial tissues, where they provide epithe-
lial cell polarization and control paracellular transport. Cell-to-cell junction provides
anchoring, communication, and also barrier between the neighboring cells. Besides
cell-to-cell junctions, in multicellular organism, also cell-to-extracellular matrix
adhesions and non-junctional adhesion mechanisms exist (Fig. 1).
Anchoring cell-to-cell junctions are desmosomes and adherens junctions, which
are the protein structures between the cells, and hemidesmosomes and focal adhe-
sions that connect cells to extracellular matrix (i.e., basal lamina in the epithelium).
The intermediate cytoskeletal proteins, cytokeratin or desmin, are part of desmo-
somes, where cadherins are the proteins that form the anchor. In hemidesmosomes
that connect polarized epithelial cells to extracellular matrix, the cytokeratins are
anchored to the extracellular matrix by the involvement of transmembrane integrins.
Adherens junctions are composed of bundles of actin filaments. They can appear as
bands encircling the epithelial and endothelial cell. Actin bundles are connected
across the plasma membrane with the cadherins, like in desmosomes. In focal
adhesions, which are adhesion junctions of cell to extracellular matrix in polarized
and non-polarized cells, the actin filaments are anchored to the substrate by integrins,
like in hemidesmosomes.
Cell-to-cell communication is enabled by gap or communication junctions, which
enables cell in the epithelium to be in direct contact, where the diffusion of
molecules can occur directly, without passage through extracellular matrix. The
cytoskeletal proteins are not involved in the formation of the junction. The main
proteins that form the gap junction are connexins, which form a pore in the cell
membrane called connexon. These junctions are especially important in the heart
muscle cell, where they enable heart beating through action potential propagation.
Tight junctions are present only in epithelial cells. Their role is dual; they prevent
the apical membrane proteins from moving to basolateral surface, thus enabling
polarization of the epithelium; and they seal the spaces between epithelial cells to
prevent the movement of molecules between the cells. Actin filaments are involved
in the formation of the tight junction, together with transmembrane proteins
claudins, occludins, and junction adhesion molecules (JAM) (Wickstead and Gull
2011; Backert et al. 2013).
312 M. Čemažar
The Role of Cytoskeleton in Cell Electrofusion
Cell electrofusion was a topic on numerous studies, due to the important application
of cell fusion in biotechnology and medicine, mainly for production of hybridomas
and preparation of cell vaccines. Most of the studies were focusing on the evaluation
of different electrical parameters, as well as on other parameters that could influence
the cell fusion, such as the composition of the fusion medium, osmolarity of the
fusion medium, temperature, and post-pulse incubation of cells. Namely, in the cell
electrofusion experiments, the biggest obstacle is the difference in size of the cells to
be fused. In fact, in hybridoma technology, small lymphocytes need to be fused with
large myeloma cells in order to get the production of antibodies (Jaroszeski et al.
1994; Pucihar et al. 2011; Rems et al. 2013). The study that addressed the cytoskel-
eton changes after electrofusion was performed by Blangero et al. (Blangero et al.
1989). Chinese hamster ovary (CHO) cells, a clone WTT, have been selected for the
experiments. This clone is less anchorage dependent than the parental cell line and
can grow also in suspension. However, the plated cells were used for electrofusion
experiments, and the cells were immunohistochemically stained for tubulin and actin
filaments at different times posttreatment. The protocol for electric pulses used in the
study for electrofusion was as follows: five square wave electric pulses of 1200 V/cm
and 100 μs duration were applied to the attached cells via two parallel stainless steel
electrodes with 5 or 18 mm distance in between. No particular change in the
organization of actin filaments was observed during the observation time of
60 min at 37 C. On the other hand, the organization of microtubules was signifi-
cantly altered already 10 min after the treatment. The fluorescently labeled micro-
tubules were present only around the nucleus, which were clustered in the middle of
the fused cell. The observed effect was reversible, and after 60 min the microtubule
network was morphologically similar to the network in control untreated cells.
In conclusion, the experiments on the involvement of cytoskeleton in cell
electrofusion showed that microtubules were transiently depolymerized during cell
fusion, while actin filaments were not affected.
The Effects of Electroporation on Cytoskeleton and Intercellular

Connections
Due to the important role of electroporation in the biomedical research and espe-
cially in the medical treatments (electrochemotherapy, gene therapy, DNA vaccina-
tion), numerous of preclinical studies are devoted to the elucidation of the molecular
changes that electroporation alone or in combination with cytotoxic drugs and
nucleic acids (DNA, RNA) is triggering in the cells. These preclinical studies are
needed for better planning of electroporation-based treatments. Almost 25 years ago,
the study by Rols and Teissie provided experimental evidence that predominantly
microtubules are altered by electroporation and that they play a role in the stabili-
zation process of permeabilized cell membranes, presumably due to their intercon-
nections with plasma membrane. These experiments were done on attached CHO
WTT cells and on suspension of erythrocytes, using ten square wave electric pulses
of 100 μs duration and different electric field intensities ranging from 500 to
2400 V/cm (Rols and Teissie 1992; Teissie et al. 1994). Shortly after that, the
phenomenon of microtubule depolymerization was demonstrated in chick embryo
corneal fibroblast following one exponentially decaying electric pulse of varying
voltage-to-distance ratio 500–1000 V/cm and capacitance 125–960 μF. Besides
depolymerization of microtubules, perinuclear collapse of intermediate filament
vimentin was observed, and, similarly to CHO WTT cells, actin filaments were not
affected (Harkin and Hay 1996).
The first study on endothelial cell was performed by Kanthou et al. (Kanthou
2006) evaluating the role of cytoskeleton of endothelial cells in the observed
vascular effects of electroporation in vivo. In vivo vascular effects of electroporation
and electrochemotherapy were demonstrated in tumors and also in muscles after
electroporation alone. In vivo studies showed that application of electric pulses
causes profound reduction in blood flow, which was reversible. The return of
blood flow to pretreatment values was quicker for the normal tissue (muscle) than
for tumors (Jarm et al. 2010). Primary human umbilical vein endothelial cells were
used in the study. Besides cytoskeletal proteins, microtubules, actin filaments, and
vimentin, endothelial monolayer permeability was determined and, also for the first
time, the intercellular connections adherens junctions, which constitute a major type
of endothelial junction responsible for the control of vascular permeability. Endo-
thelial cells were grown in Transwell inserts, with a pore size of 0.4 μm till they
reached confluence, when the experiments were performed. In situ electroporation
was performed by a cuvette-type electrode that was designed to encompass the cells
growing in the inserts. Due to this setup, a lower electric field strength was needed to
effectively permeabilize cells; three square wave electric pulses of 10–80 V with a
duration of 100 μm were applied to the cells via the electrodes with 4 mm distance.
Immunofluorescence microscopy was used to determine the morphological changes
of cytoskeletal filaments and Western blot analysis to determine the phosphorylation
status of myosin light chain indicating on the contractility of endothelial cells and
passage of fluorescently labeled dextran through the endothelial monolayer to
measure the permeability of endothelium. The results of the study showed that
electroporation of adherent endothelial cells causes immediate but transient disrup-
tion of microtubules and actin filaments. In addition, the loss of contractility and
vascular endothelial cadherin (VE-cadherin) from the adherens cell junction was
also observed. All these changes lead to the increase in endothelial monolayer
permeability.
In contrast to previous studies on CHO WTT cells and corneal fibroblast, in the
study on endothelial cell, actin filaments were altered. This can be explained by the
use of different pulse conditions in the study on fibroblasts and lack of anchorage
dependence in CHO WTT cells, while in endothelial cells, actin filaments play a
crucial role in maintenance and control of vascular permeability. This is done
through the cell contractility that is regulated by the actin-myosin interaction requir-
ing actin polymerization and phosphorylation of myosin light chain and through the
integrity of cell-to-cell adherens junction (Kanthou et al. 2006).
314 M. Čemažar
The impact of experimental setup, i.e., adherent, clustered, or cells in suspension

and density of the cells and their shape, on the effectiveness of permeabilization and
intercellular connections was a topic of several theoretical and experimental studies
(Pucihar et al. 2006, 2007a, b, 2008a, b). CHO cells, either parental strain or WTT
clone, were used in these studies. A numerical computation of induced transmem-
brane voltage needed to successful permeabilization of the irregularly shaped cells
was performed and the results verified experimentally on CHO cells exposed to five
electric pulses of 150 ms duration at 100 V/cm. The finite element numerical model
proved to be a valuable tool, especially for computing the induced transmembrane
voltage needed for successful permeabilization of irregularly shaped adherent cells
or cells in tissue (Pucihar et al. 2006). The importance of cell density on the
effectiveness of permeabilization was demonstrated in a study by Pucihar et al.
(Pucihar et al. 2007a). CHO WTT cells in suspension were used in these experi-
ments, the density of the cells varied from 1 106 to 400 106 cells/ml, and the
authors used different parameters of electric pulses; they applied eight square wave
electric pulses of 100 μs duration with electric field strength of 400 or 700 V/cm.
Flow cytometric measurement of number of propidium iodide-positive cells, as well
as mean fluorescence intensity of these cells, was used to measure the fraction of
permeabilized cells and amount of loaded propidium iodide (electroloading). The
increased cell density resulted in ~50% lower fraction of permeabilized cells.
Furthermore, the decrease of electroloading was even larger with increased cell
density than expected from the difference in the permeabilized cell fraction. This
was attributed to the cell swelling that occurs after permeabilization, also due to the
rearrangement of cytoskeletal filaments, and reduced availability of propidium
iodide to the permeabilized membrane. The same phenomenon affected the resealing
of the membrane of cells that was slower in dense cell suspension compared to dilute
one (Pucihar et al. 2007a).
Furthermore, transport of propidium iodide was studied in adherent cells com-
pared to cells in suspension. Again, CHO WTT cells were used in experiments. The
experiments were performed at the same cell densities in both cases (1 105 cells/
ml). Cells were either grown in suspension and just before the experiments spinned
down and plated in a chamber slide or they were plated in the chamber slide one day
before the experiment, so that they formed attached confluent monolayer. Fluores-
cence of propidium iodide was used to measure the kinetics of transport into the cells
and fluorescence microscope equipped with sensitive photomultiplier (PM) tube.
Fluorescence signal entering PM tube was then transformed to a voltage signal,
amplified and filtered with a custom-made differential amplifier, and stored in a
transient recorded, which was afterward digitalized and processed on a computer
using suitable software program. The cells in chamber slides were exposed to single
square wave pulse of various amplitudes (350, 500, 650, 800 V) and/or durations
(0.1, 0.5, 1.0, 3.0 ms) that were applied through two parallel wire electrodes
(diameter 0.5 mm, length 10 mm, interelectrode distance 5 mm). The results of
experimental part of the study demonstrated that at the same pulse parameters,
transport into the cells growing as confluent monolayer was considerably lower
than transport into the cells in suspension (Pucihar et al. 2008a). This result was in
agreement with previously published numerical and experimental results on dense

cell suspension, further supporting the important role of the induced transmembrane
voltage in different types of cell culture conditions, where cells have different
arrangements of cytoskeleton filaments as well as intercellular connections.
The first study that addressed the role of intercellular connections with regard to
the induced transmembrane voltage and consequently the effectiveness of electro-
poration was theoretical and experimental study on adherent CHO cells grown in
clusters (Pucihar et al. 2007b, 2008b). Two types of modeling were performed. In the
first one, the cells in the cluster were modeled as electrically connected, so the
electric current between the cytoplasm of the cells in the contact was not impeded by
the plasma membranes separating them. In the second model, the cells in the cluster
were considered as electrically insulated, so that the cells would behave as individual
cells. Calculated regions of increased membrane conductivity corresponded to the
experimentally observed electroporated regions but only if the cells were modeled as
electrically insulated. The cells were exposed to one pulse of 400 V and 200 μs
duration. On the contrary, when the cells were exposed to low, nonporating electric
field, they behaved as electrically connected, implicating on the role of communi-
cation channels between the cells, i.e., gap junctions, which were demonstrated that
exist in CHO cells (El-Fouly et al. 1987). Further studies are needed to undoubtedly
demonstrate the role of gap junctions in the electroporation process.
Recently, a destabilization of plasma membranes was analyzed by atomic force
microscopy, which enables a direct observation of plasma membranes of fixed and
live cells. CHO cells were grown on coverslip for fixed cell experiments and in Petri
dishes for living cells observation. Cells were exposed to eight square wave electric
pulses of 5 ms duration at 400 V/cm at 1 Hz repetition frequency through stainless
steel electrodes that were applied directly onto the glass coverslip or plastic Petri
dish. The results of this study for the first time demonstrated that atomic force
microscopy can be used for electroporation process investigations. Transient rip-
pling of the membrane surface was visualized, and a decrease in membrane elasticity
by 40% was measured (Chopinet et al. 2013). Further atomic force microscopy
studies on live CHO cells growing in Petri dish, exposed to the same electric pulses
condition as in previous study, demonstrated that electroporation affects actin
filaments also in CHO cells, which was in contrast to previously published results
in the same cell line (Chopinet et al. 2014; Rols and Teissie 1992). The results of the
study showed that only one form of actin filament is affected, namely, the actin
filament that forms the cell cortex, while other cytoplasmatic actin filaments are not
destabilized by electroporation. Other types of cytoskeletal filaments were not
investigated in this study (Chopinet et al. 2014).
As already mentioned, different cell types, as well as different growing condi-
tions, can greatly influence the electroporation-induced response of cells with respect
to the involvement of cytoskeletal filament and intercellular connections. Further
insights on the specific response of different cell types provided two studies that
were performed on different tumor cells and fibroblasts (Xiao et al. 2011;
Pehlivanova et al. 2012). In the study of Xiao et al, hepatocellular carcinoma cells
HepG2 were selected. This cell line is commonly used in genotoxicological studies
316 M. Čemažar
as it is one of few hepatic cell lines that have retained the activity of metabolic
enzymes. The attached cells grown in a glass microscopic chamber were exposed to
30 electric pulses of 450 ns duration, frequency 1 Hz at the field intensity of 8000 V/
cm. It should be noted that in this study, the pulsing condition was different,
compared to the studies evaluating effects of electroporation mentioned above.
Nevertheless, in this first study on tumor cells evaluating the role of cytoskeleton,
only actin cytoskeletal filaments were studied, in a way that they were disrupted
before electroporation by treatment of cells with cytochalasin B, which causes actin
filaments depolymerization. The actin filaments were not directly observed, but
rather apoptosis, necrosis, propidium iodide uptake, and mitochondrial transmem-
brane potential were measured after exposure of cells to electric pulses. The results
of this study demonstrated that depolymerization of actin filaments protects the cells
from electroporation-induced apoptosis and necrosis, presumably due to the lack of
signal transduction into the cell to mitochondria. On the other hand, depolymeriza-
tion of actin filaments had no effect on the electroloading of propidium iodide, i.e.,
permeabilization of the membrane (Xiao et al. 2011).
Further support for different responses of different types of cells to electropora-
tion provided a study by Pechlivanova et al., who used in the experiments two
different types of human breast cancer cells, MDA-MB-231, which has loosely
adhesive properties, and MCF-7, as well as 3T3 fibroblast cell line. Cells were
grown either on glass coverslips or directly on the bottom of the 24-well plates.
Cells were exposed to eight biphasic electric pulses of 50 μs duration, with 20 μs
interval between both phases and 880 μs pause between the pulses. The amplitudes
of applied electric pulses were between 200 and 1000 V, and the electric pulses
were delivered through two parallel stainless steel electrodes with a 10 mm
distance between them. Cell adhesion and survival of cells after electroporation
were measured by crystal violet and 3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, while
cytoskeletal rearrangements and cellular interconnections were visualized by immu-
nofluorescence. The main conclusion of this study, based on the results obtained, is
that cell adhesion and the type of adhesions (cell to cell or cell to matrix) is cell-type
specific, namely, cell adhesion and survival of 3T3 fibroblast and MCF-7 breast
carcinoma cells, were not affected significantly by the applied electroporation. On
the other hand, the electroporation of invasive MDA-MB-23 cells results in
increased adhesion at lower electroporation voltages and decreased cell adhesion
at 1000 V/cm. Actin filaments were transiently depolymerized in fibroblasts, while
in both types of cancer cells, actin filaments were not depolymerized, but were
rearranged in stress fibers, lamellipodia, and filopodia. Furthermore, based on the
electroporation-induced adhesion changes, in MDA-MB-231 cells, cell-to-matrix
adhesion dominates, while in MCF-7 cells, cell-to-cell adhesion. However, the types
of intercellular connections were not studied (Pehlivanova et al. 2012).
Only in a few studies, investigators have exposed cells to nanosecond electric
pulses electroporation (nanoporation, nanosecond pulsed electric fields) and studied
their influence on cytoskeleton, intercellular gap and tight junctions, nuclear mem-
brane, and telomeres (Stacey et al. 2011; Pakhomov et al. 2014; Steuer et al. 2016).
Three different types of cells were used in the study by Stacey et al., Jurkat leukemia
cells, which grow as suspension, and two adherent cell lines, HeLa human epithe-
lioid cervical carcinoma and SV40 fibroblasts. The cells were exposed to one electric
pulse of 60 ns duration at the field intensity of 60,000 V/cm. Jurkat cells were more
severely damaged by application of electric pulses, demonstrated by induction of
breakdown of actin filaments, localization of telomeres out of the nucleus, and
shredded nuclear membrane, compared to adherent cells. The authors suggested
that the minimal protection of the cytoskeleton offered to cell in suspension allows
the effects of the nanosecond pulsed electric fields to be more readily felt at the
nucleus, whereas in adherent cell, much of the energy may be absorbed by the
extensive cytoskeleton, with no apparent effect on the nucleus (Stacey et al. 2011). In
a second study, Pakhomov et al. showed that actin filaments are disrupted by
exposure of CHO cells to four 600 ns long pulses of 19,200 V/cm (Pakhomov
et al. 2014). In a third study by Steuer et al., rat WB-F344 liver epithelial cells were
used (Steuer et al. 2016). Cells grown in monolayers were exposed to 20 nanosecond
pulses of 100 ns duration with varying amplitudes between 5,000 and 35,000 V/cm,
which are sublethal conditions for nanosecond pulses. Cell viability, actin filament
morphology, and gap and tight junctions were studied. The results of the study
showed that exposure to nanosecond pulsed electric fields temporarily changes the
ability of cells to communicate through gap junctions in a time and field strength-
dependent manner. The recovery was observed within 24 h. The immunohistochem-
ical staining of Cx43 protein, a component of gap junctions, demonstrated disas-
sembly of these junctions, while staining of ZO-1 protein in tight junctions
demonstrated that tight junctions are not significantly affected. Actin cytoskeleton
was reorganized and not depolymerized as demonstrated in a study by Stacey et al.
(Steuer et al. 2016; Stacey et al. 2011)
The Effects of Electrochemotherapy and Gene Electrotransfer

on Cytoskeleton and Intercellular Connections
Electrochemotherapy is a cancer therapy, where electroporation is used to enhance

the delivery of cytostatic drugs into the cells. It is already widely used in European
cancer centers for treatment of mainly cutaneous cancers. Several mechanisms are
involved in the antitumor effectiveness of electrochemotherapy, among others, also
vascular disrupting action. To further elucidate the observed vascular effects in vivo
on preclinical tumor models and in treatment of patients with electrochemotherapy, a
study was performed comparing the effect of electroporation and electroche-
motherapy with bleomycin in human endothelial cells. In that study, human micro-
vascular endothelial cells HMEC-1 were selected, which are a good model for
human tumor endothelium. The cells were grown in eight chamber slides for
determination of morphological changes and in Transwell inserts with pore size of
3 μm for determination of endothelial monolayer permeability. The electrodes used
in this study had a gap of 7.3 mm to fit into the chamber of the slide, and the
following parameters of electric pulses were used: 50–500 V/7.3 mm and eight
318 M. Čemažar
Fig. 2 The effect of electrochemotherapy with 300 nM bleomycin at different times posttreatment
on actin cytoskeleton of attached HMEC-1 cells exposed to eight 100 μs long pulses at various
amplitudes. The electric pulses were applied via two stainless steel electrodes 7.3 mm apart. Note
that the actin filaments are not affected at 100 V; some cell can recover after electrochemotherapy
applied at 300 V, while at 500 V all the cells die within 24 h. The experiment and acquisition of the
images were done by Dr. Cecil Meulenberg
pulses of 100 μs duration with repetition frequency of 1 Hz. In the electroche-

motherapy experiments, 3–1000 nM bleomycin was used. The results of this study
confirmed the results on electroporation-induced changes in cytoskeleton of previ-
ous study performed on human umbilical vein endothelial cells. In addition, for the
first time, electrochemotherapy-induced changes in cytoskeleton of endothelial cells
and permeability of endothelial monolayer were demonstrated. Electroche-
motherapy induced quicker onset and more pronounced increase in endothelium
monolayer permeability compared to electroporation alone. In addition, also the
changes in cytoskeletal filament morphology had quicker onset and were
unrecoverable (Fig. 2), while when using electroporation alone, the changes of
cytoskeletal filaments were reversible. This study provided further insight into the
cellular changes of the vascular disrupting action of electrochemotherapy
(Meulenberg et al. 2012).
Gene electrotransfer is a method, where electroporation is used to enable the entry
of nucleic acids, mainly plasmid DNA, to enter the cells in vitro and also in vivo.
This method is used for gene therapy and DNA vaccination. The therapies are not in
routine clinical practice as electrochemotherapy, but the progress in this field is very
quick. The role of actin filaments during gene electrotransfer was studied in CHO
cells growing either in suspension or attached. The cells in suspension were exposed
to ten electric pulses of 5 ms duration and field strength of 700 V/cm, while for the
attached cells, lower field strength was used, 400 V/cm. The results of the study
demonstrated that actin patches colocalized with the DNA at the plasma membrane
and thus provide, for the first time, evidence that actin filaments are involved in DNA
electrotransfer through the plasma membrane (Rosazza et al. 2011). Further studies
from the same group, using the same cell line CHO and the same electrical
parameters, demonstrated that for the intracellular trafficking of DNA, microtubule
filaments with motor proteins kinesins and dyneins are responsible for fast
(200–1600 nm/s) and long-range transport of DNA toward the nucleus. Actin
filaments together with the myosin might be responsible to transfer the DNA, either
in endocytotic vesicle or free plasmid DNA toward the microtubule network
(Rosazza et al. 2013).
Conclusions
In conclusion, electroporation alone or in combination with cytostatic drug or

plasmid DNA affects the cytoskeleton and intercellular connections. These effects
differ dependent on the cell type. Using reversible electroporation, with electric
pulses of up to 100 μs duration with voltages up to 2000 V/cm, the effects are
reversible and, for example, in endothelial cells, cause increase of endothelial
monolayer permeability which contributes to the vascular effect of electroporation
in vivo. In combination with cytostatic drug bleomycin, these effects are irreversible
and resulted in vascular disrupting action of electrochemotherapy. Nanosecond
pulsed electric fields have more pronounced effects on cytoskeleton leading to cell
death. Sublethal nanosecond pulsed electric fields cause disassembly of gap junction
intercellular communication. The same junctions are involved in the observed
behavior of clustered cells, where they behave as one giant cell, if exposed to
nonporating electric pulses. Furthermore, cytoskeletal filament microtubules and
actin filament are also involved in the gene electrotransfer. Altogether, studies on
molecular mechanisms that are involved in the process of electroporation are very
important; thus, further studies are needed to fully elucidate the involvement of
cytoskeleton and intercellular connection in different types of electroporation appli-
cation, which will possibly lead to further development and improvement of treat-
ment options based on electroporation.
Acknowledgements This work was conducted within the scope of LEA EBAM (French-
Slovenian European Associated Laboratory: Pulsed Electric Fields Applications in Biology and
Medicine) and COST Action.
Cross-References

320 M. Čemažar
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Nucleic Acid Electrotransfer in Mammalian
Cells: Mechanistic Description 19
Muriel Golzio and Marie-Pierre Rols
Abstract
The development of new drugs depends greatly on their successful, efficient, low
cost, and safe delivery into target cells or tissues. In the case of highly charged
macromolecules such as nucleic acids, the therapeutic effectiveness is mainly
limited by their bio-distribution within the tissue and the poor permeability of the
plasma membrane of cells. For this purpose, electroporation appears as a prom-
ising method for nucleic acid delivery. Electroporation is a physical method of
vectorization that consists of application of electric pulses on cells or tissues.
Optimization of the pulses’ parameters leads to the transient permeabilization of
the plasma membrane for molecules which otherwise cannot enter the cell.
Therefore, the understanding of different principles of drug and gene delivery is
necessary and needs to be taken into account according to the specificity of their
delivery to tumors and/or normal tissues. This approach has been routinely used
in cell biology for more than 30 years for cell transfection and in medicine in a
number of clinics and hospitals through Europe to treat cutaneous cancers by
increasing the toxicity of anticancer drugs (electrochemotherapy); it is also now
under clinical trials for nucleic acid delivery (electrogenotherapy, electro-
vaccination). The present chapter focuses on electrotransfer of nucleic acids,
the nature of nucleic acids (plasmid DNA, mRNA, siRNA, LNA, etc.) which
can be electrotransferred, and the mechanism of their electrotransfer.
M. Golzio (*)
Toulouse, France
e-mail: muriel.golzio@ipbs.fr
M.-P. Rols (*)
Institut de Pharmacologie et de Biologie Structurale, IPBS/CNRS UMR 5089 and University of
Toulouse, Toulouse, France

DOI 10.1007/978-3-319-32886-7_21
324 M. Golzio and M.-P. Rols
Keywords
Delivery • Electroporation • Nucleic acid • Plasmid DNA • siRNA
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
Why Choosing Electroporation for Nucleic Acid Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
Electropermeabilization of the Cell Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
Plasmid DNA Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
Electric Field Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328
DNA/Membrane Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
Internalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
Intracellular Trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
RNAi-Based Small Oligonucleotide Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
siRNA Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332
Modified Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332
Toward In Vivo Delivery of Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
Introduction
The development of new drugs depends greatly on their successful, efficient, and safe
delivery into target cells or tissues. Physiological barriers and toxicity of carrier systems
create multiple challenges to bring molecules to the clinic. An ideal method efficiently
delivers therapeutic molecules while being biocompatible, safe, and targeted.
In this context, electroporation (EP) is a promising non-viral biophysical method for
in vitro and in vivo delivery of various molecules such as drugs (Mir et al. 1998) and
nucleic acids (Rols 2010). EP was introduced in 1970s and consists of the direct
application of external electric field pulses on target cells or tissues that transiently
destabilize the plasma membrane and cause its permeabilization (Neumann et al. 1982).
The efficiency and convenience (i.e., easiness, low cost, and speed) of this technique
lead to an increasing number of in vivo applications on a large number of both internal
and surface organs and tissues (Golzio et al. 2010). Moreover, very few side effects have
been reported (mostly superficial burn) emphasizing the innocuousness of this method
for clinical use. In addition, no change in the expression profile of major tumor
suppressor genes or oncogenes, of genes involved in the stability of DNA, and no
promotion of tumor genesis were detected. The expression of metastasis-promoting
genes was not increased after electrochemotherapy. To date, several preclinical and
clinical studies using EP for cancer treatment have demonstrated encouraging results
showing antitumor effectiveness (Sersa et al. 2008; Bodles-Brakhop et al. 2009).
In this chapter, the mechanism of nucleic acid delivery is described according to
the nature of nucleic acids which can be electrotransferred and the associated clinical
applications.
19 Nucleic Acid Electrotransfer in Mammalian Cells: Mechanistic Description 325
Why Choosing Electroporation for Nucleic Acid Delivery
Several options are available for nucleic acid delivery. Broadly, these can be
categorized into local or systemic deliveries. Systemic delivery should be avoided
as it is associated with the following problems: intravascular degradation (serum
endonuclease cleavage, phagocytosis by scavenger macrophages), activation of
complement, nonspecific targeting (distribution takes place to all tissues, except
for the brain), fast elimination through renal filtration, and potential toxicity
(Aigner 2007). In addition, cancer tissue is difficult to reach intravenously as,
compared to normal capillaries, tumor blood vessels are immature and display
functional defects that lead to restricted intra-tumor blood flow (Jain 2005). Finally,
intravenously injected nucleic acids must extravasate and move up to the tissue core.
In contrast, using EP method, nucleic acids can be delivered locally and can thus
bypass all the mentioned limitations. Moreover, it has been shown that the applica-
tion of electric pulses on the skin in vivo resulted in a rapid increase in vascular
permeability that gradually recovered to basal levels at different times posttreatment,
depending on dextran size (Bellard et al. 2012). Simultaneously, the immediate
constriction of the blood vessels occurred that was more pronounced for arterioles
compared to venules. This vasoconstriction of arterioles results in a transient “vas-
cular lock” (Jarm et al. 2010). The increased permeability of small vessel walls
whatever the dextran size associated with delayed perfusion explains the improved
delivery of the intravenous injected molecules (i.e., drugs, gene delivery) into the
tissues induced by electropermeabilization in vivo (Fig. 1).
Systemically injected nucleic acids must avoid intravascular degradation (I), renal
clearance (II), and nonspecific targeting (III); access the accessible tissues (IV);
extravagate (V); diffuse through the complex extracellular matrix (VI); be up
Fig. 1 EP overcomes physiological barriers to delivery (Adapted from Chabot et al. (2015))
taken by the cell (VII); and escape the endosome (VIII). On the contrary,
electrotransferred nucleic acids are injected locally (1), migrate rapidly through the
extracellular matrix (2) thanks to the electric field (E) and can also exit from the
blood vessel (20 ), enter the cell through the permeabilized membrane (3), and have
access to the cytoplasm (4).
One other EP advantage is that the delay between the nucleic acid injection and
the targeted delivery is very short (Rols 2010). In fact, cells are embedded into a
more or less complex extracellular matrix that can prevent nucleic acid diffusion.
Therefore, they cannot reach and enter cells easily as they are retained in the stroma
where they are more exposed to degradation. By dragging nucleic acids through the
extracellular matrix, EP first increases access to target cells and then protects nucleic
acids from degradation by the nucleases present, namely, in tumor fluids (Golzio
et al. 2010).
Nucleic acids must enter into the cells, but the hydrophobic cell membrane
creates a barrier for intracellular delivery of highly negatively charged molecules
such as nucleic acids. To overcome these delivery hurdles, non-viral and viral
strategies have been developed (Zuhorn et al. 2007; Aagaard and Rossi 2007). EP
utilizes naked material, which makes it more cost-effective and improves its safety
profile compared to carrier systems and virus production. Moreover, the size of
the nucleic acid is less limited using EP method than in the case of the virus
approach.
Although many nucleic acid carriers have been successfully used for in vitro
applications, these delivery systems are usually inappropriate for in vivo use due to
their high intra- and extracellular toxicity or unsatisfactory tissue delivery. Most of
the carrier systems enter cells through endocytosis (Bumcrot et al. 2006). Thus,
molecules must undergo endosomal escape to reach its target before acidification in
lysosomes. Additionally, retention in endosomes must be avoided as nucleic acids
could activate immune response through Toll-like receptors (TLRs) which are
present in these sites. Electric pulse application results in the formation of transient
permeable structures (Rols and Teissie 1990) also described as pores at the cell
plasma membrane level causing transient membrane permeabilization which may
avoid the endocytotic pathway at least for RNAi-based small oligonucleotide deliv-
ery (as shown in Fig. 1). For larger nucleic acids such as plasmid DNA, a more
complex multistep mechanism is involved in the electrotransfer process, as described
in detail below.
Electropermeabilization of the Cell Membrane
The exposure of living cells to short and intense electric pulses induces position-
dependent changes of the transmembrane potential difference (Neumann et al. 1982;
Hibino et al. 1991). Cells initially have a resting transmembrane potential difference
which is uniform all along their plasma membrane. The application of an electric
field superimposes an electro-induced transmembrane potential, Δψi, to the resting

transmembrane potential. The value of Δψi is given by the equation of Schwan
considering the cell membrane as a thin spherical dielectric shell:
Δψi ¼ f gðλÞ r E cos ðθÞ;
where f is a factor depending on the cell shape; g(λ) a parameter depending on the
conductivities λ of the membrane, of the extracellular medium, and of the cytoplasm;
r the radius of the cell; E the electric field strength; and θ the angle between the
electric field direction and the normal to the membrane at the considered point of the
cell surface. Being dependent on the angle θ, the electric field effect is not uniform
along the cell membrane, and its maximum effects are present at the poles of the cells
facing the electrodes (Rols and Teissie 1990).
When the resulting transmembrane potential reaches a threshold value (close to
200–400 mV), transient permeant structures (TPS) are generated at the cell mem-
brane level. The lifetime of these permeant structures also called “electropores” is in
the order of minutes (Rols and Teissie 1990).
The surface area of the cell membrane and the density of these TPS are controlled
by the electric field parameters (Rols and Teissie 1990). The molecular structure of
TPS in terms of lipids and/or proteins is not yet elucidated. The basic model suggests
the formation of pores which would give the external molecules a direct access to the
cytoplasm (Neumann et al. 1982). However, if this model is in agreement with the
observed post-pulse-free diffusion of molecules smaller than 4 kDa into the cyto-
plasm, the delivery of larger molecules such as plasmid DNA cannot be simply
described by “electropores” (Rols 2010).
Plasmid DNA Delivery
Plasmid DNA electrotransfer was first published in the early 1980s (Neumann
et al. 1982) and is, up to now, the most studied and used nucleic acid in electropo-
ration application. It basically consists in mixing naked DNA in an appropriate
buffer together with cells and exposing them to a series of well-controlled electric
pulses. Plasmid DNA-encoded protein is expressed within a few hours after the
electroporation. Whereas initial procedures could cause considerable cell damage,
technical developments have led to improvements of the equipment (pulse genera-
tors, electrodes) and optimization of protocols. The use of square wave pulse
generators allows for the control of the electrical parameters (intensity, duration,
frequency, number) independently. The user can therefore adapt them according to
the molecule, the cell, and the tissue to load. The possibility to optimize the
parameters of the pulses makes the approach very versatile. The number of publi-
cations showing the efficiency of the technique has grown exponentially (Rols
2010). Despite the fact that the gene expression level does not reach the one of the
viral methods, EP is one of the most efficient non-viral approaches.
Fig. 2 Schematic
presentation for the
electrotransfer of small
molecules. Schematic cartoon
showing the processes
occurring during application
of electric pulses for the
delivery of molecules of small
molecules (<4 kDa)
However, the gene expression efficiency for different tissues can vary dramati-
cally. Tumors are very difficult to transfect compared to muscle fibers. There is no
clear explanation for such dramatic differences since all the cells submitted to
electric pulses are efficiently permeabilized. Although the exact mechanism
governing DNA electrotransfer is still not well understood, it has been described
as a complex multistep process (Fig. 2).
Electric Field Effects
DNA electrotransfer involves a step of DNA/membrane interaction which implies

cell membrane permeabilization (Fig. 3). The interaction of the DNA with the
permeabilized membrane, and thus also the level of the associated protein expres-
sion, is directly proportional to the membrane area which is permeabilized (Escoffre
et al. 2011). The electric field strength controls the area of the membrane which is
permeabilized. It has to be high enough to permeabilize as much cell surface as
possible, but it has to be as low as possible to preserve the cell viability (Golzio
et al. 2002). For successful electrotransfer, the DNA must be present before the
application of the electric pulses. If the cell membrane stays permeabilized for
several minutes, the DNA/membrane interaction cannot occur when DNA is added
even immediately after pulse application. Moreover, pulses have to be sufficiently
long to yield DNA electrotransfer (milliseconds range) (Golzio et al. 2002) even if
100 μs pulses have proven to efficiently transfect cells in vitro and in vivo (Daud
et al. 2008). In fact, the higher the pulse duration is, the denser the TPS are on the
membrane allowing more DNA molecules to interact. In addition, DNA has to be
electrophoretically pushed to the membrane to be transferred. The negatively
charged plasmid DNA migrates toward the positive electrode until it encounters
the cell membrane. The pulse duration controls the electrophoresis of the DNA
(Neumann et al. 1982; Golzio et al. 2002; Escoffre et al. 2011).
Fig. 3 Schematic presentation of DNA electrotransfer in cells. During the application of the
electric field, the plasma membrane is permeabilized, and the DNA (in yellow) is electrophoretically
pushed on the cell membrane and inserted as clusters. After the electric field application, the DNA
aggregates are internalized and migrate through the cytoplasm to reach the nucleus. The protein is
afterward expressed in the cytoplasm
DNA/Membrane Complexes
DNA distribution on the electropermeabilized membrane is not homogeneous

(Fig. 3). It is located in yet undefined “competent sites” where it forms aggregates
of various sizes (from 0.1 to 0.5 μm). These aggregates are formed only during the
electric field application, but they stayed at the membrane for several minutes
(Escoffre et al. 2011; Golzio et al. 2002). DNA interacting with the membrane is
not simply accumulated as adsorbed aggregates but instead is inserted in it. The
application of electric fields with opposite direction (inverted polarity) at high
frequency (higher than 10 Hz) can indeed remove DNA from the membrane
(Escoffre et al. 2011). The DNA/membrane complexes need about 1 s to become
stable enough to resist to an inverted polarity electric field. In conclusion,
DNA/membrane complexes can be divided in two classes: (i) metastable complexes
from which DNA can leave and return to the external medium and (ii) stable
complexes from which DNA cannot be removed. Only complexes of the second
class remain present after 1 s and lead to effective DNA expression.
Internalization
After DNA/membrane interaction, a slow transmembrane traffic occurs as the DNA

is detected in the cytoplasm 30 min after electric pulse application. The way the
DNA crosses the membrane remains unclear. However, two main models are
proposed: (i) DNA aggregates move toward electropores and arrive naked in the
cytoplasm, or (ii) DNA is internalized by an endocytosis-like process and is enclosed
in a vesicle when in the cytoplasm. Experiments performed on giant unilamellar

vesicles (GUVs) show that giant electropores or flaps (several μm large) may be
created and support the direct entrance of plasmid DNA into the lipid vesicles or
lipid bilayers (Portet et al. 2011). However, to date such models have not been
supported by experimental data performed on cells. Indeed, it would mean that DNA
would cross the membrane in a relatively fast and free process. Instead, DNA shows
no free diffusion, stays immobile at the membrane for minutes, and is somehow
isolated from the external medium (Golzio et al. 2002). The model of electroen-
docytosis has been suggested by several groups (Rosazza et al. 2011). The stimula-
tion of fluid-phase endocytosis has been reported by most of them. An increase of
fluid-phase marker uptake such as lucifer yellow or dextran is shown to occur after
the application of an electric field. Moreover, the internalization of membrane
phospholipids is increased, and the fluid-phase marker uptake shows temperature
dependence (Antov et al. 2005). In addition, the cytoskeleton is also involved in the
macromolecule uptake. A recent study shows formation of actin patches that
colocalize with the DNA aggregates at the membrane (Escoffre et al. 2011). More-
over, an alteration of the actin network shows a decrease in both the amount of DNA
interacting with the cell membrane and the gene expression level. Up to date, the
DNA translocation mechanism is not yet fully characterized; it is possible that both
suggested models occur at the same time as DNA has been detected in both
aggregated and free forms in the cytoplasm (Escoffre et al. 2011).
Intracellular Trafficking
The DNA trafficking through the cytoplasm is not yet described, and very few
investigations have been performed so far. Nevertheless, some works show a
possible involvement of the cytoskeleton in the process (Rosazza et al. 2011). A
pre-pulse stabilization of the microtubule network, using the taxol drug, enhances
plasmid DNA expression. In addition, plasmid DNA seems to interact with the
dynein motor proteins (Vaughan et al. 2006). In the latter work, the plasmid DNA
carries nuclear localization sequences (NLS) on specific sequences which could
have a role in the intracellular trafficking. Older works show an enhancement of
gene expression when all post-pulse steps are performed at 37 C and that ATP is
one of the key partners for efficient DNA electrotransfer (Rols 2010). The trans-
fection is therefore a cell-mediated process. Given that the free diffusion of a
plasmid DNA in the cytoplasm is highly improbable, active transport of the DNA
to the nucleus should take place. The DNA has been detected at the outer part of the
nucleus. It accumulates all around it mainly in an aggregated but also in a more
diffusive form (Fig. 3). The DNA remains in form of clusters while crossing the
cytoplasm. The free diffusive form observed can come from either the diffusion of
DNA already free while crossing the membrane or from DNA that escapes from the
cluster.
Expression
Gene expression can be detected a few hours following pulse application (Fig. 3).
The nuclear envelope represents a barrier that affects its success. Only a small
fraction of the electrotransferred DNA can indeed reach the nuclear compartment
as it is the case for all non-viral delivery methods. The way the DNA gets into the
nucleus is poorly understood. Cells undergoing mitosis show better gene expression,
as the loss of the nuclear envelope integrity during the cell division may help the
DNA to overcome this barrier. DNA transfer, especially in nondividing cells, is
supposed to occur through the nuclear pore complex (NPC) (Rols 2010; Escoffre
et al. 2011). Therefore, plasmid DNA carrying NLS sequences should have higher
affinity for the NPC and have a better chance to get into the nucleus. This could be a
strategy to increase the gene delivery efficiency. Another possibility would be to
permeabilize the nuclear envelope by the application of electric fields with
nanosecond-range durations. In fact, nanosecond-range pulses can theoretically
permeabilize intracellular organelles such as nucleus without affecting the plasma
membrane (Beebe et al. 2003).
As a summary, plasmid DNA delivery mediated by electric fields is a complex
multistep process:
(i) The plasma membrane is permeabilized by the electric field. The efficiency of
permeabilization depends on the electrical parameters (field strength, pulse
duration, and number).
(ii) DNA is electrophoretically pushed to the cell membrane. DNA is inserted in
the permeabilized area of the membrane and accumulates in metastable
complexes until the electric field is switched off.
(iii) DNA/membrane complexes are stable for minutes after pulse application.
(iv) DNA is translocated into the cytoplasm through electropores and/or by
endocytosis-like process.
(v) DNA migrates through the cytoplasm via a cell-mediated process that may
involve the cytoskeleton.
(vi) DNA accumulates at the nucleus envelope where it could be translocated
through the NPC and/or wait for the loss of nuclear envelope integrity
occurring during the cell division.
(vii) Gene expression.
RNAi-Based Small Oligonucleotide Delivery
Difficulties encountered with inhibiting expression of therapeutic targets using

conventional approaches have prompted many researchers to consider using RNA
interference (RNAi) as a new antitumor strategy (Bumcrot et al. 2006), because it
offers the possibility of targeting any pathological protein. The RNAi pathway is
mediated endogenously by micro(mi)RNAs and experimentally by small silencing

RNAs (siRNAs). Both are small (~22 nt) noncoding RNAs that, once loaded into the
RNA-induced silencing complex (RISC), bind to their target messenger RNA
(mRNA) impairing its translation which subsequently suppresses gene expression.
The scientific community believes that miRNAs are the next important class of
therapeutic molecules after siRNAs. Indeed, the role of miRNAs as key players in
human cancer was highlighted by the fact that the loss of miRNA regulation is a
crucial factor in the development and progression of a wide variety of tumors.
miRNAs act as either tumor suppressors which help in controlling cellular growth
or oncogenes (OncomiRs) which promote rapid cell growth. In many cases, tumor
suppressor miRNAs’ reactivation by synthetic miRNA mimics or OncomiR inhibi-
tion by antisense DNA (antagomirs) leads to a significant antitumor response. Thus,
emerging therapeutic applications involving RNAi-based oligonucleotides include
miRNA mimics, antagomirs, and siRNAs (Chabot et al. 2015).
siRNA Delivery
The electrotransfer of RNAi-based oligonucleotides using fluorescently labeled

siRNA showed that a direct transfer into the cell cytoplasm of the negatively charged
siRNA was present across the plasma membrane exclusively on the side facing the
cathode. When added after electroporation, the siRNA was inefficient for gene
silencing because it did not penetrate the cells (Paganin-Gioanni et al. 2011; Chabot
et al. 2015). 48 h after the electrotransfer, siRNA was found distributed homoge-
neously in the cytoplasm of cultured tumor cells (Paganin-Gioanni et al. 2011). No
electrotransferred siRNA was seen in the nucleus of viable cells. Similar localization
was observed in vivo after siRNA intra-tumoral injection followed by EP. Thus,
upon EP, siRNA had an immediate, free access to the cytoplasm allowing its direct
interaction with its mRNA target. The EP mechanism, by bypassing the endocytotic
pathway, presents an enormous advantage in comparison to the other non-viral
chemical delivery methods (Fig. 4).
Modified Oligonucleotides
Effective RNAi-based therapy also depends on oligonucleotide stability and speci-

ficity. To improve these factors, chemically modified oligonucleotides have been
developed (Corey 2007) including 20 -O-methyl, 20 -methoxyethyl, locked nucleic
acids (LNA), and phosphorothioate linkages. Functional benefits of these backbone
modifications are that they increase the thermodynamic properties of oligonucleo-
tides, their base-pairing specificity, and their resistance to nucleases and hence their
efficacy. Interestingly, it has been observed that LNA/DNA chimeras could be
efficiently electrotransferred in vitro and in vivo (Chabot et al. 2012). The number
or the position of LNA into the DNA sequence was not interfering with the
electrotransfer efficiency. The mechanism of LNA electrotransfer appears to be
Fig. 4 Schematic presentation of siRNA electrotransfer in cells. Upon EP, siRNA has imme-
diate, free access to the cytoplasm allowing its direct interaction with the enzymatic machinery
(RISC) and its mRNA target. Transmembrane translocation of small interfering RNA (siRNA)
occurs through the plasma membrane of cells during the electric pulse application (Golzio
et al. 2002), which implies that siRNA does not penetrate the cells by diffusion as just described
for small molecules. The amount of uploaded siRNA is under the control of the cumulative pulse
duration time and the electric field strength
Fig. 5 Schematic
presentation of LNA/DNA
electrotransfer in cells.
LNA/DNA oligomers enter
into the cell cytoplasm driven
by electrophoretic forces as
shown for siRNA but rapidly
reach the nucleus. Therefore,
LNA/DNA oligomers have a
free access to the cytoplasm
and the nucleus and can then
target the mature miRNA or
the pri-miRNA, respectively
very similar to the one of siRNA. Therefore, EP offers the same attractive advantage
for LNA/DNA chimera as for siRNA, in contrary to lipid-mediated transfection in
which LNA/DNA chimeras were shown to be localized in the nucleus and at its
periphery in a punctate way suggesting an endosomal distribution (Chabot
et al. 2012, 2015; Fig. 5).
Since the discovery of the RNAi pathway, there has been an explosion of interest
and knowledge in using this technology for clinical application. Although highly
attractive as a therapeutic approach, several hurdles must be overcome to successfully
introduce RNAi-based therapies into the clinic. Some of these include efficient, safe,
and localized delivery to diseased cells and tissues. In this context, EP is a promising
non-viral strategy to target RNAi-based oligonucleotides to the correct tissue, to
facilitate their cellular uptake, and to give direct access to their intracellular target.
Toward In Vivo Delivery of Nucleic Acids
The use of electroporation for introducing naked plasmid DNA in vivo is an

expanding field. The first studies reported encouraging results of electrically assisted
delivery of DNA into easy-to-access tissues such as the skin, liver, tumors, and
muscle in mice. Subsequently, electroporation using plasmid DNA was also obtained
in almost all tissues in rodents. The feasibility of electroporation as a nucleic acid
delivery system was demonstrated using different reporter genes as well as thera-
peutic genes. Various therapeutic genes were used in electroporation protocols.
Several different diseases were treated by electrogene therapy, including cancer;
muscle disorders, such as muscle dystrophy; blood disorders; and arthritis (Rols
2010). Skeletal muscles were used as a pool for the various molecules that were
expressed after electrotransfection and were then shed into the bloodstream and act
as therapeutic proteins. So far studies have mainly concerned diabetes, anemia
(erythropoietin), and DNA vaccines (Bodles-Brakhop et al. 2009).
RNAi-based oligonucleotide electroporation was first used in vivo in 2003 by the
delivery of synthetic siRNA in ovo into chick embryos. siRNA electrotransfer into a
mammalian organ was described, and numerous reports have been published dem-
onstrating in vivo RNAi-based oligonucleotide electrotransfer potency (Aigner
2007). In fact, siRNA electrotransfer has been validated in the rodent muscle, skin,
eyes, brain, joint tissue, kidney, and tumor (Chabot et al. 2015). For instance, using a
siRNA against the green fluorescent protein (GFP), it has been demonstrated in
mouse muscle that low amount of siRNA can be electrotransferred specifically and
led to the downregulation of the targeted protein expression without any tissue
damages, independently of the age of the animal (Chabot et al. 2015).
Conclusions
Clear differences of processes by which nucleic acid molecules of different sizes

translocate across the electropermeabilized membrane have been observed. While
small soluble molecules cross the permeabilized membrane rather freely for much
longer time than the duration of the electric pulse application, plasmid DNA transfer
involves complex steps including interaction with the membrane. While the effects
of the electric field parameters are about to be elucidated (pulse strength higher than
a threshold value, long pulse duration for efficient gene expression), the associated
destabilization of the membrane which is a stress for the cells and may affect the cell
viability still has to be clearly described. The transfer kinetics of siRNA differs from
other molecular electrotransfer, such as the electrotransfer of small molecules (e.g.,
propidium iodide) or plasmid DNA. The electrophoretic drag allows direct access of
siRNA into the cytoplasm where its enzymatic machinery and target are present. In
vitro, mechanisms involved are clearly specific for the physicochemical properties of
the electrotransferred molecules. Additional studies are also necessary in order to
understand the cascade of events triggered by cell exposure to electric pulses at the
cell and tissue levels where new constraints coming from tissue organization are
present, such as the inhomogeneity of the electric field strength and the intercellular
distribution of nucleic acids. Moreover, the knowledge of the effect of the cells
themselves and environment according to their origin (tumor, skin, muscle, etc.) and
characteristics (cell density, packing, membrane elasticity, etc.) on these processes
will help to find new strategies to abolish the existing bottlenecks of the method.
Clinical applications have been successfully developed for antitumoral drug deliv-
ery, and clinical trials for gene electrotransfer are currently underway. The true
potential of electrotransfer as a safe and efficient non-viral method will be known
with the publications of the results of these clinical trials.
Cross-References
▶ Biomedical Applications of Electroporation

▶ Electroporation Phenomena on Tissue Level and Systemic Effects
▶ Modeling of Electroporation and Related Phenomena
▶ Preclinical Development for Biomedical Application of Electroporation
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Biophys J 36:349–362
Different Cell Sensitivity to Pulsed Electric
Field 20
Olga Pakhomova, Elena C. Gianulis, and Andrei G. Pakhomov
Abstract
One of the most anticipated benefits of medical applications of electric pulses
(EP) in cancer therapy is their potential to selectively ablate tumors while sparing
healthy tissues. The reason for such selectivity is the differential sensitivity of
diverse cells and tissues to killing by EP. Early studies observed profound
differences in sensitivity of various tumor and normal cell lines when exposed
to either 100 or 10 μs pulses. The recent studies using short EP with pulse
durations ranging from 10 to 300 ns have also reported a differential sensitivity
of various cell lines. Although nanosecond EP applications require more energy
to kill the cells, they provide a more distinct separation in cell sensitivities than
conventional longer pulses thus demonstrating the better potential for selective
elimination of the cell lines and tissues.
Life or death decisions in response to EP exposure depend on many processes
determining an ability of the cell to reseal the plasma membrane instantly after EP
treatment and ability of the resealed cells to recover from EP insult. The mediators
regulating cell sensitivity to EP are under investigation, and the role of some of
them (lipid content, actin cortex) will be discussed in this chapter. While the data
indicate a potential of EP for selective elimination of certain types of cells, there
are no clear criteria for prediction of cell sensitivity to EP. Effect of electric pulses
in cells partially can be predicted on the bases of presently known parameters but
O. Pakhomova (*)
e-mail: opakhomo@odu.edu
E.C. Gianulis • A.G. Pakhomov
e-mail: egianulis@odu.edu; egianulis@gmail.com; apakhomo@odu.edu; 2andrei@pakhomov.net

DOI 10.1007/978-3-319-32886-7_22
338 O. Pakhomova et al.
requires experimental verification on specific sets of cell lines and EP parameters

(pulse duration, amplitude, frequency of their delivery).
Keywords
Electric pulses • Electroporation • Membrane permeabilization • Electro-
sensitivity • Cell survival
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
Methods for Cell Sensitivity Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
Clonogenic Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
Metabolic Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
The Assays Based on Disruption of the Plasma Membrane Integrity . . . . . . . . . . . . . . . . . . . . . . 340
Other Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
Methods for EP Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
Cells in Suspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
Adherent Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
Three-Dimensional (3D) Cell Culture Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
Diversity of Cell Sensitivity to Microsecond-Duration EP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Cell Sensitivity to Nanosecond Electric Pulses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
The Selectivity of the Cell Killing by EP Is Higher for Shorter Pulses . . . . . . . . . . . . . . . . . . . . . . . . 347
Possible Mechanisms Underlying Cell Sensitivity to EP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
Cell Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
Membrane Susceptibility to Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
Anchor Dependence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
Cancer Versus Noncancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
The Role of the Plasma Membrane Lipid Composition, Actin Cortex, Membrane
Elasticity, and Other Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350
The Role of External Calcium and Trypsinization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
Introduction
EP of high amplitude can compromise the integrity of the cell plasma membrane, the
process referred to as electroporation or electropermeabilization. EP of 0.1–10 ms
duration were first used for living cell membrane disruption and now are widely
employed in biology and medicine to introduce impermeable molecules into cells,
including DNA, RNA for gene regulation, or cytotoxic agents such as bleomycin to
kill tumors. More intense EP treatments cause irreversible damages in the membrane
and are used for ablation of cancerous tissue (irreversible electroporation, IRE).
About two decades ago short nanosecond duration EP became available for
research. Multiple studies were focused on their physical properties, interaction
with cells and tissue, and benefits for cancer therapy. Nanosecond EP (nsEP) can
permeabilize not only the plasma membrane but also intracellular membranous
structures, such as the mitochondria, nucleus, and endoplasmic reticulum.
20 Different Cell Sensitivity to Pulsed Electric Field 339
Downstream effects of nsEP include morphological modification (disassembly of

actin structures, cell swelling, and blebbing), triggering signaling pathways (calcium
or phosphoinositide 3-kinase), and induction of different cell death pathways (auto-
phagy, apoptosis, or necrosis). Killing cancer cells by nsEP has been extensively
explored in vitro, followed by successful tumor ablation trials in animals and humans
(Garon et al. 2007; Nuccitelli et al. 2014), without recurrence and with minimal side
effects. The use of nsEP to ablate tumors presents a promising therapeutic modality.
With ablation therapies in mind, in this chapter cell sensitivity (or susceptibility)
to EP is defined by the lethal effect of EP treatments. Knowing cell sensitivity is
crucial for prediction of the tumor behavior and correct planning of the tumor
treatment strategy. It was studied for multiple cell lines and various treatment
conditions in vitro. The current state of knowledge will be presented in this chapter.
Methods for Cell Sensitivity Assessment
This section will be focused on in vitro methods most commonly employed to assess the
cytotoxic efficiency of EP exposures. It is not intended to be comprehensive, and
additional details can be found in another chapter of this handbook (Different Cell
Viability Assays Following Electroporation in vitro). Measuring cell survival is a
standard approach to evaluating the EP sensitivity (Riss et al. 2004). A relationship
between the insult-producing factor and the proportion of cells that survive is usually
described by cell survival curve. Avariety of techniques are currently available. Each cell
viability assay has its set of advantages and disadvantages, which will be reviewed below.
Clonogenic Assay
This assay determines the cell’s ability to proliferate indefinitely to form a large
colony or clone. The cells damaged by EP exposure may retain their ability to
metabolize and go through one or two mitoses but are unable to divide and produce
a large number of descendants. This state can be measured for cells in the culture and
commonly denoted as loss of reproductive integrity, reproductive death, mitotic
death, or mitotic catastrophe. The cells that retain their capacity to proliferate
indefinitely produce a large clone that grows on a dish surface as a colony visible
by the naked eye. This method is recognized as one reporting about “true” cell
survival, however cannot be used with primary or suspension cell cultures.
Metabolic Assays
Metabolic assays principle is based on the ability of living cells to utilize the assay
substrate, which usually is added to cell culture and is converted by live cells into
reduced form, which can be recognized based on distinct color or fluorescence. The
assays are reporting about metabolic activity of the entire cell population and do not
differentiate healthy and dying cells.
One group of the assays is based on the reduction of colorless tetrazolium salts to
colored formazan products. The exact mechanism of the reduction is not well
understood, but likely involves the succinate-tetrazolium reductase system which
belongs to the respiratory chain of the mitochondria. Some formazan products, such
as in the popular MTT assay, get accumulated as insoluble precipitate inside the cells
or on the external surface of the cell membrane and must be solubilized before
absorbance recording. MTT assay is cytotoxic and can only be employed for the
end-point measurement. In another group of assays (XTT, MTS, and WST) tetrazo-
lium salt can be converted to soluble formazan in media. The assays are less toxic
than MTT and allow for multiple readings within 1–2 h.
Another group of metabolic assays is designed based on redox indicator
resazurin. The resazurin is blue and nonfluorescent. The viable cells convert it to
resofurin, which is fluorescent and also pink. The assay shows an excellent correla-
tion with formazan-based assays but is more sensitive and more linear when used
with fluorescence detection. The assay is less toxic than formazan-based ones, can be
used for continuum monitoring or end-point measurement of the cell metabolism,
and can be multiplexed with other assays.
The Assays Based on Disruption of the Plasma Membrane Integrity
Trypan blue is one of the earliest and widely used methods for dead cells staining.
The reagent enters the cells with impaired membrane and cell viability must be
determined by counting the unstained cells. The assay is fast, inexpensive, and
compatible with light microscopy. However, it has drawbacks and is reliable in a
cell population with high viability (70–80%). In a typical electroporation experi-
ment, the trypan blue dye provides poor reproducibility and higher dead cell count
than other techniques. Blue staining cannot be used to distinguish between the
healthy cells and the cells that are alive but losing cell functions.
Propidium iodide (PI) is traditional dead cell indicator. The propidium ion is
membrane impermeable, can enter only the cells with an impaired membrane. The
propidium ion, the fluorescence of which is negligibly small, becomes brightly
fluorescent upon binding to nucleic acids inside the cells.
Calcein-AM is live cell indicator. Acetoxymethyl (AM)-form of the dye is cell
permeable and nonfluorescent. In live cells, the dye is converted to a fluorescent and
membrane impermeable form by intracellular esterase that cleaves the AM-ester.
Membrane integrity is needed to retain the fluorescent form in the cells. Therefore,
calcein-AM is the indicator of enzymatic activity and membrane integrity.
Because PI and calcein emit in different ranges of the spectra, they can be
combined in one assay. Both probes are compatible with flow cytometry, fluores-
cence microscopy, and with fluorescence microplate readers.
Acridine orange (AO) and PI is another good combination of fluorescent dyes for
detection of live and dead cells. AO is cell permeable, binds nucleic acid in nucleated
cells (live and dead), and upon binding emits a green fluorescence. PI enters only in
cells with compromised membranes and absorbs AO signal due to Förster resonance
energy transfer (FRET). As a result, dead cells emit only red light, while live cells
emit in the green range.
Other Assays
Adenosine triphosphate (ATP) assay. ATP content in the cells is recognized as a

valid marker of viable cells. When cells lose membrane integrity, they rapidly drop
in ATP content: ATP leaks outside or gets destroyed by endogenous ATPases. The
detection signal is luminescent; it stabilizes within 10 min after addition of reagent
and typically glows for several hours. The ATP assay is fast and very sensitive (can
detect fewer than 10 cells per well). The assay requires cell lysis and is designed for
end-point measurement.
Protease activity assay. The protease activity measurement can serve as a marker
of the cell viability because the proteases are active only in viable cell population and
degrade quickly after the cell death. The Cell-permeable fluorogenic protease sub-
strate enters live cells, where proteases recognize and cleave it. The cleaved substrate
generates a fluorescent signal proportional to the number of viable cells. The assay is
not toxic, very sensitive, and enables many possibilities for multiplexing other
assays.
A real-time viability assay. A recently developed RealTime-Glo assay is available
for continuous data measuring for at least 72 h. The key components are shrimp
luciferase enzyme and an inactive substrate. They are added directly to the culture
media. Live cells convert the substrate into an active form that can be recognized by
luciferase to generate a luminescent signal. The active substrate form is used rapidly
by the luciferase. Thus the luminescent signal diminishes soon after cell death. The
assay is nonlytic, nontoxic, and permitting multiplexing with other assays. Also, it
can be used for end-point measurement.
Methods for EP Delivery
EP can be delivered to the cells using several approaches.
Cells in Suspension
Exposure in electroporation cuvettes is the most common method used in vitro.

The cuvettes have parallel plates of the electrodes, separated by 1, 2, or 4 mm. The
cuvettes produce mostly uniform electric field in the space between the electrodes.
Cuvettes are offered by several manufacturers and usually are sterile, with elec-
trodes made of aluminum (in the past, cuvettes with gold-plated and stainless steel-
made electrodes were also available on the market). This technique is designed for
cells in suspension; adherent cell lines also can be used after the detachment from
the substrate.
Adherent Cell Culture
Many investigators reported using different custom-made and commercially avail-

able electrodes for gene electrotransfer in adherent cells, as well as for studying time
dynamics and physiological consequences of electroporation in individual cells. The
design of such electrodes ranges from an array of parallel-plate electrodes which can
be lowered into a Petri dish or a well with cells attached to the bottom, to
manipulator-driven coplanar tungsten rods which deliver the electric field to indi-
vidual selected cells (Mazeres et al. 2009; Pakhomov et al. 2009). However, all these
methods have limited utility to study the cytotoxic effect of EP. The shortcomings
include one or more of the following: a very small number of cells exposed to a
uniform electric field, whereas survival requires a cell population study; significant
nonuniformity of the electric field applied, with survival test indiscriminately
collecting data from areas with different electric field; mechanical damage and
killing of cells by lowering the electrodes, which is difficult to separate from the
EP effect; and nonsterile conditions. As a result, the majority of studies which
focused on the cytotoxic effect of EP in cell lines were performed using electropo-
ration cuvettes, which required the detachment of the adherent cells from the
substrate and their exposure in suspension.
A technique which enables survival studies in adherent cells without their
detachment has been introduced recently (Pakhomova et al. 2014). Cells are
grown on glass coverslips with an optically clear but electrically conductive indium
tin oxide (ITO). These coverslips can be exposed in electroporation cuvettes to
deliver uniform electric field to most of the adherent cells under sterile conditions.
Cell survival can be analyzed on the EP-exposed coverslips with light or fluorescent
microscopy or with a microplate reader.
Three-Dimensional (3D) Cell Culture Models
Cell sensitivity to EP can also be analyzed in 3D environment when cells are

cultured within a dense matrix. The cells are grown as spheroids (Matrigel) or are
placed in a hydrogel made of collagen or agarose. 3D cell culture models (also called
3D in vitro tumor model) can be exposed to EP by insertion of needle electrodes into
the matrix (Arena et al. 2012; Muratori et al. 2016). The region of ablation is
determined primarily by electric field distribution in the matrix and can be visualized
and quantified with live/dead staining. This approach helps to predict EP treatment
outcomes by obtaining the spatial information on electric field distribution in the
tissue and data on the electric field threshold for cell death at the specific set of pulse
parameters (duration, number, repetition rate).
Diversity of Cell Sensitivity to Microsecond-Duration EP
In the early work by O’Hare et al. (1989), fifteen cell lines derived from normal and
malignant tissues were tested for sensitivity to 10 μs pulses delivered at 1 Hz; pulse
amplitude and a number of pulses varied. The cells were grown in identical settings.
Membrane permeabilization was determined soon after exposure using flow
cytometry. The cells were labeled with fluorescent probes for detection of live
cells (fluorescein diacetate) and cells with permeabilized membrane (propidium
iodide). Cell survival was determined in 12 h after EP exposure by trypan blue
exclusion assay. This work demonstrated that different types of cells showed mark-
edly different electrosensitivity. The authors pointed out that cell sensitivity did not
correlate with cell size or threshold values for membrane permeabilization. Lym-
phoid cells were the most sensitive, followed by epithelial cells and fibroblasts,
whereas endothelial cells were the most resistant.
Another comprehensive study on cell sensitivity to EP was reported by M. Cemazar
et al. (1998). Twelve cell lines were included in the study. Eight electric pulses of 100 μs
duration at the field strength of 100–1800 V/cm were applied at 1 Hz. The efficiency of
different electric field intensities for electropermeabilization was studied “soon after
exposure” (about 30 min) by measuring PI uptake using flow cytometry. Cell viability
was determined by either a clonogenic or MTT assay. This study demonstrated persua-
sively that different cell lines responded differently to the same EP treatment. Electric
field intensity causing 50% reduction in cell survival (IF50) differed about threefold
between the most sensitive and most resistant cell lines (ovary carcinoma cells IGROV
1 and mouse carcinoma cells EAT, respectively). Noncancerous Chinese hamster lung
fibroblasts had an intermediate sensitivity (Table 1). The sensitivity varied profoundly
even though the threshold for permeabilization was approximately the same
(400–600 V/cm). In contrast to earlier research, this study did not observe a correlation
between the electrosensitivity and the tissue origin of cells. Remarkable differences in
electrosensitivity were measured even between subclones of the same cell line (B16
mouse melanoma) and parental and subclone lines of IGROV (Table 1).
The classic theory of electroporation predicts the potential induced on the cell
membrane by the externally applied field is proportional to the cell diameter. There-
fore, the authors introduced an electrosensitivity index (EI) intended to test the effect
of cell diameter on the cell survival after the EP insult. It was calculated as: EI =
1/(IF50 2r), where IF50 is the electric field intensity (V/cm) at which cell survival is
reduced by 50%, and 2r is the cell diameter (in cm). These calculations did not reveal a
correlation between cell sensitivity and cell diameter or the electroporation threshold.
Cell Sensitivity to Nanosecond Electric Pulses
Short nanosecond EP (nsEP) are distinguished by high electric field strength, wide
bandwidth, and fast rise time. They can affect both the plasma membrane and
intracellular membranous structures, thereby impairing organelle functions. Disrup-
tion of the endoplasmic reticulum or mitochondrial membrane may cause calcium
Table 1 Electrosensitivity of tumor cellsa (Reproduced with permission from Cemazar et al.
(1998))
Name IF50b (V/cm) 24 ( 104 cm) AM SEc EId (V-1)
IGROV l 750 19.8 1.8 0.68
SA-1 1320 12.8 1.7 0.59
DC3F 1300 18.6 0.5 0.41
MCF7 1280 20.6 2.0 0.38
B16FO 1360 19.7 2.5 0.37
TBL. Cl2 1340 20.4 2.0 0.37
TBL. Cl2 PT 1310 21.0 1.5 0.36
HeLa 1450 21.5 2.9 0.32
IGROV l/DDP 1640 20.3 2.1 0.30
B16Fl 1850 18.6 1.3 0.29
MM46T 1780 20.8 1.9 0.27
EAT NDe 21.2 6.5 NDe
a
Cell lines are ordered according to their electrosensitivity index, from most to least electrosensitive
cells
b
IF50 = electric-field intensity at which cell survival was reduced by 50%
c
AM SE = arithmetic mean SEM
d
EI = electrosensitivity index
e
ND = could not be determined, due to the technical limitations of the electropulsator
and reactive oxygen species influx into the cytoplasm and/or changes in the cyto-
plasm redox potential, thus engaging new pathways, which lead cells to death and
are not engaged after conventional “long-pulse” EP exposures.
Stacey et al. used the clonogenic assay and trypan blue exclusion to compare the
sensitivity to 60 ns, 60 kV/cm EP in 11 cell lines (Table 2). The anchor-dependent
cells were more resistant to nsEP exposure than cells that are propagated in suspen-
sion. The only notable exception was mouse 3T3 fibroblasts which were as sensitive
as suspension cells.
A study by Ibey et al. compared the sensitivity to shorter 10-ns EP for five
commonly studied cell lines (Ibey et al. 2011). This work determined the EP doses
which killed 50% of the cell population (LD50), as well as the “effective dose”
(ED50) for PI uptake and phosphatidylserine (PS) externalization in 50% of cells
(Fig. 1). The absorbed dose (AD, mJ/g) for EP treatment was calculated as
AD = 106 δ τ E2/ρ, where τ is pulse duration (s), E is the electric field strength
in the medium (kV/cm), ρ is the density of the solution (g/cm3), and δ is the
conductivity of the solution (mS/cm) (Ibey et al. 2009).
The authors found that the LD50 differs by almost 20-fold across the cell lines
studied. The most sensitive cell line was Jurkat (T-cell leukemia), and the epithelial
adenocarcinoma HeLa cells were the most resistant (Fig. 1). The authors did not find
a direct correlation between cell sensitivity and membrane permeabilization (Fig. 2).
The mechanism responsible for the difference in cell line sensitivity was explored by
evaluating cell size, calcium concentration in the exposure medium, and effect of
trypsin treatment prior to exposure. None of these features correlated with the
Table 2 Percentage survival (standard error) of cells exposed to 60 ns (60 kV/cm) pulses
normalized to nonexposed cells (Reproduced with permission from Stacey et al. (2003))
Cell line Cell type 1 pulse 5 pulses 10 pulses
♦◘Jurkat (n = 4) Human T-cell 14.8 2.5a 8.3 1.7a 4.2 1.7a
leukemia
♦◘ HL60 (n = 3) Human 11.7 3.9a 3.7 0.5a 2.0 0.4a
promyelocytic
leukemia
♦AT-LCL (n = 3) Human B-cell 18.0 5.6a 0.8 0.4a 0.7 0.5a
lymphoblastoid
line from a patient
with AT
♦Bloom-LCL (n = 3) Human B-cell 64.1 11.2 9.8 7.4a N/A
lymphoblastoid
line from a patient
with BS
3T3 (n = 3) Mouse fibroblasts, 5.6 1.5a 5.5 1.5a 3.1 1.7a
preadipocytes
SV-Normal (n = 6) Human SV40 100.7 29.0 63.2 17.9 N/A
transformed
normal fibroblasts
SV-AT (n = 4) Human SV40 106.4 8.2 119.0 17.4 N/A
transformed
fibroblasts from a
patient with AT
SV-FA (n = 3) Human SV40 79.2 12.5 39.9 8.9 N/A
transformed
fibroblasts from a
patient with FA
SV-XP (n = 3) Human SV40 65.1 3.1 70.1 19.2 N/A
transformed
fibroblasts from a
patient with XP
◘ HS578T BrCa (n = 3) Human ductal 84.2 6.7 67.1 15.9 N/A
breast carcinoma
◘ HeLa (n = 3) Human 92.7 1.9 75.1 6.7 64.6 8.9
adenocarcinoma of
the cervix
n number of experiments, N/A not available
AT ataxia telangiectasia, XP xeroderma pigmentosum, FM Fanconi’s anemia, BS Bloom syndrome
♦ Suspension cell lines
◘ Malignant cell lines
a
Cell lines where significant differences (P = 0.01) in cell survival were observed between pulsed
and nonpulsed cells
cellular susceptibility to injury and death by 10-ns EP, suggesting that it was largely
determined by cell physiology.
The property of nsEP to target selectively certain cell types was illustrated by an
experiment where the mixture of Jurkat cells (most sensitive to nsEP) and HeLa cells
Fig. 1 Cell survival as determined by dose in several cell types. Mean survival values (+/ s.e.,
in % relative to sham-exposed control, n = 3–5) are plotted against the dose delivered to the
cuvette. Logarithmic fit lines (dashed) reveal significant differences between cell populations. LD50
values (J/g) are displayed in the legend for each population as calculated using the logarithmic fit
(Reproduced from Ibey et al. 2011)
Fig. 2 Comparison of ED50 for PS externalization and propidium uptake with LD50 in
different cell lines. Error bars represent the 95% confidence intervals, as calculated from best fit
using logarithmic function (Reproduced from Ibey et al. 2011)
(the least sensitive to nsEP) was exposed to 10-ns EP (280 J/g) and the death was
observed only in Jurkat cells (Fig. 3). This finding proves that short 10-ns pulses can
be employed for selective targeting of certain cell types, a property that may
potentially be beneficial for cancer ablation.
A recent study compared the nsEP sensitivity of four cancer and two normal human
cell lines under strictly controlled experimental conditions (Gianulis et al. 2016). This
was the first study that compared nsEP susceptibility of different adherent cells without
detaching them from the substrate, by using ITO-coated glass coverslips (Pakhomova
et al. 2014). The cell lines tested were: MRC-5 normal lung fibroblasts; BJ normal skin
Fig. 3 Dose-dependent
changes in survival are
retained in a heterogeneous
cell mixture. Jurkat and HeLa
cells were exposed in mixture
to a 100 pulse train of 10-ns
EP at 150 kV/cm, resulting in
efficient killing of Jurkat but
little effect on HeLa cells
(mean +/ s.e, n = 3)
(Reproduced from Ibey et al.
2011)
fibroblasts; HEP G2 hepatocellular carcinoma; HPAF-II pancreatic adenocarcinoma;

IMR-32 neuroblastoma; and HT-1080 fibrosarcoma. All these cell lines were propa-
gated in the uniform conditions, using the same medium and batch of serum. Individ-
ual coverslips were exposed in 1-mm gap electroporation cuvettes and treated with
different numbers of 300 ns pulses (from 0 to 2000) at 1.8 kV/cm, 50 pulses/s. Cell
survival was assessed 20 h later by a resazurin-based metabolic assay. The experiments
revealed striking differences in nsEP susceptibility. The difference in LD50 between
the most sensitive cell line (neuroblastoma) and the least sensitive one (pancreatic
adenocarcinoma) was almost 80-fold, with intermediate results for other cell types.
The cell killing efficiency showed no apparent correlation with cell size, tissue origin,
or other morphological or physiological features.
The Selectivity of the Cell Killing by EP Is Higher for Shorter Pulses
Another study by Ibey et al. (2010) explored the cytotoxic effect of 10-ns EP
(quantitation, mechanisms, efficiency, and specificity) in comparison with 300-ns,
1.8- and 9-μs EP. The experiments were performed in Jurkat and U937 cells. The EP
effects were characterized by survival assays, DNA electrophoresis, and flow
cytometry. The study established that 10-ns EP caused apoptotic or necrotic death
within 2–20 h after the treatment. Survival (S, %) followed the absorbed dose
(D, J/g) as S = αD(K), where coefficients K and α determined the slope and the
“shoulder” of the survival curve. The coefficient K was similar for both types of cells
and all pulse durations, whereas α was cell type- and pulse duration-dependent.
Long pulses caused immediate propidium uptake and phosphatidylserine
(PS) externalization, whereas 10-ns pulses caused PS externalization only.
The authors concluded that the longer 1.8- and 9-μs EP caused cell death
efficiently but indiscriminately (LD50 1–3 J/g in both cell lines). 10-ns EP were
less efficient but very selective: LD50 was 50–80 J/g for Jurkat, but 400–500 J/g for
U937; 300-ns EP showed intermediate effects. Shorter EP opened propidium-
impermeable, small membrane pores (“nanopores”), triggering different cell death
mechanisms as compared to longer pulses.
Thus, the reduction of the EP duration from micro- to nanoseconds decreased the
cytotoxic efficiency of the treatment but improved its selectivity against certain cell
types. At the primary mechanisms level, this difference can be related to the
transition from the conventional electroporation to nanoelectroporation (which is
defined as the formation of stable plasma membrane pores not exceeding 1–2 nm in
diameter). Cell death triggered by the opening of nanopores and, potentially, also by
damage to organelles, can be either necrotic or apoptotic. In either case, the ability to
repair the primary damage and the pathways to full recovery or death are determined
by intrinsic biological properties and physiology of the cell. This large “biological
component” may explain why physical parameters of exposure have limited predic-
tive power for nsEP treatments and why different cell types can vary so much in
nsEP sensitivity.
The results of the study suggested that the ability of nanosecond EP to target
selectively certain cell types could be beneficial for medical applications such as
tumor ablation.
Possible Mechanisms Underlying Cell Sensitivity to EP
Cell Size
The transmembrane voltage (Vm) induced by an externally applied electric pulse is

directly proportional to the cell radius:
Vm ¼ Vapp K cos ðθÞ r,
where Vapp is the applied field, r is the cell radius, K is a constant (1.5 for spherical
cells in physiological conditions), and θ is the angle between the electric field
direction and any given membrane site (O’Hare et al. 1989). Then, the applied
electric field will cause smaller polarization of the cell membrane in smaller cells,
which should render them less vulnerable to EP. However, experimental data did not
confirm this conjecture. Some small cells proved to be highly sensitive to killing by
EP, including Nalm-6 β-cell precursor (O’Hare et al. 1989), SA-1 sarcoma (Cemazar
et al. 1998), and T-lymphocyte Jurkat cells (Ibey et al. 2011). Therefore, the
difference in cell size did not determine the difference in sensitivity to EP.
Membrane Susceptibility to Electroporation
Differences in the ability of the plasma membrane to withstand high transmem-

brane potential (due to unspecified chemical or structural differences) may
potentially explain the different susceptibility to EP. The cells with a low electro-
poration threshold will develop more extensive membrane damage and are less
likely to recover from it, resulting in reduced survival. However, the difference in
cell survival could not be linked directly to the threshold value for electro-
permeabilization (O’Hare et al. 1989). The same conclusion was made by Cemazar
et al. based on the fact that the threshold value was approximately the same
between all cell lines tested, whereas their sensitivity to EP treatments was
different (Cemazar et al. 1998). Likewise, no correlation between the cell sensi-
tivity and the electroporation threshold was observed with 10-ns EP (Fig. 2 (Ibey
et al. 2011)).
Anchor Dependence
Higher sensitivity of suspension-grown cells as compared to anchor-based cells was

reported in many studies that tested different exposure conditions: microsecond-
duration pulses at different pulse number and amplitude (O’Hare et al. 1989); two
hundreds of 20 ns pulses at 35 kV/cm (Garon et al. 2007); 60-ns pulses at 60 kV/cm
(Stacey et al. 2003); and 10 to 100 of 10-ns pulses at 60–285 kV/cm (Ibey et al.
2010, 2011). Cell lines derived from solid tumors and endothelial cells are typically
more resistant than lymphoid lines. However, the suspension-based U937 cells are as
resistant as many adherent cells and make a notable exception to this rule. Likewise,
some adherent cells were reported to be as sensitive as lymphoid cells, e.g., the
pancreatic cancer AsPC-1 (Garon et al. 2007) and preadipocytes 3T3 (Stacey et al.
2003).
Cancer Versus Noncancer Cells
Several studies reported a higher sensitivity of malignant cells. A basal cell carci-
noma was more sensitive to 30-ns pulses than a paired normal (fibroblast-like) cell
line (Yang et al. 2011). When grown in spheroids and exposed to EP in the presence
of a high concentration of Ca2+ ions, a fibroblast cell line was more resistant to
100-μs pulses (8 pulses at 1 Hz) than three carcinoma cell lines (Frandsen et al.
2015). A burst of high-frequency bipolar pulses of 1 μs duration was reportedly more
effective in four malignant cell lines than in four normal cell lines (Ivey et al. 2015),
although it is not entirely clear why PC12 pheochromocytoma cell line was regarded
as normal. In this study, larger nuclei of cancerous cells were thought as the reason
for their higher vulnerability. In contrast, other reports found no relation of EP
susceptibility to the cancerous or noncancerous cell type (e.g., see Table 2). A
substantial difference in EP sensitivity was found between subclones of the same
cell line (Table 1, B16 and IGROV) (Cemazar et al. 1998). Normal cell lines (skin
and lung fibroblasts) were less vulnerable to 300-ns EP than neuroblastoma but more
vulnerable than fibrosarcoma, hepatocellular carcinoma, and pancreatic adenocarci-
noma (Gianulis et al. 2016).
The Role of the Plasma Membrane Lipid Composition, Actin Cortex,

Membrane Elasticity, and Other Factors
The importance of cholesterol content in the plasma membrane and its link to cell
sensitivity to EP was studied by Cantu et al. using CHO and Jurkat cells exposed to
10- or 600-ns EP (Cantu et al. 2016). Depletion of membrane cholesterol increased
the membrane fluidity, consequentially, increased the nsEP-induced influx of cal-
cium ions and propidium uptake and lowered the threshold for PS externalization
and PI uptake. Concurrently, cholesterol depletion sensitized the cells to nsEP as
manifested by lowering of the LD50. However, the basal cholesterol level is similar
in Jurkat and CHO cells, but Jurkat cells are far more sensitive to EP. Hence, the
authors concluded that the basal cholesterol content per se is not a sole determinant
of cell sensitivity to EP, although the membrane lipid composition and cholesterol
content, in particular, are important determinants of the response to EP. The authors
proposed that lipid rafts rather than lipids per se and also raft-associated proteins and
sphingolipids, which control many aspects of the cell physiology, could play a
substantial role in nsEP response pathways.
Stacey et al. (2003) proposed that cytoskeletal structures may be responsible for
lower sensitivity of adherent cells to nsEP, acting as distributed capacitances which
prevent a build-up of high electric fields at the membrane. The role of cytoskeletal
structures was also studied in relation to cell elasticity, which could be a factor
affecting cell sensitivity to nsEP (Thompson et al. 2014). Membrane elasticity was
measured by the atomic force microscopy in CHO cells that were exposed to 10-ns
pulses (150 kV/cm) or/and treated with latrunculin A to disrupt the actin cortex. The
application of latrunculin A decreased cell elasticity and increased membrane
damage after nsEP exposure. In the cells not treated with latrunculin A, nsEP
exposure caused the disruption of actin filaments, and cell elasticity decreased in a
manner similar to latrunculin treatments. These results suggested an association
between cell susceptibility to nsEP exposure, cell stiffness, and the cytoskeletal
integrity. For the first time, mechanical properties of the cell were put in correlation
with sensitivity to nsEP treatments; however, further research is needed to identify
the mechanisms involved.
The Role of External Calcium and Trypsinization
Cell trypsinization is commonly employed to detach adherent cells from the sub-
strate, to expose them to EP in suspension, using electroporation cuvettes.
Trypsinization may cause damages to proteins located on the membrane surface
and likely disrupts actin filaments throughout the cells (Pietuch and Janshoff 2013).
Ca2+ is an important second messenger ion and can enter electroporated cells
through either membrane pores or voltage-gated Ca2+ channels (the loss of the
resting membrane potential due to electroporation will cause activation of these
channels). The overload of cells with Ca2+ is a well-established cause of cell death,
by either necrosis or apoptosis. The role of trypsinization and Ca2+ concentration in
the electroporation medium was tested with 10-ns EP, but no impact or correlation
with cell survival was observed (Ibey et al. 2011).
Conclusions
Diverse studies, with EP of different durations, reveal profound differences in the

susceptibility of different cell types to this insult. The EP selectivity against different
cell types appears to increase when the pulse duration is decreased. However,
cellular determinants or markers of the susceptibility to EP have not been
established. The present level of knowledge is not sufficient to predict the suscep-
tibility to EP in any given cell type without experimentation. The existing data do not
necessarily prove that cancer cells are more vulnerable than normal cells, and the EP
ablation will not necessarily be as efficient and selective against some cancers
compared to other cancers and healthy tissues. Nonetheless, knowledge-based
manipulation of exposure parameters (pulse duration, amplitude, and rate), proper
choice of the pulse delivery protocols, and combining EP with other treatments (like
cytotoxic chemical agents and temperature changes) are the tools to enable high
selectivity and efficiency of cancer ablation by EP.
Cross-References

▶ Different Cell Viability Assays Following Electroporation in vitro
▶ Mathematical Models Describing Cell Death Due to Electroporation
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Electric Pulse Repetition Rate:
Sensitization and Desensitization 21
Claudia Muratori, Maura Casciola, and Olga Pakhomova
Abstract
In most applications of electroporation in biology, medicine, and industry, the
desired effect is achieved by the delivery of multiple pulses. The pulse repetition
rate (PRR) is one of the key factors which determine the efficiency of such
treatments. Despite intense research, the impact of PRR has not been fully
understood and remains one of the most controversial topics. Depending on the
endpoints and specific conditions, increasing the PRR may increase, decrease, or
make no difference in the effects of an electric pulse (EP) treatment. This
complexity results from a combination of physical factors and biological effects.
Electric modeling has been widely employed to explain PRR effects, but even the
most advanced models showed limited predictive power. Recently it has been
reported that mammalian cells exposed to electric pulses become more sensitive
to subsequent EP treatments, a phenomenon that has been called electro-
sensitization. The sensitized state typically develops within tens of seconds
after the initial EP exposure and reaches a maximum within minutes. When the
overall EP treatments are made long enough for sensitization to develop, their
effects increase two- to threefold. Electrosensitization is engaged when the
overall treatment duration is made sufficiently long, by either lowering the PRR
or by splitting the complete treatment into fractions. Understanding the role of
PRR is critical for tuning various electroporation-based technologies and treat-
ments to achieve the highest efficiency.
Keywords
Electroporation • Membrane permeabilization • Pulse repetition rate •
Electrosensitization
C. Muratori • M. Casciola • O. Pakhomova (*)

e-mail: cmurator@odu.edu; mcasciol@odu.edu; opakhomo@odu.edu

DOI 10.1007/978-3-319-32886-7_23
354 C. Muratori et al.
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
Effects of Pulse Repetition Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
High Pulse Repetition Rate Is More Efficient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
Low Pulse Repetition Rate Is More Efficient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
Other Effects of Pulse Repetition Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
Possible Mechanisms Responsible for Diverse Effects of PRR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
Electrosensitization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
Introduction
Numerous studies have focused on optimization of electroporation conditions to

produce the maximum desired effect while minimizing side effects. However, with
multiple parameters to consider (electric field strength, pulse duration, shape, num-
ber, and repetition rate), these studies have been laborious, the optimization process
remains mostly empirical, while quantitative and mechanistic principles that deter-
mine the outcome of EP exposures are debated.
Among the different EP parameters, the impact of the PRR (sometimes referred to
as PRF – pulse repetition frequency) is the least understood and remains the most
controversial topic, despite being the subject of intense research. Aside from the
trivial heating effect that increases with increased PRR, experimental and theoretical
studies reported larger bioeffects at higher PRR (Table 1), larger effects at lower
PRR (Table 2), biphasic or more complex behavior, or a relatively little role of PRR
(Table 3). Only a few studies had been specifically focused on the role of PRR. The
majority of studies used various endpoints, and PRR was one of the variables
evaluated alongside other parameters of interest. The shortage of comprehensive,
Table 1 Selected reports on larger biological effects of higher PRR

Pulse Pulse Pulse intensity, Studied object/
PRR, Hz duration, μs number kV/cm endpoint Reference
10–10,000 0.004 30 80 Jurkat cells/ Vernier et al.
permeabilization (2006)
100–4,000 0.012 100 (16–30) 10 3
Nociceptor/ Jiang and
threshold Cooper
excitation (2011)
1–7 0.1 2,000 30 Animals/B16 Nuccitelli
tumor reduction et al. (2010)
1–5,000 100 8 1–1.3 Patient/overall Marty et al.
treatment response (2006)
0.1–77 1,000 6 0.8 CHO cells/ Faurie et al.
viability (2010)
21 Electric Pulse Repetition Rate: Sensitization and Desensitization 355
Table 2 Selected reports on larger biological effects of lower PRR

Pulse Pulse
duration, Number intensity,
PRR, Hz μs of pulses kV/cm Studied object/endpoint Reference
0.1–500 0.06–9 150 1.8–13.3 U-937 and CHO Pakhomova
cells/permeabilization et al. (2011)
and viability
1–5,000 20 8 0.05–0.4 SKOV3 cells/viability Yang et al.
(2009)
2–30 40 500 13 Jurkat cells/ Lamberti
permeabilization and et al. (2015)
viability
1–5,000 100 8 1.3 Animals/SA-1 tumor Sersa et al.
growth (2010)
0.01–7,000 100 4 0.3–0.8 Potato tissue/ Silve et al.
permeabilization (2014)
0.03–3 100, 8, 80 2.5 Animals/C57BL6 Tumor Al-Sakere
1,000 regression et al. (2007)
Table 3 Selected studies, which reported negligible or complex nonlinear effects of PRR
Pulse Pulse
duration, Number intensity, Studied object/ Effect of
PRR, Hz μs of pulses kV/cm endpoint Reference PRR
1, 10, 1000 0.005 5 50–60 Chromaffin Craviso Negligible
cells/ et al. (2012)
intracellular
calcium
concentration
5–500,000 0.6 10 3.1 CHO cells/ Steelman et Negligible
fluorescent dyes (al. 2016) for
uptake propidium
iodide,
V-shaped
for YoPro-
1
1–5,000 20 8 0.25 Animals/ Yang et al. Negligible
SKOV3 tumor (2009)
size
0.5–100 50 200 0.9 CHO cells/ Vernhes Bell-
viability and et al. (1999) shaped
permeabilization
1–8,300 30, 100 8, 26 40–200 DC3F cells/ Pucihar V-shaped
permeabilization et al. (2002)
wide-scale studies may explain, at least partially, the controversy concerning PRR
impact and mechanisms involved.
The sensitivity of cells and tissues to the EP is often viewed as a physical
phenomenon in which the EP acts on the cell plasma membrane. Continuum models
of electroporation have been employed to explain and study this process. Usually,
these models take into account the electric parameters of the membrane (conductiv-
ity, permittivity) and employ them to study the processes happening in the plasma
membrane during the electric field application and shortly after that, for instance,
changes in transmembrane potential, number and size of pores, and molecular
transport. However, this methodology has limitations, and many studies recognized
that the experimental data cannot be explained solely by physical descriptions. Even
the most advanced models do not account for factors such as variation in membrane
lipid composition, lipid oxidative state, and/or an immediate biological response of
the cells.
Recently, the role of the PRR has been further challenged by the discovery that
cells exposed to EP develop a delayed and profound increase in the sensitivity to
subsequent EP treatments, a phenomenon named electrosensitization (Pakhomova
et al. 2011). The sensitized state typically develops within tens of seconds after the
initial EP exposure and reaches a maximum within few minutes. Studies on electro-
sensitization demonstrated that the cytotoxicity of an EP treatment increases when
the overall treatment duration allows for the onset of sensitization. The treatment
duration, rather than a particular PRR, is responsible for the enhanced cytotoxicity.
The prolonged exposure can be accomplished by either applying pulses at a low rate
or by splitting a single high-rate train into separate fractions with an interval long
enough to engage sensitization. The sensitization effect can only partially be
explained by physical mechanisms of EP interaction with the membrane and is
currently viewed as a biological response.
The following sections are focused on the reported effects of PRR and discussion
of the possible underlying mechanisms, including sensitization and desensitization.
Effects of Pulse Repetition Rate
High Pulse Repetition Rate Is More Efficient
Increased efficiency of higher PRR is usually accredited to the temporal summation

of small lesions, which can disappear without consequences if the interval between
pulses is sufficiently long.
In the work of Vernier et al. (2006), significant uptake of impermeable dyes
(propidium iodide and YoPro-1) was reported in human T-lymphocyte Jurkat cells
when they were exposed to 4-ns pulses at 1 and 10 kHz rates (total 30 pulses,
80 kV/cm). No dye influx was detected at the lower rates of 10 and 100 Hz. Jiang and
Cooper (2011) showed a reduction of the nociceptor excitation threshold from 30 to
24 and to 16 V/cm as the PRR increased from 100 Hz to 1 and 4 kHz, respectively
(for a train of 100 12-ns pulses).
The 24 h survival of Chinese hamster ovary (CHO) cells decreased from 60 to
about 20%, while PRR increased from 0.1 to 77 Hz (six pulses of 1 ms duration at
800 V/cm) (Faurie et al. 2010). Similarly, a train of 2,000 pulses (100 ns duration,
30 kV/cm) was more efficient in eliminating murine melanomas at PRR of 5 and
7 Hz when compared to 1 or 3 Hz (Nuccitelli et al. 2010), though the statistical

significance of this finding was not evaluated.
The studies reporting increased efficiency with increasing PRR are summarized
in Table 1.
Low Pulse Repetition Rate Is More Efficient
In contrast to the above studies, several publications reported about more efficient
cell killing at lower PRR, both in vitro and in vivo. A progressive enhancement of the
cytotoxic effect as the PRR decreased from 5 kHz to 1 kHz, 60 Hz, and 1 Hz was
reported in human ovarian carcinoma SKOV3 cells exposed to exponentially
decaying EPs (Yang et al. 2009). In a study by Al-Sakere et al., the tumor regression
was more efficient at 0.03 Hz than at 0.3 Hz (eight pulses of 1 ms at 2.5 kV/cm) or at
0.3 Hz than at 3 Hz (80 pulses of 100 μs at 2.5 kV/cm; see Fig. 2 in Al-Sakere et al.
(2007)). In these experiments, the delivered energy was kept constant, with longer
exposures at a greater number of pulses and a lower electric field being typically
more efficient, despite a lower temperature rise.
The studies reporting increased EP efficiency at lower PRR are summarized in
Table 2.
Other Effects of Pulse Repetition Rate
A number of studies reported little or no effect of PRR (Table 3). Other studies, also
included in Table 3, reported more complex dependences which could not be
attributed to simple “increase” or “decrease” categories.
Pucihar and coauthors (2002) found that the uptake of Lucifer yellow dye was the
same at PRR of 1Hz and 8.3 kHz in Chinese hamster fibrosarcoma cells (DC3F)
exposed to 26 pulses of 30 μs duration. However, it required about 1.5 times higher
electric field for the higher PRR. The dependence was similar for 100 μs pulses,
except for a slightly higher dye uptake at 10 Hz compared to both lower (1 Hz) and
higher PRR (1 and 2.5 kHz). Another study by Steelman and coauthors (2016)
reported that the PRR may affect the transport of specific dyes across the membrane
in a molecular-specific manner. The uptake of three membrane impermeable dyes
was monitored across a wide range of PRR (5 Hz–500 kHz, 600 ns duration,
3.1 kV/cm), and the frequency trends in the uptake were found to be unique to the
identity of each fluorophore. The uptake of propidium iodide (PI) shows essentially
no frequency dependence, YoPro-1 uptake demonstrated a V-shape dependency with
a maximum at 50 kHz, and the uptake of FM 1–43 had a negative trend as PRR
increases. An involvement of transmembrane protein channels was proposed as a
possible explanation for the specificity in the frequency trends.
In another study by Vernhes et al., the cytotoxic effect in CHO cells had a bell-
shaped dependence on PRR (200 pulses of 50 μs duration at 0.9 kV/cm). The effect
was lower at the frequency of 10 Hz and gradually enhanced when the rate decreased
to 0.5 Hz or increased to 100 Hz (Vernhes et al. 1999). At the same time, membrane
permeabilization effect assessed by PI uptake was the same in the range 0.5–20 Hz
and increased at 50 and 100 Hz. The authors hypothesized that the slow rotation of
cells in suspension is responsible for the increased cytotoxicity at the lowest PRR.
Different portions of cell membrane get exposed to the field, and more membrane is
porated during slow EP treatment. Later, this idea was extended into a complex
model that related random statistical rotations of cells in suspension to EP efficiency
(Schoenbach et al. 2009).
Possible Mechanisms Responsible for Diverse Effects of PRR
As noted above, the most trivial effect of high PRR is a possible increase of the
temperature. A heating curve from a train of electroporating pulses has a sawtooth
shape, with abrupt temperature rise during each pulse, and gradual cooling between
the pulses. When PRR is increased, shorter intervals between pulses leave less time
for heat dissipation, and the overall temperature rise is faster and more significant
than with lower PRR. In many applications of electroporation, reducing PRR is
necessary to prevent thermal effects and damages.
High PRR may result in temporal summation of transient and otherwise sub-
threshold bioeffects. For example, a single EP near the threshold for electroporation
can cause a small (10–20 nM) rise in cytosolic Ca2+ concentration which, depending
on the cell type, may last a few seconds. If the next EP is applied after the Ca2+
transient is over, no physiological effects will follow. However, if the next pulse(s)
come with short intervals, the evoked Ca2+ responses may overlap and reach a
physiologically significant level (usually about 200 nM), producing a variety of
downstream biological effects.
The resealing speed of the membranes is recognized as a factor that can contribute
to higher effects of low PRR. Setting an extra time between pulses or between trains
of pulses allows for the membrane repair and rebuilding of the transmembrane
voltage to the level where a new population of pores can be generated. The higher
efficiency of low PRR has been explained in few numerical modeling studies. These
studies take into account properties of the membrane and the extracellular medium
(conductivity and permittivity) and the pulse characteristics (shape, amplitude,
count, rise/fall time, interpulse interval). The models allow to track the primary
events occurring at the membrane and can predict the number, the size and density of
pores, and the rate of transmembrane transport. The numerical approach used by Son
et al. (2016) investigates the number and size of pores created in response to a train
of 20 identical, evenly spaced (1 Hz) electric field pulses (1 kV/cm, 100 μs duration).
This model shows that once the pores are created, subsequent pulses do not con-
tribute to further pore creation because the existing pores increase the membrane
conductance and reduce the transmembrane voltage to values, which are lower than
the electroporation threshold. This behavior is expected to have direct consequences
on the transport of ions and molecules. The authors demonstrated how the applica-
tion of additional pulses does not significantly increase the uptake of Ca2+ into the
cell (5–12%). Slow PRR provides time for the membrane to reseal, thus allowing for
the generation of a sufficient transmembrane voltage to produce a new population of
pores. The study did not account for the effect of different pulse durations, the PRR,
and the entry of solutes larger than Ca2+ which limit the possibility to generalize
these conclusions. In line with this work, the numerical analysis by Lamberti et al.
demonstrated a negative trend in pulse efficiency as PRR increases (train of the ten
40 ns pulses delivered at 2, 5, 10, 30 Hz, 13 kV/cm (Lamberti et al. 2015)).
Thus, an argument raised against the efficiency of high PRR is that a “leaky”
electroporated cell membrane will not be charged by the applied EP as efficiently as
an intact membrane. This gradual decrease in the EP efficiency as the cellular
membrane gets more and more electroporated has been appropriately named “desen-
sitization” (Silve et al. 2014; Lamberti et al. 2015). This mechanism explains why
low PRR treatments, which allow more time for membrane resealing between
sequential pulses, may be more efficient.
Electrosensitization
Electrosensitization is a phenomenon by which cells exposed to EP develop a

delayed and substantial increase in the sensitivity to subsequent EP treatments.
Two- to threefold increase in cytotoxicity can be obtained by dividing an EP dose
into two segments with an interval long enough to engage sensitization (Pakhomova
et al. 2011): the first segment makes cells more sensitive to the cytotoxic effect of the
second segment, thereby resulting in more efficient overall treatment. The treatment
protocols engaging electrosensitization have been shown to increase the volume of
the ablation zone in different three-dimensional (3D) tumor cultures (Muratori et al.
2016), suggesting that sensitization may benefit cancer ablation by irreversible
electroporation. Sensitization profoundly enhanced the uptake of PI and bleomycin,
a cell-impermeable cytotoxic agent used for electrochemotherapy (Pakhomova et al.
2013). These findings proved that plasma membrane permeabilization is increased in
sensitized cells. The enhanced electroporative uptake may assist electro-
chemotherapy and gene electrotransfer.
The study that led to the discovery of electrosensitization was initially conceived
to learn how the PRR affects cell survival (Pakhomova et al. 2011). Figure 1 (left
column) shows the results of multiple experiments that investigated the cytotoxic
efficiency of EP trains delivered at different PRR. Each plot represents an experi-
ment where the electric field, the pulse duration, and the number of pulses were kept
constant, while the PRR was the only parameter that varied. The studies were
performed in two cell lines (human monocyte lymphoma U 937 and Jurkat) and
under diverse exposure conditions: 300 ns to 9 μs pulse duration, 1.8–9 kV/cm
electric field, 2–500 pulses per train, at 0.001–1,000 Hz PRR. The data consistently
showed that, regardless of the specific conditions tested, maximum cell killing in all
experiments was achieved at the lowest tested PRR. As the PRR increased, the effect
gradually weakened and reached a plateau at higher frequencies. Surprisingly, the
level at which the plateau was reached differed more than 1,000-fold from one set of
Fig. 1 The effect of PRR (left column) and of the total duration of the treatment (right column) on
cell survival. Each panel (except those at the bottom) represents a separate series of experiments
where cells were exposed to a fixed number of pulses of a given amplitude and duration (see legends
within the figure). The only variable in each series was the PRR and, consequently, the total duration
of the treatment. Other data in the legends are the cell type and the time point after exposure when
the cell survival was measured. Each data point is the mean s.e. for 3–12 independent
experiments to another (Fig. 1, bottom graph in the left column). The difficulty to
explain such a diversity suggested that perhaps the PRR was not responsible for the
increased efficiency of the slower treatments. Indeed, when the same data were
replotted against the total duration of the treatment (Fig. 1, right column), the
different sets of experiments came to an agreement. In all cases, the cytotoxic
efficiency started to increase when the treatment duration neared 10 s (Fig. 1, the
bottom graph in the right column). These data prompted that the first EP train
somehow conditioned the cells, making them more sensitive to the second train
delivered after a proper delay. The treatment duration is explicitly tied to the PRR,
being simply the ratio of the pulse number to the PRR. Therefore, to assign the
enhancement of EP effects merely to the treatment duration, PRR was eliminated as
a variable: a high-rate EP train was split into separate fractions separated by a
quiescent period (Fig. 2a). The results of such exposure fractionation are shown in
Fig. 2b. The train of 150 pulses was far more efficient for cell killing at 1 Hz than at
1,000 Hz. However, when the 1,000 Hz train was split into two fractions (75 pulses,
0.075 s each) separated by a 150 s interval, the cell survival dropped to the level
achieved at 1 Hz in 150 s of exposure. This finding unequivocally demonstrated that
it was indeed the treatment duration, rather than a particular PRR, that determined
the enhancement of the cytotoxic effect.
The higher efficiency of fractionated EP treatments (or split-dose treatments) has
been established in multiple cell lines (CHO, murine melanoma B16, U 937, Jurkat,
murine squamous cell carcinoma KLN 205), for EP durations ranging from 60 ns to
100 μs and pulse amplitudes from 1.8 to 13.3 kV/cm. Sensitizing cells by split-dose
exposure caused a significant reduction of the lethal dose. In U 937 cells, lethal doses
that killed 50 (LD50) and 90% (LD90) of cells were 2–2.5 times lower for the
fractionated exposures (Pakhomova et al. 2011). This result can potentially be
further improved by careful optimization of different exposure parameters.
Several earlier studies attributed the enhanced effect of low PRR to slow, random
rotation of cells in suspension so that different portions of the cell membrane faced
the electrodes and were permeabilized by EP (Vernhes et al. 1999). However, the
increased cytotoxicity of fractionated exposures has been reported not only for cells
in suspensions but also for substrate-attached cells, cells embedded in agarose gel,
and cell spheroids (Pakhomova et al. 2011, 2013; Muratori et al. 2016). Such
universality helped to exclude the possible impact of sedimentation and random
rotation of cells in suspension.
Several studies have shown that a more efficient permeabilization of the plasma
membrane is associated with split-dose treatment. In particular, it has been reported
that fractionated treatment improves the electroporative uptake of bleomycin
Fig. 1 (continued) experiments. For the bottom plots, the curves from all series of experiments were
collapsed together; for clarity, only the connecting lines are shown. Note that regardless of the
treatment regimen, the cell survival started to decrease when the treatment duration neared 10 s (the
right bottom panel) (Reproduced from Pakhomova et al. 2011)
Fig. 2 Enhancement of the EP cytotoxic effect by exposure fractionation. (a) Schematic represen-
tation of a train of ten EP (single-dose protocol) and the same ten pulses delivered as two identical
trains of five pulses each separated by a quiescent period (split-dose protocol). (b) U 937 cells were
exposed to a train of 150, 300 ns pulses (9 kV/cm) using PRR indicated in the legend. The exposure
to EP was far more effective at 1 Hz than 1,000 Hz. However, splitting the 1,000 Hz train in two
fractions of 75 pulses each, with a 150 s interval, made it as efficient as 1 Hz (Modified from
Pakhomova et al. 2011)
(Pakhomova et al. 2013). Also when comparing the PI uptake in CHO cells exposed
to two trains of EP ((2 + 2) pulses, 600 ns, 20 kV/cm, Fig. 3), it was shown that the
second train elicited a stronger PI uptake than the first one. This data indicates that
the second EP exposure causes more membrane damages than the identical first one
by forming more pores, larger pores, or a combination of the above (Gianulis and
Pakhomov 2015).
The electrosensitization phenomenon has been shown to be dependent on the
treatment parameters (Pakhomova et al. 2011; Muratori et al. 2016), the cell type
(Pakhomova et al. 2013), and the electroporation buffer composition (Dermol
et al. 2016). Electrosensitization only develops within a certain range of pulse
parameters, and the optimal conditions may be different for different cell types.
Dermol et al. demonstrated that the amount of PI uptake after two trains of EP
((4 + 4) pulses, 100 μs, 0.75 kV/cm) is dependent on the electroporation buffer
composition (Dermol et al. 2016). In a high-conductivity buffer, the sensitization
effect was lost, and the PI uptake for single- and split-dose protocols was similar.
When using low conductivity or hyperosmotic buffer, the split-dose protocol was
less effective than the single-dose protocol, suggesting the occurrence of a desensi-
tization rather than a sensitization effect.
Several experimental and numerical studies ascribed the higher efficiency of split-
dose exposures to membrane repair mechanisms occurring during the interval
Fig. 3 Propidium iodide (PI) uptake is increased after the second fraction of EP. CHO cells were
exposed to 2 pulses (2p, gray symbols), or two series of 2 pulses (2p + 2p groups, black symbols)
separated by 150 s. Arrows identify the exposures to a pair of 600 ns, 20 kV/cm pulses. Mean
s.e. for 18–23 cells per group. Error bars are shown in one direction only for clarity. The difference
between the groups is significant at p < 0.01. Blue and orange triangles are added to the original
figure: right catheti show the magnitude of PI uptake after the first two pulses (blue) and after
second two pulses (orange). Note the increased response to the second dose of EP (Modified with
permission Gianulis and Pakhomov 2015)
between exposures (Silve et al. 2014; Lamberti et al. 2015; Son et al. 2016). A
resealed plasma membrane restores the transmembrane voltage to the level suitable
for the production of a new pore population. Consequently, the second EP fraction,
which likely does not contribute to the pore formation when delivered immediately
after the first fraction, regains its influence and participates in pore creation with an
efficiency similar to the first EP fraction. This hypothesis is suitable for cases where
the efficiency of the first and second fractions is equal, but it does not provide an
explanation for the situations where the response to the second EP fraction is
significantly larger (Fig. 3, Gianulis and Pakhomov 2015; Dermol et al. 2016).
This effect can only be explained by the occurrence of a biological response that is
not usually taken into account in modeling work.
The present body of knowledge identifies the delayed electrosensitization as a
biological phenomenon responsible for the high efficiency of fractionated EP treat-
ments, even though the underlying physiological mechanisms remain elusive. Listed
below are some mechanisms that may be involved in sensitization, although their
role has yet to be proven:
1. EP-induced colloid-osmotic cell swelling: Permeabilization of cells leads to water

uptake and cell swelling due to the so-called colloid osmotic mechanism. The
increase of cell diameter may cause a higher EP-induced voltage across the cell
and increases electroporation. Swelling takes tens of seconds, which may explain
the delayed onset of sensitization.
2. The influx of Ca2+ and its downstream effects: Among ions that can enter cells
through electropores, calcium ion regulates multiple key processes in the cells.
The link between the increased Ca2+ concentration and the delayed cell suscep-
tibility to EP is not clear. However, it should come as no surprise that prolonged
time intervals when the internal Ca2+ is elevated (due to a longer EP treatment)
may be unfavorable for cells and make them more vulnerable.
3. Oxidative damage to the membrane: Intense EP may increase the level of reactive
oxygen species (ROS) (Bonnafous et al. 1999), and a recent study found that
oxidative damage to the membrane increases its susceptibility to electroporation
(Vernier et al. 2009). Hence, EP-induced ROS production makes the cell mem-
brane more sensitive to EP.
4. Cytoskeleton rearrangement: Disruption of the cytoskeleton structures was
reported as one of the particular effects of EP and may affect the susceptibility
of mammalian cells to EP (Thompson et al. 2014).
5. Adenosine triphosphate (ATP) leakage and depletion: Once cells are porated, ions
from the outside diffuse in, whereas the intracellular ions leave the cells. The cells
attempt to restore ion gradients activating ion pumps and repairing the membrane.
High energy demand for these processes may be aggravated by ATP leakage out
of cells through the electropores. Therefore, a prolonged high demand for ATP
combined with ATP loss during longer exposures could be a factor responsible for
sensitization.
Taking the phenomenon of electrosensitization into account could benefit EP

applications, even before the exact mechanisms of sensitization are established.
Electrochemotherapy (ECT) is a nonthermal ablation procedure, accepted in
clinical practice, and effective on solid tumors. Bleomycin is a low-permeant
cytotoxic drug commonly used for the treatment of cancer with ECT. Electro-
permeabilization facilitates bleomycin entry into cells and increases its cytotoxicity
by orders of magnitude. Engaging electrosensitization has been shown to improve
the electroporative uptake of bleomycin (Pakhomova et al. 2013). The study was
done using pulse parameters which resemble the standard ECT treatment regimen
(8 pulses, 100 μs duration, 100 Hz). B16 and CHO cells were sensitized by splitting
a single train into two trains (with 5 min interval), and the LD50 decreased by 1.5–2
times. The engagement of sensitization increased the electroporation-assisted
bleomycin uptake and caused an increase in lethality comparable to a tenfold
increase in bleomycin concentration when using a single EP dose. In addition to
bleomycin, sensitizing cells by split-dose exposure increased the electroporative
uptake of propidium ion suggesting that in sensitized cells, membrane permeabi-
lization is more efficient. Perhaps electrosensitization may assist other EP applica-
tions that rely on electroporation for intracellular delivery such as gene
electrotransfer and electroporation-mediated siRNA delivery.
Recently the effect of electrosensitization has been visualized in 3D culture
(Muratori et al. 2016). In KLN 205 cells grown as spheroids in Matrigel matrix,
Fig. 4 Split-dose treatments increase cell death in KLN 205 spheroids. (a) KLN 205 spheroids
grown in Matrigel were exposed to either 200 pulses or two trains of 100 pulses each (300 ns, 600 V,
50 Hz). PI uptake was measured at 24 h after treatment. Scale bar: 0.5 mm. (b) Shows the PI uptake
(left y-axis) and the percentage of cell death (right y-axis). Mean s.e., n = 4. * p < 0.01
(Reproduced from (Muratori et al. 2016)
fractionated treatments increased the ablation volume and cell death up to two to
threefold compared to single-train treatments (Fig. 4). The maximum enhancement
of ablation was achieved when the two fractions were separated by 100 s. Similar
results were obtained for KLN 205 cells embedded in agarose. Cells in suspension
and embedded in matrices responded to fractionated treatments very similarly,
suggesting that sensitization should occur in vivo as well. Indeed, the delivery of
51 pulses (50 μs, 600 V, 10 Hz) in three trains of 17 pulses each separated by 30 s has
been shown to increase tumor ablation in a 3D hind limb tumor model (Jiang et al.
2015).
Irreversible electroporation (IRE) has recently appeared as a new minimally
invasive procedure for cancer ablation (Rubinsky 2010). In IRE applications,
tumor cells are killed by applying high-intensity EP which cause irreparable cell
damage. The efficiency of the treatment depends on the coverage of the entire tumor
volume with a sufficiently high electric field. However, devices approved for IRE
have limited voltage output limiting the ablation region. Multiple electrode insertion
and multiple exposures are needed for treatment of large tumors, making the
procedure longer and risky for patients and challenging for physicians. The limited
size of the ablation zone is a major drawback to a wider use of IRE.
Engaging electrosensitization may allow for ablation of larger tumors or tissue
volumes without increasing pulse amplitude or dose. For larger tumors, this may
require a single insertion of electrodes and a single treatment instead of multiple
insertions. Lower dose and slower EP delivery translate into reduced tissue heating,
which helps the preservation of the extracellular scaffold and assists the recovery.
Moreover, lowering the EP voltage reduces side effects of ablation, such as pain and
muscle contractions, and minimizes the chance of heart arrhythmias.
Conclusions
Changing PRR has a complex impact on the biological efficiency of exposure, with
the direction of changes dependent both on the specific EP parameters and on
endpoints studied. The higher efficiency of higher PRR can be caused by a temporal
summation of small and transient bioeffects which would have little impact if pulses
are spaced far enough to let such effects expire. Higher PRR can also cause a greater
temperature rise, by allowing less time for heat dissipation. On the contrary, higher
efficiency of lower PRR can be explained by allowing enough time for the cell
membrane to reseal between pulses (i.e., by removing the desensitization factor), as
well as by a biological phenomenon of delayed increase of EP sensitivity following a
conditioning EP treatment (sensitization). The exact mechanisms underlying sensi-
tization have yet to be established. The overall effect of PRR change depends on the
combined impact of the above mechanisms and possibly on some other unknown
factors. Taking PRR into account can be a powerful and flexible approach to achieve
desirable results in medical and biotechnological EP applications.
Cross-References

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Lightning-Triggered Electroporation as a
Mechanism for Horizontal Gene Transfer 22
Tadej Kotnik
Abstract
Horizontal gene transfer (HGT) plays a prominent role in evolution and genetic
variability of life. Five biotic mechanisms of HGT among prokaryotes have so far
been extensively characterized: conjugation, competence, transduction, gene
transfer agent particles, and transitory fusion with recombination; but it seems
questionable whether they can account for all ongoing HGT, and it is even less
clear how HGT could have proceeded before any of these mechanisms – them-
selves products of evolution – had developed. An alternative and perhaps more
general path to HGT is offered by non-biochemical, yet natural mechanisms of
destabilization of the membranes enveloping the genetic material: freeze-thaw
cycles, abrasive action of gravel and sand, and electroporation triggered by
lightning strokes. This chapter focuses on the latter mechanism of gene transfer
– DNA uptake and heritable expression based on reversible electroporation
(electrotransformation), which is by far the most efficient technique of artificial
HGT, reported to date for bacteria from at least 13 of their 29 currently recognized
taxonomic phyla, archaea from at least two of their five phyla, microalgae from at
least three of their six phyla, and yeasts from both their phyla. As a complement,
irreversible electroporation is a mechanism of DNA release (electroextraction),
although less efficient in the laboratory than chemical extraction. It is shown that
conditions for electroporation-based DNA release, uptake, and transformation are
present in many natural habitats exposed to lightning strokes, with quantitative
estimates that the number of microorganisms subjected to conditions for
lightning-triggered HGT, particularly in freshwater habitats, may well exceed
1017 per year.
T. Kotnik (*)
Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia
e-mail: tadej.kotnik@fe.uni-lj.si

DOI 10.1007/978-3-319-32886-7_25
370 T. Kotnik
Keywords
Horizontal gene transfer • Evolution • Electroporation • Electroextraction •
Electrotransformation • Lightning strokes • Lightning-triggered transformation
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
Reversible Electroporation as a Mechanism of DNA Uptake and Transformation
in Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
Irreversible Electroporation as a Mechanism of DNA Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
Lightning-Triggered Electrotransformation as a Contributor to HGT in Microorganisms . . . . 375
Emergence of the Hypothesis and Tentative Empirical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
Theoretical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
Assessing the Feasibility and Importance of Lightning-Triggered HGT . . . . . . . . . . . . . . . . . . . . . . 379
Delivery and Propagation of Electric Current . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
Electric Pulse Waveform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
Microorganisms, DNA, and Exposure Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
Introduction
DNA sequencing has revolutionized our understanding of evolutionary relationships

between organisms, yet it also revealed that evolution does not only proceed solely
by gradual divergence of species due to random mutations and their natural selection
but also by interchange of genetic material between species (horizontal gene transfer
– HGT). This started to emerge in the early 1990s from comparison between
genomes, which revealed bacterial genes present in some eukaryotes yet absent
from any archaea, though eukaryotes are phylogenetically closer to archaea than to
bacteria, and it was corroborated by comparison between nucleotide sequences in
individual essential genes, from which it emerged that phylogenetic trees inferred
from different such genes can differ drastically.
Horizontal gene transfer is now widely recognized as a major contributor to
genetic variability of prokaryotes, with at least five natural mechanisms extensively
documented:
• Competence: uptake by an organism of DNA from its surroundings

• Conjugation: transfer of DNA between two organisms in direct contact
• Transduction: transfer of DNA from one organism into another via an infection by
a virus (phage)
• GTA-mediated transfer: transfer of DNA by gene transfer agents (GTAs),
viruslike particles synthesized by some bacteria
• Transitory fusion with recombination: pairwise fusion of some archaea into a
hybrid, followed by homologous recombination of their DNA and the hybrid’s
fission back into two archaea
22 Lightning-Triggered Electroporation as a Mechanism for Horizontal. . . 371
As all these mechanisms are biotic, i.e., based on biomolecules synthesized and
utilized by the organisms involved in HGT, it follows that these evolution-
accelerating mechanisms are themselves products of evolution. Thus, though it is
now widely accepted that HGT has been ongoing since the earliest stages of
evolution, it is unclear whether – and how – HGT could have proceeded before
any of its biotic mechanisms had developed.
It also seems questionable whether each occurrence of HGT identified to date can
be explained by the five biotic mechanisms outlined above. Namely, although
competence, conjugation, and transduction are found in both prokaryotic kingdoms,
i.e., both in archaea and in bacteria, it is questionable whether at least one of these
mechanisms can act in every archaeal and bacterial species. Competence is thus
estimated to occur naturally in only 1% of all bacterial species, and likely in an
even lower fraction of archaeal species, and hindering the efficiency of competence
further is rapid degradation of free DNA in natural habitats. For conjugation,
efficiency quickly decreases with increasing genetic distance, as the proteins utilized
in conjugative coupling are highly adjusted to a particular organism’s envelope.
Regarding transduction, most viruses performing this mechanism of HGT (phages)
infect selectively, only transferring genes among genetically very close organisms –
often even only within a single strain of a single species. Moreover, evidence is
rapidly accumulating for HGT in eukaryotic organisms, in particular microalgae and
yeasts, although neither conjugation nor competence exist in eukaryotes, and they
are also in general not infectable by bacterial or archaeal phages.
As so often in biology, at least some of these limitations may have exceptions.
The bacterium Acinetobacter baylyi can take up and express even highly fragmented
and degraded DNA (Overballe-Petersen et al. 2013), and this ability may be present
in other naturally competent bacteria. A mechanism somewhat resembling conjuga-
tion is used by the bacterium Agrobacterium tumefaciens to transfer its genes into
cells of some flowering plants, inducing tumorigenesis (Zupan et al. 2000). Also,
several viruses with a broad host range and/or adaptive host specificity are known
both in bacteria (Koskella and Meaden 2013) and eukaryotes (Bandin and Dopazo
2011). As a consequence, it is highly questionable whether there is a case of HGT for
which it could be inferred that none of the known biotic HGT mechanisms can
explain it. While our knowledge and understanding of HGT are rapidly increasing,
we are thus still far from a reliable assessment of the relative importance of each of
the known biotic HGT mechanisms and farther still from a conclusion whether there
are no additional such mechanisms of relevance.
An alternative and perhaps more general path to HGT, in particular among
unicellular organisms – both prokaryotes and eukaryotes – is offered by abiotic, i.
e., non-biochemical, yet natural mechanisms of destabilization of the membranes
enveloping the genetic material: freeze-thaw cycles, abrasive action of gravel and
sand, and electroporation triggered by lightning strokes (Kotnik and Weaver 2016).
This chapter discusses the latter mechanism – lightning-triggered electroporation of
microorganisms’ envelopes, causing DNA release, uptake, and transformation, thus
acting as a natural abiotic mechanism of HGT among them. Following this intro-
ductory section, section “Reversible Electroporation as a Mechanism of DNA
372 T. Kotnik
Uptake and Transformation in Microorganisms” discusses reversible electroporation

as a mechanism of DNA uptake and transformation in microorganisms (electrotrans-
formation), and section “Irreversible Electroporation as a Mechanism of DNA
Release” treats irreversible electroporation as a mechanism of DNA release
(electroextraction). Section “Lightning-Triggered Electrotransformation as a Con-
tributor to HGT in Microorganisms” outlines the emergence of the hypothesis of
lightning-triggered HGT, several experiments providing indirect empirical support
to its feasibility, and theoretical considerations further corroborating that conditions
for both electroextraction and electrotransformation are present in natural habitats
subjected to lightning strokes. Finally, section “Assessing the Feasibility and Impor-
tance of Lightning-Triggered HGT” outlines some guidelines for improved assess-
ment of the feasibility and importance of lightning-triggered HGT.
Reversible Electroporation as a Mechanism of DNA Uptake

and Transformation in Microorganisms
Artificial electroporation-induced uptake of DNA with subsequent expression,

termed gene electrotransfer, is, if properly designed and optimized, achievable in
most biological cells; in those of multicellular eukaryotes, however, such transfer is
in most cases not heritable (electrotransfection), while in prokaryotes, as well as
many unicellular eukaryotes, it is generally possible to achieve both expression and
heritability (electrotransformation). Electrotransfection was first achieved in mam-
malian cells in the early 1980s (Wong and Neumann 1982), while in bacteria the first
studies suggested any form of gene electrotransfer to be achievable only after a
complete removal of the cell wall. This was, however, largely due to insufficient
available electric field amplitudes, and subsequent development of more powerful
pulse generators quickly led to successful electrotransformation of a broad range of
microorganisms with an intact wall, including not only bacteria and archaea but also
unicellular eukaryotes – both microalgae and yeasts. To date, successful electrotrans-
formation has thus been reported for bacteria from at least 13 of the 29 currently
recognized taxonomic phyla, for archaea from at least 2 of their 5 phyla, for
microalgae from at least 3 of their 6 phyla, and for yeasts from both their phyla
(Table 1).
Electrotransformation is effective both in Gram-negative and Gram-positive
bacteria, although the latter generally require higher fields and/or yield lower
transformation efficiencies, which is attributed to the thicker peptidoglycan layer
of the cell wall in Gram-positive bacteria; thus, with plasmid DNA, the optimized
efficiencies are typically up to 108–1010 transformants per μg DNA for Gram-
negative bacteria and up to 106–107 transformants per μg DNA for Gram-positive
bacteria. In the same vein, bacteria possessing in addition an outer polysaccharide
capsule are generally electrotransformed with even lower efficiencies, but these can
still exceed 104 transformants per μg DNA (again with plasmid DNA) for bacteria in
the exponential growth phase, in which the capsular synthesis rate decreases.
Table 1 A sample of successfully electrotransformed microorganisms (Reproduced from Kotnik

et al. (2015) with permission. References to most reports summarized in this table are given in
Kotnik 2013a)
Phylum Species
Archaea
Crenarchaeota Metallosphaera sedula, Sulfolobus acidocaldarius, Sulfolobus islandicus,
Sulfolobus solfataricus
Euryarchaeota Methanococcus voltae, Pyrococcus furiosus
Bacteria
Actinobacteria Brevibacterium lactofermentum, Corynebacterium diphtheriae,
Mycobacterium smegmatis
Bacteroidetes Bacteroides fragilis, Bacteroides uniformis, Prevotella ruminicola
Chlamydiae Chlamydia psittaci, Chlamydia trachomatis
Chlorobi Chlorobium vibrioforme
Cyanobacteria Arthrospira platensis, Fremyella diplosiphon, Synechococcus elongatus
Deinococcus- Deinococcus geothermalis, Thermus thermophilus
Thermus
Firmicutes Bacillus cereus, Clostridium perfringens, Enterococcus faecalis,
Lactobacillus casei, Streptococcus pyogenes
Fusobacteria Fusobacterium nucleatum
Planctomycetes Planctomyces limnophilus
Proteobacteria Campylobacter jejuni, Escherichia coli, Salmonella enterica,
Sinorhizobium meliloti, Yersinia pestis
Spirochaetes Borrelia burgdorferi, Serpulina hyodysenteriae
Tenericutes Mycoplasma pneumoniae
Thermotogae Thermotoga maritima
Unicellular algae (microalgae)
Chlorophyta Chlamydomonas reinhardtii, Chlorella ellipsoidea, Chlorella vulgaris,
Dunaliella salina, Scenedesmus obliquus
Heterokontophyta Nannochloropsis sp. W2J3B, Phaeodactylum tricornutum
Rhodophyta Cyanidioschyzon merolae
Unicellular fungi (yeasts)
Ascomycota Candida maltosa, Ogataea polymorpha, Pichia pastoris, Saccharomyces
cerevisiae, Schizosaccharomyces pombe
Basidiomycota Cryptococcus neoformans, Pseudozyma antarctica, Pseudozyma flocculosa
Efficiency of electrotransformation in bacteria also depends strongly on the

molecular form of DNA being transferred. Generally, the efficiency is the highest
for supercoiled circular double-stranded DNA (the indigenous form of plasmid and
chromosomal DNA in many prokaryotes), somewhat lower for relaxed circular
double-stranded DNA, much lower for circular single-stranded DNA (indigenous
to most ssDNA viruses) and linear double-stranded DNA with homologous ends
(indigenous to eukaryotes), and lower still for linear double-stranded DNA with
non-homologous ends (Kimoto and Taketo 1996).
374 T. Kotnik
For DNA concentrations spanning from pg/ml up to μg/ml, efficiency of

electrotransformation in bacteria is roughly constant, implying that within this
range and under fixed experimental conditions, for each bacterium the probability
of being transformed increases roughly proportionally to the concentration of DNA
surrounding it. The optimal parameters of the electric pulses used to achieve
electrotransformation vary with bacterial species and even strain, but generally,
pulse amplitudes (electric fields) range from 2 to 30 kV/cm and pulse durations
from milliseconds to tens of milliseconds.
In eukaryotic cells, the efficiency of gene electrotransfer, particularly at low
DNA concentrations, is improved if the electroporating pulse is followed by a
contiguous, longer but much weaker pulse (tens or hundreds of milliseconds, tens
of V/cm) that exerts an electrophoretic drag on the DNA molecules, and while this
effect does not seem to have yet been investigated in bacteria or archaea, it is worth
noting that with the now prevailing rectangular-pulse generators, the electropho-
retic “tail” of the pulse has to be formed by appending a second pulse of lower
voltage, while with the simpler exponential-decay-pulse generators, it was inherent
to the pulse shape, which is also the case for natural discharges, including lightning
strokes.
Efficiency of electrotransformation in bacteria can also be improved by
hyperosmolarity of the medium, typically achieved by dissolving sorbitol or man-
nitol at 0.5–1.5 M concentrations; in nature, hyperosmolarity of aqueous media is
generally a consequence of high concentrations of salts, but the efficiency of
electrotransformation in high-salinity media does not seem to have yet been studied,
likely due to the fact that for lab transformation protocols, similarity to natural
conditions is less important than efficiency and practical feasibility. With respect
to the latter, unlike sorbitol or mannitol, added salts increase the electric conductivity
of the medium considerably; for a fixed electric field, as delivered by the prevalent
generators of pulses with a fixed voltage, this increases the electric current and the
heating of the medium, while for a fixed electric current, this reduces the electric
field induced by this current.
As described above, the highest efficiencies of electrotransformation in bacteria
are generally achieved with plasmid DNA. Among strains of the same species,
transfer can be efficient also with unaltered indigenous plasmids, but between
distant species, and particularly between phyla, the highest efficiency is usually
obtained with artificially engineered chimeric plasmids (shuttle vectors). The
principal aim of implementing gene transfer in bacteria is generally to achieve
the most efficient transformation possible; furthermore, if the transferred gene
originates from a eukaryote, using the most efficient form of DNA for transforma-
tion – plasmids – is only possible if this gene is inserted into a plasmid. As a
consequence, in most reports on electrotransformation in prokaryotes, the result is
obtained with pre-engineered plasmid DNA, and there has not been much motive
for a systematic investigation of feasibility of interspecies electrotransfer of
unaltered natural – either plasmid or chromosomal – DNA. Still, as Table 1
testifies, electrotransformation can in general occur in species of many archaeal
and bacterial phyla.
Irreversible Electroporation as a Mechanism of DNA Release
Perhaps the earliest reported use of high-voltage electric pulses for killing of
microorganisms dates back to 1896, when the Louisville Water Company studied
various methods of purifying river water (Fuller 1896). The first scientific study of
destruction of bacteria by irreversible membrane electroporation was published in
1967, showing that the lethal effect is nonthermal and results from extensive
membrane disruption and leakage of intracellular contents, including DNA (Hamil-
ton and Sale 1967). Since then, irreversible electroporation has become a well-
known method for nonthermal inactivation of microorganisms, as well as for
extraction of biomolecules, termed electroextraction (Kotnik et al. 2015).
Electroextraction of DNA has been demonstrated from both prokaryotes and
eukaryotes (particularly from microalgae and yeasts), but as a laboratory technique,
it was long considered inferior in efficiency to the standard DNA extraction method
of alkaline lysis with purification in CsCl-ethidium bromide density gradients. As
such, it was mostly used in those applications where low yields were acceptable, yet
the extraction had to be fast and with limited contamination by debris (DNA can also
be extracted rapidly by intense vortexing with glass microbeads or by intense
ultrasonication, but these techniques disintegrate the exposed microorganisms, so
the extract is a highly heterogeneous mix of very diverse biomolecules and
multimolecular fragments of the cell’s components). Still, the fact that irreversible
electroporation results in release of DNA – both chromosomal and plasmid – is
undisputable, and furthermore, even regarding the yields, it was recently shown that
with electroextraction, they can, at least for plasmid DNA and sufficient optimiza-
tion, be comparable or even superior to alkaline lysis (Haberl et al. 2013).
Lightning-Triggered Electrotransformation as a Contributor

to HGT in Microorganisms
Emergence of the Hypothesis and Tentative Empirical Support
Perhaps the first mention in scientific literature of the possibility that lightning
strokes could cause both DNA electroextraction (from microorganisms
electroporated irreversibly) and subsequent electrotransformation (of nearby micro-
organisms electroporated reversibly), thus resulting in HGT, occurred in 1990. In
July of that year, James Pfau and Philip Youderian from the University of Southern
California published a brief report showing that the standard approach to laboratory
electrotransformation, where alkaline lysis is used for DNA extraction and then
electroporation for transformation, can be simplified by mixing the DNA-donor and
DNA-recipient microorganisms and then applying electric pulses both for extraction
and transformation. They showed that a yield of transformants, albeit highly
suboptimal, is obtained even when a single electric pulse is applied to such a
mixture, and in the closing sentence of their report, they referred to the possibility
of natural gene transfer triggered by lightning strokes as their “speculation” (Pfau
376 T. Kotnik
and Youderian 1990). Five years later, a review of molecular evolution in bacteria
referred to lightning-driven HGT – again only in passing – as an “interesting
concept” (Trevors 1995). Six further years then passed until this topic was investi-
gated again by a group of researchers from the University of Lyon I in France
(Demanèche et al. 2001); by delivering pulses not through a discharge arc but
through electrodes in direct contact with the bacteria-containing sample, this study
did not differ much methodologically from earlier investigations and applications of
electrotransformation, but it was the first in which its authors declared as their goal to
investigate experimentally the feasibility of lightning-triggered HGT.
Still, at least four experimental studies published in 1990–1992, including that by
Pfau and Youderian already discussed, and one predating it by 4 months, can be
viewed as providing empirical support to the feasibility of lightning-triggered HGT –
at least to the extent achieved a decade later in the work of Demanèche and
coworkers cited in the preceding paragraph, i.e., with a single pulse applied to a
mix of DNA-donors and DNA-recipients, although with delivery of the pulse
without a discharge arc.
In the first such study submitted for publication in March 1990, David Summers
and Helen Withers from the University of Cambridge reported exposing a mix of two
Escherichia coli strains to a single pulse of 12 kV/cm and 4.6 ms, obtaining transfer
of plasmid DNA in about 1/106 exposed bacteria (Summers and Withers 1990).
Pfau and Youderian, in their abovementioned report written in July 1990, exposed
a mix of E. coli and Salmonella typhimurium – species belonging to the same family
(Enterobacteriaceae), but different genus (Escherichia, Salmonella) – to a single
pulse of 20 kV/cm and 5 ms, getting transfer of plasmid DNA in both directions: at
1/1500 from S.t. to E.c. and 1/500,000 from E.c. to S.t. (Pfau and Youderian
1990).
In June 1991, John Kilbane and Barbara Bielaga from the Institute of Gas
Technology in Chicago reported exposing a mix of E. coli and Pseudomonas
aeruginosa – same class (Gammaproteobacteria), different order/family (Enterobac-
teriales/Enterobacteriaceae, Pseudomonadales/Pseudomonadaceae) to a single pulse
of 12.5 kV/cm, obtaining detectable transfer of both plasmid and chromosomal DNA
in both directions (Kilbane and Bielaga 1991).
Finally, in May 1992 a group of researchers from the Pasteur Institute in France
reported exposing a mix of E. coli and Mycobacterium smegmatis – same kingdom
(Bacteria), different phylum/class (Proteobacteria/Gammaproteobacteria,
Actinobacteria/Actinobacteridae) to a single pulse of 12.5 kV/cm, getting transfer
of plasmid DNA in 1/106 exposed bacteria (Baulard et al. 1992).
The main aim of these studies was to assess the possibility of simplifying the
apparatus and streamlining the protocols for electrotransformation, and the single-
pulse approach was largely dismissed as too inefficient for practical applications;
namely, efficiencies achievable in single-pulse exposures were by at least 2–3 orders
of magnitude lower than if the DNA-donors were electroporated and the supernatant
transferred to the DNA-recipients that were then electroporated separately and by a
further order of magnitude inferior to the standard procedure of DNA extraction by
alkaline lysis and pulses only delivered to electroporate DNA-recipients. Still, these
studies can be viewed as providing tentative support for feasibility of lightning-

triggered HGT among prokaryotes, largely unaffected as the genetic distance
between them increases.
Theoretical Analysis
With cloud-to-ground lightning strokes, even after the air ionization is completed
and the electric current’s path through the air is fully established, the electrical
resistance of this path (typically several km long) dominates over the resistance of
the ground through which the electric current’s propagation then continues; as a
result, the magnitude of the lightning stroke’s electric current is largely independent
of the local composition of the ground it enters (be it highly resistive dry soil or sand,
moderately resistive freshwater, or highly conductive seawater). Limiting the anal-
ysis to aquatic habitats in which DNA diffuses the most easily, it is also reasonable to
assume the stroke’s electric current (I) spreads out roughly radially outward and
downward from its point of entry, so that the resulting electric current density (J) and
the electric field strength it induces (E) decrease roughly inversely proportionally to
the square of the distance (r) from this point (Kotnik 2013a):
I J I
J¼ ,E ¼ ¼ ; (1)
2πr 2 σ 2πσr 2
where σ denotes the electrical conductivity of the medium through which the current
is propagating. Denoting by Imax the peak current, we thus have
I max J max I max

J max ¼ 2
, Emax ¼ ¼ : (2)
2πr σ 2πσr 2
The electric field thus decreases radially in a continuous and monotonic manner,
so there is generally an innermost region where this field is sufficient for irreversible
membrane electroporation, causing DNA release, and adjacent to it outward is a
region where the field is insufficient for irreversible yet sufficient for reversible
electroporation, allowing for DNA uptake, expression, and heritability (Fig. 1).
Assuming for the lightning stroke the statistically determined median of its peak
current, Imax 30 kA, and for the medium the average electrical conductivity of
seawater, σ 40 mS/cm, an electric field exceeding 9 kV/cm (sufficient for
reversible electroporation of most microorganisms) is induced at radial distances
up to 3.6 cm; hence, in seawater hit by a typical lightning stroke, electroporation
can occur in a hemispherical volume of at least 100 cm3. Similarly, an electric field
exceeding 30 kV/cm (sufficient for irreversible electroporation of most microorgan-
isms) is induced at radial distances up to 2 cm, so that within the 100 cm3 where
electroporation can occur, in the inner 20 cm3 it is predominantly irreversible and
in the rest of the volume largely reversible. With 3 109 cloud-to-ground strokes
per year, and with 1% of them striking the seas, this corresponds to 2400 m3 of
seawater per year subjected to conditions for reversible electroporation, which at the
378 T. Kotnik
Fig. 1 Lightning-triggered HGT. Molecules of DNA – both chromosomal (red) and plasmid
(violet) – are released from organisms in the region of irreversible electroporation (yellow) and
plasmid DNA to a more limited extent also in the region of reversible electroporation (green). For
host organisms without natural competence, transformation is restricted to the region of reversible
electroporation, while naturally competent hosts can also be transformed by the released DNA in the
region without electroporation (blue). For both electroextraction and electrotransformation, DNA
has to traverse all layers of the organism’s envelope, pictured here as consisting of a plasma
membrane (gray) and a wall (orange), but in some organisms additionally comprising an outer
membrane and/or a capsule (Reprinted from Kotnik (2013a) with permission)
microorganisms’ concentrations typically exceeding 1011 per m3 of seawater (see

Table 1 in Whitman et al. 1998) implies that at least 1014 microorganisms per year
are subjected in seawater to conditions under which electrotransformation can occur.
In freshwater lakes, due to their much lower electrical conductivity, the volumes
subjected to irreversible and reversible electroporation at the same peak electric
current are about three orders of magnitude larger: assuming the average electrical
conductivity for freshwater lakes, σ 2 mS/cm, an electric field of 9 kV/cm is
exceeded in a volume of 9000 cm3 and 30 kV/cm in 1500 cm3. As 3 109
cloud-to-ground strokes (99% of all such strokes) strike continents and islands,
and freshwater lakes cover more than 0.5% of the total continents’ surface area,
assuming that lightning strokes are roughly uniformly distributed over the land as the
lakes, at least 1.5 107 cloud-to-ground strokes hit the lakes, with a volume of
106 m3 of freshwater per year subjected to conditions for reversible electropora-
tion, and with microorganisms’ concentrations typically exceeding 1011 per m3 also
in freshwater (see Table 1 in Whitman et al. 1998) implies that at least 1017
microorganisms per year are subjected in freshwater to conditions under which

electrotransformation can occur.
To summarize, these rough estimates suggest that at least 1017 microorganisms
per year are subjected to conditions suitable for lightning-triggered electrotrans-
formation, the majority of them in freshwater habitats (Kotnik and Weaver 2016).
Assessing the Feasibility and Importance of Lightning-

Triggered HGT
The quantitative estimates outlined above suggest that sufficiently close to the
surface of natural aquatic habitats, lightning-triggered HGT may well be feasible.
A comprehensive theoretical analysis of the dependence between the abundance of
microorganisms in those habitats (expressed, e.g., as their number per unit volume)
and the probability of DNA released from one of them by electroporation to come
into contact with another capable of its uptake and transformation due to either
reversible electroporation or natural competence would contribute significantly to
the quantitative understanding of this issue. Still, from the fact that transformation
by means of natural competence occurs in many aquatic bacteria it follows that at
least in some such habitats, bacteria are sufficiently abundant for DNA released
from one bacterium to come into contact with another bacterium in a manner
allowing this DNA to be taken up by this bacterium and transform it. Lightning-
triggered electroporation is one such mechanism of DNA release (irreversible
electroporation for both chromosomal and plasmid DNA, reversible electropora-
tion for plasmid DNA and to a more limited extent), and in regions with frequent
thunderstorms, it is perhaps also not insignificant compared to other natural causes
of bacterial death.
In addition, and perhaps of particular importance from the aspect of the early
evolution, a recent study has provided some convincing arguments why early life is
likely to have evolved in shallow ponds (Mulkidjanian et al. 2012), which, unlike the
deep-sea hydrothermal vents that are also considered a possible habitat of the earliest
organisms, are not accessible to lightning strokes.
The experimental studies employing a single electric pulse for both electroex-
traction of donor microorganisms and electrotransformation of recipient microor-
ganisms, discussed in section “Emergence of the Hypothesis and Tentative
Empirical Support,” can also be viewed as providing tentative support for feasibility
of lightning-triggered HGT among microorganisms, and the general applicability of
electrotransformation to a very broad range of microorganisms is apparent from
Table 1. Still, a rigorous and critical assessment leads to some clear misgivings
against treating these results as a proof-of-principle for feasibility of lightning-
triggered natural HGT, and based on these misgivings, the guidelines for a proper
(re)assessment of such feasibility can be formulated, as outlined in the following
subsections.
380 T. Kotnik
Delivery and Propagation of Electric Current
All currently commercially available electric pulse generators developed for

electrotransformation of microorganisms are designed as to deliver electric pulses
through electrodes in direct contact with the sample; this provides a well-controlled
and thus easily reproducible and optimizable exposure, but compared to the light-
ning strokes and arc discharges in general, such an exposure lacks the acoustic
shockwave and the ultraviolet light burst that accompany arc discharges. As the
results presented in Table 1 and section “Emergence of the Hypothesis and Tentative
Empirical Support” were all obtained with commercial generators, none featured an
arc discharge, but these accompanying phenomena can influence both the microor-
ganisms’ viability and their ability for DNA uptake. Thus, it was recently reported
that compared to electric pulses delivered in direct contact with the biological
sample, arc discharges result in a more efficient DNA uptake and expression
(Broderick et al. 2011) and also cause a more extensive release of intracellular
molecules (Boussetta et al. 2013), although it should be noted that both these studies
were performed on eukaryotic cells.
For a reliable assessment of lightning-triggered HGT, the electric pulses should
be delivered as an arc discharge, with the current entering the sample from above,
traversing an air gap separating the sample from the current-emitting electrode. To
achieve also radial propagation of the electric current from its point of entry, thus
emulating what occurs with lightning stroke’s current upon its entering the ground
(be it solid or liquid), the receiving electrode can be designed as a ring (for
two-dimensional radial propagation) or a hemispherical shell (for three-dimensional
radial propagation) surrounding the sample. A recently designed experimental
system for controlled delivery of arc discharges to biological samples is depicted
in Fig. 2.
Electric Pulse Waveform
The electric current of lightning strokes is characterized by a rapid rise to the peak
value (median zero-to-peak time of 5 μs) and subsequent exponential decrease
(median time constant of 100 μs or, equivalently, peak-to-half time of 70 μs).
Incidentally, the waveforms of pulses used in commercially available electric pulse
generators for electrotransformation are also exponentially decreasing, partly
because such pulses are the simplest to form, requiring only a discharge of a
precharged capacitor and perhaps also due to the inherent “tail” of such pulses that
exerts an electrophoretic drag on DNA, thus possibly improving the efficiency of
DNA uptake (see section “Reversible Electroporation as a Mechanism of DNA
Uptake and Transformation in Microorganisms”). Still, the generators used for
electrotransformation typically deliver electric pulses with a time constant of expo-
nential decrease in the range of 1–5 ms, while those of typical lightning strokes are,
as mentioned in the preceding paragraph, an order of magnitude shorter. As the
results presented in Table 1 and section “Emergence of the Hypothesis and Tentative
Fig. 2 An exposure system for emulating exposures of biological samples to lightning strokes. (a,
b) Major components in solid (a) and wireframe (b) representation: (1) base, (2) dock guide, (3)
sample loading dock, (4) receiving electrode connector, (5) transparent tube container, (6) emitting
electrode tip, (7) emitting electrode encasement, (8) core, (9) emitting electrode guide, (10) upper
stabilizer, (11) emitting electrode connector, (12) central cogwheel, (13) stepper motor with its
cogwheel, (14) stepper motor slot. (c) A conical emitting electrode. (d) A ring-shaped receiving
electrode (for two-dimensional radial propagation of the electric current). (e) A hemispherical-
bucket-shaped receiving electrode (for three-dimensional radial propagation of the electric current)
(Reprinted with permission from Marjanovič and Kotnik (2013); see this reference for further
images, including photographs of the whole actual system and its use in arc delivery into various
biological samples)
Empirical Support” were all obtained with commercial generators used for
electrotransformation, none delivered exponentially decreasing pulses with a time
constant below 1 ms, and this cannot be viewed as a proper empirical support for the
feasibility of lightning-triggered HGT. At least one study did report attaining
electrotransformants, in non-negligible yields, with a pulse of 100 μs duration,
but of a rectangular waveform (Grenier et al. 2008).
For a reliable assessment of lightning-triggered HGT, the electric pulse used for
electrotransformation should thus, in addition to its delivery as an arc discharge (see
section “Delivery and Propagation of Electric Current”), also resemble the time
course of the electric current – both its waveform and overall duration – of a typical
lightning stroke. As none of the contemporary electroporation-based technologies
and treatments utilizes such pulses, such an assessment calls for development of
custom-designed generators. While downscaling of the amplitude of the electric
current is almost unavoidable, it causes no essential loss of emulation consistency; as
the lightning stroke’s current always dissipates away from its point of entry into the
ground, a downscaling of its amplitude in exposure systems merely reduces the size
382 T. Kotnik
Fig. 3 Schematic diagram of the pulse generator for lightning emulation. U, grid power supply. T,
isolation transformer. B, bridge rectifier. HV DC–DC, high-voltage DC–DC converter. P, linear
potentiometer. R, resistors (200 kΩ each). C, capacitor (1 μF, 5 kV). S, high-voltage relay. V, digital
voltmeter. The 1 μF capacitance C was chosen for use with the system depicted in Fig. 2, for which
the impedance (including the biological sample in a 90-mm Petri dish) was measured at 100 Ω
(Reprinted with permission from Reberšek et al. (2015); see this reference for further images,
including photographs of the arc delivery into various biological samples and its effects as well as of
the short-circuiting artifact mentioned in the body text and the elimination of this artifact)
of concentric areas subjected to various ranges of current density and electric field
strength it induces. Certainly, the very highest current densities of lightning strokes
are absent in downscaled exposure systems, but those are lethal to all living
organisms and thus of no interest to investigation of HGT, while the electric field
sufficient for both irreversible and reversible electroporation – and thus for both
DNA electroextraction and electrotransformation – is consistently reached even with
downscaling of the lightning stroke’s current by a factor of 1000 (Kotnik 2013b).
A schematic diagram of a simple generator satisfying both the requirement of
electric current waveform resembling that of a lightning stroke (5 μs zero-to-peak
time, 100 μs time constant of exponential decrease) and the requirement of
delivery as an arc discharge is outlined in Fig. 3. A capacitor discharge circuit
generates an output current with a waveform very similar to those of lightning
strokes, while the output of the generator is isolated from the line voltage by an
isolation transformer both for safety reasons and to minimize the leakage current. A
high-voltage direct current converter supplies the output stage of the generator, while
the output voltage of the converter is set by a potentiometer and monitored by a
voltmeter. The two resistors separate the converter from the output, while the
capacitor stores the energy for the pulse during the charging phase, and the high-
voltage relay releases this energy into the load during the discharging phase. The
capacitance of the capacitor is chosen so that together with the impedance of the
exposure system to which the pulse generator is connected (including the biological
sample), the discharge generates an arc current with a time constant of exponential
decrease of 100 μs, similar to those of lightning strokes (e.g., if the impedance of
the exposure system is measured at 100 Ω, a capacitor of 1 μF should be used in the
generator).
A possible alternative to the relay used in this design would be an insulated-gate
bipolar transistor (IGBT), but these are generally more limited in their peak current
and its risetime and much more prone to irreparable damage. With exposure systems
such as the one depicted in Fig. 2, a problem encountered empirically is the gradual
lateral drift of the initially vertical arc discharge from the emitting electrode into the
sample, which for discharges exceeding 10 μs often results in short-circuiting
between the emitting and the receiving electrode, but this artifact can be eliminated
by incorporating an insulating cylinder concentrically between the emitting electrode
and the receiving electrode, so that it forms a tight contact with the surface of the
sample and thus precludes the discharge from evading the sample from above and
short-circuiting the electrodes (Reberšek et al. 2015).
Microorganisms, DNA, and Exposure Conditions
For a reliable assessment of lightning-triggered HGT, the microorganisms of primary

interest are those for which natural habitats are accessible to lightning strokes.
Similarly, the DNA used in such assessment, be it plasmid or chromosomal, should
be natural and devoid of artificial modifications often introduced in the laboratory to
improve transfer and/or expression or to prevent DNA degradation by the host;
successful lightning-triggered transformation with a shuttle vector or another type of
engineered or altered DNA molecule does not necessarily imply that natural plasmid
or chromosomal DNA can be transferred in the same manner.
The medium in which the microorganisms are exposed to electric pulses should
resemble their natural habitat, free of conditions and substances used to adjust
osmolarity, conductivity, microorganisms’ permeability and/or viability, as well as
DNA stability and/or transferrability. And in the same vein, also other experimental
conditions absent from natural environments, such as centrifugation and filtration,
should be avoided throughout all the relevant stages of the experiment – at least from
the start of the exposure to electric pulses to the evaluation of the resulting transfor-
mation and expression. The experimental studies in which the adequate choice of
microorganisms, DNA molecules (if not electroextracted), as well as the nature-
emulating medium and experimental conditions will all be respected are currently in
their initial stages, and most of the work still lies ahead.
Conclusions
The above considerations suggest that under contemporary conditions, per year, at
least 1017 microorganisms are subjected to lightning strokes that generate conditions
suitable for electrotransformation. To estimate the actual number of lightning-
induced electrotransformants per year in natural habitats, this number, or an
improved estimate thereof, would have to be multiplied by the transformation
efficiency at naturally occurring concentrations of environmental DNA suitable for
transformation, and to complicate matters further, these concentrations are combi-
nations of DNA released by the mechanism under consideration and DNA released
due to other causes of cell death. Clearly, uncertainties in such estimates are large,
perhaps spanning many orders of magnitude, and reliable answers will certainly
require both extensive and elaborate measurements and experiments.
384 T. Kotnik
Furthermore, such an assessment, if sufficiently perfected, may elucidate impor-

tance (or unimportance) of lightning-triggered HGT for evolution of current micro-
organisms under contemporary conditions, but it will not say much about the
potential role of this mechanism during early evolution, when the biotic mechanisms
had not yet evolved. For example, primordial microorganisms’ envelopes may have
differed considerably from modern ones in their intact permeability to nucleotides
and their polymers, and the lightning stroke rates were likely much higher during
periods with intense volcanic activity.
An even harder question to answer is that of the relative importance of lightning-
triggered HGT compared to biotic HGT; even the assertions on the relative impor-
tance of each biotic HGT mechanism still vary wildly, with some studies claiming
that competence, conjugation, and transduction explain virtually all-known HGT in
prokaryotes, and others positing that GTAs are the predominant mechanism of
HGT in oceans. Thus, perhaps the only aspect that the existing evidence suggests
rather clearly is that in the sense of attainable phylogenetic distance between the
DNA-donor and DNA-recipient microorganisms, electrotransformation in general
acts more broadly than any of the biotic mechanisms. Other than this, to assess
what fractions of the natural HGT are due to each individual mechanism, and
particularly which HGT mechanism dominated during the early evolution, will
likely require substantial progress in our knowledge of HGT – both biotic and
abiotic.
Acknowledgment This work was supported by the Slovenian Research Agency (Grant P2-0249)
and conducted in the scope of the European Laboratory of Pulsed Electric Fields Applications (LEA
EBAM) and within networking efforts of the COST Action TD1104 – European Network for
Development of Electroporation-Based Technologies and Treatments (EP4Bio2Med).
Cross-References
▶ Gene Delivery by Electroporation in Vitro: Mechanisms
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313x.2000.00808.x
Gene Delivery by Electroporation In Vitro:
Mechanisms 23
Marie-Pierre Rols
Abstract
Cell membranes can be transiently permeabilized by application of electric pulses.
This process, called electropermeabilization or electroporation, allows hydrophilic
molecules, such as anticancer drugs and nucleic acids, to enter into targeted cells
and tissues. The knowledge of the processes involved in membrane permeabi-
lization and in gene transfer is mandatory for this promising method to be effi-
ciently and safely used. The behavior of the membranes and the cells both while the
electric field is on and after its application has therefore to be addressed. The
description of the full mechanisms takes benefit from studies performed on differ-
ent biological models (lipid vesicles, cells in 2D and 3D culture) and from different
microscopy tools that allow to visualize the processes. Single-cell imaging exper-
iments revealed that the uptake of molecules (antitumor drugs, nucleic acids) takes
place in well-defined membrane regions and depends on their chemical and
physical properties (size, charge). Small molecules can freely cross the electroper-
meabilized membrane and have a free access to the cytoplasm. Heavier molecules,
such as plasmid DNA, face physical barriers (plasma membrane, cytoplasm
crowding, nuclear envelope) which engender a complex mechanism of transfer.
Gene electrotransfer indeed involves different steps, occurring over relatively large
time scales. As will be presented in this chapter, these steps include the initial
interaction with the electropermeabilized membrane, the crossing of the membrane,
the transport within the cell toward the nuclei, and finally gene expression.
Keywords
Electroporation • Electropermeabilization • Gene transfer • Gene expression •
Nucleic acids • Optical imaging
M.-P. Rols (*)


DOI 10.1007/978-3-319-32886-7_26
388 M.-P. Rols
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
Membrane Electropermeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
The Basics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
The Way to Conduct an Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
Kinetics of Membrane Permeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
Electrotransfer of DNA Molecules into Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392
What Is Known About the Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392
DNA/Membrane Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
Dynamic of the Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
DNA Transfer Through the Cytoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394
Passage Through the Nuclear Envelope and Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
Active Transport of DNA Aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
New Challenges to Increase Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
Lipid Vesicles and Spheroids as Other Models to Study Gene Electrotransfer . . . . . . . . . . . . 398
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
Introduction
Gene therapy is a treatment option for a number of diseases as inherited disorders and
cancer. Despite the fact that a lot of methods of vectorization have been developed
during the last decades, the technique has still to be improved to be both efficient and
safe (Verma and Weitzman 2005). Among the different approaches, electroporation
appears as the most promising one. This physical method can be efficiently used for
the targeted delivery of molecules in a wide range of cells and tissues (Yarmush
et al. 2014). Electroporation is nowadays a well-known technique of cell transfection
used in the laboratories. Vaccination and oncology gene therapy are major fields of
application of DNA electrotransfer in clinics (Lambricht et al. 2016; Sersa
et al. 2015). Translation of preclinical studies into clinical trials has started. The
first clinical trial of plasmid electroporation carried out in patients with metastatic
melanoma has shown hopeful results (Daud et al. 2008). The method has also been
successfully used for the treatment of companion animals. However, despite the fact
that the pioneering work on plasmid DNA electrotransfer in cells was initiated more
than 30 years ago (Neumann et al. 1982), many of the mechanisms underlying
membrane electropermeabilization and DNA electrotransfer remain to be elucidated.
Even if in vitro electrotransfer is efficient in almost all cell lines, in vivo gene
delivery and expression in tumors can be not as efficient as in the case of viral
vectorization. It is therefore mandatory, for increasing gene transfer and expression
while preserving safety, to increase knowledge about the mechanisms. This chapter
aims to describe the basic aspects of what is known about plasma membrane
electropermeabilization and associated gene delivery processes in cells and by
doing so to give some tips to perform experiments and optimize protocols.
23 Gene Delivery by Electroporation In Vitro: Mechanisms 389
Membrane Electropermeabilization
The Basics
Cells have a resting transmembrane potential which is uniform all along their plasma
membrane. Exposure of living cells to short and intense electric pulses induces
position-dependent changes of this transmembrane potential. Being dependent on
the angle between the electric field direction and the normal to the membrane, the
electric field effects are not uniform along the membrane. Maximum effects are
present at the poles of the cells facing the electrodes when the resulting transmem-
brane potential reaches a threshold value. Above this threshold, permeabilization of
the cell membrane occurs. Electropermeabilization of the plasma membrane is a
prerequisite for gene electrotransfer since nucleic acids are highly charged and large
molecules cannot enter cells.
The Way to Conduct an Experiment
Electropermeabilization can be performed in different manners depending on the way

cells are grown. For cells grown on Petri dish, culture medium can be removed and
replaced by a low ionic, iso-osmotic buffer. This pulsation buffer allows to limit the
Joule effect and therefore helps to preserve the cell viability. The composition of this
medium is generally a 10 mM phosphate buffer, 250 mM sucrose, and 1 mM MgCl2.
On a practical point of view, the bottom of the Petri dish can serve as an
electropulsation chamber. For cells in suspension, culture medium can be removed
by centrifugation and cells resuspended in the pulsing buffer and placed in purchased
cuvettes or in “home-made” chambers that can be easily obtained by placing the
electrodes on the bottom of the Petri dish. In such a configuration, cells placed between
the electrodes will be submitted to electric pulses; cells out of the electrodes will serve
as control cells (see Fig. 1). The electric pulses are delivered through a set of electrodes
connected to the pulse generator. In most experiments, square wave electric pulse
generators are used. Contrary to exponential decay generators, they allow the inde-
pendent control of the amplitude of the electric field pulses E and their duration T. This
is important specially for mammalian cells which have no cell wall and therefore are
more affected by electric pulses than bacteria and yeast. The electric pulse parameters
have to be carefully selected considering the characteristics of the cells in particular
their size and geometry. One key step to further ensure DNA electrotransfer and
expression is to determine the best electric conditions allowing both the permeabi-
lization of the plasma membrane and the preservation of the cell viability.
The use of video microscopy allows visualization of the permeabilization phe-
nomenon at the single-cell level. ▶ Chap. 68, “Fluorescent Indicators of Membrane
Permeabilization Due to Electroporation”, such as propidium iodide (PI), are very
convenient to detect the electrotransfer of molecules into the cytoplasm. They can
390 M.-P. Rols
Fig. 1 Tips for your experiments. Cells are pulsed on Petri dish or on cuvettes. Permeabilized and
viable cells are plotted to define the optimum conditions ((1) E < Ep or just above, no or poor
permeabilization; (2) E> > Ep, viability loss; (3) best values)
simply be added to the cells before application of the electric pulses. In aqueous
solution, their fluorescence is very low. When present into the cells, the fluorescence
quantum yield of PI increases in such a way that it is not necessary to remove the
solution to detect the molecule inside the cell. Once cells are placed on the stage of
the microscope and a region of interest is selected (where the electric field will be
applied, thanks to the electrodes connected to the pulse generator), the experiments
can start. The uptake of the fluorescent dye into the cells will be the signature of
membrane electropermeabilization. As explained in more details in other chapters of
this handbook and whatever the value of the pulses duration T, permeabilization only
appears above a threshold value of pulse intensity E, called Ep. Therefore, the first
experiment to perform consists of submitting the cells to increasing values of E and
determining the permeabilization efficiency (i.e., the percentage of cells that have
been electropermeabilized, cells which nuclei become fluorescent). For E < Ep,
which in the example of Fig. 1 is equal to 0.4 kV/cm, no permeabilization occurs.
Above E, increasing E leads to the progressive permeabilization of the whole cell
population that is obtained at 0.8 kV/cm. Then, the next step is the determination of
the cell viability. Cell viability is indeed affected when membranes are electroper-
meabilized due to different reasons including the release of intracellular compounds
such as ATP. For field values higher than 0.9 kV/cm, viability is affected. Once
obtained, such kind of results easily allows to define the best conditions for mem-
brane permeabilization and also for gene electrotransfer. In the example shown in
Fig. 1, the electric field values that can be used range from 0.6 to 1.0 kV/cm.
Kinetics of Membrane Permeabilization
Electropermeabilization of cells is a fast process that can be detected immediately

after the application of electric pulses. Usually, transport across the membrane is not
Fig. 2 The different steps involved in membrane electropermeabilization and gene

electrotransfer. (1) As directly visualized under a microscope a few seconds following pulses
delivery; (2) as drawn from 1. Left: Before pulse application, the membrane is a barrier that prevents
the passage of plasmid DNA (blue spots) toward the cell. Middle: During electric pulse application,
the plasma membrane is electropermeabilized facing the two electrodes and DNA aggregates are
formed. This interaction takes place only on the membrane facing the cathode. Right: 2 h after
electric pulse application, DNA molecules have begun to reach the nucleus and expression can be
detected
homogeneous on the whole cell membrane. It occurs at the sides of the cells
facing the electrodes in an asymmetrical way where it is more pronounced at the
anode-facing side of the cells than at the cathode (Fig. 2), i.e., in the hyperpolarized
area than in the depolarized area, which is in agreement with both theoretical
and experimental considerations as explained in other chapters of this Handbook
of Electroporation (▶ Chaps. 59, “Transmembrane Voltage Induced by Applied
Electric Fields”; ▶ 2, “Critical Electric Field and Transmembrane Voltage for
Lipid Pore Formation in Experiments”). Electropermeabilization can be described
as a three-step process in respect to electric field: (i) before electropulsation, the
plasma membrane acts as a physical barrier that prevents the free exchange of
hydrophilic molecules between the cell cytoplasm and external medium;
(ii) during electropulsation, when pulses parameters have been correctly defined,
E > Ep, the formation of transient permeable structures facing the electrodes
allows the exchange of molecules; propidium iodide is observed to rapidly access
the cell interior in the region of the cells facing the electrodes, mainly at the
anode-facing site; and (iii) after electropulsation, the membrane can stay perme-
able before resealing occurs (Golzio et al. 2002). Lifetime of permeabilization
can be assayed by adding the fluorescent dyes at various times following the
pulses. If the cell membrane is still permeable, then the cell will be fluorescent.
Resealing varies from a few seconds (when cells are put at 37 C just after
pulsation) to several hours (when cells are maintained on ice) according to the
experimental conditions (temperature and pulse parameters). However, one has to
take into account that viability can be affected since ATP release will occur. It is
therefore better to avoid to maintain the cells at low temperature after pulse
delivery.
392 M.-P. Rols
Whatever the molecules used to detect permeabilization (if they are small enough
and charged), a direct transfer into the cell cytoplasm is observed. When added after
electropulsation, molecules can still penetrate into the cells but less efficiently
because electric field acts on both the permeabilization of the membrane and on
the electrophoretic drag of the charged molecules from the bulk into the cytoplasm.
The electrotransfer mechanism involved is indeed specific for the physicochemical
properties of the molecule (Paganin et al. 2011).
Progress in the knowledge of the involved mechanisms, in particular in the
elucidation of membrane structures that are responsible for molecules transfer, is
still a biophysical challenge. Hydrophilic pores have been proposed to be created
and their formation confirmed by molecular dynamics modeling. But their existence
in permeabilized cells has still to be proven. Phospholipid scrambling and changes
on lateral mobility of proteins have been observed suggesting that part of the
membrane surface is occupied by defects or pores and that these structures propagate
rapidly over the cell surface (Escoffre et al. 2014). The fact that the entire cell surface
is affected was not so obvious since permeabilization is only induced in specific
regions of the cells. So, even if the entire mechanisms of membrane electropermea-
bilization/electroporation are not fully understood, and the existence of the exact
structures responsible for molecules uptake is still a debate, this physical method of
vectorization has become one of the most efficient for gene delivery.
Electrotransfer of DNA Molecules into Cells
What Is Known About the Process
The first electroporation-mediated gene transfer experiment was published more

than 30 years ago (Neumann et al. 1982). Translation to the clinic benefited from
increased knowledge of the mechanisms involved in the electrotransfer of nucleic
acids during the last three decades. As for electropermeabilization, single-cell studies
aided in describing the process of DNA electrotransfer.
In addition to membrane permeabilization, DNA electrotransfer is dependent on
DNA electrophoresis. The oligonucleotide must indeed be present during the pulse
to be later on transferred in the cytoplasm. The electrophoretic mobility of pDNA is
not dependent on its number of base pairs. Short pulses with high field strength can
be used but are less effective than long pulses with lower field strength. Therefore,
pulse parameters have to be determined to lead the membrane to be permeable
(E > Ep) while preserving as much as possible cell viability (above 30–50%).
Reporter genes (as the green fluorescent protein or β-galactosidase) are useful to
optimize the protocol. As for electropermeabilization, single-cell microscopy and
fluorescent plasmids can be used to visualize and determine the different steps of
electrotransfection. Plasmids can be labeled with fluorescent dyes (as toto, yoyo,
yopro) to allow visualization of its electrotransfer. DNA molecules, which are
negatively charged, migrate electrophoretically when submitted to the electric
field. Under electric fields which are too small to permeabilize the membrane (E < Ep),
the DNA simply flows around the membrane in the direction of the anode. Beyond
the critical field value, above which cell permeabilization occurs (E > Ep), the DNA
interacts with the plasma membrane.
DNA/Membrane Interaction
Interaction only occurs at the pole of the cell opposite the cathode, and this
demonstrates the importance of electrophoretic forces in the initial phase of the
DNA/membrane interaction. When the DNA/membrane interaction occurs, the
formation of “microdomains” whose dimensions lie between 0.1 and 0.5 μm is
observed (Fig. 2). Also seen are clusters or aggregates of DNA which grow during
the application of the field. However, once the field is cut, the growth of these
clusters stops. DNA electrotransfer can be described as a multistep process: the
negatively charged DNA migrates electrophoretically toward the plasma membrane
on the cathode side where it accumulates. This interaction, which is observed for
several minutes, lasts much longer than the duration of the electric field pulse.
Translocation of the plasmid from the plasma membrane to the cytoplasm and its
subsequent passage toward the nuclear envelope takes place with a kinetics ranging
from minutes to hours.
Dynamic of the Process
DNA/membrane interaction and as a direct consequence gene expression depend on

electric pulse polarity, repetition frequency, and duration. Both are affected by
reversing the polarity and by increasing the repetition frequency or the duration of
pulses. These observations revealed the existence of two classes of DNA/membrane
interaction: (i) a metastable DNA/membrane complex from which DNA can leave
and return to external medium and (ii) a stable DNA/membrane complex, where
DNA cannot be removed, even by applying electric pulses of reversed polarity. Only
DNA belonging to the second class leads to effective gene expression (Faurie
et al. 2010). Dynamics of membrane/complexes formation has been poorly under-
stood because direct observations have been limited to time scales that exceed
several seconds. However, experimental measurement of the transport of plasmid
DNA and propidium iodide with a temporal resolution of 2 ms has been performed,
thanks to the high speed and sensitive camera, and allowed the visualization of the
DNA/membrane interaction process during pulse application (Escoffre et al. 2011).
Plasmid complexes, or aggregates, start to form at distinct sites on the cell membrane
during the first pulse. Increasing the number of pulses does not lead to the creation of
new sites, but to the increase in the amount of DNA. The formation of plasmid
complexes at fixed sites suggested that membrane domains may be responsible for
DNA uptake, and their lack of mobility (as directly observed under the microscope
or quantify by fluorescence return after photobleaching (FRAP) measurements)
could be due to their interaction with the actin cytoskeleton. As will be described
394 M.-P. Rols
Table 1 Kinetics of the different steps involved in gene delivery

Time
scale Steps involved in DNA electromediated delivery References
μs Plasma membrane facing the electrodes is permeabilized Golzio et al. (2002)
ms Electrophoretic migration of DNA toward the membrane Escoffre
DNA/membrane complex formation et al. (2011)
Golzio et al. (2002)
s Conversion of the metastable form of the DNA/membrane Faurie et al. (2010)
complex to a stable one
min DNA translocation/diffusion across the membrane Escoffre
et al. (2011)
hour DNA migration toward the nucleus along the cytoskeleton Rosazza
et al. (2011; 2013)
day Gene expression Rosazza
et al. (2016)
later in this chapter, several publications reported evidences for the involvement of
cytoskeleton (Rosazza et al. 2011; 2013). The dynamics of the entire process is
reported in Table 1. If pulse delivery occurs in a relative short time scale (μs to ms),
the subsequent traffic of plasmid DNA occurs during the minutes and hours follow-
ing pulse delivery.
DNA Transfer Through the Cytoplasm
The process of plasmid transfer through the cellular cytoplasm to the nuclear
envelope is a complex process (Lechardeur and Lucaks 2006). In principle
micro-sized aggregates of DNA or vesicles filled with DNA could be too large to
pass through the pores formed by electroporation. However, individual DNA
molecules, while they can pass through electropores, have a limited mobility
within the cell and may well be totally degraded before reaching the nucleus. It
is possible and worth investigating the possibility that the actin cytoskeleton reacts
to the presence of DNA aggregates and plays an important role in the subsequent
intracellular transport. It seems reasonable that only aggregates beyond a certain
size (a few hundred nanometers) can induce a biological cellular response and can
be transported by the cell. In addition, the fact that the DNA is in aggregate form
means that the DNA in the center of the aggregate is relatively protected from
degradation. Therefore, for gene therapy purposes, it is optimal for DNA to enter
the cell as single molecules, but the subsequent transport toward the nucleus is, for
biological (possibly by inducing a response of the actin cytoskeleton) and physical
(diminishing enzymatic degradation) reasons, optimized if the DNA is in a micro-
sized aggregate form.
Even if the first stage of gene electrotransfection, i.e., migration of plasmid DNA
toward the electropermeabilized plasma membrane and its interaction with it,
becomes understood, guidelines to improve gene electrotransfer can not only result
from the way pulse parameters have been selected. Expression of the pDNA is
controlled by the viability of the pulsed population, and successful expression of the
plasmid depends on its subsequent migration into the cell. Therefore, the intracellu-
lar diffusional properties of plasmid DNA, as well as its metabolic instability and
nuclear translocation, represent cell-limiting factors that must be taken into account.
The cytoplasm is composed of a network of microfilament and microtubule systems,
along with a variety of subcellular organelles present in the cytosol. The mesh-like
structure of the cytoskeleton, the presence of organelles, and the high protein
concentration mean that there is a substantial molecular crowding in the cytoplasm
which hinders the diffusion of plasmid DNA. These apparently contradictory results
might be reconciled by the possibility of a disassembly of the cytoskeleton network
that may occur during electropermeabilization and are compatible with the idea that
the cytoplasm constitutes an important diffusional barrier to gene transfer. In the
conditions induced during electropermeabilization, the time a plasmid DNA takes to
reach the nuclei is significantly longer than the time needed for a small molecule
(hours compared to minutes). Therefore, plasmid DNA present in the cytosol after
being electrotransferred can be lost before reaching the nucleus, for example,
because of cell division. Finally, after the cytoskeleton, the nuclear envelope will
represent the last, but by no means the least important, obstacle for the expression of
the plasmid DNA.
Passage Through the Nuclear Envelope and Gene Expression
A high transport does not always result in a high level in expression. The relatively
large size of plasmid DNA (2–10 MDa) makes it unlikely that the nuclear entry
occurs by passive diffusion. Single-particle tracking (SPT) experiments of individual
DNA aggregates in living cells showed how electrotransferred DNA is transported in
the cytoplasm toward the nucleus. The modes of DNA aggregate motion in CHO
cells have been analyzed. Fast active transport of the DNA aggregates occurs over
long distances. Tracking experiments in cells treated with different drugs affecting
both the actin and the tubulin networks clearly demonstrate that this transport is
related to the cellular microtubule network (Fig. 3, Rosazza et al. 2016).
Active Transport of DNA Aggregates
Several studies point toward the contribution of endocytosis in the electrotransfer of

DNA, but more investigations have to be performed in order to understand what type(s)
of endocytosis would be involved. It is necessary to understand as well how electric
fields could stimulate such processes. Also notably, any endocytosis model would
only explain the internalization of large molecules as it does not support the
free membrane crossing of small molecules. It has therefore to be considered to
occur in parallel to another model valid for small molecule transmembrane
exchange. One model that could reconcile all the DNA internalization models
396 M.-P. Rols
– Clathrin Caveolin Adapter protein

Plasmid DNA
Myosin VI Dynein Trajectories
E
Myosin V Kinesin Expressed protein
+
2 Macropinocytosis
4
1 3 Clathrin-ME Caveolin/Raft-ME
Actin
filaments
Rab11
Rab5
Early endosome 5
Recycling endosome
Rab9
Lamp1
Late endosome
6 Lysosome
Microtubules 7
8
Nucleus
Fig. 3 Schematic representation of the mechanism of DNA electrotransfer. During the electric
pulses, (1) the plasma membrane is permeabilized, (2) DNA is electrophoretically pushed onto the
cell membrane, which results in (3) DNA/membrane interactions. After resealing of the membrane,
(4) DNA is internalized by endocytosis and other means where actin may take shape of bursts of
polymerization. (5) While being actively transported in the cytoplasm by actin and tubulin net-
works, DNA aggregates pass through the endosomal compartments. Free DNA interacts with
adapter protein in order to be transported by motor proteins. For gene expression to occur, (6)
DNA has to escape from endosomal compartments. Once in the perinuclear region, (7) DNA
crosses the nuclear envelope to be expressed and (8) yield proteins released. Reprinted with
permission from Rosazza et al., Current Gene Therapy 2016
would be that DNA accumulates where pores are formed and that its electrophoret-
ically driven insertion in the membrane pulls the pore and the plasma membrane
around. This would generate membrane curvature that could be recognized as an
emerging endocytic vesicle and induce a similar response from the cell as for an
endocytic process, with the recruitment of actin, clathrin, caveolin, dynamin, and
other endocytic regulators (Rosazza et al. 2016; Rems and Miklavcic 2016).
Electrotransferred DNA trajectories possess portions of active transport
interrupted by phases of nearly immobility (Rosazza et al. 2013). During the phases
of active transport, DNA aggregates featured a motion on average having a velocity
of 250 nm/s, persisting for 6 s and leading to a displacement of 1.3 μm. However, the
distributions were rather broad with velocities from 50 to 3400 nm/s, displacements
from 0.1 to 12 μm, and active transport durations from 2 to 30 s. These ranges are in
agreement with other types of intracellular particle dynamics as observed for viruses,
polyplexes, lipoplexes, receptors, endosomes, and mitochondria. Lower velocities
were shown to correspond to actin-associated transport. Indeed, after disruption of
the microtubules using the nocodazole drug, active transport of the DNA still
occurred, and the measured velocities were in the range expected for myosin motors
operating on actin – between 50 and 300 nm/s for myosin VI and between 250 and
500 nm/s for myosin V. In addition to motor-driven transport, actin-related move-
ment could be also due to bursts of actin polymerization which were reported to
drive viruses, bacteria, or endosomes from the plasma membrane to the cytosol with
mean velocities ranging from 50 to 600 nm/s.
New Challenges to Increase Gene Expression
As mentioned above, the dense latticework of the cytoskeleton impedes free diffu-
sion of DNA in the intracellular medium. Electrotransferred plasmid DNA,
containing specific sequences, could then use the microtubule network and its
associated motor proteins to move through the cytoplasm to the nucleus (Vaughan
and Dean 2006). Clear limits of efficient gene expression using electric pulses are
therefore due to, in addition to the passage of DNA molecules through the plasma
membrane, the cytoplasmic crowding and transfer through the nuclear envelope.
One of the key challenges for electromediated gene therapy is to pinpoint the rate-
limiting steps in this complex process and to find strategies to overcome these
obstacles. One of the possible strategies to enhance DNA uptake into cells is to
use short (10–300 ns) but high pulse (up to 300 kV/cm) induce effects that primarily
affect intracellular structures and functions. As the pulse duration is decreased,
below the plasma membrane charging time constant, plasma membrane effects
decrease and intracellular effects predominate. An idea, to improve transfection
success, is thus to perform classical membrane permeabilization allowing plasmid
DNA electrotransfer to the cell cytoplasm and then after, when DNA has reached the
398 M.-P. Rols
nuclear envelope, to specifically permeabilize the nuclei using these short strong
nanopulses. Thus, when used in conjunction with classical electropermeabilization,
nanopulses gave hope to increase gene expression (Beebe et al. 2003). However this
work was not yet replicated. Another idea is to combine electric pulses and ultra-
sound assisted with gas microbubbles. Although electroporation induced the forma-
tion of DNA aggregates into the cell membrane, sonoporation induced its direct
propulsion into the cytoplasm. Sonoporation can therefore improve the transfer of
electro-induced DNA aggregates by allowing its free and rapid entrance into the cells
(Delalande et al. 2013).
Lipid Vesicles and Spheroids as Other Models to Study Gene

Electrotransfer
Coming back to a mechanistic point of view and due to the complexity of the
composition of the plasma membrane, other experimental tools can be useful to
characterize the membrane domains observed during gene electrotransfer. For that
purpose, giant unilamellar vesicles (GUV) represent a convenient way to study
membrane properties such as lipid bilayer composition and membrane tension
(Riske and Dimova 2005). They offer the possibility to study and visualize membrane
processes due to their cell-like size in the absence of any constraint due to cell
cytoskeleton. They can be obtained by simple methods such as electroformation,
and their composition can be very simple (one type of phospholipids) or more complex
(several lipids including cholesterol). Experiments showed a decrease in vesicle radius
which was observed as being due to lipid loss during the permeabilization process.
Three mechanisms responsible for lipid loss were directly observed: pore formation,
vesicle formation, and tubule formation, which may be involved in molecule uptake.
However, no interaction between plasmid DNA and the GUV membrane could be
observed; a direct transfer of DNA into the GUVs took place during application of the
electric pulses (Portet et al. 2011). That gives clear evidence that “lipid bubble” is not
always relevant as a cell and a tissue is not a simple assembly of single cells.
Therefore, it is necessary to develop and use different models, from simple lipid
vesicles to tumor multicellular tumor spheroids more closed to the in vivo situation,
for the understanding of the membrane permeabilization and DNA electrotransfer
process in tissues. Each of these models has advantages and limits. Together combined
they can help in the study of the full processes (Table 2).
Even if the high majority of studies underlying molecule transfer by electric fields
have been performed on 2D cell culture in Petri dish or in cells cultured in suspension,
3D multicellular spheroids represent a nice, relevant, cheap, easy-to-handle in vitro
model. Upon growth, spheroids display a gradient of proliferating cells. These prolif-
erating cells are located in the outer cell layers and the quiescent cells are located more
centrally. This cell heterogeneity is similar to that found in avascular micro-regions of
tumors (Sutherland 1988). Confocal microscopy allowed to visualize the repartition of
permeabilized cells in spheroids submitted to electric pulses. Results revealed that cells
were efficiently permeabilized, whatever their localization in the spheroid, even those
Table 2 What models can address about electropermeabilization and gene delivery processes
Model Membrane permeabilization DNA electrotransfer
GUV Direct visualization of membrane Failed to address DNA/membrane
permeabilization and its consequences interaction (DNA is directly transferred
(deformation, lipid loss) inside the vesicle)
2D Kinetics of permeabilization and its Visualization of DNA/membrane
Cell consequences (lateral and transverse complex formation and access to DNA
culture mobility of lipids and proteins) traffic into the cells
3D Molecule diffusion and transfer that Allow to address DNA delivery in 3D
Cell mimic in vivo complex situation (contacts and mimic what happens in vivo
culture between cells, junctions, and extracellular (decrease in gene expression from the
matrix) periphery to the core)
in the core, mimicking previously observed in vivo situations. Propidium iodide

uptake was observed to be present but spatially heterogeneous within the 3D
multicellular spheroid after electroporation, with a progressive decrease from periph-
eral to interior cells. In the case of large molecules as plasmid DNA, spheroids allowed
showing that electrophoresis, and not tissue deformation or electroosmosis, is the
driving force for interstitial transport. In addition, and at the opposite of cells in 2D
cultures, only cells on one side of the outer leaflet expressed the reporter gene (Gibot
and Rols 2013). This low expression is in fair agreement with in vivo experiments on
tumors. Close contacts between cells and extracellular matrix (ECM) may act as
physical barrier that limit/prevent (uniform) DNA distribution and explain the absence
of gene expression in the inner region of spheroid. The limited access of plasmid DNA
to the central region of spheroid remains a significant barrier to efficient gene delivery
in tissues. Taken together, these results, in agreement with the ones obtained by the
group of R. Heller (Marrero and Heller 2012), indicate that the spheroid model is more
relevant to an in vivo situation than cells cultured as monolayers and therefore can be
useful to address the mechanisms of DNA electrotransfer. In order to assess the effects
of the extracellular matrix composition and organization, as well as intercellular
junctions and communication, other 3D reconstructed human connective tissue
model can be used. Cell sheets, reconstructed in vitro by a tissue engineering
approach, presents multiple layers of primary dermal fibroblasts embedded in a native,
collagen-rich extracellular matrix (ECM) and can be a useful tool to study skin DNA
electrotransfer mechanisms. Cells within this standardized 3D tissue can be efficiently
electropermeabilized by millisecond electric pulses. A better comprehension of gene
electrotransfer in such a model tissue would help improve electrogene therapy
approaches such as the systemic delivery of therapeutic proteins and DNA vaccination.
Conclusions
Classical theories of electropermeabilization present some limits to give a full

description of the transport of molecules through membranes. Certain effects of
the electric field parameters on membrane permeabilization, and the associated
400 M.-P. Rols
transport of molecules, are well established, but a great deal of what happens at the
molecular level remains speculative. Molecular Models of Lipid Bilayers and
Electropore Formation are giving interesting new insight into the process. Electro-
induced destabilization of the membrane includes both lateral and transverse redis-
tribution of lipids and proteins, leading to mechanical and electrical modifications
which are not yet fully understood. One may suggest that such modifications, which
may vary according to the microenvironment, can be involved in the subsequent
transport of molecules interacting with them such as the DNA molecules. Experi-
mental verification of the basic mechanisms leading to the electropermeabilization
and other changes in the membrane, cells, and tissues remains a priority given the
importance of these phenomena for processes in cell biology and in medical appli-
cations. In vivo gene electrotransfer (▶ Chap. 81, “Principles of Electroporation for
Gene Therapy”) will face other challenges such as the necessity to control electric
field distribution and gene expression both in space (targeted DNA delivery to the
cells) and in time. Guidelines for successful DNA delivery are still required but we
can be optimistic that further working to improve gene electrotransfer mechanisms
will yield effective treatments.
Acknowledgment This research was performed in the scope of the EBAM European Associated
Laboratory (LEA) and is a result of networking efforts within COST TD1104. It was supported by
the Centre National de la Recherche Scientifique (CNRS), the Agence Nationale de la Recherche
(ANR), Projet PIERGEN ANR-12-ASTR-0039, and the Direction Générale de l’Armement (DGA).
Cross-References
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Electroporation of Biofilms
24
Flavien Pillet
Abstract
The extraordinary resistance of bacterial biofilms against the antimicrobials and
human immune system raises a grave issue in the medical field. This resistance is
due to the confinement of bacteria into a highly dense extracellular matrix. This
matrix serves as a filter and increases the concentration needed to kill bacteria
from 100 to 1000 times, in comparison with free-floated bacteria (planktonic).
Within context, the development of new therapeutic strategies is required. To
achieve this aim, the pulsed electric fields (PEFs) are a promising technology. The
purpose of this chapter is to introduce the complexity of the biofilms and the
phenomenon involved in their formation, maturation, and proliferation. The
mechanisms of adhesion and the roles of extracellular matrix are specifically
discussed. Then, the chapter considers two different strategies which use the PEF
technology to inactivate the biofilms. The first approach is direct disinfection of
biofilms by PEF. In this case, strong electric fields are applied during a short time.
The aim is to induce a bacterial inactivation by irreversible damages of the cell
envelope by the production of reactive oxygen species. This method is efficient to
prevent in vivo, the biofilm formation on burned wound. The second strategy is to
expose biofilm with low electric fields during a long period and in combination
with antimicrobials. This technique is called the bioelectric effect and allows
removing of the antibiotic resistance of biofilms. The mechanism is not fully
understood, but the observed effect is probably due to mass transfer electropho-
resis and disruption of the extracellular matrix.
Keywords
Bacterial biofilms • Extracellular matrix • PEF disinfection • Bioelectric effects
F. Pillet (*)
CNRS; IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France
Université de Toulouse; UPS; IPBS, Toulouse, France
e-mail: flavien.pillet@ipbs.fr; pillet@ipbs.fr

DOI 10.1007/978-3-319-32886-7_27
404 F. Pillet
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404
The Biofilms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404
The Biofilm Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
Membrane Permeabilization of Planktonic Bacteria by Pulsed Electric Fields . . . . . . . . . . . . . . . . 407
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407
Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408
Pulsed Electric Field Disinfection of Biofilms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
Applications In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
Applications In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
Bioelectric Effect on Biofilms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
Introduction
Pulsed electric fields (PEFs) are now currently used in cancer therapy to induce a
reversible permeabilization of the plasma membrane in cancer cells and induced an
entrance of cytotoxic molecules. This treatment is named the electrochemotherapy,
and until now, thousands of patients were treated in Europe and the USA. In the food
industry, PEFs are employed for the sterilization of pathogenic bacteria, obtained by
the irreversible permeabilization of the plasma membrane and the cell-wall disrup-
tion. Interestingly, PEFs are not still exploited in medicine to inactivate the bacterial
biofilm. Through a few significant examples, the aim of this chapter is to investigate
the feasibility of PEF to treat the biofilms.
The Biofilms
Background
Biofilms are one of the most primitive living form and existing for more than 3.2
billion years. In the classical definition, a biofilm is a community of microorganisms
densely self-organized in an extracellular matrix and adhered to an inert or biological
support. Most of microorganisms (bacteria, yeasts, and algae) are able to form
biofilms and can be found in all environments, even the most hostile, like in extreme
conditions of temperatures, pH, or in nutrient-poor medium.
This high resistance raises serious problems in the industrial sector, the food
industry, and the medical field. In the industrial field, biofilms can be involved in
biofouling and biodegradation of metals on pipelines and ships (Schultz et al. 2011).
24 Electroporation of Biofilms 405
In some instances, the biofilms may be highly pathogenic for human. For example,
the Legionella pneumophila colonization in water systems (domestic water supply,
cooling towers) causes legionellosis, a fatal respiratory disease. In the food industry,
the biofilms promote survival and growth of pathogenic bacteria on food contact
surfaces, such as Listeria monocytogenes, the causative agent of listeriosis, a serious
infection lethal in 20–30% of cases. In the medical field, biofilms are responsible of
80% of hospital-acquired infections associated with the implantation of medical
device (e.g., catheter, stents, orthopedic instrumentation, prosthetic heart valves,
pacemakers). Staphylococcus epidermidis and S. aureus are the most frequent
origins of these nosocomial infections, and the human and economic cost is consid-
erable. For instance, S. epidermidis causes each year in the USA 100,000 deaths and
a cost of $45 billion (Harris et al. 2016). In addition, biofilms may also be involved in
chronic lung diseases. Biofilms of Pseudomonas aeruginosa can be found in patients
infected by the cystic fibrosis, and the presence of Mycobacterium tuberculosis
biofilms is suspected in the case of tuberculosis. These few examples are illustrative
of the scale of problem of biofilms in various sectors. The eradication of the biofilms
is therefore a critical challenge. In the area of medicine, the highly antibiotic
resistance of biofilms is the main obstacle to their eradication. This resistance is
due to the secretion of extracellular matrix which acts as a filter. Therefore, the
bacteria within biofilms are 100–1,000 times more resistant to antimicrobial agents
than bacteria in suspension (Ceri et al. 1999). Consequently, the concentration of
antibiotics required is far too high to be used correctly without risk to the patients.
An alternative is to remove the infected material, but the surgical operation may be
dangerous for the patient, and the exposure to new infection is high. For chronic
diseases, like the cystic fibrosis, the ablation of infected tissues is clearly not an
option thus no treatment is available.
The Biofilm Life Cycle
Biofilm formation starts with the attachment of free bacteria in suspension (plank-
tonic) to a biotic or abiotic surface (Fig. 1). According to the adhesion forces
between the cells and the surface, the attachment can be reversible or irreversible.
In this latter case, bacteria forms microcolonies and the production of the extracel-
lular matrix is initiated. During the maturation step, the division of bacteria takes
place within the biofilm community protected against environment by the matrix.
The proliferation step is characterized by the detachment of planktonic bacteria and
biofilm fragments, leading to colonization of new surfaces and/or a systemic
infection.
The adhesion of planktonic bacteria is a complex process that may vary greatly,
depending to bacterial species and surface. Bacterial adhesion is mediated by a
large diversity of molecular interactions, according to the support. In the initial step
(from seconds to minutes), the attachment of bacteria is facilitated by nonspecific
interactions (van der Waals forces, electrostatic forces, and hydrophobicity). For
example, several proteins contribute to the hydrophobic properties of bacteria, like
406 F. Pillet
Fig. 1 Steps in biofilm formation
AtlE and Bap in S. epidermidis. However, these nonspecific interactions are not the
primary driving force toward adhesion, and specific interactions are required to
initiate the formation of biofilms. The second step (from minutes to hours) of
adhesion is due to a multitude of molecular recognitions between receptors and
ligands. To name a few, the family of cell-anchored proteins in S. epidermidis are
able to recognize specific molecules on the surface targeted, like the protein Sbp
which binds the abiotic surfaces and epithelial cells or the protein SdrF which
interacts specifically with the human collagen of type I. In addition, the bacterial
appendages (pili, fimbriae) are also involved in specific interactions with the
surface to be colonized.
During the maturation (few hours to few days), the extracellular matrix is secreted
(Flemming and Wingender 2010). Also known as extracellular polymeric substances
(EPS), the matrix is a three-dimensional heterogeneous network mostly composed of
proteins, carbohydrates, lipids, and extracellular DNA (eDNA). In an infectious
context, the main role of the matrix is to induce a high resistance to antimicrobials
and immune system. However, the matrix is involved in other phenomena required
for the growth and the development of biofilms. The extracellular matrix is crucial to
maintain the cell adhesion, the cohesion, the microbial attachment on the surface and
provide the mechanical stability of the biofilm. Furthermore, the matrix allows the
retention and the accumulation of nutrients and prevents the desiccation. In few
cases the biofilms’ matrix operates as an external digestive system by enzymatic
activity. Last but not least, the matrix is primordial for bacterial communication
inside the biofilm. The most remarkable phenomenon is the quorum sensing; this
process is defined as the range of mechanisms of stimuli and the response to control
and coordinate the gene expression between bacteria, according to the cell density.
An outstanding example is the bacterial programmed cell death observed in biofilms
of Escherichia coli. This phenotypic manifestation of self-destruction of bacteria
allows the production of extracellular matrix and nutrients to sibling’s bacteria.
Another particularity of matrix is also related to improve the horizontal gene transfer
between bacteria, leading to acquisition of new antibiotic resistances.
The proliferation is the last step required to the biofilm life cycle and the systemic
infection. This process is the result of a balance between the biofilm growth and the
release of free bacteria, under the regulation of gene expression. However, these
mechanisms are not well understood yet, and only few examples are described in the
literature. In Staphylococcus family, the upregulation of Agr gene increases the
persistence and the virulence of the biofilm. By contrast, the downregulation of
this gene induced the dissemination of planktonic bacteria.
As a conclusion, the microbial biofilms are communities of bacteria highly
organized and dynamic, whose growth and survival depend on a complex machin-
ery, involving the bacterial adhesion, the biofilm maturation including the secretion
of extracellular matrix, and eventually the bacterial proliferation. For decades, the
research has been focused on the comprehension of these mechanisms, but no
effective treatments have been yet found, and the eradication of biofilm infections
in human is currently a huge challenge. Within context, new therapeutic approaches
must be found.
Membrane Permeabilization of Planktonic Bacteria by Pulsed

Electric Fields
Background
The first example of bacterial inactivation by electric field was described by Fuller in
1898 for the purification of river water. Then, the first industrial application was
reported in 1919, with the Electro-Pure Process device developed by Anderson and
Finkelstein for pasteurization of milk by ohmic heating. In 1967, Sale and Hamilton
observed a nonthermal inactivation by pulsed electric field (PEF) on bacteria by
application of micropulses at 25 kV/cm. This surprising observation led the authors
to formulate the hypothesis of irreversible PEF permeabilization of bacteria, as a
consequence of the alteration of the membrane by electrical breakdown. This
hypothesis was improved by Neumann and Rosenheck in 1972 with the demonstra-
tion of ATP leakage from vesicles permeabilized by PEF (Neumann and Rosenheck
1972). Today, this versatile method is used in many domains as diverse as cancer
treatment (Rols et al. 1998; Marty et al. 2006) (▶ Part VIII, “Biomedical Applica-
tions of Electroporation”) or food industry (Saldaña et al. 2014) (▶ Chap. 134,
“Pulsed Electric Field Treatment for Fruit and Vegetable Processing”).
Principle
Membrane electropermeabilization, also called electroporation, is a nonthermal

physical process based on the application of pulsed electric fields to induce a
controlled permeabilization of the plasma membrane of eukaryote cells and
prokaryotes.
408 F. Pillet
Membrane permeabilization is directly correlated with transmembrane voltage

(Escoffre et al. 2009) (▶ Chap. 59, “Transmembrane Voltage Induced by Applied
Electric Fields”). In the absence of electric field, the resting transmembrane potential
(ΔΨ0) is homogenous on the cell. In the presence of electric field, the induced
transmembrane potential (ΔΨE) depends on the cell shape (f), the medium and cell
conductivity (g), the cell radius (r), the intensity of electric field (E), the orientation
of the cosθ angle, the duration of exposure (t), and the charging time of the plasma
membrane (τ). When the sum in absolute value of ΔΨ0 + ΔΨE is above 200 mV, the
plasma membrane is permeabilized. In this case, there is no permeabilization on the
membrane exposed perpendicularly to the electric field. By contrast, there is a
maximum permeabilization on the membrane exposed in parallel to the electric
field. Furthermore, according to the electrical parameters applied (number of pulses,
electric field intensity, frequency, etc.), the permeabilization is reversible or
irreversible.
However, the cell membrane permeabilization is not only the only phenomenon
observed during PEF exposure. In a recent study, a cell-wall disorganization was
mentioned after PEF in Bacillus pumilus (Pillet et al. 2016). Morphological and
mechanical damages as well as a loss of hydrophobicity were observed at the nanoscale.
Applications
The reversible permeabilization is not lethal for bacteria and allows the transfer of
biomolecules, like the nucleic acids. This technique, also called electrotrans-
formation, is classically used in molecular biology to transfer exogenous DNA
into bacteria. The electric field-mediated transfer is a two-step process, with a fast
anchored of DNA during PEF exposure, pursued by a slow crossing of the plasma
membrane. Furthermore, it has been proved in E. coli that this mechanism is
accompanied by a bacterial orientation parallel to the field lines and a considerable
cytoplasm ion leakage (Eynard et al. 1998).
The irreversible permeabilization leads to the death of bacteria exposed. There is
a wide range of applications. First, the PEFs are currently used in the food industry to
inactivate pathogenic bacteria. There are many advantages compared to thermic
sterilization: the energetic cost is lower and the preservation of nutrients is better.
Indeed, the absence of thermal increase prevents the Maillard reactions which are
responsible for the carbohydrate and protein degradation during cooking. For exam-
ple, orange juices sterilized by PEF are commercialized in many countries, like in the
Netherlands (McHugh and Toepfl 2016). Second, the irreversible permeabilization
of the cell membrane improves the entrance of antimicrobials molecules. In Bacillus
cereus, the PEF exposure facilitates the entry of nisin and increases by 60 times the
inactivation rate. Third, PEF may be used for the electroextraction of molecules
present in the cytoplasm, like the proteins.
To conclude, the electropermeabilization of planktonic bacteria by PEF has
developed substantially over the past decades, particularly in molecular biology
Fig. 2 Electropermeabilization principle
and food industry. By contrast, the inactivation of bacteria by PEF is still not used in
a medical context. This is the consequence of two major reasons. The first is that the
cell radius of eukaryotic cells is larger than the bacteria, resulting in a higher
sensitivity to PEF (Cf. the equation in Fig. 2). In this context, the PEF treatment to
kill bacteria in vivo may be dangerous for the patient. The second reason is that there
is still a lack of studies to understand the effects of PEF exposure on bacteria in a
three-dimensional conformation, like biofilms.
The aim of this chapter is to give an overview of the promising studies which
described the PEF to prevent and kill bacterial biofilms. This chapter will be focused
on two strategies: (i) direct disinfection of biofilms by irreversible permeabilization
of the bacterial cell envelope and (ii) the bioelectric effect to enhance antimicrobial
penetration within the biofilms.
Pulsed Electric Field Disinfection of Biofilms
Principle
PEF disinfection of biofilms is based on the bacterial inactivation induced by

the exposure to moderate or strong electric fields (between 1.5 and 5 kV/cm),
with a wide range of pulse duration and frequency. The mechanism of inactivation
is not fully understood, but probably due to irreversible permeabilization of the
bacterial cell envelope and/or production of reactive oxygen species (ROS). This
part focuses on the few studies on biofilm eradication in two contexts, in vitro and
in vivo.
410 F. Pillet
Applications In Vitro
One of the most common problems of biofilms is their ability to induce the
accumulation of microorganisms and biological material on wetted surface. This
phenomenon is called the biofouling and concerns various domains in industry and
medicine. A previously work described the effects of pulsed electric fields on
biofouling, particularly on the development and inhibition of Pseudomonas
aeruginosa biofilms (Perez-Roa et al. 2006). The authors used interdigitated
electrodes with 29 μm spacing between 22 μm wide electrodes and a voltage
between 0.5 and 5 V. These are approximately electric fields from 200 V/cm to
2 kV/cm. Furthermore, alternating current, square-wave pulses were applied. The
effects of applied voltage, the frequency, and the pulsing duty ratio (percentage of
pulsing time during PEF exposure) were evaluated on the inhibition of biofilm
formation. The best result was obtained with a combination of short pulses and
high voltages. A 50% reduction of the electrode area covered by the biofilm was
measured at 2 kV/cm, 200 Hz, and 1% of duty ratio (i.e., pulses of 50 μs).
However, the mechanisms involved are still unknown, and several reasons may
be considered: the disruption of the cellular adhesion process, the electrolyte
formation, and biocidal effects of the electric fields. This promising study demon-
strates the efficiency of PEF to prevent the colonization of bacteria, leading to
biofilm formation and biofouling.
On the other hand, the eradication of formed biofilm is also a huge challenge.
Indeed, in the human body, the majority of bacterial infections are caused by
biofilms. Unfortunately, the inactivation of biofilm is further complicated by their
resistance to antimicrobials. A recurrent issue is the mesh infection by biofilm. This
is a classical pathology which may occur after a ventral hernia repairs and represents
a morbidity in 4–16% of patients. In a recent study, the PEF effect on eradication of
biofilm-infected mesh was investigated (Khan et al. 2016). For this, the viability of
P. aeruginosa on mesh was evaluated after PEF by bioluminescent imaging. The
biofilm disruption on mesh was visualized by scanning electron microscopy (SEM).
A concentric electrode system was used to apply various PEFs at different electric
field strengths and numbers of pulses. The frequency was fixed at 2 Hz and the pulse
duration at 50 ms. In the area treated, the conditions required to inactivate 100–80%
of bacteria were 300 pulses at 1.2 kV/cm or 150 pulses at 2.3 kV/cm. Furthermore,
the inactivation area between the electrodes was proportional to the number of pulses
delivered. This work is encouraging to develop a tailored device to treat infected
mesh in human.
Applications In Vivo
The PEF technology is already used in medicine, as a nonthermal process to destroy

solid tumors by irreversible permeabilization (Jiang et al. 2015) ▶ Chap. 35, “Effects
of Reversible and Irreversible Electroporation on Endothelial Cells and Tissue Blood
Flow”). Recently few studies applied the PEF to eradicate, in mice model, bacteria
Fig. 3 PEF disinfection in vivo of a burned wound. A third-degree burn area was inflicted on the
mouse skin. Immediately after injury, the infection was initiated with multidrug-resistant bacteria.
Next, the PEF was applied on the wound infected, using two-plate electrodes (Golberg et al. 2014,
2015)
deposited on burned wound (Golberg et al. 2014, 2015). The principle is summa-
rized in Fig. 3.
The authors describe the inactivation of Acinetobacter baumannii, a bacteria
involved in hospital-acquired infections on the lung, wound, and burn. The infected
areas were treated 30 min after the infection, with pulses of 70 μs at 5 kV/cm and a
frequency of 1 Hz. The viability of bacteria on the skin treated was evaluated by
bioluminescence. The inactivation rate is correlated with the number of pulses. After
40 pulses, 2.0 log10 of inactivation was measured, whereas 80 pulses led to 5.5
log10 of inactivation. For 80 pulses, the inactivation rate was almost identical after
3 h, with 4.9 log10. It shows the long-term efficacy of PEF to inactivate bacteria on
burned wound. These results are interesting and suggested a bright alternative to
prevent the biofilm formation in burned wound. Further experiments should be done
to evaluate the efficiency of this method to kill already well-established biofilm
in vivo.
Bioelectric Effect on Biofilms
Principle
The main restriction for the biofilm inactivation in vivo is their high resistance to
antibiotics and immune system. This resistance is correlated with the presence of
extracellular matrix which prevents the entrance of antibiotics and biocides into the
biofilm. Within context, an interesting method, named the bioelectric effect, is
discussed in this part. It consists to exposition during long periods (few hours to
several days) of low electric fields (less than 10 V/cm), in combination with
antimicrobials. The treatment induced phenomenon of electrophoresis results in a
412 F. Pillet
Fig. 4 Principle of bioelectric effects. The drug delivered into the biofilm is facilitated by the
disruption of the extracellular matrix due to PEF electrophoresis. The black sticks show the bacteria
and the red dots indicate the antimicrobials
disruption of the extracellular matrix and increases the antimicrobial concentration

delivered into the biofilms. Thus, this method improves the antibiotic efficiency
against bacteria packed in biofilm. The principle is described in Fig. 4.
Mechanism
The electrical enhancement of efficacy of antimicrobials to eradicate biofilm bacteria

was described for the first time in 1992 by the Costerton team (Khoury et al. 1992).
The aim was to enhance the antimicrobial penetration into the biofilm, in order to
reduce the antibiotic concentration required to kill bacteria. For this, the authors
produced biofilms of S. epidermidis and P. aeruginosa with a homemade device
composed by a central flow channel, a peristaltic pump, and stainless steel studs
considered as electrodes. The biofilms were exposed until 12 h to an electric field of
1.5 V/cm with a current density of 15 μA/cm2. To limit the reactive oxygen species
production, the polarity was inverted each 64 s. The bacterial inactivation rate was
calculated by colony counting at different exposure times. The combination of low
PEF with an antibiotic (tobramycin) was measured for each bacterial strain tested.
For S. epidermidis, the exposure to 2.5 mg/ml of tobramycin during 12 h does not
lead to significant bacterial inactivation. By contrast, when the tobramycin was
combined with the PEF exposure, all bacteria were inactivated after 8 h. Similarly,
with P. aeruginosa, the presence of 8 mg/ml induced a loss of viability around
1 log10 after 12 h, whereas this viability decreased by almost 7 log10 with antibiotic
within electric field. These results shown that the low voltage fields can be employed
to decrease the necessary inhibitory concentration of antibiotics in biofilms. How-
ever, the effects of PEF alone versus the bioelectric effect should be evaluated. To
answer, the authors made a complementary study on P. aeruginosa (Costerton et al.
1994). They compared during 48 h the viability of biofilm bacteria exposed to
electric fields, in the absence or presence of tobramycin. After 48 h, bacteria are
not inactivated by the PEF alone. In comparison, the combination of PEF and low
concentration of tobramycin (5 mg/ml) led to a bacterial eradication of more than
6 log10. These observations suggested a synergic effect of antibiotics with the pulsed
electric fields. To explain this phenomenon, several hypotheses could be formulated.
The first evidence is that electric field can facilitate the displacement of charged
molecules across the extracellular matrix by electrophoresis, thus inducing a mass
transfer (Jass et al. 1995). Another explanation is that the extracellular matrix
becomes permeable after PEF exposure.
However, the bioelectric effects are not necessarily generalizable for all biofilm
organisms and antimicrobial agents. To illustrate this point, a previous study inves-
tigated the bioelectric effect of 11 antimicrobial agents from different categories to
improve the inactivation of P. aeruginosa, methicillin-resistant Staphylococcus
aureus, and S. epidermidis biofilms (del Pozo et al. 2009). The biofilms were
cultivated in a continuous flow biofilm growth reactor during 36 h at 37 C. The
PEF exposure was applied 24 h at room temperature in the absence or presence of
antimicrobials. The inactivation rate was quantified by colony counting. When the
antimicrobial agents were used in the absence of electric fields, there was no
significant biofilm inactivation, except with the trimethoprim-sulfamethoxazole for
S. epidermidis. In the presence of electric fields and antimicrobials, the bioelectric
effect was very variable. For example, a bioelectric effect was observed with the
daptomycin (an anionic antibiotic) against S. epidermidis but not with methicillin-
resistant Staphylococcus aureus. On the other hand, the efficacy of vancomycin
(an uncharged antibiotic) was enhanced by electrical fields with methicillin-resistant
Staphylococcus aureus and S. epidermidis. The overall conclusion is that the bio-
electric effect is not generalizable for all bacterial biofilms and antimicrobials. This is
a complex process which included not only the mass transfer by electrophoresis
because the bioelectric effect seems independent to the charge of antibiotics.
Another critical phenomenon during the electric field exposure is the presence of
oxygen intermediates or oxygen gas and the influence of pH. In an earlier study, the
impact of these components was evaluated on the bioelectric effect with tobramycin
against P. aeruginosa biofilms (Stewart et al. 1999). First of all, the bioelectric effect
was measured. In the absence of electric fields, a 5 μg/ml of tobramycin led to 2.9
log10 of bacterial inactivation. The inactivation rate increased until 5.6 log10 when
an electric field was applied in combination with tobramycin. Then, the influence of
pH was tested. Indeed, the pH decreased during PEF, from 7.2 to 4.5 in this study. No
increase of antibiotic efficiency was measured when the buffer composition was
changed to obtain a pH similar to PEF exposure. Therefore, the low pH during PEF
exposure was not responsible for the bioelectric effect. Next, the sodium thiosulfate
was used to neutralize oxygen intermediates during PEF exposure. The inactivation
rate has not been reduced in the absence of reactive oxygen species. On the other
hand, when gaseous oxygen was added in the absence of electric current, the
tobramycin efficacy to eradicate biofilms was increased by almost 2 log10. This
study demonstrates that the oxygen gas may improve the bioelectric effect.
More recently, a very interesting study focused on the gene expression in
S. aureus biofilms exposed to gentamicin within electric fields (Zhang et al. 2014).
As previously described in the literature, the antibiotic used alone has limited effects
in bacterial viability within biofilms. Furthermore, the biofilm inactivation cannot be
414 F. Pillet
done by increasing the antibiotic concentration. After 3 days of exposition, only an

inactivation of 2 log10 was measured with 256 mg/L of gentamicin. By contrast, in
the presence of low electric fields (5.6 V/cm, 4 h per day), a concentration of 2 mg/ml
of gentamicin was sufficient to obtain 6 log10 of inactivation after 3 days of
treatment. To get a better understanding of this bioelectric effect, gene expression
was evaluated. The authors have shown that the electric fields (alone or with
gentamicin) caused an upregulation of positive autolytic regulator genes and
downregulation of negative autolytic regulator genes. But, no effect was observed
on cell-cell interaction genes. These results suggested that the electric fields are
responsible for bacterial autolysis and then increase the sensitivity to gentamicin.
The S. aureus autolysis by electric field exposure was confirmed by scanning
electron microscopy.
Applications
Bioelectric effect applications are still in its infancy and examples are limited. A
recent study described the eradication in vitro by electric effect of a dual biofilm
which can be involved in periodontitis (Lasserre et al. 2015). This disease may cause
the damaging of the tooth-supporting tissue. The infection is provoked by a dual
biofilm of Streptococcus gordonii and Porphyromonas gingivalis. In this work, the
viability of P. gingivalis was investigated with dual-species biofilms of 7 days old.
The inactivation rate was 79.1% with 0.2% of chlorhexidine and 98.9% when the
chlorhexidine was supplemented with low electric current (10 mA). This study is
promising to develop a new treatment against oral biofilm bacteria.
Finally, a very exciting study concerns the inactivation in vivo by bioelectric
effects of Pseudomonas aeruginosa in lung infection (Giladi et al. 2010). Mice were
infected in the lung by intranasal administration of P. aeruginosa. The authors
demonstrate the efficiency to inactivate bacterial growth in vivo of electric fields
(12 V/cm) as monotherapy or in combination with ceftazidime. This proof of
concepts is promising to treat the pulmonary infections by bioelectric effects.
Conclusions
The use of pulsed electric fields is a promising method to inactivate bacterial biofilm
in vitro and in vivo. In order to achieve this aim, two different strategies may be
employed. The PEF disinfection is the same principle to that used in the food
industry and consists to induce an irreversible permeabilization of the bacterial cell
envelope. This strategy is efficient to inactivate bacteria in burned wound but may be
more complicated to treat in internal tissue, due to the higher sensitivity of the
eukaryotic cells. For the bioelectric effect, the main target is the extracellular matrix
which protects the bacteria against the antimicrobials. This strategy could be more
secure because the electric field strength applied is very low. However, the
mechanisms involved in the combination of electric fields and antimicrobials against

biofilms are not well known. Further studies should be done to develop the bioelec-
tric effect and inactivate biofilms in the medicine field. In addition, a major part of
biofilms are composed of different bacteria which cohabit in symbiosis, thereby
increasing the difficulty to develop effective treatments.
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doi:10.1016/j.biomaterials.2014.05.069
Bacterial Cell Envelopes: Composition,
Architecture, and Origin 25
Didier Zerbib
Abstract
The bacterial envelope is a complex layered structure, whose primary function is
to protect the cell from the environment. The Gram-staining procedure has been a
fundamental method to classify the bacteria for more than 100 years. It was based
on the effect of the structure and composition of the bacterial envelope on the
staining procedure. It has made it possible to classify the bacteria in two main
categories: the Gram-positive, which bind the Gram stain, and Gram-negative
bacteria, which do not. Currently, the prokaryotes are classified in 30 phyla of
Bacteria and in 5 phyla of Archaea. This classification does not consider the
Gram-staining properties which was an excessive simplification. The growing
amount of data concerning the genomic sequences of bacteria has significantly
amended the view of the bacterial phylogeny. In addition, the access to the
genetics of the biogenesis of the cell envelope has also allowed envisioning the
bacterial Tree of Life in a different way. The bacterial envelopes are now defined
with the number of membranes they contain: the cells are either diderms or
monoderms. In addition, the presence or the absence of lipopolysaccharides
(LPS) in the diderm envelopes is also a fundamental criterion. This chapter is
an overview of current knowledge about the composition and architecture of
bacterial envelopes in light of recent data showing that the diversity of structures
allows reaching the same main objective, the survival and the protection of the
bacterium from its environment.
Keywords
Bacterial envelopes • Diderm • Monoderm • Cell wall
D. Zerbib (*)
LISBP, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France
IPBS, Université de Toulouse, CNRS, UPS, Toulouse, France
e-mail: didier.zerbib@insa-toulouse.fr

DOI 10.1007/978-3-319-32886-7_28
418 D. Zerbib
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418
Diderm-Lipopolysaccharide Bacteria Cell Envelope (Glycobacteria) . . . . . . . . . . . . . . . . . . . . . . . . . 420
The Outer Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421
Diderm Cell Wall: The Peptidoglycan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
The Periplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424
The Inner Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424
Monoderm Bacteria Cell Envelope (Firmicutes) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424
Monoderm Peptidoglycan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426
Surface Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426
Teichoic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427
The Cytoplasmic Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427
The Corynebacterineae Envelope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427
“A Phylum Level Perspective on Bacterial Cell Envelope Architecture” . . . . . . . . . . . . . . . . . . . . . 428
Dominance of the Diderm-LPS Cell Envelope Architecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 428
Phyla with Typical Monoderm Cell Envelopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
Diderm Exceptions in the Monoderm Phyla . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
Diderm Alternative to Diderm LPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
Exceptions in Phyla Spirochaetes, Proteobacteria, and Actininobacteria . . . . . . . . . . . . . . . . . . 429
The Phylum Deinococcus-Thermus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430
The Phylum Thermotogae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430
Bacterial S-Layer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430
The Archaeal Cell Envelope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
The Origin of Diderm Bacteria; Selective Pressure or Endosymbiosis? . . . . . . . . . . . . . . . . . . . . . . . 431
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434
Introduction
The main reasons for the application of electric fields to bacteria are either to cause
their complete destruction after cell lysis or to induce a transient and selective
permeability of the envelope to allow introduction or extraction of molecules or
macromolecules. To achieve these objectives, even when the electroporation of the
membrane is effective, it is necessary to take account of the architecture of the
bacterial envelope developed to protect the bacteria from their hostile environment.
From a “bacterial point of view,” life is associated to the inside and the death to the
outside: the envelope must thus be a very effective protective barrier.
The bacterial envelope, whose primary function is probably to protect the cell
from the environment (Silhavy et al. 2010; Dufresne and Paradis-Bleau 2015), is an
active component; it is essential for survival, division, adaptation, morphogenesis,
and pathogenesis. In addition to osmotic pressure protection and bacterial shape
control, it enables the entry of nutrients, vitamins, and cofactors and the efflux of
toxins and unwanted metabolites (Silhavy et al. 2010). The energy production is
controlled by the envelope. Electron transport chains induce the electrochemical
proton gradient needed to yield the proton motive force in the bacterial membrane
(Taylor 1983) together with cytoplasmic ATP production by oxidative phosphory-
lation. The cell envelope is able to sense and transduce signals allowing the
25 Bacterial Cell Envelopes: Composition, Architecture, and Origin 419
bacterium to resist and adapt to environmental stresses and changes (Jordan et al.
2008). The bacterial envelope represents the interface between bacteria and hosts; it
plays roles in motility, adherence, and secretion (Silhavy et al. 2010).
In addition to its multiple active roles, the envelope represents a physical barrier
that protects the bacteria from the environment (Holtje 1998; Silhavy et al. 2010).
The fence, or the grid, is mainly constituted by the cell wall, giving the bacterial
shape, the bacterial resistance to osmotic pressure, and preventing the envelope from
rupture. The fence is “porous” and these holes are chemically filled with many
constituents like proteins or polysaccharides organized in supramolecular networks.
The fence is coated and reinforced by one or two hydrophobic selective barriers: the
bacterial membranes. Very schematically, like mentioned above, electroporation
generated by electric fields have been used to perform two types of actions on
this bacterial barrier: either their destruction to kill bacteria (see, for example,
▶ Chaps. 20, “Different Cell Sensitivity to Pulsed Electric Field,” or ▶ 63, “Math-
ematical Models Describing Cell Death Due to Electroporation”) or their transient
and localized disruption to allow either the input (of DNA, for example, ▶ Chap. 23,
“Gene Delivery by Electroporation In Vitro: Mechanisms”) or output (▶ Chap. 128,
“Impact of Pulsed Electric Field Treatment on Must and Wine Quality”) of interest-
ing compounds. It is clear that the structure, the chemical composition, the organi-
zation, and the biophysical and biochemical properties of the fence will greatly
influence the effectiveness of the electric field attack. Even if the main wanted effect
when applying electric field is to render the membrane permeable, the entire
envelope organization has to be taken into account when the global effect of electric
field has to be evaluated in terms of effectiveness (Pillet et al. 2016). The electric
field is a sort of unique entity facing manifold envelope types. Fortunately, electric
field parameters can be adjusted at several levels (intensity, voltage, number of
pulses, pulses frequency, pulses sources, pulses shapes) in order to find specific
electroporation or lysis protocols. If the bacterial envelope could be summed up
simply by one or two lipid membranes that pulsed electric fields (PEF) should
destroy or simply make permeable, only one or two universally efficient electropo-
ration protocols would exist. This is very far from being the case.
To simplify bacterial cell envelope descriptions, it is important to give clear
definitions of their different types in light of microbiological data but also of the
bacterium phyla properties and of the evolution of our knowledge of bacterial
phylogeny (Sutcliffe 2010). This vision was greatly enlarged when the amount of
genome sequencing data increased in databases. The staining procedure developed
by Christian Gram (1884), in addition to being a fundamental microbiological
diagnostic method for over a century, allowed very early and very conveniently to
classify bacteria into two large groups: the Gram-positive and the Gram-negative
bacteria. During years, textbooks associated each group to the actual Phyla of
Firmicutes (Gram-positive) or Proteobacteria (Gram-negative), each represented
by the models Bacillus subtilis (or Staphylococcus aureus), with a single membrane
and a cell wall, and Escherichia coli (or Salmonella typhymurium), with two
membranes, respectively. The principle of Gram staining lies in the effect of the
structural differences of the cell envelope of these two main types of bacteria.
420 D. Zerbib
Firmicutes are Gram positive because they possess a thick layer of peptidoglycan
(PG) that retains the primary dye. Proteobacteria are Gram negative because the
thin layer of PG, which is protected by an outer membrane (OM), binds only
very few dye molecules that are easily removed when the decolorant used in the
Gram-staining procedure disrupted the OM (Beveridge 2001). It is now largely
admitted that bacterial envelope architecture has to be defined by referring to the
number of cellular membrane and not by Gram-staining properties: diderm-
bacteria have two membranes, while monoderms have only one (Gupta 1998).
Even if the vast majority of diderms are Gram negative (Proteobacteria) and
monoderms are often Gram positive (Firmicutes), there are many examples of
monoderm Firmicutes displaying aberrant staining (negative or variable) and of
some diderm Firmicutes exceptions (Corynebacterineae) that stain as Gram
positive.
Bacteria are called diderm bacteria when they have two cellular membranes,
regardless of lipid composition (Gupta 1998). Among diderm bacteria, diderm-LPS
bacteria contain LPS in their OM and have been called Glycobacteria (Cavalier-
Smith 2006, 2010). Cavalier-Smith (2006) also defined Eobacteria as being the
counterpart of Glycobacteria, i.e., diderm bacteria without LPS. However, because
diderms like Thermotogae or Fibrobacteres were not present in Eobacteria
(Cavalier-Smith 2006) while monoderms like Chloroflexi (see below) were indeed
included (Cavalier-Smith 2010), the denomination “Eobacteria” will not be used
here for non-LPS diderms.
In this chapter a brief summary of the overall composition of the envelopes of
typical monoderm and diderm bacteria together with selected examples of partic-
ular and atypical species with a phylum perspective is presented. It is more focused
on the cell wall composition and organization than on the lipid membranes
description and dynamics. Electric field effects on membrane are also beyond the
scope of this review and will be discussed in detail in other chapters. This chapter
provides a simplified scheme of the different bacterial envelope structures as they
have to be taken into account when biotechnologists and biophysicists need to
evaluate or improve the effects of electric field on bacterial envelopes of various
species.
Diderm-Lipopolysaccharide Bacteria Cell Envelope

(Glycobacteria)
Typical diderm-LPS bacteria, largely represented by Gram-negative members of

Proteobacteria phylum, have a cell envelope composed of three layers (Fig. 1). The
OM and the inner membrane (IM), also called cytoplasmic membrane (CM), both
delineate the hydrophilic periplasm that contains essentially a thin layer of PG
constituting the cell wall (CW). The envelopes of typical diderm have a thickness
of 35 nm in average, the membrane thickness varies from 5 to 8 nm, and the PG
between 6 nm (in E. coli) and 2.4 nm in Pseudomonas aeruginosa (Vollmer and
Fig. 1 Typical diderm-LPS bacteria envelope. The three layers of an envelope of typical diderm-
LPS (Proteobacteria) are shown schematically here. Phospholipids and Lipid A moiety of the
lipopolysaccharides are represented in blue. The transmembrane porins and the lipoproteins of outer
membrane are in green. The periplasmic proteins and proteins of the inner membrane are in orange.
The carbohydrate units of peptidoglycan are either in blue (MurNAc) or in red (GlyNAc). LPS core
and O-antigen are both represented in gray. Peri periplasm, OM outer membrane, IM inner
membrane, PG peptidoglycan, LPS lipopolysaccharide
Seligman 2010). Both membranes are hydrophobic and semipermeable. In addition

to lipids and carbohydrates, the three layers contain proteins.
The Outer Membrane
The OM of Proteobacteria is an asymmetric lipid bilayer, with phospholipids only

found in the inner leaflet. In E. coli, phospholipids of the OM consist of 90% of the
zwitterionic phospholipid phosphatidylethanolamine (PE), 6% of the anionic phos-
pholipid phosphatidylglycerol (PG), and 4% of the anionic phospholipid cardiolipin
(CL) (or diphosphatidylglycerol) (Lugtenberg and Peters 1976). In comparison to
422 D. Zerbib
the IM the inner leaflet of the OM contains more saturated fatty acids, is more rigid,
and has a smaller net negative charge.
In Glycobacteria, i.e., diderm-LPS bacteria (Cavalier-Smith 2006, 2010), the
outer leaflet of the OM is mainly composed of the Lipid A, the lipid moiety of
anionic glycolipids: LPS (Raetz and Whitfield 2002). LPS is mainly responsible
for the human immune system recognition of bacterial infections. Lipid A is also
called the endotoxin, and LPS induces endotoxic shock associated with Gram-
negative bacteria septicemia. The LPS itself is a glucosamine disaccharide bearing
six or seven acyl chains (lipid A), a polysaccharide core, and an extended poly-
saccharide chain called the O-antigen (Raetz and Whitfield 2002). LPS plays a
critical role as an antigen through the O-antigen. Pathogenic E. coli are classified
through their O-antigen properties together with their flagellin content, the major
protein constituent of flagella, that is referred as H giving, for example, the E. coli
pathogenic strain O157:H7. LPS is also involved in the surface net charge of the
envelope and in the overall hydrophobicity of diderm-LPS bacteria. The acyl
chains of LPS are saturated and thus favor LPS packing, which is reinforced by
the neutralization of LPS negative charges with divalent cations like Mg++ and Ca+
+
(Nikaido 2003). Because of its properties, the LPS leaflet is a very effective
barrier for hydrophobic molecules, a property that is increased by the hydrophilic
selectivity of embedded porins for molecules larger than 700 Daltons (Nikaido
2003).
Only two main types of proteins are present in the OM: the lipoproteins and the
β-barrel proteins, (Silhavy et al. 2010). There are more than 100 different
OM-lipoproteins in E. coli (Nakayama et al. 2012) comprising the very abundant
tri-acylated Braun’s lipoprotein (Lpp). Lipoproteins have an N-terminal cysteine that
is modified by thioether-linked diacylglycerol and amino-linked acyl chains that
allow the embedding of the lipid moieties of lipoproteins in the inner leaflet of
the OM.
β-Barrel proteins are integral transmembrane proteins called outer membrane
proteins (OMPs). OMPs are composed of β-sheets that form cylinders. Some
OMPs function as porin allowing the passive diffusion of small molecules and
defining part of the OM selectivity (Klebba 2005). Porin like OmpF and OmpC
are trimers of 16 transmembrane β-strands monomers. They are not specific and
allow the diffusion of mono- and disaccharides but also of amino acids. PhoE,
having the same topology, is specific for phosphate. Another specific porin is
LamB, which is a trimer of 18 transmembrane β-strands that allow the specific
diffusion of maltose and maltodextrins across the OM. An interesting example is the
one of OmpA, a monomeric conditional porin. OmpA can adopt two different
conformations and be either a porin or participate to the structure of the envelope.
Finally, some larger OMPs with 20–24 transmembrane β-strands allow the transport
of very large compounds like some vitamins and iron chelates (Nikaido 2003). In
comparison to OM enzymes, the protease OmpT (Vandeputte-Rutten et al. 2001) or
OMPs like the porins OmpF and OmpC are very abundant (around 250,000 copies/
cell) but are not essential. However, the OM is essential by itself and its essentiality
is believed to rely on its protective function.
Diderm Cell Wall: The Peptidoglycan
The essential peptidoglycan (murein) is the rigid, bag-shaped (sacculus) exoskeleton

of bacteria (Weidel and Pelzer 1964) that determines the cell shape (Turner et al.
2014). It is made of glycan strands cross-linked by short peptides (Vollmer and
Seligman 2010). PG layers are covering the CM of nearly all bacteria. However, PG
is absent in Mycoplasma from Tenericutes, in some Rhickettsia from Proteobacteria
(Tamura et al. 1995), and Planctomyces from Planctomycetes, and even if biosyn-
thetic genes are present, it has never been detected in members of the phylum
Chlamydiae (Chopra et al. 1998).
The integrity of PG prevents bacteria from cell lysis in low osmolarity media.
Enzymatic digestion of PG by lysozyme induces the loss of the cell shape and
provokes the formation of spheroplast sensitive to cell lysis in low osmolarity media
or in water. Any inhibition of PG biosynthesis by mutation or antibiotic action
during bacterial growth will provoke cell lysis.
The linear glycan strands of PG are made up of alternating N-acetyl glucosamine
(GlcNAc) and N-acetyl muramic acid (MurNAc) linked in β-1 ! 4. MurNAc is
substituted by unusual pentapeptide containing rare D-amino acids. In nascent PG,
the pentapeptide is often L-Ala—D-Glu—mA2pm (meso-diaminopimelic acid)—D-
Ala—D-Ala. The last D-Ala is generally absent from mature PG. Cross-linking of the
linear glycan strands occurs between those peptides, generally between the penulti-
mate D-Ala and the mA2pm at position 3, and form a peptide bridge of seven amino
acids. This organization, the length of the glycan strands and the degree and nature of
cross-linkage are the most common. However, variations exist, from species to
species, and depending on strains and growth conditions. Those variations have
been used, since more than 40 years, as a taxonomic criterion. PG is attached to the
OM through a small lipoprotein (58 residues) anchored in the inner leaflet of the OM:
the Braun’s lipoprotein (Lpp) or “murein lipoprotein” (Braun 1975). The N-terminus
of Lpp is linked to the lipids embedded in the OM whereas its C-terminal end is
covalently linked to the diaminopimelic acid of the PG pentapeptide. Lpp is the most
abundant protein in E. coli with more than 500,000 molecules per cell. Lpp mutants
produce extracellular OM vesicles indicating that Lpp is the real anchor of the OM on
PG. OmpA also provides an additional link between PG and OM.
The chemical composition of PG of numerous species is now very well known.
The challenge is to observe the real 3D organization of PG, for which several models
have been proposed (Vollmer and Seligman 2010). The classical “horizontal layers
model” propose an architecture in which the glycan strands are parallel to the CM
(Pink et al. 2000). Conversely, in the “vertical scaffold model,” the peptides are
parallel to the membrane whereas the glycan strands run perpendicular to the long
424 D. Zerbib
axis of rod-shaped bacterium (Meroueh et al. 2006). Even if the layer model is
favored, the actual 3D architecture of PG in different bacteria is still under investi-
gations (Turner et al. 2014).
The Periplasm
The periplasm is defined as the aqueous compartment located between the OM and
the CM. It consists in a hydrophilic and oxidizing space between the two mem-
branes, containing the cell wall (Mullineaux et al. 2006). The periplasm is rich in
proteins such as degradation enzymes (RNase or alkaline phosphatase); periplasmic
binding proteins, involved in sugar and amino acid transport and in chemotaxis; and
“chaperones” involved in envelope biogenesis. An interesting view of the periplasm
has recently been presented as being an OMP quality control space (Lyu and Zhao
2015). It is indeed a challenge for OMP proteins that are synthesized in the
cytoplasm to cross the hydrophilic periplasm without any energy producing system:
a role of E. coli periplamic proteins like Skp, SurA, and DegP that help OMPs in
such a process has been proposed (Mogensen and Otzen 2005).
The Inner Membrane
The IM or cytoplasmic membrane (CM) of diderm is a symmetric phospholipid

bilayer. In E. coli, the CM is composed of 75% of the zwitterionic phospholipid
phosphoethanolamine (PE), 20% of the anionic phospholipid phosphatidylglycerol
(PG), and 5% of the anionic phospholipid cardiolipin (CL) and small amounts of
phosphatidylserine (PS). The CM of the Diderm E. coli has a smaller negative charge
than the one of the monoderm B. subtilis that is rich in PG (van der Does et al. 2000).
In Proteobacteria and in monoderm, other minor lipids are present like the
polyisoprenoid lipid carrier undecaprenyl-P that transports activated sugar for enve-
lope biogenesis. Moreover, the diderm CM contains integral α-helical proteins
together with lipoproteins. In contrary to IM of monoderm, the diderm CM does
not contain any teichoic acid (Silhavy et al. 2010). All the membrane associated
functions performed by eukaryotic organelles have to be achieved by the bacterial
CM: that is, energy production, lipid biosynthesis, protein transport and secretion; all
the proteins involved in those functions are found in the CM.
Monoderm Bacteria Cell Envelope (Firmicutes)
The main difference between monoderm and diderm bacteria cell envelopes is the
absence of OM in the former (Fig. 2). This type of envelope was previously defined
as specific of Gram-positive bacteria. This simple view has been modified and the
recent discovery of a real OM in the Gram-positive Firmicutes Mycobacteria is a
proof of concept attesting that Gram positive and even Firmicutes are not all
Fig. 2 Typical monoderm bacteria envelope. The two layers of an envelope of typical
monoderm (Firmicutes) are shown schematically here. Phospholipids are depicted in blue. Inner
membrane proteins are in orange. Carbohydrate basic units from the peptigoglycan are in blue
(MurNAc) and red (GlyNAc). Wall teichoic acids (WTA) are in pink with the decorations by
carbohydrates in pale blue and by alanine in black. Lipoteichoic acids are in green and their
modifications are symbolized as for WTA. Covalently attached or non-covalently attached proteins
are depicted in green. PG peptidoglycan, CM cytoplasmic membrane, CW cell wall
monoderm bacteria. This section is a description of the main features of the structure
of the model Gram-positive Firmicutes envelope represented by B. subtilis or
S. aureus and designed as “monoderm bacteria” (Sutcliffe 2010).
In Gram-negative diderm-LPS bacteria, the presence of the OM barrier stabilizes
the CM, and the PG is relatively thin. In monoderm bacteria, the OM is absent and to
protect the cell from the turgor pressure, the layers of PG are several times thicker.
The thickness of monoderm envelopes varies from one species to another. For
example, it is 40 nm in Staphylococcus aureus and 60 nm in Bacillus subtilis
(Vollmer and Seligman 2010). The thickness of the membrane is, as in diderm,
relatively constant and close to 6 nm. It is especially the thickness of the PG that is
variable: 20 nm in S. aureus to 34 nm in B. subtillis, for example (Vollmer and
Seligman 2010 9). In Firmicutes, the cell wall is reinforced by the presence of long
anionic polymers, the teichoic acids (TA) (Swoboda et al. 2010). TA are formed by a
disaccharide linkage unit and an anionic chain of polyglycerolphosphate or polyribi-
tolphosphate repeats decorated with saccharides and positively charged D-alanyl
esters. Wall teichoic acids (WTA) are covalently attached to PG (Brown et al.
2013), while lipoteichoic acids (LTA) are linked to the head groups of CM lipids.
TA represent about 60% of the cell envelope mass and are mainly responsible,
together with PG, for the envelope structure and function. Like LPS in
426 D. Zerbib
Proteobacteria, WTA in Firmicutes are critical determinants of the envelope surface

net charge and hydrophobicity.
The extracellular proteins of monoderm bacteria, because of the absence of OM,
are either covalently attached to the PG, partially inserted in the CM, linked to CM
lipids anchors, or attached to teichoic acids (Dramsi et al. 2008). Several extracel-
lular proteins of monoderm are orthologues of diderm periplasmic proteins.
Monoderm Peptidoglycan
The PG of monoderm Firmicutes is made of glycan strands composed of disaccharide

repeats linked together with pentapeptide units. The classical monoderm PG chemical
composition is identical to the one of diderm bacteria. The linear glycan strands of PG
are made up of alternating N-acetyl glucosamine (GlcNAc) and N-acetyl muramic acid
(MurNAc) linked in β-1 ! 4 and are cross-linked together with short specific pep-
tides. The major difference with diderm PG is in the PG thickness; the Gram-positive
PG can be 30–100 nm thick whereas the Gram negative is only a few nm thick.
Among the Gram-positive bacteria, the differences between PG structures are mainly
based on the type of cross-linking of the glycan strands by peptides (Vollmer 2008). In
contrary to B. subtilis, S. aureus and many other Gram-positive Staphylococcaceae
contain branched stem peptides in their PG that are involved in β-lactam resistance
(Chambers 2003). In S. aureus, a pentaglycine branch is present on the third amino
acid of the stem peptide and involved in glycan strands cross-link. The presence of this
specific type of branched peptide is responsible for the S. aureus resistance to β-lactam
antibiotics because the target protein, the transpeptidase that performs this specific
peptide cross-link (PBP2A) is structurally different in these bacteria and therefore
insensitive to inhibition by b-lactams. Branched peptides are also attachment site of
specific PG covalently associated proteins.
Surface Proteins
The adherence of pathogenic bacteria like S. aureus is an important pathogenicity

factor. TA are involved in bacterial adherence together with adhesins, proteins that
are able to bind components of the extra cellular matrix like fibronectin, fibrinogen,
and elastin. Some adhesins are only interacting with PG or TA, but most of them are
covalently attached to the stem peptide of PG (Dramsi et al. 2008). Those proteins
are secreted by sortases through a pentapeptide motif present in their C-terminal end.
Sortases catalyze the modification of PG precursors by surface proteins prior to their
incorporation in mature PG (Dramsi et al. 2008). In S. aureus, sortase A (StrA)
possesses more than 20 protein substrates involved in adhesion, which escape from
the immune system, phage binding and internalization. Sortase B (StrB) allows the
surface display of an iron acquisition protein. The sortases represent the architects of
the bacterial surface. Other surface proteins are anchored in the CM through helices
inserted in the lipid leaflet. Finally, other surface proteins involved in cell wall
modeling are non-covalently linked to either PG or TA.
Teichoic Acids
These long anionic polymers are found in many Gram-positive bacteria including the
models S. aureus and B. subtilis. They are responsible for the negative surface net
charge of those bacteria. The WTA are coupled to PG, and the LTA are anchored in
the CM (Fig. 2). WTAs are linked on the C6 of the MurNAc residues of PG glycan
strands. The most common WTA are formed by disaccharides modified by more
than 60 repeat units of either polyribitolphosphate (polyRboP) or polyglycerol-
phosphate (polyGroP). WTAs are found “perpendicular” to the PG layers.
Depending on species or strains, the RboP or GroP units can be modified with
saccharides and positively charged D-alanyl esters. Those variations affect the
functions of the WTA. LTA are similar to WTA, they are formed by polyGroP
modified by either a D-alanine or a sugar moiety. They are anchored in the CM
through embedded glycolipids. Since they contain fewer repeat units than WTA,
they do not span beyond the PG. The negative surface net charge of the Gram-
positive envelope is essential, and when TA are absent from a given species, they are
replaced by other polyanionic polymers containing either carboxylate or sulfate
groups to provide the negative charges. LTA or WTA are not essential separately,
but it is not possible to delete both pathways simultaneously because their primary
function is to be anionic and to play a role in cation homeostasis. Cations binding on
TA induce the formation of a cationic network that increases the rigidity of the
envelope. Moreover, the density of TA negative charges influences the sensitivity of
bacteria to cationic antibiotics and to host lytic enzymes.
The Cytoplasmic Membrane
The monoderm CM is a symmetrical phospholipid bilayer. The proportions of

zwitterionic phospholipid phosphatidylethanolamine (30% PE) and anionic phos-
pholipid phosphatidylglycerol (70%, PG) are different than in diderm giving the
monoderm CM a higher negative surface charge (van der Does et al. 2000). This
bilayer contains undecaprenyl-phosphate necessary for the envelope biogenesis
together with α-helical anchored lipoproteins and the saccharide moiety of LTA.
The Corynebacterineae Envelope
The suborder of Corynebacterineae, from the phylum Actinobacteria, includes the

family of mycobacteriaceae (Mycobacterium), corynebacteriaceae (Corynebacte-
rium), and nocardiaceae (Nocardia). These bacteria were defined as monoderm high
G + C Gram-positive bacteria, but they display properties of both Gram-positive and
Gram-negative bacteria (Fu and Fu-Liu 2002). The cell envelope of M. tuberculosis
has indeed characteristics of both Gram-positive and Gram-negative bacteria
(Jankute et al. 2015).
428 D. Zerbib
A common CM is located under a typical thick monoderm PG, but the PG is

covalently attached to arabinogalactan (AG) itself linked to very long chain specific
fatty acids: the mycolic acids (MA, (Marrakchi et al. 2014)). This giant macromol-
ecule called the PG-AG-MA complex fills the very large periplasm of Mycobacteria.
The mycolic acids are β-hydroxylated, α-branched fatty acids and can contain up to
90 carbons. They are responsible for the waxy appearance of mycobacteria; they are
involved in the impermeability of the envelope and play an important role in the
pathogenicity of Mycobacteria. MA biosynthesis relies on multiple protein com-
plexes (Veyron-Churlet et al. 2004; Carel et al. 2014) that represent targets for
efficient antibiotics (Nataraj et al. 2015). Finally, the mycobacterial envelop contains
a variety of non-covalently bound cell envelope lipids and glycoconjugates as
recently reviewed (Daffé et al. 2014). It has been demonstrated by cryo-electron
microscopy of vitreous sections (CEMOVIS) that Corynebacterineae have a
OM. The asymmetric OM is supposed to be formed by an inner leaflet of mycolic
acids and an outer leaflet of MA comprising inserted extractible lipids (Daffé et al.
2014). The precise structure of the OM remains unclear (Zuber et al. 2008). Like
Gram-negative bacteria, mycobacteria have porins in the OM, but those are struc-
turally different from the one of E. coli. Corynebacterineae can be designed as
Gram-positive diderm-non-LPS bacteria.
“A Phylum Level Perspective on Bacterial Cell Envelope

Architecture”
The present understanding of phylogenetic tree allowed classifying prokaryotes in

30 phyla in the domain Bacteria and 5 phyla in the domain Archaea (http://www.
bacterio.net/-classifphyla.html) (Sutcliffe 2010). The increasing amount of data
concerning the genome sequences together with the precise knowledge of the
genetics of the cell envelope biogenesis allowed seeing the bacteria in the Tree of
Life in a different manner. It is now clear that the Gram-based classification was an
oversimplification and that bacterial envelopes are now to be defined with the
number of membranes that they contain (Sutcliffe 2010): bacteria are divided into
either monoderm or diderm, and the presence or absence of LPS in diderm is also a
fundamental criterion. Among Bacteria, there is a clear dominance of diderm LPS
(Glycobacteria) that are not restricted to Proteobacteria but represented in 17 phyla.
Typical monoderms exist in Firmicutes and Actinobacteria phyla but also in
Tenericutes and Choroflexi. Some other exceptions exist and are briefly listed below.
Dominance of the Diderm-LPS Cell Envelope Architecture
Based on 16S rRNA gene sequencing, it has been shown that in addition to the three
most known phyla (Firmicutes, Proteobacteria, Actinobacteria), 21 additional phyla
could be defined (Cavalier-Smith 2006, 2010; Pace 2009). The enormous amount of
data concerning Proteobacteria explains why Gram-negative envelope was
synonymous of diderm-LPS envelope. It is true that many phyla have been shown to
contain LPS, as shown either by biochemical characterization or by identifying LPS
biosynthesis genes in the genome. This architecture is typical of 17 out of 24 phyla
and is thus not restricted to Proteobacteria. Some LPS-containing bacteria do not
possess any known component of the LPS biosynthesis pathway (Chlamydiae,
Bacteroidetes, Chlorobi, Cyanobacteria) (Sutcliffe 2010).
Phyla with Typical Monoderm Cell Envelopes
Only Firmicutes and Actinobacteria are mainly composed of monoderm bacteria

with some very important exceptions in the suborder of Corynebacterineae. In
addition to the thick cell wall, as mentioned above, the presence of polyanionic
polymers is typical of monoderm. The distribution of teichoic acids or teichuronic
acids in those two phyla suggests that they are determinants of monoderm
polyanionic envelopes. The CM of representative of these phyla also contains either
lipoteichoic acids or lipoglycans. The Tree of Life has been modified and other
monoderm phyla have been defined. For example, in the 2nd edition of Bergey’s
Manual of Systematic Bacteriology, the class Mollicutes (Gram-negative bacteria
devoid of cell wall) has been excluded from the phylum Firmicutes and classified in
the phylum Tenericutes. In addition, monoderm representatives of a previously
proposed phylum (Ktedonobacteria) are now a class of the phylum Chloroflexi
that might be a typical monoderm phylum (Sutcliffe 2010).
Diderm Exceptions in the Monoderm Phyla
Within the Firmicutes phylum, there are several diderm-LPS bacteria, from the class
of Clostridia, and some of them even possess a genuine LPS biosynthesis pathway.
As mentioned above, within Actinobacteria, the suborder of Corynebacterineae has
the same PG-AG-MA complex even if the mycolic acids are very different from one
species to another. Thus, contrary to textbook definitions, Actinobacteria are not all
monoderms.
Diderm Alternative to Diderm LPS
Exceptions in Phyla Spirochaetes, Proteobacteria,

and Actininobacteria
Several genera of Spirochaetes phylum have no LPS but have a diderm cell envelope
with a specific lipid replacing lipid A in the outer leaflet of the OM. In the
Proteobacteria phylum, example exists (Shingomonas) with no LPS and an OM
made of glycosphingolipids. In the Actinobacteria phylum, the Corynebacterineae
430 D. Zerbib
possess an asymmetric OM like the diderm-LPS Gram-negative bacteria and can be

considered as diderm-MA bacteria (Sutcliffe 2010).
The Phylum Deinococcus-Thermus
Deinococci have a complex envelope with a lipid rich layer without LPS and
covered by a protein S-layer. LPS seems to be replaced by an unusual lipoglycan
(Rothfuss et al. 2006). The S-Layer is formed by a lipoprotein (Hpi) anchored
directly in the outer lipid layer. Layered envelope with apparent OM and an
S-Layer also exists in Thermus species. Proteins typical of OM like β-barrel proteins
and Bam family proteins (β-barrel assembly machinery) have been identified in
those bacteria (Brosig et al. 2009).
The Phylum Thermotogae
Bacteria from the phylum Thermotogae have an outer envelope (toga) that is
essentially composed of proteins rather than lipids. Thermotogae members are
Gram negative, contain porins, and do not have any LPS biosynthetic genes
(Sutcliffe 2010).
The take home message is that there are only few typically monoderm phyla and
that the very large majority of known diderm bacteria are diderm-LPS bacteria with
some exceptions when LPSs are replaced by other lipid molecules in the
OM. Actinobacteria and Firmicutes, which represent 40% of the recognized bacte-
rial taxa, are not pure monoderms. It seems that only Chloroflexi might be a typical
monoderm phylum.
Bacterial S-Layer
S-layers (Surface layer) are self-assembled paracrystalline protein lattices found in

many bacteria and almost all archaea. As an important component of the bacterial
cell envelope, S-layers can fulfill various biological functions and are usually the
most abundantly expressed proteins in a cell. In Archae, S-Layer is the only cell-wall
component. S-Layers are found in both Gram-positive and Gram-negative bacteria.
In Gram positive, the S-Layer is non-covalently attached to PG or other cell-wall
components. In Gram negative, the S-Layer is commonly attached to the LPS of
the OM.
S-layers are mostly composed of one protein or glycoprotein species. In glyco-
proteins, the degree of glycosylation and glycan composition greatly varies. Single
strain can express different S-layers depending on culture conditions. The S-layer
proteins are poorly similar; they are usually acidic and hydrophobic. The S-Layer
proteins form regular crystalline lattices, which may have an oblique (p1, p2), square
(p4), or hexagonal (p3, p6) symmetry. The S-layer proteins possess a heavier domain
that forms the core of the morphological unit cell and a lighter domain that provides
connectivity between the units. The central core-forming region is usually oriented
toward the cell envelope, giving rise to an overall corrugated inner surface. By
contrast, the outer surface appears smooth despite highly variable and species-
specific ultrastructures. Between 30% and 70% of the unit cell volume is occupied
by the protein, which leads to the formation of identical and well-defined pores with
a diameter of 2–8 nm (Pavkov-Keller et al. 2011).
The Archaeal Cell Envelope
The composition of the archaeal cell envelope is very different from the bacterial cell
envelope (Albers and Meyer 2011). With the only exception of Ignicoccus, which
exhibits an OM and a very thick periplasm, Archaea possess only a single mem-
brane. The CM is enclosed by an S-layer. Archaeal lipids consist of repeating
isoprenyl groups linked to a glycerol backbone through an ether linkage. These
lipids form diether bilayer membranes. Hyperthermo-acidophiles contain tetraether
lipids forming a monolayer membrane. These membranes are very impermeable to
protons and enable these Archaea to survive in highly acidic conditions. Another
specificity of Archaea is that the extracellular proteins are glycosylated with N- and
O-glycosylation. Finally, Archaea do not produce any murein, with the exception of
some methanogenic species that produce pseudo-murein.
The Origin of Diderm Bacteria; Selective Pressure or

Endosymbiosis?
As written in an article’s summary of Norman Pace (Pace et al. 2012), “. . .In 1977,
Carl Woese and George Fox published a brief paper in the Proceedings of National
Academy of Sciences (USA) that established, for the first time, that the overall
phylogenetic structure of the living world is tripartite. . .”. The Tree of Life has been
presented as having three main branches: eubacteria, Archaea, and Eucarya (Woese
et al. 1990). Since this fundamental discovery based on the analysis of the rDNA,
many scientists have collected and interpreted phylogenetic data such as the com-
parison of the sequences of ribosomal proteins, bacterial physiology, bacterial
morphology, conservation of key proteins, and presence of specific indels. in order
to grow more and more the universal Tree of Life. While retaining the same
backbone than that of the original tree (Woese et al. 1990), a consensual Tree of
Life (Fig. 3a) has recently been presented by P. Forterre (2015).
The most studied phyla are represented on this tree with an attempt to consensual
rooting and grouping in super-phyla (Fig. 3b). The phyla comprising members with
LPS-containing envelopes are distributed in all the different super-phyla. On the
other hand, they are less represented in the super-phylum Cyanobacteria. However,
it is striking to see that the phyla containing the pure monoderms and the atypical
diderms are more grouped in the super-phylum Cyanobacteria. In the quest of the
432 D. Zerbib
Fig. 3 Schematic and simplified Universal Tree of Life redrawn with simplifications and
modifications from P. Forterre (2015). (a) The backbone of the Universal Tree of Life. The three
main clades organized in super-phyla are indicated: Bacteria are in green, Archaea in pale blue, and
Eucarya in pale red. Potential rooting on common ancestors (Forterre 2015) are indicated: LUCA
Last Unknown Common Ancestor, LBCA -Bacterial-, LACA -Archaeal-, LARCA -Arkaryal-,
LECA -Eukaryotic-. (b) Schematic simplified bacterial branch of the Universal Tree of Life from
P. Forterre (2015). The names of the most studied phyla are indicated together with the names of
super-phyla (in bold). The names of phyla containing a majority of typical diderms are in black,
those of phyla containing a majority of typical monoderms are in red. Phyla containing
LPS-bacteria are underlined. Phyla in green corresponds to atypical alternative to diderm-LPS
bacteria (see text)
last common ancestor (LCA) in bacteria, the division of the Bacteria empire into
diderm or monoderm should probably be seen more as a phylogenetics criterion. The
origin of diderm bacteria is still debated. Two main theories are opposed in the
literature: either their endosymbiotic origin or their selection under antibiotics pres-
sure. The first is supported by James A. Lake (2009) and the second by Radhey
Fig. 4 A cartoon of the development of outer bacterial envelope redrawn with simplifications
and modifications from R.S. Gupta (2011). This cartoon of the development of the outer envelope
of different bacterial phyla was based on a study of the distribution of inserts in the Hsp60 protein
(occurring “between” the simple and typical diderms) and inserts in the Hsp70 protein (occurring
“between” typical diderm and Chloroflexi monoderm) (Gupta 2011). IM Inner membrane in red,
LPS lipopolysaccharide in either green or grey, PG peptidoglycan in pale green, OM outer
membrane in green, MM mycomembrane containing mycolic acids (MA) in blue, T outer toga in
grey dashed line. The noncharacterized outer layer of Chloroflexi is in grey
S. Gupta (1998, 2011). James Lake has proposed that diderm came from a former
endosymbiosis between an Actinobacteria and a Clostridia (Lake 2009). Radhey
Gupta has presented very convincing evidence, based on the study of insertions and
deletions in the genes of Hsp60 and Hsp70, showing that probably, the endosymbiosis
does not explain the origin of the diderms. Diderms might have evolved from an
ancestral monoderm under the selection pressure of antibiotics. It is very interesting
because this theory allows understanding how the bacterial envelopes could have
evolved (Fig. 4). The typical monoderms may have acquired an outer layer (still not
characterized in Chloroflexi) and then an outer cell envelope devoid of LPS
(Deinococcus-Thermus). Finally, the presence of LPS in the OM would have given
a large part of the typical phyla of diderm-LPS bacteria (Fig. 4). The same ancestral
typical monoderm could have acquired either a toga (Thermotogae), either an OM
containing mycolic acids (Corynebacterineae) or an atypical external envelope bear-
ing LPS (Fusobacteria, Negativicutes, Synergistetes, Elusimicrobia) (Fig. 4).
Trying to develop and analyze the Gupta’s theories in deep phylogeny is far from
the purpose of this review. However, it might be interesting, in the aim of under-
standing more clearly the complexity and variability of bacterial envelopes, to try to
434 D. Zerbib
read the Universal Tree of life as it is interpreted today by Patrick Forterre (2015)
(Fig. 3b), in light of the theory on the origin of the diderms of R. Gupta (1998, 2011)
(Fig. 4), after an in-depth analysis of the data which have helped to build this chapter
and with the knowledge and the vision of the bacterial phylogeny like presented by
Iain Sutcliffe (Sutcliffe 2010).
Conclusions
The bacterial cell envelopes are complex, dynamic, and play fundamental roles in the
adaptation and the protection of bacteria. The peptidoglycan is the most preserved
component of bacterial envelopes. It is essential in the maintenance of the rigidity and
the shape of bacteria. It is also essential to protect the cell from the very strong osmotic
pressure of the cytoplasm. In addition to the PG, the asymmetry of the OM of diderm
bacteria, the teichoic acids or their equivalents anionic in monoderm bacteria, and the
complex PG-AG-MA of Corynebacterineae ensure a perfect integrity of the cell
envelope. The architecture of the cell envelopes of many bacteria is well known,
and the biosynthesis of their components is also perfectly decrypted.
The challenge is probably to assess the impact of the electrical fields at the
molecular level, on each type of envelope, and taking into account all of the
biochemical, structural, and architectural characteristics in each bacterium which
must be pulsed for a given application on specific species of bacteria.
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190:5672–5680
3D Culture Models to Assess Tissue
Responses to Electroporation 26
Abstract
Cell and tissue responses to external stimuli are difficult to study in vivo. Tradi-
tional monolayer culture conditions allow for observation of cellular response to
stimuli in vitro with a great degree of control and manipulation of experimental
conditions; however, many studies have shown that cells exhibit different gene
expression patterns, drug resistance, and mechanical stress responses in
two-dimensional environments, than when cultured in three-dimensional
(3D) culture environments or in vivo. Cell-cell and cell-matrix interactions
determine many aspects of cellular behavior, including proliferation, metabolism,
differentiation potential, and viability. Therefore, many biomimetic strategies
exist for 3D cell culture for various applications. This chapter describes 3D
culture methods for assessing tissue response to exogenous stimuli, specifically
electroporation. These methods include spheroid culture, cell culture on electro-
spun scaffolds, and cell culture on decellularized human dermal matrices. Spher-
oid culture is generally recognized as a model system for tumor development and
has been used extensively to study electroporation effects. Other 3D culture
techniques include using electrospun scaffolds for various tissues such as oral
mucosa and head and heck squamous carcinoma, and can be readily adapted to
studying electroporation effects. Decellularized human dermis has been recently
demonstrated as an excellent substrate for recapitulating human skin and used for
electroporation applications.
A.A. Bulysheva (*)

e-mail: abulyshe@odu.edu
R. Heller
School of Medical Diagnostics and Translational Sciences, Old Dominion University, Norfolk, VA,
USA
e-mail: rheller@odu.edu

DOI 10.1007/978-3-319-32886-7_29
438 A.A. Bulysheva and R. Heller
Keywords
Electroporation • Electrotransfer • Gene therapy • 3D cell culture • Biomimicry •
Spheroid • Extracellular matrix • Electrospinning • Cryogenic electrospinning
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438
Spheroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439
Spinner Flask Suspension Prostate Cancer Spheroid Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439
Microgravity Melanoma Spheroid Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
Hanging Drop Spheroid 3D Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441
Electrospun Scaffolds for 3D Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443
Electrospinning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443
Cryogenic Electrospinning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445
Cryogenic Electrospun Scaffolds for Oral Mucosa and Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446
Recellularized Human Dermis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
Introduction
Traditional in vitro tissue and cell culture has a long history starting with Harrison’s
report of tissue culture of frog nerves in 1907 with the hanging drop method for
maintaining neurons for up to 4 weeks (Freshney 2005). It was not until introduction
of the proteolytic enzyme trypsin in 1916 that cell culture free of the extracellular
matrix was possible resulting in culture and subculture of adherent cells (Freshney
2005). The first human cell line (HeLa) was established in 1952 from a cervical
carcinoma (Freshney 2005). Adherent mammalian cell culture has provided a large
body of knowledge about cell structure and function. It is a convenient experimental
platform for investigating cellular response to their environment and to various
perturbations. This two-dimensional (2D) environment, however, does differ from
the native environment that various cell types experience in the body. Extracellular
cues from the extracellular matrix, cell-cell interaction, and mechanical forces in
three dimensions are absent in traditional cell culture of adherent cells to tissue
culture plastic or other coated two-dimensional surfaces (Griffith and Swartz 2006).
Many studies have demonstrated variability in gene expression profiles of the same
cells grown in 2D vs. 3D cultures, including differentiation, proliferation, drug
response, and survival. It is hypothesized that tissue specific in vitro 3D culture
systems match cell response to perturbations more closely to in vivo responses than
can be observed in 2D culture (Griffith and Swartz 2006). Therefore, many efforts
have been dedicated to creating 3D culture environments for assessment of cells
responding to perturbations such as electroporation.
Various 3D culture systems have been developed largely for tissue engineering
application to mimic skin, mucosa, blood vessels, liver, bone, and other tissues. The
guiding principles remain similar for constructing a suitable model system. A
scaffold is created for mimicking the extracellular matrix, and the appropriate cell
26 3D Culture Models to Assess Tissue Responses to Electroporation 439
type is selected and then seeded on the scaffold. The cells are allowed to replicate
their microcellular environment for a set amount of days and the system is tested for
specific markers of the desired tissue and cell viability. Once the model is validated,
it can then be used for studying tissue responses. This chapter will discuss 3D culture
systems and techniques available for studying tissue responses to electroporation,
including spheroid culture for tumor modeling, electrospinning techniques for scaf-
fold fabrication, and 3D culture systems of skin and oral mucosa and their potential
utility.
Spheroids
Spheroid culture has been ubiquitous throughout many disciplines. The hanging
drop method, which remains the main method for creating spheroids, has been
established initially for developmental biology and embryology studies (Freshney
2005). It was later adapted by cancer biology, stem cell biology, and tissue engi-
neering disciplines (Freshney 2005). Multiple groups have used spheroid culture
methods to evaluate effects of electroporation on cells particularly tumor cellular
microenvironment, uptake of molecules of various sizes (including dyes, drugs, and
DNA), effects on membrane integrity, and cellular viability (Canatella et al. 2004;
Mellor et al. 2006; Wasungu et al. 2009; Marrero and Heller 2012; Gibot et al. 2013;
Gibot and Rols 2013; Chopinet et al. 2012; Frandsen et al. 2015). Several distinct
spheroid models have been developed for studying effects of applied electric fields
on tumors in vitro.
Spinner Flask Suspension Prostate Cancer Spheroid Model
Canatella et al. (2004) reported using spheroid culture of prostate cancer cells to
study the effects of electroporation on molecular uptake in a multicellular environ-
ment (Canatella et al. 2004). DU145 cells were suspended in siliconized spinner
culture flasks and allowed to form spheroids in suspension culture over a period of
multiple days. Spheroids were filtered via nylon meshes and centrifuged to ensure
uniform spheroid sizes were used for further experiments. All electroporation exper-
iments on uniformly sized spheroids were performed in 4-mm gap cuvettes. The
spheroids were dissociated into single cell suspensions for analysis of molecular
uptake after application of electric pulses. Flow cytometry was used for quantitative
analysis of fluorescence intensity. Propidium iodine (PI) was used to label and
identify nonviable cells. Cells close to the periphery of the spheroids were identified
by higher uptake of Hoechst 33342 or calcein blue-AM. Green fluorescence intensity
was used to quantify calcein molecular uptake occurring due to electroporation.
Molecular uptake by cells in spheroids was reduced compared to molecular uptake
by individual cells in suspension, in a manner proportional to spheroid size, with
larger spheroids taking up fewer molecules of calcein. Cell uptake of calcein was
inversely proportional to the distance from the outer surface of the spheroid. Larger
field strengths, increased pulse length, and pulse width could enhance molecular
uptake by cells residing in the spheroid (Canatella et al. 2004). Modeling of the
electric field and predicted molecular uptake profiles were also correlated to exper-
imental results. This study concluded that cells densely packed in three dimensions
respond heterogeneously to pulsed electric fields. Cell location, cell size, and
extracellular space solute concentration all contribute to this heterogeneity
(Canatella et al. 2004).
Microgravity Melanoma Spheroid Model
Another example of spheroid culture was developed under simulated microgravity

conditions for modeling melanoma tumors (Marrero et al. 2009). The culture system
consists of a coculture of keratinocytes and B16F10 melanoma cells or SKMEL-5
melanoma cells that form a large viable spheroid of approximately 1 cm in diameter
(Marrero and Heller 2012; Marrero et al. 2009). HaCaT cells were trypsonized and
resuspended at a high cell concentration in a rotary microgravity chamber. The
chamber was maintained under constant rotation, simulating microgravity condi-
tions. The rate of rotation was adjusted to account for the size of the forming
spheroids. After 2 days of culture, the cells adhere to each other forming large
spheroids. These ~1 cm in diameter spheroids were then injected with melanoma
cells to form a coculture model. This spheroid model was tested for cell viability
with a TdT-FragEL DNA fragmentation detection assay. Presence of basement
membrane extracellular matrix components was evaluated with immunofluorescence
staining for fibronectin. Cell proliferation was assessed with staining for the prolif-
eration marker Ki67. The spheroids injected with melanoma cells remain stable for
up to 15 days. Cell viability was determined to be 80%, while cell proliferation was
confirmed with positively stained nuclei for Ki67. Keratinocytes were able to
produce their own extracellular matrix indicated by positive immunoreactivity
with fibronectin antibody (Marrero et al. 2009). This in vitro model of melanoma
is particularly useful for evaluating gene electrotransfer parameters for melanoma
tumors in vitro due to its macroscopic size enabling DNA injection to the center of
the spheroid similar to in vivo protocols (Marrero and Heller 2012). Other important
features include presence of keratinocytes, which provide cell-cell interactions and
extracellular matrix, which account for cell-matrix interactions, together providing a
similar microenvironment normally experienced by the melanoma cells in vivo. It
was demonstrated that electrotransfer electrodes that are typically used for in vivo
electrotransfer can be easily tested using this in vitro model. Six plate, four plate,
6-needle, and 4-needle electrodes were used to deliver green fluorescent protein
(GFP), or interleukin 15 (IL-15) encoding plasmid DNA. GFP and IL-15 expression
enhancement was confirmed in electrotransfer groups compared to no pulse controls.
Therefore, this spheroid in vitro model of melanoma growth and development can be
used to establish treatment regimens for translational therapies of melanoma in vivo
(Marrero and Heller 2012; Marrero et al. 2009).
Hanging Drop Spheroid 3D Models
The classic hanging drop method for spheroid formation has been explored with
human carcinoma and mouse sarcoma cell lines due to their ability to grow in
monolayer cultures and form spheroids (Wasungu et al. 2009). Hanging drops of
20 μL containing 500 cells were allowed to aggregate in a handing drop for 72 h.
Then the cell aggregates were transferred to wells coated with agar and filled with
culture media. The spheroids were allowed to grow for up to 10 days when they
reached ~500 μm in diameter. Electric pulse induced permeabilization and molecule
delivery efficiency experiments were then conducted with delivery of PI and plasmid
DNA encoding the reporter gene GFP. Spheroids were resuspended in pulsing
buffer. Both PI and plasmid DNA were dissolved in the pulsing buffer for their
respective experiments and were introduced to the spheroids from the outside of the
spheroid, in contrast to Marrero et al. (2009), where the spheroids received plasmid
DNA injections into the center of the spheroid. Parallel plate electrodes were used to
deliver electric pulses establishing a relatively uniform electric field. This study
observed that while cell permeability appeared homogenous, gene expression was
not homogenously distributed throughout the spheroid. This observation is consis-
tent with observations made by Canatella et al. (2004), with cells located on the outer
edges of the spheroids expressing GPF, while inner cells remaining largely unaf-
fected. After a certain threshold, application of higher electric field strengths resulted
in lower gene expression, attributed to increased cell death. Cell-cell interactions
were attributed to the differences between transfecting single cells compared to cells
aggregated in spheroid culture. While the importance of the extracellular matrix role
was suggested, no indication of its presence in the spheroid was experimentally
presented (Wasungu et al. 2009). This spheroid model offers a well-controlled
environment for studying mechanisms of electrotransfer mediated molecule delivery
(Wasungu et al. 2009).
The hanging drop method for spheroid formation was subsequently used to
evaluate gene transfer under common electrotransfer conditions (Chopinet et al.
2012). Human colorectal carcinoma cells were used to form spheroids in the same
manner as the prior study (Wasungu et al. 2009). Cells were suspended in a drop for
72 h and transferred to agar-coated wells with media, to allow maturation and growth
of large spheroids. The effects of electric pulses on permeabilization and gene
transfer were compared between single cells in suspension and cell aggregated in
3-day old spheroids. Spheroids were exposed to PI or plasmid DNA encoding GFP
dissolved in pulsing buffer on the outside of the spheroids. Different pulsing
conditions were empirically tested to delineate pulse effects on cell permeability,
viability, and gene expression. It was shown that detectable DNA interaction with
cells occurred only on the external surface of the spheroids. Less than 1% of the cells
were found to express the reporter gene indicating low transfection efficiency
possibly due to high cell mortality. It was also noted that only the external surface
cells interacted with plasmid DNA and therefore internally located cells within the
spheroid were shielded from efficient DNA uptake. In conclusion, it was reported
that this spheroid model can be adapted to various tumor cell lines in order to study
potential electrotransfer treatments without the use of animals (Chopinet et al. 2012).
The hanging drop in vitro tumor spheroid model was further used to evaluate the
relationship between tumor size and sensitivity to applied pulsed electric fields
(Gibot and Rols 2013). Two distinct spheroid generation methods were tested, the
hanging drop method as described above, and plating the same cell type (human
colorectal carcinoma cells) in a nonadherent culture well. Cells were plated in ultra-
low attachment wells of a 96-well plate then centrifuged at 300 g for 5 min. After
centrifugation induced cell aggregation, the cells were cultured for 5 days forming
spheroids. Different starting cell numbers yielded different sized spheroids using
both methods. Electric pulses were applied with parallel plate electrodes. Spheroid
growth curves were meticulously established as a measure of cell viability post
electropermeability treatments compared to untreated controls. Spheroid age and
size as variables for sensitivity to electric pulses were controlled by varying the
spheroid size with different spheroid formation methods, while maintaining the same
age. It was determined that spheroid size was the main variable in spheroid sensi-
tivity to electric pulses. Smaller spheroids were found to be more sensitive to
equivalent electric pulses than larger spheroids. A higher degree of growth delay
was demonstrated by normalized growth curves in smaller spheroids independent of
spheroid age (Gibot and Rols 2013). This study serves as another example of how a
spheroid model can be used to study effects of electric pulses on cells in a three-
dimensional environment.
The nonadherent spheroid formation technique was used to study tumor sensi-
tivity to calcium electroporation (Frandsen et al. 2015). This technique was
expanded to other tumor cell lines, including a human bladder transitional cell
carcinoma and a human breast adenocarcinoma. The nonadherent method for spher-
oid formation was also applied to fabricating human dermal fibroblast spheroids for
modeling normal tissue as a control for tumor spheroids modeling tumors (Frandsen
et al. 2015). Spheroids were subjected to calcium electroporation or bleomycin
electroporation conditions normally used in vivo for tumor treatments. Spheroid
size was measured daily after treatment and was normalized to the untreated
controls. It was determined that spheroids derived from tumor cell lines were more
sensitive to calcium and were more sensitive to bleomycin electroporation treatment
than spheroids derived from normal fibroblasts in terms of molecular uptake and
viability (Frandsen et al. 2015). It is unclear what tissue a human dermal fibroblast
spheroid can represent. Dermal fibroblasts seldom form closely packed clusters
in vivo especially in the dermis, where fibroblasts are separated by large collagen
bundles with minimal cell-cell contact. However, this study does demonstrate
another example of using tumor spheroids in vitro for modeling tumor tissue
in vivo and evaluating electroporation effects on tumor cells.
Spheroid models are excellent choices for studying electroporation effects on
solid tumors of various types due to structural and functional similarities to solid
tumors in vivo. Cell-cell interactions can closely resemble those present in tumors
in vivo, it is also known that extracellular matrix components are produced in the
microcellular environment within the spheroids. However, depending on the tissue
to be studied, the spheroid model may or may not be the appropriate in vitro model.
For studies interested in structures such as skin, blood vessels, skeletal muscle, heart
muscle, or other sites, it may be beneficial to explore other in vitro models that more
closely resemble the structure and function of the tissue of interest.
Electrospun Scaffolds for 3D Culture
Electrospinning
Electrospinning is a tissue engineering technique frequently used for fabricating

scaffolds that are biomimetic, extracellular matrix analogues. These ECM mimick-
ing scaffolds are often used for 3D cell culture in vitro and for developing implant-
able medical devices for regenerating damaged or dysfunctional tissues and organs.
The process of electrospinning depends on electrostatic field application to a charged
polymer solution. Figure 1 shows a diagram of the basic process of electrospinning.
Typically, a syringe filled with a viscose polymer solution of choice is placed into a
static electric field. A static electric field is formed by applying voltage across the tip
of the metal needle and a metal collecting target. A typical applied voltage is on the
order of 25 kV. Factors such as the distance between the polymer solution and
collecting target, the shape of the collecting target, and the amplitude of the applied
voltage determine the electrostatic force that acts on the polymer solution. Under
optimized conditions, the electrostatic force overcomes the surface tension forces at
the tip of the needle and a polymer jet extrudes from the needle towards the
collecting target. Dry polymer fibers collect on the collecting target if the solvent
is able to evaporate during the travel, therefore the distance to the collecting target
Fig. 1 Traditional electrospinning process

has to also be long enough to allow enough time for solvent evaporation. For this
reason, solvents that readily evaporate at ambient temperatures are preferred. Fibrous
scaffolds that form may consist of nonwoven fibers with diameters in the range
between 50 nm and 10 μm. Factors that contribute to scaffold fiber diameters are
polymer molecular composition, solvent type, polymer concentration, electro-
spinning solution viscosity, air gap distance, magnitude of applied voltage, relative
humidity during electrospinning, and temperature during electrospinning (Barnes
et al. 2007). Natural polymers such as type I collagen, type II collagen, type IV
collagen, laminins, elastin, fibrinogen, gelatin, chitosan, and silk fibroin have been
successfully electrospun in addition to popular synthetic polymers such as poly-
caprolactone, polydioxanone, polylactic acid, and polyglycolic acids (Barnes et al.
2007). Silk fibroin, a natural fiber with structural similarities to collagen and
excellent biocompatibility and mechanical properties, is frequently used for tissue
engineering applications (Bulysheva et al. 2012). Silk fibroin readily electrospins at
various concentrations and is soluble in many commonly used solvents. Silk fibroin
serves as a favorable substrate for cell adhesion, particularly for keratinocyte-
fibroblast cocultures (Bulysheva et al. 2012, 2013).
Electrospun scaffolds can be optimized to have specific mechanical properties
with fibers mimicking extracellular matrix fibers in fiber size and cell attachment
sites. Fiber size is dependent on the concentration of the polymer, higher concen-
tration resulting in larger fiber diameters. Cell attachment sites are provided by the
polymer molecular composition. Natural polymers normally found in the mamma-
lian extracellular matrix have abundant mammalian cell binding sites. Synthetic
polymers generally lack those binding sites but can be synthetized to mimic cell
adhesion sites. Alternatively, synthetic scaffolds may be coated with desired natural
polymers to improve cell adhesions. However, traditional electrospinning often
results in highly compacted mats of nanofibers with limited void space between
fibers. Highly compacted microstructure inhibits cell infiltration into electrospun
scaffolds, limiting in vitro and in vivo applications where cell infiltration and
vascular ingrowth are desirable, such as 3D cell culture or skin grafts (Brauker
et al. 1995; Leong et al. 2009, 2010). Increasing cell infiltration directly without
increasing spacing between fibers can be accomplished during the electrospinning
process by electrostatically spraying cells into nascent scaffolds (Stankus et al.
2007). This method requires complex protocols with sterile conditions during
electrospinning and electrospraying and further considerations for electric field
generated force on the fibers and cells. Cell viability is also a major concern.
While more cells get integrated throughout the scaffolds, the void space between
fibers remains limited. Cell migration and vascular ingrowth remain poor, limited by
the densely compacted fibers surrounding the cells (Brauker et al. 1995).
Increasing porosity can be accomplished in multiple ways. Inclusion of sacrificial
fibers has been proposed as a way to increase porosity. The sacrificial polymer is
electrospun in parallel with the desired polymer. Subsequently, sacrificial fibers are
selectively dissolved with a solvent that does not dissolve the main fiber material (Jin
et al. 2004). This approach does significantly increase porosity but not sufficiently
for adequate cell infiltration (Jin et al. 2004) perhaps due to the relatively small size
of the fibers that are washed away, thus leaving relatively small gaps between
remaining fibers. A more effective way to increase porosity and cell infiltration
has been the cotton-ball electrospinning method. It has been shown that this method
leads to scaffolds with high cell infiltration after 1 week of culture (Blakeney et al.
2011). Another effective method for increasing void space between fibers, cell
infiltration, and blood vessel ingrowth into electrospun scaffolds is
low-temperature electrospinning, which is also known as cryogenic electrospinning
and cold-plate electrospinning (Leong et al. 2009, 2010; Simonet et al. 2007).
Cryogenic Electrospinning
Low-temperature or cryogenic electrospinning was first described by Simonet et al.

(2007) as a simple process aimed at increasing void space between electrospun
fibers. The electrospinning process which is shown in Fig. 2 remains largely the
same with a small modification to the collection target. The target is maintained at a
very cold temperature usually with dry ice, while the rest of the process is maintained
at ambient temperature. Relative humidity is maintained at 30% or higher. High
humidity (>30% relative humidity) and low temperatures of the collecting target
causes quick condensation and ice crystal formation on the collecting target. Ice
accumulation and electrospinning are deliberately synchronized to occur simulta-
neously, thereby, ice crystals prevent tight packing of electrospun fibers into dense
scaffolds. Once the scaffold has formed and the electrospinning process is complete,
ice crystals can be removed either by freeze drying, allowing them to melt, or
washing them away with a warm solvent (Marrero and Heller 2012; Wasungu
et al. 2009). Care must be taken to ensure that only water insoluble polymers are
Fig. 2 Cryogenic electrospinning process

Fig. 3 Electrospinning process. Photograph of a standard electrospinner with a collector, and

polymer syringe controlled by a syringe pump inside an insulated cabinet, and a power supply
outside of the cabinet (a). Standard electrospun silk (b) compared to cryogenic electrospun silk (c)
raised to melting temperature. Water-soluble polymers must be freeze-dried prior to

cross-linking. Scaffolds must remain frozen until sublimation can occur, and freeze
thaw-cycles must be avoided. Any melting of the ice crystals prior to cross-linking
will result in full or partial destruction of the scaffold, such as electrospun silk
fibroin. Once the ice crystals are completely removed, only the polymer fibers
remain in the scaffolds, with large void spaces between the fibers. Figure 3 demon-
strates a typical electrospinner system.
Cryogenic Electrospun Scaffolds for Oral Mucosa and Skin
Cryogenic electrospinning of silk fibroin was first described by Bulysheva et al.

(2012) with applications to 3D in vitro culture of epithelial lined tissues such as oral
mucosa and head and neck carcinomas (Bulysheva et al. 2012, 2013). Silk fibroin is
a natural material widely used clinically as a biocompatible material for implantable
devices such as nonresorbable surgical suture. It is also widely used in tissue

engineering due to its similarity in structure to collagen I and therefore biomimetic
properties for cell attachment, mechanical properties, and nonimmunogenicity
(Bulysheva et al. 2012, 2013). However, conventional electrospun silk supports
low cell infiltration due to compacted fiber structure and therefore is difficult to
use for true three-dimensional cell culture. Cryogenic electrospun silk is consider-
ably more porous with significantly wider spaces between fibers. This feature was
shown to significantly enhance cell infiltration and therefore expanded the applica-
tions for electrospun silk (Bulysheva et al. 2012). Conventional and cryogenic
electrospun silk scaffolds were fabricated with the same electrospinning parameters,
including silk fibroin concentration, applied electric field, distance between the
collecting target and the polymer source, as well as translation and rotation speeds
of the target. Cryogenic electrospun scaffolds were then freeze-dried to remove any
residual water. Both scaffold types were cross-linked and disinfected in gradient
ethanol submersion and then washed with PBS and media prior to cell culture.
Immortalized dermal fibroblasts were seeded onto conventional and cryogenic
electrospun scaffolds and cultured for up to 28 days. It was shown that cryogenic
electrospun scaffolds facilitate dermal fibroblast infiltration throughout the entire
depth of the scaffold, compared to conventional silk scaffolds, which maintained
fibroblasts on the seeding surface only. Cell viability was measured with a Live/Dead
stain, quantifying the percentage of viable and dead cells contained within each
scaffold. Both electrospun scaffolds maintained high cell viability for the duration of
the study up to 4 weeks. Mechanical properties such as strain at break, elasticity
modulus, and peak stress were significantly reduced in cryo-spun scaffolds com-
pared to controls, indicating limitations to applications where mechanical strength is
not a virtue. Cryogenic electrospun scaffolds were seeded with oral keratinocytes
and dermal fibroblasts in a coculture system modeling oral mucosa. In this case,
fibroblast were seeded first and allowed to infiltrate throughout the scaffolds for
several days prior to keratinocyte cell seeding. Cryogenic electrospun scaffolds
supported high cell viability in the coculture system for up to 4 weeks. Proliferation
was evaluated with Ki67 staining, while keratinocyte differentiation was evaluated
with staining for involucrin. It was shown that keratinocytes derived from normal
oral mucosa were able to proliferate and differentiate to terminal differentiation
when cultured on cryogenic electrospun scaffolds with dermal fibroblasts at
air-liquid interface (Bulysheva et al. 2012). It was also shown, that oral carcinoma
cells behaved differently on cryogenic electrospun silk scaffolds from normal oral
mucosal keratinocytes, with high proliferation rates, high infiltration into the scaf-
folds, and low differentiation potential, as normally seen in vivo. Therefore, it was
concluded that cryogenic electrospun silk scaffolds provided a suitable 3D micro-
environment for oral mucosa studies (Bulysheva et al. 2012).
This model was modified to mimic head and neck carcinoma in 3D
culture. Studying the morphology of the tumor extracellular matrix under scanning
electron microscopy allowed for mimicking its morphology with electrospinning
parameters and assembling a silk electrospun scaffold that duplicated the native
tumor extracellular matrix in terms of fiber size, orientation, and spacing (Bulysheva
et al. 2013). Subsequent scaffold seeding with human head and neck carcinoma cells
was performed and cell infiltration, viability, proliferation, and differentiation
marker expression analysis were performed on these cells grown in 3D culture, in
classic 2D culture, and in vivo in a mouse host. Using the same cell line in all three
models allowed for a direct comparison between in vitro culture models and in vivo
situation. The 3D culture model mimicked in vivo tumorigenesis more closely than
the traditional 2D culture, in terms of gene expression profile as well as cell
proliferation. This observation was further supported by chemoresistance studies.
Three-dimensionally cultured carcinoma cells were largely resistant to up to 25-fold
increase in Taxol concentration that would completely inactivate the same cells
cultured in traditional 2D culture (Bulysheva et al. 2013).
Cryogenic electrospun silk scaffolds were subsequently used for epidermal
keratinocyte coculture with dermal fibroblasts (Sheikh et al. 2015) for modeling
skin similar to previously established mucosal keratinocyte coculture with dermal
fibroblasts (Bulysheva et al. 2012). This skin model may behave similar to the oral
mucosal model in terms of fibroblast infiltration and keratinocyte proliferation and
differentiation; however, no data was provided to prove presence of keratinocytes
after seeding (Sheikh et al. 2015). Further validation would be needed to determine
whether different epidermal strata were formed and were distinct from the dermal
strata, in order to indicate whether this 3D culture model is representative of normal
human skin. These scaffolds can easily be manufactured to a particular thickness
(e.g., 200 μm) and cut to convenient pieces for well-plate culture (e.g., 12 mm in
diameter for a 24-well plate). The mechanical properties of these scaffolds are such
that each disk with cultured cells can be transferred from well to well or from well to
electroporation cuvette. It would also be relatively easy to apply electroporation
electrodes such as parallel-plate, pin, or needle electrodes typically used for in vivo
applications to this in vitro electrospun silk model. While this is a potentially useful
3D culture in vitro model, it has not yet been used for studying electroporation
effects.
Recellularized Human Dermis
A more complete 3D culture of human skin was recently developed for evaluating
tissue response to electroporation and gene delivery (Bulysheva et al. 2016). Donor
decellularized human dermis was recellularized with human keratinocytes (HaCat
cells) and primary human dermal fibroblasts. Dermal fibroblasts were seeded at a
higher concentration on the reticular surface of the dermis and were allowed to
infiltrate the dermis for 1 week after seeding. The grafts were flipped over and
keratinocytes were then seeded on the papillary surface. There was evidence of an
intact basement membrane between the epidermis and the dermis demonstrated by
collagen IV staining and supported by distinctly separate epidermal and dermal
layers. The epidermis was further shown to contain all distinct layers characteristic
of normal human thin skin. This model was used for luciferase encoding plasmid
DNA delivery with electroporation to demonstrate its utility in studying electropo-
ration of human skin ex vivo (Bulysheva et al. 2016).
Recellularized dermis or cryogenically electrospun constructs seeded with cells

can be readily, manually manipulated to be moved with sterile forceps from well to
well, or into an electroporation cuvette or between electrodes for electroporation,
without damage to the constructs. The size of these constructs is typically chosen to
fit into the wells of a 48 or 24-well plate out of convenience considerations. Thus the
constructs are often 10–12 mm in diameter and ~0.2–0.3 mm in thickness.
Recellularized dermis constructs can also be injected with ~50 μL of DNA similar
to intradermal injections in vivo for studying gene delivery to the skin. These
considerations are important for evaluating the practicality of using these 3D
in vitro models for studying tissue response to electroporation. It would be difficult
to use the standard organotypic skin model for the same experiments with the culture
system lacking mechanical integrity.
Conclusions
Three-dimensional cell culture is becoming more popular for studying various

tissues of the human body. In particular, many 3D culture models have been utilized
for studying effects of pulsed electric fields. Primarily, the spheroid models have
contributed to improved understanding of electroporation effects on tumors. While
spheroid models are wonderful tools for studying solid tumor responses to electro-
poration, other normal tissues like skin are not well represented by spheroid cell
culture methods. Cryogenic electrospun silk scaffolds have been used for 3D culture
of oral mucosa, head, and neck squamous carcinoma and skin. These 3D culture
systems can be easily used for evaluating tissue response to electroporation. The
constructs are representative of their respective tissues, mimicking cell-cell and cell
matrix interactions while recapitulating the necessary cellular microenvironment.
Another 3D skin model was developed specifically for studying gene transfer
parameters to the skin. The recellularized human dermis contains all appropriate
epidermal and dermal layers of the skin, as well as dermal extracellular matrix. This
3D skin culture system and many other tissue appropriate models are currently
available for studying specific tissue responses to electroporation or gene
electrotransfer.
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ijpharm.2009.03.035
Mechanistic Description of Membrane
Electropermeabilization 27
Justin Teissie
Abstract
Classical cell membrane electropermeabilization of cell membrane is the result of
the delivery of electric field pulses on cells. The electric field pulse lasts from
submicro- to several milliseconds. The electric field intensity is large enough to
induce a dramatic structural local alteration of the cell membrane organization.
This results in an enhanced permeabilization of the target cell membrane for
molecules otherwise poorly transportable. This structural alteration is indeed a
complex process, and its molecular characterization remains an intense field of
investigations. The new transient organization of the cell membrane supports a
massive transport due to electrophoretic forces and diffusion-driven gradients.
This chapter describes the fast events inducing electropermeabilization or the
immediate consequences of the field-induced alteration of the membrane and
cellular organization. The methods suited to monitor these fast events are criti-
cally described as they are key factors in the accuracy of the informations. Three
steps are present in cell membrane electropermeabilization: trigger, expansion,
and stabilization. The experimental results are discussed in terms of structural
information on the new transient membrane organization. Most informations are
related to the massive enhanced molecular transport across the membrane and its
modulation by the electric field pulse delivery.
Keywords
Membrane • Transmembrane Voltage • Transport • Membrane Structure
J. Teissie (*)
Toulouse, France

DOI 10.1007/978-3-319-32886-7_39
452 J. Teissie
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
The Electric Field Induces a Transmembrane Voltage (TMV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453
The Resulting TMV Induces Membrane Permeabilization (Electropermeabilization) . . . . . . . . 454
Methods Used in Detecting and Follow-up of Membrane Electropermeabilization . . . . . . . . . . . 455
Conductance Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
Transport Assayed by Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456
Permeabilization during the Pulse Delivery Depends on the External Field Strength and
Pulse Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
Conductance Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
Induced Transport across the Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
Permeabilization as Described by its Post-Pulse Consequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
Control of Cell Membrane Electropermeabilization by the Electrical Parameters . . . . . . . . . 460
Biochemical Definition of Permeabilized Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
Structural Arrangements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
Biphotonic Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
Nuclear Magnetic Resonance (NMR) Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464
Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464
Fluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464
Lateral Mobility of Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
Atomic Force Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
Interfacial Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
Modes of Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466
Modulation of Electropermeabilization by Physicochemical Factors . . . . . . . . . . . . . . . . . . . . . . . . . . 467
Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467
Buffer Osmotic Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467
Buffer Ionic Strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468
Nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468
Cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468
Membrane Order . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471
Introduction
This chapter describes the events occurring along classical electropermeabilization

(electropermeabilization). This is what is observed with a field range of
0 < E < 10 kV/cm for mammalian cells (diameters between 10 and 30 μm) and
pulse durations larger than submicros (therefore classical electropermeabilization
included some experiments called nsPEF (nanosecond pulsed electric field) when
the duration is larger than 100 ns) (Deng et al. 2003; Son et al. 2014). This is
obtained with pulse generators with square wave profile rising time larger than
0.1 μs.
Electric pulses when delivered to a cell suspension or a tissue result in a
membrane permeabilization (electropermeabilization). Classical electroperme-
abilization results in an enhanced transport of poorly permeable ions and molecules
27 Mechanistic Description of Membrane Electropermeabilization 453
across the membrane and in a change in its electrical conductivity. Electro-

permeabilization can be reversible if the pulsing parameters are selected in a proper
way. This reversibility is called resealing (resealing). This means that an enhanced
membrane permeabilization is transiently induced while preserving the cell viability.
More drastic pulsing conditions can make this permeability irreversible (cell lysis
would result) (Rems and Miklavcic 2016). This chapter is an attempt to make a state
of the art in the field by pointing out the most recent observations. More classical
information can be read in the works of the leading groups in the field during the last
40 years (see “Cross-References” at the end of the chapter).
The Electric Field Induces a Transmembrane Voltage (TMV)
When a cell is subjected to an external electric field, the cell (where the plasma
membrane can be considered as a dielectric shell) can be considered as a spherical
transmembrane voltage (TMV) (transmembrane voltage). During the electric field
application (electropulsation) (electropulsation) on the cell, an induced transmem-
brane voltage (ΔVi) is created which is locally associated with the dielectric prop-
erties of the plasma membrane. Using a physical model based on a thin, weakly
conductive shell (the membrane, conductivity λm), full of an internal conductive
medium (the cytoplasm, conductivity λi), and bathed in an external conductive
medium (conductivity λe), solution of Laplace’s differential equation gives ΔVi as:

ΔViðM, Ε, tÞ ¼ f gðλÞrE cosθðMÞ 1 expðt=τmÞ (Eq:1)
where M is the point on the cell that is considered, t is the time after electropulsation
is turned on, f is a factor depending on the cell geometry (for a sphere, f = 1.5), r is
the radius of the pulsed cell, E is the electric field strength, and θ(M) is the angle
between the direction of the field and the normal of the cell surface in M. g(λ) is
related to the different conductivities as (Zimmermann et al. 1947)
h i
g ð λÞ ¼ 2λe 2λm þ λi þ ðλm λiÞðr d=rÞ3 3λmðr d=rÞ
h i (Eq:2)
= ð2λe þ λmÞð2λm þ λiÞ þ 2ðr d=rÞ3 ðλi λmÞðλm λeÞ
where d is the thickness of the membrane (a few nm). τm is the characteristic time
constant of the membrane charging and can be written as (Kinosita and Tsong 1977):
τm ¼ rCmð2λe þ λiÞ=ð2λeλiÞ (Eq:3)

454 J. Teissie
Fig. 1 Modulation of the TMVof a spherical cell by an applied external field. The TMV point from
negative to positive and their lengths are proportional to the voltage. The large arrow on the top
represents the electric field lines. On the left, a cell is pictured with its homogeneous resting voltage;
the middle is indicative of the field-induced TMV that is highly position dependent along the cell
surface. On the right, the resulting modulation of the TMV when the field is present, a highly
complex position-dependent TMV, is observed
If the plasma membrane is considered to be a pure spherical dielectric (λm = 0), we

obtain g(λ) = 1. In these conditions and when the steady state is reached (t >> τm)
as for millisecond pulses, Eq. 1 simplifies to:
ΔViðM, EÞ ¼ 1:5rEcosθðMÞ (Eq:4)
When living cells are electropulsed, ΔVi adds to the resting one ΔV0. This gives
an additive effect on one side of the cell and a subtractive one on the opposite. The
electric field effect is strongly position dependent (Fig. 1).
The Resulting TMV Induces Membrane Permeabilization

(Electropermeabilization)
When the new membrane electric potential difference ΔVm (ΔV0 + ΔVi) locally
reaches a critical value (ΔVc) (nowadays estimated between 0.25 and 0.6 V for
living cells) (Rems and Miklavcic 2016), a local alteration of the membrane structure
leads to membrane permeabilization. Molecular processes supporting classical
electropermeabilization (electropermeabilization) remain poorly understood (Teissie
et al. 2005) in spite of more than 40 years of effort (Neumann and Rosenheck 1972).
The membrane is called “porous.” There is a general agreement that it cannot be
described by the occurrence of reversible hydrophilic holes, so-called pores (“elec-
troporation”) (electroporation), in the lipid bilayer. A membrane of a cell is a more
complex system. It is an out of equilibrium complex from a thermodynamic point of
view. It forms a complex with the extracellular matrix and the cytoskeleton. Its
transmembrane voltage is dependent on the cellular energy reserves.
If at any point ΔVm gets high enough to induce sufficient number of conductive
defects, the defects allow enhanced ionic transport through the membrane and
increase the membrane conductivity by several orders of magnitude. This in turn
partially discharges the membrane and thereby reduces ΔVm during the pulse. In
such case, the membrane conductivity has to be considered as a function of ΔVm,
which will be in more details described in other chapters (see “Cross-References” at
the end of the chapter).
Electropermeabilization is described as a local event in the cell surface.
Membrane permeabilization is associated by an enhanced transport of water-
soluble small molecules (that are of a low molecular weight, say less than 2 kD) and
ions. It is a time-dependent process, and its kinetics can be observed by monitoring
the electrical conductivity change in the membrane or the change in the transport
parameters. It can be assayed by the inflow of dyes (trypan blue, propidium iodide,
and similar) by observation under the microscope or with a flow cytometer.
Permeabilization can then be quantified by two parameters: the number of cells in
the population where the uptake can be detected and the amount of uploaded dyes
(i.e., the distribution of fluorescence intensity in each single cell in the population).
To follow the onset of electropermeabilization which occurs during the pulse
delivery, the observation is on the submillisecond time range. But in most experi-
ments, it results in an enhanced transport (transport) during the post-pulse step that is
observed. But this is affected by the resealing (see “Cross-References” at the end of
the chapter). Different biophysical approaches monitor the transport as mentioned
above. They are shortly described in a first part of this chapter. They are used to
analyze the processes and their control by biophysical and physiological parameters.
A lot of studies have been developed on pure lipid assemblies (planar lipid
membrane, liposomes). Processes on such physical models are relevant of soft
matter physics and would not be considered in the present chapter. Only events
occurring in living cell membranes will be approached.
Computer-assisted simulations of transvoltage effect on lipid bilayers are not
considered relevant of classical electropermeabilization (different pulse duration,
much higher equivalent field strength).
Methods Used in Detecting and Follow-up of Membrane

Electropermeabilization
Conductance Changes
Direct Assays by Electrical Measurements

access to the conductance of the suspension (conductance). Under steady-state
condition, the conductance of a diluted intact cell suspension can be considered
controlled by the conductance of the external solution. An ohmic behavior is
observed when a voltage pulse is delivered on the cell suspension (as long as the
Joule heating is small).
456 J. Teissie
(i) The dielectric shell has conductive defects that increase during the pulse
(ii) There is leak of the internal (cytoplasmic) solution
Both events result in an increase in the cell suspension conductivity. This is a

very fast process that can be detected on the submicrosecond time scale after
correcting from the interfacial electrochemical reactions on the electrodes (Kinosita
and Tsong 1979).
Indirect Approaches by Fluorescence

Membrane-embedded fluorescent probes have their emission controlled by the
transmembrane voltage by a direct effect of the transmembrane field (Hibino et al.
1991). The probe emission change linked to the alteration of the membrane conduc-
tance (that affects the transmembrane field) is very fast and closely follows the
change in conductance. A very fast detection is needed where short (ns to μs) laser
flashes triggered at delayed times after the onset of the field pulses are used to
stimulate the emission (Frey et al. 2006; Hibino et al. 1991).
Transport Assayed by Fluorescence
Membrane permeabilization is associated to an enhanced diffusion-driven transport

of water-soluble molecules and ions following the physical laws of diffusion (Fick’s
equation) (diffusion). This is a time-dependent process, and its kinetics can be
observed by monitoring the inflow of a fluorescent molecules. In most cases,
molecules such as propidium iodide are used as their fluorescence emission is
strongly enhanced in the cytoplasm due to their binding to nucleic acids. Transport
is assayed online with time resolution down to the us (Pucihar et al. 2008) or imaging
on the ms range as shown by video imaging in the late 1990s (Table 1).
In most cases, it is the post-pulse transport that is assayed as it is a result of the
structural alterations present during the pulse delivery (see “Cross-References”).
Table 1 Synthetic view of the different techniques which are used to investigate membrane
electropermeabilization (strength and limits, main results)
Methods Temporal resolution Spatial resolution Molecular information
Conductance Fast (microsecond) Low (on population) average size of defects
Patch camp Slow (second) Low (on single cell) molecular definition
of defects
Fluorescence Fast (microsecond) 1 μm on single cell transport
NMR very slow (hours) Average information tilt of lipid headgroups
SEM very slow High (but fixed sample) ultrastructural defects
AFM very slow High ultrastructural defects
Permeabilization during the Pulse Delivery Depends

on the External Field Strength and Pulse Duration
Conductance Changes
Electrical Measurements
Conductance changes during the pulse can be observed on cell suspension only
when the field is larger than a threshold (critical value Ec) (Kinosita and Tsong 1979)
(conductance). The increase is time dependent during the delivery of the pulse. Its
magnitude is dependent on the electric field intensity. The relative conductivity
changes during the application of a rectangular field pulse between 0.75 and
1.2 kV/cm, respectively, with a duration of 1 ms to a mammalian cell dense
suspension (pellet) in a low conductivity isosmotic buffer sucrose are also a very
fast increase. The conductance increase can be detected within the first μs of the
pulse delivery after correcting for the electrodes interfacial contribution to the
electrical signal. A plateau in the conductance change is observed for long pulses
in conductive buffers.
Imaging Observations
The use of a very fast fluorescence imaging microscopy connected to a pulse generator
gives access to the conductance changes (Hibino et al. 1991) (imaging). A voltage-
sensitive fluorescent dye was used an indicator of the time-dependent transmembrane
change induced by the external electric field on sea urchin eggs. But when the field
was over a critical value, the transmembrane voltage kept locally a constant value on a
cap facing the electrodes. A high membrane conductance of the order of 1 S/cm2 is
therefore present within 2 μs after the onset of the external field. The size of the
conductive cap is determined by the strength of the external field. This high local
membrane conductance affects the electric field-induced transmembrane voltage as
predicted by Eq. 1. A steady value is observed in the cap where the transmembrane
conductance is obtained (Hibino et al. 1993). Only 0.5 microseconds after the onset of
the rectangular electric pulse, the two sides of the cell facing the electrodes are
affected, and a high membrane conductance in the order of 1 S/cm2 is measured; an
asymmetric process is observed; a higher conductance is detected on the positive side.
Along the pulse delivery (the external field remaining constant), a further increase in
the conductance is detected, reaching the order of 10 mS/cm by 1 ms. The kinetics of
the conductance increase is different on the two opposite sides of the cell, being faster
on the negative-electrode side; as a result at the end of a 1 ms pulse, the conductance
on the negative side is more than twice that on the positive side. Different processes
clearly support electropermeabilization on the opposite caps of a pulsed cell.
Induced Transport across the Membrane
The transport of a water-soluble dye (propidium iodide, PI) induced by electro-

permeabilization was monitored under an ultrafast fluorescence microscope
458 J. Teissie
(bandwidth larger than 1 MHz) during the electric pulse on a cell population of
mammalian cells as well as at the single cell level (Pucihar et al. 2008) (propidium
iodide). The transport became detectable as early as 60 μs after the start of the pulse
(transport). Transport is delayed as compared to the enhanced membrane conduc-
tance. After the delay of 60 μs, the time course of fluorescence during the pulse was
approximately linear, suggesting mainly electrophoretic transport of molecules
during the pulse delivery lasting up to 1 ms.
Transport through these pathways can occur by different mechanisms during and
after a pulse. To determine the time scale of transport and the mechanism(s) by
which it occurs, efflux of a fluorescent molecule, calcein, across erythrocyte ghost
membranes was measured with a fluorescence microscope photometer with a fast
(millisecond) time resolution during and after electric pulses that lasted several
milliseconds (Prausnitz et al. 1995). Only a reduced leak was observed during the
pulse, and most of the efflux from the ghost was after the pulse. Transport caused by
electropermeabilization occurred predominantly by electrophoresis and/or electro-
osmosis during a pulse, although transport occurred in part or almost completely by
diffusion within milliseconds to seconds after the pulse (electrophoresis,
electroosmosis).
On a shorter time scale (microsecond), the course of electropermeabilization was
analyzed during the electric field application using a rapid single cell fluorescent
imaging system. Interaction and penetration of propidium iodide, with and across
cell membrane, was asymmetrical during electropermeabilization. Localized
enhancement of the dye fluorescence was observed during the pulsation on the cell
surface and in the cytoplasm. But this was observed only when the amplitude of the
external field was higher than a critical value. Specific staining of a limited anode-
facing cell volume was observed during the first milliseconds when the pulse was
applied. The membrane fluorescence level increased linearly during the pulse deliv-
ery, whereas the geometry of the staining was unchanged. The fluorescence in the
cap increased linearly during the pulse delivery (lasting up to 20 ms), as expected
from an electrophoretic drift. The fraction of the membrane on which structural
alterations occurred, Ap, was defined by the electric field strength E. It was a linear
function of the reciprocal of the electric field strength.
Ap ¼ A0ð1 Ep=EÞ (Eq:5)
where Ep is the critical field strength needed to trigger membrane

The size of the cap where permeabilization is present increased with an increase
of the field strength as soon as the field was larger than the critical field Ep (Fig. 2).
The density of defects supporting the transport across the permeabilized mem-
brane cap was governed by the pulse duration (Fig. 3).
Electropermeabilization is a localized but asymmetrical process (electro-
permeabilization). Transport was not the same on the two cell sides. The membrane
defects (defects) are created unequally on the two opposing sides of the cell during
the pulse, implying a vectorial effect of the electric field on the membrane.
Fig. 2 Effect of the field strength on the size of the electropermeabilized cap. Cells are pictured as a
sphere. Field pulses are delivered at a constant pulse duration. No permeabilization is present before
the pulse delivery (left side of the cartoon). Low field has no effect on the membrane organization.
As soon as the field induces a permeabilizing TMV, i.e., the field strength is larger than Ep, a cap is
brought to the “permeabilized state” (a gray cap on the cartoon). The size of this permeabilized cap
increases with a further increase of the field strength
Fig. 3 Effect of the pulse duration on membrane electropermeabilization. Cells are pictured as a
sphere. No permeabilization is present before the pulse delivery (left side of the cartoon). As soon as
the field induces a permeabilizing TMV, a cap is brought to the “permeabilized state” (the gray cap
on the cartoon) (trigger step). As long as the field is present with a constant strength, an increase in
the defects supporting the permeabilization occurs shown by darker levels in the gray levels of the
cap. This is called the expansion step
Exchanges of calcium ions through electropermeabilized membrane of Chinese

hamster ovary cells were found to be asymmetrical. Entry of calcium ions during a
millisecond pulse occurred on the anode-facing cell hemisphere. The exchanges
during the pulse were mostly by an electrophoretic drift. The membrane regions
stained by propidium iodide uptake were the same as those where calcium exchanges
occurred. Positively charged species are electrophoretically pushed in the
cell cytoplasm on the anode-facing cell hemisphere. Electropermeabilization is
facilitating the electrophoretic movement of any kind of small species across a
membrane cap controlled by the field strength during the pulse delivery.
460 J. Teissie
Water transport was detected resulting in huge osmotic swelling of the cell when
using ms pulses (a high level of permeabilization) (swelling). This is easily pre-
vented by adding large water-soluble additives in the pulsing buffer (such as sucrose
or polyethylene glycol, PEG).
Permeabilization as Described by its Post-Pulse Consequences
Most studies are not on the events during the pulse but on the observations of cells that
had just been pulsed. The consequence is that it is not the direct effect of the onset of
electropermeabilization that is observed but its synergy with the resealing process that
follows. What is then observed is the percentage that have been permeabilized and the
level of the inflow of the reporter molecules (Canatella et al. 2001; Pucihar et al. 2008).
The last parameter is indeed strongly controlled by the resealing. A more relevant
approach of the onset of permeabilization during the pulse is to observe the subpop-
ulation of permeabilized cells in the total population of pulsed cells. One limit is of
course the sensibility of the assay to detect permeabilized cells. Results were observed
to be in many cases different by using different assays. The inflow is under the control
of the permeability coefficient of the reporter molecule (roughly dependent on the
size). The number of molecules that can be detected inside the cytoplasm is controlled
by the method of detection. For example, detection is obtained under less stringent
conditions when using PI penetration (fluorescence detection) rather than trypan blue
labeling (colorimetric method) (detection).
Control of Cell Membrane Electropermeabilization by the Electrical

Parameters
Field Strength
The electric field strength of the pulses delivered on the cell suspension was observed
to be a critical parameter to trigger cell membrane permeabilization (detected in most
cases by the uptake of a fluorescent dye). Transport is indeed controlled by the field
strength E. Fluorescent-positive cells (i.e., permeabilized) induced by the pulse are
detected only when the intensity of the pulse is larger than a critical threshold
(Ep) (note: in a cell population, a percentage of cells has a leaky membrane where
the dye uptake spontaneously takes place. They are observed in the control cells. This
must be corrected from the observed permeabilization). For a given cell (size r) and a
given pulse duration (T), Ep induces the permeabilization on a cap with a critical size,
across which transport can be detected. An accurate definition should be Ep(r,T).
A fairly linear relationship is observed between permeabilization and the recip-
rocal of E (Eq. 5). This supports the online observation that a cap on the cell surface
is brought to the permeabilized state by the pulse with a size controlled by the field.
This cap is where the critical permeabilizing TMV was reached along the pulse. At
high field, all cells are permeabilized, as Ep(r) is obtained for all values of r in the
population.
Pulse Duration
When the field strength of the delivered pulses is larger than this critical strength Ep,
it is observed that the percentage of cells detected as permeabilized is increased with
an increase in the pulse duration. Transport is controlled by the pulse duration T
(duration). This increase is detected to level off at a plateau value close to 100% (all
cells are detected to be permeabilized when long pulses are delivered).
Interestingly, the critical parameter Ep is observed to decrease with an increase in
the pulse duration. A linear relationship is observed between Ep(T) and the recipro-
cal of the pulse duration (T). This gives access by extrapolation to a limiting value of
Ep (for an infinite duration). This may result from several factors such as:
– The density of defects increases with the pulse duration in the cap where the
critical TMV is triggered (defects). The inflow increases as a consequence and
detection is obtained with a very limited permeabilized cap.
– Due to the electrophoretic drift of membrane proteins, a microdomain is created
that is more sensitive to the field-induced TMP.
– Due to the electromechanical stress on the cell, membrane stretching occurs, and
the cap facing the electrode has a forced curvature making it more fragile.
– The transport by the electrophoretic forces during the pulse is high enough to give
a detectable accumulation of the reporter dye.
Repetition of Pulses
In most studies, not only a single pulse is delivered to the cells, but a train of pulses is
applied where two complementary parameters are to take into account: the number
of successive pulses and delay between each single pulse (frequency) (delay).
Conductance measurements give a direct access of the effect of the repetitive
pulses. It is observed that the membrane conductance is further increased by
the repetitive pulses but that a partial resealing occurs during the delay (Kinosita and
Tsong 1979). More conducting defects are induced along a repetition of pulses.
The local defects are considered part of the structural membrane reorganization
induced by the external field. They remain present for a few seconds after each single
pulse. Their density is dependent on the number of pulses. Cumulative effects are
shown by an enhanced transport and are observed when repeated pulses are applied.
Transport therefore increases with the number of pulses. More cells are then detected
as permeabilized. Again a plateau level is detected when a large number of pulses are
delivered. Indeed, this brings a result where all cells are permeabilized.
Delay between Pulses

The delay plays a role in the final state of pulsed cells. This is a complex process that
is not clearly explained at the present state of our knowledge (so-called electro-
sensitization) (electrosensitization).
At a constant number of pulses/pulse duration product, transfer of molecules is
strongly affected by the time between pulses. Permeabilization is observed to be
higher when a low number of long pulses are delivered with a long delay rather than
a repetition of short pulses.
462 J. Teissie
Orientation of Pulsing
The electric field being a vector has a given orientation (perpendicular to the
electrodes when they are plane parallel). Cell electropermeabilization is triggered
by an electric field; its characteristics should depend on its vectorial properties. The
direction of the field is easily changed by moving the position of the parallel
electrodes relative to fixed cells (either plated or anchored to a dish). Four different
pulse sequences were compared: normal (pulses all in one given direction), crossed
(pulses delivered in two perpendicular directions), inverted (pulses with opposite
polarities), and crossed-inverted (pulses delivered in two perpendicular directions
with opposite polarities). Under non-saturating conditions, the normal condition was
observed to be the less effective conditions, while the highest effect was obtained
under the crossed-inverted one. Permeabilized caps are formed only on one side of
the cell under the less effective conditions (field strength close to Ep), while they
appear on four different places on the cell surface under the most effective setting.
Multiple studies have shown that delayed bipolar (BP) electric pulses in the
microsecond range are more effective at permeabilizing cells as compared to mono-
polar (MP) pulse equivalents. To study permeabilization effectiveness, MP or BP (with
no delay in the inversion) pulses were delivered to single Chinese hamster ovary
(CHO) cells, and the response of propidium iodide (PI) or calcium uptake was
measured by confocal microscopy. Results showed that BP pulses were less effective
at increasing intracellular calcium concentration or PI uptake than MP pulses. Flow
cytometry analysis of CHO cells after exposure (at 15 min) revealed that to achieve
positive PI transport, ten times more BP pulses were required than MP pulses. Overall,
unlike longer pulse exposures, BP nsPEF exposures proved far less effective at both
membrane permeabilization than MP nsPEF (Ibey et al. 2014). The uptake of calcium
ions was decreased by more than an order of magnitude by the delivery of a second
one of opposite polarity immediately after a first pulse. This effect reflects the vectorial
character of the electrophoretic transport of ions through the electropermeabilized caps
in the membrane during the two parts of the bipolar pulse. This observation supports
the conclusion that electrophoresis is the dominant transport mechanism during the
pulse delivery (where permeabilization is created), rather than diffusion that is driven
by the concentration difference. By delivering the second pulse with a delay after the
first one, the effect of reverse electrophoresis disappears (electrophoresis). This delay
is very short (in the hundred microseconds range) and illustrative of a very fast
recovery of the membrane perturbation supporting the electrophoretic transport.
Biochemical Definition of Permeabilized Structures
Lipids
Lipid unilamellar vesicles (LUV) (vesicles), physical models of cells but composed
of pure lipids, are quite suitable systems as their size is close to the one of cells. Their
molecular packings are similar to what one expects to be present in the plasma
membrane. When submitted to short (where stretching is avoided) high field pulses,
LUVare transiently permeabilized. Leakage of trapped sucrose can be detected when

the field strength is larger than a critical value (Ep). No global and permanent
damages to the vesicle bilayer are detected. Electropermeabilization is therefore
obtained in a lipid assembly. Lipid domains when present in cell membrane are
putative target of electropermeabilization.
Proteins
Electropermeabilization of red blood cells was supported by the associated cell

osmotic swelling. It was observed that in a low ionic medium, at least 35% of the
conductive defects were related to the opening of Na+/K+ ATPase channels. This
conclusion was supported by the partial blockage of the membrane conductance
generated by the externally applied electric field pulse by a specific inhibitor,
ouabain. A similar inhibition of the conductance was obtained when using a specific
cross-linking reagent, Cu++ phenanthroline, of the ATPase. Interestingly this
effect was not observed in a high ionic medium as expected for the effect of the
cross-linker. A large fraction of the voltage-induced defects that occurred are still
unidentified. The effects of large magnitude transmembrane voltage pulses on
voltage-gated Na and K channel behavior were studied in frog skeletal muscle
membrane of isolated fibers (Chen and Lee 1994). A 4 ms transmembrane potential
pulse of 600 mV reduces both Na and K channel conductivities and the ionic
selectivity of the K channels against Na + ions. This reflects an electro-
conformational damage to ionic channels in parallel to the induction of defects in
the lipid bilayer. Classical effectors such as tetrodotoxin (TTX) and tetra-
ethylammonium (TEA) binding effects were unaltered. By the whole-cell patch-
clamp method investigation on neuronal cells, currents of voltage-gated
(VG) Ca(2+) and Na(+) channels (I(Ca) and I(Na)) in cultured GH3 and NG108
cells were observed to be inhibited when cells were pulsed with submicrosecond
fields at or above 1.5–2 kV/cm. The field pulses caused prolonged inhibition of
I(Ca) and I(Na). A “leak” current (I(leak)) was present indicative that other field
targets were present in the plasma membrane.
Structural Arrangements
Biphotonic Microscopy
Molecular center asymmetry is required for the creation of second harmonic

generation (SHG) (second harmonic generation) signals. It makes this spectroscopy a
powerful technique for visualizing changes in interfacial layers in the
plasma membrane of biological cells. Lipophilic SHG probes, such as Di-4-
ANEPPDHQ (Di-4)(pyridinium, 4-(2-(6-(dibutylamino)-2-naphthalenyl)ethenyl)-
1-(3-sulfopropyl)-, hydroxide), can detect instantaneous/immediate perturbations in
the plasma membrane. Rapid changes in membrane symmetry could be detected
using SHG (Moen et al. 2014). Following pulsed electric fields exposure, an
464 J. Teissie
instantaneous drop of ~50% in SHG signal was detected only from the anodic pole of
the cell. This dramatic loss in SHG signal reflects a local dramatic perturbation in the
interfacial nature of the membrane.
Nuclear Magnetic Resonance (NMR) Spectroscopy
31P NMR spectroscopy gives structural informations on the lipid polar head region
(nuclear magnetic resonance). Chinese hamster ovary (CHO) cells were reversibly
permeabilized by submitting them to short, high-intensity, square wave pulses
(1.8 kV/cm, 0.1 ms). Due to these pulsing conditions (high field, short duration), a
large cap was electropermeabilized covering the majority of the cell surface, but the
population viability was preserved. It was taken advantage from the observation that
cells remained in a permeable state without loss of viability for several hours at 4 C.
A differential method in the NMR pulse sequence was used in 1987 to analyze the
phospholipid head groups and to get rid of the signals coming from other phosphorus
groups. Control cells displayed the classical bilayer signature. A new anisotropic
peak with respect to control cells was observed on 31P NMR spectroscopic analysis
of the phospholipid components. This peak is only present when the cells are
permeable, and normal anisotropy is recovered after resealing. This field-induced
anisotropic peak was located downfield from the main peak associated to the
phospholipids when organized in bilayers, but its localization peak is very different
from the one of a hexagonal phase. This new transient signature associated to the
electropermeabilized state can be explained by a new erected orientation of the lipid
polar heads or by a random distribution of their orientation. This reorganization of
the polar head group region results in a weakening of the hydration layer.
Electron Microscopy
Ultrastructural changes of the plasma membrane were detected just after the pulse
delivery by scanning electron microscopy (scanning electron microscopy). Cells
were chemically fixed a few seconds after their exposure to electric pulses in order to
investigate the ultrastructural changes associated with the reversible permeabi-
lization and observed by electron microscopy. By scanning electron microscopy,
numerous microvilli and blebs were observed immediately after application of the
field. Blebbing was facilitated under hypoosmolar conditions suggesting that they
were assisted by the osmotic swelling induced by the membrane permeabilization
(Gass and Chernomordik 1990). The appearance of osmotic pressure-dependent
“blebs” was indicative of local weakening of the plasma membrane.
Fluorescence Microscopy
Studies using fluorescence microscopy with 1-palmitoyl-2-{6-[(7-nitro-2-1,3-

benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (C6-NBD-PC),
a phospholipid analog, monitored phospholipid scrambling of mammalian cells

(fluorescence microscopy). Millisecond permeabilizing pulses induced membrane
disorganization by increasing the translocation of phosphatidylcholines according to
an ATP-independent process. These pulses resulted in a rapid phospholipid flip/flop
within less than 1 s and were exclusively restricted to the regions of the
permeabilized membrane. The electrically mediated local membrane disorganization
was not associated with a loss of cell viability. The occurrence of such a flip-flop
supports the existence of direct interactions between the movement of membrane
zwitterionic phospholipids and the electric field.
Lateral Mobility of Membranes
Lateral movement in each layer of the plasma membrane is present in all cell
membranes. They are measured by using fluorescence recovery after photo-
degradation (FRAP). Electropermeabilization by ms long pulses affects the fast
lateral mobility of a glycosylphosphatidylinositol (GPI)-anchored protein (Rae-1).
The mobile fraction is indicative that 10–20% of the membrane surface is affected by
defects under these conditions (where only a moderate field intensity was used to
preserve the cell viability). These structures, which support the membrane perme-
ability, propagate rapidly (<1 min) over the cell surface after the pulse.
Atomic Force Microscopy
Atomic force microscopy (AFM) directly visualized the consequences of electro-

permeabilization in terms of membrane reorganization (atomic force microscopy). The
classical approach is to obtain morphological changes, and transient rippling of membrane
surface was detected in the minutes following the pulse delivery. A more sophisticated
approach gave information on the changes in nanorheology as one can locally measure the
membrane elasticity. Permeabilizing pulses lasting several milliseconds with field intensity
preserving the cell viability were delivered on CHO cells. The morphology of cells was
preserved. A decrease in membrane elasticity by 40% was measured (Chopinet et al.
2013). This was observed both on chemically fixed and living CHO cells. This observation
supports the occurrence of a modification affecting the entire cell surface suggesting that a
cytoskeleton (a key feature in membrane stiffness) destabilization took place. A puzzling
conclusion was that the mechanical change was homogeneous on the cell surface in the
few minutes following the pulse delivery with no correlation with the part of the membrane
that was detected as electropermeabilized by the fluorescence assay.
Interfacial Modifications
The organization of the membrane water interface gives rise to a set of different
forces that are called hydration forces (Leikin et al. 1993). They are responsible for
the non-spontaneous fusion of two cells when their respective membranes are
466 J. Teissie
brought in close contact. A strong repulsion is detected when the intermembrane

space is reduced to a few nanometers. They are described as resulting from a local
organization of the interfacial water. Water dipoles are sensitive to the local electric
field, and as a result, a regular organization of the water takes place. The associated
dipolar fields on the two membranes are in opposite directions during the close
approach. When cells are electropermeabilized, these forces vanished, and a spon-
taneous fusion is detected (Sowers 1986). This is observed during the pulse delivery
but remains present in the few seconds following the pulse delivery. This is indic-
ative that the structures supporting these repulsive forces, i.e., the regular
organization of interfacial water molecules, have been altered under the electro-
permeabilization processes. This interfacial modification is restricted to the electro-
permeabilized part during and in the few seconds following the pulse delivery.
Modes of Transport
Transport during the onset of electropermeabilization (when the external field is

present) occurs by electrophoretic forces acting on charged molecules (Prausnitz
et al. 1995) (transport). Indirect evidences (transdermal delivery) are indicative that
the transport of neutral molecules (mannitol) during the pulse delivery is very low.
Different patterns are observed depending on the size of the molecule that is
transferred.
For small molecules (using PI as a model), observed during the very first
milliseconds during the pulse, the field strength controlled the part of the cell surface
where the permeabilized state was detected. These caps are where transport occurs.
After a short delay, a linear increase in fluorescence is observed during the pulse
illustrative of an electrophoretic drift across the electropermeabilized cap of the cell
membrane. After the pulse, transport occurs by diffusion.
Small interfering ribonucleic acids (siRNA) are 20–22 bp oligonucleotides that
can achieve a transient silencing in protein expression. Electrotransfer of siRNA is a
very efficient method for its introduction in the cytoplasm to obtain silencing.
Electrotransferred siRNA passed through the plasma membrane of cells only during
the pulse leading to electropermeabilization. The siRNA, being negatively charged,
migrated against the field direction by electrophoresis and penetrated the cytoplasm
(Paganin-Gioanni et al. 2011). The transfer results from the electrophoretic drift in
the buffer and across the electropermeabilized cap of the cell membrane. This
conclusion was further supported by quantification of the intracellular siRNA-
associated fluorescence that is a linear function of the number of successive pulses
in a train and by the observation that inversion of the electric field polarity led to a
penetration of the siRNA on opposite sides of the cell in the cytoplasm. After the
pulse delivery, a free diffusion is present in the cytoplasm but no further entry
(electrophoresis).
In the case of deoxyribonucleic acid plasmid (pDNA), the field pushes the
oligonucleotides (a charged polymer) toward the membrane where their interaction
on the electropermeabilized part of the cell membrane results in the formation of
long-lived localized spots. This pDNA/membrane interaction occurs on the side

where they are accumulated by the electric field-associated electrophoretic drag.
This local accumulation, present only during the pulse, is therefore controlled by the
pulse duration and by the number of pulses. No transfer occurs during the pulse.
Translocation within the cytoplasm was another step that happened several minutes
after the pulse train.
Modulation of Electropermeabilization by Physicochemical

Factors
Temperature
Electropermeabilization is a collective structural change in the membrane organiza-

tion and therefore is temperature dependent. Results on the temperature effect during
pulse application on cell membrane permeabilization were determined in two dif-
ferent cell lines: V-79 and B16F-1 (Kanduser et al. 2008). Cells were trypsinized to
be brought in suspension. The cell membrane electropermeabilization was deter-
mined by the toxicity following the uptake of bleomycin (clonogenic assay). Via-
bility was preserved only on non-permeabilized cells due to the high cytotoxicity due
to bleomycin uptake. A train of eight rectangular pulses lasting 0.1 ms were
delivered at 1 Hz with increasing amplitudes between 500 V/cm and 900 V/cm.
The cells were incubated at the experimental temperature and pulsed, and immedi-
ately after the pulse application, the treated cell suspension was maintained at room
temperature in order to allow cell membrane resealing. The main conclusion was that
chilling of cell suspension, during the pulse train delivery from physiological
temperature (of 37 C) to 4 C, led to a lower percentage of cell membrane
permeabilization (a higher cell survival). The differences were the highest under
the more drastic conditions where a large number of cells were affected by the
electrical treatment (electric pulse amplitude of 900 V/cm). A loss of membrane
dynamics (obtained at low temperature) makes cell membrane less sensitive to
Buffer Osmotic Pressure
The effects of pulsing buffer osmotic pressure on electric field-induced permeabi-

lization were analyzed on Chinese hamster ovary cells growing either in monolayers
or in suspension. Osmotic pressure has no effect on the induction step of permeabi-
lization, but its increase was shown to inhibit the expansion step for pulses lasting
0.1 ms (osmotic pressure).
When cells were subjected to long (ms) square wave pulses, a condition mediat-
ing a high gene transfer, no effect on cell permeabilization for a small molecule such
as propidium iodide was observed. A cell swelling is detected under such long pulse
conditions. Its magnitude is controlled by the osmotic pressure. But the use of a
468 J. Teissie
hypoosmolar buffer during pulsation allows more efficient loading of cells with
macromolecules such as beta-galactosidase, a tetrameric protein. This is indicative
that the swelling was associated with an increase in the structural changes during the
pulse.
Buffer Ionic Strength
The impact of external medium conductivity on the efficiency of the reversible

permeabilization caused by pulsed electric fields is complex. Of course a trivial
effect is the Joule heating during the pulse delivery that increases with the conduc-
tivity. Temperature effects on electropermeabilization are therefore present.
Increasing the ionic strength of the pulsing medium results in an increase in
sieving of the permeabilizing electrodefects (expansion step). For permeabilization
linked to the delivery of one single pulse of 0.1 ms, media of lower conductivity
induced more efficient permeabilization to bleomycin independently of the medium
composition (Silve et al. 2016). Treatment of cells with trypsin or pronase before
application of the pulses abolishes the ionic modulation of both electro-
permeabilization. A similar rate of expansion of permeabilization is obtained what-
ever the ionic content of the pulsing buffer. The proteolytic manipulation of the
extracellular matrix modulates the process of electropermeabilization suggesting its
control by surface membrane proteins. It stresses that the conductivity of the medium
affects permeabilization by several ways. As the energy delivered by pulses
increases with the ionic content of the buffer, electropermeabilization cannot be
related to the energy delivered to the cells.
Nucleotides
The structure of a biological membrane is dependent on the energy reserves in the

cytoplasm, which are controlled by the nucleotide level. Permeabilization is associ-
ated to a leak of adenosine triphosphate (ATP). The permeabilization by ms long
pulses efficiency remains unaffected by the ATP and adenosine diphosphate (ADP)
cytoplasmic levels.
Cytoskeleton
The cytoskeleton is an internal framework of a eukaryotic cell, built by a dynamic

network of protein filaments that provide structural support and drive the movement
of the cell internal components. Three categories (microfilaments, intermediate
filaments, and microtubules) are present. Colchicine, known to depolymerize the
microtubules in CHO cells, induced no modification on the first two steps of the
electropermeabilization process, creation and expansion (microtubules). Disruption
of the actin cytoskeleton structures was reported as one of the characteristic effects of
submicrosecond-duration pulsed electric field (nsPEF) in both mammalian and plant

cells (Pakhomov et al. 2014). CHO cells modified to get expression of a fluorescent
protein (mApple)-tagged actin were used to analyze if nsPEF modifies the cell actin
by a direct action (during the pulse delivery) or as a result of the events following cell
membrane permeabilization (actin). A train of repetitive high field short pulses
resulted in cell membrane electropermeabilization (YO-PRO ®-1 dye (Thermo
Fisher) uptake) and swelling. Actin filaments were replaced by dimmer and uniform
distribution of diffuse actin. To block the PEF-induced swelling, the pulsing buffer
was isoosmotically supplemented with a solute (sucrose), which remains
unpermeant along membrane electropermeabilization. Disintegration of the actin
cytoskeleton was a result of cell swelling, which, in turn, was caused by cell
electropermeabilization and the resulting transmembrane diffusion of solutes
which led to the osmotic imbalance.
Membrane Order
Cell electropermeabilization resulting from the delivery of pulses lasting 0.1 ms is

detected only under more stringent conditions when cells have been treated by
ethanol; lysolecithin is observed to facilitate cell electropermeabilization. More
precisely, these molecules that modify membrane order and the related membrane
fluidity, when used in concentrations compatible with cell viability, are shown to
affect the expansion step, not the critical field that triggers electropermeabilization.
Electropermeabilization is inducing a transition in the membrane organization.
Membrane order is modulating the energy barrier needed to evoke the slow step in
this membrane transition which occurs when cells are submitted to a field larger than
a characteristic threshold (expansion step). Less order would increase the magnitude
of this energy barrier; more order would decrease it.
When millisecond square wave pulses are delivered, no effect was observed on
cell permeabilization for small-sized molecules when cells were pretreated by
lysolecithin. Only little change on electroloading of proteins such as
R-phycoerythrin was obtained in the presence of ethanol. Cell membrane fluidity,
which is controlled by the membrane order alterations, does not have significant
effect on reversible electroporation (Kanduser et al. 2006).
Conclusions
Cell membrane electropermeabilization is a local effect in the cell surface. The size
of the permeabilized area is controlled by the field strength. This means that
permeabilization was triggered locally as soon as the induced transmembrane volt-
age reached a critical permeabilizing threshold (0.2–0.6 V). Defects supporting the
transmembrane transport are induced (defects). The density of defects (membrane
conductance, transport efficiency) within the cap was controlled by the pulse dura-
tion in a complex way. The behavior of the permeabilized caps was different on the
470 J. Teissie
two opposite cell sides facing the electrodes. The onset of the membrane structural
changes associated to the increase in conductance was in the submicrosecond range.
Transport was delayed after the membrane conductance changes. Osmotic swelling
is very large with long (ms) pulses. The local membrane alteration induces a
collective reorganization of the cell. The molecular organization of the conductive
defects remains poorly characterized, but they result in an interfacial reorganization.
The critical value of the field-inducing permeabilization is under the control of the
size of the targeted cell. In a mixed population (such as blood), the field effect can
therefore be targeted on the subpopulation of large cells. Electropermeabilization
and resulting electroloading of phagocytes was obtained on a whole blood sample
without hemolysis of the smaller red blood cells (electropermeabilization). Further-
more, leucocyte membrane integrity was recovered and was maintained for at least
24 h. This was one more convincing evidence that the driving force to induce
electropermeabilization is the field-induced transmembrane potential. This factor is
under the control of the cell size. It is delayed from the onset of the pulse delivery by
the membrane charging time by less than 1 μs in a conductive buffer. The triggering
of permeabilizing (conductive) defects in the pulsed cell membrane is very fast (less
than 1 μs) as observed from the conductance experiments, when corrected for the
electrolytic artifacts on the electrode surface.
The onset of electropermeabilization follows the onset of the external field pulse.
It can be described in three main steps:
1. “Induction step.” The electric field induced the membrane voltage increase which
results/creates local defects (may be due to kinks in the lipid chains) when it
reached a critical value (about 200 mV). A mechanical stress was present with a
magnitude that depended on the buffer composition. This is a very fast event
(submicrosecond range) under the control of the external electric field.
2. “Expansion step.” The conducting structural defects expanded as long as the
electric field was present and with a strength larger than a critical value (observed
during the induction). Again, an electromechanical stress remained present
mainly with pulses in the ms time range.
3. “Stabilization step.” As soon as the field intensity was lower than the threshold
value, a relaxation process was taking place within a few milliseconds, which
brought the membrane to the permeabilized state for small molecules.
Cross-References
Experiments
▶ Measurement of Molecular Transport after Electropermeabilization

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14:881–899
Involvement of Reactive Oxygen Species in
Membrane Electropermeabilization 28
Justin Teissie
Abstract
Cell membrane electropermeabilization results in the creation of structural defects
in the membrane. Cell membrane electropermeabilization is a stress for the cell.
For its defense, the cells exposed to pulsed electric fields are the site of reactive
oxygen species generation. This is observed on all cell systems where it has been
investigated. Reactive oxygen species are partly generated by electrochemical
reactions at the level of the electrodes and diffuse to the cell surface. But an
endogenous oxidative burst is present at the cell surface. The amplitude of the
oxidative burst is controlled by the pulsing parameters (field strength, pulse
number, and duration). This is directly correlated to the extent of the
permeabilized cap on the cell surface. This is indeed a major site of its emission.
Different biochemical reactions follow this oxidative burst depending on the cell
system that was pulsed. Lipid peroxidation is one of the major effects. The loss
of cell viability is directly associated with the amplitude of the oxidative burst
and its control by scavengers. It strongly affects electric pulse-mediated gene
transfer by its damaging effect on plasmids. This can be controlled by the addition
of scavengers. Under stringent electric conditions, reactive oxygen species appear
to be present in the cytoplasm, where they affect the organelles such as mito-
chondria with apoptotic consequences.
Keywords
Membrane • Transmembrane voltage • Membrane structure • Reactive oxygen
species • Cell viability • Oxidative burst
J. Teissie (*)
Toulouse, France

DOI 10.1007/978-3-319-32886-7_40
474 J. Teissie
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474
Detection of Reactive Oxygen Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475
Direct Chemiluminescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475
Chemiluminescence Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476
Fluorescent Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476
Phagocytes Exposure to Pulsed Electric Field Triggers ROS Generation . . . . . . . . . . . . . . . . . . . . . 477
Exposure of Nonphagocytic Cells to Pulsed Electric Field Triggers ROS Generation . . . . . . . . 478
Exposure of Nonmammalian Cells to Pulsed Electric Field Triggers Reactive Oxygen
Species Generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480
Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480
Exposure of Prokaryotic Cells to Pulsed Electric Field (E. coli) . . . . . . . . . . . . . . . . . . . . . . . . . . . 481
Exposure of Yeast Cells to Pulsed Electric Field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481
Pulsed Electric Field Generated Reactive Oxygen Species Enhanced Lipid Peroxidation . . . . 482
Pulsed Electric Field F Generated Reactive Oxygen Species Control Electrotransfection . . . . 482
Reactive Oxygen Species Generation by Cells Exposed to Nanosecond Pulsed Electric
Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485
Introduction
Reactive oxygen species (ROS) are highly reactive ions and “free radicals”
(chemicals containing atoms with an unpaired electron in its outer orbit) involving
oxygen molecules (Reactive oxygen species). They are known to be (a) short lived,
(b) unstable, and (c) able to react with other molecules to achieve the formation of
stable derivatives. Reactive oxygen species (ROS) are highly reactive chemical
forms of oxygen, such as superoxide anion and hydroxyl radical. They form chain
reactions, where a free radical obtains an electron from a nearby compound to form a
new free radical. Free radicals may steal electrons from cellular structures or
molecules. The family of reactive oxygen species is complex (see Apel and Hirt
2004 as a reference). They originate from different pathways but may be synthesized
by enzyme systems as demonstrated in phagocytic cells, neutrophils, and macro-
phages (Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase),
myeloperoxidases). NADPH oxidase (Nox) has a dedicated function of generating
reactive oxygen species (ROS). The knowledge on NADPH oxidase (Nox) is largely
derived from studies in phagocytic leukocytes but NADPH oxidase activity also
exists in nonphagocytic cells and plant cells (Jiang et al. 2011) (NADPH oxidase).
Unlike Nox in phagocytic cells, the subcellular localization of other Nox isoforms
in nonphagocytic cells is mainly but not restricted to the cell plasma membrane, the
first target of electropermeabilization. Genes encoding Nox subunits only exist
in eukaryotes (including yeasts or microalgae). Nox can be activated by many
chemical, physical, environmental, and biological factors. Superoxide anion is a
highly reactive species that can damage cells. Antioxidants compounds must provide
electrons to free radicals to neutralize them. A key requirement is that these
28 Involvement of Reactive Oxygen Species in Membrane Electropermeabilization 475
compounds are able to lose an electron (that is given to the ROS) without becoming
reactive to stop the chain reaction. They are either chemicals (Vitamin E, Vitamin C,
beta-carotene, coenzyme) or enzymatic systems (superoxide dismutase (SOD),
catalase, glutathione peroxidase). As an example, for their defense, cells have
scavenging and antioxidant mechanisms that can rapidly remove the superoxide
anion (scavengers). Superoxide dismutase (SOD) forms hydrogen peroxide (H2O2)
from the superoxide anion. Catalase then converts hydrogen peroxide to water
(Orozco-Cardenas and Ryan 1999). ROS generated by cellular processes do become
harmful if antioxidant defenses are not present. A balance between ROS production
and removal is required to get their inactivation. If such is not the case, then damage
may occur.
Increased reactive oxygen species (ROS) generation seems to be a common
response in cells exposed to stresses. In response to wounding, potato tubers are
observed to generate reactive oxygen species (ROS). This is a multistep response but
immediately following wounding, an initial burst of ROS occurs, reaching a max-
imum within 30–60 min. This initial oxidative burst was too fast to be dependent on
new protein synthesis. In addition to this initial oxidative burst, several other
massive bursts occur at later times postwounding. These latter bursts are associated
with wound healing and are probably involved to a specific response of intact plant
cells. The source of ROS is likely to be a plasma membrane NADPH-dependent
oxidase. The initial burst appears as a spontaneous and poorly regulated production
of ROS that provides a first line of defense against plant injury.
Electropermeabilization results in a transient membrane damage (new transport
pathways are present) resulting from an electrical stress (the transmembrane voltage
is locally brought to critical threshold inducing a structural and dynamical reorga-
nization of the plasma membrane) and from electromechanical constraints (electro-
permeabilization, defects). This is not only a membrane alteration but involves
a more general cell response. It is well accepted that cell recovery after membrane
electropermeabilization is an active cellular process in which exocytosis plays an
important role. Resealing of the membrane is not a simple mechanical event but
involves the cellular machinery. Cells are stressed due to plasma membrane electro-
permeabilization. Analysis of the cell response to this electrical stress is a key
component of electropermeabilization.
Detection of Reactive Oxygen Species
Direct Chemiluminescence
Reactive Oxygen Species (ROS) can be detected by a direct weak fluorescence when
using very sensitive devices (Quickenden et al. 1985). Emission can then occur from
excited carbonyl groups to give a photon (in the blue region) with the formation of
the ground state carbonyl group. In addition, excited singlet oxygen molecules can
decay with the production of far red photons (around 780 nm). Other singlet O2
emissions lie far into the infrared region (Table 1).
476 J. Teissie
Table 1 Synthetic view of the different techniques which are used to detect ROS
Method Equipment Specificity of detection Sensitivity
Direct chemiluminescence Expensive Very high Low
Chemiluminescence
reagents
Lucigenin Luminometer Superoxide radical Fair,
anions population
Luminol (not water Luminometer Superoxide radical Fair,
soluble) anions population
Fluorescence assay
20 ,70 -Dichlorofluorescein Microscope, Low High, single
FACS cell
Dihydroethidium Microscope, Low High, single
FACS cell
The excited singlet O2 molecules may also excite a secondary emission by

reacting with unsaturated lipids to produce excited carbonyl groups. Emission is
therefore associated with the oxidation of lipids, i.e., reflecting a membrane damage.
A possible emission is that arising after dimerization of excited singlet oxygen.
In aqueous, in vitro model systems, the emission comprises several bands, the
strongest are centered in the red and far red (600–700 nm). A weaker broad emission
is found between 450 and 550 nm (blue and green).
Chemiluminescence Reagents
A more sensitive and easy to use protocol is the addition of reporter chemicals that
react with ROS with the formation of photons (chemiluminescence). Sensitive
luminometers can be found in most Biochemistry laboratories. They are slow for
the detection and cannot give access to the early stages of the ROS generation.
Luminol (its salt form is soluble in water) or lucigenin dependent chemilumines-
cence (CL) are very popular for the detection of superoxide radical anions in
biological systems. Luminol reacts in its univalently oxidized form and lucigenin
reacts in its univalently reduced form with O2. In both cases, light production
depends on the formation of an unstable derivative, which decomposes to an
electronically excited product. This excited form releases its energy as a photon as
it falls to the ground state. ROS identification by CL measurements is complex. CL
reagents selectively detected O(2)(*-). However, no CL reagent had selectivity on the
detection of H2O2, ClO(), (1)O(2), and NO (Yamaguchi et al. 2010).
Fluorescent Reagents
One of the techniques for detecting intracellular ROS is to use the ROS-mediated
oxidation of the nonfluorescent substrate 20 ,70 -dichlorofluorescein (H2DCF) to a
green highly fluorescent product, 20 ,70 -dichlorofluorescein (DCF). Esterified forms

of H2DCF (20 ,70 -dichlorofluorescein diacetate (DCFH-DA)) can enter cells, as cell
membranes are freely permeable to such neutral derivatives (fluorescence). In the
cytoplasm, deacetylation by intracellular esterases results in the formation of the acid
form. This acidic form becomes trapped intracellularly and detects the ROS in the
cell. The level of ROS can be detected by a fluorimeter, fluorescence microscopy, or
flow cytometry. Due to its reactivity to cellular processes, it appears that its use for
ROS detection is controversial and H2DCF should be considered as an indicator of
the degree of general oxidative stress. Dihydroethidium (DHE) can freely permeate
cell membranes. It is used extensively to monitor superoxide production. Oxidation
of DHE by superoxide anions forms a red fluorescent product (ethidium or
2-hydroxyethidium) with a bright fluorescence after intercalating with DNA
(Owusu-Ansah et al. 2008; Gomes et al. 2005).
Phagocytes Exposure to Pulsed Electric Field Triggers ROS

Generation
Nox were first detected on phagocytes as its response to pathogens is the major way
for the cell defense. The effect of high voltage electric field pulses on phagocytes
(human blood neutrophiles and rat peritoneal macrophages) was investigated in the
earliest investigation of cell electropermeabilization (Malinin et al. 1989). One single
capacitive discharge pulse (exponentially decay time τ = 90 μs, E = 1–7 kV/cm)
induces a luminol-dependent chemiluminescent (CL) response. The signal was
associated with a respiratory burst in the phagocytes. This was a slow signal
which reached a maximum amplitude only approximately 2 min after the delivery
of the electric pulse to neutrophils (chemiluminescence). The process was even
slower on macrophages. The chemiluminescent response of electropulsed phago-
cytes was observed to be a function of the electric field strength. No response was
detected if the strength was lower than about 1 kV/cm when delivered on neutrophils
while macrophages appeared more sensitive. The burst amplitude then increased
with the field intensity but reached a maximum at 4–5 kV/cm and then decreased.
A similar behavior was observed with macrophages but with lower field strengths.
The difference in field sensitivity was proposed to result from the different diameters
of neutrophiles and macrophages, 10 and 20 μm, respectively. The decrease at high
fields was proposed to result from the occurrence of an irreversible permeabilization
(irreversible electropermeabilization).
However, the burst from phagocytes mediated by the electric pulse was detected
only when Ca2+ ions were present in the pulsing medium. 1 mM of EDTA in the
incubation buffer completely prevented the CL response. The signal was dependent
on the Ca2+ concentration, and at each given field strength, an optimized Ca2+
concentration was obtained where the highest CL signal was obtained. A delayed
addition of Ca2+ was not effective for detecting the CL burst. Mg2+ ions played a
positive role in the electric field mediated CL response and were detected to be
competitors for Ca2+. The respiratory burst was supposed to be the result of an influx
478 J. Teissie
of Ca2+ and Mg2+ ions into the cytoplasm linked to the electropermeabilization
of the phagocyte plasma membranes. Classical (by pathogens) or artificial (by Ca2+
ionophores) activation of phagocytes was described to result from an increased Ca2+
ion concentration in the cytoplasm. Intracellular Nox responses were not a direct
consequence of membrane electropermeabilization but a consequence of the asso-
ciated transmembrane transport.
Exposure of Nonphagocytic Cells to Pulsed Electric Field Triggers

ROS Generation
Using the chemiluminescent probe lucigenin, electropulsation of Chinese hamster

ovary (CHO) cells induced generation of reactive oxygen species when electro-
permeabilization was observed (Gabriel and Teissié 1994) (chemiluminescence).
No signal was observed for pulse with an amplitude lower than the critical value
required to obtain cell membrane permeabilization. The ROS emission then
increased with an increase in the field strength (Bonnafous et al. 1999). A peak in
emission was then observed followed by a decrease with a further increase in field
strength. No electrochemical reaction at the level of the electrodes was observed
under these conditions of classical electropermeabilization (field about 0.7–1.5 kV/
cm, duration in the ms range). The electroinduced cell process was activated by Ca2+
and Mg2+ ions and by exogenous adenosine 50 -triphosphate (ATP). The ROS
generation was inhibited by adding chelating agent EDTA, or the hydroxyl radical
scavenger dimethylsulfoxide. The emission was linked with the electro-
permeabilized state of the membrane of the pulsed cells. ROS emission was long
lived after the pulse delivery but decays during the postpulse period. It was observed
all along the membrane resealing (described in another chapter of this handbook)
(resealing). The emission was a function of the pulse duration. Long (ms) pulses
elicited a strong signal at low field but the field inducing the strongest emission was
at a higher strength with a shorter pulse duration. The oxidative jump yield was
controlled by the extent of alterations allowing permeabilization (under the control
of the pulse duration) within the electrically affected cell area (under the control of
the field strength), but showed a saturating dependence on the pulse duration over
1 ms (oxidative jump).
Exposure to electric fields of erythroleukemia K562 cells also enhanced up to
fivefold the spontaneous emission of light detected in cell membranes (Maccarrone
et al. 1995a). The emitted photons were mainly in the red edge of the spectrum,
suggesting the involvement of singlet oxygen. The presence of antioxidants during
electroporation did not affect membrane permeability but quenched the lumines-
cence. Again the signal was a function of the field intensity and of the pulse duration
(decay time as a capacitive discharge system was used). A critical field must be
delivered. The emission increased with a further increase in field intensity up to a
peak and then decreases.
The transient electrically induced membrane restructuration which supports
enhanced membrane permeability is topologically well defined on the surface of
the cell (Hibino et al. 1991) (electropermeabilization). Exchange of polar species

only takes place on the permeabilized part of the cell surface which is controlled by
the field intensity. Electropermeabilization is a stress for the cell and metabolic
responses associated with this stress must be studied. A key point is to discover
whether this stress affects the whole cell surface or especially the permeabilized part.
This generation was directly associated with the part of the membrane surface which
was electrically restructured (permeabilized), as predicted by its dependence on
electric parameters (dependence on the reciprocal of the field strength). The field
strength controlled the part of the cell surface that was affected by electro-
permeabilization in the same way that it controlled the ROS generation under
nondestructive conditions. A direct evidence was provided by monitoring the
catalytic effect of ROS on oxidation process during CHO cell electro-
permeabilization (Gabriel and Teissié 1995). Imaging was given by a photochemical
time-dependent methodology. 5-(N-hexadecanoyl)-aminofluorescein (HEDAF), an
interfacial light-sensitive fluorescent probe, was inserted into the Chinese hamster
ovary cell membrane. Oxidation of this reporter is observed by a “bleaching” (loss)
of the emission. This effect occurred on the external layer of the cell where the
reporter was inserted. This is a surface effect. Analysis of the photooxidation
reaction by the fluorescence decay showed that the rate of reaction was faster
when the cell population was electropermeabilized. This increase in the rate constant
depended on the field strength. The modulation of the photooxidation reaction
appeared to be restricted to the electropermeabilized part of the cell surface (photo-
oxidation). Using fluorescence digitized videomicroscopy imaging on a single cell,
spatial heterogeneity of the photooxidation reaction was indeed directly observed
when the cell was electropermeabilized. There were heterogeneous compartmenta-
tions of the membrane reaction into two independent domains with specific reactiv-
ity. The major part of the cell surface was associated with a photooxidation rate
constant similar to the one observed on control cells. From the photochemical
properties of the reporter HEDAF, the difference of reactivity was associated with
an activator effect of oxygen-reactive species generated by an electroinduced
oxidative jump. Higher reactivity was detected on a cap at the cell surface.
The conclusion was that these species were localized in the buffer in front
of the permeabilized part of the plasma membrane. The direct observation is that
long-lived electropermeabilization only affects a restricted part of the pulsed
cell surface, from both a structural (enhanced transport) and a metabolic point of
view (Nox activation).
The modulation of the oxygen species electroinduction and the use of antioxidant
products (dimethylsulfoxide, sodium L-ascorbate) showed that cell survival after
electric treatment was directly correlated to the oxidative jump intensity (scaven-
gers). This observation had to be associated with the cell-damaging action
of oxygen-reactive species (Bonnafous et al. 1999). Cell electropulsation induced
reversible and irreversible alterations of the membrane assembly. The oxidative
stress was only present when the membrane permeabilization was reversible.
High field and long pulses were known to trigger a loss in cell viability due to
the absence of resealing. Irreversible membrane electropermeabilization inhibited
480 J. Teissie
Fig. 1 ROS are generated on the part of the cell surface that is electropermeabilized. When no field
is delivered and as long as electropermeabilization is not present (field strength smaller than the
critical field Ep), there is no detected ROS generation. When the field strength is larger than Ep, a
cap on the cell surface is electropermeabilized, where ROS are generated on the external side of the
cell. The magnitude of the ROS generation is proportional to the size of the
electropermeabilized cap
the ROS generation by electric pulse delivery by an unknown chemical or physical

pathway (irreversible electropermeabilization). The oxidative jump was not a simple
feedback effect of the local membrane electropermeabilization. It was strongly
associated with long-term cell survival (Fig. 1).
Enhanced production of intracellular reactive oxygen species (ROS)
was observed on Ehrlich ascites carcinoma cells (EAC) (Shil et al. 2005). The
intracellular generation of ROS was determined by fluorescence using
20 ,70 -dichlorodihydrofluorescein diacetate (DCH-FDA) (fluorescence). But such a
response was not observed on C2C12 myocytes where the response was specifically
on the cell surface (Markelc et al. 2012).
Exposure of Nonmammalian Cells to Pulsed Electric Field Triggers

Reactive Oxygen Species Generation
Plants
Plant cells are eukaryotes where the cell membrane is surrounded by a wall layer
(wall). They are known to be easily electropermeabilized. Electropermeabilization
of intact black Mexican sweet (BMS) maize cells induced a generation of reactive
oxygen species (oxidative jump) (Sabri et al. 1996). Using the nonpermeant
chemiluminescent probe lucigenin, it was observed that the electro-induced chemi-
luminescent response depended on the electric parameters (electric field intensity,
duration, and number of pulses) (chemiluminescence). An electric field of intensity
larger than a critical value must be delivered to observe the oxidative jump. The
wall did not prevent the ROS generation. While the electroinduced cell
permeabilization had a short life (fast resealing), the oxidative jump that was
triggered by this electropermeabilization was a much longer-lived response. The
electroinduced loss in viability was linearly correlated to permeabilization. But
there was no such correlation between the oxidative jump and the loss in viability.
The modulation of oxygen species field induced generation by antioxidant prod-
ucts (dimethylsulfoxide, sodium L-ascorbate, and glutathione) was not associated
with any improvement in cell viability (scavengers). This behavior was radically
different from what was observed with mammalian cells. The associated metabolic
response was not the same and might reflect signals arising from the wall in the
plant.
Lentil (Lens culinaris) root protoplasts (plant cells where the walls have been
biochemically removed) have been subjected to electric field pulses suitable for gene
transfer (Maccarrone et al. 1995b). Exposure to electric fields resulted in an enhance-
ment up to fivefold of the spontaneous emission of light. The spectral localization of
the emitted photons was in the red edge of the spectrum, reflecting the involvement
of singlet oxygen. The presence of antioxidants during the pulse delivery did not
reduce the permeabilization but quenched the luminescence.
Exposure of Prokaryotic Cells to Pulsed Electric Field (E. coli)
Electric pulse delivery on prokaryotic cells of Escherichia coli also enhanced light
emission, indicative of the generation of single oxygen (Maccarrone et al. 1995b).
Strong fields of the order of 10 kV/cm suitable for gene transfer in bacteria were
delivered. The signal was higher in lipoxygenase-expressing clones. The ROS
generation appeared to be intracellular.
Exposure of Yeast Cells to Pulsed Electric Field
Yeast cells are eukaryotes where the cell membrane is surrounded by a wall layer
(wall). The wall organization in yeasts is different from the one in plant cells. A weak
lucigenin chemiluminescence (CL) was present when the reactive was mixed with
yeasts. When the cells were subjected to high electric field pulses inducing their
electropermeabilization, the CL light emission increased with a dependence on
the field intensity and pulse number (at a given pulse duration) (Galutzov and
Ganeva 1997) (chemiluminescence). Its kinetics was different from the one observed
on control cells. The electrically induced CL was strongly influenced by the meta-
bolic state and by the addition of KCN, an inhibitor of the mitochondrial respiratory
chain. The yeast membranes must be intact during the electric shock as CL did not
appear when yeasts were pretreated with detergent. It was proposed that the chemi-
luminescence was due to the action of metabolically generated reactive oxygen
species (ROS) with lucigenin inside the cells. The role of electric field treatment
was to facilitate the transport of lucigenin to the mitochondrial sites of ROS
generation.
482 J. Teissie
Pulsed Electric Field Generated Reactive Oxygen Species

Enhanced Lipid Peroxidation
Lipid hydroperoxide formation was observed in the membranes of animal and plant
cells that survived the electric pulse delivery (Maccarrone et al. 1995a). Exposure of
animal and plant cells (Erythroleukemia K562 cells and lentil (Lens culinaris) root
protoplasts (wallless)) to electropermeabilizing field pulses enhanced the amount of
conjugated hydroperoxides detected in cell membranes in parallel with the previous
observation of up to fivefold spontaneous emission of light. The spectral analysis of
the light emission suggests the involvement of singlet oxygen. The presence of
antioxidants during the pulse delivery quenched the luminescence but did not reduce
the permeabilization and the formation of hydroperoxides. The amount of hydro-
peroxides formed in cell membranes of both cell-types was a function of field
strength applied (Maccarrone et al. 1995b). A greater membrane fluidity resulted
from electropermeabilization maybe as a result of the electropermeabilization-
induced lipid peroxidation formed upon pulsed electric fields (lipid peroxidation).
Lipoxygenase activity appeared to be involved in the process of formation of
membrane hydroperoxides suggesting an intracellular effect.
Results from both computer simulations of lipid bilayer molecular dynamics
and experiments with living cells showed that the oxidation of membrane compo-
nents enhanced the fragility of the membrane to electropermeabilization (Vernier
et al. 2009). This was described on an oxidation sensitive lipid with two double
bonds (1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine). This appears to
result in an augmented hydrophobic mismatch induced by the oxidatively truncated
phosphatidylcholine in the native lipid matrix. Such mismatches are indeed sponta-
neous defects that make the membrane assembly fragile to the transmembrane
voltage increase along the pulse delivery (defects).
One should mention an effect of low magnitude electric fields on lipid systems.
Lipid acyl chains were peroxidized as a side effect of electroformation of giant lipid
unilamellar vesicles (GUV). This peroxidation of a wide range of polyunsaturated
phosphatidyl-choline lipids resulted in a dramatic change in the phospholipid com-
position and material properties of the synthesized vesicles (Zhou et al. 2007). Lipids
with high unsaturation levels formed higher levels of peroxidation byproducts.
Peroxidation byproducts depended linearly on the strength of the electric field. As
lipid peroxidation controlled membrane properties such as domain formation and
mechanical stability, this electrochemical reaction must be taken into account when
playing with electroformed GUVs containing polyunsaturated lipids.
Pulsed Electric Field F Generated Reactive Oxygen Species

Control Electrotransfection
Electrically mediated gene transfer is an efficient method to transfect cells in vitro or

in different tissues (gene electrotransfer). However plasmids (pDNA) are highly
sensitive to oxidation by ROS. Indeed, they are considered as ROS scavengers.
During C2C12 myoblasts electropermeabilization under conditions optimized for

gene transfer, ROS were generated on the surface of the cells as described previ-
ously. Their level was increased with an increase in the field strength as previously
described. No long-term genomic DNA damage was induced. But in electrically
mediated gene transfer, pDNA was observed to form aggregates on the pulsed
cell surface for a few minutes. Plasmid DNA for transfection being present outside
the cells after the pulse delivery neutralized the generated ROS (decrease in the
induced lucigenin chemiluminescence) and improved the pulsed cell survival
(Markelc et al. 2012). A similar role was played when artificial antioxidants
(vitamin C or tempol) were present rather than pDNA (scavengers). When antiox-
idants were present during gene electrotransfer, the transfection efficiency of C2C12
myoblasts was significantly increased (1.6-fold when tempol, a soluble antioxidant,
was added). Also in vivo (M. tibialis cranialis in mice), the transfection efficiency
was increased 1.4-fold by a tempol injection.
A similar improvement was obtained on plant cells. Intact black Mexican sweet
(BMS) maize cells could be electrotransfected if cell plasmolysis was applied before
electropulsation (Sabri et al. 1998). Transient expression (β-glucuronidase (GUS)
and chloramphenicol acetyltransferase) and stable expression (phosphinotricin
acetyltransferase) of foreign genes were obtained. Different amounts of ascorbic
acid (a ROS scavenger) were added just after a very short delay (less than 5 s) after
the pulse delivery. The pulse-induced Lucigenin chemiluminescence was slightly
decreased by ascorbate addition in the absence of pDNA. A 100% increase in
transient GUS expression in intact BMS cells, a crop difficult to transform, was
obtained by simply adding 10 mM ascorbate just after electropulsing the plasmo-
lyzed cell–pDNA mixture. The cell viability was preserved.
Addition of antioxidants in pulsing buffers has been reported to improve the
transfection yield such as glutathione (van den Hoff et al. 1992). As antioxidants
were in many cases toxic for the cells (Ascorbate), only limited amounts could be
added. As they could affect the pulsing buffer conductivity, they were added after the
pulse delivery as just described with BMS cells. Some macromolecules (serum) were
effective as ROS scavengers but as they were competitors for pDNA electric field
mediated interaction with the plasma membrane, they were added just after the pulse
delivery (Delteil et al. 2000; Roche et al. 2008). Expression was improved as well as
cell survival.
Reactive Oxygen Species Generation by Cells Exposed

to Nanosecond Pulsed Electric Fields
Nanosecond pulsed electric field (nsPEF) is a novel modality for permeabilization of

membranous structures (plasma membrane and organelles) (Pakhomov et al. 2007).
Electropermeabilization is obtained by the delivery of trains of nano- to sub-
microsecond pulses (ranging from a few ns to 600 ns) with very high field intensities
(several tens of kV/cm). It could result not only from a charging effect but also from
a dielectric effect when the pulses are very short (electropermeabilization).
484 J. Teissie
NsPEF were observed to trigger ROS when they were delivered on a cell free
buffer (Pakhomova et al. 2012). This was observed by the observation of H2DCF
oxidation by nsPEF in cell-free RPMI buffer (Roswell Park Memorial Institute
medium) or in a simple salt medium (electrochemistry). Samples were exposed to
50, 100, 200, or 300 ns pulses at 4.9 or 9.8 kV/cm, 1 or 0.3 Hz. ROS generation was
increased with an increase in the pulse parameters and with the longer delay
(0.3 Hz). Formation of H2O2 was suggested by an electrochemical effect of ns
PEF on the electrodes. A short lived current is present with a very high intensity.
Formation of H2O2 in nsPEF-treated media was confirmed by increased oxidation of
Amplex Red (N-Acetyl-3,7-dihydroxyphenoxazine) specifically by H2O2 in the
presence of horseradish peroxidase (HRP) that produced highly fluorescent resorufin
(fluorescence). Such ROS generation by pulsing the buffer was not reported when
longer pulses that are usually used in electroporation were used.
In Jurkat cells, delivery of 300 ns pulses at 4.5 kV/cm increased the conversion
of H2DCF in DCF. The delivery of increasing numbers of electric pulses caused an
increased effect. This is reflected by the creation of more cell damages (including
alterations to mitochondria) following nsPEF exposure (mitochondria). More ROS
were formed with time after exposure in nsPEF-sensitive Jurkat cells, but not in
nsPEF-resistant U937 cells. It is worth mentioning that U937 cells are easily
permeabilized by classical PEF. ROS increase within individual cells exposed
to nsPEF was visualized by oxidation of dihidroethidium (DHE), a dye trapped
in the cytoplasm of CHO cells (fluorescence). ROS were present in the cell after
nsPEF delivery. nsPEFs generated both extracellular (in part electrochemical) and
intracellular ROS, among which H2O2.
Electropermeabilizing electric pulse train of 100 ns long and 30 kV/cm triggered
an increase in reactive oxygen species (ROS) in human pancreatic carcinoma cells
(Nuccitelli et al. 2013). This condition of nsPEF was known to have multiple effects
(electropermeabilization of plasma membrane and organelle membranes, resulting in
an immediate increase in intracellular Ca2+, a long-term DNA fragmentation and
caspase activation) (apoptosis). Again increasing the number of pulses delivered
increased the level of ROS. Antioxidants (Troxol) prevented the formation of ROS.
But Ca2+ appeared as a key effector as Ca2+ chelators prevented the oxidative burst
being present either in the buffer or inside the cell. Increase in intracellular Ca2+ was
needed for the ROS generation.
Conclusions
Electropermeabilization is not limited to a transient increase in transmembrane

transport. The stress on the membrane triggers a global cell response. This results
in a defense mechanism through the generation of ROS. The larger is the level of
permeabilization, while keeping the cell viable, the higher is the level of ROS
generation. The mechanism is complex. Under some conditions, only plasma Nox
appears to be stimulated. In other conditions (such as with nsPEF), the increase in
cytoplasmic Ca2+ was present and appeared as an active factor in the ROS
generation.
This ROS generation can have direct effects on the cell viability and may trigger
an apoptotic death. A puzzling observation is that pulsing cells under irreversible
conditions does not induce ROS generation. An indirect effect on electric field
mediated gene transfer is due to the ROS damaging effect on pDNA.
Bioeffects of the delivery of electropermeabilizing pulses are not limited to the
creation of transient permeant structures in the plasma membrane. The transient
blebs formation on the plasma membrane (described in the chapter on mechanisms
of electropermeabilization) may result from the oxidation of lipids. The PEF asso-
ciated ROS formation clearly is responsible of oxidative cell damage and involved in
the induction of cell death.
Cross-References

Experiments
▶ Different Cell Sensitivity to Pulsed Electric Field
▶ Modeling of Electrochemical Reactions During Pulsed Electric Field Treatment
▶ Stress Induction and Response, Inactivation, and Recovery of Vegetative Micro-
organisms by Pulsed Electric Fields
▶ Stress Response of Plants, Metabolite Production due to Pulsed Electric Fields
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Plant Cell Wall: Description, Role in
Transport, and Effect of Electroporation 29
Cristèle Delsart
Abstract
Although the electropermeabilization of a plant cell involves crossing the cell
wall and the plasma membrane, research on the effect of electroporation has been
more focused on the study of the plasma membrane. This chapter aims to improve
knowledge of the effect of electroporation on the plant wall. To facilitate the
understanding of this topic, the description of plant cell wall and its role in
transport is first described. This review showed that there are disagreements
between the authors. Some argue that the electroporation has no effect on the
wall, while others observe an increase or decrease in the permeability of the wall.
A decrease in the permeabilization of the cell wall would be due to the establish-
ment of a cell protection system under the action of stress induced by the pulsed
electric fields. An increase in the permeabilization of the cell wall after pulsed
electric field treatments would be linked to the modification of the polysaccharide
and lignin composition. Others point to the importance of the electric charge of
the wall. These disagreements may be related to the fact that electroporation of the
wall differs based on the parameters of the pulsed electric field and the type of
treated cell (thickness, electrical charge, and constituents of the cell wall). Dif-
ferent methodologies to understand the effect of electroporation on the walls are
described in this chapter and should be followed by additional studies to lift the
veil on this issue.
Keywords
Plant cell wall • Electroporation • Pulsed electric fields • Permeabilization
C. Delsart (*)
Laboratoire Transformations Intégrées de la Matière Renouvelable (UTC/ESCOM, EA 4297
TIMR), Sorbonne Universités, Université de Technologie de Compiègne, Centre de Recherche de
Royallieu, Compiègne, France
e-mail: cristele.delsart@utc.fr

DOI 10.1007/978-3-319-32886-7_143
490 C. Delsart
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490
Plant Cell Wall: Description and Role in Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
General Definition of the Cell Wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
Chemical Composition of the Primary Wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493
The Functions of the Cell Wall and Focus on the Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496
Summary of the Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
Effect of Electroporation on Plant Cell Wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
Introduction
The cells of plants are different from animal cells in that they are larger in size, that
they generally have an angular shape and are geometric, and that original and
specific structures including the cell wall are present. Without their cell walls, plants
would be very different organisms. Indeed, the plant cell wall is essential to their
growth, nutrient transport, maintenance, protection against pathogens, reproduction,
etc. The cell wall is responsible for the size and shape of cells, the reversible
(elasticity) and irreversible (plasticity) cell expansion, cell permeability, the retention
of many cations, and the mechanical property of the tissue and is also the source of
signaling molecules.
The plant cell is a constituent unit of the multicellular organism that is a plant.
Each plant cell covers its plasma membrane by an extracellular production consti-
tuting the rigid cell wall. This differential production of the wall in terms of
composition, organization, and thickness creates areas of cell specialization in the
plant. The wall does not isolate the cells as there are a multitude of canaliculi that run
through and allow an intracellular communication. Thus, a cytoplasmic continuum
extends through the whole plant from the roots to shoots. The structure and compo-
sition of the wall prevent the passage of large molecules through its mesh. But cell
walls remain permeable to water and to most small water-soluble molecules.
Electroporation is the process of creating pores within the cell membrane through
the application of an electric current. The cell can be reversibly (when the pores close
again after the stop of applied voltage) or irreversibly (when the pores cannot be
closed or afterward due to loss of homeostasis, etc.) electropermeabilized. The ability
to extend or retract molecules across a cell membrane via the application of an electric
current has rapidly led to many technological interests for various industrial sectors.
One example is the use of reversible electroporation in the field of genetics for the
introduction of plasmid DNA or exogenous RNA in cells (▶ Chap. 23, “Gene
Delivery by Electroporation in Vitro: Mechanisms”); another use in the medical
field is for the introduction of drug molecules in targeted cells (electrochemotherapy).
There has also been a strong interest in the food industry in reversible electroporation
29 Plant Cell Wall: Description, Role in Transport, and Effect of. . . 491
to increase kinetics and extraction yields of interest intracellular compounds in plants

(▶ Chap. 37, “Selective Extraction of Molecules from Biomaterials by Pulsed Elec-
tric Field Treatment”), to improve the process of drying, or to inactivate the micro-
organisms in food products (▶ Chap. 66, “Modeling Microbial Inactivation by Pulsed
Electric Field”). The phenomenon of electroporation on cell membranes is unknown,
but it has been shown by microscopic observation that pores are created in the cell
membranes (▶ Chap. 69, “Atomic Force Microscopy to Explore Electroporation
Effects on Cells”). The effect of electric current on the cell walls also is unknown
and moreover is very little documented. Existing literature on the disruption of the
cell wall of the microorganisms following the application of electrical current is larger
than that for plants. This chapter aims to link existing information on the effects of
electroporation on the cell walls of plants, and with the aim to improve the under-
standing and analysis of these data, this part will be preceded by a description of the
cell wall and its involvement in intercellular communication.
Plant Cell Wall: Description and Role in Transport
General Definition of the Cell Wall
A characteristic of all plant cells is that they are surrounded by an extracellular

matrix called the cell wall. This is composed of several interconnected networks of
polysaccharides, glycoproteins, and polyphenols. However, its nature and chemical
composition vary widely depending on the particular species and its tissue struc-
tures. Indeed, differences may appear depending on the age of the cells, their level of
differentiation, or functionality. The cell wall structure changes with the stage of
development of the cell and is generally characterized by three levels of organization
(Fig. 1).
The primary wall is a loose network of cellulose microfibrils, encompassed in a
highly hydrated amorphous matrix of hemicellulose and pectin. The secondary wall
(mainly consisting of cellulose, hemicellulose, and lignin) has three layers, distin-
guished by the orientation of their cellulose fibers which confer resistance to
the wall.
The Middle Lamella and the Primary Wall

The first level of organization corresponds to the middle lamella that forms during
mitosis and corresponds to the common portion between two adjacent cells to ensure
cohesion. As a “primitive” wall, it consists mainly of pectin substances.
Then, the primary wall is formed. It is produced by meristem cells and charac-
terizes the cells in their growth phase. It remains thin, flexible, and stretchable during
growth to allow cell elongation. This is a very hydrated structure (up to 90% water)
consisting of a loose network of cellulose microfibrils embedded in an amorphous
matrix of pectin substances, hemicellulose, and protein (Albersheim et al. 2010).
492 C. Delsart
Fig. 1 Schematic representation of the cell envelopes
The Secondary Wall

Finally, in some tissues, a secondary wall may build up gradually between the
plasma membrane and the primary wall. It is constituted by the development of
multiple and varied thickenings, when cell growth is complete. This is a rigid
structure, inextensible and weakly hydrated (maximum 20% water) consisting
essentially of crystalline cellulose microfibrils arranged in concentric layers disposed
parallel to each other and various hemicelluloses (Fig. 1). This secondary structure is
generally strengthened by deposits of hydrophobic lignins. This is, for example, the
case of conducting tissue (xylem) or support tissue (sclerenchyma).
Lignins are phenolic polymers resulting from the copolymerization of three kinds
of aromatic alcohols: the coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol,
generally designated by the term monolignols. Their polymerization is carried out by
oxidative dehydrogenation under the influence of laccase and peroxidase enzymes
(Albersheim et al. 2010). Lignification occurs at the end of cell growth and does not
occur uniformly in the different layers of the wall. The inlay starts in the middle
lamella and then progresses through the primary wall and then the different layers of
the secondary wall. At the end of cell growth, lignin encrusts cellulose and hemi-
cellulose, ensuring a certain rigidity to the wall.
Finally, the secondary wall can also be mineralized by silica or calcium carbonate
deposits or receive lipid appositions of cutin or suberin conferring imperviousness to
the wall (Ghanati et al. 2005).
Chemical Composition of the Primary Wall
Carpita and Gibeaut (1993) consider the existence of two models for organizing the
primary wall. Type 1 is representative of most dicotyledonous (plants whose seeds
has two embryonic leaves or cotyledons) and some monocotyledonous (seeds
contain only one embryonic leaf) plants. In this model, xyloglucans are the major
hemicellulose, with a high content of pectin, which can reach 35% of the mass
parietal. Type II, specific to most monocotyledons, is characterized by a low content
of pectin (about 5%). The main fraction hemicellulosic often linked with phenolic
groups is represented by the glucuronoarabinoxylans which may represent up to
more than 70% of the mass parietal.
The primary wall is thus composed of three large classes of polysaccharides
defined according to their extractability and composition: cellulose, hemicelluloses,
and pectins. These polymers are interconnected and form a veritable network. The
cellulose microfibrils are associated with hemicellulosic polysaccharides by numer-
ous noncovalent bonds (hydrogen bonds), forming the first mesh level, which
ensures and controls the mechanical strength of the assembly. This first structure is
itself impregnated with a more or less dense network of pectins conferring a certain
mechanical flexibility and regulating the parietal dynamic. In addition to the poly-
saccharides, the cell wall is composed of proteins (Fig. 2).
Fig. 2 Schematic diagram of the major structural components of the primary cell wall and their
likely arrangement
494 C. Delsart
Cellobiose
H OH CH2OH
H OH CH2OH O
O
OH H H H H (...)
O
OH H H H H
O H H O OH H
H
H H OH H H
O O
O CH2OH H OH
H OH
CH2OH Glucose chain
Microfibril
Microfibril
= 36 glucose chains
Fig. 3 Cellulose molecule and modeling of the assembly of cellulose fibers
Cellulose
Cellulose is considered to be the most abundant polymer on Earth (more than 50% of
the terrestrial biomass). This polymer is particularly abundant in the secondary walls
and accounts for between 20% and 30% of the dry matter of most primary walls. The
cellulose molecule is synthesized at the level of the plasma membrane by cellulose-
synthase enzymatic complexes. Its basic structure consists of long linear chains of
residues of β-D-glucose linked by (1–4) and stabilized by intramolecular hydrogen
bonds. The repeating unit composed of the association of two glucoses is called
cellobiose (Fig. 3).
In the primary wall of higher plants, these chains of glucose combine together by
intermolecular hydrogen bonds to form microfibrils (arrangement of 36 parallel
chains of β-1,4-glucose) that can in turn be associated, again via hydrogen bonds,
to form cellulose macrofibrils (Fig. 3) conferring on this polymer remarkable
mechanical strength properties (Albersheim et al. 2010).
Hemicelluloses (Linked Glycans)

Hemicelluloses represent all wall polysaccharides, which are not cellulosic or pectic
and are alcalino solubles although some are also soluble in water. They can represent
up to 30% of the dry mass of the primary walls of dicotyledon (Carpita and Gibeaut
1993). Hemicellulosic polymers associate to the cellulose by noncovalent bonds and

enable cellulosic microfibrils to be indirectly but firmly connected to each other
(Fig. 2). They can also establish covalent bonds with pectins through the ramifica-
tions of arabinans.
These polysaccharides are chemically very varied. However, the most abundant
and studied hemicellulosic polymer in the primary wall of dicotyledons is
xyloglucan. This polysaccharide is composed of a skeleton of β-(1,4)- glucose and
has a conserved structure, based on the repeating units consisting of 7–10 mono-
meric units. Depending on the stage of development and plant species, other
hemicellulosic polymers may be encountered such as xylan and mannan.
Pectins
The pectins, which represent about 35% of polymers of the primary wall of dicot-
yledons, are the most diversified parietal macromolecules (Carpita and Gibeaut
1993). They include complex heterogeneous polysaccharide rich in galacturonic
acid residues, more or less negatively charged. Another peculiarity of pectins is their
ability to form a highly hydrated matrix organized in gel (Fig. 2).
Among these polysaccharides are distinguished arabinans, galactans, and
arabinogalactans forming generally the side chains. But the most abundant, typical,
and studied pectins are the homogalacturonans and rhamnogalacturonans, which are
the main channels. Homogalacturonans and rhamnogalacturonans may be linked
together by covalent bonds, and their biosynthesis in the Golgi apparatus requires
some 50 types of enzymes of glycosyltransferases (Albersheim et al. 2010).
Proteins
In addition to the polysaccharides described above, the cell wall is made of structural
proteins (Fig. 2) and enzymatic proteins involved in its development, remodeling,
and protection against pathogens.
Structural Proteins
These proteins, which have no known enzymatic activity, are immobilized in the wall
via covalent bonds involving other proteins or polysaccharide compounds (Carpita
and Gibeaut 1993). There are several classes of structural proteins which may be
classified according to their amino acid compositions and their degree of glycosyla-
tion. There are essentially the glycine-rich proteins or the hydroxyproline-rich proteins
(Josè-Estanyol and Puigdomènech 2000). The glycine-rich proteins are associated
with lignified cells suggesting a possible interaction with the lignins. Furthermore,
these proteins can be expressed in response to biotic or abiotic stress (Albersheim
et al. 2010). The hydroxyproline-rich proteins include three protein families: the
proline-rich proteins, the extensins, and the arabinogalactan proteins. As the proline-
rich proteins, the extensins appear to play a role in the defense against pathogens. In
addition, these proteins are clearly involved in cell wall architecture by their ability to
establish inter- and intramolecular covalent bonds to each other but also with the wall
polysaccharides. These proteins may play a role in the stiffening walls. Finally, the
arabinogalactan proteins are associated with both the wall and the plasma membrane.
496 C. Delsart
Many roles have been proposed for these glycoproteins such as involvement in the
division and cell differentiation, adhesion, and/or intercellular communication,
programmed cell death, or plant defense to pathogens (Albersheim et al. 2010).
Enzymatic Proteins
Many enzymes are also present in the wall and carry out various functions. For
example, chitinases and glucanases are involved in a plant’s defense to pathogens.
Invertase, peroxidases, phosphatases, dehydrogenases, endoglucanases, xylosidases,
and pectinases are involved in modifying cell wall polysaccharides. Expansins are
involved in the regulation of release and extension of the cell wall during cellular
growth by allowing relaxation of cellulose-hemicellulose network. The xyloglucan-
endo-transglucosidases/hydrolases are also involved in controlling the release of the
wall but also in remodeling of the cell wall architecture by cleaving xyloglucans
chains or by participating in the incorporation of xyloglucans between cellulose
microfibrils. As a final example, the pectin-methylesterases are wall enzymes that
catalyze the demethylesterification of methylesterified galacturonic acid, releasing
methanol and protons. This enzyme would therefore be implicated in acidification of
the pectins and also in promoting the action of endopolygalacturonases contributing
to the relaxation of the cell wall (Albersheim et al. 2010).
The Functions of the Cell Wall and Focus on the Transport
Diverse Roles of the Cell Wall

As described in the preceding paragraphs, the plant cell wall is a wrapper both strong
and stretchy that surrounds the plasma membrane. This is a protective structure, but
it is also dynamic and metabolically active which ensures diverse and vital functions
in the plant (Carpita and Gibeaut 1993; Albersheim et al. 2010).
First, it allows the growth of young cells and ultimately determines their mor-
phology (size and shape of cells) and, therefore, the ability of plants to stand upright.
Cell growth is only possible if there is a certain degree of freedom between the
components of the wall; the weak bonds or simple entanglement of cell wall poly-
mers facilitate magnification. In contrast, covalent bonds between molecules
severely limit growth opportunities.
From a physicochemical point of view, the wall forms a buffer system between
the outside environment and the cytoplasmic content; it is in the parietal compart-
ment as the cytoplasm draws the elements necessary for its metabolism and rejects
certain compounds it develops. Furthermore, the wall ensures cell cohesion and
opposes osmotic shock by resisting sometimes significant cell turgor pressure
(Carpita and Gibeaut 1993). However, the wall usually remains permeable at the
level of punctuations traversed by plasmodesmata, allowing intercellular
communications.
The protective role of the wall against the external environment makes sense
when the cell is attacked by microorganisms and other pathogens or when subjected
to various environmental stresses. Indeed, when the plants are subjected to biotic or
Cell wall
Vacuole
Cytoplasm
Symplasmic pathway
Apoplasmic pathway
Fig. 4 Mapping of apoplasmic and symplasmic pathways
abiotic stress, their cells are able to generate the metabolic reactions. These are
generally classified into three major categories of reactions: (1) the production of
defense proteins, (2) the stimulation of certain secondary metabolic pathways via
elicitor molecules from the wall (i.e., oligosaccharins, “signal molecules” for the
production of phytoalexins) and (3) the quantitative and qualitative change of cell
walls (the parietal reinforcement (defense mechanism to seal the walls and limit the
entry of harmful elements to the plant) can be performed by macromolecule deposits
such as proteins and glycoproteins, polysaccharides, or aromatic polymers of lignin)
(Vorwerk et al. 2004).
Transport Through Cell Walls

Although the wall literally binds the cells together, it does not insulate absolutely and
it does not prevent exchanges or opportunities for interaction between them. There
are two possible pathways: the apoplastic pathway where the molecules circulate
between cells in the wall and the intercellular spaces and the symplasmic pathway
where the compounds circulate from cell to cell in the cytoplasm via small channels
which pass through the cell walls (Fig. 4).
Apoplastic Pathway
The apoplast is made up of cell walls and intercellular spaces. In other words, it is the
“extracellular matrix.” From another point of view, it is still “the internal physio-
logical environment of the plant.” Indeed, external stimuli are not received directly
by cells, but via exchanges with the internal environment. The movement of solutes
is related to the properties of different layers of the apoplast. So these are the walls
properties that control ion diffusion in the apoplast (Albersheim et al. 2010).
498 C. Delsart
Electronegativity Walls
Due to the predominance of free carboxylic groups of galacturonic acids present in
the middle lamella and the primary wall, the outer surface of the cell wall is
negatively charged. This electronegativity excludes from the apoplast all negatively
charged ions while providing the accumulation of the cations in the apparent free
space (AFS). This flow of cations is a passive, physical process: the kinetics of these
cations in the apoplast is controlled by diffusion and by the mass flow governed by
the flow of transpiration. The fixing of cations on the walls can affect not only the
acquisition of nutrients but also the tolerance of the plant regarding the toxicity of the
ion (Sattelmacher 2001).
Cation Exchange Capacity of Roots

Immobile anions in root walls are quantified by the cationic exchange capacity
(CEC). The CEC has an influence on the flow of charged ions in the root. CEC is
higher in dicotyledons than in monocotyledons (Sattelmacher 2001). It also varies
according to the species and age of the root: young zones have higher CEC than
older areas. Finally, the CEC can be affected by various environmental parameters.
The Porosity of the Cell Walls

In the primary wall as in the secondary wall, the cellulose and hemicellulose network
contains interfibrillary and intermicellar spaces ranging in size from 3.5 to 14 nm,
which shows that the walls do not represent a major diffusion barrier even for larger
molecules. But this permeability can be reduced due to environmental stresses such
as the presence of toxic ions (Sattelmacher 2001). In addition, the porosity of the
walls changes according to ontogeny and cell differentiation. Indeed, the apex cells
would present immature walls where intermicrofibrillar pores are not yet formed,
which would explain the impermeability of their walls for ions and fluorescent dyes.
Similarly, the deposits of suberin and lignin during the maturation of the endoderm
and hypodermis would reduce the porosity of the walls. The pore diameter in the
walls suberized is still unclear. The solutes of low molecular weight (i.e., hydrated
ions) have diameters not exceeding a few tens of nanometers; the ions cannot be
selected by height in the absence of barriers like apoplast. In contrast, large chelates
are excluded. Thus, metals such as Zn or Cu are preferentially absorbed in their ionic
form rather than their chelated form. Generally, molecules larger than about 6000 Da
require longer times and/or higher concentration gradients to diffuse through “nor-
mal walls.”
Symplasmic Pathway
The symplasm is a continuum of cytoplasm of adjacent cells, interconnected by
plasmodesmata (Albersheim et al. 2010). These plasmodesmata are structures with
diameters of around 50 nm. Each plasmodesma contains a “ring” cytoplasmic with a
diameter of a few nm (3–6 nm) by which the transport symplasmic can take place.
Each plasmodesma is lined by plasma membrane that is continuous from cell to cell
and usually contains a rodlike structure derived from the endoplasmic reticulum that
is called the desmotubule. The number of plasmodesmata by μm2 of membrane

surface is from 1 to 10. The latter are the zone where an intense exchange of water,
ions, and metabolites between the cells (in particular, saccharose) takes place. The
maximal size of molecules that diffuse freely is about 1000 Da. Plasmodesmata are
also the crossing point for viruses during infection; in this case, it is necessary for the
channel to widen significantly.
The extent of interconnection, and conversely the extent of isolation, depends on
the developmental patterns of formation and occlusion of plasmodesmata. Although
plasmodesmata normally form in the cell plate at cytokinesis (primary plasmodes-
mata), they can be sealed off later and they can also develop de novo in existing cell
walls (secondary plasmodesmata). Transport through plasmodesmata is probably
subject to several kinds of regulatory mechanism, apart from total occlusion. Sec-
ondary plasmodesmata in leaves can develop the capacity to transport molecules
some 20-fold larger than the exclusion limit that applies to primary plasmodesmata,
i.e., up to a molecular weight of about 15,000 Da. They do so under the influence of
movement proteins, which are coded for by systemic viruses (able to spread from
cell to cell in the infected plant). Plants may have their own homologues of viral
movement proteins to facilitate intercellular transport of informational macromole-
cules in special situations.
Summary of the Key Points
• Three levels of organization: (1) the middle lamella (mainly consisting of pectin
substances), the common portion between two adjacent cells to ensure cohesion;
(2) the primary wall, a loose network of cellulose microfibrils, encompassed in a
highly hydrated amorphous matrix of hemicellulose and pectin; and (3) the
secondary wall (mainly consisting of cellulose, hemicellulose, and lignin), three
layers distinguished by the orientation of their cellulose fibers which confer
resistance to the wall.
• Main functions: growth, protection, exchanges.
• Role in transport:
1. The apoplastic pathway where the molecules circulate between cells in the wall
and the intercellular spaces; the outer surface of the cell wall being negatively
charged, easier diffusion for positively charged ions/small molecules; the pres-
ence of interfibrillar and intermicellar spaces in the cellulose and hemicellulose
network ranging in size from 3.5 to 14 nm; molecules larger than about 6000 Da
require longer times and/or higher concentration gradients to diffuse through
wall cell.
2. The symplasmic pathway where the compounds circulate from cell to cell in the
cytoplasm via small channels (plasmodesmata with a diameter of 3–6 nm) which
pass through the cell walls; the maximal size of molecules that diffuse freely is
about 1000 Da.
500 C. Delsart
Effect of Electroporation on Plant Cell Wall
Table 1 presents the studies that directly or indirectly discuss the phenomenon of
electroporation on the walls of plant cells and these are presented in this chapter. In
this section the methodology for the study of the parietal system and the conditions
for pulsed electric field (PEF) treatments have been described as well as the treated
vegetal material.
Although there are few specific studies about the effect of electroporation on the
cell wall (Table 1), there is much analytical data from studies which aim to
electroporate the envelopes in plant cells in order to extract their intracellular
compounds (▶ Chap. 37, “Selective Extraction of Molecules from Biomaterials by
Pulsed Electric Field Treatment”). Table 2 summarizes the different examples of
PEF-assisted treatment applied to plants (Barba et al. 2015).
The extracted molecules vary in size (from ions to polymers such as cellulose), in
electric charges (from uncharged for the sucrose to electrically charged for the
proteins), in hydrophobicity (from hydrosoluble for the anthocyanins to hydrophobic
for the lipids), and in organ origin (root, bark, leave, fruit). Although the extraction
of these compounds is improved by the application of pulsed electric fields to the
plants, this phenomenon is not understood. It could be due to a direct effect of the
PEF technique on the wall of the cells or it just be due to increased permeabilization
of the membranes (and thus an indirect effect). However, the following hypothesis
can be made: the permeabilization of cell wall is increased after a solid/liquid
extraction assisted or not by pulsed electric fields. Seeing the extraction of
non-permeable molecules to the wall such as cellulose (large molecule), some pro-
teins (negatively charged), or lipids (non-hydrosoluble) is made possible.
The characterization of the permeabilization of the wall after PEF is possible
when the purpose is to get the genetic material into a plant cell. Indeed, the aim is to
create temporary pores (which close after the application of electric current) in the
cell envelopes (walls and membranes) of a plant in order to introduce the plasmid
DNA or RNA in the cell and to check the success of this transfer by measuring the
expression of introduced gene (▶ Chap. 23, “Gene Delivery by Electroporation in
Vitro: Mechanisms”). Knowing that one DNA base pair is 660 Da (in average), this
means that the introduced molecules can be very large and therefore cannot readily
cross the cell wall. This is why, in order to obtain plant cell transformation, it is
always necessary to overcome the plant cell wall barrier by various pretreatments.
This is a time-consuming step during which cell viability can be affected. Some
scientists have tried to introduce DNA into intact plant cells (with their cell wall). For
example, Morikawa et al. (1986) successfully introduced tobacco mosaic virus
(TMV) RNA in the mesophyll cells of Nicotiana tabacum var. Samsun by using
PEF (a single pulse of 0.65 kV/cm at 0.002 μF) without a prior destruction of the
wall. The introduced gene was successfully expressed in the recipient cells as judged
by the assay for the virus coat protein using immunofluorescence and by the virus
infectivity assay of the homogenate of the electrotransformed cells for local lesions
on tobacco leaves. With this electroporation method, cell survival before and after
the electric pulse treatment was relatively low (about 30% after culture for 3 days).
Table 1 Presentation of existing studies on the effects of PEF on cell wall

Products PEF conditions Methods References
Mesophyll cells 1 pulse of 1.2 kV/cm at Immunofluorescence Morikawa
of Nicotiana 0.003 μF, of 1.3 kV/cm at technique with TMV et al. 1986
tabacum 0.003 μF, of 2.7 kV/cm at (Tobacco Mosaic Virus)
0.001 μF, 2 pulses of fluorescent antibody
2.7 kV/cm at 0.001 μF
with an interval of 5 s and
8 pulses of 2.5 kV/cm at
0.001 μF with the same
intervals
Black Mexican 750 V/cm; one 15 ms Transient expression Sabri et al. 1996
sweet maize pulse; square pulse (13-glucuronidase and
chloramphenicol
acetyltransferase) and
stable expression
(phosphinothricin
acetyltransferase) of
exogenous genes
Black Mexican Several kV/cm during Transformation efficiency Eynard
sweet maize several ms; square pulses et al. 1997
Dried apple 1 kV/cm; 60 pulses (pulse Mercury porosimeter Bazhal
width of 300 ms); 1Hz; (pores, pore distribution, et al. 2003b
rectangular shaped porosity, bulk density, and
monopolar pulses skeletal density)
Apple, potato, 0–1500 V/cm, 1–1000 Conductivity Bazhal
cucumber, pulses (pulse width of disintegration index Z; et al. 2003a
aubergine, 10 5–10 3 s); 0.33 Hz; Estimation of the
pear, banana, monopolar pulses of characteristic damage
carrot rectangular shape times τ
Potato 30–500 V/cm with a single Diffusion of the Galindo
1 ms rectangular pulse fluorescent dye FM1-43 et al. 2008
through the cell wall
observed with
epifluorescence
microscope
Intact tobacco 0.25–7.5 kV/cm; 10–20 Staining with vital dye Janositz and
cells and pulses (pulse width of Phenosafranine and Knorr 2010
protoplasts 2–8 μs); 2206–1985 J/g; microscopy; cell
from tobacco 2 Hz; exponential pulses disintegration index Zp
cells (conductivity)
White 5 kV/cm; 20 pulse (pulse Light microscopy of lignin Janositz
asparagus width of 400 μs); 2 Hz; with phloroglucinol dye; et al. 2011b
16 kJ/kg; exponential lignin content
pulses determination via the
detection of acid detergent
fiber and acid detergent
lignin
(continued)
502 C. Delsart
Table 1 (continued)
Products PEF conditions Methods References
Potato 5 kV/cm; 20 pulses (pulse Microscopic visualization Janositz
width of 100 μs); 2Hz; of tissue with cell walls et al. 2011a
exponential pulses pectins stained with
ruthenium red
Glandular 50–150 V/cm with a time Light and scanning Sukardi
trichome cells of 2–3 s electron microscopy et al. 2013
of leaves
(patchouli
plant)
Carrot 1–4 kV/cm; 100–500 Water proton mobility by Aguiló-Aguayo
pulses (pulse width of time domain nuclear et al. 2014
20 μs); 0.20–15.09 kJ/kg; magnetic resonance
treatment (TD-NMR) relaxometry
time = 2–10 ms; 20 Hz,
bipolar mode; square
pulses
Grape berry PEF1 (4 kV/cm; total Light microscopy of tissue Delsart
skins treatment time of 1 ms; sections with cell walls et al. 2014
4 Wh/kg) and PEF2 polysaccharides stained
(0.7 kV/cm; total with periodic acid Schiff
treatment time of 200 ms;
31 Wh/kg)
Grape berry PEF1 (4 kV/cm; total Light and transmission Cholet
skins treatment time of 1 ms; electron microscopy; et al. 2014
4 Wh/kg) and PEF2 biochemical analysis of
(0.7 kV/cm; total pectic fractions of cell
treatment time of 200 ms; walls
31 Wh/kg)
Dry yellow 5 kV/cm; pulse width of Microscopy of tissue Baier et al. 2015
peas 10–15 μs; 667 pulses; sections with cell walls
2 Hz; 125 kJ/kg stained with toluidine blue
Immunofluorescence shows that as much as 50% of the cells that survived 24 days
had expressed genetic information encoded on the introduced gene. Ten years later,
Sabri et al. (1996) demonstrated that cell plasmolysis before electropulsation (one
square wave pulse of 15 ms at 750 V/cm) is an efficient approach to DNA delivery
into intact plant cells (black Mexican sweet maize). They demonstrated that osmotic
changes, which create a void between the cell wall and membrane, allowed macro-
molecules to pass through the cell wall, maximizing the contact of macromolecules
with cell membrane. They suggest that what hinders direct gene transfer in intact
cells is not the presence of the cell wall but the low transfer of DNA molecules to the
cell membrane. In 1997, Eynard et al. confirmed this result that on intact plant cells
(black Mexican sweet maize), the wall may prevent the transfer and that if plasmid
was pulled through the cell wall to the membrane before the pulse by a simple hyper
osmotic treatment, the electropulsation of the intact plant cell led to transformation.
Genetics was also used by Golberg et al. (2012) trying to understand the effect of
electroporation on the cell wall. More precisely, Listeria monocytogenes cells have
Table 2 Different examples of PEF-assisted treatment applied to plants

Products Extracted molecules
Rapeseed stems and leaves Proteins, polyphenols
Grape skins Polyphenols
Grape seeds Polyphenols
Vine shoots Proteins, polyphenols
Borage leaves Polyphenols
Spearmint leaves Polyphenols
Stevia rebaudiana Bertoni Chlorophyll, carotenoids, polyphenols, proteins
Orange peel Polyphenols
Sesame cake Proteins, polyphenols
Hemp seed cake Polyphenols
Papaya seeds Proteins, polyphenols, carbohydrates
Papaya peels Proteins, polyphenols
Mango peels Proteins, polyphenols
Potato peels Steroidal alkaloids (triterpenoid)
Olive kernel Protein, polyphenols
Flaxseed hulls Polyphenols
Wood chip and switch grass Cellulose
Norway spruce Polyphenols
Barks Polyphenols
Sugar beetroot Sucrose
Red beet Betalain
Chicory Inulin
Carrot Beta-carotene
Paprika Carotenoids, vitamin C
Red cabbage Anthocyanins
Podophyllum peltatum Podophyllotoxin (lignan)
Fresh tea leaves Polyphenols
Saffron Crocin safranal, picrocrocin
Aconitum coreanum Guanfu base A (alkaloid)
Corn silk Polysaccharides
Tibetan spiritual mushroom broth Exopolysaccharides
Maize Oil, phytosterols
Soybeans Oil, isoflavonoids
Rapeseeds Oil, tocopherols, polyphenols, phytosterols
been genetically modified in order to modify the components and the load of the cell
wall. Through this approach, they observed that the sensitivity of the bacteria was
not the same according to the mutations, and they concluded that the charge on the
bacterial cell wall affects electroporation. Even if this study has been done on
microbial cells, the employed technique to determine the impact of PEF on cell
wall is very interesting and should be tested on plant cell.
Other authors took physical measurements to characterize plant tissue after PEF
treatment. Bazhal et al. (2003b) have characterized porous structure by using a
504 C. Delsart
mercury porosimeter to analyze pores, pore distribution, porosity, bulk density, and
skeletal density of the apple tissues. The results show that electroplasmolysis affects
not only cell membranes but also the cell wall integrity of samples. The sizes of the
PEF-induced pores were comparable with the tissue cell wall thickness. Through
conductivity measurements allowing the calculation of conductivity disintegration
index Z and the estimation of the characteristic damage times τ, Bazhal et al. (2003b)
demonstrated the optimal electric field strength depends on the type of tissue and is
higher for cells having developed secondary cell wall, aubergine (500–600 V/cm)
and pear and banana (900–1100 V/cm), whereas for apple, potato, carrot, and
cucumber, the value falls in the range from 200 to 400 V/cm. Another technique
proposed by Aguiló-Aguayo et al. (2014) is the study of the water distribution in
different cellular compartments (vacuole, cytoplasm, walls) after pulsed electric field
treatment. They suggested increases in both cell wall and vacuole permeability as a
result of exposure to pulsed electric fields.
An alternative approach is the observation by microscopy of the cell wall before
and after PEF. The different techniques and dyes are specified in Table 1. The cells of
several plants have been observed by microscopy after applying PEF, but the
photographs are a very limited number. In 2008, Galindo et al. examined by
microcopy the diffusion of the fluorescent dye FM1-43 through the cell wall in the
potato tissue. The diffusion of the dye FM1-43 through the cell wall of pulsed tissue
slowed, suggesting a significant decrease in cell wall permeability. Experimental
evidence links those changes with the production of H2O2 by cell wall-associated
peroxidases. This is a fast response that is already detected within 30 s after the
delivery of the electric field. In 2011 (2011a), Janositz et al. observed by microscopy
PEF-treated potato tissue stained with ruthenium red. The dye ruthenium red binds to
deesterified carboxyl groups and stains pectin in the cell wall and middle lamellae.
They observed that the tissue compartment is slightly changed. The difference form
of PEF treatments used on the potato could be an explanation for the contradictory
conclusions of these two studies. Janositz et al. worked with an electric field strength
of 5 kV/cm (20 pulses, pulses duration of 100 μs) while Galindo et al. used a single
1 ms rectangular pulse at 30–500 V/cm.
In 2010, protoplasts from cultured tobacco cells and cultured plant cells with cell
walls were observed by microscopy after staining with vital dye phenosafranine
(Janositz and Knorr 2010). Visual observation showed higher sensibility of pro-
toplasts to the electric field compared to cells with cell walls. The elimination of cell
walls leads to a loss of structural support. A noticeable decrease of protoplast cell
area was already shown after the first pulses at quite low treatment level (0.5 kV/cm,
10 pulses, pulse width of 2–8 μs, 8824 kJ/kg). This is in contrast to cells with cell
walls, where the phenosafranine uptake, which indicates irreversible pore formation,
was only registered at higher PEF intensities (1.2 kV/cm, 20 pulses, pulse width of
2–8 μs, 2541 kJ/kg). The extent of protoplast cell rupture was nearly twice as high
compared with cells with cell walls under the same treatment conditions, demon-
strating the protective effect of plant cell walls.
The observation of glandular trichome cells within the leaves of patchouli plant
by light microscopy and scanning electron microscopy showed that the cell walls
Fig. 5 Skin cross sections of (a, b, c) untreated berries (0 kV/cm), of (a’, b’, c’) PEF1-treated
berries (4 kV/cm; 1 ms, 4 Wh/kg), and of (a*, b*, c*) PEF2-treated berries (0.7 kV/cm; 200 ms,
31 Wh/kg) observed at day 0, under transmission electron microscopy: (a, a’, a*) outer hypodermis,
(b, b’, b*) inner hypodermis, (c, c’, c*) outer hypodermis. CW cell wall, o organelles, ml middle
lamella, m mitochondria, VPC vacuolar phenolic compounds, p plastid, iH inner hypodermis, oH
outer hypodermis (Reprinted (adapted) with permission from (Cholet et al. 2014). Copyright (2014)
American Chemical Society)
were broken down (rupture) after PEF (100 V/cm, 2 s of treatment) application
(Sukardi et al. 2013).
Cytological preparation for light and transmission electron microscopy has been
performed on grape skins after PEF (day 0) and on fermented skins after PEF and
alcoholic fermentation (day 14) (Delsart et al. 2014; Cholet et al. 2014). Depending
on the PEF parameters, cell wall structure was differently affected (Figs. 5 and 6).
At day 0, it was possible to observe that the cell walls of untreated berries showed
no signs of deterioration (Fig. 5a–c). At 14 days, the contrast under transmission
electron microscopy of the cell walls of untreated berries was seen to be homoge-
neous and electron dense (Fig. 6a, b). Immediately after PEF treatment at 4 kV/cm,
small spherical structures, which were localized only at the middle lamella level in
the control batch (Fig. 5c), were observed over a large portion of the hypodermis cell
wall thickness (Fig. 5c’). At 14 days, the cell walls of the outer hypodermis showed
perforations (Fig. 6a’, arrowed). At the inner hypodermis, fiber cell wall
506 C. Delsart
Fig. 6 Skin cross sections of (a, b) untreated berries (0 kV/cm), (a’, b’) PEF1-treated berries (4 kV/
cm; 1 ms, 4 Wh/kg), and (a*, b*) PEF2-treated berries (0.7 kV/cm; 200 ms, 31 Wh/kg) observed at
day 14, under transmission electron microscopy: (a, a’, a*, b*) outer hypodermis; (b, b’) inner
hypodermis. CW cell wall, eE external epidermis, iH inner hypodermis, ml middle lamella, oH outer
hypodermis, p plastid, VPC vacuolar phenolic compounds (Reprinted (adapted) with permission
from (Cholet et al. 2014). Copyright (2014) American Chemical Society)
polysaccharides were more distended than in the control batch (Fig. 6, panel b
vs. panel b’). Immediately after PEF treatment at 0.7 kV/cm, the outer hypodermis
cells (Fig. 5a*), and even more so those of the inner hypodermis (Fig. 5b*), were
disorganized, showing destructuring of the middle lamella of cell walls (arrowed). At
highest magnification, the aspect of the cell wall appeared unstructured, with the
presence of small spherical structures throughout the whole thickness (Fig. 5c*). At
14 days, cell angles were observed to be rather distended with high-contrast deposits
and perforations that evoke meatus (Fig. 6a*). Tangential walls of contiguous layers
of cells were asymmetrical on either side of the middle lamella (Fig. 6b*). Moreover,
the middle lamella was observed under transmission electron microscopy as being
more highly contrasted and electron dense (Fig. 6b*), which could be one of the
consequences of changes in the wall that contained more sites where the
counterstaining metal salts of the different sections could lodge.
Microscopy of tissue sections of yellow peas have been also studied after
electrical treatment (Baier et al. 2015). Cells of the control tissue are completely
surrounded by an intact cell wall. Cell walls in peas subjected to pulsed electric fields
appeared thinner or are locally disrupted indicating that cells are at least partially
disintegrated. The intercellular space covers a larger area, which also reflects rupture
of the cell wall and a loosening of the connections between single cells. These
observations indicate that opening of the cells is not only due to a permeabilization
of the membrane but also a consequence of disrupted cell walls.
In their aim to determine the impact of PEF on the cell walls, some scientists have
chosen to analyze the chemical composition of the cell walls of vegetal tissues before
and after the application of the electric fields. Hence, Janositz et al. (2011b) have
quantified the lignin of white asparagus. The application of PEF has an influence on
lignin content in asparagus. The amount of raw lignin decreased from 12.6%
(0.08) in untreated asparagus samples to 10.2% (0.34) in the PEF-treated aspar-
agus base section. According to the authors, this effect might be attributed to the
PEF-induced leakage of lignin-cellulose bonds, enabling the start of delignification
resulting in softer texture spear. Cholet et al. (2014) characterized the cell wall
polysaccharidic fractions of grape berry skins. Depending on the PEF parameters,
cell wall polysaccharides were differently affected. After PEF treatment of high
intensity, short duration, and low energy (4 kV/cm, 1 ms, 4 Wh/kg), pectic cell wall
organization was only slightly but measurably affected further to the treatment:
external fractions tended to increase, which probably increased the number of
weak bonds, and the quantity of internal fractions was decreased, which also
decreased the number of strong bonds. It can be hypothesized that the disappearance
of internal fractions had disturbed the microfibrillar interactions of cell walls, thus
freeing new space at the heart of the cell walls into which water may penetrate, thus
inducing a stretching thereof. After PEF treatment of higher strength, longer dura-
tion, and higher energy (0.7 kV/cm, 200 ms, 31 Wh/kg), the analysis of the
polysaccharide fractions revealed that the entire pectic skeleton had been disrupted.
The whole polysaccharide mesh was greatly weakened, only one internal fraction
being strengthened. Authors have demonstrated that cell wall structure was differ-
ently affected depending on the PEF parameters, providing variable performance in
terms of extraction.
After reviewing these articles, it seems that the majority of authors seem to
converge toward the conclusion that the application of pulsed electric fields has an
effect on the cell wall. However, there are some authors who do not agree. Sabri
et al. (1996) and Eynard et al. (1997) indicate that the transfer of plasmid DNA from
the outside to the inside of the plant cell would not be a change in the cell wall but a
better contact between the genetic material with the plasma membrane after appli-
cation a state of pre-PEF plasmolysis. However, other authors believe that the
508 C. Delsart
permeability of the cell wall can be changed by an electric field pulse. All these
authors observed an increase in the permeabilization of the wall, with the exception
of a study conducted by Galindo et al. (2008) who observed a decrease. From the
results of these studies, different hypotheses about the effects of pulsed electric fields
can be stated:
• Effect on cell wall components (polysaccharides, lignins, etc.). Still, it is not clear
whether cell wall components are changed directly due to the PEF treatment or due
to cell membrane disintegration and the release of cytoplasm. Is there an effect on
enzymes in the wall or on other intracellular enzymes released in large numbers?
• Effect on the electric charge of the wall that would induce a structural change.
• Effect of stress on the cell that would induce a reactive oxygen species (ROS)
production and the strengthening of the wall as if a pathogen or environmental
stress was attacking.
Conclusions
The plant cell wall can be both rigid and impermeable and also flexible and permeable.
Function and cell age determine the structure and the composition of the cell wall. The
environmental conditions which plants are subjected to can also affect the characteristics
of the cell wall. The application of an electric field pulses (PDF) to the cell wall has to be
considered as an external stress that can affect it. Although most studies show an effect, a
small number of researchers believe that there is no impact. In addition, there is also
disagreement on whether the pulsed electric fields increase or decrease the permeability
of the cell wall. Indeed, scientists observed a decrease in the permeabilization of the cell
wall that would be, according to their hypothesis, due to the establishment of a cell
protection system under the action of stress induced by the PEF. However, most authors
observed an increase in the permeabilization of the cell wall after PEF treatments.
Scientists have linked this increased permeability to the modification of the polysaccha-
ride and lignin composition. Others point to the importance of the electric charge of the
wall. It also was shown that PEF processing parameters and the type of treated cell
(thickness and constituents of the cell wall) influences the permeabilization of the cell
wall. The number of studies on this subject is low and requires special attention in future
years to resolve the many remaining questions.
Cross-References
▶ Atomic Force Microscopy to Explore Electroporation Effects on Cells

▶ Modeling Microbial Inactivation by Pulsed Electric Field
▶ Selective Extraction of Molecules from Biomaterials by Pulsed Electric Field
Treatment
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Regulated and Apoptotic Cell Death After
Nanosecond Electroporation 30
Stephen J. Beebe
Abstract
This chapter will focus on how nanosecond pulsed electric fields (nsPEFs) induce
apoptosis and regulated cell death (RCD) in cells in vitro. When studies analyzing
nsPEF-induced cell death (CD) began, apoptosis was the only RCD mechanism
known and was contrasted with primary “necrosis” or accidental cell death
(ACD), when plasma membranes (PMs) immediately lose integrity. Now that
necrosis is known to be programmed, the term “necrosis” has become ambiguous.
Several RCD mechanisms are now known and nsPEFs induce apoptosis as well
as other non-apoptotic RCD mechanisms in a cell-type-dependent manner. This
chapter will emphasize apoptotic CD because it is a hallmark of cancer. Cell fate
is determined by a balance between anti-apoptotic survival mechanisms and
pro-apoptotic CD mechanisms. These schemes will be outlined. Extrinsic and
intrinsic apoptosis mechanisms will be defined and a review of methods to
determine apoptosis, including their caveats and limitations, will be discussed.
Examples will be given of some cellular “sensors” and how nsPEFs might
activate them. Molecular mechanisms that induce apoptosis and other RCD
mechanisms will be presented. Overall, what is presently known about nsPEF-
induced apoptosis and RCD will be covered.
Keywords
Regulated cell death (RCD) • Intrinsic apoptosis • Extrinsic apoptosis • BCL2
family proteins • Caspases • Cytochrome c • Regulated necrosis • Accidental cell
death (ACD) • Primary necrosis
S.J. Beebe (*)

e-mail: sbeebe@odu.edu

DOI 10.1007/978-3-319-32886-7_146
512 S.J. Beebe
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
Apoptosis Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
Intrinsic Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515
Extrinsic Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515
Measurements of Apoptosis in Response to nsPEFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517
Phosphatidylserine Externalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517
Caspase Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517
Cleavage of Caspase Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518
Cytochrome c Release and ΔΨm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518
DNA Fragmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519
Changes in BCL2 Family Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519
What Causes nsPEF-Induced CD? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 520
Some Effects of nsPEFs on Apoptosis and Other RCD Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . 521
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 524
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 526
Introduction
At the “end of days,” cell death (CD) mechanisms will destabilize survival mecha-
nisms and eukaryotic cells will die. This natural CD is programmed by genetically
encoded molecular mechanisms that can be altered by pharmacologic and/or genetic
intervention(s) that target key components of CD machinery. Such regulated cell death
(RCD) that occurs in microenvironmental stresses, (post)embryonic development,
tissue homeostasis, and immune responses is referred to as “programmed cell death”
(PCD) (Galluzzi et al. 2012). PCD is nature’s euthanasia for removing unwanted or
damaged cells without damaging surrounding clonal mates and adjacent tissues. In
reality, cell fate is determined by anti-apoptotic survival signals from external sources,
such as growth factors, and internal sources, such as NFκB (nuclear factor kappa-light
chain enhancer of activated B cells) and BCL2 (B-cell CLL/lymphoma 2), and
pro-apoptotic death signals from the external environment, such as Fas (apoptosis
signaling factor), TNFα (tissue necrosis factor alpha), and the internal milieu, such as
BAX (BCL2-associated death promoter X) and t-BID (truncated BAX-like BH3
protein). These cell fate signals are integrated at mitochondrial membranes, that may
or may not involve the mitochondrial permeability transition pore (mPTP), which is
located between the inner and outer mitochondrial membranes (IMM and OMM),
allowing transport of molecules up to 1.5 kDa into and out of the mitochondrial matrix.
When CD is decided, pro-apoptotic signals overrule anti-apoptotic signals and cyto-
chrome c is released. Mitochondria are involved in making euthanasia decisions by
apoptotic or sometimes non-apoptotic RCD mechanisms.
Defining CD is a more complex exercise than originally conceived in the late
twentieth century (Kerr et al. 1972). However, to simplify classifications, it is
expedient to define CD as “regulated” or “accidental.” In the late 1990s when CD
responses to nsPEFs were first investigated, RCD was synonymous with apoptosis,
30 Regulated and Apoptotic Cell Death After Nanosecond Electroporation 513
and accidental cell death (ACD) was equivalent to “necrosis.” PCD and apoptosis
were sometimes used interchangeably; however PCD, as indicated above, is RCD
that occurs as part of developmental programs or maintenance of normal homeo-
static functions. Apoptosis is now known to be one of several RCD mechanisms
(Galluzzi et al. 2012). Several non-apoptotic RCD mechanisms have been shown to
be programmed as in “programmed necrosis” and distinct from primary necrosis or
ACD. Thus, the term “necrosis” is now indefinite and can refer to primary necrosis
or nonregulated CD with immediate loss of plasma membrane (PM) integrity or refer
to one of several RCD programs as in non-apoptotic, caspase-independent CD. If
CD is not immediate, it is highly likely to include some regulated aspects, either pro-
and/or anti-survival. While there are many RCD mechanisms (Galluzzi et al. 2012),
apoptosis will be the primary RCD mechanisms discussed in detail in this chapter in
response to nsPEFs. However, other RCD mechanisms that could be induced by
nsPEFs include necroptosis, which includes programmed lytic CD by pore forma-
tion in the PM; pyroptosis, which is characterized by caspase-1 activation and leads
to programmed lytic CD; and parthanatos, which is CD induced by PAR (poly
(ADP-ribose)) formation related to excessive DNA damage. Autophagy has also
arguably been considered a CD mechanism. However, autophagy is primarily a
survival mechanism that serves homeostatic functions for protein and organelle
turnover. What has been considered CD by autophagy is better defined as CD with
autophagy Thus, when autophagy fails, it leads to CD by interfacing with RCD
mechanisms such as apoptosis or other RCD mechanism(s).
Apoptosis occurs in three phases. First is induction, which determines the kinetics
of CD and depends on the strength-duration conditions of nanosecond pulses. The
next phase is the effector phase – when executioners are activated and the death
commitment is reached – the point of no return. This constitutes time from the first
mitochondrial change to caspase activation, which can occur quickly. The final
phase is cell degradation, when caspase substrates are cleaved and morphological
features of apoptosis appear. To ensure that CD is complete, there are feedback loops
that amplify apoptosis signaling through extensive mitochondrial release of cyto-
chrome c. Once the CD commitment is reached as executioners are sufficiently
activated and inhibitors of executioners are sufficiently inactivated, CD in vitro
occurs when PM integrity is breached. Thus, CD may appear as ACD or primary
necrosis when an apoptotic program was actually running.
Apoptosis was initially defined in vivo by morphological characteristics that
differentiated dying cells from cells undergoing or having undergone primary
necrosis. Apoptotic cells or cells during the “evolution of apoptosis” were defined
as those cells that exhibited cytoplasmic shrinkage, chromatin condensation, nuclear
fragmentation, PM blebbing, and formation of apoptotic bodies with intact mem-
branes (Kerr et al. 1972). In vivo, dying apoptotic cells are phagocytized before they
loss PM integrity, and the final stages of apoptosis occur within phagocytes.
However, in vitro where phagocytosis does not occur, apoptosis may have been
activated, but cells will eventually lose PM integrity, appearing morphologically as
primary necrosis if their molecular history was not investigated. This is referred to as
post-apoptotic necrosis. Thus, as indicated above, artificial in vitro environments can
514 S.J. Beebe
pose problems for analysis of apoptosis and RCD in general. It is for this reason that
morphologic characteristics of apoptosis in vitro can be misleading concerning RCD
identification.
For apoptosis induction by nsPEFs, low conditions may be lethal to some cell
populations, while other populations will just be stressed. In response to stress (see
▶ Chap. 17, “Cell Stress Responses to Pulsed Electric Fields”), simultaneous pro-
and anti-apoptotic signaling must be integrated. Also, autophagy may be induced in
attempts to repair and survive. These situations are also cell-type dependent and will
have an impact on the timing of appearances of apoptosis markers should apoptosis
occur. As indicated above, depending on in vitro conditions, it is possible that PM
integrity may be lost before the appearance of some apoptosis markers, such as a
DNA ladder, the largest cellular structure to degrade. Also, the appearance of more
than one RCD mechanism is not uncommon in response to nsPEFs. For example,
caspase-dependent and caspase-independent apoptosis were observed at low and
high nsPEF conditions, respectively (Ren and Beebe 2011; Ren et al. 2012; Beebe
et al. 2013). Also, RCD mechanisms can depend on post-pulse incubation conditions
(Pakhomova et al. 2013; Morotomi-Yano et al. 2014). Thus, circumstances other
than electric field settings can affect RCD mechanisms (see section “Measurements
of Apoptosis in Response to nsPEFs”).
Initial studies analyzing apoptotic CD in response to nsPEFs (Beebe et al. 2002,
2003) began about the time evading apoptosis became known as a hallmark of cancer
(Hanahan and Weinberg 2000). The finding that nsPEFs induced apoptosis suggested
this technology could bypass this cancer hallmark as a new treatment strategy for
cancer. It is also noteworthy that the major cell lines used in those early studies, Jurkat
and HL-60 cells, respond to nsPEFs by apoptosis induction; not all cell types
do. Apoptosis was also of great interest because it was shown to be important in
numerous physiological and pathological circumstances. Now, other RCD mecha-
nisms are known to be important for these same reasons (Galluzzi et al. 2012).
Apoptosis Pathways
Apoptosis can occur by either intrinsic (intracellular) or extrinsic (extracellular)

mechanisms. In this specific case, the nsPEF stimulus is applied externally. So are
electric fields “sensed” and transmitted by intrinsic and/or extrinsic signals? In
extrinsic apoptosis, the death ligand or stimulus comes from the extracellular
media; however, these death ligands act on cognate death receptors on the
PM. There is no evidence that nsPEFs actually stimulate death receptors. However,
the PM itself can be considered a receptor for nsPEFs. An initial hypothesis
addressing nsPEF cell “sensors,” based on a simple electric model of a biological
cell, was that sub-microsecond pulses would have greater interactions with intracel-
lular structures (Schoenbach et al. 2001). While this was found to be valid (Beebe
et al. 2013; Schoenbach et al. 2001), the PM was not impervious as shown by
nsPEF-induced nanopores in PM smaller than propidium iodide, which was used to
determine membrane permeability (Vernier et al. 2006; Pakhomov et al. 2009). The
hypothesis from the simple biological cell model did not address whether intracel-
lular effects were responsible for apoptotic CD. However, since nsPEFs had intra-
cellular effects (Schoenbach et al. 2001) and induced apoptosis (Beebe et al. 2002,
2003), it was tempting, and still is, to consider that nsPEFs induce apoptosis due to
intracellular effects. However, this does not exclude necessary effects on PMs for
apoptosis or other RCD mechanisms. The groundwork for defining intrinsic and
extrinsic apoptosis and how they are measured (see section “Measurements of
Apoptosis in Response to nsPEFs”) will be addressed before outlining how nsPEFs
induce apoptosis (see sections “What Causes nsPEF-Induced CD?” and “Some
Effects of nsPEFs on Apoptosis and Other RCD Mechanisms”).
Intrinsic Apoptosis
Intrinsic apoptosis is induced by intracellular stresses that directly or indirectly lead to

release of cytochrome c and other pro-apoptotic factors from mitochondria. Intracellular
stresses induced by nsPEFs can include organelle or DNA damage, oxidative stress,
cytosolic/mitochondrial calcium overload, and/or unfolded proteins in the endoplasmic
reticulum (ER), among other possible internal effects. These become cellular stress
sensors linked to signaling cascades that are all wired to mitochondria-centered control
(Galluzzi et al. 2012; Gogvadze et al. 2006). Cell death is initiated here by opening the
mPTP and/or mitochondrial outer membrane permeabilization (MOMP) with cyto-
chrome c release as well as release of other factors such as SMAC/DIABLO (second
mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein
with low pI), which promote apoptosis by blocking IAPs (inhibitors of apoptosis). This
action on mitochondria requires pro-apoptotic BCL2 family members such as BAX or
BAK (BCL2 homologous antagonist/killer), which are held in check by anti-apoptotic
BCL2 family members such as BCL2 or BCLxl (B-cell lymphoma extra large). The
functions of these pro- and anti-apoptotic BCL2 proteins are regulated by pro-apoptotic
BH3-only BCL2 family members such as BAD (BCL2-associated death promoter),
which sequesters anti-apoptotic BCL2 proteins and thereby releases pro-apoptotic
BCL2 proteins such as BID, which directly activates pro-apoptotic BCL2 proteins.
Both mechanisms lead to MOMP and cytochrome c release. Cytochrome c, deoxy-
ATP, and initiator caspase-9 bind to APAF1 (apoptotic protease activating factor 1),
forming the apoptosome, one of the major scaffold-mediated activation mechanisms for
initiator caspase cleavage. Initiator caspase-9 is activated here, which then activates
executioner caspase-3, and the apoptotic degradation phase begins.
Extrinsic Apoptosis
Extrinsic apoptosis initiates CD through CD receptors, such as Fas receptors in

collaboration with FADD (FAS-associated death domain) that together activate
caspase-8 at the cytoplasmic side of the PM in the death-induced signaling complex
(DISC), the other major scaffold-mediated activation mechanism for initiator caspase
516 S.J. Beebe
cleavage. Type I cells (lymphocytes) express lower levels of XIAP, DISC formation is
rapid, and caspase-8 directly activates caspase-3, independent of mitochondrial
involvement. In contrast, type II cells (hepatocytes) express higher levels of XIAPs,
and DISC formation occurs more slowly and is insufficient to directly activate
caspase-3 (Jost et al. 2009). In this case, DISC-activated caspase-8 cleaves BID to
generate t-BID, a pro-apoptotic BH-3 only protein, which leads to MOMP and release
of cytochrome c as well as SMAC / DIABLO from mitochondria. This then leads to
activation of caspase-3 by caspase-9, like that occurring in intrinsic apoptosis.
There are several models proposed for release of cytochrome c and other
pro-apoptotic proteins from mitochondria (Gogvadze et al. 2006). One mechanism
activates the mPTP that forms between the IMM and OMM allowing transport of ions
and larger molecules. This is called mitochondrial permeability transition (MPT). This
causes swelling of the IMM and rupture of the OMM. This is likely related to calcium
overload of mitochondria and can occur in apoptosis and programmed necrosis. A
second mechanism for cytochrome c release is through a channel formed by oligo-
merization of BAX/BAK, forming pores large enough for transport of proteins from
the inner mitochondria space. t-Bid induces oligomerization of BAX/BAK and
enhances pore formation. The mitochondrial membrane potential (ΔΨm) can be lost
if this is a pervasive event or maintain if it occurs in a limited number of mitochondria
and/or maintained by of glycolytic ATP. A third mechanism for cytochrome c release
is by positive feedback of caspase activity on cytochrome c release including loss of
ΔΨm (Ren and Beebe 2011), permeabilization of OMM, and disruption of mitochon-
drial morphology. The mechanism(s) whereby nsPEFs induced cytochrome c release
remains to be determined. However, loss of ΔΨm occurs at lower electric fields than
cytochrome c release (Jost et al. 2009) and most likely at earlier times (Beebe et al.
2003) after nsPEF treatment. Relationships between cytochrome c release and ΔΨm
are likely cell-type dependent. NsPEF-stimulated E4 squamous carcinoma cells (SCC)
released cytochrome c (Ren and Beebe 2011) and B16f10 cells did not (Ford et al.
2010), yet both exhibited decreases in ΔΨm.
A recent published review assessed effects of high voltage nanosecond electric
pulses on eukaryotic cells (Batista Napotnik et al. 2016). Apoptosis in vitro was
detected in 47 papers, and only 5 failed to detect it. However, based on some
methods used to demonstrate apoptosis, this may be an overestimation of apoptosis
occurrences. Methods to determine apoptosis are not trivial. Below is a discussion of
a number of different techniques that can be used and should not be used to
determine apoptosis in nsPEF-treated cells in vitro.
Measurements of Apoptosis in Response to nsPEFs
There are a number of cellular “markers” that have been generally used to define
apoptosis, but a single identifier of apoptosis is insufficient. Apoptosis, or any RCD
program, should be defined by multiple markers. Not all markers used are necessar-
ily valid, especially in response to nsPEFs, and specific considerations should be
given to methods and protocols employed.
Indicators, but not necessarily definitive identifiers, for nsPEF-induced apoptosis

in vitro include morphological changes, phosphatidylserine (PS) externalization,
caspase activation, PARP (poly(ADP-ribose)polymerase) cleavage, cytochrome
c release from mitochondria, DNA fragmentation, and changes in pro- and anti-
apoptotic proteins. Of fundamental importance for these analyses is a strength-
duration response (dose response) and/or time course studies. Apoptosis is a time-
dependent process, so not all markers occur concurrently. Studies can be performed
efficiently by first doing a 24 h “dose” response for viability and then doing a
relatively broad time course for apoptosis markers with conditions that cover
10–90% CD. Below, the various apoptosis markers will be considered and some
examples of how these have been used with nsPEF studies.
Morphology
While apoptosis was initially defined by cell morphology, much has subsequently been
discovered indicating overlapping morphologies with different RCD mechanisms. For
several reasons, the Nomenclature Committee on Cell Death (NCCD) recommends that
morphology should not be used as an apoptosis or other RCD indicator (Galluzzi et al.
2012). First, similar CD morphologies exhibit a wide range of functional and bio-
chemical heterogeneity. Second, morphological identification is subjective and
difficult to quantify. Also, it is possible to block apoptosis, and cells exhibit
morphological features of necroptosis. In addition, in vitro conditions can play a
significant role in morphological features of RCD. As indicated above, in vitro
conditions leading to post-apoptotic necrosis can be misidentified as non-apoptotic
RCD. Finally, biochemical apoptosis markers are objective and quantitative and
express a range of conditions that are hidden behind morphological characteristics.
Phosphatidylserine Externalization
Externalization of Phosphatidylserine (PS) is an early apoptosis marker for phago-

cytosis (Galluzzi et al. 2012). During apoptosis, PS is externalized from the inner to
the outer leaflet of the PM. However, under the influence of electric fields, PS can be
pulled through nanopores in this same way (Vernier et al. 2006). Allowing PS to
re-equilibrate for at least 30 min after electric field exposure may resolve this
concern (Batista Napotnik et al. 2016). PS externalization that is sensitive to caspase
inhibition can also be considered to be apoptosis mediated (Beebe et al. 2003).
Caspase Activation
Caspase activation is a good indication that apoptosis is active. Active caspases can
be shown by increased catalytic activity as well as by demonstrating antibody-
specific active caspases by western blots. To identify intrinsic versus extrinsic
518 S.J. Beebe
apoptosis, initiator caspase-8 and caspase-9 can also be determined in the same
ways. Catalytic assays with good specificity for caspase-3/caspase-7, caspase-8, and
caspase-9 were demonstrated with Jurkat clones that did and did not express
caspase-8, FADD, or APAF-1 (Ren et al. 2012). Example of caspase activation as
apoptosis markers occurred as increased caspase-3 catalytic activity in Jurkat cells
(Ren et al. 2012; Beebe et al. 2003, 2013) and U937 cells (Pakhomova et al. 2013)
and in Jurkat and HL-60 cells as cleaved caspase-3 on western blots (Morotomi-
Yano et al. 2014; Estlack et al. 2014). NsPEFs have also induced increases in
caspase-8 and caspase-9 catalytic activity (Ren et al. 2012) and cleaved caspase-
8 (Estlack et al. 2014). However, neither catalytic nor western blot assays allow
determination of percentages of caspase-positive cells. Although constitutively
present as inactive, pro-caspases, increased expression of caspase mRNAs may be
needed where apoptosis is inhibited by high levels of anti-apoptotic proteins often
overexpressed in cancer cells. For example, increases in mRNA for caspase-3 may
have been needed for nsPEF-induced CD in U937 cells that expressed high levels of
anti-apoptotic cFLIP (cellular FLICE-like inhibitory proteins) (Estlack et al., 2014).
Cleavage of Caspase Substrates
Active caspases lead to proteome changes including appearances of apoptosis

cleavage products, such as caspase-specific cleavage of PARP, a DNA repair
enzyme, that appears on western blots (Pakhomova et al. 2013; Morotomi-Yano
et al. 2014). Caspases also cleave major structural elements in the cytosol, mito-
chondria, and nucleus as well as survival enzymes such as protein kinases.
Cytochrome c Release and DCm
Release of cytochrome c is a valid marker for apoptosis and is sometimes called a

point of no return for apoptotic CD. However, type I cell-extrinsic apoptosis is
independent of mitochondria, and cytochrome c is not released. Mechanisms for
cytochrome c were presented previously (see section “Extrinsic Apoptosis”). In
response to nsPEFs, loss of ΔΨm occurs fairly soon after treatment (Beebe et al.
2013; Batista Napotnik et al. 2012). Nevertheless, since all normal cells have
cytochrome c within their mitochondria, cytochrome c release into the cytoplasm
can be demonstrated by western blots of isolated cytoplasm that contains cyto-
chrome c, but is devoid of mitochondrial proteins, such as cytochrome c oxidase
(Beebe et al. 2003). Another cytochrome c release assay permeabilizes cells to
remove cytochrome c from the cytosol and then measures cytochrome c in fixed
cells that remains in mitochondria with primary cytochrome c antibody and fluores-
cent secondary antibody. Here, a decrease in cytochrome c after treatment indicates
release (Ren and Beebe 2011; Ren et al. 2012).
While loss of ΔΨm can occur, it is not a prerequisite for apoptosis (see section
“Extrinsic Apoptosis”). Recall that loss of ΔΨm could occur as a result of other RCD
mechanisms involving MPT and will definitely occur before cells lose PM integrity,
such as in post-apoptotic necrosis. For nsPEF effects, it is reasonable to consider that
the mechanism for dissipation of ΔΨm is nanoporation of the IMM (Batista
Napotnik et al. 2012). While this can be true, there appears to be other effects on
ΔΨm at lower electric fields. This is presented in section “What Causes nsPEF-
Induced CD?”
DNA Fragmentation
Fragmented DNA that is apoptosis-specific forms a ladder of ~180–200 base pair

DNA fragments on agarose gels. DNA is cleaved by endonucleases at sites between
nucleosomes, the protein-containing structures that occur in chromatin every
180–200 bp. This forms a DNA ladder instead of a DNA smear that indicates
nonspecific DNA cleavage. In contrast to many reports and claims that the
TUNEL (terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling)
assay provides an apoptosis marker, it exhibits no specificity for internucleosomal
DNA stand breaks. The TUNEL assay cannot differentiate apoptosis-mediated from
non-apoptosis-mediated DNA cleavage, including primary necrosis (Grasl-Kraupp
et al. 1995). TUNEL-positive cells could be a result of non-apoptotic RCD or ACD
instead of apoptosis. It does indicate that DNA has been damaged. Sub-G0 DNA
upon cell cycle analysis can also indicate DNA damage, but not necessarily from
apoptosis.
Changes in BCL2 Family Proteins
Changes in pro- and anti-apoptotic proteins (BAX, BAK, t-BID and BCL2, BCLxl,
respectively) do not specifically show that apoptosis is present. Apoptosis is deter-
mined by the balance between pro- and anti-apoptotic proteins, not their absolute
changing levels. When the BAX/BCL2 ratio is greater than 1, apoptosis can be
initiated. Thus, changes in BCL2 family proteins indicate a shift in the balance of
factors that determine apoptosis, and survival has occurred, but it does not neces-
sarily indicate that the shift is sufficient to trigger apoptosis. However, the presence
of Puma and/or Noxa, two BH-3 protein expressed in response to increases in p53,
indicates the presence of DNA damage.
In a nutshell, understanding how to effectively and convincingly demonstrate
these basic apoptosis features should help design basic experimental strategies to
demonstrate how nsPEFs induce CD either by extrinsic or intrinsic apoptosis. If CD
is delayed and apoptosis markers are not present, non-apoptotic RCD or
programmed necrotic CD is present. For both internal and external stimuli, caspase
activation is a hallmark of apoptosis. Recall that caspases are regulated by simulta-
neously opposing mechanisms that activate and inhibit them. Apoptosis requires that
caspase inhibitors are turned off and caspase activators are turned on. While the
presence of active caspases is a good indicator of apoptosis, other apoptosis markers
520 S.J. Beebe
should always be included, such as cytochrome c release, PARP cleavage, DNA

ladders, or PS externalization, when used with caution. Other indicators such as
changes in BCL2 family proteins can provide support, but are not definitions of their
own. TUNEL and morphological indicators can be uncertain indicators. The NCCD
favor biochemical markers and discourage the use of morphology for defining CD
(Galluzzi et al. 2012).
What Causes nsPEF-Induced CD?
Because pulse durations are short, thermal effects can be neglected in spite of the
extreme electrical power levels (Schoenbach et al. 2001). Electric field-specific CD
is independent of thermal effects. Heat provides an additional stimulus and another
variable. Long pulse durations, high electric field intensities, long treatment times,
and/or high repetition rates can cause significant thermal effects when heat cannot be
dissipated. Temperatures above 43 C can cause thermal damage. Relationships
among these nsPEF parameters are incomplete and complicated by heterogeneous
effects on cell structures and functions and dependence on cell type, cell cycle, and
in vitro conditions. Heat should be excluded from the mix.
NsPEF-induced cellular effects have been described by considering the nonther-
mal energy density (absorbed dose) (Ibey et al. 2009, 2010) or by considering
charging effects that impact cells (Schoenbach et al. 2009). Energy density as a
metric for efficacy of nsPEFs on cells can be simply expressed as E2τN, where E is
the electric field, τ is pulse duration, and N is pulse number. This works well at a
given pulse duration as electric fields or pulse numbers are increased, but not when
pulse durations are varied (Beebe et al. 2002, 2003; Ibey et al. 2010; Schoenbach
et al. 2009). The electric charge transferred through cellular membranes and cellular
structures of suspended cells is a valuable determinant that holds well for defining
efficacy of nsPEFs on PM and subcellular membrane effects as well as secondary
biological responses including RCD (Schoenbach et al. 2009). This paper demon-
strated linear relationships for viability and platelet aggregation in response to
nsPEFs with different pulse durations, electric fields, and pulse numbers used the
simplified formula Eτ(N)1/2, where Eτ and N were defined above. Scaling with the
square root of pulse number considers a statistical motion of cells between applied
electric fields as an extension of random walk statistical result due to random rotation
of cells in solution during multiple pulses. In early experiments, when energy density
was held constant for different pulse durations, electric charge transferred into cells
scaled with caspase activation in Jurkat and HL-60 cells (Beebe et al. 2002, 2003),
cytochrome c release in Jurkat cells (Beebe et al. 2003), cytochrome c release and
increases in BAX in HCT116 colon carcinoma cells with and without p53 (Hall et al.
2007), and loss of PKA (protein kinase A) catalytic activity (Beebe 2015). The
movement of charged ions through all cell structures would be expected to cause
mechanical effects and is proposed to modulate cell functions including apoptosis
induction and changing gene expression (Roth et al. 2016).
It was suggested that nanopores in all cell membranes may be responsible for
RCD induction (Gowrishankar et al. 2006). The finding that gadolinium blocked PM
permeabilization and reduced CD in Jurkat and GH3 cells in response to nsPEFs
suggested that CD was mediated by PM permeabilization (André et al. 2010). Other
studies supported this hypothesis, but suggested there was a mitochondrial mecha-
nism downstream of the PM that appeared to be important. In N1-S1 hepatocellular
carcinoma (HCC) cells, dissipation of ΔΨm was calcium dependent at lower and
intermediate electric fields and calcium independent at much higher electric fields
(Beebe et al. 2013; Beebe 2015). The calcium-dependent component for the loss of
ΔΨm suggests that a mechanism other than IMM permeabilization may be involved,
since membrane permeabilization does not require calcium. Calcium was necessary,
but not sufficient on its own for loss of ΔΨm and loss of viability, which were
coincident and electric field dependent. NsPEFs that were at and significantly above
the threshold for loss of ΔΨm required an influx of calcium across the PM and a
square pulse waveform with fast rise-fall times, for optimal effects. N1-S1 cells
exposed to nsPEFs waveform that exhibited a slow rise-fall time and a miss-matched
load (bipolar at pulse end) exhibited an influx of calcium, but not a loss of ΔΨm or
loss of viability. Since calcium functions (almost) always require a protein, a
calcium-binding protein may be necessary. Alternatively, nsPEFs may more readily
promote mitochondrial calcium overload. A number of inhibitors of proteins that are
or may be associated with mPTP, which include calcium- and voltage-dependent
proteins, did not significantly alter the calcium-dependent loss of ΔΨm (Beebe
2015). This suggests effects on mPTP may not be involved. Additional studies are
required to determine how nsPEF dissipates ΔΨm (Fig. 1).
Some Effects of nsPEFs on Apoptosis and Other RCD Mechanisms
For nsPEFs to induce apoptosis, some cellular sensors must be modulated, such as
the PM, ER, mitochondria, nucleus/DNA, and cytoskeleton or other cellular struc-
tures that leads to executioner mechanisms that can be pharmacologically or genet-
ically modified (Galluzzi et al. 2012). Once a sensor is modulated, time-dependent
mechanisms initiate activation of RCD machinery (inactive pro-caspases) that are
constitutively expressed in all cells. NsPEFs can induce stress responses (see
▶ Chap. 17, “Cell Stress Responses to Pulsed Electric Fields”). It is also likely
that adaptive responses such as autophagy may be activated depending on the
stimulus intensity. For example, nsPEFs (10 ns, 150 kV/cm, >100 pulses) activated
autophagy in U937 and CHO-K1 cells as a mechanism to repair cellular damage
until conditions overwhelm these processes resulting in CD (Ullery et al. 2015). An
example of time dependence of caspase activation as an apoptosis marker was shown
as HL-60 cells were exposed to 10 ns and 60 ns pulses with near equivalent energy
densities. Caspase activation occurred earlier with the 60 ns pulse (25 min), while
caspase activity reaches a similar level later with the 10 ns pulse (40 min) (Beebe
et al. 2003). Likewise, 40 minutes after treatment, cytochrome c release was greater
for 60 ns pulse than 10 ns pulses. Cytochrome c release occurred earlier (1.5–3 h)
522 S.J. Beebe
Fig. 1 Hypothesis for nsPEF-induced apoptosis – nsPEFs affect PMs and intracellular mem-
branes and structures as indicated by the lightning bolts. While there is no evidence that nsPEFs act
on the Fas receptor (bolt I), they do affect the PM (an extrinsic receptor), endoplasmic reticulum
(ER), mitochondria, and nucleus (bolts II–V, respectively). Activation of the extrinsic pathway in
type I cells causes direct activation of caspase-8 and caspase-3. In type II cells, caspase-8 cleaves
BID and the product t-BID causes pro-apoptotic proteins like BAX/BAK to induce cytochrome
c release. The pathway downstream of t-Bid is shared with the intrinsic pathway. nsPEF-induced
permeabilization of the PM causes an influx of Na+ and Ca2+. Ca2+ can lead to activation of calpains
that can cleave BID (Ren and Beebe 2011; Beebe et al. 2013). In combination with Ca2+, through
the intrinsic pathway, electric fields may act on the mitochondria to promote a loss of ΔΨm and
cytochrome c release (Ren and Beebe 2011) leading to activation of caspase-9 at the apoptosome
and then activation of caspase-3 (Ren et al. 2012). Electric fields can also activate the intrinsic
apoptosis pathway by acting on the ER and/or nucleus, signaling mitochondria to release cyto-
chrome c. DNA damage can increase p53, which induces Puma and/or Noxa to promote mitochon-
drial release of cytochrome c. Apoptosis is induced when the BAX/BCL2 ratio favors BAX for
cytochrome c release. Caspases also damage DNA acting at nucleosomes to form 180–200 bp
fragments. Direct effects on mitochondria may also induce CD by other RCD mechanisms
(Modified and reprinted with permission Ref. (Beebe et al. 2013))
than caspase activation (3–4.5 h) in Jurkat cells (Ren et al. 2012). Time dependence
of apoptosis was also shown in U937 cells as caspase-3/caspase-7 (1 h) and PARP
cleavage (2 h) (Pakhomova et al. 2013).
NsPEFs have been reported to induce both intrinsic and extrinsic apoptosis. The
PM serves as the receptor for extrinsic apoptosis. Consistent with this scheme,
caspase activation appeared before or without cytochrome c release (Ren and Beebe
2011; Ford et al. 2010). The differences between type I cells with readily activated
DISCs and low IAP levels and type II cells with low DISC activation and high IAP
levels explain differences between type I and type II cell-extrinsic pathways (see
section “Extrinsic Apoptosis”). The extrinsic pathway was also activated indirectly
by nsPEF-induced release of FAS ligand (FASL) in type I cells with caspase-8 and
caspase-3 activated, but not cleavage of BID, cytochrome c release, or activation of
caspase-9. (Estlack et al. 2014). In the same study, U937 cells expressed high levels
of cFLIP. These results resolved why U937 cells are more resistant than Jurkat cells
to nsPEFs.
For intrinsic apoptosis, nsPEFs are hypothesized to affect subcellular structures
(usually membranes). Organelles are considered to be intracellular “receptors” or
“sensors.” The ER has been shown to be a sensor for nsPEF-induced release
intracellular calcium (White et al. 2004; Semenov et al. 2013). nsPEFs have also
been shown to affect intranuclear structures including permeabilization (Thompson
et al. 2016) and damage to the nuclear membrane, inducing genotoxicity, which
severely compromises telomere sequences and affects the cell cycle (Stacey et al.
2011). Small nuclear ribonucleoprotein particles (snRNPs) were also affected,
suggesting effects on nuclear RNA-protein complexes (Chen et al. 2007). NsPEFs
caused disintegration of the actin cytoskeleton as a result of cell permeabilization,
cell swelling, and an osmotic imbalance (Pakhomov et al. 2014). Mitochondria are
also nsPEF sensors for apoptosis induction. Mitochondria are hubs in the wheel of
life with ATP production and CD with regulation of apoptosis as well as other RCD
mechanisms. Essentially all apoptosis mechanisms invoke mitochondrial involve-
ment (Galluzzi et al. 2012; Gogvadze et al. 2006).
NsPEFs induce heterogeneous CD mechanisms including caspase- and calcium-
dependent and caspase- and calcium-independent mechanisms in diverse set of
responses in E4 SCC including mechanisms involving caspases and calpains, quan-
titative and reciprocal changes in BID and t-BID, flow cytometric analysis of
cytochrome c release, and roles for intracellular and extracellular calcium (Ren
and Beebe 2011). Caspase activation without cytochrome c release at early times
and at lower electric fields suggested type 1 cell-extrinsic apoptosis. However, at
higher electric fields, cytochrome c was released (~50% of cells) by caspase-
independent (~40%) and caspase-dependent (60%) mechanisms. Increases in
t-BID and decreases in BID were caspase dependent (55–60%, apoptosis) and
calcium dependent (40–45%). The caspase-independent, calcium-dependent BID
cleavage was shown to be sensitive to calpain inhibition (z-LLY-fmk) indicating
that it was calpain mediated. Caspase-independent BID cleavage (40–45%) that was
calcium-dependent and calcium-independent demonstrated roles for calcium-
sensitive and calcium-insensitive calpain activity as well as for extracellular (70%)
and intracellular (30%) calcium sources. Activation of caspases and calcium-depen-
dent and calcium-independent calpains suggested apoptosis as well as non-apoptotic
RCD mechanisms occurring coincidently. These data demonstrate nsPEF-induced
cross talk among three different protease families making use of two calcium sources
524 S.J. Beebe
at the critical BID cleavage point connecting extrinsic and intrinsic apoptotic and
non-apoptotic RCD pathways provided beneficial repetitive mechanisms to maxi-
mize cytochrome c release and multiple RCD programs.
NsPEF stimulus intensity can be adjusted by pulse conditions. A single pulse at a
given pulse duration with a very low versus very high electric field can be perceived
as distinct stimuli by cells. Jurkat cells deficient in APAF-1 and unable to initiate
apoptosis through the apoptosome and caspase-9 exhibited APAF-1 and caspase-
dependent apoptosis at relatively low electric fields (ten 60 ns pulses, <40 kV/cm),
but APAF-1- and caspase-independent RCD at higher electric fields (ten 60 ns pulses
>40 kV/cm) (Ren et al. 2012; Beebe et al. 2013). This is in agreement with data
showing apoptotic and non-apoptotic CD mechanism(s) in Jurkat cells exposed to
10 ns pulses using internucleosomal DNA fragmentation (Ibey et al. 2010). As
electric fields were increased, caspase-independent RCD increased and coincided
with caspase-dependent apoptosis. However, it is not possible in either study to
determine whether one mechanism predominated over the other. Using Jurkat cells
deficient in FADD and caspase-8, nsPEFs induced caspase activation through
caspase-independent cytochrome c release through the intrinsic pathway, demon-
strating that the extrinsic pathway is not necessary for apoptosis induction in Jurkat
cells. However, this does not mean that extrinsic apoptosis cannot occur in these and
in fact do (Estlack et al. 2014).
In U937 cells, nsPEF can induce two modes of CD depending on the presence or
absence of sucrose in the extracellular media. Without sucrose, cells swell and die in
1–2 h by “necrosis,” but given the delay in CD, this is likely programmed necrosis as
opposed to primary necrosis, which would be immediate. In the presence of sucrose,
cells die by apoptosis as defined by caspase activation and PARP cleavage in 1 and
2 h after treatment, respectively. Sucrose apparently “unmasked” apoptosis
mechanisms.
The presence or absence of extracellular calcium determined nsPEF-induced CD
mechanisms depending on the cell type (Morotomi-Yano et al. 2014). In the pres-
ence of extracellular calcium, HeLa S3, HEK293, and K562 cells underwent RCD
characterized by PAR formation, a non-apoptotic RCD mechanism called
parthanatos or PAR-mediated CD (Galluzzi et al. 2012). In the absence of calcium,
HeLaS3 cells were less susceptible to nsPEFs, but exhibited apoptotic proteolysis of
caspase and PARP-1. In the presence of calcium or irrespective of calcium, HL-60
cells and Jurkat cells respectively exhibited caspase-associated apoptosis.
Conclusions
Apoptosis or caspase-mediated RCD is nature’s primary means of euthanasia when

cells are no longer needed during homeostasis, embryonic development, and
immune system function. NsPEFs induce RCD in a cell-type-dependent manner,
including CD through intrinsic and extrinsic apoptosis pathways as well as
non-apoptotic RCD mechanisms, which have not been well characterized.
Non-apoptotic CD defined as “necrosis” is confusing since necrosis can be regulated
Table 1 Methods of determining apoptosis

Apoptosis marker Method Advantages Disadvantages
Morphology Microscopy Initial definition of Subjective
apoptosis Difficult to quantify
In vitro artifacts
Regulated cell death with
overlapping morphologies
Not recommended
Phosphatidylserine Flow Typical apoptosis nsPEFs externalize PS
(PS) externalization cytometry feature Analysis 30 min after
Microscopy Easy to quantify exposure
Caspase activation Catalytic Quantitative Does not identify % positive
activity Can be isozyme cells
specific
Identifies specific
pathways
Western blot Quantitative Does not identify % positive
Specific cells
Time intensive
Caspase-substrate Western blot Quantitative Does not identify % positive
cleavage Specific cells
Downstream of Time intensive
active caspase
Cytochrome Flow Identifies % of Requires care
c release cytometry positive cells Does not work with type I
Identifies type II extrinsic pathway
extrinsic and intrinsic
pathways
Cytosol Specific Time intensive
western blot Identifies type II Requires care
extrinsic and intrinsic Mitochondria-free cytosol
pathways Does not identify % positive
cells
DNA Agarose gel Specific Time intensive
fragmentation electrophoresis Late event – may not occur
(~180–200 bp) before in vitro post-apoptosis
necrosis
Change in BCL2 Western blot Supportive Non definitive
proteins
and is different than primary necrosis, which includes immediate cell rupture. When
PAR is present, the RCD mechanism is likely parthanatos. If cell death is time
dependent, occurs in the absence of apoptosis markers (Table 1), and is not further
characterized, it is best defined as non-apoptotic or caspase-independent RCD rather
than necrosis. nsPEF-induced extrinsic apoptosis is most likely induced with the PM
as the “receptor” as opposed to death receptors like Fas. Intrinsic apoptosis origi-
nates from intracellular “sensors” including mitochondria, ER, nucleus/DNA, cyto-
skeleton, and/or other intracellular structures less well explored. Intrinsic apoptosis
526 S.J. Beebe
may (intrinsic and extrinsic type II cells) or may not (extrinsic type I cells) include
cytochrome c release. nsPEFs have also been shown to induce autophagy, which is a
survival mechanism and shares components with apoptosis and other RCD mecha-
nisms. Post-autophagic CD is best described as CD with autophagy, most likely
occurring by some RCD mechanism.
Unlike heat, cold, radiation, and any other cell stresses, no living cell or organism
on earth evolved defense mechanisms against nsPEFs, because they do not exist in
nature. Cell fate is controlled by a balance between CD signals and survival signals.
NsPEFs disrupt this balance by overwhelming cell survival mechanisms. Recall also
that the in vitro extracellular milieu complicates defining CD mechanisms and that
nsPEFs may induce more than one CD mechanism simultaneously.
Future in vitro studies will more specifically define events downstream of cellular
“sensor” or “receptors” activated by nsPEFs, for example, analysis of effects on ER,
chaperones, and protein unfolding responses; mitochondria, cytochrome c release,
ΔΨm, and electron transport system; DNA and intranuclear perturbations and
transcription; responses emitted from actin cytoskeletal disruption and lysosomes;
and effects on metabolic and enzymatic mechanisms that regulate proliferation. In
addition, non-apoptotic RCD mechanisms presently described as “necrosis” require
better characterization. As this field broadens, new investigators will strategize
approaches beyond these to widen our understanding of how nsPEFs induce apo-
ptosis and RCD.
Cross-References
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Part III
Electroporation Phenomena on Tissue Level
and Systemic Effects
Boris Rubinsky
Electroporation in Scars/Wound Healing
and Skin Response 31
Laure Gibot and Alexander Golberg
Abstract
Wounds are an essential part of the life cycle of organisms due to their interaction
with environment, which can sometimes be very harsh. Although wound healing
has puzzled humanity from early days and has gradually emerged from art and
witchcraft to modern medical procedures, the detailed mechanisms underlying
what results in normal and abnormal wound healing are not well established. This
gap in knowledge exists because of the tremendous complexity of the overall
wound-healing process. Despite this gap in our understanding of detailed mech-
anisms, new methods to treat wounds continue to appear, bringing promising new
solutions to patients suffering from acute or chronic wounds. Because of its
specific action on cell membrane without extensive heating and with no observed
effects on the extracellular matrix, electroporation found application in multiple
aspects of treating wounds. In this chapter, the authors explain the basics of what
the wounds are and what is known about wound-healing process. The use of
electroporation-based technologies for various applications for treating wounds,
wound disinfection, gene electrotransfer for the treatment of diabetic chronic
wounds, and electrostimulation of wound healing, is discussed in this chapter. In
addition, the skin response to electroporation, especially with emerging applica-
tions of electroporation in skin rejuvenation and scars and keloid treatments, is
discussed.
L. Gibot (*)
Toulouse, France
e-mail: gibot@ipbs.fr
A. Golberg (*)
The Porter School of Environmental Studies, Tel Aviv University, Tel Aviv, Israel
e-mail: agolberg@gmail.com; agolberg@tauex.tau.ac.il

DOI 10.1007/978-3-319-32886-7_64
532 L. Gibot and A. Golberg
Keywords
Electroporation • Wound healing • Human skin • Wound disinfection • Scar
treatment • Rejuvenation
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532
Fundamentals of Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533
Why Electroporation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535
Skin Response to the Externally Delivered Pulsed Electric Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535
Skin Rejuvenation with Pulsed Electric Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 536
Gene Electrotransfer (GET) as a Tool in Wound-Healing Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538
Electrostimulation of Healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541
Electrochemotherapy Stimulates Aesthetic and Functional Wound-Healing Process . . . . . . . . . 541
Burn Wound Disinfection with Pulsed Electric Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 542
Applications of Electroporation in Surgery: Mesh Disinfection and Scar Treatment . . . . . . . . . 545
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 546
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547
Introduction
Wounds are an essential part of the life cycle of organisms due to their interaction
with the harsh environment. The development of new tissues in place of a wound is a
complex set of processes controlled by a myriad of internal and external factors.
Understanding and exercising control over this overall set of processes could
transform a huge segment of patient care. Although wound healing has puzzled
humanity from early days, the detailed mechanisms underlying what results in
normal and abnormal wound healing are not well established. This gap in knowledge
exists because of the tremendous complexity of the overall wound-healing process
with results from interactions among the fields of biology, chemistry, mechanics, and
electrics all occurring in space and time. Despite this gap, new methods to treat
wounds continue to appear, bringing promising new solutions to patients. In recent
years, electroporation-based therapies have been suggested to improve various
aspects of wound-healing process.
Electroporation of skin is a unique process, when short, high-voltage pulsed
electric fields induce temporary or permanent permeabilization of the tissue. This
is a nonthermal, chemical-free method to increase permeability of skin tissues and
skin individual cells to various molecules, through reversible electroporation path-
way. It is also a method to control cell viability, through irreversible electroporation
pathway. To achieve electroporation, a specific electric field strength is required. A
precise control over electric field distribution in the skin through electrode position-
ing or conductivity manipulations provides a convenient tool for procedure man-
agement. This control, possible in time and space, is rarely achievable by other
physical therapies. This chapter discusses the fundamentals of wound healing; the
current challenges in the field and insights on how electroporation-based
31 Electroporation in Scars/Wound Healing and Skin Response 533
technologies can address these challenges. Electroporation-based applications

described here could potentially lead to new devices and methods to improve patient
care in this challenging field of medicine.
Fundamentals of Wound Healing
A living system at any level, cell, tissue, organ, or organism, is defined by the boundary
with the external environment. The disruption of this boundary by environmental
hazards is a wound (Fig. 1). Once the injury takes place, all organisms are mobilized
to close the wound and restore the boundary; otherwise, the bacterial contamination or
the leakage of water and nutrients and imbalance of energetic expenditures will lead to
organism death. A process of this boundary restoration between the cell, organ, or
organism and its environment is known as a wound-healing process (Fig. 1).
In mammals, wound healing is a dynamic, chronic process that is divided into
four overlapping phases: hemostasis, inflammation, proliferation, and remodeling
(Robson et al. 2001). During hemostasis, constriction of the damaged vessels and
clot formation physically limit blood loss. During the inflammatory phase, leuko-
cytes and then monocytes accumulate to combat infection in the wounded tissue. In
this phase, multiple cytokines and growth factors are released to the wound bed and
contribute to the fibroblast migration, differentiation, and activity. During the pro-
liferative phase, fibroblasts deposit a new and specific extracellular matrix and
differentiate into myofibroblasts. In the final, i.e., remodeling phase, reorganization
of the closed wound environment occurs until repair is completed. Inflammation
plays an important role in the organism response to the injury and in the control of all
stages of the wound healing (Koh and DiPietro 2011; Fig. 2).
To describe this complex dynamic process in tissues, Robson et al. (2001) intro-
duced the concept of wound-healing trajectory (Fig. 3), which demonstrates the
time-dependent cumulative effects of these multiple processes that occur from injury
through healing. According to the healing trajectory curve, normally healed tissues
are characterized by complete restoration of functions and structure (Fig. 3). In
contrast, chronic wounds are characterized by incomplete restoration of structure
and functions. In proliferative, hypertrophic scarring, however, the healing process
Fig. 1 Organism injuring pathways

Fig. 2 The pattern of leukocyte infiltration into the wound. In hemostasis the tissue is populated
with rare mast cells and macrophages. After the injury, at the early inflammation period, neutrophils
are rapidly recruited from the circulation. They are followed by macrophages and then lymphocytes
and mast cells at the late inflammation period. At the resolution and remodeling phase, the wound is
mostly populated by mast cells and rare macrophages again (Duffield et al. 2012)
Fig. 3 Wound-healing
trajectories (Figure adapted
with permission from
(Golberg et al. 2013).
Copyright # 2013 Wiley
Periodicals, Inc)
does not stop as it should, and the tissue fails to reach a normal cell density and a
balance between collagen deposition and degradation (Robson et al. 2001). Prolif-
erative scarring and chronic wounds in human have adverse physical, aesthetic,
functional, psychological, and social consequences. The major aim of all wound-
healing technologies and treatments is to restore completely the functions and
structure of the wounded site to its initial condition.
Why Electroporation?
In cases of abnormal healing, to reverse the healing trajectory to normal, there is an

obvious need for intervention in cell metabolism, life cycle, and density. Electric
fields provide a convenient method for intervention as there is no need in external
chemicals, which by themselves can induce milliards of unknown by-reactions.
Physical methods such as light and ultrasound have limited depth penetration
properties in the skin; in contrast, electric field distribution is mostly predicated by
the electrical properties of an organ. Once delivered to the cell membrane, externally
applied electric fields can modulate the natural transmembrane potential and thus
modify cell behavior.
By modulating transmembrane potential of cells, calibrated pulsed electric field
application leads to a local and transient cell membrane permeabilization, i.e.,
electroporation phenomenon (Neumann and Rosenheck 1972). The main advantage
of this physical approach is that cell perturbation is perfectly controlled in time since
the membrane electropermeabilization is transient and space since solely the tissue
located in between electrodes is affected.
The developments of this methodology are multiple both in food, environmental,
and human health areas (Yarmush et al. 2014).
Skin Response to the Externally Delivered Pulsed Electric Fields
When external electric fields are applied to the normal skin, several phenomena take
place at the same time. Electric fields affect skin on tissue and cellular levels. On a
tissue level, it was shown in multiple studies that electric fields increase the perme-
ability of small molecules, DNA, and RNA into the skin (Yarmush et al. 2014). This
long investigated property of increasing skin permeability suggested in the early
studies the use of electroporation for needless drug delivery and later for DNA
vaccination applications. An additional tissue-level effect of electric fields on the
skin is the modulation of the blood flow. Studies on electrochemotherapy of tumors
and normal skin (Fig. 4) showed that pulsed electric fields cause temporary vaso-
constriction and then vasodilation at the areas where electrodes were applied (Jarm
et al. 2010). In addition, recent studies showed that pulsed electric field applied in
electroporation mode could kill cells, but did not affect the structure of extracellular
matrix (Jiang et al. 2015).
On the cellular and molecular level, the application of pulsed electric fields leads
to cell reversible electroporation, cell irreversible electroporation, and death most
probably through both necrosis and apoptosis, degranulation of mast cells, and
release of multiple molecular factors to the treated areas at the time of treatment
and up to days after the electric field was removed (Fig. 5). Proteins and DNA
synthesis were showed to be promoted by non-permeabilizing pulsed electric field,
but its fine characterization needs to be performed in skin electroporation context to
use this effect in electro-induced healing strategy. The threshold for each phenom-
enon depends on the cell type and its specific location –“niche” in the skin. This
Pulse generator 1 min 6h 12h

Current probe
80
Blood Flow Change (%)

60
40
with baseline
20
0
−20
−40
−60
−80
−100
0 1 2 3 4 5 6 7 8 9 10 11 12
Time (h)
Fig. 4 Pulsed electric field effects on the normal rat skin. Schematic representation of the
experimental setup and digital image of electrodes applied on the dorsal rat skin (left panel).
Electroporation caused temporary vasoconstriction at the treated site (right panel). The blood
flow returned to the basal levels around 6 h after pulsed electric field (PEF) application. In the
following hours, the increase of the flow at the PEF-treated areas was observed. Figures show the
images taken using Moor LDI instrument. The plot shows the ratio between the flow at the center of
the PEF-treated area and the flow measured at the same area before PEF. Black squares on the
images show the regions of interest at which the flow was measured before and after PEF. PEF
protocol applied: 500 V, 200 pulses, 70 μs pulse duration, 3Hz, 2 mm gap between the electrodes
(Figure adapted with permission from (Golberg et al. 2015). Copyright # 2015 Rights Managed by
Nature Publishing Group. Distributed under a Creative Commons CC-BY license)
“niche” is important as it defines the local conductivity and, thus, electric field
distribution near and at the cell membrane.
These complex responses of the skin to the applied pulsed electric field are only
partially described. Precise control of the delivered electric fields in time and space
to the skin opens new opportunities to treat wounds. In the following sections, the
current state of the art of wound healing by pulsed electric fields will be described,
and further studies in this field will be suggested.
Skin Rejuvenation with Pulsed Electric Fields
Aging, trauma, and chronic metabolic diseases, including diabetes, often lead to
alterations in skin color, texture, and barrier function. Loss of skin function leads to
atrophy, infection, chronic wounds, laxity, and rhytides. Epidermal atrophy – char-
acterized by a thinning of the epidermis and an increase in fragility – is observed in
~32% of individuals over 60 years of age (Mengeaud et al. 2012). Thus, it is not
surprising that 2,156,075 skin rejuvenation procedures were performed in 2013 in
the United States alone.
MCP-1
*
VEGF *
Time after PEF
IP-10
* 5weeks
2weeks
IL-10
* 2h
EGF *
IL-1b
*
0 2 4 6 8 10 12 14
Fold change in comparison with normal skin
Fig. 5 Secreted molecules in the normal rat skin after electroporation. Factors secreted to the
area of normal rat skin exposed to PEF protocol of 500 V, 200 pulses, 70 μs pulse duration, 2Hz,
2 mm gap between the electrodes. The bars show fold increase in the normalized total protein
concentration at the treated areas in comparison to controls. The bars show the average from
measurements between six treated areas in three different animals (Figure adapted with permission
from (Golberg et al. 2015). Copyright # 2015 Rights Managed by Nature Publishing Group.
distributed under a Creative Commons CC-BY license)
Because these skin diseases are often associated with poor reepithelialization,
poor blood supply, reduced collagenesis, and a loss of collagen functional properties,
current rejuvenation therapies are focused on the removal of nonfunctional tissue
and the induction of de novo growth of healthy dermal cells, blood vessels, and
extracellular matrix. Various physical and chemical methods are used for skin
rejuvenation. However, the major disadvantage of current physical rejuvenation
methods is that they deliver external energy to the whole tissue bulk, affecting
both cells and extracellular matrix; this changes the function and architecture of
treated tissue. The major disadvantage of chemical rejuvenation therapies is that
even though they target only cells, they involve the delivery of external molecules
that can cause an off-target tissue response. This uncontrollable subsequent reaction
might result in clinical complications such as burns, skin vascular malformation,
tumors, keloids, hypertrophic scarring, skin contraction, paralysis of facial muscles,
necrosis, intravascular penetration, and infection.
As electroporation affects precisely cell membrane and does not affect extra-
cellular matrix, it was suggested that it could lead to cell-based therapy for skin
rejuvenation. The cell-based therapy concept is that the energy is delivered to the
skin cells and not to the bulk tissues as done by other physical methods. The
application of pulsed electric field on the normal rat skin led to >72% increase of
total collagen content 3 weeks after the treatment in comparison with untreated
skin (Table 1). This amount reduced to ~50% increase of total collagen content
2 months after the treatment in comparison with untreated skin (Table 1; Golberg
et al. 2015).
Table 1 The impact of PEF on total collagen content in the normal rat skin. Based on data
from (Golberg et al. 2015)
Number 3 weeks 2 months
Voltage Duration of pulses (mg/100 mg (mg/100 mg
(V) (μs) N tissue) (n = 3) STDEV tissue) (n = 3) STDEV
100 10 200 136 22 178 18
200 10 400 217 36 147 29
300 10 600 197 25 143 3
400 10 800 205 15 144 14
500 10 1000 210 9 178 2
100 30 400 153 26 95 14
200 30 600 152 22 151 13
300 30 800 180 43 206 7
400 30 1000 213 25 147 2
500 30 200 194 25 174 1
100 50 600 143 28 150 21
200 50 800 177 12 154 7
300 50 1000 192 23 156 20
400 50 200 163 4 170 16
500 50 400 171 12 122 9
100 70 800 143 26 145 11
200 70 1000 137 21 183 6
300 70 200 223 11 186 23
400 70 400 197 35 164 18
500 70 600 193 41 153 17
100 90 1000 165 18 111 14
200 90 200 183 14 136 21
300 90 400 222 14 153 13
400 90 600 185 7 143 13
500 90 800 205 5 167 15
Control 129 9 129 9
In addition, the same study showed that the PEF protocol consisting of applied
voltage, 500 V; pulse duration, 70 μs; number of pulses, 200; and frequency of
pulses delivery, 3 Hz, leads to the increased epidermis thickness (up to 3 weeks after
treatment) – important for infection prevention and angiogenesis, important for
nutrient supply and waste removal, and important for increased metabolism, mea-
sured by the rate of glucose uptake (Fig. 6).
Gene Electrotransfer (GET) as a Tool in Wound-Healing Therapy
Electroporation is a reliable technique for nucleic acid delivery in targeted organs

and tissues: it is the gene electrotransfer, or GET. At skin level, for example, this
technique allows to safely and locally deliver therapeutic genes to stimulate wound
healing (Gibot and Rols 2016).
Stratum corneum Thickness (µm)

80 25
Epidermis thickness (µm)

*
*
70
20
60 *
50 * 15
40
*
30 10
20
5
10
0 0
1 l
1 day
1w ys
3w es
m s
s
tro
2 ee
th
da
e
on
on
C
b 1 min 6h 24h 3w 2m
200
Blood Flow change (%)
150 *
with base line
*
100
* *
50
0
−50
*
-100
6h e 0
12 urs
24 urs
72 urs
1w urs
3w eks
8w eks
ks
ee
tim
o
ho
ho
ho
e
e
c 200
% Increase of 18FDG uptake
180 *
*
160
*
140
120
100 *
80
60
40
20
0
12 hours 1 day 3 days 1 week
Fig. 6 Skin rejuvenation with pulsed electric fields. (a) Dynamics of thickened skin epidermis
and resolution to the baseline levels after PEF. Images show H&E staining of the PEF-treated
epidermis. The plots show the average thickness of the epidermis and stratum corneum. Yellow
arrows show occasional apoptotic basal cells (*) p-val < 0.001. (b) Angiogenesis in the skin
detected with Doppler blood flow imaging (left panel) and immunohistochemistry of the newly
formed capillaries (right panel) with detection of angiogenesis marker Nestin. Increase in the Nestin
expression in the papillary dermis capillaries (black arrows) in comparison to untreated skin was
observed from 1 day to 3 weeks after PEF treatment. The intensity of staining 2 months after PEF
was very similar to control, suggesting the maturation of the vessels. (c) The increased metabolism
of skin areas treated by PEF as detected using 18FDG uptake (Figure adapted with permission from
(Golberg et al. 2015). Copyright # 2015 Rights Managed by Nature Publishing Group. Distributed
under a Creative Commons CC-BY license)
Gene electrotransfer was first developed for DNA vaccination and gene therapy,
but it also shows great promise in the field of regenerative medicine since it allows
the continuous and local production of therapeutic factors involved in wound-
healing process.
While acute wounds occur suddenly and heal at a predictable rate according to the
classical wound-healing process, chronic wounds are associated with impaired/
delayed healing and include pressure ulcer, diabetic foot ulcer, and venous stasis
ulcer. Numerous changes associated with diabetes at the cellular, molecular, and
genetic levels have been identified and promote this delayed wound healing
(Blakytny and Jude 2006). For these situations of nonhealing wounds, the develop-
ment of efficient therapeutic strategies is urgent. Indeed, chronic wounds are becom-
ing a major societal issue with the aging of the population and represent a significant
burden to patient and health-care system.
While the topical application of specific recombinant growth factors was shown
to benefit cutaneous healing, it requires large amounts of peptide because of multiple
administrations due to their short half-life which make this approach complex and
expansive. A promising and alternative approach consists in the local and transient
administration of a gene encoding the growth factor of interest. This is where gene
electrotransfer (GET) is highly valuable. Indeed, GET is a safe and efficient way to
deliver therapeutic genes which make it an attractive approach for the treatment of
wound healing and regenerative medicine in general. Several recent reviews compile
in vitro and in vivo studies on gene electrotransfer in wound healing (Gibot and Rols
2016).
The vast majority of studies on GET in wound healing were performed in vivo.
The pig is the most relevant animal model for studying healing process because its
mechanisms are comparable to those found in humans. However, for convenient
reasons, it is the murine model which is the most widely used. It offers the
advantage of working with available mutants, who allows to study GET for
wound healing in both normal context and within diabetic models. Indeed, leptin
receptor-deficient diabetic mice Leprdb/Leprdb are a relevant model of delayed
wound healing since they recreate a biochemical environment found in diabetic
patients. The choice of a model is based on many factors such as availability, cost,
and ease of use and on its anatomic/functional similarity to human tissue. Small
mammals such as rodents are conventionally used for studies on cutaneous phys-
iopathology. However, murine skin differs from the human skin from the view-
point of its architecture and its cellular composition. In addition, a transcriptomic
study recently showed that only 30% of genes associated with the skin are identical
between human and mouse (Gerber et al. 2014). Thus, it is of utmost importance
for the next years to develop human tissue models, especially by tissue engineering
approaches, in order to study gene electrotransfer in human tissue context. In order
to stimulate wound-healing process, therapeutic genes have been successfully
electrotransferred to injured skin. Plasmids encoding growth factors known to
promote cell proliferation, migration, and differentiation and to stimulate angio-
genesis were applied. Vascular endothelial growth factor (VEGF), keratinocyte
growth factor (KGF), and transforming growth factor (TGF) were nowadays the
most used in experiments.
Interestingly, using electroporation type of pulses to deliver therapeutic gene in
diabetic wound murine model, Lee et al. demonstrated that the control group “electric
field alone” significantly improves cutaneous wound closure (Lee et al. 2004).
This observation underlines the electrostimulation potential of electrical parameters

used in electroporation context.
Electrostimulation of Healing
Normal human epidermis possesses a transepithelial potential which is established

via an asymmetrical distribution of Na + ions in the different compartment of the
epidermis. Skin injury disrupts this transepithelial potential and induces an endog-
enous electric field directed toward the center of the wound. This electric field seems
to facilitate reepithelialization of the wound. Indeed, cutaneous cells are sensitive to
this electric field that directs their migration during wound healing. This phenome-
non is named galvanotaxis. In vitro studies led on monolayers showed that cell
exposure to external electric field affects protein distribution and synthesis and cell
orientation and migration through phosphatidylinositol-3-OH kinase-gamma and
PTEN pathways (Zhao et al. 2006).
When standard wound care alone fails to heal chronic wounds, electric field
application has been shown to improve healing and wound closure: It is electrosti-
mulation (Kloth 2014). A meta-analysis recently published demonstrated that
electrostimulation is efficient in improving wound healing, even if electrical param-
eters applied are empirically chosen (Koel and Houghton 2014).
Evidences obtained from experimental data and clinical trials efficiently support
the benefic role of electrostimulation for promoting wound-healing process, espe-
cially in a context of pathologic chronic wounds. Thus, American Centers for
Medicare and Medicaid Services (CMMS) have officially recommended the use of
electric fields for the treatment of chronic wounds (Kloth 2014).
Electrochemotherapy Stimulates Aesthetic and Functional

Wound-Healing Process
In oncology, electroporation ensures targeted and massive influx of cytotoxic mol-

ecules in tumor cells. This clinical application, called electrochemotherapy (ECT), is
nowadays used in several European countries for the treatment of skin and subcu-
taneous cancers. By potentiating the cytotoxicity of drugs, ECT reduces the doses
injected into patients and thus limits their side effects. Furthermore, clinicians
commonly observed, but cannot explain, aesthetic and functional wound healing
of the sites treated with ECT. Molecular assumptions regarding the mechanisms
underlying this phenomenon will be proposed in the section devoted to ECT and
wound healing. When tumors are located in head and neck sites, it is of utmost
importance that the treatment promotes an aesthetic and functional healing, which is
always observed by the clinicians with ECT (Fig. 7).
The common assumption to explain this observation is that normal cells located at
the margin of the tumor are spared because of their quiescence, which makes them
less sensitive to genotoxic effect of drugs used in ECT such as bleomycin and
Basocellula
carcinoma
Temple
Melanoma
Scalp
Fig. 7 Aesthetic and functional wound healing observed after tumor treatment by ECT
(Adapted from (Glass et al. 1997; Marty et al. 2006). Copyright # 2016 Elsevier with permissions)
cisplatin. New data obtained recently allow proposing a novel molecular hypothesis
that needs to be experimentally confirmed. Calvet et al. demonstrated that both ECT
with bleomycin and electric field alone induced the externalization of calreticulin
(Calvet et al. 2014). Calreticulin is usually an endoplasmic reticulum-resident
protein. When externalized, it becomes a danger-associated molecular pattern mol-
ecules (DAMP) associated with the induction of immunogenic cell death. However,
interesting studies showed that calreticulin externalization also plays a major role in
cutaneous wound healing by stimulating fibroblast and keratinocyte proliferation,
migration, and differentiation (Nanney et al. 2008). These effects were shown to pass
through TGF-β3 secretion, a growth factor well known to induce collagen and
hyaluronan remodeling, to accelerate wound-healing process, and to improve cell
migration.
Burn Wound Disinfection with Pulsed Electric Fields
Approximately 500,000 people seek medical treatment for burns every year in the
United States; infection remains a major cause of morbidity and mortality in these
patients. In addition to the extent and nature of the thermal injury affecting the
susceptibility to infection, the type and amount of the microbial burden colonizing
the wound appear to influence the risk of morbidity and mortality. Pathogens that
infect burn wounds are primarily Acinetobacter baumannii, methicillin-resistant
Staphylococcus aureus, Pseudomonas, and Klebsiella – pathogens that are increas-
ingly resistant to various antimicrobial agents. To address this problem, based on
Fig. 8 IRE disinfection in vivo: C57BL/6 black mice model. (a) Schematic illustration for
procedures used in the study. The 1 cm2 burn injury was followed by dispersion of A. baumannii on
part of the wound. Next pulsed electric field was applied using two plate electrodes. A. baumannii
infection load was quantified using bioluminescent imaging. (b) Left panel shows digital photog-
raphy of the burned (white frame) and infected (orange frame) areas of the skin. Central panel
shows digital photography of the applied electrodes. Right panel shows the images of the mice as
observed inside the dark imaging box. Orange frame shows the infected area as detected by a strong
bioluminescent signal emitted from bacteria. (c) The performed experiment timeline
(Figure adapted with permission from (Golberg et al. 2014). Copyright # 2014 World Scientific
and Imperial College Press Co Pte Ltd. Illustration schemes by Inna Desyatnik)
successful disinfection of food, IRE was applied to treat the bacterial wound
infections (Fig. 8). Using 80 pulses of 5000 Vcm 1, stable disinfection with
4.91 0.71 Log10 reduction of Acinetobacter baumannii, 3 h after treatment,
was achieved (Figs. 8, 9, and 10) (Golberg et al. 2014).
The magnitude of disinfection was correlated with both the electric field strength
and the number of delivered pulses. The increase in pulse number led to a larger
reduction in bacterial load and bioluminescent signal immediately after treatment, as
compared to the increase in the field strength. Increasing the pulse number from 40 to
80 led to a 255% increase in the reduction of bacterial load in the wound, from
1.49 0.07 Log10 to 5.30 0.85 Log10. Increasing the field, however, from
2500 V cm 1 to 5000 V cm 1, while keeping the number of pulses at 40, led to
only a ~37% increase in the log reduction of bacterial load in the wound, from
1.49 0.07 Log10 to 2.04 0.29 Log10.
a 500V b 1000V
10min 35min 45min 55min 10min 35min 45min 55min
500V, 70µs, 1Hz 1000V, 70µs, 1Hz

1.E−07 p=0.0096 1.E−07 p=0.0052
p=0.03 1.E−06 p=0.095
1.E−06
Survival Fraction
Survival Fraction
1.E−05 1.E−05
1.E−04 p=0.0097 1.E−04 p=0.0052

1.E−03 1.E−03
1.E−02 1.E−02
1.E−01 1.E−01
1.E+00 1.E+00
)
)
=3
=3
=3
=3
=3
=3
=3
=3
(N
(N
(N
(N
(N
(N
(N
(N
in
in
in
in
s
se
se
se
se
m
m
ul
ul
ul
ul
10
35
10
35
0p
0p
0p
0p
,4
,8
,4
,8
in
in
in
in
m
m
45
55
45
55
c Control d 500V, 80pulse e 1000V, 80pulse
10min 35min 240min 35min 55min 240min 35min 55min 240min
p=0.179
p=0.055
1.E−02 1.E−07 1.E−07
p=0.0237
Survival Fraction
Survival Fraction
Survival Fraction
1.E−05 1.E−05
1.E−01 1.E−03 1.E−03
1.E−01 1.E−01
1.E+00
m =3)
m =3)
)
0m =3
=3
m =2)
0m =3
=3
0m =2
=2
(N
(N
(N
(N
(N
(N
(N
(N
(N
in
in
in
in
in
in
in
in
in
m
m
35
55
35
55
24
10
35
24
24
Fig. 9 The effect of pulse number and electric field strength on A. baumannii infection load
reduction. (a) Applied voltage 500 V, 2 mm gap between electrodes. (b) Applied voltage 1000 V,
2 mm gap between electrodes. (c) Control: not treated, burned, and infected skin. (d) Applied
voltage 500 V, 2 mm gap between electrodes. (e) Applied voltage 1000 V, 2 mm gap between
electrodes. Top panel shows the post-burn time when the images were taken. The bottom panel
shows the survival fraction of microorganisms as detected by the top panel images (logarithmic,
inversed scale). N shows the number of animals per group. Error bar standard deviation of the
mean (Figure adapted with permission from (Golberg et al. 2014). Copyright # 2014 World
Scientific and Imperial College Press Co Pte Ltd)
Fig. 10 Summary of
A. baumannii survival 3 h
after IRE, the effect of
electric field intensity.
N shows the number of
animals per group. Error
bar standard deviation of
the mean. Gap between
electrodes 2 mm. Pulse
duration: 70 μs
(Figure adapted with
permission from (Golberg
et al. 2014). Copyright #
2014 World Scientific and
Imperial College Press Co Pte
Ltd)
Applications of Electroporation in Surgery: Mesh Disinfection

and Scar Treatment
Two million Americans undergo abdominal surgery annually with a postoperative

incisional hernia rate of 10–23% (Khan, Blumrosen et al. 2016). About 400,000
ventral hernia repairs are performed each year in the United States alone, with
reported hernia recurrences in 40–50% of cases (Khan, Blumrosen et al. 2016).
Synthetic mesh reinforces hernia repairs and has been shown to decrease recurrences
compared to primary repair alone. However, morbidities related to mesh infection
can limit efficacy. Reported mesh infection rates range from 4% to 16%. Antibiotics
alone are not an effective treatment for mesh infections. Khan et al. (2016) investi-
gated the effect of pulsed electric fields (PEF) on biofilm-infected mesh (Khan,
Blumrosen et al.). In that study, Prolene mesh was infected with bioluminescent
Pseudomonas aeruginosa and treated with PEF using a concentric electrode system
to derive, in a single experiment, the critical electric field strength needed to kill
bacteria. The effect of the electric field strength and the number of pulses on bacterial
eradication was investigated. For all experiments, biofilm formation and disruption
were confirmed with bioluminescent imaging and scanning electron microscopy
(SEM). It was found that the critical electric field strength needed to eradicate
100–80% of bacteria in the treated area was 121 14 V/mm when 300 pulses
(50 μs, delivered at 2 Hz) were applied and 235 6.1 V/mm when 150 pulses were
applied. The area at which 100–80% of bacteria were eradicated was
50.5 9.9 mm2 for 300 pulses and 13.4 0.65 mm2 for 150 pulses. Eighty
percent threshold eradication was not achieved with 100 pulses.
Hypertrophic scarring (HTS) after trauma and burn injury remains a major
clinical challenge that leads to physical, aesthetic, functional, psychological, and
social stresses in thousands of patients. Current data show that alterations in coag-
ulation, inflammation, angiogenesis, fibroplasia, contraction, remodeling, and
mechanical tension correlate with the formation of HTS. But as of yet, the mecha-
nisms that induce HTS are not well understood. This gap in knowledge leads to
limited clinical success in therapeutic procedures. Various techniques such as surgi-
cal excision, intralesional steroid or interferon injection, cryotherapy, laser therapy,
electron-beam irradiation, mechanical compression dressing, silicone sheet applica-
tion, and combinations thereof have been tested to treat scars over the years. Despite
all of these efforts, a recent metareview shows that there are only modest improve-
ments in the healing outcomes among all these treatments (Leventhal et al. 2006).
Recent study used electrochemotherapy for keloid and hypertrophic scar treatment in
the clinical trials (Manca et al. 2013). Twenty patients with keloids or hypertrophic
scars were treated with one or more sessions of electrochemotherapy with
bleomycin. Treatment was well tolerated by patients, and no serious adverse events
were observed with one recurrence observed after 18 months. A median reduction of
87% was observed in volume size, and 94% of lesions showed a volume reduction of
more than 50%. Scar pliability and erythema scores were also significantly reduced.
A reduction in hitching was observed in 89% of patients, and a reduction in pain was
observed in 94% (Manca et al. 2013).
Conclusions
In the case of the normal wound healing, no intervention might be needed. However,
in cases of abnormal healing, both chronic wounds and hypertrophic scarring, there
is an obvious need for intervention. For many decades, wound care methods have
been developed to improve healing by addressing cutaneous cell stimulation, reduc-
tion of bacterial contamination, and regulation of cytokines and proteases or by
promoting exogenous growth factors expression. It is important to remember that
wound healing is an extremely complex processing, and interventions that could
achieve the system effects are needed to advance this field. Manipulations of cell
metabolism, life cycle, and density could provide partial solution to improve the
patient care. Electroporation-based technologies can obviously help achieve all these
goals. It has been already shown that electroporation-based procedures can manip-
ulate cell metabolism and gene expression in wounds; they can control cell density
and infection and can directly be applied to the skin to both induce skin rejuvenation
and reduce hypertrophic scarring. Each of these applications requires its own
optimization for the applied parameters as sometimes the required effect is on a
single cell-type level and sometimes overall skin organ level. Future work should
address both specificity of electric field parameters for various applications and
integration of electroporation-based devices and procedures to the different existing

protocols and workflows of clinical wound care.
Cross-References

▶ Electroporation-Based Applications in Biotechnology
▶ Gene Electrotransfer for Ischemic Tissue
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Principles and Use of Magnetic Resonance
Electrical Impedance Tomography in Tissue 32
Electroporation
Eung Je Woo and Matej Kranjc
Abstract
Electroporation is a phenomenon caused by externally applied electric field of an
adequate strength and duration to cells that results in increase of cell membrane
permeability to various molecules which otherwise are deprived of transmem-
brane transport mechanism. As the membrane electroporation is a consequence of
an induced transmembrane potential which is directly proportional to the local
electric field, magnetic resonance electrical impedance tomography (MREIT)
was proposed for reconstruction of electric field distribution during
electroporation. MREIT enables determination of electric field distribution by
measuring the electric current density distribution and electric conductivity of the
treated subject during application of electric pulses by using MRI scanner and
numeric algorithms. MREIT yields an electric field distribution, which is a time
average of its altering time course so that all the consequences of conductivity
alteration of the treated tissue due to electroporation are not neglected within
obtained electric field distribution. Feasibility of this method has been demon-
strated by determining electric field distribution during electroporation in silico,
in agar phantoms, plant tissues, and animal tissues ex vivo and in vivo. In this
chapter, mathematical framework of MREIT and the concept of monitoring
electric field distribution are provided together with fundamentals of electrical
conductivity imaging. Maps of electric field distribution established during elec-
troporation of various tissues that were obtained by means of MREIT are also
included.
E.J. Woo (*)

Department of Biomedical Engineering, College of Medicine, Kyung Hee University, Seoul,
Republic of Korea
e-mail: ejwoo@khu.ac.kr
M. Kranjc (*)
e-mail: matej.kranjc@fe.uni-lj.si

DOI 10.1007/978-3-319-32886-7_65
550 E.J. Woo and M. Kranjc
Keywords
Electric field • Magnetic resonance imaging • Electrical impedance • Current
density
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 550
Electrical Behavior of Biological Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551
Assessing Electrical Conductivity of Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 552
Electrical Impedance Tomography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 552
Magnetic Resonance Electrical Impedance Tomography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 552
MREIT in Tissue Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564
Introduction
An accurate coverage of the cell with sufficiently large electric field (see ▶ Chap. 56,
“Electric Field Distribution and Electroporation Threshold”) presents one of the
most important conditions for successful electroporation. A method that would allow
determination of the electric field strength within tissue would thus be of great
importance for electrochemotherapy as well as for other electroporation based
applications. A method capable of determining electric field distribution during the
pulse delivery seems to be useful as electroporation depends on local electric field.
This would enable detection of insufficient electric field coverage before the end of
the treatment, thus increasing and assuring its effectiveness. As the electric field
distribution inside the observed tissue cannot be measured directly, an indirect
approach of obtaining it needs to be evaluated. By means of Ohm’s law, the electric
field distribution can be determined when an electric current density and an electrical
conductivity of the tissue are obtained. The electric current density inside a conduc-
tive tissue can be obtained by magnetic resonance imaging (MRI) using current
density imaging technique (CDI) (see ▶ Chap. 33, “Current Density Imaging as
Means to Follow Tissue Electroporation”) by measuring magnetic field changes
caused by applied current. Whereas tissue conductivity can be obtained by magnetic
resonance electrical impedance tomography (MREIT), a technique used for recon-
struction of electrical conductivity inside a tissue by means of current density. The
use of MREIT for conductivity imaging has advanced rapidly in the last decade and
has now reached the stage of in vivo animal and human imaging experiments. As the
method does not present additional cost to the conventional MRI procedure, it could
become a part of an MRI system and provide additional and valuable contrast
information.
This chapter is divided in two parts. In the first part, mathematical framework of
MREIT is provided together with fundamentals of electrical conductivity imaging.
The second part of the chapter is dedicated to results of monitoring electric field
32 Principles and Use of Magnetic Resonance Electrical Impedance. . . 551
distribution during application of electric pulses in agar phantom, tissue ex vivo, and
mouse tumor in vivo and in silico.
Electrical Behavior of Biological Tissue
In this chapter, we deal with bioelectromagnetic phenomena at dc or low frequency

so that we can ignore the effects of electric polarization and magnetic induction.
When exposed to such an electric field, a biological tissue conducts electrical
current. Given the electric field, the amount of the current depends on a passive
material property called the electrical conductivity. In Ohm’s law, we can express the
relation as
JðrÞ ¼ σ ðrÞEðrÞ ¼ σ ðrÞ∇vðrÞ (1)
where J is the current density, σ the conductivity, E the electric field intensity, v the
electric potential or voltage, and r ¼ ðx, y, zÞ a position vector.
In an electrolyte, concentrations and mobility of charge carriers, predominantly
ions, determine the conductivity σ. A biological tissue consists of cells, extracellular
matrix, and extracellular fluid. Inside the cell, there exist numerous organelles in
intracellular fluid. The complicated structures and compositions of various tissues
influence their electrical conductivity values producing an inhomogeneous conduc-
tivity distribution inside the human body (Grimnes and Martinsen 2014).
The cell has a membrane which behaves electrically as a thin insulator. At low
frequency, the membrane blocks electrical current from flowing into the cell and the
entire cell appears as a solid insulator when neglecting ion movements through ion
channels. At high frequency, a displacement current may flow through the cells since
the thin cellular membranes behave as capacitors. The conductivity at a macroscopic
scale may exhibit anisotropy at low frequency depending on the structure of the cells
and extracellular matrix materials. When the conductivity is anisotropic, it can be
represented as a tensor. Note that in this case, the direction of the current density
differs from the direction of the applied electric field.
The electric field can stem from an endogenous neuronal current or externally
injected current (Malmivuo and Plonsey 1995). In this chapter, we study the effects
of the electric field subject to an externally injected current. For the case where we
are interested only in the internal current density produced by the externally injected
current, the current density J satisfies
∇ Jð r Þ ¼ 0 (2)
since there is no such current source inside the body.

From Eqs. 1 and 2, the externally injected current produces a voltage distribution
v inside the body satisfying the following boundary value problem:

∇ σ ðrÞ∇vðrÞ ¼ 0 in Ω
(3)
σ∇v n ¼ g on @Ω
where Ω is the three-dimensional domain of the body, @Ω its boundary, n the outward
unit normal vector on @Ω and g the normal component of the current density on @Ω
(Neumann boundary condition). Note that on the portion of the boundary @Ω
excluding the current-injection electrodes, g ¼ 0 since the air outside Ω is an
insulator. Choosing a voltage reference position r0 where vðr0 Þ ¼ 0, the boundary
value problem in Eq. 3 has a unique solution v in Ω.
Assessing Electrical Conductivity of Tissue
Electrical Impedance Tomography
Electrical impedance tomography (EIT) is an imaging modality to produce cross-

sectional images of the conductivity distribution inside an electrically conducting object
such as the human body (Holder 2004). An EIT system may consist of a single or
multiple current sources that can produce sinusoidal currents of a safe amplitude of less
than a few mA at a single or multiple frequencies in the range of 1–500 kHz. Such
currents are injected into the human body through a chosen pair or plurality of surface
electrodes attached around an imaging plane of interest. Typical EIT systems use 8–32
electrodes. From those electrodes, the EIT system measures the induced boundary
voltages using a single or multiple voltmeters. The measured boundary current–voltage
data, so called the Neumann-to-Dirichlet map (NtD map), is determined by the
conductivity distribution inside the human body and also its boundary shape and
electrode positions. Using the measured boundary data, an image reconstruction
algorithm, which is basically based on Eq. 3, produces conductivity images (Seo and
Woo 2013). There have been numerous studies of EIT in terms of its theories,
algorithms, system developments, imaging experiments using phantoms, animals and
human subjects, and also clinical applications (Holder 2004). EIT produces images
with a low spatial resolution of about 5–10 mm pixel size in most cases. Its temporal
resolution is superb with more than 50 frame/s using a fast parallel EIT system. Since
the system is portable and can be less expensive than other medical imaging modalities,
EIT is finding clinical applications where image-based real-time monitoring is desired.
Magnetic Resonance Electrical Impedance Tomography
Noting that EIT using only boundary measurements has a technical limitation to
achieve a high spatial resolution, magnetic resonance electrical impedance tomog-
raphy (MREIT) has been suggested (Zhang 1992; Woo et al. 1994; Seo et al. 2014).
The basic idea is to utilize the internal information of the magnetic field induced by
an externally injected current. From the Biot-Savart law, the induced magnetic flux
density B is
ð
μ0 0 r r
0
0
BðrÞ ¼ J r 0 3
dr þ BX ðrÞ (4)
4π jr r j
Ω
where J is the current density inside the domain Ω, and BX is the magnetic flux
density produced by the currents outside the domain such as lead wires. Note that
Laplacian of BX inside the domain Ω is zero (Lee et al. 2003). Ampere’s law
provides the relation between the current density and the magnetic flux density
inside the domain Ω as
1
Jð r Þ ¼ ∇ BðrÞ: (5)
μ0
MREIT utilizes a MRI scanner to noninvasively measure the induced magnetic

flux density B inside the human body. MR imaging is based on the magnetization of
the protons inside the human body by a strong dc magnetic field of 3 T, for example.
Aligned magnetic dipole moments of the protons precess at the Larmor frequency
that is 127.74 MHz at 3 T. Application of an electromagnetic wave at the Larmor
frequency flips the protons to an unstable state. When this radio frequency
(RF) pulse disappears, the protons return to their original magnetized state and
emit an electromagnetic wave, which can be detected using RF coils. Adding
spatially varying magnetic fields called the gradient fields, the resonance frequency
of the protons varies depending on their positions inside the human body. This kind
of position encoding allows formation of a cross-sectional image. A pulse sequence
includes such RF and gradient pulses to affect the protons with position encoding.
Different pulse sequences may produce MR images with various contrast
information.
The magnetic flux density in Eq. 4 perturbs the main magnetic field of the MRI
scanner (Joy et al. 1989; Seo and Woo 2014). The magnetization of the protons
occurs in the direction of the main magnetic field, which is the z direction
in this
chapter. This means that only the z-component Bz of B ¼ Bx , By , Bz influences the
magnetization. Note that Bz varies with position, and its spatial variation is deter-
mined by the conductivity distribution σ.
Figure 1 is a typical pulse sequence used in MREIT. It is a basic spin-echo
(SE) pulse sequence with added current pulses, which are synchronized with the RF
pulses. The current injection is repeated twice using the current pulses with the same
amplitude and opposite polarities denoted as I . This kind of MREIT pulse sequence
produces two sets of complex MR data S , so-called the k-space data, as follows:
ðð

S ðm, nÞ ¼ Mðx, yÞeiδðx, yÞ eiγBz ðx, yÞT c eiðxmΔkx þynΔky Þ dxdy (6)
Fig. 1 Typical MREIT pulse sequence based on the spin-echo pulse sequence. Injection current
pulses are inserted between RF pulses and reading gradient
where are for the current injections of I , respectively, M the MR magnitude

image, δ the systematic phase artifact, γ the gyromagnetic ratio of hydrogen, Bz the
z-component of the induced magnetic flux density, and Tc the width of the injection
current pulses (Joy et al. 1989; Seo and Woo 2014).
Applications of the Fourier transform result in two complex MR images Ψ as
Ψ ðx, yÞ ¼ Mðx, yÞeiδðx, yÞ eiγBz ðx, yÞT c : (7)
Note that Bz appears in the phase part of the complex MR images. To remove the
systematic phase artifact δ due to the main field inhomogeneity, these two complex
images are divided to get the phase difference of

Ψþ ðx, yÞ
Φðx, yÞ ¼ arg ¼ 2γBz ðx, yÞT c : (8)
Ψ ðx, yÞ
After applying proper preprocessing methods including phase unwrapping and

scaling, the induced magnetic flux density Bz is obtained as
Φðx, yÞ
Bz ðx, yÞ ¼ : (9)
2γT c
Figure 2 shows a typical example of a phantom imaging experiment in MREIT. Note
that the conductivity anomaly at the center of the phantom refracts the Bz signal.
To compute the internal current density
J using Ampere’s law in Eq. 5, all
three components of B ¼ Bx , By , Bz should be measured. This can be done by
a b
-8
x 10 [T]
c d 6
-2
-4
-6
Fig. 2 (a) Conductivity phantom including an agar object with 0.5 S/m conductivity at the center
of the phantom with a background saline of 2 S/m conductivity. (b) Its MR magnitude image at the
middle of the phantom. (c) Phase image at the same imaging slice. (d) Bz image computed by using
Eq. 9
repeating the data collection process twice more while rotating the imaging
object inside the MRI scanner (Joy et al. 1989). Once B is available, J is
computed using Eq. 5, and images of the conductivity distribution can be
obtained by using a method such as the J-substitution algorithm (Kwon
et al. 2002). Though there exist numerous studies of utilizing B in MREIT and
also in magnetic resonance current density imaging (MRCDI) as reviewed in
Woo and Seo (2008), this object rotation is difficult or impractical using a
currently available clinical MRI scanner.
To avoid the impractical object rotations, it is highly desirable to reconstruct
current density and conductivity images using the measured data of Bz. Using only
Bz data, computation of only an approximate J is possible since Bz does not contain
any information about Jz. Noting that J is strongly influenced by the three-
dimensional geometry of the imaging object as well as its internal conductivity
distribution, a model-based approach may produce a best achievable current density
JP, so-called the projected current density (Park et al. 2007). Using a three-
dimensional model of the imaging object, numerical solutions of Eq. 3 and Eq. 1
assuming a homogeneous conductivity distribution of 1 S/m inside Ω provide the
internal current density JH (Lee et al. 2003). Assuming that the z-component Jz of the
true current density J ¼ J x , J y , J z is a perturbation of the z-component JH
z of the

computed current density J ¼ J x , J y , J z , the projected current density can be
H H H H
computed as a best approximation of the true current density one can compute by
using the measured Bz data. In addition to the estimation of the internal current
density from the measured Bz data, the projected current density can be utilized in
conductivity image reconstructions using a similar approach as the J-substitution
algorithm (Kwon et al. 2002). Figure 3 shows an example of the projected current
density experimentally obtained from an in vivo canine brain.
For conductivity image reconstructions using only Bz, the induced magnetic flux
density Bz can be expressed from Eqs. 1 and 4 as
0 dv 0
0 dv 0
ð σ ðrÞ x x r yy r
μ dy dx 0
Bz ðrÞ ¼ 0 0 3
dr (10)
4π jr r j
Ω
to emphasize the fact that it includes the information about the conductivity σ. Here,
we ignore the magnetic flux density originated from the external lead wires since its
Laplacian is zero in the domain Ω. Direct reconstruction of a conductivity image
from the measured Bz data requires at least two current injections in orthogonal
directions as much as possible (Seo and Woo 2011). For isotropic or equivalent
isotropic conductivity image reconstructions, two current injections are commonly
used. In this kind of Bz-based MREIT, the most widely used conductivity image
reconstruction method called the harmonic Bz algorithm is based on the following
observation (Seo et al. 2003):

1 2 dσ dσ dv dv dσ dv dσ dv
∇ Bz ðx, yÞ ¼ , , ¼ (11)
μ0 dx dy dy dx dx dy dy dx
at the position (x, y) in a chosen imaging slice. Since Eq. 11 is satisfied for both
current injections I with the same conductivity distribution σ, we can get
2 3
dv1 dv1 2 dσ 3

6 dy
6 dx 776 dx 7 1 ∇2 Bz1
4 dv2 dv2 54 dσ 5 ¼ μ0 ∇2 Bz2 : (12)

dy dx dy
This provides a pixel-by-pixel relation between the two-dimensional gradient of

the conductivity and the Laplacian
h iof the measured Bz data subject to two current
dσ dσ
injections. After computing dx , dy by solving the linear system of equations in
Eq. 12, applications of numerous integration techniques can produce a conductivity
a ε1+ ε+2
400
ε2− ε1−
0
17.5 [nT]
b
-0.5
c 0.55 [A/m2]
Fig. 3 (a) MR magnitude image of a canine head with two pairs of current injection electrodes.
(b) Two images of the measured Bz data subject to two current injections. (c) Magnitude of the
projected current densities subject to the current injections at two different directions
image σ (Oh et al. 2003). At least one boundary voltage data or at least one known
conductivity value preferably on a position at the boundary should be incorporated
into the image reconstruction process to produce a unique conductivity image
(Seo and Woo 2011; Seo and Woo 2013).
Fig. 4 MR magnitude images versus MREIT conductivity images of animals and human subjects:
(a) canine brain, (b) canine chest, (c) canine abdomen, (d) canine pelvis, (e) human leg, and (f)
human knee
There have been numerous experimental studies in MREIT using conductivity

phantoms, animals, and human subjects (Woo and Seo 2008; Seo and Woo 2014).
These results indicate that MREIT is capable of producing multi-slice cross-sectional
images of a conductivity distribution inside an electrically conducting object such as
the human body with a spatial resolution of an adopted MRI scanner. Conductivity
image reconstructions of animals and human subjects with about 1 mm pixel size
have been demonstrated using clinical 3 T MRI scanners. Figure 4 shows numerous
conductivity images of animals and human subjects reconstructed by using the
harmonic Bz algorithm.
As mentioned earlier, some biological tissues such as the muscle and white matter
exhibit anisotropy in their conductivity values. Though MREIT can reconstruct
anisotropic conductivity tensor images in theory, its experimental demonstration
has been deferred primarily due to the requirement of the measured Bz data with a
very high signal-noise ratio (SNR) (Seo et al. 2004). To overcome this practical
technical difficulty, Kwon et al. (2014) lately proposed a method called DT-MREIT
to combine diffusion tensor imaging (DTI) with MREIT.
The major technical hurdle remained in MREIT as a diagnostic imaging tool is the
weak measurement sensitivity of Bz using a clinical MRI scanner for a given local
conductivity change. The induced Bz is in the range of tens of nT when the injection
current amplitude is a few mA. From Eqs. 7 and 8, the amount of the phase change
subject to the externally injected current is proportional to the product of Bz and Tc

that is the time duration of the current injection. Since Bz is directly proportional to
the amplitude of the injection current, the phase change is basically proportional to
the product of the amplitude and duration of the injection current pulse. Though
there is no safety standard yet for this kind of imaging current pulses, the pulse
amplitude and duration should be kept within a certain limit such as the threshold of
perception or the threshold of muscle contraction. To improve the image quality in
MREIT, multi-channel RF coils with better sensitivity, innovative MREIT pulse
sequences, pre- and post-processing methods, and better image reconstruction algo-
rithms without noise amplification are needed in addition to the performance
improvement of the adopted MRI scanner itself.
If a three-dimensional conductivity distribution is available for an electrically
conducting object, one may numerically solve Eq. 3 to compute the voltage distri-
bution v for any given current injection or voltage application. This leads to
computations of the internal current density J as well as the electric field intensity
E in Eq. 1. For those cases of electric stimulations, such as electroporation, cardiac
defibrillation, transcranial dc stimulation (tDCS), deep brain stimulation (DBS), RF
ablation, and so on, the knowledge of the internal conductivity distribution can be
utilized in the treatment planning (see ▶ Chap. 54, “Treatment Planning for
Electrochemotherapy and Irreversible Electroporation of Deep-Seated Tumors”)
(Chauhan et al. 2013; Jeong et al. 2016).
MREIT in Tissue Electroporation
Electroporation is used for therapeutic purposes and electric pulses can reach up
to 3000 V, and they can establish electric field distribution with strength up to
150 kV/m depending on electrodes geometry and distance between them. As CDI
and MREIT have been developed for diagnostic purpose and there used to be a lack
of reports where electric pulses that are normally used in electroporation applications
would be used. Still, in the last 5 years it was demonstrated that both, CDI and
MREIT, can be applied for obtaining electric field with such high field strength.
Determination of electric field distribution during tissue electroporation was at
first demonstrated both experimentally and numerically on homogeneous and het-
erogeneous agar phantom with electrical properties similar to human tumor and
surrounding tissue. A good agreement between experimental and numerical results
was obtained in homogeneous and heterogeneous agar (Fig. 5) and for different
pulse sequences, i.e., for different number and amplitude of pulses (Kranjc
et al. 2011).
Determination of electric field distribution during tissue electroporation was also
successfully demonstrated on ex vivo chicken liver tissue (Kranjc et al. 2012) and in
porcine muscle as shown in Fig. 6. Furthermore, experimental and numerical
investigation on the anisotropy ratio of ex vivo tissue was also performed (Essone
Mezeme et al. 2012). Alteration of anisotropy ratio of the conductivity tensor was
detected when reversible electroporation threshold was exceeded. Experimental
|EMREIT| [V/m] × 104

a b c 18
+ 14
CDI MREIT
σ 10
Uapp
6
|Enum| [V/m] × 104

d 18
14
10
e f g |EMREIT| [V/m] × 106

5.5
5
4.5
4
3.5
σ2 Uapp CDI MREIT 3
2.5
σ1 2
+ 1.5
1
0.5
h |Enum| [V/m] × 106

5.5
5
+ 4.5
4
σ2 CDI 3.5
3
σ1 Uapp 2.5
2
1.5
1
0.5
Fig. 5 Homogeneous (a) and heterogeneous phantom (e) exposed to four 100 μs long high voltage
pulses of 1000 V delivered between diagonal electrodes. The initial phase images (b, f) were
acquired by the two-shot rapid acquisition with relaxation enhancement (RARE) RF based CDI
sequence. The electric field distributions (c, g) were calculated using MREIT J-substitution
algorithm from the current density distribution. Numerical simulation of electric field distribution
(d, h) in the phantom are shown for comparison below the corresponding experimental results (The
figure is adapted from Kranjc et al. (2011))
results agreed with numerical and were also consistent with experimental investiga-
tions performed by other research groups.
Electric field distribution was also successfully applied in monitoring of electric
field distribution during application of electric pulses in potato tubers (Kranjc
Fig. 6 Electric field mapping during current injection between a pair of needle electrodes inserted
into a chunk of porcine muscle: (a) MR magnitude image where the inserted needle electrodes
appear as two black dots, (b) measured Bz image subject to injection current between the pair of
needle electrodes, (c) computed image of the projected current density, and (d) image of the
magnitude of the electric field intensity
Fig. 7 Electric field distribution in a potato tuber (a), in an agar phantom (b), and in a tissue ex vivo
(c). Pulses were delivered between two needle electrodes (marked with + and ˗)
et al. 2016). For comparison, electric field distribution in an agar phantom and in
ex vivo tissue are shown together with the distribution in potato tuber on Fig. 7. The
study was performed on potato tubers cultivar “Agata” using eight electric pulses
with an amplitude of 1000 V, pulse duration of 100 μs, and repetition frequency of
5 kHz. Electric pulses were delivered between two needle electrodes.
Electric field in the potato tuber was not distributed symmetrically (A) as in the
agar phantom (B), presumably due to the heterogeneous potato structure and con-
sequent heterogeneous electrical conductivity, which resulted in observed asymmet-
ric distribution. More inhomogeneous distribution than in agar was also obtained in
an ex vivo tissue (C), where heterogeneous distribution can also be observed,
although not as apparent as in the potato tuber. Since MREIT can be used in
reconstruction of electric field in tissues, there are no limitations in applying it on
food samples undergoing the PEF treatment. Monitoring of electric field can be used
either for general investigations on distribution of electric field in various food
Fig. 8 (a) The electric field distribution in the tumor obtained by MREIT superimposed to
the T1-weighted image acquired before the application of electric pulses. A white contour line
encloses an area exposed to an electric field strength between reversible (400 V/cm) and irreversible
(900 V/cm) electroporation threshold values. Tumor cells located outside the area are either
irreversibly electroporated (the area close to the electrodes) or remain unelectroporated (the area
toward the tumor boundary). (b) Scatterplot of the coverage of five tumors with the electric field of
reversible electroporation CMREIT and Gd-DOTA cell entrapment FGd-DOTA
samples or for fine adjustments of PEF treatment parameters, such as amplitudes of

electric pulses or changing their number, during the PEF treatment.
Investigation of the feasibility of MREIT for in situ monitoring of the electric field
distribution during in vivo reversible electroporation was performed on mouse tumors
(Kranjc et al. 2015). Animal experiments were conducted in accordance with the
guidelines for animal experiments of the European Union directives, and permission
was obtained from the Ministry of Agriculture and the Environment of the Republic
of Slovenia (permission no. 34401-43/2011/5). Electroporation was performed by
applying two sequences of four high voltage electric pulses with an amplitude of
500 V, duration of 100 μs, and at a pulse repetition rate of 5 kHz via two needle
platinum-iridium electrodes inserted into the tumor. Electric field distribution in the
tumor during pulse delivery was obtained by means of MREIT J-substitution
algorithm. For post-treatment assessment of reversibly electroporated areas in the
tumor, the contrast agent gadolinium-tetraazacyclododecane tetraacetic acid
(Gd-DOTA) was injected before the application of pulses. After 24 h, T1-weighted
images were acquired for observation of tumor areas where Gd-DOTA molecules
were entrapped inside the reversibly electroporated tumor cells.
An example of electric field distribution obtained by means of MREIT is shown
in Fig. 8a where it is overlaid to T1-weighted image acquired just before the
application of electric pulses. As expected, the electric field was the highest around
the electrodes where it exceeded irreversible electroporation threshold (900 V/cm),
while it remained under the reversible electroporation threshold (400 V/cm) in the
areas away from electrodes.
The average coverage of tumors with electric field leading to reversible electro-
poration of tumor cells CMREIT was calculated by dividing the predicted surface area
of the reversibly electroporated tumor cells with the surface area of the entire tumor.
Whereas tumor fraction FGd-DOTA was calculated by dividing the surface area of
reversibly electroporated tumor cells as obtained by entrapped contrast agent with
the surface area of the entire tumor. CMREIT SD and FGd-DOTA SD were
38 9% and 41 13%, respectively. Correlation was evaluated between CMREIT
and tumor fractions with FGd-DOTA with linear Pearson correlation analysis. Cover-
age of tumors with electric field in the range of 400–900 V/cm, where reversible
electroporation is expected, had good correlation with Gd-DOTA cell entrapment
(r = 0.956, P = 0.005) as shown in Fig. 8b.
A concern whether proposed method for determination of electric field distri-
bution can be implemented in electroporation applications was addressed by a
simulation in the case of a three-dimensional (3-D) numerical model designed for
the purpose of electrochemotherapy treatment of deep-seated liver tumors. The
treatment was done as part of an on-going Phase I/II clinical study (EudraCT
number 2008-008290-54; clinicaltrials.org – NCT01264952). The study was
approved by Institutional Medical Board and Ethical Committee of the Republic
of Slovenia. Briefly, a model of a patient with a metastasis located between the
inferior vena cava and the main hepatic veins was studied. The model included a
3-D geometry of the metastasis that was built by means of segmented MRI
images of the patient. As shown on Fig. 9, it was demonstrated that it is possible
to obtain sufficiently accurate information on electric field distribution in the
targeted and surrounding tissue by measuring only one component of magnetic
flux density and thus enable detection of areas with insufficient electric field
coverage before the end.
b
E [kV/cm]
a 2
1.8
1.6 +
1.4
–
1 1.2
5 1
0.8
3
4
0.6
2
0 mm 5 mm 10 mm 0.4
6
0.2
y
x 0
z 0 1 2 3 4 5
tumor-liver boundary x [mm]
Fig. 9 A 3-D numerical model of a deep-seated tumor in a liver (a). The tumor (in red) was located
between the inferior vena cava and main hepatic veins (all in blue). The liver tissue, surrounding the
tumor and veins, is not shown for the purpose of visualization. Electrodes (in grey) are labeled with
numbers from 1 to 6. An electric field distribution across the tumor-liver region (between electrodes
no. 4 and 6) obtained by means of the MREIT algorithm using only one magnetic field component
(solid line) and the corresponding true electric field calculated by the numerical model (dashed line)
(b). Tumor-liver boundary is marked with x. (The figure is adapted from Kranjc et al. (2012))
Conclusions
Exposure of the treated tissue to a sufficiently large electric field presents one of the most
important conditions for successful electroporation. A monitoring method that would
allow determination of the electric field would be of great importance for electroporation
based applications such as electrochemotherapy, irreversible electroporation tissue abla-
tion, and pulsed electric field processing. In recent studies, it was demonstrated by means
of experimental and numerical approaches that magnetic resonance electrical impedance
tomography together with current density imaging indeed can be used for determination
of electric field distribution during electroporation pulse delivery. As there is a lack of
tissue-specific experimental data on tissue properties for reliable numerical treatment
planning of electroporation based clinical applications, MREIT could be of significant
help in obtaining more accurate electrical conductivity values.
Still, the main difficulty of using MREIT to determine electric field distribution
during electroporation is associated with the limited capability of MRI scanners for
their use during application of electroporation such as electrochemotherapy and
irreversible electroporation tissue ablation. Hopefully, this could be surpassed in the
near future using open MRI scanners. Conductivity changes that occur during the
pulse are at the moment also too fast to be estimated with MREIT as a function of time.
Nevertheless, it is important to be aware that the cumulative effect of electric current
on the MRI signal phase is measured. Therefore, this technique yields an electric field
distribution, which is a time average of its altering time course so that all the
consequences of conductivity alteration, which affect electric current, are not
neglected within obtained electric field distribution.
Cross-References
▶ Current Density Imaging as Means to Follow Tissue Electroporation

▶ Electric Field Distribution and Electroporation Threshold
▶ Treatment Planning for Electrochemotherapy and Irreversible Electroporation of
Deep-Seated Tumors
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Current Density Imaging as Means to
Follow Tissue Electroporation 33
Igor Serša and Franci Bajd
Abstract
An expanding use of electroporation in everyday clinical practice necessitates
development of accurate and preferably noninvasive methods for assessment of
electroporation outcome. Electroporation outcome is highly dependent on tissue
coverage with sufficiently high electric fields that can be reconstructed by the
magnetic resonance electric impedance tomography, which uses current density
distribution data and electric potentials at the electrodes for reconstruction of
electric field in the sample. Current density imaging (CDI) is a magnetic reso-
nance imaging (MRI) modality for noninvasive assessment of current density
through phase information and is therefore a method of choice for monitoring of
electroporation efficacy. CDI consists of the current encoding part in which
electric current due to a set of delivered electric pulses is encoded in a signal
phase and of the imaging part in which the encoded signal is acquired. In the
overview, CDI is presented as an efficient method for a use in electroporation
applications. With respect to these applications, different k-space traversing
strategies employed in the imaging part of CDI are discussed. While the most
common strategies are based on standard spin-echo and gradient-echo pulse
sequences in which each k-space line traversal is preceded by a new set of electric
pulses, advanced strategies employing single-shot rapid acquisition with relaxa-
tion enhancement and echo planar imaging include minimal number delivered
electric pulses for entire k-space traversal. The strategies are discussed also with
respect to parameters, such as an effect of cumulative duration of electric pulses
on CDI sensitivity, SAR, SNR performance, and susceptibility of CDI to artifacts.
In addition, a reconstruction algorithm for current density distribution using
Ampere’s law and the corresponding limitations associated with the need for
I. Serša (*) • F. Bajd (*)

Jožef Stefan Institute, Ljubljana, Slovenia
e-mail: igor.sersa@ijs.si; franci.bajd@ijs.si

DOI 10.1007/978-3-319-32886-7_66
568 I. Serša and F. Bajd
sample rotations for a determination of three current density spatial components

are discussed as well.
Keywords
Magnetic resonance imaging • Current density imaging • Electric currents •
Tissue conductivity • Electroporation
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568
Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
From Electric Current Pulses to CD Image . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
DC-CDI Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574
CDI Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575
Electroporation CDI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
Electroporation CDI Implementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
Introduction
In electrochemotherapy (ECT) (Marty et al. 2006, Miklavcic et al. 2014) and

irreversible electroporation (IRE) tissue ablation (Lee et al. 2010, Neal
et al. 2011), which are two widely used clinical applications of electroporation for
solid tumor treatment, temporary cell membrane permeabilization, induced by
applying short high-intensity electric field pulses, is followed by the cell death due
to the cell uptake of an anticancer drug (ECT) or due to the cell exposure to an
excessively high electric field (IRE) (Yarmush et al. 2014). The efficacy and duration
of permeabilization are dependent on parameters such as duration, frequency, and
number of the pulses as well as on electrode geometry, number of the electrodes, and
their placement. Accurate coverage of the treated tissue with sufficiently large
electric field that is established during an application of electroporation pulses
plays an important role for a successful electroporation outcome (Miklavcic
et al. 1998). Therefore, effective and preferably noninvasive methods are needed
for assessment of the parameters maximizing tissue coverage with electric field. For
a reconstruction of electric field distribution, magnetic resonance electrical imped-
ance tomography (MREIT) was suggested and recently demonstrated in an agar
phantom (Kranjc et al. 2011), in liver tissue ex vivo (Kranjc et al. 2014), in silico
(Kranjc et al. 2012), as well as during electroporation of mouse tumor in vivo
(Kranjc et al. 2015). The MREIT method is based on current density imaging
(CDI), which is an MRI modality developed for a detection of current density
33 Current Density Imaging as Means to Follow Tissue Electroporation 569
distribution inside a conductive sample. The detection is enabled through temporary

current-induced magnetic field spatial variations that are manifested in MR phase
alterations. From the alterations, current density is calculated by using Ampere’s law.
In the last two decades, CDI has become an increasingly important tool for studying
electrical conductivity properties of different biological tissues in various frequency
ranges (DC, AC, and RF). Recently, the CDI method along with the MREIT
algorithms was successfully applied also for measurements of tissue conductivity
tensor (Mezeme et al. 2012, Kwon et al. 2014b). Moreover, the combined approach
was successfully applied as a monitoring tool and for guidance in treatment by RF
ablation (Kwon et al. 2014a).
CDI is enabled by the use of electric pulses that are synchronized with an
imaging sequence. During the electric pulses, electric current flows through the
sample between the electrodes that are attached to the sample or inserted in it. The
CDI sequence can in principle be of any type of a MR sequence with a charac-
teristic that it can hold phase information accumulated during application of
electric pulses. In the simplest form, the CDI sequence can be a modified spin-
echo sequence (Joy et al. 1989), or it can be a sequence based on gradient-echo
imaging (Kwon et al. 2014a). In these two forms of the sequences, the k-space
(Callaghan 1991) is traversed in consecutive lines, and for each line or a few lines,
a new signal excitation is needed with a new electric pulse or a set of electric
pulses. More sophisticated sequences include a spiral acquisition (Yan
et al. 1997), echo planar (EPI) acquisition (Serša et al. 2015), or rapid acquisition
with relaxation (RARE) (Sersa 2008). Last three forms of the CDI sequence are of
a fast acquisition type and can be run also in a single-shot mode (spiral and EPI) or
in a two-shot mode (RARE). Therefore, only one signal excitation or at most two
signal excitations followed by an application of an electric pulse or a set of pulses
is needed. In CDI, the phase information is related to the magnetic field created by
currents flowing through the sample during the application of electric pulses. The
information is then used to calculate magnetic field change induced by the
currents and finally to calculate current density from the magnetic field change
by using Ampere’s law.
The outcome of CDI depends highly on the conductivity properties of the sample.
With highly conductive samples, sufficient MR phase differences are obtained with
relatively low voltages in the range from few volts up to few tens of volts and with
moderated pulse durations of typically few tens of milliseconds. However, such
conductivities are obtained mostly only in electrolytes, where conductivity of a
sample can be adjusted simply by changing concentration of salts or by changing
pH of the sample. Unfortunately, most tissues are not that conductive, so voltages of
the pulses must be increased or if that is not possible their duration must be
increased. The first can damage the sample and cause pain if applied in vivo,
while the second results in poor SNR of the obtained images due to the spin-spin
NMR relaxation of the sample. The theory of CDI sensitivity presented in Scott
et al. (1992) and Sersa et al. (1994) reveals that the optimal sensitivity of the CDI
experiment is obtained when the current application time is equal to T2 relaxation
time of the sample. In reality, for most tissues, T2 is of the order of 100 ms. This
Fig. 1 The sensitivity curve, i.e., sensitivity of a generic CDI pulse sequence as a function of
cumulative electric pulse duration tc. Relatively low sensitivities are obtained with short and high-
voltage pulses (tc < T2) that are typically used in electroporation applications (ECT and IRE) as
well as with long and low-voltage pulses (tc > T2) that are exploited for noninvasive determination
of electrical conductivity properties of tissues in vivo. In CDI, the optimal sensitivity is obtained
with cumulative duration of electric pulses equal to spin-spin relaxation time (tc = T2)
dictates the use of voltages of one order of magnitude higher than in electrolytes
(from few tens of volts to few hundred volts) for a sufficient effect of the currents on
the signal phase shift (Beravs et al. 1997, Sersa et al. 1997) and consequently on a
sufficient CDI quality.
Another extreme of the CDI use is electroporation. In electroporation, electric
pulses are short, and they are usually used in series of few pulses. For example, a
common configuration is eight pulses of 100 μs with voltages from 500 V to 1500 V
separated by 100 μs long pauses in between the pulses (Yarmush et al. 2014).
Obviously, in electroporation applications, electric pulses are cumulatively about
two orders of magnitude shorter than when used in a non-damaging configuration for
tissue imaging, and their voltage is approximately one order of magnitude higher. A
use of CDI in electroporation is therefore in a low sensitivity regime, however on the
other side of the sensitivity curve as in standard in vivo CDI applications (Fig. 1). An
advantage of CDI in electroporation is that there is practically no effect of signal
relaxation during application of electric pulses, which reduces imaging noise.
However, electroporation pulses cannot be applied during image acquisition many
times as they significantly alter electrical properties of the sample during imaging.
For that reason, spin-echo- and gradient-echo-based CDI sequences are not appro-
priate for electroporation CDI experiments. However, RARE, EPI, or spiral
sequences are due to their single- or two-shot imaging ability appropriate candidates
for the use. There is also another limitation that could prevent the use of the fast CDI
sequences and that is NMR relaxation. All three sequences (RARE, EPI, and spiral)
require a long T2 relaxation time of the sample for their successful performance. This
is because they all need long signal to enable entire images signal acquisition after
just one excitation.
a b
Fig. 2 (a) Schematic presentation of an electric current-carrying straight wire and magnetic field
around it. Magnetic field Bc makes a circular path around the wire, and its amplitude decreases
inversely proportional with a distance from the wire. In CDI, similarly, currents between the
electrodes create magnetic field variations that are encoded into phase information. (b) In CDI,
only magnetic field variations in the direction of the static magnetic field B0∙ez contribute to current-
induced phase variations. By using Ampere’s law, 2D current density j = ( jx, jy) within a slice
perpendicular to the static magnetic field can be calculated from these phase variations. Information
on electric current distribution is therefore most efficiently stored into phase variations when the
electroporation electrodes are inserted in parallel with the static magnetic field and the imaging slice
is perpendicular to the static filed and the electrodes
In this overview, typical CDI pulse sequences for determination of electric current
distribution in tissues during delivery of electroporation pulses are presented. The
sequences are classified according to their SNR performance, which highly deter-
mine the quality of calculated electric fields and electric conductivity maps through
their reconstruction by MREIT algorithms, as well as according to their specific
absorption rate (SAR) performance and adequacy for monitoring of different elec-
troporation applications (ECT vs. IRE).
Theory
From Electric Current Pulses to CD Image
One of the effects caused by electric currents is also a change of the magnetic field
around a current-carrying sample (Fig. 2a). Current I that flows along a straight wire
produces a magnetic field Bc that makes a circular path around the wire. Its amplitude
is inversely proportionally to the radial distance from the wire
μ0 I
Bc ð r Þ ¼ : (1)
2πr
In a CDI experiment, a sample is in the MRI magnet, and it has electrodes attached to
it or inserted in it. Then, voltage is applied to the electrodes which results in currents
flowing through the sample. The pattern of the currents in the sample can be of
course much more complex than in Fig. 1, but the end effect is similar; in the sample,
additional magnetic field Bc is created. The additional field is superimposed to
the statistic magnetic field of the magnet B0. As B0 » Bc, other components of Bc
than the component along B0 have practically no effect on the precession frequency
of the nuclei, while the z-component changes their precession (Larmor) frequency by
Δω ¼ γBcˍ B0 : (2)
If the currents are constant, then the corresponding magnetic field shift is constant
too. Therefore, the currents applied in a pulse of duration tc will induce a precession
phase shift of
φB0 ¼ γBcˍ B0 tc : (3)
The phase shift can then be used to calculate the corresponding magnetic field
change. As the magnetic field change is a vector, it has therefore three components.
All three can be measured in separate experiments by rotating the sample along with
the electrodes to three perpendicular orientations so that in a coordinate system
attached to the sample in each of the orientations, one of the three coordinates is
aligned with the static magnetic field
φx φy φz
Bcˍ x ¼ Bcˍ y ¼ Bcˍ z ¼ : (4)
γtc γtc γtc
Once all three components of the magnetic field change are measured, current
density in the sample can be calculated by using Ampere’s law
⇀ 1 ⇀
j¼ ∇ Bc: (5)
μ0
Rotating the sample to the perpendicular orientations to obtain all three components
of the magnetic field change is very impractical and also often not possible.
Therefore, it is desirable to use in CDI such arrangement of the electrodes with
respect to the image orientation that there is no need for the rotations and repeated
experiments. One such arrangement is obtained with a sample where currents flow in
a relatively thin slice which is also the imaging slice (Fig. 2b), and the electrodes
are positioned perpendicularly to the slice. With the arrangement, Bc_x 0 and
Bc_y 0 so that current density in the slice is calculated only from the
component Bc_z

1 @Bcˍ z @Bcˍ z
jx , jy , : (6)
μ0 @y @x
The procedure of CDI experiment, measurement and data processing, is presented in

Fig. 3.
Fig. 3 Magnitude MR image of a conductive phantom shows no indication for electric current
flowing between the electrodes that are inserted close to the phantom boundary. However, the effect
can be clearly seen in the corresponding MR phase image (with modulus 2π) by a characteristic
nonuniform pattern of phase variation. The pattern is a consequence of encoded magnetic field
variations due to electric current flowing through a conductive material under a potential difference
(voltage V and duration tc) between the inserted electrodes. From the phase pattern, experimentally
obtained current density distribution is calculated in a pixelwise manner by using Ampere’s law.
Finally, the experimentally obtained current density is compared with the simulated electric current
density
DC-CDI Sequence
As already mentioned in the introduction section, CDI is enabled by the use of an

imaging sequence that during the CD encoding part triggers a current pulse to the
sample and then enables registration of the phase shift produced by the currents
(Eq. 3). The simplest sequence which meets these criteria is a spin-echo sequence
with superimposed two electric pulses (Fig. 4a). The first electric pulse is applied
Fig. 4 (a) Standard spin-echo-based CDI pulse sequence with electric pulses inserted between the
excitation and refocusing RF pulses. Each electric pulse is of duration of tc/2, thus resulting to
cumulative duration of tc. Polarity of the second electric pulse is reversed due to a sign-reversing
character of the refocusing RF pulse. A drawback of the sequence is that a new pair of electric
pulses is needed prior to acquisition of each k-space line. (b) Two-shot RARE CDI pulse sequence,
in which the current encoding part is followed by multiple signal acquisitions traversing the entire
k-space. In order to eliminate image artifacts, the final MR phase image is obtained by co-adding
signals of two single-shot RARE CDI sequences, each with different phases of the refocusing pulses
([x] and [y])
before the refocusing RF pulse, and the second electric pulse of the same duration
and amplitude but of an opposite polarity is applied after it. The second pulse must
have the opposite polarity because the refocusing RF pulse in the sequence reverses
the phase shift of the first electric pulse. The reversed phase shift of the first pulse can
then co-add with the phase shift of the second pulse. The spin-echo CDI sequence in
Fig. 4a belongs to a groups of DC-CDI (direct current) or LF-CDI (low frequency)
sequences due to relatively long application time of currents tc in only two electric
pulses.
In Fig. 4b another version of a DC-CDI sequence is shown, namely, the two-shot
RARE CDI sequence. With the two-shot RARE CDI sequence, equal electric pulses
can be used as with the spin-echo CDI sequence; however, signal acquisition
strategy with this sequence is significantly different. In the spin-echo CDI sequence,
image signal is scanned in the k-space line by line. For each line, a signal is excited,
and both current pulses are applied. For a standard image with N = 256 lines,
256 pairs of electric pulses are delivered to the sample. This is not only time-
consuming but could also result in alteration of sample conductivity if the applied
voltage of current pulses is high. To avoid this problem, the number of delivered
electric pulses must be reduced. Ideally, only one electric pulse delivered to the
sample is sufficient to obtain one CD image. In the single-shot RARE sequence, all
image signal is acquired only after one signal excitation, which is enabled by
repetitive scanning of k-space lines in consecutive echoes. Unfortunately, as
shown in (Sersa 2008), the sequence becomes unstable when current-induced
phase shift is encoded at the beginning of the sequence. However, the problem of
instability can be solved by running the sequence twice, which is followed by a
co-addition of the corresponding signals. The sequence is therefore called the
two-shot RARE CDI sequence. In the first run, phases of the refocusing RF pulses
are identical to the phase of the excitation RF pulse, while in the second run, the
phases of the refocusing RF pulses are increased by 90 .
CDI Sensitivity
Sensitivity of a CDI sequence can be considered as the reciprocal noise of the

measured current density (Scott et al. 1992; Sersa et al. 1994). The noise has an
origin in noise of the conventional image, but also depends on the signal magnitude
(Signal). In the complex plane, the MR signal can be represented by a point.
However, due to noise, the point is smeared in both directions, in real and in
imaginary direction (Fig. 5). Distribution profile of the signal along the two direc-
tions is a normal one with a standard deviation (RMS noise) equal to σ. The ratio
between the RMS noise and the signal magnitude therefore represents uncertainty in
the signal phase and can therefore be considered as the phase noise
pffiffiffi
2 σ 1
σφ ¼ ¼ ; (7)
f Signal f SNR
Fig. 5 In the complex plane,

MR signal can be represented
by a point of which
probability is due to RMS
noise of its real and imaginary
signal components smeared in
two-dimensional normal
distribution with a standard
error σ. The uncertainty of the
point’s angle in the complex
plane, i.e., the phase noise, is
therefore equal to
σ φ ¼ σ=Signal, where Signal
is the signal magnitude
where SNR is a signal-to-noise ratio of the conventional magnitude image and factor
f comes into the formula due to possible different signal manipulations needed to
eliminate the background phase. This can be eliminated by subtracting a phase
image without application of currents ( f = 1) or with reversed currents ( f = 2)
from the phase image obtained with currents; if no subtraction is used, f = √2. The
phase noise determines also the magnetic field noise
1
σ Bc ¼ (8)
γ tc f SNR
and ultimately, via the relation for calculation of a current density component in
discrete space
N
jzˍ i, j ¼ Byˍ iþ1, j Byˍ i, j Bxˍ i, jþ1 þ Bxˍ i, j (9)
μ0 FOV
also the current density noise
2N
σj ¼ : (10)
μ0 FOV f γ tc SNR
Here FOV is image field of view and N by N is image matrix. From Eq. 10 can clearly
be seen that CDI sensitivity, i.e., reciprocal σ j, is proportional to a product of the
current application time (cumulative time of all current pulse durations) tc and signal-
to-noise ratio of the conventional magnitude image
1
Sensitivity / tc SNR: (11)
σj
For the DC-CDI pulse sequences (Figs. 4a, b), SNR is proportional to exp(tc/T2) so
that CDI sensitivity is therefore proportional to
Sensitivity / tc expðtc =T 2 Þ: (12)
Equation 12 reaches maximum when tc = T2, which is also the condition for the
optimal current application time in DC-CDI experiment as well as in all other CDI
experiments where signal magnitude decays exponentially with a decay rate equal to
T2. Dependence on CDI sensitivity on the current application time is presented in
Fig. 1. The graph in the figure clearly shows how CDI sensitivity decreases when tc
is either longer or shorter than the optimal value of tc = T2. The first case is often
found in in vivo CDI experiments where current amplitudes are limited to avoid
tissue damage and pain, while electroporation CDI, where current application times
are very short, is a good example of the second case.
Current density signal-to-noise ratio SNRj can be considered as the ration between
the current density in the samples and current density noise. According to Eq. 11, this
is equal to
j f γ μ0 FOV j tc SNR
SNRj ¼ : (13)
σj 2N
From Eq. 13 can be seen that SNRj is proportional to pixel size FOV/N (inversely
proportional to image resolution) to a product of current density and current appli-
cation time and to the signal-to-noise ratio of the conventional magnitude image.
Electroporation CDI
As can be seen from Eq. 13, to keep reasonable level of SNRj in CD images, a
product of current density and current application time must be kept at least constant
or better increasing with tc to compensate T2 relaxation signal losses. This means that
in in vivo low-current CDI applications, tc needs to be long. Best SNRj is then
obtained when tc = T2; however, in electroporation experiments, this is not possible
as tc is fixed by the electroporation protocol, usually to time below 1 ms. With
current application times that short, T2 relaxation can be neglected so that SNRj can
be kept constant when a product of current density and current application time is
constant. The effect of current application time shortening can then be compensated
by increasing current density proportionally.
As application of each electroporation pulse can change tissue properties addi-
tionally, progress of electroporation treatment can be followed only with those
methods that enable immediate analysis of electroporation effects on the treated

tissue. In case of CDI, this implies that a CD image must be obtained after only a few
electroporation pulses delivered to the sample. This condition can be met by the use
of the two-shot RARE CDI sequence (Fig. 6a). Note that Figs. 4a and 6a, which both
show the two-shot RARE CDI sequence, differ only on different electric pulses used
in the sequence. The sequence in Fig. 6a uses electroporation pulses which are all of
the same polarity, and therefore all need to be included in the same interval, between
the excitation and refocusing RF pulse of after the refocusing RF pulse and before
formation of the first echo. Another important aspect of the two-shot RARE CDI
sequence is the k-space traversal order. The lines can be ordered very differently
(sequentially, centric, reversed centric, etc.), which in conventional MR image leads
Fig. 6 (a) Modified RARE CDI pulse sequence with the current encoding part containing a set of
eight high-voltage electric pulses that are typically used in electroporation applications. (b) EPI CDI
pulse sequence with a set of eight high-voltage electric pulses included in the current encoding part
of the sequence that is followed by the EPI imaging part. In comparison to the two-shot RARE CDI
sequence, this sequence exhibits comparatively lower SAR, but is more susceptible to artifacts
Fig. 7 A graphic presentation of two different k-space traversing strategies of the two-shot RARE
CDI sequence: sequential k-space traversal with maximal/minimal MR signal at the ky edges and
centeric k-space traversal with maximal MR signal in the center of the k-space and minimal MR
signal at the ky edges. A decay of the MR signal due to spin-spin relaxation is indicated by a gradual
color transition from black to light gray
to different image contrasts as, for example, proton-density-weighting vs.

T2-weighting. In case of CDI, the contrast is not important; however, accuracy of
the measured phase shift is important. The latter can be maximized by using proton-
density-weighting, which corresponds to centric ordering of the scanned k-space
lines (Fig. 7). With the centric ordering, lines from the k-space center that contribute
the most signal to the image are scanned first, i.e., immediately after the excitation
RF pulse, and are therefore not affected much by the T2 relaxation. Therefore, the
obtained image has more signal, and phase error is the lowest. The centric k-space
ordering is especially important with samples that have short T2 relaxation times.
Another sequence that can be used in electroporation CDI is the EPI CDI
sequence (Fig. 6b). In an EPI sequence, k-space is traversed in a “zigzag” trajectory,
thus enabling acquisition of the entire image signal after just one signal excitation.
The sequence with such signal sampling is known as a single-shot EPI sequence.
Electroporation pulses in the EPI CDI sequence can be identical to those used in the
two-shot RARE CDI sequence and are added between the excitation and refocusing
RF pulse. Unlike in the RARE sequence, the additional phase shift produced by the
electroporation pulses does not destabilize the single-shot EPI sequence so that the
EPI CDI sequence, i.e., a standard single-shot EPI sequence with superimposed
electroporation pulses, is a single-shot sequence. Therefore, with the EPI CDI
sequence, double number of images can be acquired with the same number of
electroporation pulses as with the two-shot RARE CDI sequence. This is an impor-
tant advantage when high temporal resolution of electroporation monitoring is of a
high priority. In addition, the EPI CDI sequence uses only two RF pulses: an
excitation RF pulse and a refocusing RF pulse. This is convenient for in vivo
applications where SAR is an important issue. A standard EPI sequence can be
used in two versions: with the refocusing RF pulse or without it. The first version
yields a T2-weighted image due to the spin-echo formation during reading the signal
from the k-space center, while the second version yields a T2*-weighted image. For
the CDI use, the spin-echo EPI version is better as it gives more signal and therefore
produces results with lower current density noise. Despite all undeniable advantages
of the EPI CDI sequence in comparison to the two-shot RARE sequence, it has also
an important drawback, namely, it is highly susceptible to different imaging artifacts.
Methods
Sample Preparation
The performance of the two-shot RARE CDI and EPI CDI sequences was tested on a
beef liver tissue. The liver tissue was obtained from a slaughterhouse, as meat
product for human consumption. The slaughterhouse operates in accordance to
Slovenian law, and the process of slaughtering is regulated by rules on animal
protection and welfare at slaughter (Ur. l. RS, N. 5/2006), which ensures ethical
standards of slaughtering procedure and is in compliance with European Union
Council directive on the protection of animals at the time of slaughter or killing
(93/119/EC). Temperature of the liver tissue was maintained at 4 C before begin-
ning of the experiment and then allowed to warm to the room temperature. The liver
tissue was sectioned to flat cylindrically shaped samples with a diameter of 21 mm
and height of 10 mm and then placed in a plastic container (Fig. 8). Two needle
platinum-iridium electrodes with a 1 mm diameter were inserted at an inter-electrode
distance of 14 mm in the sample in orientation perpendicular to the sample slice and
in parallel to each other. The sample was exposed to high-voltage electric pulses that
are typically used in irreversible electroporation (IRE). Altogether 90 pulses of
3000 V amplitude, 1 Hz repetition rate, and 100 μs (RARE-type of sequence) or
300 μs (EPI-type of sequence) duration and were delivered to the sample. Electric
pulses were delivered using customized Cliniporator Vitae (IGEA, Carpi, Italy)
pulse generator. The current of electric pulses was measured with an oscilloscope
(WavePro 7300A, LeCroy, USA) using current probe (AP015, LeCroy, USA). All
experiments were repeated three times. The sample was replaced with a fresh one
after each electroporation pulse delivery to ensure identical initial conditions in all
electroporation experiments.
Fig. 8 A schematic presentation of a CDI sample comprised of a chamber with inserted conductive
material through which two electroporation electrodes, parallel to the static magnetic field B0, are
inserted. The sample is placed inside a radio-frequency MRI probe (segmented wire around the
sample)
Electroporation CDI Implementation
Electroporation CDI experiments for IRE monitoring were performed on a 2.35 T

horizontal bore small animal MRI scanner. The scanner was based on an
Oxford superconducting magnet (Oxford Instruments, Abingdon, UK), an
Apollo NMR/MRI spectrometer (Tecmag Inc., Houston TX, USA), and MRI
probes for MR microscopy (Bruker, Ettlingen, Germany). The sequences were
run with identical geometric and resolution parameters: field of view 30 mm,
imaging matrix 64 by 64, slice thickness 4 mm, and repetition time 1 s,
while echo-time parameters were different: inter-echo time in the two-shot
RARE CDI sequence was 2.64 ms and spin-echo time in the EPI CDI
sequence was 35 ms. To maximize CDI sensitivity, k-space traversal ordering
was centric for the RARE-type of sequence and was sequential for the EPI CDI
sequence.
Results
CDI results of the two-shot RARE CDI sequence and EPI CDI sequence on the liver
sample are shown in Fig. 9 by vector field maps of CD distributions (left column
images) as well as by CD magnitude images (right column images). In the RARE
CDI experiment, 90 electroporation pulses were delivered to the sample, and 45 CD
images were calculated as delivery of two electroporation pulses was needed for
calculation of one CD image. Results obtained with the two-shot RARE CDI
sequence are presented with CD images corresponding to the first two electropora-
tion pulses and to the last two (89th and 90th) electroporation pulses. As can be seen
from the vector field images (first vs. second row in Fig. 9), an area with higher
current density (larger than 10 kA/m2) was established around the electrodes during
the application of first two pulses, whereas during the last two pulses, this area has
expanded toward the area between the electrodes. The direction of current path did
not change with repeated electric pulses significantly; it only got more focused to the
direct path between the electrodes. Thus, the current density in the middle of the
sample increased from first to last electric pulses by 60%, i.e., from 8 kA/m2 to
13 kA/m2. These results were confirmed also by measurements of electric current
during delivery of electroporation pulses using an oscilloscope shown in Fig. 10a.
Currents calculated from CD images (red circles) are well in the range of the
oscilloscope readings (blue crosses). Figure 10b shows a typical current profile
during application of an electroporation pulse; the profile was measured with the
last electroporation pulse. From the profile can be seen that current was increasing
during the pulse. CDI results shown in the bottom row of Fig. 9 were obtained by the
EPI CDI sequence on a parallel liver sample to the one used in the RARE CDI
experiment. In the experiment, 90 electroporation pulses were delivered, and the
same number of CD images was obtained. The presented images correspond to CD
distribution at the end of the EPI CDI experiment. The results confirm that a CD
Fig. 9 Cumulative effect of repetitive electroporation pulses on current density distribution in the
electroporated beef liver tissue sample measured by the two-shot RARE CDI pulse sequence and
EPI CDI sequence. A clear difference in both in-plane current density vector field j = ( jx, jy)
calculated by using Ampere’s low and the corresponding current density magnitude field can be
seen between the first two delivered electric pulses (relatively smaller values) and the last two
delivered pulses (relatively higher values) of totally 90 electric pulses of 3000 V. For comparison,
EPI CDI results of a parallel beef liver tissue sample are also shown
distribution can be imaged also by the EPI CDI sequence. The magnitude CD image
has the highest current density next to the electrodes and then current density
decreased as distance from the electrodes increased. However, streamline of the
highest current was not a straight line connecting the electrodes, but was shifted
sideways.
Fig. 10 Measured current increase during the delivery of 90 electroporation pulses (a) and current
profile during the delivery of the last one electroporation pulse (b)
Discussion
Current density imaging is an efficient tool for monitoring electroporation. Electro-

poration CDI is enabled by fast MRI sequences modified by addition of electropo-
ration pulses. All the sequences have in principle two parts, the first current encoding
part in which a magnetic field change induced by electroporation currents is encoded
in the magnetization precession phase and the second part where image signal is
acquired preferably from the entire k-space. The obtained magnetic field change
maps are then converted to the corresponding current density maps using Ampere’s
law. The current density maps along with electric potentials at the electrodes are
inputs to the MREIT algorithm (MREIT, ▶ Chap. 32, “Principles and Use of
Magnetic Resonance Electrical Impedance Tomography in Tissue Electroporation”)
for reconstruction of the electric field between the electrodes and for calculation of
the sample conductivity maps. In the overview, two CDI sequences designed for
electroporation monitoring are presented, the two-shot RARE CDI sequence and the
EPI CDI sequence. Experiments performed on a liver test sample confirmed that
with both sequences, images of current distribution can be obtained, however, with
different limitations.
The two-shot RARE CDI sequence produces results that have fewer artifacts than
the results obtained by the EPI CDI sequence. However, the two-shot RARE CDI
sequence requires application of many refocusing RF pulses and has therefore a
considerable SAR value, which can prevent its use in clinical or other in vivo
applications. SAR is a measure of the rate at which energy is absorbed by the
human body when exposed to an RF electromagnetic radiation. As there are strict
regulations regarding SAR in clinical MRI, the performance of the two-shot RARE
CDI sequence could be impeded when used in human MRI. For comparison, in the
presented study, for acquisition of one CD image, 130 refocusing RF pulses were
delivered to the sample with the two-shot RARE CDI sequence, while only one
refocusing RF pulse was delivered to the sample with the EPI CDI sequence.
Another disadvantage of the two-shot RARE CDI sequence is that it requires double
the number of electroporation pulses for obtaining the same number of images as the
EPI CDI sequence. Based on these limitations, it can be concluded that the two-shot
RARE CDI sequence is a more appropriate technique for electroporation of tissue
samples ex vivo, while for in vivo electroporation studies, EPI CDI or perhaps also
spiral CDI (Yan et al. 1997) is better suited.
Artifacts in EPI CDI have often origin in poor magnetic field inhomogeneity of
the scanned samples. These can be to some extent reduced by using parallel imaging
technology, which allows a reduction in number of scanned k-space lines and
therefore allows a reduction of the EPI signal acquisition train length on account
of the use of multiple receiver coils (Pruessmann et al. 1999). The technology could
also contribute to better results of the EPI CDI sequence with fewer artifacts.
Another artifact contributing problem that is common to all CDI experiments are
electrodes. These need to be conductive and therefore inevitably produce MR signal
voids in their vicinity. In the study, platinum-iridium electrodes were used as they are
biocompatible and produce relatively low artifacts. The metal electrodes were
needed because the electrodes were implanted into the liver sample. If the electrodes
could be attached to the sample, then perhaps a better solution would be a use of
hydrogel (nonmetal) electrodes. These have a conductivity closer to that of the tissue
and therefore produce very limited or almost no artifacts.
Electroporation monitoring was enabled in the study not only by the use of fast
imaging methods but also by the use of a specific electrode/slice arrangement that
produces reasonable current distribution maps without any sample rotations to
orthogonal orientations. A need of these would at least impede the electroporation
monitoring by CDI if not even make it impossible. For now, no easy solution is
known that would overcome the problem associated with CDI in a general slice
orientation.
As shown in Fig. 1, CDI has its peak sensitivity at tc = T2, and, unfortunately, in
practice most biomedical applications of CDI are such that there is a demand for
either application of very short- and high-current pulses or long- and low-current
pulses. The first case is associated with CDI applications in monitoring electropora-
tion, while the second case is associated with attempts to image physiological
currents, which are low due to low conductivity of biological tissues and also due
to low voltages that living systems can bear (Joy et al. 1989). The most challenging
of these is undoubtedly CDI of neuronal currents, which is still mostly at the level of
theoretical concepts (Hagberg et al. 2006, Cassara et al. 2009). The CDI sensitivity
may become problematic in IRE experiments, where the standard IRE protocol uses
only 100 μs long electric pulses repeated 90 times at the frequency of 1 Hz. The
frequency of 1 Hz is too low to enable execution of more than one electric pulse in
one CDI sequence. This limit is posed by the T2 relaxation time of biological tissues,
which is of the order of 100 ms. Only one 100 μs pulse is four times shorter than
what was used in a standard electroporation experiment on the same test sample,
where four 100 μs were delivered in intervals of 100 μs (Kranjc et al. 2011). A way to
compensate the fourfold loss of sensitivity loss due to shortening the electric pulses is to
increase the signal-to-noise ratio of the image, which can be done by increasing
imaging field of view and slice thickness or to decrease image resolution. Alternatively,
instead of one 100 μs electroporation pulse, a packet of up to four 100 μs electropo-
ration pulses delivered in short intervals or one 400 μs electroporation pulse can be
used. If none of these is an option, then the only solution would be to perform
experiments in a higher field magnet. Namely, image signal in theory increases
approximately proportionally with the magnetic field squared. Signal averaging is not
an option as the temporal resolution is a priority in electroporation monitoring by CDI.
In the presented study, the highest obtained electric current was around 5.5 A,
while in clinical cases of in vivo IRE, the current can reach up to 50 A (Bertacchini
et al. 2007) due to electrode depth of insertion. Application of CDI during IRE
in vivo would therefore enable measurement of current density distributions with a
better SNR than was obtained in this study.
Conclusions
In the overview, two sequences designed for electroporation monitoring by CDI,

namely, the two-shot RARE CDI and EPI CDI sequence, are presented. With both
sequences, it was confirmed that current distribution imaging during delivery of
short high-voltage electric pulses is feasible. The sequences require only one (EPI)
or two (RARE) short electric pulses repeated in an interval of approximately 1 s for
acquisition of one current density image. This enables sequential imaging of current
distribution during delivery of the electric pulse train, which could potentially be
used in MREIT-enabled monitoring of tumor coverage by electric field during IRE
tissue ablation.
Cross-References
▶ Diffusion Weighted Magnetic Resonance Imaging for Detection of Tissue Elec-

troporation In Vivo
▶ Medical Imaging of Electroporation
▶ Principles and Use of Magnetic Resonance Electrical Impedance Tomography in
Tissue Electroporation
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Mass Transfer of Electrolytic Species
During Electric Field-Based Tumor 34
Treatments
Guillermo Marshall
Abstract
To attain a reliable outcome in electric field-based tumor treatments, dose plan-
ning is a must, and dose planning, in turn, requires establishing the dose–response
relationship. But finding reliable dose and response parameters implies analyzing
the electric field–tissue interaction, in particular, the inevitable appearance of
complex electrolytic mass transfer processes and the inherent tissue damage. This
review of electric field-based tumor treatments highlights the fundamental role
played by the mass transfer of electrolytic species in the dose–response relation-
ship. During the electrolysis process in electric field-based tumor treatments,
electrochemical reactions take place at the electrodes, producing at the anode
oxygen, chlorine, and protons as the main by-products, while hydrogen and
hydroxide ions are released at the cathode. Proton and hydroxyls generation
yields strong pH fronts. Since these fronts are the main product of electrolytic
reactions, it is reasonable to assume that they are the main cause of tissue damage.
The amount of electrolytic products emerging from chemical reactions is propor-
tional to the amount of electric charges or Coulomb dose passing through the
tissue; thus, Coulomb dose is proportional to tissue damage. Theory shows that in
a constant electric field such as in electrolytic ablation, an optimal dose–response
relationship is the minimum Coulomb dose necessary to achieve total tumor
destruction while minimizing healthy tissue damage. In a pulsed electric field
such as in gene electrotransfer, unwanted damage due to electrolysis is
non-negligible; here, an optimal gene electrotransfer treatment is predicted as
the critical Coulomb dose yielding maximum electroporated area with minimum
G. Marshall (*)
Laboratorio de Sistemas Complejos, Departamento de Computación, Facultad de Ciencias Exactas
y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
Instituto de Física del Plasma, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos
Aires and Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina
e-mail: marshalg@retina.ar; marshall.guillermo@gmail.com; marshallg@arnet.com.ar

DOI 10.1007/978-3-319-32886-7_68
590 G. Marshall
damage. Theory shows that when tissue natural buffer is taken into account,
damage is attenuated (though still remaining non-negligible), and the critical
Coulomb dose for optimal gene electrotransfer increases.
Keywords
Electrolytic ablation • Electrochemotherapy • Gene electrotransfer • Irreversible
electroporation • Dose planning methodology • Dose–response relationship •
Buffer natural system • Mathematical modeling
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590
Electric Field-Based Tumor Treatments: Electrolytic Ablation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
Electric Field-Based Tumor Therapies: Electroporation-Based Treatments . . . . . . . . . . . . . . . . . . . 597
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605
Introduction
The use of electric field (EF)-based tumor treatments triggers complex electrolytic
mass transfer processes underlining these treatments, thus constituting a paradig-
matic example of biomedical applications of electrochemistry (Marshall 2014).
Depending on the type of EF being applied, direct or pulsed, these treatments can
be classified into electrolytic ablation (EA) and electroporation (EP)-based tumor
treatments. Electrolytic ablation (EA) of tumors, also called electrochemical treat-
ment of tumors (EChT), is a nonthermal ablative method consisting in the applica-
tion of a low constant electric field (long pulse) through two or more electrodes
inserted in the tissue generating electrolytic products that induce tumor necrosis.
This treatment was pioneered by Nordenström (1983) and has been widely used in
China with good clinical results (Li et al. 2006). Some of the characteristics of EA
are its simplicity, effectiveness, low cost, and negligible side effects. In EA typical
clinical applications, 6–8 V/m are used during periods from minutes to hours. There
are several groups working in Australia, China, Cuba, Japan, Sweden, and the USA;
a review can be found in Nilsson (2000).
Electroporation (EP)-based tumor treatments emerged during the last years as a
new cancer treatment. EP perturbs cell membrane integrity by the application of
pulsed electric fields. EP-based techniques, such as electrochemotherapy (ECT),
gene electrotherapy (GET), and irreversible electroporation (IRE), were
implemented for medical purposes. ECT combines reversible EP with poorly
permeant anticancer drugs, such as bleomycin, to potentiate their entry to the cell,
thus their intrinsic cytotoxicity. Since its beginnings in the late 1980s, ECT has
evolved into a clinically verified treatment for tumors of different origin in Europe
(▶ Chap. 93, “Electrochemotherapy and Its Clinical Applications”). GET uses the
same EP mechanism as in ECT to transfer plasmidic DNA instead of a drug. GET
34 Mass Transfer of Electrolytic Species During Electric Field-Based. . . 591
has a great potential as a nonviral gene delivery system as it achieves the introduc-
tion of plasmids or oligonucleotides into the cells. After the development of EP
devices, this technique became widespread for delivering molecules inside the cell.
If GET is applied to muscle cells, it works in favoring the antigen production within
the skeletal muscle and in the activation of pro-inflammatory pathways and in the
recruitment of cells involved in antigen presentation. This mechanism turns GET
into a useful strategy in DNA vaccination protocols and a promising approach for the
introduction of foreign antigens into the host for inducing an immune response not
only against infectious diseases but also against malignant tumors (Chiarella et al.
2010). A recent derivation of ECT is IRE, introduced by Davalos et al. (2005), an
irreversible EP (electric pulses above the irreversible threshold), without thermal
effects, that leaves intact main tissue structures. A combination of EA with reversible
EP has been shown recently to significantly increase the extent of tissue ablation in
comparison to that obtained with EA alone (▶ Chap. 91, “Combining Electrolysis
and Electroporation for Tissue Ablation”). A possible explanation is that reversible
EP potentiates the entrance of toxic electrolytic products into the cell increasing
tissue ablation.
The use of a high-intensity nanosecond pulsed electrical fields (nsPEF) or
nanosecond EF is another type of tissue electroporation gaining momentum
(▶ Chaps. 94, “Tissue Ablation Using Nanosecond Electric Pulses” and ▶ 80, “Preclinical
Studies on Nanosecond Pulses”). It has the effect of reaching the nucleus of the cell,
thus allowing a direct manipulation of the nucleoplasm apparently leaving intact the
membrane cell. The effects on plasma membrane are to permeabilize it after a few
nanoseconds, while the effects in subcellular membranes are to permeabilize intra-
cellular organelles producing a massive calcium release to the cytoplasm which
triggers cell death mechanisms.
Although having less undesired effects than traditional cancer therapies like
radiotherapy or chemotherapy, EF therapies still have some side effects (pain during
pulse delivery, swelling of the treated area, necrotic ulcers when over treating the
area, plasmid damage) that it is necessary to minimize. In silico modeling validated
with in vivo and in vitro measurements can greatly contribute to elucidate funda-
mental aspects of electric field–tissue interaction, thus contributing to minimize side
effects through dose planning. In this context, it is worth observing that in spite of
the wide differing scales of the electric parameters involved in EF therapies, there is
a close electrochemical analogy between EA- and EP-based treatments. This
becomes evident imagining an EA treatment with a very short pulse similar to one
pulse of an EP-based treatment. To a greater or lesser extent, both are affected by the
underlying electrolytic process, a process that is a function of the applied electric
charge, i.e., Coulomb dose.
Knowledge of the dose–response relationship leading to an optimal treatment
implies the analysis of the electrolytic mass transfer processes and tissue damage.
This analysis is firstly introduced for electrolytic ablation, whose aim is tissue
destruction, and then extended to EP-based tumor treatments, in particular to GET,
in which tissue destruction is an unwanted effect. In EA it is shown that an optimal
dose–response relationship is the minimum Coulomb dose necessary to achieve total
592 G. Marshall
tumor destruction while minimizing healthy tissue damage. An optimal GET is the
critical Coulomb dose that yields maximum electroporated volume (assuming
uptake proportional to electroporated volume) while minimizing healthy tissue
damage.
The plan of the chapter follows: section “Electric Field-Based Tumor Treatments:
Electrolytic Ablation” presents a brief review of constant EF tumor treatments, the
mass transfer processes underlying this treatment, and its dose planning; section
“Electric Field-Based Tumor Therapies: Electroporation-Based Treatments”
describes a brief review of pulsed EF therapies whether combined or not with
anticancer drugs or plasmids, the associated mass transfer underlying these thera-
pies, and its dose planning; and finally, section “Conclusions” draws some general
conclusions.
Electric Field-Based Tumor Treatments: Electrolytic Ablation
In the following, a brief review of experimental and theoretical modeling of EA

protocols with a differing degree of complexity is presented. It emphasizes the
underlying ion transport process leading to tissue damage and its role in obtaining
the dose–response relationship. Tissue damage in EA is mainly produced by
necrosis. This is because, during the electrolysis process, electrochemical reactions
take place at the electrodes, producing at the anode oxygen, chlorine, and protons
as the main by-products, while hydrogen and hydroxide ions are released at the
cathode. Proton and hydroxyls generation yields strong pH variations that are
named pH fronts. Since pH fronts are the main product of electrolytic reactions,
it is reasonable to assume that pH fronts are the main cause of tissue damage.
According to Faraday’s law, the amount of electrolytic products emerging from
chemical reactions is proportional to the amount of electric charges or Coulomb
dose passing through the tissue. As a consequence, Coulomb dose is proportional
to tissue damage.
In a pioneering series of papers discussed in Nilsson (2000), in silico modeling of
an EA protocol applied to a tumor tissue was presented. The tissue matrix was seen
as an electrolyte and the EA protocol as an electrolysis process. The ion transport in a
zone near one of the electrodes (cathode or anode) was described by a quasi-three-
dimensional model (spherical symmetry model) using the Nernst-Planck equations
for ion transport. Results show pH profiles at the anode to be strongly correlated with
the size of in vitro measured necrosis, thus confirming the effect of pH (the spreading
of hydroxyl ions) in tissue necrosis and suggesting that the model could be used for
predicting EA tumor treatment.
More realistic models were presented in Colombo et al. (2007). The EA model-
ing, based on first principles and taking into account the most relevant variables of
the phenomenon, consists in the one-dimensional Nernst–Planck equations for ion
transport in a four-component electrolyte (Na+, OH , Cl , and H+) and the Poisson
equation for the electrostatic potential under galvanostatic conditions in which the
full cathode–anode ion transport interaction is described; the model was solved
numerically and validated with in vivo and in vitro modeling using two electrodes
with a separation of 3 cm between them. The main finding was that, whether in vivo,
in vitro, or in silico, an initial condition with almost neutral pH evolves between
electrodes into extreme cathodic alkaline and anodic acidic fronts moving toward
each other, leaving the possible existence of a biological pH region between them
(if sufficiently long time has not been elapsed); toward the periphery, the pH falls to
its neutral values. In particular, in vivo measurements of necrotized tissue as a
function of the Coulomb dose were presented. Here a point platinum electrode
(anode) with a diameter of 1 mm was inserted in the center of the tumor, while the
cathode was placed subcutaneously far away from it. The macroscopic necrotic area,
defined by a dark, almost spherical colored zone around the anode, was measured for
different Coulomb doses (10, 30, and 50 C) scaling linearly with the Coulomb dose.
Toward a more realistic in vitro modeling for studying pH effects, in Olaiz et al.
(2010a) a new gel model composed of a matrix of collagen into which sodium
chloride (main salt present in tissues), NaHCO3 (incorporating, together with CO2,
buffering capacity), and egg yolk (organic matter similar to tissue composition) were
added was presented. The model was called the collagen–macronutrient gel model,
and as previous agar–agar models, it is basically a porous and hydrated elastic gel
structure. In this work, the concept of electrodenaturation front was introduced,
showing that it can be easily followed by a color virage, thus, used for mimicking
tumor tissue destruction by an EA. Since the main objective of EA is tumor tissue
destruction, electrodenaturation front tracking turns out to be an appropriate measure
of its determination. In particular, front tracking reveals that the electrodenaturation
front grows under a diffusion-controlled regime, a result allowing in principle, to
predict the time needed for tumor destruction without compromising healthy tissue.
To show the effects of pH fronts in a different electrode configuration in Olaiz
et al. (2010b), a one-probe two-electrode device (OPTED) containing the cathode
and the anode very close to each other (1 mm) was introduced. Main advantages of
the OPTED are the insertion of one applicator rather than two or more (thus
minimizing tissue intrusion, for instance, in the nervous system), the ability to
reach tumors beyond capabilities of conventional surgery, and the minimization of
electric current circulation through the treated organ. Since the effects of the
exposure of a 3D gel model during OPTED-EA can be assessed by the volume
covered by pH fronts, in the abovementioned work, a pH front tracking by means of
pH indicators’ color change was performed. Results of the OPTED-EA 3D gel
modeling show that starting from an initial uniform condition, two half-spherical pH
fronts, one basic and the other acid (from cathode and anode, respectively), expand
toward the periphery configuring a distorted full sphere. Between electrodes, the two
fronts collide and are neutralized.
A 2D space and time representation of the OPTED-EA experiment showing the
evolution of the acid and basic pH fronts and the measurement of the damage area
are depicted in Fig. 1. The left image in Fig. 1 is a sequence of four snapshots at
different times extracted from a video of 1200-s duration in which the camera is
viewing the experimental process from above. Acid and basic pH fronts are
represented by bright and dark pixels, respectively. The central image named a
594 G. Marshall
Fig. 1 Time (s) sequence of digital snapshots (taken from above) showing the evolution of the acid
and basic pH fronts in a 3D gel model during OPTED-EA with a constant current of 4 mA. Left
image, from top to down: (a) 100 s, (b) 300 s, (c) 700 s, and (d) 1200 s. Center image: reslice from
the sequence of snapshots. Lines joining left and center panes indicate where the left snapshot is
located in the stack. Right image: calculated trajectory of both pH fronts (From Olaiz et al. 2010b)
reslice is constructed from digital gray scales made from a sequence of snapshots
such as those shown in the left image (using imaje from the National Institutes of
Health, NIH) as follows. Snapshots made each second are averaged over 1% of the
cell width (direction parallel to the y-axis) to reduce each image to a line. Then these
lines are stacked to yield the space-time image spanning the duration of the exper-
iment (1200 s). The right image depicts the trajectory of both fronts and is the result
of a border detection algorithm applied to the central image.
Dose planning methodology is critical for a reliable treatment outcome. Since the
late 1970s, Coulomb dose according to tumor size was the guideline for the optimal
choice of electric parameters (Nilsson 2000). Later on, searching for optimal dose
planning, several authors investigated the dose–response relationship between the
applied current, treatment time, Coulomb dose, and necrotized tissue volume (NVT).
Some authors (Nilsson 2000) used different Coulomb doses and found a linear
relationship between damage and Coulomb dose. This result was also experimen-
tally corroborated in Colombo et al. (2007) though it was not checked with a
theoretical model.
In Luján et al. (2015) dose planning in EA was addressed for the first time through
in silico modeling using the Nernst–Planck equations for ion transport validated with
data taken from Olaiz et al. (2010b). Here, the dose planning methodology is
presented in detail. It is recalled that in EA, Coulomb dose and tissue damage are
reliable dose and response parameters, respectively. For dose planning, there is a
need to obtain the dose–response relationship, i.e., the Coulomb dose–tissue dam-
age. The Coulomb dose is obtained for a fixed electric current as a function of the
time elapsed during the EA experiment. Damage is indirectly obtained, tracking the
pH front in space and time during the EA experiment (this front is put into evidence
with the tracking technique previously shown). Clearly, the volume covered by pH
fronts (phenolphthalein and methyl red with transition pH ranges 8.0–9.6 and
4.8–6.2, respectively) determines tissue damage. Here, damage is obtained with
the necrotized tissue volume (NTV) defined as the volume of the tumor necrotized
by EA. With these two parameters, it is possible to construct the NTV vs. Coulomb
dose graph thus experimentally establishing the functional relation between them.
This graph is presented in Fig. 2 (marker lines) showing the experimentally obtained
linear relation between dose and response.
How the dose–response relationship is predicted and their functional relationship
theoretically established? The Coulomb dose is obtained for a fixed electric current
as a function of the time elapsed through the numerical simulation of the experi-
mental EA model previously described. The numerical simulation uses the
Poisson–Nernst–Planck equations describing electric field and ion transport in an
electrolyte with four components; see details in Luján et al. (2015). Damage is
indirectly obtained numerically tracking the pH front in space and time during the
EA simulation. The volume covered by pH fronts determines the predicted damage.
Figure 3 shows predicted acid (blue circle) and basic (red circle) pH fronts trajecto-
ries in space (horizontal) and time (vertical). The light green background corre-
sponds to the gel matrix. Shadowed spheres indicate two ideal tumors whose volume
596 G. Marshall
Fig. 2 NTV experimental 2mA, in silico

4mA, in silico
(marker lines) and predicted 8mA, in silico
(continuous lines) 2mA, in vitro
vs. Coulomb doses for 4mA, in vitro
8mA, in vitro
different electric currents
NTV/(10–3 m)3
(From Luján et al. 2015)
1000
1 10
Q/C
Fig. 3 Predicted acid (blue

area) and basic (red area)
determined by pH fronts
trajectories in space 200
(horizontal axis) and time
(vertical axis downwards).
The light green background 400
corresponds to the gel matrix.
Shadowed spheres indicate
t/s
two ideal tumors 600
800
1000
1200
is covered by the equivalent red blue pH front. With these two parameters, it is
possible to construct the necrotized tissue volume vs. Coulomb dose graph, thus
theoretically establishing the functional relation between them. This is presented in
Fig. 2 (continuous lines) for different electric currents. Results show that predictions
follow experimental trends and there is a nonlinear relationship between NTV and
Coulomb dose, that is, NTV scales as Q 1.4.
The question arises whether this predicted dose–response relationship between
the Coulomb dose and NTV is an optimum for clinical use. The answer requires
some basic assumptions: the ideal generic tumor to be ablated by the physician (plus
a security margin) is contained in a sphere of radius R, thus, a fixed volume size, and
the OPTED electrode is located at the center of the tumor. Therefore, when this
generic tumor is subjected to an OPTED-EA treatment, its volume is gradually

covered in time by a spherical pH front of radius R (I, t) that determines NTV as
illustrated in Fig. 3 (for two tumors in different positions). Clearly, when NTV
reaches the sphere of radius R, the tumor is totally destroyed with a minimum
Coulomb dose.
Electric Field-Based Tumor Therapies: Electroporation-Based

Treatments
While in EA tumor necrosis induced by pH fronts is the main goal of the treatment,
in reversible EP-based treatments such as in ECT, IRE, or GET, it must be
minimized because of its collateral effects. With the aim of illustrating the role of
pH fronts in EP-based tumor treatments, a brief review of experimental and theo-
retical models of EP treatments follows.
The effect of electric pulses in pH changes in a cell suspension was analyzed in
Saulis et al. (2005). Using NaCl solutions buffered with different amount of sodium
phosphate under high-voltage electric pulses and stainless steel anode and different
materials in the cathode, authors found that although variations in the whole volume
of the electrolytic chamber can exceed 1–2 pH units in average, near the cathode this
variation was significantly greater. It was conjectured that the change of pH, in some
cases, might be one of the factors causing cell death.
In Turjanski et al. (2011), the EP process was seen from a new angle apparently
overseen in the literature, the role of pH in ECT modeling based on ion transport
during the treatment. The analysis was developed through in silico modeling vali-
dated with in vitro gel measurements drawing from previous experience of the
authors in electrolytic ablation. For conditions typical to many ECT clinical studies
found in the literature, experimental results, as shown in Fig. 4, unveil the presence
of strong pH fronts emerging from both electrodes. Moreover, theoretical predictions
of the comparison of ECT pH fronts with those arising in EA showed a striking
result: anodic acidification is larger in ECT than in electrolytic ablation, suggesting
that tissue necrosis could also be greater. Since they might give rise to tissue
Fig. 4 Evolution in space

(horizontal axis, anode at the
left, cathode at the right) and
time (vertical axis up–down)
of the anodic acid (pink) and
cathodic basic (red) pH fronts
in an ECT gel model
(8 pulses, 400 V, 300 μs,
1 Hz). Total time length: 8 s
(From Turjanski et al. 2011)
598 G. Marshall
necrosis, an unwanted condition in clinical applications of ECT as well as in GET, it

was deemed relevant to quantify their extent and evolution.
Experimental results from literature disclose that in EP protocols consecutive
pulses increase tissue electric conductivity and the electroporated area, effects not
taken into account in previous theoretical modeling. With the aim of including these
effects, in Suárez et al. (2014), a theoretical model describing the electric field with a
new formulation of the electric conductivity and its validation with measurements in
a vegetable tissue model (potato slice) was presented. The theoretical model solves
the nonlinear Laplace equation with a variable conductivity depending on the
electric field, the temperature and the number of pulses, and the Penne’s bioheat
equation for temperature. Theory predicts and experiments confirm that, increasing
pulse number, the current density and the electroporated area (potato-blackened
area) increase, a fact that can only be explained by an increase in conductivity due
to higher pulse numbers. In the context of this review, the unaccounted conductivity
increase due to consecutive pulses can be described in terms of pH effects as follows.
Pulse number is proportional to Coulomb dose; therefore, at a constant applied
electric field, as pulse number increases Coulomb dose increases, leading to higher
pH effects. Naturally, higher pH effects increase conductivity and therefore electric
current and electroporated area.
Toward more realistic models of EP-based tumor treatments, in Maglietti et al.
(2013), the role of pH fronts in EP-based tumor treatments was experimentally
measured in an ex vivo model. The aim was to show that the pH fronts generated
produce non-negligible pH changes in a tissue regardless of the presence of natural
buffers. Experimental measurements are based on the application of EP for different
sets of pulse parameters (corresponding to IRE, ECT, or GET modalities) to an
ex vivo model (chicken muscular tissue) for conditions found in many studies.
Results show the existence of pH fronts emerging from both electrodes and that
these fronts are immediate and substantial. pH fronts are indirectly measured through
the evanescence time (ET) of a pH indicator. The evanescence time is defined as the
time required for the tissue buffer to neutralize them. Muscular tissue samples were
sliced from fresh chicken. To estimate the hydroxyl production around the cathode
inside the tissue, a pH indicator dye (phenolphthalein, C20H14O4, transition pH
range of 8.0–9.6, from colorless to red) was used. One drop of phenolphthalein was
applied over the surface of the samples, and two electrodes (parallel surgical steel
needles 0.8 mm thick, 2.5 cm long, their surfaces separated from each other by
0.4 cm) were laid horizontally over the region of interest. Only the hydroxyl
production at the cathode was studied as it is equivalent to the proton production
at the anode.
Immediately after the onset of pulse delivery, the pH indicator dye in the sample
changes from colorless to red. After the end of the pulse delivery and as hydroxyls are
neutralized by the tissue buffer, the sample turns to colorless again, indicating a
neutralizing phenomenon. This evanescence time, which is an indirect measure of
the amount of hydroxyls produced, is estimated by the time needed for the tissue buffer
to turn the pH value to less than 8 (nearly neutral). Figure 5 presents a chart of the
evanescence time for each experimental series. Here, standard pulse parameters stand
180,00
ET/s
160,00
140,00
120,00
100,00
80,00
60,00
40,00
20,00
0,00
V
P
T
D
D
D
P
T
T
/2
/2
/2
x2
x3
x2
x2
x3
x3
ST
ST
ST
x1
x1
x1
ET
ET
ET
ET
ET
ET
ET
T
E
ET
ET
ET
EC
IR
G
G
G
G
G
G
Experimental Series
Fig. 5 Chart of the mean evanescence time ET (s) for each experimental series. The standard pulse
parameters for IRE, ECT, and GET are depicted in blue. Pulse parameters corresponding to half,
double, and triple standard GET pulse amplitudes are shown in red. Pulse parameters corresponding
to half, double, and triple standard GET pulse lengths are depicted in green. Pulse parameters
corresponding to half, double, and triple standard GET pulse numbers are shown in orange.
Standard IRE, standard ECT, and all GET parameter variations are significantly different from
standard GET (p<0.01, N= 6). By standard optimal GET parameters are meant four pulses of
80 V/cm, 100 ms at 5 Hz. All experimental series are markedly different from each other (p<0.01,
N = 6), the bar represents mean evanescence time, and error bars show standard deviation (From
Maglietti et al. 2013)
for optimal pulse parameters for IRE, ECT, and GET (IRE: 90 pulses of 1500 V/cm,
100 μs at 1 Hz; for ECT eight pulses of 1000 V/cm, 100 μs at 10 Hz, 1–5000 Hz
(according to literature); and for GET four pulses of 80 V/cm, 100 ms at 5 Hz). These
standard parameters are depicted in Fig. 5 as well as the different experimental series
(more details are presented in Maglietti et al. (2013)). It is observed that pH changes in
GET last longer than in ECT and IRE, ranging from 30 s to about 2 min. Even when
standard GET pulse parameters are considered, plasmid exposure time at these
extreme pH conditions is enough to produce their denaturation. When any of the
pulse parameters (pulse length, pulse amplitude, or a number of pulses) is halved, the
evanescence time is substantially lowered as compared with standard GET. This result
may have relevant implications for treatment efficiency since less exposure time
results in less plasmid damage and less cell loss. Also, there is a significant increase
in the evanescence time when doubling or tripling pulse amplitude, pulse length, or the
number of pulses, as compared with standard GET.
Higher Coulomb doses correlate with stronger pH changes and thus with longer
evanescence times. This could contribute to less tissue sparing when treating healthy
margins in ECT and less plasmid expression in GET. These results suggest that the
effect of pulse parameter variation determining the Coulomb dose and thus pH fronts
must be taken into account to improve GET efficiency and make more accurate
treatment planning and design models that predict the outcome of different GET
protocols better.
600 G. Marshall
Fig. 6 Graph of the evanescence time vs. Coulomb dose, for GET protocols. Squares represent the
evanescence time for standard GET parameters; circles represent mean evanescence time for a half,
double, and triple pulse amplitudes, respectively. Triangles represent mean evanescence time for a
half, double, and triple pulse lengths, respectively. Rhombuses represent mean evanescence time for
a half, the double, and a triple number of pulses. Error bars show standard error (From Maglietti
et al. 2013)
Figure 6 presents a log–log scale of the evanescence time as a function of the

applied Coulomb dose corresponding to GET protocols. A linear regression analysis
shows that evanescence time scales almost linearly with the Coulomb dose. This
may be relevant in clinical applications, since knowing the Coulomb dose allows
estimation of the evanescence time value and thus a priori knowledge of whether pH
effects are admissible.
Figure 5 also shows that the evanescence time induced by standard ECT and IRE
lasts a very short time, usually less than 5 s, though enough to contribute to tissue
necrosis near the electrodes. This might be irrelevant in IRE (all tissue near
electrodes is intended to be ablated), but not in ECT where the treatment must be
as specific to tumor cells as possible. As an example, Fig. 7 shows minor tissue
injuries observed 7 days after a standard ECT procedure (eight pulses, 1000 V/cm,
100 us, 1 Hz) was applied to a canine patient (an 8-year-old male Doberman) with a
squamous cell carcinoma located in the anterior part of the mouth. The area depicted
in the figure corresponds to healthy tissue margins treated to prevent tumor recur-
rence (security margins). Tissue injuries are evidenced as light red halos surrounding
central red spots in the mucosa (corresponding to points where electrodes were
placed). These injuries are mainly due to extreme pH changes induced by the
Coulomb dose applied.
Fig. 7 Minor tissue injuries,

seen as red halos around
central red spots in the mucosa
(encircled in yellow) of a
canine patient mouth,
observed 7 days after ECT
treatment. The dog owner’s
hand is shown in the picture
(From Maglietti et al. 2013)
While in EP-based tumor treatments such as ECT or IRE, the ET is very short
(though enough to cause minor injuries) due to electric pulse characteristics and
biological buffers present in the tissue, in GET, ET is much longer, enough to
denaturalize plasmids and produce cell damage. When any of the electric pulse
parameters is doubled or tripled, the ET grows, and, remarkably, when any of the
pulse parameters in GET is halved, the ET drops significantly. Reducing pH fronts
has relevant implications for GET treatment efficiency, due to a substantial reduc-
tion of plasmid damage and cell loss.
One step forward toward more realistic EP-based protocol models was presented
in Olaiz et al. (2014) studying the role of pH in tissue damage in a hyaluronidase +
GET protocol application through in vivo and in silico modeling. Since GET is also
associated with some tissue damage that must be minimized, optimal GET requires a
compromise between maximum electroporated area (assuming uptake proportional
to electroporated area) and minimal tissue damage. In McMahon et al. (2001), it was
shown that GET in skeletal muscle can be improved by pretreatment with hyal-
uronidase. Even if the use of pretreatment by hyaluronidase reduces muscle fiber
damage, traces of necrosis still remain visible in the tissue with a concomitant loss of
muscle fibers and a reduction in transgenic gene expression. As previously discussed
in Maglietti et al. (2013), damage and loss of efficiency may be strongly dependent
on pH alterations induced by electrolysis. Accordingly, in Olaiz et al. (2014), these
effects were addressed through in vivo modeling using a transparent chamber
implanted into the dorsal skinfold of a mouse and intravital microscopy (a visual
technique that enables imaging of biological processes in live animals) and in silico
modeling using the Poisson–Nernst–Planck equations for ion transport. Experimen-
tal results, as vividly shown in Fig. 8, unveil the presence of pH fronts and that these
fronts are immediate and substantial thus giving rise to tissue damage. Theory
confirms these experiments and shows that in GET protocols whether with or
without hyaluronidase pretreatment, pH fronts are the principal cause of muscle
damage near the electrodes.
In the following, using data from (Olaiz et al. 2014) the methodology
for obtaining a GET dose–response functional relationship is explained in detail.
602 G. Marshall
Fig. 8 Anodic and cathodic pH fronts induced by GET in mice observed by intravital microscopy
through a dorsal skinfold chamber: anodic acid front (left electrode) marked with phenol (red, light
yellow area) and cathodic basic front (right electrode) marked with phenolphthalein (pink–red area)
(From Olaiz et al. 2014)
In GET, as in EA dose planning, there is a need to establish reliable dose and

response parameters. While in GET, the Coulomb dose and damage area (unwanted
damage) continue being reliable dose and response parameters, respectively (here
the Coulomb dose is defined as the product of electric pulse intensity, pulse duration,
and pulse number), there is an additional response parameter that must be consid-
ered, the electroporated area (here it is assumed that uptake is proportional to
electroporated area). Hence, two dose–response relationships emerge in GET:
Coulomb dose-damage and Coulomb dose-electroporated area, respectively, com-
peting among themselves. Since the main goal of GET is to maximize electroporated
area and minimize tissue damage, an optimal GET protocol would be the Coulomb
dose that yields maximum electroporated area while minimizing tissue damage.
How these dose–response relationships are predicted and their functional rela-
tionship established? The Coulomb dose is obtained, for a given pulse number, as a
function of the time elapsed, through a numerical simulation with data from Olaiz
et al. (2014). The numerical simulation uses the Poisson–Nernst–Planck equations
describing electric field and ion transport in a four-component electrolyte (proton,
hydroxide, chloride, and sodium). By computing the pH distribution in a given
region, the damaged area in that region is obtained, thus the healthy area (assuming
that the healthy area is the complement of the damaged area). The damage area is
defined as the area in which pH values are above 8.5 or below 5.5. The healthy area
falls within the interval 5.5 and 8.5. The electroporated area is obtained neglecting
ion transport, assuming ohmic behavior and solving the nonlinear Laplace equation
governing the electric field in the standard way, details in Olaiz et al. (2014).
With these three parameters, it is possible to construct the Coulomb dose
vs. damaged area and the Coulomb dose vs. electroporated area, respectively,
thus theoretically establishing the functional relation among them. Figure 9 shows
the normalized healthy tissue (NHTA) (gray line) and normalized electroporated area
(NEPA) (black line), respectively, vs. Coulomb dose. It is clear that while electro-
porated area increases with Coulomb dose, healthy tissue area decreases. In other
words, as expected, damage area increases with Coulomb dose.
Fig. 9 Normalized healthy 1

tissue (NHTA) (continuous
gray line), normalized healthy
tissue with buffer (dotted gray 0.8
line), and normalized
NEPA and NHTA

electroporated area
(continuous black line), 0.6
respectively,
vs. Coulomb dose
0.4
0.2
0
0 1 2 3 4 5 6
Q/mC
The next question is how to determine an optimal GET. A quick answer is shown
in Fig. 9 where the point of intersection of the two continuous curves determines the
optimal GET protocol. It shows the two competing dose–response relationships in a
GET protocol. In other words, an optimal efficiency in GET protocols, that is, a
compromise between obtaining maximum electroporated area and minimal tissue
damage, is given by the critical Coulomb dose determined by the point of intersec-
tion of both curves.
Though previous theoretical models of ion transport under EP-based protocols
advanced our knowledge of electrolytic effects in tissue damage, the presence of
tissue natural buffering was not taken into consideration raising the question of
whether natural buffer attenuation would significantly diminish tissue damage due to
pH fronts. Natural buffering modeling was firstly treated in a series of papers in EA
protocols (Nilsson 2000); in the first EA model, the tissue was treated with an
aqueous solution of sodium chloride; in the second model, a bicarbonate buffer
system was taken into account; and in the third model, the transport of chlorine and
chlorinated species was added.
The open question of whether natural buffer attenuation would significantly
diminish tissue damage due to pH fronts in GET was theoretically elucidated in
Marino et al. (2014) who presented an in silico modeling of tissue damage in a GET
taking into account the presence of tissue natural buffering. The model is based on
previous works of the authors in EA and EP and in the works of Nilsson (2000) in
EA. Ion transport, assuming a tissue with a bicarbonate buffering system, is
described by the Nernst–Planck equations in a seven-component electrolyte (H+,
OH , Cl , Na+, HCO3, CO2, and CO3) (details in Marino et al. (2014)); the model is
validated with experimental data from Olaiz et al. (2014). Results shown in Fig. 9
depicting the normalized healthy tissue with buffer (dotted gray line) vs. Coulomb
dose predict that in GET protocols, regardless of the presence of tissue natural buffer
604 G. Marshall
attenuation, tissue damage remains substantial and pH fronts are the principal cause
of tissue damage near the electrodes.
Obtaining an optimal GET follows the same line of reasoning as in the previous
GET modeling without buffer attenuation. The answer is shown in Fig. 9 where the
point of intersection of the healthy tissue with buffer (dotted gray line) with the
electroporated area (continuous black line) determines the optimal GET protocol.
Comparing the intersection points determined by the models with and without
buffering, respectively, shows that the optimal GET model with buffering is shifted
upward and to the right relative to the model without buffering. In other words, for an
optimal GET, the Coulomb dose is larger when buffering is present.
Conclusions
The role played by the mass transfer of electrolytic species in the dose–response
relationship in an electric field-based tumor treatments has been elucidated, partic-
ularly the role of the Coulomb dose due to the appearance of pH fronts. To have a
thorough understanding of the effects of pH fronts in EA tissue damage, numerous
in vivo, in vitro, and in silico models of electrolytic ablation, whose aim is to
eliminate tumors mainly by necrosis, were presented. In these models, the immediate
appearance of strong pH fronts emerging from the electrodes was demonstrated.
These fronts are the principal cause of tissue damage in electrolytic ablation. A
detailed exposition of the methodology for obtaining the EA dose–response rela-
tionship through the establishment of reliable dose–response parameters and pH
front tracking was presented. The Coulomb dose and the damaged area are shown to
be reliable dose and response parameter, respectively. In EA, an optimal
dose–response relationship is the minimum Coulomb dose necessary to achieve
total tumor destruction while minimizing healthy tissue damage.
Using the electrochemical analogy between EA- and EP-based tumor treatments,
it was possible to extend EA methodology to EP-based tumor treatments. Numerous
in vivo, in vitro, and in silico models of EP-based tumor treatments, whose aim is to
maximize electroporated area and minimize tissue damage, were presented. Again,
in these models the immediate appearance of strong pH fronts emerging from the
electrodes was demonstrated. Thus, to a greater or lesser extent, EP-based tumor
treatments are affected by the underlying electrolytic process, a process that is shown
to be a function of the applied Coulomb dose. In an EP-based treatment such as in
GET, unwanted tissue damage due to electrolysis is non-negligible. Here, the
Coulomb dose and electroporated and damage areas (unwanted damage) are
shown to be reliable dose and response parameters, respectively; thus, two
dose–response relationships emerge Coulomb dose-damage and Coulomb dose-
electroporated area, respectively, competing among them. Since the main goal of
GET is to maximize electroporated area and minimize tissue damage, it was shown
that an optimal GET is predicted as the critical Coulomb dose yielding maximum
electroporated area with minimum damage. When tissue natural buffer is taken into
account, damage is attenuated (though still remaining non-negligible), and the
critical Coulomb dose for optimal GET increases. Optimal GET protocol hopefully
increases treatment efficiency, leading to a substantial reduction of plasmid damage
and cell loss.
Cross-References
▶ Combining Electrolysis and Electroporation for Tissue Ablation

▶ Preclinical Studies on Nanosecond Pulses
▶ Tissue Ablation Using Nanosecond Electric Pulses
References
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pone.0017303
Effects of Reversible and Irreversible
Electroporation on Endothelial Cells 35
and Tissue Blood Flow
Bostjan Markelc, Maja Čemažar, and Gregor Serša
Abstract
Electroporation/electropermeabilization (EP), achieved by application of electric
pulses to cells or tissues, induces reversible permeabilization of cell membranes
under suitable conditions, thus facilitating entry of exogenous molecules into
cells. If excess pulses are applied, and the cell cannot recover, this leads to
irreversible electroporation (IRE). EP and IRE of tissues in humans are feasible,
efficient, and tolerable, and its most advanced routine clinical uses are electroche-
motherapy (ECT), where cytotoxic drugs are delivered to cells to treat tumors and
nonthermal ablation of tumors with IRE. Also, reversible EP is gaining momen-
tum as an efficient method for gene electrotransfer and DNA vaccination. The use
of IRE, where the application of electric pulses results in the creation of a tissue
lesion without a dependence on thermal processes or the requirement of adjuvant
drugs, is currently intensively researched for treatment of various types of cancer.
When EP, either reversible or irreversible, is applied to tissues in vivo or in
patients, it also has tissue-specific effects. It was shown that EP affects the
vasculature in tumors and in normal tissues such as muscle and skin. Application
of reversible EP pulses increases the permeability of affected blood vessels,
causes a transient vascular lock, i.e., decrease in perfusion, and modulates the
B. Markelc
ÜRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of
Oxford, Oxford, UK
e-mail: bostjan.markelc@oncology.ox.ac.uk
M. Čemažar
e-mail: mcemazar@onko-i.si; maja.cemazar@fvz.upr.si
G. Serša (*)
e-mail: gsersa@onko-i.si

DOI 10.1007/978-3-319-32886-7_70
608 B. Markelc et al.
diameter of affected blood vessels. Similarly, IRE increases the permeability of

the affected blood vessels. Both IRE and reversible EP preserve the normal
function of affected blood vessels in the long term which is important when
they are used in well-perfused organs such as the liver or brain.
Keywords
Vascular lock • Vasoconstriction • Permeability • Electroporation • Normal blood
vessels
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608
Effects of EP on Endothelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609
Vascular Effects of Reversible EP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
Vascular Effects of IRE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 618
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 618
Introduction
Electroporation/electropermeabilization (EP), achieved/caused by application of

electric pulses to cells or tissues, induces reversible permeabilization of cell mem-
branes under suitable conditions, thus facilitating entry into the cells of
non-permeant or poorly permeant molecules (Rems and Miklavcic 2016). The
delivered pulses generate an electric field between the electrodes capable of inducing
a rapid buildup of charge across the plasma membrane of cells, commonly referred to
as a transmembrane voltage (TMV). Once the TMV reaches a critical value, it is
thought that electrically conductive pores form in the membrane in an attempt to
prevent permanent damage by shunting current and limiting further TMV rise (Rems
and Miklavcic 2016). If the pulse amplitude and duration are tuned to permit pore
resealing, and cell viability is maintained following exposure, the process is catego-
rized as reversible electroporation. Biological responses and associated techniques
have a broad spectrum of biomedical applications, such as gene (gene
electrotransfer) and cytotoxic drug delivery (Yarmush et al. 2014). Reversible EP
of tissues in humans is feasible, efficient, and tolerable, and its most advanced
routine clinical use is electrochemotherapy (ECT) (Yarmush et al. 2014). If excess
current is applied, and the extent of pore formation is such that the cell cannot
recover, this leads to irreversible electroporation (IRE). Macroscopically, this results
in the creation of a tissue lesion without a dependence on thermal processes or the
requirement of adjuvant drugs (Arena et al. 2011). The use of IRE for the treatment
of different cancer types is currently intensively researched, and several clinical trials
are being conducted to show its safety and efficacy (Wagstaff et al. 2016).
In the clinical setting, besides efficacy of treatment, the safety of the technique
and its effects on the exposed tissue are of considerable importance, especially when
35 Effects of Reversible and Irreversible Electroporation on Endothelial. . . 609
the healthy tissue is also affected. In the case of gene electrotransfer, where plasmid
DNA encoding a therapeutic gene, or DNA vaccination, where plasmid DNA
encoding the desired antigen, is introduced into the cells, the target tissue is most
commonly either the skin or muscle (Gothelf and Gehl 2012; Sällberg et al. 2015).
EP can also be used for transdermal delivery of molecules, where EP is exploited to
enable systemic access of small molecules without physically breaking the dermis, in
which case the skin is again the target tissue (Zorec et al. 2013). The majority of ECT
in clinical settings is nowadays performed on cutaneous and subcutaneous tumors,
where, although the target is the tumor, the surrounding skin is also exposed to the
electric field (Yarmush et al. 2014). Recently, ECT has also been used in the
treatment of visceral and deep-seated tumors such as colorectal cancer and colorectal
liver metastases as well as in the treatment of brain metastases and bone metastases
(Miklavcic et al. 2014). On the other hand, IRE is predominantly used in the
treatment of internal tumors such as liver, pancreatic, kidney, and prostate tumors
(Wagstaff et al. 2016).
For a tissue to retain its normal functionality, it is essential to have a constant and
unperturbed blood flow which supplies the tissue with nutrients and oxygen. Thus,
especially when treating well-perfused organs, such as the liver and brain, the effects
of EP on blood flow are of considerable importance. The effects of EP on blood flow
have been first observed in tumors (Sersa et al. 1998) and later confirmed in various
other tissues. In this chapter, the blood flow modifying effects of EP as well as the
effects of EP on endothelial cells in vitro will be addressed.
Effects of EP on Endothelial Cells
Although EP is a physical method and the response of exposed cells is similar

between different cell types even in cell assemblies that try to mimic the complexity
of a tissue (Rems and Miklavcic 2016), there are some effects of EP that can only be
observed on a tissue level. After the first observations of blood flow modifying
effects of EP in tumors, liver, and muscle (Sersa et al. 1998; Ramirez et al. 1998;
Gehl et al. 2002), a mathematical model of electric field distribution in and around
blood vessels revealed not only that the endothelial cells lining the blood vessels are
electroporated as the rest of the cells but are actually exposed even to up to 40%
higher electric field than the surrounding tumor cells (Sersa et al. 2008).
The first study to focus on in vitro effects of EP on endothelial cells used human
dermal microvascular endothelial cells (HMEC-1) and measured their electroper-
meabilization (uptake of propidium iodide) and electro-sensitivity (survival of cells)
after exposure to field intensities between 0 and 1,800 V/cm. To achieve electropo-
ration, the cells were placed between two parallel stainless steel electrodes (2 mm
distance between electrodes) connected to a Jouan GHT 1287 electroporator and
subjected to 8 square-wave electric pulses (100 μs, 1 Hz). EP of HMEC-1, at an
electric field of 1,000 V/cm, was found to induce 30% of cells to be permeabilized,
with ~85% cell survival, and at the highest amplitude of 1,800 V/cm, only 85% of
cells were permeabilized with 57% of surviving cells (Cemazar et al. 2001). This
indicated a relatively high cellular resistance of HMEC-1 to EP.
The effects of EP on endothelial cells in vitro were then more thoroughly
investigated using human umbilical vein endothelial cells (HUVECs) (Kanthou
et al. 2006) as well as using HMEC-1 (Meulenberg et al. 2012). In order to study
the permeability and integrity of endothelial monolayers, HUVEC or HMEC-1 were
grown on a 0.4 μm pore size polycarbonate transwell inserts coated with human
fibronectin. Once a confluent monolayer was formed, HUVECs were exposed to
3 square-wave electric pulses (10–80 V/voltage-to-distance ratio 25–200 V/cm;
1 Hz; duration 100 μs; 4 mm distance between electrodes) (Kanthou et al. 2006),
whereas HMEC-1 were exposed to 8 square-wave electric pulses (0–500 V/voltage-
to-distance ratio 0–685 V/cm; 1 Hz; duration 100 μs; 7.3 mm gap between elec-
trodes). The difference in the low electric fields required for electropermeabilization
of HUVECs compared to the other studies was attributed to the specific geometry of
the experimental system that was used by the authors (Kanthou et al. 2006). In both
cell lines, EP increased the permeability of endothelial monolayers, and the increase
in the permeability was dependent on the electric field strength (Kanthou et al. 2006;
Meulenberg et al. 2012). The integrity of endothelial monolayer is controlled by cell-
to-cell junctions. Adherens junctions are a specialized type of cell-to-cell junction
found in endothelial cells and contribute toward the maintenance of the barrier
function, and one of the main proteins in these junctions is VE-cadherin (Dejana
2004). In HUVECs exposed to 3 square-wave electric pulses (10–60 V/voltage-to-
distance ratio 25–150 V/cm; 1 Hz; duration 100 μs; 4 mm distance between
electrodes), loss of VE-cadherin was evident from the junctions between adjacent
endothelial cells immediately after electroporation. On a structural level, HUVECs
responded to electroporation by dissolution of actin fibers and microtubules, and
these effects were evident immediately (within 5 min) after electroporation. Both
actin fibers and microtubules were progressively dissociated in response to increas-
ing voltage applications. At the cell periphery, diffuse actin staining assumed a
honeycomb-like appearance, with fine processes extending from the cell body.
However, unlike microtubules and actin filaments, vimentin intermediate filaments
remained relatively resistant to EP, although some damage at peripheral areas was
evident (Kanthou et al. 2006). Similar observations were made with HMEC-1 that
were grown on chamber slides and exposed to 8 square-wave electric pulses
(0–500 V/voltage-to-distance ratio 0–685 V/cm; 1 Hz; duration 100 μs; 7.3 mm
gap between electrodes). Here, only actin filaments were studied (Meulenberg et al.
2012). When parameters for reversible EP were used, the observed changes were
transient in both cell lines and the cytoskeletal networks recovered to pretreatment
state within 1–2 h after EP (Kanthou et al. 2006; Meulenberg et al. 2012). In
HUVEC line a burst in myosin light chain phosphorylation activity, most evident
at 30 and 60 min after EP, correlated with active cytoskeletal remodeling and the
reestablishment of contractile actin filamentous structures. Total levels of actin,
h-tubulin, and vimentin proteins were not downregulated by EP for up to 16 h
after treatment (Kanthou et al. 2006). Additionally, morphological investigation of
HMEC-1 revealed that at electric field intensities above 274 V/cm, large
polykaryonic cells with nuclei clustered centrally were present, demonstrating cell
fusion that contrary to the effects observed on cytoskeleton did not resolve within
24 h after EP (Meulenberg et al. 2012).
Altogether, these data suggest that electroporation can permeabilize endothelial
cells; however, they seem to be less sensitive to EP compared to other cell types. EP
immediately disrupts the organization of actin or tubulin monomers into three-
dimensional filamentous structures but does not result in any degradation of the
corresponding monomeric proteins. Moreover, EP disrupts the barrier function of the
endothelium by interacting with the cytoskeletal organization and junctional integ-
rity of endothelial cell-to-cell junctions.
Vascular Effects of Reversible EP
When the use of EP moved from in vitro to in vivo setting, it became evident that,
besides the expected increased uptake of therapeutic molecules by the cells or
increased expression of a transgene, when gene electrotransfer was performed, EP
also has a tissue-specific effect. Early reports of blood flow modifying effects of EP
were published in studies of ECT on tumors in mice and rabbits (Sersa et al. 1998;
Ramirez et al. 1998); however, the authors did not explore the effects of EP on
normal blood vessels.
The first in-depth study conducted on normal blood vessels and blood flow
investigated changes to permeabilization of cells, membrane resealing, and effects
on perfusion after EP in a mouse muscle tissue (Gehl et al. 2002). Three different
electrode geometries were compared. Needle electrodes consisting of two arrays of
each four 0.5 mm needles placed 2 mm apart, with 4 mm between the arrays and two
different plate electrodes, both consisting of two opposing metal plates, separated by
4 or 5.3 mm. In all experiments, eight pulses with a frequency of 1 Hz were
delivered. Pulse duration was varied from 10 to 20,000 μs and applied voltage-to-
electrode distance ratio from 100 to 1,600 V/cm. More than 100 different pulse
combinations were tested separately on the hind- and forelimb. Application of
electric pulses decreased the uptake of 51Cr-EDTA in muscle tissue when it was
injected intravenously (i.v.) after EP in comparison to the uptake of 51Cr-EDTA
when it was injected before EP. When 51Cr-EDTA was injected intramuscularly,
there was no decrease in uptake when the tracer was added after EP – on the contrary,
uptake was larger than before EP (Gehl et al. 2002). Depending on the time of
i.v. injection after EP, the decrease of 51Cr-EDTA uptake into muscle cells varied,
with the lowest uptake when 51Cr-EDTA was injected immediately after EP. At later
time points of injection, the uptake of 51Cr-EDTA was similar to the uptake when it
was injected before EP. Therefore, showing that when molecules are delivered to
tissue by blood vessels, EP transiently obstructs their delivery, indicating a blood
flow modifying effect of EP. The observed blood flow modifying effects were further
explored by investigating perfusion in mouse hindlimb as a function of electric pulse
applications using the dye patent violet blue. Plate electrodes were placed over the
tibia, and electric pulses were delivered followed by immediately i.v. injecting patent
Fig. 1 Perfusion after delivery of electric pulses. Plate electrodes were placed over the tibia and
electric pulses were delivered. Immediately thereafter patent violet blue was injected i.v. The
perfusion delay in the pulsed extremity was defined as the time between the paw of the unpulsed
extremity turned blue and the time the paw of the pulsed extremity turned blue (Reproduced with
permission from Gehl et al. (2002))
violet blue. The perfusion delay in the pulsed extremity was defined as the time
between the paw of the unpulsed extremity turned blue and the time the paw of the
pulsed extremity turned blue (Fig. 1).
The results showed a biphasic nature of the perfusion delays that could be seen
with a first threshold leading to a plateau of short perfusion delays followed by a
second threshold to a phase of rapidly progressing perfusion delays up to 30 min
after EP (Gehl et al. 2002). Using voltage-to-distance ratio 1,200 V/cm and 100 μs
pulses delivered with plate electrodes to the hindlimb and injecting the dye at various
times after EP, the perfusion delays were determined to be maximal at 1–3 min post-
EP, with subsequent resolution resembling the kinetics of membrane resealing. The
onset of the progressive phase of perfusion delays coincided with irreversible
permeabilization becoming a dominant phenomenon (Gehl et al. 2002). An inter-
esting outcome of this experiment was that sometimes the paw turning blue had a
demarcation line between one-half of the paw changing color and the other half
remaining uncolored. Moreover, the resolution of the shorter perfusion delays was
very fast and occasionally followed by hyperemia (Gehl et al. 2002). Therefore, the
role of the sympathetic nervous system in the response of blood vessels to EP was
investigated. Mice were pretreated with reserpine, which depletes nerve terminals of
norepinephrine and is known to lower blood pressure and heart rate and at the same
time diminish regional vasoconstriction mediated by sympathetic fibers acting on
smooth muscles. In limbs exposed to EP, reserpine increased blood flow compared
with pulsed limbs of controls, which indicated that local vasoconstriction mediated
by the sympathetic nervous system plays a major role in the observed perfusion
delays (Gehl et al. 2002).
Taken altogether, this data confirmed that EP has blood flow modifying effects on
normal blood vesselsin muscle tissue. A model for the vascular effects of EP was
proposed where the first, short perfusion delays were attributed to the permeabi-
lization of muscle fibers and/or endothelial cells induced a Raynaud-like phenome-
non, a reflexory vasoconstriction of afferent arterioles mediated by the sympathetic
nervous system. The resolution of this initial perfusion impairment depends on the
release of the vasoconstriction. In this model the subsequent longer lasting perfusion
delays were attributed to the leakage of fluids and molecules into the extracellular
volume leading to increased interstitial pressure and decreased intravascular pres-
sure. The resolution of this second phase is supposed to follow the kinetics of
membrane resealing after EP (Gehl et al. 2002). Considering drug delivery to the
electroporated tissue, when the drugs are injected i.v., the direct relevance of the
observed effects is that during the first phase immediately after EP, when the
membranes are maximally permeabilized, there is a hypoperfusion of the exposed
tissue, and therefore drug delivery to – and washout from – the EP area is minimal.
This phenomenon was termed “the vascular lock” (Gehl et al. 2002).
To provide a direct visual confirmation of the suggested blood flow modifying
effects of EP, a dorsal window chamber model in mice together with fluorescence
intravital microscopy was used. Dorsal window chamber (DWC) or dorsal skinfold
chamber is an experimental technique performed in living animals, which enables a
direct visual access to the blood vessel network of the skin by intravital microscopy.
For this purpose, two titanium frames are secured to the double layers of the dorsal
skinfold in a mouse with stainless steel screws and sutures, and thereafter one layer
of the skin with the fat and the connective tissues is excised, exposing the vascula-
ture of the remaining skin layer. If combined with fluorescently labeled vascular
markers, this approach enables quantification of changes in blood flow and perme-
ability of blood vessels. When different sizes of dextrans are used, this approach can
also simulate the effect of EP on permeability and blood flow for differently sized
molecules that are used in conjunction with EP.
The effect of the standard ECT parameters on permeability and blood flow was
studied with three different sizes of molecules: 20 , 70 , and 2,000 kDa dextrans.
Electric pulses were delivered through two parallel stainless steel rods (length 5 mm,
diameter 1.3 mm) 4 mm apart (8 square-wave electric pulses, 100 μs, voltage-to-
distance ratio 1,300 V/cm, 1 Hz). The application of electric pulses led to immediate
constriction of all affected blood vessels. The decrease in diameter of venules was
approx. 20%, and it was restored to pre-EP values within 3 min after EP. Constriction
of arterioles was more pronounced (approx. 65%) and mainly restored within 3 min
after EP. However, the total restoration of their diameter was obtained after 8 min
post-EP (Bellard et al. 2012). A similar constriction of all affected blood vessels was
observed when pulses used for gene electrotransfer to the skin and muscle, where a
combination of high-voltage (HV) and low-voltage (LV) electric pulses is used, were
delivered through two parallel stainless steel rods (length 5 mm, diameter 1.3 mm)
4 mm apart (one pulse (voltage-to-distance ratio 1,000 V/cm, 100 μs) followed by a
1 s lag and eight pulses (voltage-to-distance ratio 140 V/cm, 50 ms, 1 Hz)) (Markelc
et al. 2012). The only difference in the observed response was that when HV-LV
pulses were used, the time needed for the diameters of blood vessels to return to
pre-EP values was longer than when ECT pulses were used (Bellard et al. 2012;
Markelc et al. 2012). To determine whether the observed decrease of diameters of the
affected blood vessels is connected to the previously reported “vascular lock effect”
of EP, FITC-labeled dextrans were injected i.v. at various times after EP. Regardless
of the used electric pulse parameters or the size of the i.v. injected FITC-labeled
dextrans, a transient vascular lock was observed that lasted up to 5 min after EP
(Fig. 2); however, even up to 30 min after EP, the filling up of blood vessels was
slightly impaired compared to control mice (Bellard et al. 2012; Markelc et al. 2012).
The use of DWC and time-lapse fluorescence microscopy allowed to quantify the
changes in permeability of normal blood vessels after EP. In order to measure the
Fig. 2 Illustration of the EP-induced vascular lock. Electric pulses were delivered through two
parallel stainless steel rods (length 5 mm, width 1.3 mm) 4 mm apart (8 square-wave electric pulses,
100 μs, voltage-to-distance ratio 1,300 V/cm, 1 Hz). 20, 70, and 2,000 kDa FITC-labeled dextrans
were injected 20 s after EP. DWCs were observed by fluorescence microscopy, and images were
acquired immediately after (0 min), 2, 4, and 6 min after injection of FITC-labeled dextrans
(Reproduced with permission from Bellard et al. (2012)
leakage of FITC-labeled dextrans from blood vessels into the surrounding tissue
after EP, the FITC-labeled dextrans were injected i.v. 3 min before the application of
electric pulses and the relative variation of fluorescence intensity was determined in
the tissue surrounding blood vessels. When HV-LV pulse parameters were used in
conjunction with 2,000 kDa FITC-labeled dextran, the increase in relative variation
of fluorescence intensity was linearly dependent on time during the first 8 min after
EP, after which it reached a plateau. However, there was a detectable increase in
relative variation of fluorescence intensity even 30 min after EP (Markelc et al.
2012). This indicates that HV-LV pulse parameters increase the permeability of
blood vessels even for molecules as large as 2,000 kDa, which is roughly the size
of plasmid DNA. A more in-depth investigation of the increase in permeability of
blood vessels after EP was performed with ECT pulse parameters, where three
different sizes of FITC-labeled dextrans were used to explore whether the increase
in permeability varies for molecules with different sizes (Bellard et al. 2012). For all
three different sizes of used dextrans (20, 70, and 2,000 kDa), a high increase in
permeability of blood vessels was determined which was readily visible in the
acquired images (Fig. 3). In the case of 70 kDa and 20 kDa, FITC-labeled dextran,
the leakage, as quantified by the increase in relative variation in mean fluorescence
intensity, was detected immediately after EP. In contrast, EP did not induce an
immediate leakage of 2,000 kDa FITC-labeled dextran. However, after a delay
(4 min), a similar increase in relative variation in mean fluorescence intensity to
the 20 kDa, FITC-labeled dextrans was determined. This indicates that the perme-
ability of blood vessels is gradually increased after EP and that it has different
kinetics for different sizes of molecules (Bellard et al. 2012). Regardless of the
used pulse parameters, HV-LV or ECT, the increase in permeability of blood vessels
after EP was transient, and the pre-EP state where blood vessels are impermeable for
large macromolecules was restored.
Fig. 3 Direct imaging of fluorescence leakage from the DWC vessels into tissues after EP. 70 kDa
FITC-labeled dextran was injected i.v., and blood vessels were visualized by fluorescence micros-
copy at 25 magnification at the indicated times. Upper panel – no EP applied. Lower panel –
electric pulses were delivered through two parallel stainless steel rods (length 5 mm, diameter
1.3 mm) 4 mm apart (8 square-wave electric pulses, 100 μs, voltage-to-distance ratio 1,300 V/cm,
1 Hz) (Reproduced with permission from Bellard et al. (2012)
The same approach, intravital fluorescence microscopy imaging of a mouse skin

in a DWC, was combined with mathematical modeling to quantify the
electroporation-facilitated increase in microvascular permeability for both 70 kDa
and 2,000 kDa sizes of dextrans (Corovic et al. 2015). As previously, ECT pulse
parameters were used, and the electric pulses were delivered through two parallel
stainless steel rods (length 5 mm, diameter 1.3 mm) 4 mm apart (8 square-wave
electric pulses, 100 μs, voltage-to-distance ratio 1,300 V/cm, 1 Hz). FITC-labeled
dextrans were intravenously injected 12 min before EP, and images were acquired
before and after EP. Fitting the parameters of the constructed mathematical model to
the experimental data confirmed a higher microvascular permeability after EP for
smaller sizes of dextrans compared to the larger ones.
When EP is used in healthy tissue, an important concern is whether there are any
damaging effects to the tissue itself. A histological analysis of changes to the skin
after EP (two parallel stainless steel rods (length 5 mm, diameter 1.3 mm) 4 mm apart
(1 pulse (voltage-to-distance ratio 1,000 V/cm, 100 μs) followed by a 1 s lag and
eight pulses (voltage-to-distance ratio 140 V/cm, 50 ms, 1 Hz)) was performed 1, 12,
24, and 48 h after EP. The most prominent features that were observed were
(i) rounding up of vessel endothelial cells and (ii) narrowing of vessel lumen and
stacking of erythrocytes that were present only at 1 h after EP. Between 1 and 12 h
after EP, the extravasation of erythrocytes, increased presence and marginalization of
leukocytes, and increased infiltration of leukocytes into the tissue between the
electrodes were observed. Recovery of the epidermis and underlying tissue under
the electrodes was evident at 24 and 48 h after EP (Markelc et al. 2012).
In summary, reversible EP affects normal blood vessels in several ways. Firstly,
immediately after EP, there is a decrease in diameters of all affected vessels which is
accompanied by a “vascular lock.” This first phase is short lived and it is resolved
within 5 min after EP. Secondly, EP increases the permeability of blood vessels for
macromolecules of various sizes, and this increase in permeability can persist up to
30 min after EP, although it is primarily resolved within 10 min after EP. Lastly, on a
histological level, reversible EP does not have long-term effects on normal blood
vessels, as all observed changes were resolved within 48 h after EP.
Vascular Effects of IRE
Although reversible EP and irreversible EP (IRE) use the same physical phenome-
non, i.e., increase in permeability of cells exposed to electric pulses, their biological
mode of action is different. In the case of IRE, the pulsed electric fields are applied to
cells in order to cause irreversible damage to cells by affecting the cell membrane. In
contrast, with reversible EP the applied pulsed electric fields are meant to only
transiently increase the permeability of the cellular membrane, thus allowing the cell
to repair the damage and survive (Yarmush et al. 2014). When applied to tissue, the
goal of IRE is to destroy the cells while sparing the tissue scaffold, large blood
vessels, and other tissue structures which are important for retaining the functionality
of the treated organ in the long term (Yarmush et al. 2014).
The effects of IRE on vasculature were confirmed with magnetic resonance

imaging (MRI) of perfusion in rat brain after EP (Hjouj et al. 2012). The electric
pulses were delivered through an intracranial electrode implanted in the rat striatum
and another flat electrode pressed against the rat chest with a second electrode. The
voltages used ranged from 250 to 650 V, the number of pulses ranged from 50 to
90, the pulse duration ranged from 50 to70 μs, and the pulse delivery frequency was
4 Hz. To determine the increase in the permeability of blood-brain barrier (BBB)
after IRE, an MRI contrast agent Gd-DOTA was injected i.v. before or after IRE, and
its distribution in the tissue around the implanted electrode was measured. Applica-
tion of IRE increased the permeability of BBB, which lasted for up to 40 min after
application of electric pulses. However, the study also found that the volume of BBB
disruption is larger than the volume of tissue disruption due to IRE and that it is
extending further away from the implanted electrode (Hjouj et al. 2012; Sharabi et al.
2016). This suggests that the observed increase in the permeability of BBB after IRE
happens in the area where the electric field already substantially decreased and did
not reach the threshold for IRE; thus it could be considered that the area was exposed
to reversible EP.
Long-term preservation of function of large blood vessels after IRE was first
demonstrated in a study where the electric pulses were applied directly to the carotid
artery in rats through custom-made clamping electrodes (0.3 mm distance between
electrodes, ten pulses, voltage-to-distance ratio 3,800 V/cm, 100 μs, 10 Hz). IRE
caused an ablation of all the cells to the margin of blood vessels, including vascular
smooth muscle cells, within 7 days after the treatment. Interestingly, there were no
differences in the blood vessel wall structure in comparison to the untreated controls
24 h after IRE. Although IRE caused the ablation of the cells, it did not compromise
the blood vessel matrix, and a histological analysis of the affected vessels performed
28 days after IRE showed a repopulation with endothelial cells (Maor et al. 2007).
A different approach using specially made endovascular devices with four electrodes
on top of inflatable balloons and delivering the same parameters of electric pulses as
in the first study confirmed complete ablation of all the cells to the margin of blood
vessels while retaining the function. Within 35 days after IRE, the blood vessels
were also repopulated with endothelial cells (Maor et al. 2010). This demonstrates
that the use of IRE close to large blood vessels is safe and causes no complications.
Another study demonstrated the ability of IRE to retain the function of larger
blood vessels. IRE was performed in mouse liver by delivering the electric pulses
through a pair of 10 mm diameter circular flat-plate electrodes (90 pulses, voltage-to-
distance ratio 500 V/cm, 70 μs). The function of blood vessels in IRE-treated liver
was then confirmed by intravenously injecting Evans Blue dye and showing that
large blood vessels remained perfused after treatment (Kos et al. 2015; Bulvik et al.
2016).
Nowadays IRE is more and more often used in clinics in well-perfused organs for
treatment of different cancer types such as pancreatic adenocarcinoma, renal cell
carcinoma, and hepatic malignant tumors (Scheffer et al. 2014). Although IRE
causes ablation of all the cells to the margin of blood vessels, including vascular
smooth muscle cells, all of the performed studies indicate a preservation of the
function of large blood vessels due to the preservation of blood vessel matrix which
is in long term repopulated with endothelial cells (Narayanan et al. 2014; Chen et al.
2015; Wagstaff et al. 2016).
Conclusions
Electroporation, either reversible or irreversible, has a profound vascular effect when

it is applied to tissues. Application of reversible EP pulses increases the permeability
of affected blood vessels, causes a transient vascular lock, i.e., decrease in perfusion,
and transiently decreases the diameter of affected blood vessels. Similarly, IRE
transiently increases the permeability of the affected blood vessels. In light of the
future use of reversible EP and IRE in well-perfused organs, it is important that both
techniques preserve the normal function of affected blood vessels in the long term.
Cross-References
▶ Antiangiogenic Gene Therapy

▶ Blood Flow Modifying and Vascular-Disrupting Effects of Electroporation and
Electrochemotherapy
▶ Effect of Electroporation on Blood-Brain Barrier
▶ Preclinical Studies on Irreversible Electroporation
▶ Safety of Clinical Irreversible Electroporation
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Biophysics and Metrology of
Electroporation in Tissues 36
Uwe Pliquett
Abstract
High-voltage pulses change the electrical properties of cells and tissue. The most
sensitive targets are membrane structures charging up due to their high resistance.
With achievement of a critical transmembrane voltage, structural rearrangements
yield conductive, aqueous pores across the lipids able to transport ions but also
larger polar molecules. Moreover, due to high current density, Joule heating can
induce further reversible or nonreversible changes.
Practical applications rely on optimal pulse conditions which require objective
measures of the reaction to the stimulus. Since the electrically induced changes
and the recovery after exposure to electric pulses take place on a time scale of
microseconds up to minutes, only fast measurements are relevant. Post-
processing may be used to measure electroporation yield, time course, and level
of recovery or information about localized heating.
Here, fast electrical measurement methods before and after but also during the
presence of elevated electric field are revised from theory up to basics for data
mining. Fast impedance measurement presented here bases on time domain
approach with multisinus or step excitation rather than frequency sweep in
frequency domain.
Spatially resolved temperature measurements with time resolution compatible
with millisecond pulse duration are described. Merging electrical and thermal
action, a model for synergistic effect, is presented.
Keywords
Electrical characterization • Impedance • Time domain step response • Temper-
ature distribution • Dynamic conductance • Electroporation yield
U. Pliquett (*)
Institut für Bioprozess- und Analysenmesstechnik, Heilbad Heiligenstadt, Germany
e-mail: uwe.pliquett@iba-heiligenstadt.de

DOI 10.1007/978-3-319-32886-7_71
622 U. Pliquett
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622
Composition of Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622
Electroporation in Isotropic Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624
Electroporation in Vascularized Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625
Skin Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625
Mechanistic Studies on Solid Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626
Impedance as Guidance for Membrane Permeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 627
Interpretation of Post-pulse Impedance: Disintegration Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633
Impedance Detection Practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 634
Time Domain-Based Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 634
Multisine Excitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635
Step Excitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635
Current/Voltage Characteristics Before, During, and After High-Voltage Application . . . . . . . . 640
High-Voltage Switch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640
Very Fast Impedance Detection After High-Voltage Pulse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 641
Electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 642
Electrical Properties During High-Voltage Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 644
Voltage Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 644
Current Monitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 646
Dynamic Behavior During Changing Electric Field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 647
Conductance Evolution During the Presence of High Electric Field . . . . . . . . . . . . . . . . . . . . . . . 648
Joule Heating During Presence of Elevated Electric Field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 648
Spatially Resolved and Fast Temperature Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649
Synergistic Effect Between Heating and Electric Field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 651
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 651
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652
Introduction
The understanding of electrical effects on biological material requires information on

membrane level and basic knowledge about the structure of tissue with respect to
electrical properties.
Composition of Tissue
In comparison to single cells, tissue with its densely packed cells shows electric
field distribution that cannot be described by solving Maxwell’s equation for a
conductive sphere with insulating membrane in infinite space filled with homoge-
neous electrolyte. Moreover, cells in tissue do not have ideally insulating
membranes.
Different kinds of tissue vary considerably in their electrical properties (Alberts
et al. 2007). The liver and squamous tissue is macroscopically homogeneous
(Fig. 1a), while the muscle shows great anisotropy depending on the orientation of
the muscle fibers (Fig. 1b). The electrical conductance is highest in the direction of
the fibers, while a significantly lower conductivity is found perpendicular to it.
36 Biophysics and Metrology of Electroporation in Tissues 623
Fig. 1 Types of tissue with very different electrical properties: (a) homogeneous tissue (liver,
tumor, squamous tissue), (b) anisotropic tissue (muscle), (c) endothelium, (d) stratum corneum, and
(e) bone (Images by Antje Wolter, Baderitz, Germany)
An even more pronounced anisotropy is characteristic for epithelial and endothelial

tissue. Endothelial tissue (inner layer in Fig. 1c) has tight junctions preventing
almost all ionic and molecular transport between the apical and basal region. The
624 U. Pliquett
conductivity of epithelial tissue as found in the skin, especially the stratum corneum
(Fig. 1d), which is the outermost layer of the skin, is extremely low. This low
conductivity is overwhelmingly governed by the envelope of the corneocytes
which is a tight structure of multilayered, long-chained sphingolipids with low
water content.
Another low conductive tissue is the bone (Fig. 1e) with its low cell density in a
calcified supporting structure.
Although tissue may be homogeneous in the millimeter range, it is interrupted by
blood vessels and supporting structures like the tendon or cartilage. In particular,
blood vessels may have a considerably higher conductivity than the surrounding
tissue.
Most tissue of the human body exhibits conductivity in the frequency region
around 1 kHz on the order of 0.1 S/m, while the myocardium or blood shows about
ten times higher conductivity. The lung or gland tissue has a conductivity of less than
0.01 S/m, while the bone (Fig. 1e) is even less conductive. The best insulating tissues
are keratinized regions like the stratum corneum, nails, hair, and hoof. Highly
specialized tissue like the brain matter is very anisotropic due to the high conduc-
tivity along the nerve fibers, while the surrounding glia cells (or Schwann cells
outside the brain) act as insulators.
Despite tissue varies considerably, the main target for the electrical manipulation
with short but intense pulses is always the same. With the application of an external
electric field, membrane structures are perpendicular to the field charge up, yielding
a field inside the membrane by orders of magnitude higher than the outer one which
destabilizes the lipid structure.
Electroporation in Isotropic Tissue
Electroporation is well described on the membrane level for applied field strengths
below 1 kV/cm (Weaver and Chizmadzhev 1996) where the maximally reachable
field strength within the membrane (▶ Chap. 59, “Transmembrane Voltage Induced
by Applied Electric Fields” by Kotnik T) is only slightly higher than the critical field
strength for electroporation. The pulse duration is longer than the electrical time
constant of the tissue which may vary between 0.3 μs and 10 μs. Since pore creation
takes only nanoseconds, the tissue is thermodynamically equilibrated. With achieve-
ment of electroporation condition, pores are created which lowers the local field
strength due to a voltage divider between the permeabilized membrane and the
surrounding electrolytes. If the electrolytic conductivity is high and the field strength
within the membrane is maintained over milliseconds, significant Joule heating
occurs.
With the application of very short pulses (picosecond to nanosecond) where the
duration is significantly less than the charging time constant of the tissue, electro-
poration condition are not reached up to several kV/cm. Within nanoseconds but
sufficient field strength, pore formation does not necessarily occur. This forces
further charge to build up and yield a kind of overcharged membrane with significant
excess energy deposited within the system which is now driven away from thermo-
dynamic equilibrium and fosters fast structural changes within the lipid membranes.
Although not fully understood, these changes may range from high-density pores
(Gowrishankar and Weaver 2006) up to complete disintegration of the membrane
structure (Pliquett et al. 2007).
After the outer field ceases, recovery takes place much slower than the creation of
pores. Several phases of recovery are evident. At least one fast phase lies within less
than a millisecond, and two more proceed in the millisecond and the second range. If
a critical size of pores was exceeded, recovery is unlikely (Glaser et al. 1988).
The recovery is different for nanosecond pulses above 10 kV/cm where an
extremely high conductive state during the pulse is lost within nanoseconds after
the pulse.
Electroporation in Vascularized Tissue
With only few exceptions, all tissue of human body is vascularized including the
already mentioned “isotropic tissue.” Blood vessels are special with respect to
reaction on outer high electric field. Despite the primary target for the field are
membranes, not all membrane structures behave in the same way. The surface of
blood vessel has a high resistance due to tight junctions between the endothelial
cells. If such endothelial wall is aligned perpendicularly to the field, it charges to a
higher extent than the cell membranes within the bulk tissue and is therefore a
favored target for electroporation (Sersa et al. 2008).
Skin Electroporation
The skin consists of several layers and most of them are electrically similar to other
tissues. The most prominent exception with respect to electrical behavior but also to
molecular permeability is the stratum corneum (marked with sc in Fig. 1d). This
outermost, dead layer of the skin has a thickness between 10 μm and 30 μm and is
composed of flattened corneocytes surrounded by a multilayer system of long-
chained sphingolipids. Despite interruptions like hair follicles or sweat glands,
these about 100 lipid layers do not permit polar substances or ions to pass. This is
a natural safety function against water loss and entry of foreign substances into the
body.
Although a very efficient barrier, even the envelope of corneocytes is interrupted
by protein structures like desmosomes, responsible for mechanical strength between
the cells. While intact in the basal region, they degrade apically and become slightly
conductive.
In comparison with most tissue, the stratum corneum does not show a distinct
threshold for dramatic conductance increase but changes electrical properties over a
wide range of applied field strength. This behavior is only partially attributed to
electroporation. At low voltage, 2 < U/V < 5, electroporation is unlikely, but
626 U. Pliquett
activation of pathways through appendages has been shown to dominate ionic

transport immediately after field application. During longer exposure, electrowetting
drives electrolytes between the corneocytes which widens tortuous pathways. These
are the dominant pathways for iontophoresis which is not associated with pore
creation through membranes. At higher applied voltage, around 10 V, electropora-
tion along pathways with only one or very small number of membranes occurs.
These are mostly the walls of sweat glands or hair follicles but also preexisting
defects within the stratum corneum, for instance, degraded desmosomes. Electropo-
ration occurs above 30 V creating direct pathways through membranes and the body
of corneocytes. Other than electroporation of single membranes, pore creation is
clustered due to a synergistic electrical and thermal effect (Pliquett et al. 2008). This
yields permeable regions which recover their barrier function as long as heating did
not melt the long-chained sphingolipid structures. In the center of the permeable
region (LTRs, localized transport regions) where multilamellar structures were
vesicularized under the influence of Joule heating, even after partial recovery, a
high permeability lasts longer but recovers within hours due to the growth of new
corneocyte layers.
Pulses of several kilovolts on a skinfold (mice) between plate electrodes increased
conductance dramatically. Despite fast electrical recovery, these regions appeared
reddish and developed scar tissue within some days. It is postulated that a high
density of small LTR appeared over the entire area (Nuccitelli et al. 2006).
Mechanistic Studies on Solid Tissue
Electroporation changes mostly the permeability of membranes especially for small

ions. But the molecular transport through permeabilized membranes is much more
relevant for practical applications. In general, transport J (e.g., current density)
across a membrane area A with the permeability P occurs in the presence of a driving
force (e.g., potential difference Δφ) and a concentration c of the driven species:
cP
J¼ Δφ (1)
A
Despite concentration or pressure gradients creating a driving force as well
(governed by Nernst-Planck equation), the overwhelming part of the transport is
driven by the electric field (Δφ/dmembrane).
Although one can assess the permeability to a certain species only by measure-
ment of its flux for a given driving force and concentration, electrical measurements
are helpful for guidance. The significance of electrical measurements is their ease of
use and comparatively simple interpretation.
Another important material property with respect to electroporation is the critical
field strength or voltage where dramatic changes in membrane permeability occur.
This, often called electroporation threshold, is a thermodynamic property which
depends not only on the field strength but also on its duration and on the “history” of
electric field application. For setting the parameters of the pulse protocol, a simple
estimation of the threshold prior to treatment is helpful.
Impedance as Guidance for Membrane Permeabilization
The impedance indicates how much a material hinders the electric current due to an
applied electric field (Grimnes and Martinsen 2014). It is determined by measuring
the answer to an electric stimulus. This can be the resulting current to an applied
voltage or vice versa. The ratio between voltage U and current I is the resistance R,
while its reciprocal is called conductance G. If alternating current (ac) is applied at
energy-conserving elements like capacitors or inductors, a phase shift between
voltage and current results, which turns the voltage and current into complex
numbers U and I and yields the complex resistance, termed impedance Z:
U
_
_¼
Z ¼ R þ jX (2)
_I
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
X is the imaginary resistance, while R is real. The magnitude is jZj ¼ R2 þ X2.
The phase angle can be calculated with φ = arctan (X/R). It is negative for capac-
itors with a phase shift of 90 (or –π/2). The marked difference between real and
imaginary resistance is that a real resistance turns the electric energy, for instance,
into heat, while it is stored in a capacitor and released during discharge. No energy is
consumed because the effective power P = U I cos φ becomes zero for an ideal
capacitor with φ = 90 .
The reciprocal of the impedance is called admittance: Y = G + jB where B is the
imaginary conductance.
For material with capacitive elements, both the magnitude and the phase of the
impedance change over a wide range by varying the frequency. At least three
distinguishable dispersions, regions with pronounced frequency dependence of the
impedance, exist in cell-based material. The α-dispersion up to several Hz arises
from movement of charges at the membrane surface in the direction of the electric
field, thereby polarizing the whole cell. If the frequency increases, the time for this
kind of polarization is not sufficient, and no more energy can be deposited in this
mode. The next step is between 100 Hz and 10 MHz when cell membranes are
increasingly capacitive shortened (ß-dispersion). The orientation of dipoles (large
molecules, water) can follow up to much higher frequency but declines at several
GHz (γ-dispersion).
The most important dispersion for cells or tissue with insulating membranes is the
β-dispersion. A simple equivalent circuit (▶ Chap. 62, “Computational Approach for
Electrical Analysis of Biological Tissue using the Equivalent Circuit Model” by
Ramos A, Suzuki D) comprises a resistor for the intracellular medium
(mostly electrolyte) in series with a capacitor mimicking the membrane capacity
(Fig. 2). This RC combination is shunted by a real resistor for the extracellular
electrolytes.
628 U. Pliquett
Fig. 2 Simple equivalent scheme for cells and tissue with real resistors for the extra- (Re) and
intracellular (Ri) electrolytes and a capacitor for the cell membranes (Cm). The influence of α- and β-
dispersion is neglected
Fig. 3 Equivalent circuit

accounting for pore creation
within the plasma membrane
of cells
This simple circuit is only valid at low membrane conductivity. Else, a resistor
parallel to the membrane capacitor is necessary. The membrane conductivity of cells
is on the order of 10 μS/cm2 which is mostly governed by ionic channels. Any
creation of aqueous pores increases the membrane conductivity dramatically. Imme-
diately after the field ceases, a fast recovery decreases the conductivity in several
steps within microseconds to minutes. Thus, for modeling electroporation, the
resistor Repore related to electrically created pores needs to be variable (Fig. 3).
Given the drastic changes of the membrane conductivity due to electroporation,
impedance measurements are excellent for monitoring electrical pore creation and
recovery.
A simple way of electrical monitoring is the measurement of the resistance at dc
(direct current) before and after electroporation. Although, changes are evident, there
is no good way for quantitative analysis because of multiple influences. A critical
source of error is the transmembrane voltage. It will distort the resistance measure-
ment, if it is unknown. Even more critical is the fact that it is subject to change
as well.
For measurement of the transmembrane voltage, cells can be clamped onto a
micropipette, where the membrane inside the pipette will be broken, which gives
direct access to the electrical properties of the cell membrane except the small
fraction inside the pipette (around 1 μm2–3 μm2).
A simple calculation assuming a non-excitable membrane shows the difference. If
the resting transmembrane voltage is 60 mV and shifts with the application of a
Fig. 4 (a) Whole-cell clamp

with direct access to the cell
interior. (b) Model for cell
membrane with
transmembrane voltage ΔφM
current of 10 pA to 50 mV, the simple ratio U/I gives 50 mV/10 pA = 5 GΩ.
This, however, is wrong. It would be right to use for the voltage the difference
between transmembrane voltage under resting condition (60 mV) and the voltage
under current flow which yields only 10 mV/10 pA = 1 GΩ. Moreover, in excitable
membranes comprising voltage-sensitive ionic channels (mostly nerve and muscle
membranes), using direct current will induce conductivity changes due to opening or
closure of these channels which is independent of electroporation.
Since whole-cell clamp (Fig. 4) allows not only monitoring of voltage and current
but also the transmembrane voltage, a good interpretability of the data is given.
However, when using tissue where proper information on the membrane level is not
fully available, interpretation even by employing biophysical models is often
questionable.
The use of alternating current will circumvent the problem associated with
transmembrane voltage – at least partially. With higher frequency, the current
fraction across cell membranes increases. At low frequency (<1 kHz), electrode
polarization yields tremendous distortion of the measurement. This and additional
uncertainties like electrode contact to tissue, nonlinear effects within the cell struc-
tures or electrolytic fluctuations cause serious problems. The common solution is
taking an impedance spectrum (impedance spectroscopy) over the frequency range
of the ß-dispersion, e.g., from 100 Hz to 10 MHz. The typical spectrum of cellular
objects is a declining real part and a minimum in the imaginary part where the slope
in the real part reaches a minimum (Fig. 5). The corresponding frequency is called
the characteristic frequency. The importance of the ß-dispersion of cell-based mate-
rial lies in the overwhelming contribution of cell membranes.
Despite the spectrum of real material departs from any spectrum obtained with the
three-element circuit, quantitative analysis is quite reliable. Due to a distribution in
cell behavior within the tissue, the half circle in the Nyquist diagram is depressed or
shifted toward the first quadrant (Fig. 6). Moreover, at low frequency, diffusion
behavior within the electrolyte yields the Warburg impedance, while an additional
dispersion is often found at high frequency. This dispersion may be influenced by the
γ-dispersion but is in most cases overwhelmed by the capacitive behavior of aqueous
electrolyte in a particular measuring chamber. Since a four-electrode interface
depresses the Warburg impedance (Fig. 6b), the low-frequency dispersion is attrib-
uted to Gap junctions and α-dispersion.
630 U. Pliquett
Fig. 5 Typical impedance

spectrum of biological
material (stratum corneum,
0.72cm2), (a) real and
imaginary part, (b) Nyquist
diagram (X vs. R, also called
locus diagram, Wessels
diagram, or Cole plot)
For assessment of Repore (Fig. 3), an impedance spectrum should be taken before
and after electroporation. With the assumption that Cm, Re, and Ri are invariant,
Repore can be quantified.
An example will be given using human cadaver skin bathed at both sides in
phosphate-buffered saline (PBS). The equivalent circuit (Fig. 7) is slightly different
accounting for the unique behavior of the skin, especially the stratum corneum
(marked with sc in Fig. 1d). This part overwhelms the skin resistance and can be
modeled as a capacitor CSC for the membrane stack with a parallel resistor for
slightly conductive interruptions like hair follicles or sweat glands (Fig. 1d). The
complete rest of the skin is modeled with Rd.
A typical spectrum, measured with tetrapolar interface (see electrodes), for
untreated skin is shown in Fig. 8b (blue line). Rd can be estimated as an intersection
of the curve with the x-axis at high frequency (left side), while RSC is the difference
between the resistance at the right intersection and Rd. Using the characteristic
frequency fSC of the stratum corneum, CSC will be:
Fig. 6 Nyquist diagram of

(a) cow heart (two-electrode
interface) and (b) sheep liver
(four-electrode interface)
Fig. 7 Equivalent circuit

accounting for pore creation
across the lipid membranes of
stratum corneum
1
CSC ¼ (3)
2πf SC RSC
An initial calculation lumps the resistors associated with the stratum corneum to:
RSC Repore
Rp ¼ (4)
RSC þRepore
which is shown in Fig. 8b.

632 U. Pliquett
Fig. 8 (a) Nyquist diagram of full human skin before and after electroporation, (b) change of the
parallel resistance (Rp) of the stratum corneum which is highly related to Repore. (c) Area-specific
resistance of the electrically created pores and (d) fractional area of the stratum corneum involved in
electroporation
With Rp, and the resistance of stratum corneum before electroporation Rsc, before,
Repore can be calculated:
RSC, before Rp
Repore ¼ (5)
RSC, before Rp
With a simple biophysical model based on assumptions like pore structures created
as direct pathway through the multilayer structure with 100 single layers in series
(depending on body location) and electrolytic conductivity within the pore equaling
the conductivity of the buffer, a fractional area of pores can be calculated (Fig. 8b):
N m dm
Af ¼ (6)
Asc σ el Repore
Asc is the area of the specimen (0.72 cm2), Nm is the number of multilamellar sheets
(100), dm is the thickness of a single membrane (7 nm), and σ el is the conductivity of
the electrolyte (1.4 Sm1).
Fig. 9 Typical recovery of

low-frequency impedance
(e.g., 100 Hz) after high-
voltage application
This simple approach is often used but neglects very important features of
processes associated with electroporation, especially if living tissue is involved.
First, it is not clear what fraction of pores is still open at the time of the
measurement given the fast recovery processes ranging from microsecond up to
several hours. Moreover, when characterizing tissue, Re (corresponds to RSC for
skin) is nearly unchanged in a time frame of milliseconds but may change dramat-
ically due to cell swelling or leakage of the cytoplasm within seconds to minutes
(Schmeer et al. 2004; Pavlin et al. 2005). Another factor which cannot be neglected
because of the high-current density during electroporation is the Joule heating which
directly affects electrolytic conductivity.
To access these changes, fast impedance measurements prior and immediately after
electroporation are required. The technical challenge is the fast switching between
impedance measurement equipment, which is subject to destruction under influence of
high-voltage pulses reaching up to several kilovolts. Moreover, since changes occur
fast, measuring an entire spectrum should not take longer than a millisecond. But also
the electrode system is challenging. Since high current density distorts electrode
behavior, the use of the same electrode for pulse application and impedance measure-
ment is not favorable. Moreover, electrode polarization requires either mathematical
compensation or the use of more sophisticated electrode systems. Despite less accurate
measurement results, many practical arrangements use simple electrode systems.
Interpretation of Post-pulse Impedance: Disintegration Index
Electrical characterization is very sensitive for following electrically induced

changes on the membrane level. An objective measure is the electroporation yield,
which bases on the increase in conductivity at low frequency with respect to the
slight changes at high frequency (Fig. 9). Normalization between not electroporated
and 100% membrane permeabilization (complete diminishment of the ß-dispersion)
yields the disintegration index (▶ Chap. 76, “Techniques to Detect Electroporation
in Food Tissues” by Lebovka N, Vorobiev E) (Lebovka et al. 2002)
ðσ untreated σ treated Þ
CDI ¼ (7)
ðσ untreated σ 100% Þ
634 U. Pliquett
After pulsing, information about membrane recovery and ionic redistribution

either by water uptake or leakage of cytoplasm are detectable. Using the electrical
impedance together with biophysical models allows calculating membrane-specific
behavior like, for instance, pore density.
Impedance Detection Practice
The next paragraph introduces time domain-based electrical characterization of the

material. Besides impedance measurement with multisine excitation, assessment of
relaxation behavior after step application is emphasized. Measurements in frequency
domain, contactless measurements, or tomographic approaches are not
considered here.
Time Domain-Based Measurements
Any measurement should be faster than significant changes of the physical property
of interest. Moreover, the measurement should never influence the properties of the
material under test. This is challenging for electrical measurements at biological
material where both, the material and the electrodes, are nonlinear with respect to the
current-voltage characteristics. Even more demanding is the fact that membrane
electroporation drives the material on purpose into nonlinear region and the recovery
takes place in time intervals much faster than any impedance measurement within
the interesting frequency range, very common between 100 Hz and 10 MHz.
The “classical” method for impedance measurement is sweeping the frequency
throughout the desired spectrum (frequency domain) and determining the magnitude
and phase of the impedance at each frequency. Depending on the frequencies chosen
and the instrument settings, this can take form a few seconds up to several minutes.
Given the fast post-pulse processes, this measurement approach is not suitable for
monitoring cell recovery. Despite this, it is often used for assessment of the electro-
poration yield. Due to fast changes, the time between electroporation and measure-
ment is critical. However, changes slow down within several seconds yielding
significant measurement after this time.
Fast electrical characterization for tracing recovery processes is preferentially
done in time domain. The application of a broad bandwidth signal and monitoring its
response does not need more time than one period of the slowest frequency compo-
nent. Using transformation into frequency domain (Fourier or Laplace transforma-
tion) and calculation of the ratio between U(jω) and I(jω) yields the impedance
spectrum. Although any broad bandwidth signal is useful for excitation, only few
signals are preferred. The choice bases on properties like simplicity to generate, low
crest factor, energy within the frequency range of interest, and stable signal pro-
cessing. An interesting signal with respect to signal processing is the Dirac function,
but it is impossible to generate. Even a simplified signal with finite amplitude and
short duration requires some effort. In opposite, a step function is simple to generate,
and the resulting data are easy to process as well. Other popular excitation signals are
multisine wave, chirp, and maximum length sequence. Advantages and disadvan-
tages of different excitation signals are given in Table 1.
For characterization of biomaterials after electroporation step and multisine
function are favored, both allow high repetition rate, e.g., 1000 measurements/s
for a frequency band between 1 kHz and 10 MHz. The maximum length sequence
(MLS) is preferred for high-frequency applications.
Multisine Excitation
The signal, a sum of sine functions of arbitrary amplitude and phase, is generated as
lookup table and converted into an analog signal by a digital analog converter
(ADC). Subsequent low-pass filtering removes higher-order harmonic distortion.
Both current and voltage are sampled with at least twice of the highest frequency in
the signal (sampling theorem) for at least one period of the lowest frequency in the
signal. This means a spectrum between 1 kHz and 10 MHz requires at least 20,000
sampling points. With continuous sampling over several minutes after electropora-
tion, the creation of huge data volume is unavoidable. It should be noted that this
does not depend on the number of frequency compounds within the signal (typically
6–30).
Adding sinusoidal functions may result in intense peaks (Fig. 10a), which is
expressed as crest factor, the ratio between peak voltage and current to its RMS
value. Sophisticated routines for optimizing crest factor were developed (Min et al.
2008) adjusting phase and amplitude of the individual frequency compounds
(Fig. 10b).
The choice of the frequencies is critical for preventing leakage effects, e.g.,
misinterpretation due to non-integer number of periods sampled. It is advantageous
that all frequencies are multiples of the lowest frequency compound, and the
sampling interval is exactly one period or a multiple of it.
Due to the absence of steady state during the first period, at least one or more
periods should pass before data acquisition starts. However, once steady state is
reached, each period of a repeating signal can be used which yields the highest time
resolution (Garcia-Sanchez et al. 2015).
Step Excitation
A single step has a continuous frequency spectrum. However, for practical reason,
square wave functions, i.e., a series of steps with alternating polarity, are used.
This reduces the spectrum to discrete lines with the fundamental frequency
equaling the repetition frequency of the square wave and all odd harmonics. The
amplitude of the harmonics declines with the factor 1/n where n is the number of the
harmonic.
636
Table 1 Excitation signals used in time domain-based impedance spectroscopy

Signal Function Spectrum Advantages Disadvantages
Dirac function 1 Continuous spectrum Impossible to generate (only approximate)
0.8 Simple data
0.6 processing
0.4
normalized amplitude
0.2
0
0 50 100 150 200
f/ kHz
140
Rectangular 0.1 Simple generation Low signal energy at high frequency
120
wave 0.05 100
Direct assessment of ➔sophisticated signal processing
0
80 relaxation behavior
U/V
A / mV
60 Suitable for broadband
-0.05
40
applications (> 6 decades)
-0.1 20
0 1 2 3 4 0
0 5 10 15 20 25 30
t / ms
f / kHz
0.3 0.06
Multisine Simple processing Susceptible to cross modulation
0.2 0.05
Predefined frequencies and
0.1 0.04
amplitudes
0 0.03
U/V
U/V
-0.1 0.02
-0.2 0.01
0
0 20 40 60 80 100 0 5 10 15 20
t / ms f / kHz
U. Pliquett
6
36
Chirp 0.3 Simple processing DAC needed for generation or harmonic

0.2 5
Frequency band adjustable distortion when using square chirp trace
0.1 4
0 3
U/V
A / mV
-0.1
2
-0.2
1
-0.3
-5 0 5 10 15 20 25 0
0 5 10 15
t / ms f / kHz
0.25 15
Maximum Simple generation and Sensitive to noise
0.2
length sequence processing
10
0.15 Suitable for high frequency
U/V
A / mV
0.1
5
0.05
0 0
0 1 2 3 0 20 40 60 80 100
t / ms f / kHz
Biophysics and Metrology of Electroporation in Tissues
637
638 U. Pliquett
Fig. 10 Multisine signal (a) without optimized crest factor and (b) after optimization (39 frequen-
cies between 100 Hz and 1 MHz are used)
If a step, current and voltage, is applied to complex impedance, it relaxes to a new

state. This yields an exponential charging function in case of an RC combination.
Although it is feasible to characterize the material directly in time domain (spectrum
of time constants and relaxation strengths), most researchers expect the impedance
spectrum as measurement result. Generally, one period of current and voltage is
Fourier transformed, and the impedance is calculated as quotient of the voltage and
current in frequency domain.
Although similar to the multisine approach, some precaution for obtaining suit-
able results should be taken.
For fastest measurement repetition, exactly one complete period should be
extracted from the signal. Because of unavoidable noise due to low amplitude at
higher harmonics, any spectrum becomes very uncertain at high frequency, and
further processing is required.
There are two basic possibilities for increasing the significance of the spectrum at
high frequencies: (1) logarithmic averaging and (2) partial integration with non-
uniform sampling.
In the first approach, impedance at odd harmonics within a predetermined
frequency range around a center frequency is averaged (Fig. 11). Logarithmic
spacing of the center frequencies gives adequate line density throughout the desired
bandwidth.
Partial integration of the step response (e.g., current due to an applied square
voltage) yields a short vector for the time signal with highly significant samples. Due
to the nature of the response for biological material, which is generally the sum of
exponential functions, short integration times immediately after the step and increas-
ingly longer integration toward the end of a half period yield the best results. For
practical reason, logarithmic spacing is favorable. In principle, the exact limits of the
integration intervals may vary but should be exactly determined. Integration of the
response over a predefined range (hardware presented in Fig. 12) suppresses
uncorrelated noise, increases the signal energy, and enhances signal-to-noise ratio
(SNR).
Fig. 11 Impedance spectrum

of human body measured
between the leg and arm using
four electrodes designed for
electrocardiography (EKG
electrodes). The red curve is
without averaging, while the
circles result from logarithmic
averaging of the impedance
10 50 0.5
U/V
U / mV
I / µA
0 0 0
0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8
t / ms t / ms t / ms
−10 −50
Fig. 12 Schematic for the measurement system. The stimulus and the sampling regime are
generated digitally from the same controller. For typical biomaterial, the voltage step forces an
exponentially declining current. During the negative half period, “reset” is active and the integrator
capacitor stays uncharged. “Reset” is released together with the step, and the integrated curve is
sampled at predefined times
The number of time points for practical approach is between 4 and 10 per time
decade. This results, for instance, in 50 sample points for a frequency range between
100 Hz and 10 MHz. Because of a nonuniform spacing, discrete Fourier transforma-
tion (e.g., fast Fourier transformation) is not feasible. Therefore, an analytic approach
640 U. Pliquett
for calculation of the Fourier coefficients using partial integration between adjacent
sampling points is necessary. Precaution should be taken that the dc offset is zero and
the integration boundaries with respect to the time when the step is released are well
defined. While for the timing software algorithms or hardware delay lines are useful,
the dc offset should be compensated with integration over an entire period.
Current/Voltage Characteristics Before, During, and After

High-Voltage Application
For assessment of electrical behavior during electroporation, voltage and current

monitors but also the proper electrode systems are necessary. Moreover, when
measuring impedance before and after electroporation, high-voltage switching
should prevent damage to the low-voltage electronics but also minimize artifacts.
High-Voltage Switch
A critical part is the switch between high-voltage pulse apparatus and impedance
measurement. Any interconnection between high- and low-voltage electronics needs
to be avoided. The simplest and safest method is to measure impedance, disconnect
manually from instrumentation, and reconnect after pulsing. Although this is some-
times used for guidance, it is not feasible for quantitative studies because of the
impossibility to resolve the recovery process.
Several ways of switching have been investigated. The most common is a high-
voltage relay. This allows switching times of some milliseconds and sustains voltage up to
several kilovolts. The disadvantage is the time lag between the end of the pulse and start
of impedance measurement. A much faster switching is possible with diode (Fig. 13a) or
HEXFET switches. Sophisticated switches like H bridges handle any polarity of the high-
voltage pulse but preserve also the features of the measurement stimulus.
Important features of the measurement stimulus (rectangular wave) are the charge
balance (offset free) and the rise time of the step function. Simple diode switches are
fast and efficient, but they handle only one polarity of the high-voltage pulse and an
offset carrying measurement stimulus. This, however, is acceptable for many appli-
cations. Another interesting approach is the usage of the same power supply for
high-voltage generation and impedance measurement. In this case, for both high-
voltage pulse and measurement stimulus, separate current sources are used, one for
100 mA 10 A (pulse) and the other with 100 μA. Bipolar pulses, also for
measurement, are generated by using an H bridge (Fig. 13b).
A clamping input amplifier which is powered by the high voltage as well buffers
the signal to the subsequent electronics. This approach allows very fast measure-
ments with bipolar pulse application but is for economic reason limited to 1.2 kV due
to availability of affordable high-voltage transistors.
If the pulse voltage is very high, e.g., more than 30 kV, the use of relays is quite
problematic because available devices are expensive and comparably slow
Fig. 13 Possibilities for high

switching speed for
assessment of impedance
immediately after high-
voltage pulse (a) diode switch,
(b) parallel current sources
supplied from high voltage.
Polarity is changed via H
bridge
compared to the speed of changes immediately after high-voltage application.

Possible solution is a construction using a spring-loaded sliding carriage connecting
at both ends of the slide path (several centimeters), the impedance measurement
apparatus and in the middle the high-voltage electrode. Since the pulses are on the
order of nanosecond, even high-speed operation with 5 ms sliding time will be
adequate if it is ensured that the contact to the high-voltage electrode lasts for the
duration of the pulse (Pliquett and Schoenbach 2009). The disadvantage of this
approach is the limitation to only one pulse. Therefore, fast recovery behavior after
extremely high-voltage pulses becomes accessible.
Very Fast Impedance Detection After High-Voltage Pulse
A challenge is the impedance measurement in the presence of a voltage offset as it

appears after fast switching from pulsing to impedance measurement. Considerable
signal distortion occurs due to electrode behavior but also within the bulk of the
642 U. Pliquett
Fig. 14 Simulated current (a) as response to a voltage square wave with an offset generated at the
electrodes during high-voltage application. (b) Correction of the data using the relaxation behavior
after high-voltage application. At t = 0, the high-voltage switch disconnected the impedance
apparatus and reconnected it immediately after the pulse
material. It is important to correct each individual period of impedance measurement

for the discharging happens during up to 100 ms after pulse application (Fig. 14).
Further processing with extraction of biophysical information like, for instance,
electroporation yield or pore-sealing state is done using individual periods.
Electrodes
Electrodes close in most cases the circuit between instrumentation and the tested
material. Although alternate arrangements like capacitive or inductive coupling or
antenna structures are under investigation as well, only galvanic coupling with
electrodes is considered here.
The simplest setup is a two-electrode system where the electrodes serve for
pulsing and also for impedance measurement (Fig. 15a). The great advantage is
the simplicity. Moreover, it is for many applications the only feasible arrangement. A
clear disadvantage is a considerable distortion of the impedance measurement due to
electrochemical reactions at the electrode during high-voltage application. Particu-
larly measurements in the low-frequency range are corrupted by the unavoidable
electrode polarization. However, given the large impedance change due to electro-
poration, precise measurements are often not necessary.
If applicable, separate electrodes should be used for pulse application and for
measurement (Fig. 15b). While the pulse electrodes are chosen for high current
density, cheap material, and simple machining, measurement electrodes should have
stable electrode impedance. Often, non-polarizable electrodes like Ag/AgCl elec-
trodes (silver/silver chloride) are preferred.
With only two measurement electrodes, compensation of electrode polarization or
in general the electrode impedance is not feasible. If this is required, for instance, for
precise measurements at low frequency, tetrapolar electrode systems with separate
Fig. 15 Electrode systems for electrical characterization before and after electroporation. (a)
Two-electrode system used for both, pulsing and measurement, (b) tetrapolar system with separated
pulsing and measurement electrodes, and (c) six-electrode system with one pair used for high-
voltage application and a tetrapolar electrode system for impedance measurement (Images by
Daniel Martin, Heilbad Heiligenstadt, Germany)
current injection and voltage monitoring electrodes are chosen (Fig. 15c). It should
be noted that the current lines between the current injection electrodes and the
voltage monitor electrodes do not coincide meaning that the sensitivity between
the electrodes is not homogeneously distributed (Grimnes and Martinsen 2014).
Even regions of negative sensitivity, i.e., a region where an increase in material
impedance reduces the measured impedance at the electrodes, exist. Therefore, the
644 U. Pliquett
Fig. 16 Simple setup for assessment of electrical properties during high-voltage application
(Image by Daniel Martin, Heilbad Heiligenstadt, Germany)
measured quantity is not the impedance but should be regarded as transfer function
having the unit of the impedance (also called transfer impedance). This is especially
important for measurements at living body, for instance, when performing electro-
chemotherapy with parasitic path to ground. Comparing this rather low potential of
misinterpretation to the problems with electrode polarization using two electrode
systems makes tetrapolar arrangement still favorable.
Electrical Properties During High-Voltage Application
Due to fast recovery, impedance measurements do not reflect the electrical properties
of the treated material during the presence of the electric field. To monitor this,
voltage and current are monitored during pulsing. Often, a sampling resistor is used
for current measurement, while the voltage is monitored with high-voltage probe
(Fig. 16).
The electrode impedance may considerably distort the measurement. To avoid
this, separate voltage monitoring electrodes are favorable. Especially in experiments
where the applicator electrodes are small and therefore the current density is high,
this can prevent misinterpretation.
Voltage Monitoring
The voltage measurement during nanosecond pulses with very intense electric field
is a challenge. Resistive voltage dividers are not always suited. Well established for
very short pulses are capacitive voltage dividers (Fig. 17a). Another arrangement
employs the electrooptical effect (Pockels effect).
Fig. 17 Contactless methods for measuring voltage (a) capacitive sensing and (b) electrooptical
transducer based on Pockels effect (Images by Daniel Martin, Heilbad Heiligenstadt, Germany)
An electrooptical crystal which changes polarization behavior linearly with

electric field (linear electrooptical effect, Pockels effect) is placed between two
electrodes which are connected to the voltage monitor electrodes of the measuring
chamber (Fig. 17b). The polarization of a transmitted light beam is measured as
intensity in p- and s-plane (perpendicular, sagittal). A linear relation between the
change in polarization and the voltage across the crystal with rise time down to
femtoseconds ensures precise and fast measurements. Since the maximum voltage is
limited depending on the crystal at several thousand volts, a much smaller distance
of the sensing electrodes with respect to the applicator electrodes is used which
makes this kind of measurement also feasible for assessment of local electrode field
with a special resolution of several millimeters.
646 U. Pliquett
Current Monitor
While current-sensing resistors are very popular, they exhibit serious danger of
failure due to strong field transients. In such case, lead connection may break,
and the resistor becomes insulating which in bad cases results in full high voltage
at the current input of the measuring device. In order to avoid expensive failures,
inductive transducers like Rogowski coils (Fig. 18a) are used. The advantage of this
particular type is the open design which allows wrapping it around any wire. For
compensation of the transfer function (differentiation), an integrator is
recommended.
The Hall effect allows noncontact sensing in the immediate vicinity of a current-
carrying wire (Fig. 18b) from dc up to several MHz with the advantage of cheap and
stable design.
Fig. 18 Contactless methods

for measuring current with (a)
pickup coil (here Rogowski
type, other coils with closed
design are used as well) and
(b) with Hall transducer
(Images by Daniel Martin,
Heilbad Heiligenstadt,
Germany)
Dynamic Behavior During Changing Electric Field
An important quantity with respect to electrical treatment is the critical field strength
or in particular the voltage necessary for electroporation, Ucritical. Pioneering work
focusing on this issue used a sequence of pulses with increasing voltage and
monitored the electroporation yield (Prausnitz et al. 1993). This could be the changes
in electrical properties but also cell killing or molecular transport across cell mem-
branes. This procedure is well suited for general optimization of pulse protocols but
does not allow individual presetting of the treatment apparatus.
The application of current ramp functions with measurement of the voltage yields
important measures like critical voltage for electroporation or maximal conductance
increase. The current-voltage characteristic exhibits a discontinuity in the slope
which reflects the critical voltage for electroporation (Fig. 19a). It should be noted
Fig. 19 (a) Current/voltage

relation of sheep liver using a
current ramp with 82 A/s. The
electrode consisted of two
rows of 1 mm needles
comprising four needles with
2 mm spacing. The distance
between the rows was 4 mm.
Two additional thin needles
(0.3 mm) were placed in 3 mm
distance between the needle
rows for monitoring the
voltage. (b) Dynamic
conductance ΔI/ΔU
648 U. Pliquett
that the voltage rather than the field strength is the primary parameter of interest for a
given electrode system since this is to be set at the equipment. Although the
application of a voltage ramp with current measurement is possible as well, current
application is milder. The ramp slope is important, because at slow slope, electro-
poration condition is reached at lower current due to higher charge transfer and more
time for structural changes.
Although the primary current/voltage relationship (I/U ) yields the Ucritical, a
higher significance and further interpretation are possible using the dynamic con-
ductance (Fig. 19b). It is defined as slope of the I/U curve and calculated as:
dU ΔU
Gdy ¼ or for practical use Gdy ¼ (8)
dI ΔI
Other than the conductance G = I/U, it reflects directly the course of electropo-
ration. It can be modeled assuming a proportionality of Gdy and the number of pores
which yields:
ΔGdy ebðU Ucritical Þ

2 2
Gdy ¼ Gdy, 0 þ (9)

1 þ ebðU Ucritical Þ
2 2
Besides Ucritical, Gdy,0 is the initial dynamic conductance at U = 0. It equals the dc

conductance of the material. ΔGdy relates to the biggest change due to the high-
voltage application, e.g., the maximum of pores created within the membrane
structures. The voltage, where half of the changes occurred, equals Ucritical. The
parameter b is related to the distribution of individual Ucritical and mean time for pore
creation on membrane level but also to behavior of single pores.
Conductance Evolution During the Presence of High Electric Field
For fast assessment of electroporation success during pulse application, rectangular

pulses are preferred (Fig. 20). If the current does not change after the displacement
current ceased (few μs), electroporation was insignificant (43 V). With higher
voltage applied (100 V), the displacement current increases, but during the pulse,
also further current increase was found which is mainly due to Joule heating. Only
behind the critical voltage for electroporation (155 V), considerable higher current is
evident, overshooting even the displacement current. The further increase is only
partially due to Joule heating. Most of the effect is attributed to further pore creation
or pore expansion.
Joule Heating During Presence of Elevated Electric Field
If current flows through resistive material like electrolyte, electrical energy is turned
into heat. Given the high current density during electroporation, significant heating
occurs. It depends, besides the square of the field strength E, also on the conductivity
Fig. 20 Current due to

voltage application at the liver
with the same electrode
system as in Fig. 19
κ of the material and the time. A simple approach for square pulse (t ➔Δt, E =
const.) is given in Eq. 10:
E2 κΔt
Δϑ ¼ (10)
cel ρel
cel is the heat capacity of the electrolyte and ρel its mass density. It is simple to
calculate the heating in homogeneous medium with equally distributed field strength
like it would be expected for plate electrodes (Fig. 15a). Local heating in inhomo-
geneous material may considerably vary.
Spatially Resolved and Fast Temperature Measurement
The measurement of Joule heating during electroporation remains a challenge, since

heating occurs on a short time scale and often in a small volume. Using a miniatur-
ized thermoelement, fast temperature measurements were done in mouse tumors. In
this case, the transducer with active signal conditioning was optically decoupled
from subsequent electronics.
A pure optical temperature measurement uses the cutoff wavelength for optical
transparency of a gallium arsenide (GaAs) crystal which depends on temperature
with 0.4 nm/K. Other optical methods use the temperature-dependent Raman effect
(anti-Stokes lines). Unfortunately, commercial probes are much bigger than micro-
thermoelements (wire diameter 30 μm) and are therefore slower and not suited for
very small volume.
For assessment of the temperature distribution at the surface (e.g., skin),
temperature-sensitive liquid crystals (TLCs) are preferred (Fig. 21). Other
than thermo-cameras, they are much faster and work underwater as well. Thus,
assessment of the surface temperature of species submersed in electrolytes is
feasible.
650 U. Pliquett
Fig. 21 Skin surface (1.4 1.8 mm2) covered with TLC (LCR Hallcrest, Glenview, IL): blue area
at 45 C and 65 C during a 200 V/300 ms pulse. The voltage drop across the stratum corneum was
on average 34 V. (a) Before pulse, (b) 100 ms, (c) 300 ms after pulse onset
At the skin with the highly insulating stratum corneum, huge temperature rise
would be expected due to the high conductivity within electrically created pores and
the low heat conductivity of the lipids. Measurements, however, showed much less
temperature rise but still reaching more than 72 C (Pliquett et al. 2008; Pliquett et al.
2005), sufficient for lipid melting and dramatic structural changes within the multi-
lamellar system which is responsible for the barrier function (▶ Chap. 64, “Modeling
Transdermal Delivery by Electroporation: The Thermodynamic Approach” by
Becker S). Simulations show that on one hand, significant convection cools the
regions with high current density and on the other hand, voltage divider effect limits
the field strength within the pores (Pliquett et al. 2008).
In solid tissue like tumor, temperature rise was found to be higher than expected
from electrical measurements prior to electroporation. The reason is mainly a
dramatic increase in conductivity due to membrane permeabilization.
Excessive heating was not found for pulse protocols with medium voltage long pulses
but also for nanosecond pulses with extremely high field strength. Typical temperature
increase for single pulses is less than 1 K and for pulse trains, depending on pulse spacing
with interpulse cooling, is up to several kelvins which proves that electroporation is
basically a nonthermal phenomenon (Lackovic et al. 2009; Garcia et al. 2014).
Fig. 22 Synergistic effect of

electric field and temperature:
The probability for
electroporation is highest in a
region where the elevated
temperature meets sufficient
field strength
Synergistic Effect Between Heating and Electric Field
Onset and extent of electroporation depend on the temperature. Especially the

critical field strength for electroporation declines with temperature. If initial electro-
poration yields Joule heating due to locally elevated current flow, further electropo-
ration is forced. This explains the occurrence of localized transport regions (LTRs) in
the skin although an existing pore should lower the probability of further pore
creation due to lowering the electric field in this area. However, on the level of
single membranes within the envelope of corneocytes, the influence radius of a
permeable region of about 10 nm in diameter is only a few nanometer. Thus, high
field strength is maintained even within the immediate vicinity of initial electropo-
ration. With local heating, lowering the critical field strength in this region, the
highest probability for electroporation occurs immediately around existing pores
(Fig. 22).
In solid tissue, the extent of electroporation increased dramatically with elevated
temperature but did not show the pronounced localization as in the skin which is
mainly due to conductive pathways around the cells. With proper spacing between
pulses, excessive heating is avoidable (Pliquett and Nuccitelli 2014) due to cooling.
Conclusions
Electroporation is a strong tool for manipulating cells and tissue. Optimization of the
treatment protocol needs easy-to-measure target functions. This may be the electri-
cally driven molecular transport, but its online measurement is not simply possible.
Electrical and temperature measurements, for sensing electroporation yield, pave the
way for adaptive equipment (Pliquett et al. 2004; Cukjati et al. 2007). While the
temperature measurement can prevent overheating due to harsh pulse protocols,
electrical measurements can predict the critical voltage and electrical behavior of the
material for presetting the pulse apparatus (Pliquett et al. 2004). Moreover, after field
652 U. Pliquett
application, the recovery of membrane integrity is an indicator of electroporation

success.
Since electroporation is a nonlinear effect, non-precise measurements can yield
misinterpretation which is especially important for optimization of the method. To
prevent this, electrode systems with separate electrodes for high-voltage application
and electrical characterization are favored.
Cross-References
▶ Computational Approach for Electrical Analysis of Biological Tissue using the

Equivalent Circuit Model
▶ Modeling Transdermal Delivery by Electroporation: The Thermodynamic
Approach
▶ Techniques to Detect Electroporation in Food Tissues
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Selective Extraction of Molecules from
Biomaterials by Pulsed Electric Field 37
Treatment
Abstract
In present time, the selective extraction of molecules assisted by pulsed electric
fields (PEF) has gained growing interest. The PEF can serve as an effective tool
for improvement of traditional extraction processes in biotechnological and food
industries. The PEF-assisted extraction can be applied in two different modes by:
(a) diffusion in solvent (solvent extraction) and (b) application of pressing pro-
cedures (expression). The PEF-assisted extraction methods are suitable for the
selective recovery and extraction of sugar, inulin, starch, proteins, polysaccha-
rides, polyphenols, pigments, flavor compounds, phytochemicals, and other high-
value components. PEF can be applied for treatment of different fruits and
vegetables (apple, carrot, table beet, etc.), leaves (tea, spinach), herbs, mush-
rooms, and suspensions of cells (yeasts, microalgae, etc.). The PEF-assisted
techniques have low-operating costs and can be applied to the thermally sensitive
products in “cold” mode without deterioration of color, flavor, vitamins, and other
important nutrients of foods. “Cold” refers to the fact that only mild heat is added
during extraction procedure.
In this chapter, the main extraction procedures assisted by PEF are defined.
Different examples of PEF-assisted solvent extraction and expression of useful
E. Vorobiev (*)
Université de Technologie de Compiègne, Département de Génie des Procédés Industriels,
Laboratoire Transformations Intégrées de la Matière Renouvelable, EA 4297 - Équipe Technologies
Agro-Industrielles, Centre de Recherches de Royallieu, Compiègne, France
e-mail: eugene.vorobiev@utc.fr
N. Lebovka
Department of Physical Chemistry of Dirperse Minerals, Institute of Biocolloidal Chemistry named
after F.D. Ovcharenko, NAS of Ukraine, Kyiv, Ukraine
Laboratoire de Transformations Intégrées de la Matière Renouvelable, EA 4297, Centre de
Recherches de Royallieu, Sorbonne Universités, Université de Technologie de Compiègne,
Compiègne, France
e-mail: lebovka@gmail.com

DOI 10.1007/978-3-319-32886-7_163
656 E. Vorobiev and N. Lebovka
components from different food and biomaterials are collected. The emphasis is
placed on the selectivity of extraction. Following the handbook format, the
chapter is concise and covers various common features of PEF processing,
review on recent efforts in the field, as well as several aspects of our own
research.
Keywords
Pulsed electric fields (PEF) • Selective extraction • Extraction by diffusion •
Extraction by pressing (expression)
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 656
Actions of Pulsed Electric Fields on Biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 657
Pulsed Electric Field-Assisted Solute Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660
Pulsed Electric Field-Assisted Extraction from Fruit and Vegetable Tissues . . . . . . . . . . . . . . 660
Pulsed Electric Field-Assisted Extraction from Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . 661
Pulsed Electric Field-Assisted Extraction by Pressing (Expression) . . . . . . . . . . . . . . . . . . . . . . . . . . . 663
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 669
Introduction
The extraction of molecules from different materials of biological origin (including

foods) has a long history, and it was used for production of sugar, fruit juices, wine,
pigments and colorants, polyphenols, proteins, extracts from herbs, perfumes, oils,
and waxes (Bart and Stephan 2011). The conventional extraction techniques are
based on diffusion in solvent, extraction by cold pressing (expression), Soxhlet
extraction, maceration, and hydrodistillation (Lebovka et al. 2011). However, in
majority cases, the conventional techniques have low extraction selectivity and
separation and purification of extracted bioactive substances commonly requires
application of costly supplementary procedures. Moreover, extraction techniques
require costly and high-purity solvents and can provoke decomposition or degrada-
tion of bioactive substances. Recently, the nonconventional extraction processes
assisted by enzymes, supercritical fluids, high pressure, ultrasound, microwaves,
and pulsed electric fields have been developed (Azmir et al. 2013).
The principal advantages of PEF-assisted extraction compared to conventional
extraction techniques can be formulated as (Barba et al. 2015):
• Increased mass transfer

• Improved extraction yield
• Decreased processing time
• Decreased severity of the conventional extraction parameters (i.e., decrease of
extraction temperature, lower solvent concentration)
37 Selective Extraction of Molecules from Biomaterials by Pulsed. . . 657
• Reduction of heat-sensitive compounds degradation (e.g., flavors, proteins)

• Facilitation of purified extract (i.e., reducing grinding)
• Reduction of energy costs and environmental impact
Moreover, applications of hybrid methods combining at different steps conven-

tional and non conventional extraction can be very useful for enhancement of
extraction selectivity and efficiency.
This chapter concentrates on the analysis of recent examples of PEF-assisted
diffusion extraction and expression techniques in application to different materials of
biological origin. The special emphasis is placed on the selectivity of extraction of
bioactive or useful compounds.
Actions of Pulsed Electric Fields on Biomaterials
The action of PEF treatment on biomaterial is based on dielectric damage of cell

membranes that is the very thin barriers (h 5 nm) separating the interior and exterior
of cells. The dielectric damage requires some threshold potential across membrane,
typically of order ucm = 0.2–1 V (Weaver and Chizmadzhev 1996). At this threshold
potential, the breakdown field strength applied to membrane is of order of 106 V/cm
that noticeably exceeds breakdown field strength of mineral oil (105/cm). The
electroporation concept is very popular for explaining the dielectric breakdown of
the biological membrane. It is supposed that in the vicinity of breakdown, the external
electric field forms pores (holes) inside membranes. Transmembrane potential across
membrane in the external field E is proportional to the diameter of the biological cell d,
um dE, and electroporation of small microbial cells requires higher fields than
electroporation of larger plant cells.
For the plant tissues, efficient electroporation can be observed at the relatively
moderate electric field strengths within the interval E = 200–1000 V/cm and time of
PEF treatment tPEF = 104–101 s. The electroporation of yeasts or microalgae
commonly requires greater electric fields (E = 20–50 kV/cm) and shorter treatment
times (tPEF = 105–103 s) (Vorobiev and Lebovka 2008). Figure 1 presents the
electroporation diagram in E versus tPEF coordinates.
In a nondischarge mode, the PEF-technique is relatively nonthermal and nonde-
structive. Typically the PEF-treatment is applied without significant elevation of
temperature of order ΔT 10 C. Moreover, the PEF induces preferential or
selective damage of membranes and does not affect noticeably a structure of cell
walls. A relative mass of membrane component in fruits or vegetables is negligibly
small in compare with the total mass of tissue (0.05%).
Application of PEF in discharge mode (high voltage electrical discharges,
HVED) can induce the electroporation of membranes and damage of cell walls,
mechanical fragmentation of materials, temperature elevation, and changes in
other characteristics of treated materials. Commonly, the HVED technique is
more injurious and nonthermal. Such technique accompanies with electrical
105
Thermal
Electroporation & Ohmic heating (OH)

Electric field strength, E, V/cm
104 Non-
Thermal Thermal threshold, ΔT≈10°C
103
Electroporation
Electroporation threshold
102 E µ1/ tPEF
No electroporation
ms ms s
101
10-6 10-5 10-4 10-3 10-2 10-1 1 10 100
Duration of PEF treament, tPEF, s
Fig. 1 Schematic presentation of electroporation diagram. The dashed line corresponds to the
electroporation threshold. The solid line corresponds to the 10 C limit of temperature elevation.
The hatched area corresponds to the nonthermal electroporation (From (Maity 2015) with
permission)
breakdown, propagation of streamer, bubble formation and cavitations, light emis-

sion, appearing of localized regions with high pressure, and formation of shock and
acoustic waves. In addition, this treatment can produce a large quantity of radical
species, ozone, and provokes the fragmentation of solid particles suspended in the
treated mixtures.
The electroporation efficiency noticeably depends upon the details of pulse pro-
tocols (electric field strength, pulse duration, polarity, distance between pulses,
etc.. . .), properties of biomaterials (size of the particles and cells, degree of maturity,
electrophysical properties), temperature, conductivity, and pH of outside media
(Raso and Heinz 2006; Vorobiev and Lebovka 2008). For example, the longer
pulse durations (ti > 100–1000 μs) were found to be more effective for treatment
of plant tissues, and their effect was particularly pronounced at the room temperature
and moderate electric fields (E = 100–300 V/cm). Application of bipolar pulses
allows reduction of the food electrolysis phenomena and asymmetry of membrane
1
E=400 V/cm Orange
ti= 1000 µs
0.8
Disintegration index, Z
Apple Orange
Apple
0.6
Carrot Potato 100 mm

Potato
0.4
Carrot
Courgette Banana
0.2 Banane
Courgette
10-3 10-2 10-1 100

Treatment time, tPEF ,s
Fig. 2 Disintegration index, Z, versus time of PEF treatment, tPEF, for different plant tissues (Ben
Ammar et al. 2011)
damage in the cells. Moreover, a complex protocol with adjustable long pause
between the trains allows fine regulation of tissue disintegration without noticeable
temperature elevation during the PEF treatment.
For the evaluation of the damage degree (i.e., ratio of damaged cells to the total
number of cells), the electrical conductivity disintegration index can be used
(Vorobiev and Lebovka 2008)
Z ¼ ðσ σ i Þ=ðσ d σ i Þ (1)
Here σ is the electrical conductivity measured at low frequency (1–5 kHz), and
indexes “i” and “d” refer to the conductivities of intact and totally damaged cell,
respectively.
Figure 2 shows typical examples of Z(tPEF) dependencies for different plant
tissues (Ben Ammar et al. 2011). The Z(tPEF) curves can be rather different in
dependence of temperature T, applied electric field strength E, size of the cells, as
well as the differences in the tissue electrical conductivity contrast σ d/σ i. For
vegetable and fruit tissues (apple, potato, cucumber, aubergine, pear, banana, and
carrot), the optimal values of power consumption are within 1–15 kJ/kg. They are
noticeably lower as compared to other methods of treatment like mechanical
(20–40 kJ/kg), heating, or freezing/thawing (>100 kJ/kg).
Pulsed Electric Field-Assisted Solute Extraction
Pulsed Electric Field-Assisted Extraction from Fruit and Vegetable

Tissues
Commonly the procedures of solute extraction require a large quantity of energy and
utilize the high temperatures (hot extraction) and/or polluting solvents. Increasing
the temperature enhances extraction of the bioactive compound of interest as well as
other soluble (and undesirable) components (e.g., pigments, pectins, and polymers)
and reduces the selectivity of extraction. Moreover, at high temperature, the degra-
dation of thermolabile compounds (e.g., anthocyanins, flavonols, components of
essential oils) can occur. A prolonged extraction time results in an increased yield of
extracted compound. However, it requires more power consumption and can result
in enhanced degree of thermal degradation. The use of suitable solvent may be also
critical for degradation or coagulation of bioactive compounds or their recovery
from the target material. Moreover, the solvent consumption, its cost and toxicity,
and requirement post-extraction purification step should be also taken into account.
As example, the extraction of sugar from beetroot slices can be referred (Loginov
et al. 2011). The conventional process requires prolonged hot water diffusion at
70–75 C. Such aqueous diffusion results in a noticeable thermal degradation of the
cell wall and extraction of undesirable components such as pectins, oligo- and
polymolecular compounds into the juice. The extraction at high temperatures can
also accelerate various chemical reactions between extracted components of the juice
(e.g., color development due to Maillard reaction). Extraction of high molecular
weight compounds and formation of undesirable products of chemical reactions
result in decrease of extracted juice quality (Loginov et al. 2011). As a result, the
purification of juice requires a very complex multistaged processes and large quan-
tity of chemical reagents, e.g., lime (Loginova (Sereda) 2011).
Recent efforts have shown the promising ways for different applications of
PEF-assisted selective extraction, e.g., sucrose from sugar beetroot, betalain from
red beet, inulin from chicory, beta-carotene from carrot, phenolics from grapes,
extraction of valuable components from apple, paprika, carrot, red cabbage, etc.
(Vorobiev and Lebovka 2008). The PEF treatment can considerably enhance the
diffusivity of useful component using green solvents at ambient temperatures,
without heating (“cold” diffusion), and application of such technology allows
prevention of the thermal degradation of the cell walls and extraction of other
undesirable components.
The most important industrial expectation is related with PEF-assisted aqueous
extraction of sugar from sugar beet. Effects of electroporation in sugar beets have
been extensively investigated in laboratory scale in numerous studies (Eshtiaghi and
Knorr 1999; Loginova (Sereda) 2011; Lopez et al. 2009b). The juice from the cold
diffusion had the lowest quantity of pectin and the color of juice was systematically
3–4 times less intensive than the color of commercial juices (Loginova 2011). In
addition, the juice purity was higher for slices treated by PEF than for untreated
slices after thermal diffusion at 70 C. A scale-up study demonstrated that the
combination of mild heating at 50 C and PEF-treatment can be a useful tool for

shortening the diffusion time. The use of PEF-assisted diffusion technique in the
sugar industry is very promising, more environmentally friendly, efficient, and
thereby reducing energy consumption and economical costs.
The promising perspectives of PEF-assisted diffusion aqueous extraction of
inulin from chicory (Cichorium intybus) roots (E = 100–600 V/cm) have been
also demonstrated (Loginova (Sereda) 2011). The PEF technology noticeably accel-
erates the diffusion of inulin from chicory slices even at moderate temperatures. The
very efficient extractability of betalain (red-purple pigments) from red beet (Beta
vulgaris L.) by PEF-assisted aqueous diffusion has been also demonstrated
(Loginova (Sereda) 2011; Lopez et al. 2009a). The “cold” extraction by diffusion
(T = 30 C) attained the highest colorant yield (95%) with the lowest level of
colorant degradation (10%). The temperature contribution to electroporation effi-
ciency was similar for chicory and sugar beet tissues, and it reflected the synergistic
effect of the simultaneous thermal and PEF application (Loginova (Sereda) 2011).
Nowadays, there exist many interesting examples of PEF-assisted solute extrac-
tion of valuable food, medical, cosmetic, and pharmaceutical components such as
podophyllotoxin (rhizomes of P. peltatum), pure collagen proteins (bovine bone),
chitosan (shrimp shells), polyphenols (fresh tea leaves), crocin, safranal, picrocrocin
(saffron Crocus sativus), alkaloid Guanfu base A, GFA (Chinese medicinal herb
Aconitum coreanum), polysaccharides (corn silk), ginseng extract, and exopolysac-
charides (Tibetan spiritual mushroom broth) (see, (Barba et al. 2015) for a review).
Over the last years, the PEF-assisted diffusion has attracted considerable attention
for the valorization of high–added value compounds from by-products and food
wastes, which can be used for different purposes (e.g., food additives and/or
nutraceuticals). The PEF-assisted recovery of valuable compounds from pomace,
seeds, peels, kernels, husks of grapes, marks, and oil-cakes has been discussed
(Mahnič-Kalamiza et al. 2014). The PEF-assisted diffusion methods have been
also applied for biorefinery of agricultural, and forestry residues (stems, sawdust,
leaves, and bark), energy crops and municipal wastes, vine shoots, leafs, debris, etc.
Processed biological materials also include grape pomace (skins, pulp, seeds, and
stems), vine shoots, different leaves, seeds, peels, kernels, hulls, lignocellulosic
biomass from terrestrial plants, energy crops and crop residues, agricultural and
forestry residues (stems, sawdust, leaves and bark), and food wastes and beer waste
brewing yeasts. These materials are rich in bioactive compounds, especially poly-
phenols (anthocyanins, catechins, flavonol glycosides, phenolic acids etc.), with
antioxidant, antibacterial, antifungal, anticancer, and antiviral properties.
Pulsed Electric Field-Assisted Extraction from Microorganisms
Examples of PEF-assisted diffusion include also applications to different biological

suspensions of yeast and microalgae cells. The possibility of highly-selective and
effective extraction of different intracellular components (ions, saccharides, enzymes,
proteins, and nucleic acids) from yeast cells has been recently demonstrated (Liu et al.
2013). For example, the combination of PEF pretreatment (E = 40 kV/cm) and mild
temperature (<50 C) allowed extraction of 70% of ionic substances, 1% of proteins,
and 16% of nucleic acids. The impact of PEF on the stimulation of Saccharomyces
cerevisiae cells (Mattar et al. 2014) and reduction in their fermentation time (Mattar
et al. 2015) in an aqueous suspension was also demonstrated.
The studies of PEFeffects on aquatic microorganisms have been also initiated in a
recent time. The interest in the application of PEF-assisted solute extraction of
high–added value compounds from marine microalgae such as proteins, carotenoids,
chlorophylls, microelements, lipids, polyunsaturated fatty acids, and sterols, as well
as volatile and phenolic compounds was renovated. These compounds are of a great
interest for different applications in the food, cosmetic, pharmaceutical, and bio-fuel
industries.
The efficiency of different techniques applied to microalgae Nannochloropsis sp.
has been compared (Grimi et al. 2014). The data evidenced that electrically assisted
techniques (PEF and HVED) allowed selective extraction of ionic components and
proteins. The effect of PEF treatment (5.2%) was rather significant as compared
with supplementary contributions of HVED (1.15%) or ultrasounds (1.8%), and
the most fundamental contribution (91%) gave the high-pressure homogenization
treatment. Recently, detailed study of the effects of the composition (Parniakov et al.
2015a) and pH (Parniakov et al. 2015b) of extracellular medium on PEF-assisted
extraction efficiency of different components from microalgae Nannochloropsis was
also evaluated. The extracts were analyzed for content of pigments, proteins, carbo-
hydrates, total phenolic compounds, and antioxidant capacity. It was demonstrated
that PEF pretreatment can be used as a preliminary step for aqueous extraction of
ionic components and small size hydrophilic species. Extraction of larger species or
hydrophobic species can be blocked in the pores of cell walls. Schematic presenta-
tion of mass transfer processes between intra- and extracellular medium for
electroporated microalgae cell is shown in Fig. 3. Mass transfer includes diffusion
of different species (proteins, carbohydrates, lipids, pigments, ionics) as well as
intra- and extracellular fluids. The data evidenced the advantages of PEF-assisted
extraction at normal conditions (pH 8.5) and supplementary extraction at basic
conditions (pH 11) for selective extraction of different intracellular components
(Parniakov et al. 2015b). The diffusion of low-soluble compounds in water (e.g.,
chlorophylls, carotenoids, etc.) usually required the application of multistep extrac-
tion procedures using organic solvents (Parniakov et al. 2015a). The PEF-assisted
recovery of nutritionally valuable compounds from microalgae Nannochloropsis
spp. with the application of binary mixtures of organic solvents (dimethyl sulfoxide,
DMSO and ethanol, EtOH) and water was studied (Parniakov et al. 2015a). The two
stage PEF-assisted (E = 20 kV/cm) extraction procedure involved extraction in
water at the first step and extraction in binary mixture at the second step. The
extraction efficiency of total chlorophylls, carotenoids, proteins, phenolic com-
pounds, and value of antioxidant capacity were evaluated. It was demonstrated
that two-step procedure allows efficient extraction of proteins at the first step with
possible enhancement of extraction of pigments and other high–added value com-
pounds at the second step.
Extracellular medium: Intracellular medium

pH, binary mixtures of water
and organic solvents (dimethyl
sulfoxide, ethanol, etc…)
Mass transfer
- proteins
- carbohydrates
- lipids
- pigments
- ionics
Electroporated Porous
membrane Blocked pores cell wall
Fig. 3 Schematic presentation of mass transfer processes between intra- and extracellular medium
for electroporated microalgae cell. Mass transfer includes diffusion of different species (proteins,
carbohydrates, lipids, pigments, ionics) as well as intra- and extracellular fluids
Pulsed Electric Field-Assisted Extraction by Pressing (Expression)
Extraction by pressure is widely used in the production of fruit and vegetable juices.
In food industry the different equipment types (screw, belt, hydraulic presses, and
(or) filter presses) had been employed after pretreatment operations (fine grinding of
raw material, heating, and enzyme maceration). The preliminary works from different
laboratories demonstrated that PEF-assisted expression allows significant increase in
juice yield and quality (Praporscic 2005). The PEF-assisted expression was applied to
different fruit and vegetable tissues such as sugar beet, apples, carrots, potato,
mushrooms, grapes, and blueberries (See, (Barba et al. 2015) for a detailed review).
Figure 4 presents examples of PEF-assisted frontal batch (a) and continuous
rolling (b) pressing.
During the PEF-assisted juice expression, the applied electric field strength,
electrical conductivity of the media, and the density of pressed cake vary. So,
application of this technique requires thorough adjustment of the protocol of PEF
treatment in order to optimize the electroporation efficiency, extraction yield, and
power consumption.
In general, the PEF-assisted extraction by pressing may be good alternative to
traditional extraction by hot water or organic solvents. Similar to the above-
discussed “cold” diffusion, the efficiency of “cold” pressing of PEF-treated sugar
beet cossettes has been also demonstrated (Loginova (Sereda) 2011).
a Batch pressing b Continuos rolling pressing
Roll
electrodes
PEF generator
Slices
Juice
Electrodes
Sample:
slices
Compressing chamber
Fig. 4 Examples of PEF-assisted batch (a) and continuous rolling pressing (b) (From (Kumar et al.
2011) with permission)
The PEF-assisted pressure extraction technique is of special interest in applica-

tions for temperature or solvent sensible materials with extracts containing coagu-
lable proteins or degradable ingredients. For example, the PEF-assisted pressure
extraction from mushrooms (Agaricus bisporus) has been compared to traditional
hot water and ethanol extraction methodologies (Parniakov et al. 2014). The tradi-
tional methods gave relatively high contents of proteins, total polyphenols, and
polysaccharides. However, commonly, the extracts of mushrooms are cloudy and
their colloidal stability was low. Figure 5 compares the extracts obtained by pressure
extraction (PE), PEF-assisted pressure extraction (PE + PEF), aqueous extraction
(AE), and ethanol extraction (EE). Visual examination evidences that extracts,
obtained by PE and PE + PEF methods were more clear than extracts obtained by
traditional aqueous extraction (T = 343 K, t = 2 h) and ethanol extraction (T =
298 K, t = 24 h) techniques. In addition, the extracts, produced by PE and
PEF-assisted PE methods, have high-colloidal stability. So, application of frontal
pressing allows selective extraction of bioactive compounds with high purity, e.g.,
fresh-like proteins and polysaccharides. Moreover, application of PE + PEF tech-
nique gives noticeably higher yields and also higher nucleic acid/proteins ratio as
compared with that of the PE method.
It is interesting that supplementary ethanol extraction after application of PE,
PE + PEF, or AE techniques can be used in order to increase the yield of bioactive
compounds (total polyphenols and polysaccharides). For example, Fig. 6 compares
concentration of polyphenols obtained by PE, PE + PEF, hot water extraction AE
and EE, and supplementary yields after ethanol extraction from the solid residues
(cakes) for PE, PE + PEF techniques and exhausted slices for AE technique. The
results had shown that supplementary ethanol extraction from exhausted slices
Fig. 5 Comparison of the extracts obtained by pressure extraction PE (T = 298 K, t 1.5 h), the
PEF-assisted pressure extraction PE + PEF (T = 298 K, t 1.5 h), aqueous extraction (T = 343 K,
t = 2 h), and ethanol extraction (T = 298 K, t = 24 h) methods. The extracts obtained by PE and PE
+ PEF methods were diluted by distilled water to the same proportion as for extract obtained by
water extraction method. The applied pressure was p = 5 bar and the PEF treatment was started at
ti = 2000 s. The PEF treatment was performed at the initial electric field strength E = 800 V/cm for
the time of tPEF = 0.4 s. The extracts obtained by PE and PE + PEF methods were diluted by
distilled water to the same proportion as for extract obtained by aqueous extraction method
(Compiled from (Parniakov et al. 2013), From (Lindmark et al. 2014) with permission)
obtained by AE is ineffective and give low-additional yield of the total polyphenols

and polysaccharides. However, supplementary ethanol extraction noticeably
increased the content of total polyphenols in the extracts, obtained by PE and PE +
PEF methods, up to the level that was compatible with that in the extracts obtained
by AE method. The noticeable increase of the content of bioactive compounds in
extracts obtained by PE method was also observed. These observations reflect the
differences in the selectivity of PE and PE + PEF methods. In general, PE + PEF
allowed production of mushroom extracts with high contents of fresh-like proteins
and polysaccharides.
The efficiency of PEF-assisted solid/liquid pressure extraction (expression) of
agro-food materials can be predicted on the base of filtration/consolidation model
(Praporscic 2005). The following simplified equation can be useful for description of
time dependence of the juice extraction yield, Y(t):
t=Y ¼ τ=Y 1 þ t=Y 1 (2)
Y1 is the hypothetical maximal juice extraction yield, and τ is a parameter. In fact the
value uo = Y1/τ corresponds to the initial rate of juice expression at t = 0.
Figure 7 presents example of application of Eq. 2 to the experimental results of
batch juice expression from Muscadelle grapes (for the samples without and with PEF
+ Supplementary Frontal pressing

5 ethanol extraction
. Concentration of polyphenols C,
Mushroom
(Agaricus bisporus)
4
mg GAE/g DM
0
Pressing- Pressing- Aqueous Ethanol
extraction extraction+PEF extraction extraction
T=298 K T=298 K T=343 K T=298 K
Fig. 6 The PEF treatment of mushrooms. Concentration of polyphenols, Cph, (a) and polysaccha-
rides, Cps, (b) in the mushroom extracts after pressure extraction (PE), combined PE + PEF
extraction or hot water extraction (AE, T = 343 K, t = 2 h). The effects of supplementary ethanol
extraction (T = 298 K, 24 h) from mushroom cakes is also shown (Compiled from (Parniakov et al.
2013), From (Choi et al. 2006) with permission)
treatment) (Praporscic et al. 2007). The presentation of experimental data in t/m versus
t coordinates allowed good fitting of the experimental expression data and estimation
of the value of maximum mass of juice m1. The fitting gives Y = 55 2%, τ = 120
10 s and Y = 81 2%, τ = 246 10 s for the samples without and with PEF
treatment, respectively.
The PEF-assisted batch pressure extraction (500 V/cm, 5 bars) of carrot slices
allowed selective extraction useful for production of a “sugar-free” carrot press-cake
rich in vitamins and carotenoids. Such products can be used as an additive in diet
foods (Grimi 2009). The PEF-assisted (E = 0.1–1 kV/cm) selective extraction of
carotenoids from carrot pomace was also observed (Roohinejad et al. 2014).
Finally, we can note that the good industrial potential of PEF-assisted juice
expression has been confirmed on both laboratory and pilot scales using belt-press
equipment (Grimi 2009). The results obtained for sugar beet confirmed the improve-
ment of juice yield and purity on the application of PEF pretreatment. Visually, the
PEF-assisted pressing-extraction produced more transparent and less cloudy juices.
The potential of PEF-assisted pressing of apple mash has been also confirmed on the
laboratory and pilot scales using belt-press equipment (Turk 2010). The
PEF-assisted belt-press machine was used for processing of French cider apple
mash pumped through a collinear PEF treatment chamber (1000 V/cm) at the flow
rate of 280 kg/h.
60 Muscadelle grapes
Frontal pressing
50
Time/mass of extract, t/m, s/g
Slope=1/Y∞
40
Without PEF
30
treatment
With PEF
20 treatment
10
1/u0 0
500 1000 1500 2000 2500 3000

Time of extraction, s
Fig. 7 Example of application of filtration/consolidation model (Eq. 2) to the solid/liquid expres-
sion ( p = 5 bar, T = 298, t = 45 min) of juice from Muscadelle grapes. The PEF treatment was
performed using the electric field strength E = 750 V/cm for the time tPEF = 0.3 s (Compiled from
(Praporscic et al. 2007))
Conclusions
Recent applications of pulsed electric fields for selective extraction from foods and
biomaterials are briefly presented. The electroporation induced by application of
PEF results in acceleration of diffusion between intra- and extracellular compart-
ments. The main examples of PEF-assisted extractions include diffusion and press-
ing procedures. The traditional diffusion and pressing extraction procedures are
rather time and energy consuming, utilize high temperatures, and/or polluting
solvents. The PEF-assisted selective extraction techniques are more energy efficient
and environmentally friendly processes. Application of PEF-assisted extraction
techniques alone and/or in combination with supplementary aqueous extraction or
green solvents (ethanol or DMSO) can be a useful tool to recover valuable com-
pounds from different matrices. It results in reducing the required extraction tem-
perature and time, as well as the amount of solvents thereby increasing
the effectiveness of the process from an economical point of view. The “cold”
(i.e., without added heat) diffusion or pressure extractions also increase the eco-
nomic advantages due to lower energy consumption and open the possibility to
preserve better bioactive compounds from thermal degradation. The PEF extraction
gives promising expectations to produce new types of medical and cosmetic
products. Moreover, these techniques allow “pure” and selective extraction, thus
reducing the subsequent purification steps. The recent promising data related with
application of PEF-assisted extraction techniques for numerous foods and biomate-
rials hopes for successful industrial applications of these techniques.
Acknowledgements The authors appreciate the support from the COST Action TD1104
(EP4Bio2Med – European network for development of electroporation-based technologies and
treatments).
Cross-References
▶ Application of Pulsed Electric Energy for Grape Waste Biorefinery

▶ Application of Pulsed Electric Energy for Lignocellulosic Biorefinery
▶ Advantages of Pulsed Electric Field Use for Treatment of Algae
▶ Application of Pulsed Electric Fields for Root and Tuber Crops Biorefinery
▶ Application of Pulsed Electric Energies for Oil and Polyphenol Extraction from
Sesame Cake and Sesame Seeds
▶ Effect of Pulsed Electric Fields on Food Constituents
▶ High-Voltage Electrical Discharge-Assisted Extraction of Phenolic Compounds
from Grape Seeds
▶ Industrial Pulsed Electric Field Systems
▶ Microalgal Biorefinery for Bulk and High-Value Products: Product Extraction
Within Cell Disintegration
▶ Plant Cell Wall: Description, Role in Transport, and Effect of Electroporation
▶ Polyphenol and Protein Extraction from Rapeseed Stems and Leaves Assisted by
Pulsed Electric Fields
▶ Potential Application of Pulsed Electric Fields for Improving Extraction of Plant
Pigments
▶ Pulsed Electric Energy-Assisted Biorefinery of Oil Crops and Residues
▶ Pulsed Electric Energy-Assisted Extraction of Polyphenols from Flaxseed Hulls
and Cake
▶ Pulsed Electric Field Treatment for Fruit and Vegetable Processing
▶ Pulsed Electric Fields and High-Voltage Electrical Discharge-Assisted Extraction
of Biocompounds from Vine Shoots
▶ Pulsed Electric Fields and High-Voltage Electrical Discharges-Assisted Extraction
of Valuable Biocompounds and Biopolymers from Rapeseed By-Products
▶ Pulsed Electric Fields as Pretreatment for Subsequent Food Process Operations
▶ Pulsed Electric Fields-Assisted Extraction from Exotic Fruit Residues
▶ Pulsed Electric Field-Assisted Extraction of Pigments from Chlorella vulgaris
▶ Pulsed Electric Fields-Assisted Extraction of Molecules from Bacterial and Yeast
Cells
▶ Pulsed Electric Fields Bioproduction of Secondary Metabolites in Plant Systems
▶ Pulsed Electric Fields for Extraction of Secondary Metabolites from Plants
▶ Pulsed Electric Fields for Food Industry: Historical Overview
▶ Recovery of Primary and Secondary Plant Metabolites by Pulsed Electric Field

Treatment
▶ Selective Extraction of Biocompounds from Stevia rebaudiana Bertoni Leaves
Using Electrotechnologies
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41:135–160
Assessment of Electroporation by Electrical
Impedance Methods 38
Quim Castellví, Borja Mercadal, and Antoni Ivorra
Abstract
Electroporation causes an immediate increase in cell membrane permeability that
results in membrane conductivity increase, which has an effect in the measured
impedance of the cell suspension or the tissue. Therefore, impedance measure-
ments offer the possibility to perform real-time assessment of the electroporation
phenomenon in a minimally invasive fashion. Nevertheless, impedance measure-
ments in biological organisms depend on many factors and other processes
besides the membrane permeabilization. This lack of specificity can be an
important drawback for using impedance measurements as an electroporation
measure. An equivalent electrical model of cell suspensions and tissues is com-
monly employed to better understand how the different processes that take place
during electroporation can affect the measured impedance of a sample. This
chapter briefly overviews the information that can be extracted from impedance
measurements during and after the application of electroporation pulses. These
measurements have been widely used to observe and analyze the dynamics of the
phenomenon. Impedance has the potential to be used as a tool to assess electro-
poration effectiveness of treatment. A significant conclusion from the experimen-
tal studies on the topic is that conductivity measured shortly after treatment
appears to be correlated with electroporation effectiveness in terms of cell
membrane permeabilization. That is, it has the potential to be used as an
Q. Castellví • B. Mercadal
Department of Information and Communication Technologies, Universitat Pompeu Fabra,
Barcelona, Spain
e-mail: quim.castellvi@upf.edu; borja.mercadal@upf.edu
A. Ivorra (*)
Department of Information and Communication Technologies, Universitat Pompeu Fabra,
Barcelona, Spain
Serra Húnter Program, Universitat Pompeu Fabra, Barcelona, Spain
e-mail: antoni.ivorra@upf.edu

DOI 10.1007/978-3-319-32886-7_164
672 Q. Castellví et al.
electroporation effectiveness indicator. On the other hand, dynamic conductivity

during the electroporation pulses, which is much easier to be measured, does not
seem to be correlated with electroporation effectiveness.
Keywords
Electroporation • Bioimpedance • Electrical impedance • Membrane conductiv-
ity • Electroporation assessment • Real-time monitoring
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 672
Basic Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 673
Model for the Passive Electrical Properties of Cells and Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 674
Introducing Electroporation in the Equivalent Circuit Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 677
Experimental Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 678
During the Electroporation Pulse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 678
After the Electroporation Pulse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 681
Measurement Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 684
Pulse-Based Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 685
Small Signal-Based Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 685
Impedance Imaging Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 687
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 688
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689
Introduction
The electrical impedance of biological matter is also known as electrical

bioimpedance or simply as bioimpedance. Although it is not physiologically rele-
vant per se, electrical bioimpedance can indirectly reflect some conditions and
events.
Membrane permeabilization induced by electroporation causes an immediate
increase in cell membrane conductivity (see ▶ Chap. 13, “Electrical Conductance
of Lipid Pores”) that, in turn, causes a variation in the measured impedance of the
biological specimen (e.g., a cell suspension or a tissue). Therefore, bioimpedance
measurements have the potential to be used as a tool for real-time monitoring of the
electroporation treatments.
Nevertheless, bioimpedance depends on several physiological parameters and
processes. Membrane conductivity changes due to electroporation can be masked by
other processes leading to results that may be incorrectly interpreted. Therefore, care
must be taken in order to correctly extract conclusions from impedance
measurements.
This chapter introduces the basic concepts regarding electrical bioimpedance and
how it is affected by the electroporation phenomenon. By presenting a number of
experimental results available in the literature, observable effects of electroporation
and the limitations of this type of measurements will be explained. It will also
38 Assessment of Electroporation by Electrical Impedance Methods 673
discuss the useful information that can be extracted from impedance measurements
in terms of assessing electroporation effectiveness. Finally, the common methods
and setups that are used to perform impedance measurements of the electroporation
phenomenon will be overviewed.
Basic Concepts
According to their capacity to conduct electricity under the presence of an electric

field, materials can be, ideally, classified as conductors or dielectrics. Conductors
contain charges that under the action of an electric field can move relatively free
within the material. In contrast, the charges in dielectrics can only shift a small
distance from their equilibrium position.
Most conductive materials follow the Ohm’s law that states a linear relationship
between the electric current and the applied voltage according to the resistance, R,
which is expressed in ohms (Ω). The resistance of a piece of material depends on the
intrinsic resistivity, ρ, of the material and on its geometry. The resistance and the
resistivity or conductivity, σ, will be related by a geometrical constant, k, expressed
in (m1):
k
R ¼ ρk ¼ (1)
σ
This constant k is known as the cell constant because it depends on the geometry of
the measurement cell in which a material is characterized.
Measurement cells typically consist of two metallic flat electrodes, with area A,
across a cuvette, with a gap l, in which the material is confined (k = l/A). However,
two needle electrodes inserted into a living tissue also constitute a measurement cell.
For complex electrode geometries, it will be necessary to compute the cell constant
by numerical methods (Ivorra and Rubinsky 2007) or to find it by measuring the
resistance of a sample with a well-known conductivity (Pliquett 2010). For instance,
in the field of bioimpedance, the solution 0.9% NaCl is typically used because its
composition and conductivity (1.44 S/m at 20 C) are similar to those of blood
plasma.
When a dielectric material is placed between two conductors, the ability to store
charge of those conductors is enhanced. The charge store capacity of the system
composed by two conductors separated by a dielectric is known as the capacitance,
C, and is expressed in farads (F).
Similarly to the case of the resistance for conductive materials, the capacitance, C,
depends on an intrinsic property of the dielectric material, on the electric permittivity
e, and on the cell constant defined by the geometry of the measurement cell:
e e0 er
C¼ ¼ (2)
k k
Table 1 Expressions relating impedance and the passive electrical properties of materials
according to different notations found in the literature
Value Symbol(s) Unit(s) Expressions
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Impedance magnitude |Z| Ω jZj ¼ R2 þ X2

Impedance phase ∠Z , rad ∠Z ¼ arctanðX=RÞ
Resistance R Ω R ¼ jZj cos ð∠ZÞ
Reactance X Ω X ¼ jZj sin ð∠ZÞ
pffiffiffiffiffiffiffi
Impedance Z Ω Z ¼ R þ jX; j¼ 1
Impedivity z Ω.m z ¼ Z=k
Admittance Y Ω1, S Y ¼ 1=Z
Conductance G Ω1, S G ¼ jYj cos ð∠YÞ
Susceptance B Ω1, S B ¼ jYj sin ð∠YÞ
Admittivity y S/m y ¼ kY
Conductivity σ, κ S/m σ ¼ kG
Permittivity e F/m e ¼ kB=2πf
Complex conductivity σ, κ S/m σ ¼ y ; σ ¼ σ 0 þ jσ 00
Complex permittivity « F/m « ¼ σ=2πf ; « ¼ e0 þ je00
The electric permittivity is usually expressed as a product of the vacuum permittivity

(e0 = 8.8541 1012 F/m) and the relative permittivity of the material er that takes
values above 1.
Materials neither will be ideal conductors nor will be ideal dielectrics. That is, it
will be possible to define a conductivity value, σ, and also a permittivity value, e.
These two values are typically referred to as the passive electrical properties of the
material. Impedance is a complex magnitude that establishes a relation between the
sinusoidal voltage and the current for each frequency taking into account the
contribution of both, the resistive and the capacitive components. Therefore, from
impedance measurements performed with an electrode setup of known cell constant
(k), it is possible to extract both σ and e (Grimnes and Martinsen 2000):

1 1
Z¼Rj ¼k (3)
2πfC σ þ j2πf e
It is important to note that both σ and e may depend on the frequency. Table 1
summarizes the common notation and the expressions relating the impedance and
the passive electrical properties of the materials.
Model for the Passive Electrical Properties of Cells and Tissues
In 1925, two decades before the lipid bilayer was understood, Fricke was able to
hypothesize a reasonable value for the membrane thickness (30 nm instead of the
actual 7 nm) by analyzing the passive electrical properties of red blood cells (Fricke
Cm
Re
Ri
Fig. 1 Electrical model for a cell in suspension as seen from the electrodes. Infinitesimal portions
of the extracellular and intracellular media can be modeled as resistances, and infinitesimal portions
of the membrane can be modeled as capacitances. All those elements can be combined in an
extracellular resistance (Re) in parallel with the series combination of a membrane capacitance (Cm)
and an intracellular resistance (Ri). The same three-element model can be employed to represent the
behavior of tissues and cell suspensions
1925). His calculations were based on an electrical model for the cell and its
environment in which the cell membrane was modeled as a dielectric layer and the
extracellular and the intracellular media were modeled as conductive materials. The
electrical model employed by Fricke is still considered to be a good approximation
of the passive electrical properties of a single cell for frequencies up to several
megahertz. In this model, every infinitesimal portion of the extracellular and intra-
cellular media can be modeled as a resistance, and every infinitesimal portion of the
membrane is modeled as a capacitance.
The resistive behavior of the extracellular and intracellular media is basically due
to their contents of ions; both media are in fact ionic solutions (electrolytes). Most
abundant ions in the extracellular medium are Na+ and Cl, whereas in the intracel-
lular media, K+ is the most abundant ion. In contrast, the cell membrane consists
primarily of a thin lipid bilayer. This film (~7 nm thick) is partially permeable to
lipids and water molecules but it is almost impermeable to ions. Its intrinsic electrical
conductance is very low and it can be considered as a good dielectric. Therefore, the
structure formed by the extracellular medium, the lipid bilayer, and the intracellular
medium is a conductor-dielectric-conductor, and it behaves as a capacitance. Exper-
imentally it has been found that such capacitance has a value of about 0.01 F/m2.
The model used by Fricke can be represented with a simple circuit (Fig. 1)
consisting of a resistance representing the extracellular medium (Re) in parallel
with the series combination of a capacitance (Cm), which represents the membrane,
and another resistance which represents the intracellular medium (Ri). This model
can also be used to describe the passive electrical behavior of cell suspensions and
tissues.
Note that since the membrane acts as a capacitance, low-frequency currents will
not penetrate into the cell, whereas high-frequency currents will flow freely through
a b
Re
|Z|
Re//Ri
0.1 1 10 100 1000 10000
frequency (kHz)
Fig. 2 Low-frequency currents are restricted to extracellular spaces, whereas high-frequency

currents can flow freely through living tissues. (a) Graphic representation of the passage of
low-frequency (left) and high-frequency (right) currents through a cell suspension or tissue. (b)
Idealized graph of impedance magnitude versus frequency in a living tissue
it (Fig. 2a). Hence, the impedance magnitude will be higher at lower frequencies
(i.e., |Z| Re) than at higher frequencies (i.e., |Z| Re//Ri) because the electrical
paths at low frequencies are narrower. For intermediate frequencies, a transitional
behavior is manifested (Fig. 2b). Typically, for most animal tissues, the transition
between the low-frequency behavior and the high-frequency behavior occurs at
frequency band from about 10 kHz to about 1 MHz.
The electrical model in Fig. 1 leads to the following expression for the
impedance:
R0 R1 ΔR
Z ¼ R1 þ ¼ R1 þ (4)
1 þ j2πf τ 1 þ j2πf τ
where τ (=Cm(R0 R1)) is the time constant of the model, R1 is the parallel
combination value between Re and Ri (i.e., Re//Ri), and R0 is Re. The use of this
notation allows to express the impedance behavior in the whole frequency domain as
a function of the impedance at high frequencies (ideally an infinite frequency) R1
and the impedance at low frequencies (ideally at 0 Hz) R0. Impedance empirical data
from single cells or diluted cell suspensions can be fitted nicely with this equation
thus confirming the validity of the model (Cole 1968). However, for fitting dense cell
suspensions or living tissues, it has been found that an alternative equation – known
as the Cole equation – produces much better results:
ΔR
Z ¼ R1 þ (5)
1 þ ðj2πf τÞα
where the α parameter typically has values from 0.7 to 0.9 (note that α = 1 implies
that this equation is identical to Eq. 4). The impedance magnitude transitional
behavior of Cole compatible data is slightly more gradual than the response plotted
in Fig. 2b. The physical meaning of the α parameter is still a matter of debate (Markx
et al. 1991). Nevertheless, in the context of electroporation, it has little significance

because, as will be explained in the next section, for monitoring electroporation, it
will be required to follow the behavior of R0 rather than of the whole impedance
spectrum. Therefore, for the sake of simplicity, only the simple model Re//(Cm + Ri)
(Fig. 1) will be considered in this chapter.
From the measured impedance value and the expressions listed in Table 1,
conductivity and permittivity values at a certain frequency can be extracted.
Although both values strongly depend on frequency, from (4) it can be seen that
impedance shows an asymptotic behavior for very low frequencies (|Z| R0), as
well as for very high frequencies (|Z| R1) (see Fig. 2b). In these frequency ranges,
the contribution of the capacitance in the impedance magnitude is practically null;
therefore, constant conductivity values σ 0 and σ 1 can be extracted:
k k
σ0 ¼ , σ1 ¼ (6)
R0 R1
Introducing Electroporation in the Equivalent Circuit Model
When electroporation occurs, cell membrane becomes more permeable, and,

according to the previous model, the cell membrane conductivity will increase,
thus producing a drop in the impedance magnitude at low frequencies (i.e.,
low-frequency current will be able to flow through the cell). This indeed has been
empirically corroborated by different means. As a matter of fact, it must be noted that
the first description of the electroporation phenomenon as a disturbance of the cell
membrane permeability which can be reverted was obtained by performing resis-
tance measurements (Stämpfli 1958).
The cell membrane is not a perfect dielectric. Embedded within the lipid bilayer,
there are different sorts of protein assemblies which allow some ions to flow through
the membrane. In addition, a few ions can leak through defects in the lipid bilayer
even in the absence of embedded proteins. Hence, the electrical model displayed in
Fig. 1 needs to be modified slightly in order to include these short-circuiting effects.
In other words, R0 is not exactly equal to Re but to Re//(Ri + Rm) where Rm is the leak
resistance through the membrane (Fig. 3a). Electroporation creates extra conductive
paths across the membrane which can be modeled also as a resistance REP (see
▶ Chap. 62, “Computational Approach for Electrical Analysis of Biological Tissue
Using the Equivalent Circuit Model”).
According to the model and to empirical data (Ivorra et al. 2007), the effect of
electroporation is not immediately observable by impedance measurements at high
frequencies (at high frequencies, current flows freely across the membrane in normal
conditions). This has an important practical implication: for monitoring electropo-
ration, it will be required to perform impedance measurements at a low frequency so
that |Z| ~ R0, which is the impedance parameter directly dependent on electropora-
tion. Indeed, typical frequencies below 1 kHz or 10 kHz are employed for
a b
Re//(Ri+Rm)
Electroporation
Cm Rm REP Re//(Ri+(Rm//REP)
Re
|Z|
Ri
Re//Ri
0.1 1 10 100 1000 10000

frequency (kHz)
Fig. 3 (a) Enhanced electrical model for a living cell (and for tissues): Rm represents the short-
circuiting resistance across the membrane due to ionic channels and leakage through the lipid
bilayer, and REP represents the increased conductance due to the electroporation phenomenon (the
arrow denotes that it is variable). (b) Plot of impedance magnitude versus frequency for the
enhanced electrical model. Electroporation causes a significant drop in impedance magnitude at
low frequencies
monitoring electroporation, and it is implicitly assumed that the measured imped-

ance corresponds to R0. From this value and knowing the cell constant k, an
estimation of σ 0 can be obtained (6). Although, strictly speaking, this value does
not correspond to the actual value of σ 0, it may be a very good approximation of it if
the measurement frequency is low enough (e.g., 1 kHz) as shown in Ivorra and
Rubinsky (2007).
Experimental Measurements
During the Electroporation Pulse
During the application of the electroporation pulse, it is possible to compute a

resistance value, Rp(t), from voltage and current recordings. Then if the cell constant
of the experimental setup is known, the conductivity during the pulse, σ p(t), can be
defined. This conductivity is referred to as dynamic or quasi-dc conductivity. Strictly
speaking, the dynamic conductivity is not a true conductivity because, when dc
measurements are not possible, as it is the case, the conductivity can only be
extracted from impedance measurements at certain frequency so that both the
conductivity and the permittivity contributions can be isolated. However, after the
first very few microseconds in which cell membrane charging occurs, σ p(t) can be
considered to be a good approximation of what would be the hypothetical dc (0 Hz)
conductivity, σ 0(t). In reality, no alternatives are available: to superimpose an ac
signal to obtain an estimation of σ 0 would not be practical because low frequencies

would be required ( f < 1 kHz, T > 1 ms) and, therefore, dynamic changes during
the pulse would be not recorded because pulse durations are typically <100 μs.
Dynamic conductivity measurements have been used to study the time evolution
of membrane conductivity during an electroporation pulse. Through this type of
measurements, Kinosita and Tsong (1979) performed an early comprehensive study
on conductivity changes induced by electroporation. They observed that the con-
ductivity increase due to electroporation occurs in two clearly differentiated phases:
first, an abrupt increase that is thought to be related to the fast creation of hydrophilic
pores in the membrane and second, a slower exponential rise. The nature of this
slower increase in conductivity is still a matter of debate (Silve et al. 2014).
Fig. 4, shows a typical behavior of the measured dynamic conductance when a
high-voltage rectangular pulse is applied to a living tissue or a very dense cell
suspension. If electroporation occurs (red line), after an initial peak due to cell
membrane charging, conductance first rapidly increases, and afterward it seems to
increase much slower in a quite linear fashion or it stops increasing. The initial
abrupt change in conductance is masked by the membrane charging.
If the electroporation phenomenon does not take place (blue line), after the peak
due to the membrane charging process, tissue conductance would increase almost
inappreciably due to Joule heating. This increase is related to the fact that the
mobility of charges in ionic solutions increases with temperature therefore increasing
16
Electroporation
12
conductance (mS)
4 Without electroporation
0
0 20 40 60 80 100
time (µs)
Fig. 4 Time evolution of the measured dynamic conductance of a mice muscle during the delivery
of a 100 μs pulse of 9.3 kV/m (blue) and 55 kV/m (red). While the 55 kV/m pulse caused
electroporation and therefore an increase in conductance, the 9.3 kV/m pulse did not provoke any
observable effect in tissue conductance (Data extracted from Cukjati et al. 2007)
6 0.6 6 0.6
Liver Muscle
5 0.5 5 0.5
4 0.4 4 0.4
(S/m)
(S/m)
σp / σi
σp / σi
3 0.3 3 0.3
approx
approx
2 0.2 2 0.2
σ
σ
1 0.1 1 0.1
0 0 0 0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
|E| (kV/m) |E| (kV/m)
40 0.6 6 0.6
Skin Tumor
35
0.5 5 0.5
30
0.4 4 0.4
(S/m)
(S/m)
25
σp / σi
σp / σi
20 0.3 3 0.3
approx
approx
15
0.2 2 0.2
σ
σ
10
0.1 1 0.1
5
0 0 0 0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140
|E| (kV/m) |E| (kV/m)
Fig. 5 For four different animal tissues (rat transversal skeletal muscle, rat skin, rat liver, and
sarcoma tumor implanted in mice), relative conductivity (σp/σi) at 100 μs after the beginning of the
pulse (Data was extracted from different sources: squares, Ivorra et al. 2009; diamonds, Pavselj
et al. 2005; triangles, Cukjati et al. 2007; circles, Corovic et al. 2013; and inverted triangles, Neal
et al. 2012). Dashed lines represent sigmoid regressions of the conductivity as function of applied
electric field
the conductivity. In the particular case of physiological fluids is estimated that the
conductivity increases about 2% per each C.
For different field magnitudes and four different tissues, Fig. 5 displays the ratio
between the dynamic conductivity at 100 μs after the electroporation (σ p) pulse onset
and the original tissue conductivity (σ i) as measured before the pulse with a small
signal (see section “Measurement Systems”). It can be observed that the ratio seems
to saturate for large field magnitudes. The maximum ratio (σ p/σ i) is in all cases close
to the ratio σ 1/σ 0 measured before electroporation. This is in agreement with the
electrical model of electroporation depicted in Fig. 3: in principle, the minimum
resistance that can be reached by electroporation must be equal to the minimum
impedance magnitude that can be obtained by applying high frequencies (R1); in
both cases, the cell membrane is “short circuited.” Therefore, at 100 μs, for large
field magnitudes, cell membrane has become so permeable to ions that its resistance
is insignificant when compared to the resistance of the intracellular and extracellular
media. Such statement does not imply that membrane is completely disrupted; only a
tiny fraction of the membrane area needs to be opened (<1%) in order to achieve
such irrelevance in terms of resistance (Hibino et al. 1993).
The conductivity dependence on applied electric field, for a specific tissue, is
commonly modeled as a sigmoid function. In applications where nonuniform elec-
tric field is present (e.g., using needle electrodes), the dependence of tissue conduc-
tivity on electric field intensity affects the electric field distribution during the
electric pulses. Therefore, knowing the characteristic sigmoid function is extremely
useful to accurately predict the resulting electric field distribution by means of
numerical methods (Sel et al. 2005).
After the Electroporation Pulse
From experimental studies on the evolution of the conductance of a biological

sample after the application of the electroporation pulses or in between the applica-
tion of multiple pulses, some general observations can be extracted about the
behavior of membrane conductivity:
1. After cessation of the high-voltage pulse, conductance drops very fast (<<1 ms)
to a value which is much smaller than the conductance during the pulse (σ p) but
significantly larger than its value before the pulse (σ i) (Benz and Zimmermann
1981). This phenomenon is presumably linked to the shrinkage of hypothesized
short-lived hydrophilic pores and resembles the fast increase in conductance that
is observed when the high-voltage pulse is applied.
2. After the fast conductance drop, the conductance of the sample keeps falling
slowly toward the original conductance value (Hibino et al. 1993). This slow
recovery of the membrane resistance is presumably associated with the existence
of long-lived membrane defects which could last for minutes (Pakhomov et al.
2007). Of course, if the pulse is large or long enough, some degree of permeabi-
lization will be irreversible, and the original membrane resistance will never be
reached.
3. In the application of several pulses, post-pulse membrane conductivity increases
gradually pulse after pulse. This post-pulse conductivity increase caused by each
pulse shows a memory effect, meaning that depends on the pulses that were
applied previously (see ▶ Chap. 21, “Electric Pulse Repetition Rate: Sensitization
and Desensitization”).
Besides the above effects, there are two effects directly related to the electropo-
ration phenomenon, but not related to the membrane conductivity, that have an
impact in the impedance measurements after the pulse. First, the release of intracel-
lular contents into the extracellular medium by diffusion through the permeabilized
membrane causes an increase in the extracellular conductivity (Kinosita and Tsong
1979). Second, electroporation leads to an osmotic imbalance that causes water to
rush into the cells; this in turn causes cells to swell, and as a result the extracellular
spaces are significantly compressed. As a consequence, the resistance at low fre-

quencies increases notably (Abidor et al. 1994).
Although it may also be observed in measures on living tissues (Pliquett et al.
1995), the effect of the release of intracellular contents is particularly observable in
diluted cell suspensions. If the volume ratio between intracellular and extracellular
mediums is low, the immediate increase in conductivity related to membrane
permeabilization may be unappreciable (increase in membrane conductivity has a
small impact in the conductivity of the sample), whereas the later slow increase due
to the diffusion of ions to the extracellular medium can be observed (Pavlin et al.
2005). Up to a point, this phenomenon can be considered as an artifact and must be
avoided by performing only fast measurements shortly after the pulses.
In dense cell suspensions and particularly in tissues, the large ratio between cell
volume and extracellular volume causes the conductivity increase induced by
electroporation to be much more noticeable, whereas the post-pulse intracellular
release does not seem to have a significant impact. However, the osmotic effect does
have a significant impact in this case and may yield to conductivity values signifi-
cantly lower with respect to the original conductivity of the sample. In fact, this
effect was proposed by Abidor et al. (1994) to explain the decrease in conductivity
with respect to the initial value that was observed after performing an electroporation
treatment in cell pellets. Therefore, impedance measurements must be performed
shortly after treatment (e.g., 50 ms) for preventing that this osmotic effect masks the
conductivity increase produced by cell membrane permeabilization.
Figure 6 shows the post-pulse evolution of the resistance of the extracellular
medium Re, and the resistance of the membrane Rm, during the application of several
electroporation pulses to a cell suspension. In this example, most of the above
a b
700 2250
680 2000
1750
660
1500
640
Re (Ω)
Re (Ω)
1250
620
1000
600
750
580
500
560 250
540 0
-1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8
time(s) time(s)
Fig. 6 Evolution of (a) the extracellular medium resistance Re and (b) the membrane resistance Rm
(as a combination of the membrane resistance under normal conditions and the increase in
membrane conductivity due to electroporation) during the application of several electroporation
pulses delivered within the vertical gray lines. The individual values of the resistances were
extracted from impedance measurements by means of an electrical model (García-Sánchez et al.
2015)
a b
0.35 0.35
0.3 0.3
0.25 0.25
σ (S/m)
σ (S/m)
0.2 0.2
0.15 0.15
0.1 0.1
0.05 0.05
0 0
-0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
time(s) time(s)
Fig. 7 Two examples of conductivity evolution during in vivo electroporation of rat liver. The
points marked with circles and joined by a dashed line represent the conductivity values obtained at
the end of the electroporation pulses (in-pulse dynamic conductivity, σ p). The electroporation
protocol consisted of 8 rectangular pulses of 100 μs at 10 Hz. In (a) the magnitude of the electric
field was 45 kV/m, and in (b) it was 150 kV/m (Data extracted from Ivorra and Rubinsky 2007)
explained effects can be observed. The time course of Rm (Fig. 6b) indicates that
membrane resistance significantly decreases because of each pulse and, after that, a
slow recovery takes place. It can also be noticed that the effect of each pulse is
different (memory effect). Finally, it is also observable that after the first pulse, Re
experiences a gradual decrease during the treatment due to the release of intracellular
content to the extracellular medium.
Figure 7 displays the result of an experimental in vivo study in which rat livers
were subjected to multi-pulse electroporation (eight pulses of 100 μs at 10 Hz)
(Ivorra and Rubinsky 2007). When the electric field magnitude is 45 kV/m, the
conductivity (σ 0) slightly increases pulse after pulse up to a value 8% higher than the
original one (σ i). The dynamic conductivity at the end of the 100 μs pulses (σ p)
(represented with a single circle) is significantly higher than the conductivity just
before or just after each pulse but also shows a quite linear progressive increase. On
the other hand, when the magnitude is 150 kV/m, the dynamic in-pulse conductivity
(σ p) seems to saturate at a value (0.32 S/m) which is quite close to the pretreatment
conductivity magnitude at high frequencies (~σ 1). In this case, the conductivity (σ 0)
increase induced by each pulse is quite significant, and it accumulates up to a
conductivity value a 42% higher than the original conductivity.
The above example (Fig. 7) illustrates an important fact regarding the use of
impedance measurements for the electroporation assessment: while dynamic
in-pulse conductivity measurements do not seem to be correlated with electropora-
tion efficacy, post-pulse conductivity may be. Note that, in Fig. 7b, the in-pulse
conductivity measurement would indicate that electroporation efficacy remains
constant, or even decreases, after the third pulse, whereas the post-pulse conductivity
would indicate that electroporation efficacy increases pulse after pulse although the
incremental efficacy diminishes, as it is known to happen when electroporation
a 5 b 3
4.5 2.8
reversible treatments
irreversible treatments 2.6 reversible treatments
4 irreversible treatments
2.4
3.5
2.2
σo /σi
σp /σi
3 2
2.5 1.8
1.6
2
1.4
1.5
1.2
1 1
0 50 100 150 200 250 300 350 400 0 50 100 150 200 250 300 350 400
E (kV/m) E (kV/m)
Fig. 8 Comparison between relative increases in tissue conductivity determined from measure-
ments of the dynamic conductivity at the end of the last electroporation pulse (a) and the
conductivity 50 ms after treatment (b) for electroporation treatment known to be irreversible and
treatments known to produce reversible electroporation in subcutaneously implanted sarcomas in
mice (Data extracted from Ivorra et al. 2009)
efficacy is assessed by other methods such as dye cellular uptake or cell killing by
irreversible electroporation.
The previous message is further reinforced by the results of the study presented in
Ivorra et al. (2009) which are partially displayed in Fig. 8. Briefly, for electroporation
treatments known to be reversible and for treatments known to be irreversible in the
tissue sample under study (subcutaneously implanted sarcomas in mice), the
increase in conductivity as obtained from measurements taken 50 ms after treatment
(σ 0) and from measurements of the dynamic conductivity at the end of the last
electroporation pulse (σ p) was compared. As it can be observed, the dynamic
conductivity at the last pulse (Fig. 8a) seems to be related with the field magnitude
of the applied pulses, but at 350 kV/m it produces similar values for reversible and
irreversible treatments. That is, from those measurements, it would not be possible to
distinguish the outcome of irreversible and reversible electroporation protocols. On
the other hand, it is possible to clearly distinguish irreversible and reversible pro-
tocols from conductivity measurements taken 50 ms after treatment (Fig. 8b).
Measurement Systems
In order to measure the passive electrical properties of a material, it is required to

induce a known current flow through the material while measuring the resulting
voltage drop or vice versa. These injected signals may perturb the material properties
if excessively large (e.g., by causing electroporation or Joule heating).
Under small signal conditions (i.e., when a small voltage or current is applied for
noninvasiveness), the interface between the electrodes and the biological sample
(electrode-electrolyte interface) can be coarsely approximated by a capacitance in
the order of 10 μF/cm2 for flat metallic electrodes. Consequently, for low-frequency
signals (e.g., <10 kHz) – which are the most sensitive to the electroporation
phenomena – the undesired interface impedance may distort the measurements if
no precautions are taken.
If sufficiently large voltages are applied, electrochemical reactions are forced at
the interface of the electrodes, thereby changing the electrode behavior from capac-
itive to conductive. In this case, the impedance of the interface impedance can be
considered negligible.
In electroporation applications in which the impedance before, in between, and
after the electroporation pulses wants to be measured, two different approaches are
generally employed.
Pulse-Based Systems
This approach simply consists in applying rectangular pulses with a moderate

voltage (few tens of volts) to initiate the electrochemical reaction at the interface,
thus minimizing their undesired interface impedance. It allows quasi-dc conductance
measurements by assessing the current and voltage drop in the target sample at the
end of the pulse.
Using signal processing methods and high-performance instrumentation, this
approach can also provide the impedance at higher frequencies by observing the
dynamic behavior at the beginning of the pulse (Pliquett et al. 2000).
This is the simplest setup to measure impedance outside the electroporation
pulses, requiring a single oscilloscope to monitor voltage and delivered current
(Fig. 9). On the other hand, this approach shows significant drawbacks. First, the
voltage magnitude needs to be high enough to generate electrochemical reactions at
the interface but low enough to avoid any kind of electroporation effects over the
cells. Undesired electroporation phenomenon during inter-pulse measurements not
only can induce an error in the impedance measurement but can substantially alter
the performed electroporation protocol. Furthermore, the energy delivered during the
measurements could increase the temperature introducing errors in the measure. In
addition, the electrochemical reactions required to overcome the interface impedance
could affect the viability of the sample.
Small Signal-Based Systems
This method consists in injecting a small sinusoidal current (in the low mA range) at
the specific target frequency. At the same time that the alternating current is injected,
the voltage drop generated within the sample is measured. Processing both signals,
impedance value at excited frequency can be extracted.
Pulse generator Oscilloscope

V
CH1 CH2
I
V
Fig. 9 Schematic representation of the pulse-based approach to measure the conductivity during
electroporation treatments. In this case, the treatment consists in a single pulse, and both a
pretreatment pulse and a posttreatment pulse are delivered
As mentioned before, the interface impedance may significantly interfere in the

impedance measurements at low frequencies (<10 kHz). A first approach to avoid
that consists on performing impedance measurements at different frequencies higher
than 10 kHz. Afterward, by fitting Eq. 5 to the observed data, it is possible to obtain
an approximate value of the impedance at 0 Hz.
A second approach to minimize the effect of interface impedance at low frequen-
cies is the four-electrode method also known as tetrapolar or Kelvin method.
The tetrapolar method uses two electrodes to inject the sinusoidal current, while
the generated voltage drop is measured by two additional electrodes (Fig. 10). Since
no current flows through the measurement electrodes, the voltage drop in the
electrode-electrolyte interface is minimized – ideally canceled – preserving the
measurement integrity.
Commonly, the same electrodes employed for impedance measurements are also
used to deliver the high-voltage electroporation pulses. In order to alternate the
physical connections between the both systems, fast switching relays are commonly
employed.
By using small-magnitude alternating signals, electrochemical reactions, temper-
ature increase, and possible electroporation effects are highly minimized.
Pulse generator Oscilloscope

V
CH1 CH2
I
V
Impedance analyzer
Small signal
I+ V+ V- I- I
Fig. 10 Schematic representation of common setup in electroporation applications to measure the

conductivity before and after the pulse using small signal with a tetrapolar configuration
Impedance Imaging Systems
In most electroporation based applications it would be desirable to assess the spatial

conductivity distribution inside the biological sample. A conductivity map of the
biological sample might be used to estimate the electric field distribution or the
outcome of the electroporation treatment. Research efforts are being conducted with
this purpose.
Electrical impedance tomography (EIT) is a noninvasive medical image tech-
nique based on impedance measurements that allows obtaining cross-sectional
images of the conductivity distribution of a volume. This approach commonly
uses a large number of surface electrodes located at the periphery of the target
volume. Monitoring of electroporation in an in vivo model has been demonstrated
(Granot et al. 2009). Using this technique, it is feasible to perform fast measurements
denoting high temporal resolution but obtaining low spatial resolution images.
In order to fulfill the spatial resolution needs, magnetic resonance electrical
impedance tomography (MREIT) has been proposed as an alternative to conven-
tional EIT. This technique uses a magnetic resonance imaging (MRI) scanner to
obtain the current density image (CDI) generated by an externally injected current
(see ▶ Chap. 33, “Current Density Imaging as Means to Follow Tissue Electropo-
ration”). Once the current distribution is obtained and knowing the applied voltage
between the electrodes, the electrical conductivity and electric field distribution
maps can be inferred using numerical algorithms. This is a promising technique
that can be also employed to determine the electric field distribution during tissue
electroporation (see ▶ Chap. 32, “Principles and Use of Magnetic Resonance Elec-
trical Impedance Tomography in Tissue Electroporation”).
Conclusions
Conductance and impedance measurements allow real-time monitoring of the elec-

troporation phenomenon dynamics during and after the pulses. Different phases of
permeabilization and of posterior recovery of the cell membrane can be observed.
These data has contributed to better understanding the electroporation phenomenon.
However, since impedance is affected by a number of different processes that take
place due to membrane permeabilization, particular care is needed when inferring
conclusions from impedance measurements.
A significant conclusion that can be drawn from the experimental data presented
in this chapter is that although in-pulse quasi-dc conductivity measurements allow
detecting the electroporation phenomenon and reveal its dynamics, they do not seem
to provide reliable information regarding the impact of membrane permeabilization.
On the other hand, parameters derived from impedance measurements performed
briefly after the electroporation pulses appear to follow the electroporation effects as
observed by other means. In particular, these post-pulse measurements appear to be
useful for predicting the outcome of the treatment in terms of the reversibility or
irreversibility.
Cross-References
▶ Biophysics and Metrology of Electroporation in Tissues

▶ Computational Approach for Electrical Analysis of Biological Tissue Using the
▶ Electric Pulse Repetition Rate: Sensitization and Desensitization

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Blood Flow Modifying and
Vascular-Disrupting Effects 39
of Electroporation
and Electrochemotherapy
Gregor Serša, Maja Čemažar, and Bostjan Markelc
Abstract
Electroporation/electropermeabilization (EP), achieved by application of electric
pulses to cells or tissues, induces reversible permeabilization of cell membranes
under suitable conditions, thus facilitating entry of exogenous molecules into
cells. EP of tissues in humans is feasible, efficient, and tolerable, and its most
advanced routine clinical use is electrochemotherapy (ECT), where cytotoxic
drugs are delivered to cells to treat tumors. It is increasingly used in different
tumor types, and the technology is being adopted for the treatment of deep-seated
tumors like liver, pancreas, and bone metastases and colon tumors. Although the
primary mode of action of ECT is the destruction of tumor cells due to the
increased cytotoxicity of chemotherapeutic drugs, it also has different effects on
the tissue level. It was shown that EP and ECT have blood flow-modifying effects
on normal as well as on tumor vasculature. Application of EP pulses to normal
blood vessels increases the permeability of affected blood vessels; causes a
transient vascular lock, i.e., decrease in perfusion; and modulates the diameter
of affected blood vessels. Similarly, in tumors, application of EP or ECT increases
the permeability of affected blood vessels and causes a vascular lock. In case of
normal blood vessels, these effects are short lived, whereas in case of tumor blood
G. Serša
M. Čemažar
B. Markelc (*)
ÜRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of
Oxford, Oxford, UK
e-mail: bostjan.markelc@oncology.ox.ac.uk

DOI 10.1007/978-3-319-32886-7_165
692 G. Serša et al.
vessels, the effects are long lasting and resolve more than 24 h after ECT.
Moreover, ECT has a direct cytotoxic effect on tumor endothelial cells; thus it
has a vascular-disrupting effect. This effect is differential, destroying only tumor
blood vessels and retaining the functionality of normal blood vessels surrounding
the tumor, which is important when ECT is used in well-perfused organs such as
the liver.
Keywords
Vascular lock • Vasoconstriction • Permeability • Electroporation • Electroche-
motherapy • Vascular-disrupting therapy
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 692
Effects of Electroporation on Normal Blood Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 694
Effects of Electroporation on Tumor Blood Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 696
Effects of Electrochemotherapy on Tumor Blood Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 698
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 703
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 704
Introduction
Electroporation/electropermeabilization (EP), i.e., application of electric pulses to

cells or tissues, induces reversible permeabilization of cell membranes under suitable
conditions, thus facilitating entry into the cells of non-permeant or poorly permeant
molecules (Rems and Miklavcic 2016). The list of molecules that can be delivered to
cells and tissues with EP is ever expanding and covers a broad range of different
molecules such as cytotoxic drugs, siRNA molecules, miRNA molecules, plasmid
DNA, naked DNA, etc. (Yarmush et al. 2014). If nucleic acids are being delivered
into the cells, then the application of EP is termed gene electrotransfer (GET) and can
be used for DNA vaccination or in the treatment of cancer (Gothelf and Gehl 2012).
However, the most widespread use of EP is to deliver cytotoxic drugs to tumor cells
for treatment of cancer, termed electrochemotherapy (ECT). ECT is an effective
treatment modality for the treatment of cancer, predominantly melanoma metastases
in the skin. It is increasingly used in other tumor types, and the technology is being
adopted for the treatment of deep-seated tumors like liver and bone metastases and
colon tumors. In the case of ECT, the most commonly used drugs are bleomycin and
cisplatin, where the application of EP to cells exposed to the chemotherapeutic drugs
potentiates their cytotoxic effect (Yarmush et al. 2014). The majority of ECT in
clinical settings is nowadays performed on cutaneous and subcutaneous tumors,
where although the target is the tumor, the surrounding skin is also exposed to the
electric field (Yarmush et al. 2014). Recently, ECT has also been used in the
treatment of visceral and deep-seated tumors such as colorectal cancer and colorectal
liver metastases as well as in the treatment of bone metastases (Yarmush et al. 2014).
39 Blood Flow Modifying and Vascular-Disrupting Effects of. . . 693
Although the primary mode of action of ECT is the destruction of tumor cells due to
the increased cytotoxicity of chemotherapeutic drugs, it also has different effects on
the tissue level. One of the reported effects on the tissue level, regardless of the tissue
type, is a blood flow-modifying effect, which was first reported on tumors (Sersa
et al. 1998) and later also confirmed in different normal tissues (Gehl et al. 2002).
The growth of solid tumors is heavily dependent on blood supply; thus as the
tumor grows, it forms an ever-developing vascular network. The new tumor blood
vessels are formed during a process termed angiogenesis, which starts once the
tumor reaches a size of 1–3 mm3 and is responsible for the formation of the majority
of tumor blood vessels. The growing tumor mass relies on the newly formed
vasculature to supply tumor cells with oxygen and nutrients (Folkman 2007). Due
to the rapid growth of tumors, the newly formed tumor vasculature is abnormal and
inadequately developed. Compared to normal vasculature, it has several character-
istic features: irregular and chaotic microvascular networks and branching patterns,
irregular shape and length of capillaries, large intercapillary distances, and shunts
and dead ends, all contributing to increased resistance to blood flow and to inefficient
and slow blood flow. Furthermore, tumor blood vessel walls are structurally and
functionally abnormal. They have a reduced coverage with smooth muscle cells and
poor sympathetic innervation leading to a poor local regulation of microcirculatory
blood flow. Tumor blood vessel walls are also “leaky” for large molecules, such as
plasma proteins, due to an abnormal basal membrane as well as endothelial lining,
which have loose intercellular connections and openings between adjacent endothe-
lial cells (Barker et al. 2015). This is further recapitulated in poor perfusion, leading
to the formation of hypoxic areas in the tumor, where the local oxygen concentration
is below 1%. High tumor cell proliferation rates and the abnormal structure of the
tumor vasculature lead to both chronic, diffusion-limited hypoxia and acute, tran-
sient, perfusion-limited hypoxia (Barker et al. 2015).
By supplying oxygen and nutrients, the tumor vasculature and angiogenesis are
essential for the survival and growth of solid tumors; thus they also present an
attractive target for treatment. The rationale behind targeting tumor vasculature
comes from the idea that disrupting one vessel can lead to the death of many
tumor cells fed by this vessel. Two types of therapies are currently being developed:
antiangiogenic therapies that target newly developing tumor vasculature, i.e., angio-
genesis, and vascular-disrupting therapies that target established tumor vasculature
with vascular-disrupting agents (Clémenson et al. 2013). Another important charac-
teristic of antiangiogenic and vascular-disrupting therapies is that they specifically
target tumor blood vessels, leaving normal vasculature intact, thus sparing the
functionality of the normal tissue surrounding the tumor (Clémenson et al. 2013).
Nowadays, ECT is performed in clinical settings on cutaneous and subcutaneous
tumors, where although the target is the tumor, the surrounding skin is also exposed
to the electric field (Yarmush et al. 2014). Moreover, ECT has also been used in the
treatment of deep-seated tumors such as colorectal cancer and colorectal liver
metastases as well as in the treatment of bone metastases (Yarmush et al. 2014).
Although the main mode of action of ECT is the destruction of tumor cells due to the
increased cytotoxicity of chemotherapeutic drugs, the effects of EP and ECT on
tumor blood flow may contribute to the overall treatment outcome. The effects of EP
on blood flow have been first observed in tumors (Sersa et al. 1998) and later
confirmed in various other tissues. Moreover, destruction of endothelial cells and
tumor blood vessels after ECT has also been reported (Cemazar et al. 2001; Sersa
et al. 2008), implying that ECT may have a vascular-disrupting effect, thus classi-
fying as a vascular-disrupting therapy. In this chapter, the blood flow-modifying
effects of EP and the vascular-disrupting effect of ECT, both on normal and tumor
tissue, will be discussed.
Effects of Electroporation on Normal Blood Flow
Although EP is a physical method and the response of exposed cells is similar

between different cell types (Rems and Miklavcic 2016), there are some effects of
EP that can only be observed on a tissue level. After the first observations of blood
flow-modifying effects of EP in tumors, liver, and muscle (Sersa et al. 1998; Ramirez
et al. 1998; Gehl et al. 2002), more in-depth studies on endothelial cells in vitro and
in blood vessels in vivo were performed.
The effects of EP on endothelial cells in vitro were thoroughly investigated using
human umbilical vein endothelial cells (HUVECs) (Kanthou et al. 2006) as well as
using HMEC-1 cells (Meulenberg et al. 2012). In both cell lines, EP increased the
permeability of endothelial monolayers that were grown on 0.4 μm pore size
polycarbonate transwell inserts coated with human fibronectin. The increase in the
permeability was dependent on the electric field strength. In the study of Kanthou
et al. (2006), HUVECs were exposed to 3 square-wave electric pulses (10–80 V/
25–200 V/cm; 1 Hz; duration 100 μs; 4 mm distance between electrodes), whereas in
the study of Meulenberg et al. (2012), HMEC-1 cells were exposed to 8 square-wave
electric pulses (0–500 V/voltage-to-distance ratio 0–685 V/cm; 1 Hz; duration
100 μs; 7.3 mm gap between electrodes). On a structural level, HUVEC and
HMEC-1 cells responded to electroporation by dissolution of actin fibers and
microtubules, and these effects were evident immediately (within 5 min) after
electroporation. Both actin fibers and microtubules were progressively dissociated
in response to increasing voltage applications. Additionally, in HUVECs, a loss of
VE-cadherin, a key protein responsible for the integrity of cell-to-cell junctions, was
evident from the junctions between adjacent endothelial cells immediately after
electroporation. When parameters for reversible EP were used, the observed changes
were transient in both cell lines and recovered to the pretreatment state within 2 h
after EP (Kanthou et al. 2006; Meulenberg et al. 2012). Taken together, these data
suggest that electroporation can permeabilize endothelial cells and disrupts the
organization of cytoskeletal network. Moreover, EP disrupts the barrier function of
the endothelium by interacting junctional integrity of endothelial cell-to-cell
junctions.
On the tissue level, the blood flow-modifying effects of EP were predominantly
studied in mouse muscle and skin (Gehl et al. 2002; Bellard et al. 2012; Markelc
et al. 2012). To determine the effect of EP on blood vessels in mouse muscle, three
different electrode geometries were compared: needle electrodes consisting of two

arrays of each four 0.5 mm needles placed 2 mm apart, with 4 mm between the
arrays, and two different plate electrodes, both consisting of two opposing metal
plates, separated by 4 mm or 5.3 mm. In all experiments, eight pulses with a
frequency of 1 Hz were delivered. Pulse duration was varied from 10 to 20,000 μs
and applied voltage-to-electrode distance ratio from 100 to 1,600 V/cm (Gehl et al.
2002). A different electrode geometry, two parallel stainless steel rods (length 5 mm,
width 1.3 mm) 4 mm apart, was used to study the blood flow-modifying effects of EP
on blood vessels in mouse skin. Two different pulse parameters were studied:
standard ECT pulses (8 square-wave electric pulses, 100 μs, 1,300 V/cm, 1 Hz)
(Bellard et al. 2012) and pulses used for gene electrotransfer, a combination of one
short, high voltage (HV) and several long, short voltage (LV) pulses (one pulse
(1,000 V/cm, 100 μs) followed by a 1 s lag and 8 pulses (140 V/cm, 50 ms, 1 Hz))
(Markelc et al. 2012). Immediately after EP, a decrease in the diameters of all
affected blood vessels was observed that lasted up to 8 min post-EP (Bellard et al.
2012; Markelc et al. 2012). This was followed by an abrogation of blood flow that
was demonstrated by a slowed filling up of blood vessels with intravenously (i.v.)
injected fluorescently labeled markers (Bellard et al. 2012; Markelc et al. 2012) and
by a reduced uptake of 51Cr-EDTA in muscle tissue when it was injected i.v. after EP
in comparison to the uptake of 51Cr-EDTA when it was injected before EP (Gehl
et al. 2002). A further proof that EP is followed by a delayed perfusion was the
observed delay in the time needed for a mouse paw, which was exposed to EP, to turn
blue after i.v. injection of a patent violet-blue dye, when compared to the paw that
was not exposed to electric pulses (Gehl et al. 2002). If the mice were pretreated with
reserpine, the observed effects could be substantially mitigated (Gehl et al. 2002).
Reserpine is a drug, which depletes nerve terminals of norepinephrine, and is known
to lower blood pressure and heart rate and at the same time diminish regional
vasoconstriction mediated by sympathetic fibers acting on smooth muscles. This
indicated that local vasoconstriction mediated by the sympathetic nervous system
plays a major role in the observed perfusion delays after EP (Gehl et al. 2002).
Furthermore, the use of a dorsal window chamber (DWC) model in mice together
with fluorescence intravital microscopy revealed that EP increased the permeability
of affected blood vessels for macromolecules (Bellard et al. 2012; Markelc et al.
2012). The DWC model is an experimental technique performed in living animals,
which enables a direct visual access to the blood vessel network of the skin by
intravital microscopy. The DWC consists of two titanium frames that are secured to
the double layers of the dorsal skinfold in a mouse with stainless steel screws and
sutures. Thereafter, one layer of the skin is excised, exposing the vasculature of the
remaining skin layer. When different sizes of fluorescently labeled dextrans are
injected i.v., which serve as vascular markers, this approach enables quantification
of changes in blood flow and permeability of blood vessels. Immediately after EP,
there was an increase in the permeability of blood vessels for all sizes of dextrans
(20, 70, and 2,000 kDa) that were used. The increase in permeability after EP was
different for different sizes of molecules, with a smaller increase in permeability for
2,000 kDa dextran compared to 70 or 20 kDa and lasted for up to 15 min. However,
it could still be detected even more than 30 min after EP (Bellard et al. 2012; Markelc
et al. 2012). The increase in permeability of blood vessels after EP was transient, and
the pre-EP state, where blood vessels are impermeable for large macromolecules,
was restored.
In summary, EP affects normal blood vessels in several ways. Firstly, immediately
after EP, there is a decrease in diameters of all affected vessels which is accompanied by
a “vascular lock.” This first phase is short lived, and it is resolved within 5 min after EP
and was attributed to the permeabilization of endothelial cells/smooth muscle cells
which induced a Raynaud-like phenomenon, a reflexory vasoconstriction of afferent
arterioles mediated by the sympathetic nervous system. Secondly, EP increases the
permeability of blood vessels for macromolecules of various sizes, and this increase in
permeability can persist up to 30 min after EP, although it is primarily resolved within
10 min after EP. The resolution of this second phase is supposed to follow the kinetics
of membrane resealing after EP (Gehl et al. 2002).
Effects of Electroporation on Tumor Blood Flow
Due to the rapid growth of tumors, the newly formed tumor vasculature is abnormal
and inadequately developed. Compared to normal vasculature, it has several char-
acteristic features: irregular and chaotic microvascular networks and branching
patterns, irregular shape and length of capillaries, large intercapillary distances,
and shunts and dead ends, all contributing to increased resistance to blood flow
and to inefficient and slow blood flow. Furthermore, tumor blood vessel walls are
structurally and functionally abnormal with a reduced coverage with smooth muscle
cells and poor sympathetic innervation. Tumor blood vessel walls are also inherently
“leaky” enabling extravasation of large molecules into surrounding tumor tissue
(Barker et al. 2015). The differences between the normal blood vessels and tumor
blood vessels lead to differences in their response to EP.
The first study to directly show blood flow-modifying effects of EP in tumors
used magnetic resonance imaging (MRI) to determine the distribution of i.v. injected
albumin-(Gd-DTPA)30 in the tumor after EP. Electric pulses were delivered with
stainless steel electrodes 8 mm apart (two stainless steel strips, length 35 mm, width
7 mm, with rounded corners), and the parameters used were eight pulses, voltage-to-
distance ratio 1,300 V/cm, 100 μs, and frequency of 1 Hz. Albumin-(Gd-DTPA)30
was i.v. injected 30 min after EP, and MRI revealed a complete abrogation of blood
flow in the murine fibrosarcoma SA-1 tumors growing subcutaneously in syngeneic
A/J mice. EP had a similar effect on VX2 carcinoma tumors grown in the liver in
rabbits, where the transient abrogation of blood flow after EP was determined with
i.v. injection of a fluorescent dye fluorescein after EP and illumination of the tumor
with a fluorescent Wood’s light. In this setting, the EP induced a transient abrogation
of blood flow that lasted more than 20 min (Ramirez et al. 1998). Taken together,
experiments performed in both studies indicated that in tumors, the transient abro-
gation of blood flow after EP last more than 30 min (Sersa et al. 1998; Ramirez et al.
1998), contrary to the results obtained on normal blood vessels where it is much
shorter lived (Gehl et al. 2002; Bellard et al. 2012).
These intriguing findings were further confirmed in subsequent studies. EP
reduced the patent blue stained area of the SA-1 tumors when patent blue was
injected i.v. after EP. In this study EP was achieved by 8 square-wave electric pulses,
delivered in two sets of four pulses in perpendicular directions, voltage-to-distance
ratio 1,300 V/cm, 100 μs, and frequency of 1 Hz, were delivered by two flat, parallel
stainless steel electrodes 8 mm apart (two stainless steel strips: length 15 mm, width
7 mm with rounded corners). Within 2 h after EP, the tumor perfusion was reduced to
30% but returned to near pretreatment value within 24 h (Sersa et al. 1999, 2002,
2008; Jarm et al. 2010). A similar observation was made when 86RbCl was used
instead of patent blue dye (Sersa et al. 1999). The scale of the reduction in the
perfused area stained with patent blue dye after EP was found to be dependent on the
amplitude of the electric pulses, with greater reduction observed at higher ampli-
tudes, as well as on the number of the applied electric pulses, with each subsequent
electric pulse decreasing the perfused/stained area (Sersa et al. 1999). Using dynamic
contrast-enhanced MRI, it was further confirmed that even 24 h after EP, there is still
a slight reduction in blood volume and perfusion in tumors that were exposed to EP
(Sersa et al. 2002). The initial rapid reduction of blood flow in SA-1 tumors after EP
was closely investigated with power Doppler ultrasonographic imaging and laser
Doppler flowmetry (LDF) (Sersa et al. 2008). Power Doppler ultrasonographic
imaging is an ultrasound-based detection method that can be used to assess the
gross tumor blood flow changes when a microbubble contrast agent is injected
i.v. before the detection of the signal. LDF can be used to monitor local microvas-
cular blood flow in the tissue by measuring the spread of the wavelengths of photons
emitted by a coherent laser source when the photons scatter on moving red blood
cells in capillaries (Sersa et al. 2008). With this approach, it was again confirmed that
the reduction in blood flow after EP happens immediately after the first pulse and it is
further reduced with every subsequent pulse. The rapid and abrupt initial decrease in
perfusion of tumors after EP was followed by a gradual but only partial reperfusion
that peaked 10–15 min after the delivery of electric pulses and did not continue
afterward, during the observation time of 60 min after EP (Sersa et al. 2008).
Moreover, the decrease in blood flow was accompanied by a decrease in partial
oxygen pressure (pO2) which was measured by electron paramagnetic resonance
(EPR) oximetry. EPR is a noninvasive method (after insertion of the paramagnetic
probe), which allows monitoring of pO2 repeatedly at the same point in the tissue
over long periods of time (Sersa et al. 2002, 2008). Using this method, it was shown
that after EP, the pO2 in the tumor immediately decreased to ~20% of the control
levels and remained at lower levels for at least 8 h after EP (Sersa et al. 2002, 2008).
The reduction in pO2 after EP recapitulated in an increase in hypoxic area in the
tumor (Sersa et al. 2008; Jarm et al. 2010).
A direct visual confirmation of the blood flow-modifying effects of EP on tumor
vasculature came from a study using a DWC model in SCID mice where human
colon carcinoma HT29 tumors were grown (Markelc et al. 2013). Using 70 kDa
fluorescently labeled dextrans, coupled with fluorescent macroscopy, the direct
observation of the effects of EP on tumor and normal blood vessels was achieved.
Electric pulses were delivered with two parallel stainless steel plate electrodes
(30 mm long, 6 mm wide, 6 mm distance between electrodes), and standard ECT
pulse parameters were used (eight pulses, voltage-to-distance ratio 1,300 V/cm,
100 μs, frequency of 1 Hz). EP resulted in an immediate abrogation of blood flow
in the tumor, i.e., vascular lock, which lasted for more than 60 min (Fig. 1).
Interestingly, the tumor-supplying arterioles responded to EP in the same way as
the normal vessels, with rapid vasoconstriction and increased permeability, thus
implying that this is the main cause of the immediate vascular lock observed after
EP. Furthermore, the blood flow in the blood vessels surrounding the tumor started to
return to normal level 10 min after EP, which was in line with a small increase in
reperfusion of tumors after EP (Sersa et al. 2008; Markelc et al. 2013). EP also
resulted in an increased permeability of tumor blood vessels for macromolecules and
a partial long-lasting decrease in perfusion (Markelc et al. 2013). Thus, EP has a
differential effect on normal and tumor blood vessels which originates from the
differences between normal and tumor vasculature.
Taken together, this data suggests that EP has a differential effect on normal and
tumor blood vessels. The extent of the blood flow-modifying effects of EP on tumor
blood vessels depends on the number of electric pulses and the amplitude of the
pulses. Similar to the response of normal blood vessels, the tumor blood vessels also
respond in two distinct phases. Firstly, EP immediately induces a rapid and profound
abrogation of blood flow, which is caused primarily by the vasoconstriction of the
tumor-supplying arterioles (Jarm et al. 2010; Markelc et al. 2013). After the vaso-
constriction of tumor-supplying arterioles is finished, which is ~15 min after EP,
there is a small reperfusion of the tumors, primarily on the tumor periphery;
however, it does not lead to a complete restoration of blood flow in the tumor. The
first phase is immediately followed by a second phase, where the increased perme-
ability of tumor blood vessels for macromolecules causes an increase in the inter-
stitial tissue pressure in the tumor, which coupled with non-normal characteristics of
tumor vasculature leads to a prolonged vascular lock effect, which lasts for more
than 1 h and is fully resolved only 24 h after EP (Sersa et al. 2008; Jarm et al. 2010).
A direct consequence of this prolonged second phase is a decrease in pO2 and
increase in the fraction of hypoxic regions in the tumor (Sersa et al. 2008).
Effects of Electrochemotherapy on Tumor Blood Flow
Nowadays, ECT is performed in clinical settings on cutaneous and subcutaneous

tumors, where while the primary target is the tumor, the surrounding skin is also
exposed to the electric field (Yarmush et al. 2014). Moreover, ECT has also been
used in the treatment of deep-seated tumors such as colorectal cancer and colorectal
liver metastases as well as in the treatment of bone metastases (Yarmush et al. 2014).
For a tissue to retain its normal functionality, it is essential to have an unperturbed
blood flow which supplies the tissue with nutrients and oxygen. Thus, especially
when treating well-perfused organs, such as the liver, the effects of ECT on normal
Fig.1 Illustration of a “vascular lock” and reperfusion of tumor blood vessels after EP and ECT.
Tumor blood vessels were visualized by fluorescence microscopy at 80 magnification. Control,
mice without treatment; bleomycin, mice treated with bleomycin only; EP, mice treated with EP;
ECT, mice treated with ECT. 70 kDa fluorescently labeled dextran was injected i.v. at different times
after the therapy (1, 10, 30, 60 min), and images were taken at designated times. Scale bar is 500 μm
(Reproduced under the terms of the Creative Commons Attribution License from Markelc et al.
(2013))
and tumor vasculature are of considerable importance. Although the main mode of
action of ECT is the destruction of tumor cells due to the increased cytotoxicity of
chemotherapeutic drugs, other cell types within the tumor, as well as in the sur-
rounding normal tissue, are also affected. Moreover, some of the effects of EP and
ECT are only detectable on the tissue level and do not exist on a single-cell level. The
destruction of endothelial cells and tumor blood vessels after ECT has been reported
(Cemazar et al. 2001; Sersa et al. 2008), implying that ECT may have a vascular-
disrupting effect which could contribute substantially to the overall treatment
outcome.
In in vitro setting, ECT increased the cytotoxicity of both most commonly used
chemotherapeutic drugs, bleomycin and cisplatin in HMEC-1 cells (Cemazar et al.
2001; Meulenberg et al. 2012), thus showing that in in vivo setting, the endothelial
cells are also destroyed by ECT. A combination of a 5 min drug exposure with
electric pulses increased the cytotoxicity by~tenfold for cisplatin and~5,000-fold for
bleomycin when compared to drug alone. To achieve electroporation, the cells were
placed between two parallel stainless steel electrodes (2 mm distance between
electrodes) connected to a Jouan GHT 1,287 electroporator and subjected to
8 square-wave electric pulses (100 μs, 1 Hz) (Cemazar et al. 2001). In HMEC-1
cells, an ECT protocol, combining bleomycin and exposing cells to 8 square-wave
electric pulses (0–500 V/voltage-to-distance ratio 0–685 V/cm; 1 Hz; duration
100 μs; 7.3 mm gap between electrodes), showed that within 10 min after ECT,
cells appear swollen and the formation of honeycomb-like actin bundles begins
(Meulenberg et al. 2012). This was followed by the formation of spindle-like cells
2 h after ECT and more densely packed F-actin and beta-microtubulin, which were
dependent on the amount of bleomycin and the electric pulse amplitude (Meulenberg
et al. 2012). Moreover, ECT with bleomycin dramatically increased the permeability
of endothelial monolayer, when cells were grown on 0.4 μm pore size polycarbonate
transwell inserts coated with human fibronectin. The increase in the permeability of
the monolayer was dependent on the concentration of bleomycin and electric pulse
amplitude (Meulenberg et al. 2012). All of the observed changes had an earlier onset
compared to EP alone, and the effect was more pronounced. Contrary to the EP
alone, all the observed effects were irreversible, and the majority of endothelial cells
were destroyed after ECT with bleomycin (Meulenberg et al. 2012). This implies that
ECT with cytotoxic drugs effectively destroys endothelial cells and that in in vivo
setting, blood vessels are also a target, making ECT a vascular-targeted therapy.
A mathematical model of electric field distribution in and around blood vessels
in a tumor revealed that the endothelial cells lining the blood vessels are actually
exposed even to up to 40% higher electric field strength than the surrounding
tumor cells (Sersa et al. 2008). Additionally, when chemotherapeutic drugs are
injected systemically, then the endothelial cells lining the blood vessel walls are in
fact exposed to the highest concentration of the drug, compared to any other cell in
the tumor. In several studies, the histological analysis of ECT-treated tumors
revealed that tumor endothelial cells are destroyed within 24 h after ECT and
the vascular lesion is formed leading to necrotic areas around the destroyed blood
vessels (Ramirez et al. 1998; Sersa et al. 2008; Jarm et al. 2010). The first
observation of blood flow-modifying and vascular-disrupting effects of ECT
with bleomycin (0.5 or 1 mg/kg injected i.v.) was made on VX2 carcinoma tumors
grown in the liver in rabbits, where the transient abrogation of blood flow after
ECT was determined with i.v. injection of a fluorescent dye fluorescein after ECT
and illumination of the tumor with a fluorescent Wood’s light. In this setting, the
ECT induced an irreversible abrogation of blood flow in the tumors that later lead
to vascular lesions and necrosis (Ramirez et al. 1998). An in-depth investigation
of tumor blood flow after ECT with cisplatin (4 mg/kg was injected i.v.) was
performed, using patent blue staining as a marker of perfusion on subcutaneous
SA-1 tumors in syngeneic A/J mice (Sersa et al. 2002). Patent blue was injected
i.v. after ECT. In this study EP was achieved by 8 square-wave electric pulses,
delivered in two sets of four pulses in perpendicular directions, voltage-to-dis-
tance ratio 1,300 V/cm, 100 μs, and frequency of 1 Hz, were delivered by two flat,
parallel stainless steel electrodes 8 mm apart (two stainless steel strips: length
15 mm, width 7 mm with rounded corners). Similar to when only EP was used, 2 h
after ECT, the tumor perfusion was reduced to 30%, but contrary to EP, it
remained below 40% of the pretreatment value up to the fifth day after ECT
(Sersa et al. 2002). When bleomycin (1 mg/kg injected i.v.) was used instead of
cisplatin, ECT had the same effect, reducing the perfused area to 20% within 2 h
after ECT and remaining under 30% of the pretreatment value for more than
2 days (Sersa et al. 2008). Using dynamic contrast-enhanced MRI, it was further
confirmed that 24 h after ECT, there was still a significant reduction in blood
volume and perfusion in SA-1 tumors that were treated with ECT with cisplatin
(Sersa et al. 2002). Furthermore, when blood flow in the SA-1 tumors treated with
ECT with bleomycin was measured with power Doppler ultrasonographic imag-
ing and laser Doppler flowmetry (LDF), it was confirmed that the reduction in
blood flow after ECT happens immediately after the first pulse is further reduced
with every subsequent pulse (Sersa et al. 2008). Similar to when EP alone was
used, the rapid and abrupt initial decrease in perfusion of tumors after ECT was
followed by a gradual but only partial reperfusion that peaked 10–15 min after the
delivery of electric pulses and did not continue afterward, during the observation
time of 60 min after ECT. Interestingly, this approach did not detect significant
differences in the tumor blood flow in the first minute after ECT when compared to
EP only (Sersa et al. 2008). Similar to when EP alone was used, the pO2 measured
with EPR was reduced to ~20% of pretreatment values immediately after ECT,
regardless whether cisplatin or bleomycin was used as the drug (Sersa et al. 2002,
2008). Contrary to when EP alone was used, where the pO2 values returned to
normal within 24 h after EP, ECT had a prolonged effect, and it took more than
48 h for the pO2 values to return to 100% (Sersa et al. 2002, 2008). The
discrepancy between the tumor perfusion determined with patent blue staining,
which was still at around 30% of pretreatment value at this time point, and the pO2
values that have already returned to normal was explained by the reduced number
of tumor cells that were killed by ECT, thus reducing the demand for oxygen,
which in return increased the pO2 in the tumor (Sersa et al. 2002, 2008; Jarm et al.
2010). The initial decrease in pO2 in the tumor also led to an increase in the
fraction of hypoxic areas of the tumor 90 min after ECT with bleomycin that
returned to the values similar to control tumors within 24 h after ECT (Sersa et al.
2008). A detailed histological analysis revealed rounding up of tumor endothelial
cells 1 h after ECT and an onset of their apoptosis 8 h after ECT (Sersa et al. 2008).
Regardless whether cisplatin or bleomycin was used as a drug, ECT resulted in a
substantial increase in the necrotic areas of the tumor (Sersa et al. 2002, 2008).
Thus, the data suggests that ECT has a vascular-disrupting action and that the
destruction of tumor blood vessels leads to a long-lasting perturbation of tumor
blood flow leading to tumor necrosis.
Intriguing results were found when using the DWC model in SCID mice where
human colon carcinoma HT29 tumors were grown (Markelc et al. 2013). Using
70 kDa fluorescently labeled dextrans, coupled with fluorescent macroscopy, the
direct observation of the effects of ECT on tumor and normal blood vessels were
achieved. Electric pulses were delivered with two parallel stainless steel plate
electrodes (30 mm long, 6 mm wide, 6 mm distance between electrodes), and
standard ECT pulse parameters were used (eight pulses, voltage-to-distance ratio
1,300 V/cm, 100 μs, frequency of 1 Hz). ECT resulted in an immediate abrogation
of blood flow in the tumor, i.e., vascular lock, which lasted for more than 60 min
(Fig. 1). The tumor-supplying arterioles responded to ECT in the same way as the
normal vessels to EP alone, with rapid vasoconstriction and increased permeabil-
ity for macromolecules, which was even higher than when EP was applied
(Markelc et al. 2013). Interestingly, although the blood flow in the blood vessels
surrounding the tumor started to return to normal levels 10 min after ECT, this was
not recapitulated in the tumor. The blood flow in the tumor did not return even
24 h after ECT (Fig. 2) (Markelc et al. 2013). ECT also increased the permeability
of tumor blood vessels for macromolecules, which was evident by leakage of
trapped 70 kDa fluorescently labeled dextran from the vessels. However, due to
the pronounced long-lasting vascular lock after ECT, the pool of molecules did
not get renewed; therefore the quantification was not possible (Markelc et al.
2013). Importantly, the normal blood vessels surrounding the tumor were not
destroyed after ECT and retained their functionality after the therapy (Markelc
et al. 2013).
In summary, this data suggests that ECT has an immediate effect on tumor blood
vessels, as well as a differential effect on tumor and normal blood vessels, destroying
the first and sparing the latter. This is of a special interest as it implies that ECT is a
vascular-disrupting therapy with selective action against tumor blood vessels. Sim-
ilar to the response of tumor blood vessels to EP, their response to ECT has two
distinct phases. Firstly, ECT immediately induces a rapid and profound abrogation of
blood flow, which is caused primarily by the vasoconstriction of the tumor-supplying
arterioles (Jarm et al. 2010; Markelc et al. 2013). After the vasoconstriction of
tumor-supplying arterioles is finished, which is ~15 min after EP, there are a small
reperfusion of the tumors and increased permeability of blood vessel for macromol-
ecules. This is limited primarily to the tumor periphery; however, contrary to when
EP alone is used, there is no further increase in tumor perfusion. Instead, ECT acts on
tumor endothelial cells by direct cytotoxic action, thus destroying them, which leads
to the destruction of tumor blood vessels and complete, long-lasting abrogation of
blood flow (Sersa et al. 2002, 2008; Markelc et al. 2013). A direct consequence of
this prolonged second phase is a decrease in pO2 and an increase in the fraction of
hypoxic regions in the tumor, which in the end leads to tumor necrosis and tumor
cure (Sersa et al. 2002, 2008).
Fig.2 Illustration of reperfusion of tumor blood vessels at 4–24 h after EP and ECT. Tumor blood
vessels were visualized by fluorescence microscopy. EP, mice treated with EP; ECT, mice treated
with ECT. 70 kDa rhodamine D-labeled dextran was injected i.v. at 4, 8, and 24 h after the therapy,
and images were acquired 5 min after the injection at 80 magnification. The images are repre-
sentative of different tumors. Tumors are marked with a dashed line. Scale bar is 500 μm
(Reproduced under the terms of the Creative Commons Attribution License from Markelc et al.
(2013))
Conclusions
Electroporation (EP) and electrochemotherapy (ECT) have blood flow-modifying

effects on normal as well as on tumor vasculature. The mechanisms behind the
response of tumor and normal blood vessels to EP or ECT are different, due to
different characteristics of tumor blood vessels compared to normal blood vessels. In
both types of the vasculature, EP or ECT increases the permeability of affected blood
vessels and causes a vascular lock, i.e., decrease in perfusion. In the case of normal
blood vessels, these effects are short lived, whereas in the case of tumor blood
vessels, the effects are long lasting and resolve more than 24 h after EP or ECT.
Moreover, ECT has a direct cytotoxic effect on tumor endothelial cells; thus it has a
vascular-disrupting effect. Importantly, this is a differential effect that destroys only
tumor blood vessels and retains the functionality of normal blood vessels surround-
ing the tumor that are also affected (Markelc et al. 2013). In clinical settings, this
phenomenon was also observed as the differential effect on tumor and normal tissues
in the terms of toxicity and in even larger normal vessels surrounding tumor that
were not damaged after ECT (Gehl and Geertsen 2000; Jarm et al. 2010; Yarmush
et al. 2014). This shows that ECT can be safely used even in a situation when large
blood vessels are close to the tumor and will be exposed to the electric field during
the treatment (Marcan et al. 2015).
Cross-References
▶ Antiangiogenic Gene Therapy

▶ Effects of Reversible and Irreversible Electroporation on Endothelial Cells and
Tissue Blood Flow
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bjc.6604168
Tissue Ablation by Irreversible
Electroporation 40
Andrea Rolong, Boris Rubinsky, and Rafael V. Davalos
Abstract
The phenomenon of electroporation can be traced back to the eighteenth century
when red spots on skin (Lichtenberg figures) were observed in the areas where
electric fields were applied. Once the cause of this occurrence was understood and
controlled, induction of electric fields was achieved; the use of pulsed electric
fields was adopted in the areas of food and water sterilization to kill microbial
organisms. Applications in biomedicine soon followed where electric fields
began to play a role in the transport of and into biological material employing a
technique known as reversible electroporation in which temporary cell membrane
destabilization is achieved followed by complete recovery of membrane integrity.
Irreversible electroporation (IRE) induces cell death presumably through a loss of
homeostasis by using an energy regime substantially higher than that of reversible
electroporation. When pulse parameters are carefully designed, IRE can be used
to ablate large volumes of tissue in such a manner that it does not induce
significant traditional thermal damage. This application of IRE for the treatment
of disease has been widely investigated through in vitro studies, in vivo animal
studies, and treating human patients through clinical trials. Its nonthermal mech-
anism to induce cell death makes it an attractive modality to safely treat
unresectable tumors. When careful treatment planning is performed to account
for tumor morphology, tissue heterogeneity, and parameters for pulse delivery, it
has been shown to successfully treat cancerous tumors safely. Clinical use of IRE
includes the treatment of liver, lung, pancreas, and prostate cancer among others.
IRE has found tremendous promise for the treatment of pancreatic and prostate
A. Rolong • R.V. Davalos (*)

Department of Biomedical Engineering and Mechanics, Virginia Tech – Wake Forest University
School of Biomedical Engineering and Sciences, Blacksburg, VA, USA
e-mail: davalos@vt.edu
B. Rubinsky
Department of Mechanical Engineering, University of California, Berkeley, Berkeley, CA, USA

DOI 10.1007/978-3-319-32886-7_166
708 A. Rolong et al.
tumors. Further research and optimization of this technique point to an acceler-

ated adoption for its clinical use to treat various types of cancer and other
debilitating conditions.
Keywords
Numerical modeling • Treatment planning • Clinical trials • Cancer therapy
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 708
Irreversible Electroporation in Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 709
Prostate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 711
Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712
Lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712
Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713
Pancreas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 714
Pretreatment Planning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 715
Thermal Damage Consideration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 716
Tissue Heterogeneity and Implications for IRE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 717
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 718
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 719
Introduction
Electroporation is the phenomenon in which cell membrane permeability to ions and

otherwise non-permeant molecules is increased by exposing the cell to short, high-
electric field pulses. Such increase in permeability is related to the formation of
defects or pores in the cell membrane; this led to the adoption of the term electro-
poration to describe this occurrence. Irreversible electroporation, or IRE, is a term to
describe the effect of pulsed electric fields (PEFs) leading to cell death. The
nonthermal mechanism of inducing cell death also led to the use of the acronym
N-TIRE or nonthermal irreversible electroporation. These terms were introduced to
describe the use of pulsed electric fields as a tissue ablation modality; variations in
the parameters of these types of pulses had been in use for food and water steriliza-
tion and for transport of and into biological material for many years before identi-
fying their capability to kill undesired cells and tissues as in the treatment of
cancerous tumors. The use of PEFs to deliver drugs into cells is termed electro-
chemotherapy (ECT), the one used to introduce genetic material is known as
electrogenetransfer (EGT), and the one used to bring cells into close proximity in
order to join them as a single entity is referred to as electrofusion. All of these make
use of a set of pulse parameters categorized as reversible electroporation (RE). The
types of pulses used in food and water sterilization could be described as IRE type of
pulses in the sense that they induce cell death of microorganisms such as bacteria,
although pulses used in these areas usually employ a much higher magnitude than
the one used for pulses to treat cancerous tissue. The smaller size of bacteria,
40 Tissue Ablation by Irreversible Electroporation 709
compared to mammalian cells, makes them more difficult to kill with clinical IRE
pulses; hence, a higher magnitude is required for these cases.
R.C. Lee et al. investigated the nonthermal mechanisms of tissue death by
electrical trauma in a 1988 publication that describes cell membrane rupture and
its role in the pathogenesis of electrical trauma (Lee et al. 1988); this is only one of
many publications by R.C. Lee on the mechanisms of cell death by electric fields.
The authors noted that the transmembrane potential induced by a particular electric
field depends also on cell size and orientation in the field. The report describes
irreversible changes in the structure of skeletal muscle cells isolated from rats and
exposed to 1-ms pulses of 20–300 V/cm of electric field strength with a 30-s interval.
The researchers witnessed a drop in the resistance of skeletal muscle tissue after
exposure to electric fields similar to the cell membrane disruption and drop in
resistance produced by exposure to a permeabilizing chemical agent. Since the
temperature increase from exposure to these electric fields was negligible, they
concluded that membrane disruption was not induced by Joule heating but rather
by electroporation.
Irreversible electroporation was initially considered as the upper limit of revers-
ible electroporation and, as such, something to be avoided since the aim of reversible
electroporation is to create temporary pores in the cell membrane while maintaining
cell viability. In 2003, R.V. Davalos and B. Rubinsky described the use of classical
irreversible electroporation type of pulses (5 μs or longer) as a tissue ablation
method. With their paper in 2005, R.V. Davalos, L.M. Mir, and B. Rubinsky
published the founding paper suggesting that IRE can be applied in such a manner
to induce cell death and ablate tissue while mitigating deleterious thermal effects
(Davalos et al. 2005). This approach would avoid the deleterious effects of massive
necrosis and spare important tissue structures, such as the extracellular matrix, to
facilitate healthy tissue regeneration. This paper laid the foundation that IRE can be
applied as an independent tissue ablation modality.
This approach of delivering the IRE pulses to minimize what is traditionally
considered thermal damage marked the beginning of a series of studies in the use of
IRE as a technique to treat cancerous tumors, which are considered otherwise
inoperable due to their proximity to sensitive structures. The goal of this chapter is
to present a review of the use of pulsed electric fields to ablate tissue; in other words,
a review of the use of IRE as a minimally invasive surgical technique.
Irreversible Electroporation in Medicine
A train of pulsed electric fields causes an increase in the transmembrane potential of

cells which, above a certain threshold previously reported to be around 1 V in
magnitude, causes enough damage to the cell that it leads to cell death.
The technology is an attractive alternative for the treatment of tumors with
intricate morphologies such as those with cancer cells engulfing nerves and major
blood vessels. The nonthermal mechanism is able to kill the cancerous tissue while
preserving the structures that comprise the extracellular matrix and maintaining
viability of nerves and vascular cells.
During clinical delivery of pulsed electric fields, it has been reported that there is a
temporary decrease in blood flow in vessels that are in close proximity or within the
treatment area; this aids in the successful eradication of the malignant cells near
blood vessels as compared to the more commonly used thermal ablation therapies
which may leave some viable cancer cells as a result of the thermal sink effect from
the blood flow in a major blood vessel nearby. Thermal therapies could also either
destroy a neighboring vessel or render it unviable.
The first clinical use of irreversible electroporation involved the treatment of a
patient with prostate cancer. Today patients with liver, kidney, pancreatic, and lung
tumors have also been treated as part of various clinical trials. The advantages of IRE
over other more commonly used cancer treatment modalities such as chemotherapy,
radiotherapy, photodynamic therapy, or thermal ablation are currently under inves-
tigation. It is important to keep in mind that all of these techniques are usually
considered when surgical resection is not a viable treatment option or when a
combination of resection and other therapies is required for control of the disease.
IRE treatment has been successfully performed through laparotomy (Martin et al.
2013), laparoscopy (Cannon et al. 2013), and percutaneous incision (Narayanan
et al. 2012). The surgical procedure known as laparotomy involves making an
incision into the abdominal cavity by first cutting through the abdominal wall;
laparoscopy, on the other hand, involves a much smaller incision only big enough
to insert a fiber-optic instrument (lighted tube) in order to visualize the organs in the
abdomen – similar small incisions can be made in other locations of the abdomen to
insert the surgical instruments needed for a particular type of procedure; and lastly, in
a percutaneous procedure, access to inner organs or other tissues is gained through
needle puncture of the skin rather than by making an incision with the use of a
scalpel. From these, percutaneous treatment has shown the least amount of compli-
cations from IRE procedures due to its less-invasive nature, which in turn promotes a
faster recovery. This led to a particular study on percutaneous IRE of LAPC using a
dorsal approach (Scheffer et al. 2015a) motivated by the impediment to use the
ventral approach on the 67-year-old patient with a stage III pancreatic tumor.
Electrode placement in the 5-cm tumor located in close proximity to collateral
vessels and duodenum was performed through the dorsal approach since the most
commonly used ventral approach would pose high risk of complications from the
procedure alone. This is the first report on the success of this particular dorsal
approach and thus provides an alternative in IRE method of delivery for patients
with tumors of intricate morphologies involving critical structures.
Percutaneous IRE treatment can make use of other types of imaging modalities
that do not specifically include an endoscope. E.W. Lee et al. showed the potential of
IRE to produce an ablation zone with sharper delineation than other ablation
modalities (Lee et al. 2010) in their 2010 review of IRE procedures using image
guidance. Ultrasound can be used to place the needles in the target location;
computed tomography (CT) and magnetic resonance imaging (MRI) can be used
pre- and post-IRE to visualize the tumor region for treatment planning and to confirm
successful ablation. Histological analysis of their samples showed a demarcation

between the ablated and live tissue in the micrometer range (on the order of 1–2 cell
thickness). This characteristic of IRE makes it particularly attractive to treat tumors
in close proximity to nerves, major blood vessels, and bile ducts where all the
malignant cells can be successfully killed while maintaining these critical structures
viable.
A standard IRE procedure involves the use of general anesthesia to sedate the
patient plus administration of a neuromuscular blocking agent to prevent
uncontrolled and severe muscle contractions caused by the electric current from
PEF treatment. Electrode needles (bipolar or unipolar) are introduced into and/or
adjacent to the tumor tissue using imaging guidance. The distance between the
electrodes is selected beforehand during pretreatment planning and confirmed by
imaging right after placement to ensure proper placement – this entails proper
distance and parallel positioning between electrodes so that sufficient current flow
is generated and IRE is achieved. An ablation cycle consists of a series of high-
voltage pulses of direct current delivered with a particular interval (usually 1
s synchronized to the patient’s heartbeat to avoid interference with normal cardiac
rhythm) in between pulses to allow for recharging. A complete ablation cycle usually
lasts less than 2 min. Electrodes may be repositioned, using image guidance once
again, if this is deemed necessary to cover the full tumor region plus a margin of
error that usually ranges from 0.5 to 1 cm. Imaging is used after the procedure and
during follow-up evaluations at various time points to determine the success of the
treatment and the extent of disease recurrence or remission.
Prostate
Prostate cancer patients are ideal candidates to benefit of all the advantages provided
by an IRE procedure. Even though most men diagnosed with the disease will not die
from it, meaning it has a low mortality rate especially when detected at early stages,
they are oftentimes worried about the side effects of treatment which could include
impotence, incontinence, pain, or loss of sensation. Some patients choose to delay
treatment or even forgo completely in order to avoid detrimental side effects. IRE
offers a valuable treatment option for these patients where the negative side effects
can be circumvented and successful tumor tissue ablation can be achieved.
In 2007 G. Onik et al. applied in vivo IRE to canine prostates by means of
percutaneous needle electrodes placed under ultrasound guidance (Onik et al. 2007).
Macroscopic observation of the induced lesions revealed a very distinct fine zone of
transition from normal viable tissue to complete necrosis. Nearby structures such as
the urethra, vessels, nerves, and rectum were not affected by the IRE procedure
despite the fact that these areas were deliberately included in the region covered by
the high electric field. This seminal paper highlighted the potential of IRE to treat
tumors in areas not attainable with other focal ablation techniques or via resection.
In the first human clinical trial, 16 patients with prostate cancer were treated with
IRE. G. Onik and B. Rubinsky reported on this in 2010 (Onik and Rubinsky 2010)
where a series of outpatient procedures were performed. All patients were continent
immediately, and all patients who were potent before the procedure were still potent
after the procedure. Two patients who had bilateral areas treated required 6 months
for full return of potency. Color Doppler ultrasound showed intact flow within the
neurovascular bundle immediately after the procedure. Postoperative biopsies taken
from the area of previously known cancer in 15 patients showed no evidence of
cancer. There was one patient with a negligible PSA who refused a postoperative
biopsy and one in whom a micro-focus of Gleason 6 cancer was found outside the
treated area. This patient was successfully retreated with focal cryosurgery. An
important finding from this study is that vascular elements were patent and intact
nerve bundles were observed; these structures were surrounded by necrotic and
fibrotic tissue as a consequence of IRE treatment.
Liver
In 2011 Thomson et al. performed another clinical trial that included 38 patients in
Australia (Thomson et al. 2011). The patients presented with advanced liver, lung, or
kidney tumors where a total of 69 tumors were treated. Complete tumor ablation was
observed in 66% of the treatments with the highest percent of failure (or incomplete
treatment) observed for lung and kidney tumors. Both unipolar and bipolar elec-
trodes were used for treatments with 90 pulses of 70-μs duration, delivered in 9 sets
of 10 pulses per site of delivery. Complications of treatment included cardiac
arrhythmias; after four patients experienced this particular problem,
ECG-synchronized delivery of pulses was then adopted, but two additional patients
experienced arrhythmias despite this synchronization. 15 of 18 treatments resulted in
complete tumor ablation for patients with hepatocellular carcinoma. This early
success suggested that liver tumors may be an optimal target for IRE ablation.
Lung
In the study presented above by Thomson et al. (Thomson et al. 2011), there was one
patient with non-small-cell carcinoma (one tumor) and three patients with lung
metastases from colorectal carcinoma (five tumors). None of these patients treated
with IRE had a satisfactory tumor response, and they all presented with progressive
disease when assessed by the 3-month time point. A biopsy from one of the patients
showed coagulative necrosis of a portion of the tumor with viable cancerous tissue at
the margin of the treated lesion. All four patients experienced transient ventricular
arrhythmia, one patient presented transient supraventricular tachycardia, and one
patient required cardioversion (use of drugs or electricity to return an aberrant
cardiac rhythm to its normal state) as a response to atrial fibrillation. Pneumothorax,
or collapsed lung, was observed in two out of the four patients, but no further
treatment was required and the condition resolved spontaneously. This early study
showed some of the challenges that need to be overcome in order to improve

treatment outcome from IRE in lung cancer.
In 2012, a study by Usman et al. reported on the use of IRE to treat two patients
with lung neoplasms that had been previously deemed unresectable (Usman et al.
2012). One of the patients presented with an increase of the right suprahilar mass
with contrast enhancement CT, suggesting tumor growth reported 2 months after the
procedure. Moderate parenchymal hemorrhage was observed during the procedure,
and at the 9-month follow-up, it was suggested that the tumor had invaded the
trachea. Cancer continued to progress, and the patient succumbed to the disease
within a year post-IRE. The other patient was reported to still be alive 2.5 years after
the procedure with no major complications described. The authors explain that
challenges still remain in the use of IRE to treat lung tumor masses where a possible
reason could be an increase in impedance of current flow due to the low density of
lung tissue. Air in the lungs could further decrease energy deposition within the
tumor by providing a bypass circuit around the tumor. It is clear that further research
is needed to optimize IRE treatment of lung cancer through collaboration between
engineers and clinicians. It can be argued that these studies were limited because the
probes (i.e., electrodes) themselves were not designed for lung treatments, and thus
surgical probes need to be tailored for this particular application.
Kidney
IRE has also been used to treat renal tumors. Aside from the first study aforemen-
tioned by Thomson et al. (2011) and an almost simultaneous study also published in
2011 by Pech et al. (2011), the largest study on the use of IRE to ablate renal tumors
was reported by Trimmer et al. (2015) in which 20 patients with T1a renal carcinoma
(n = 13), indeterminate masses (n = 5), or benign masses (n = 2) underwent
CT-guided IRE. Mean tumor size was 2.2 cm 0.7. All patients were successfully
treated with no major complications although seven patients presented with minor
complications that included difficult-to-control pain, urinary retention, and self-
limiting perinephric hematomas (i.e., internal bleeding around a kidney). Incomplete
ablation caused two patients to undergo salvage therapy at 6 weeks; all 15 patients
imaged at 6 months had no evidence of recurrence; and only one patient was
observed to experience recurrence at 1 year post-IRE. This study reinforced the
safety of the use of IRE to treat renal tumors despite suffering from selection bias –
all tumors were small in size and distant from major blood vessels or critical
structures. Findings also showed that IRE procedures on the kidney can have a
risk of injury to the ureter or blood vessels in the area which indicates that additional
optimization of treatment delivery is required.
A recent review on the use of IRE to treat renal tumors was published in early
2016 (Narayanan and Doshi 2016). The authors warrant consideration when apply-
ing the same parameters used for IRE ablations of the liver to treat renal masses as
the tissue response might be very different between these. Two phase I and II
prospective clinical trials plan to assess the safety and efficacy of IRE in the
treatment of renal cancer (Wendler et al. 2015). Wendler et al. reported on the
“IRENE trial” that will evaluate completeness of ablation in renal tumors at
28 days after percutaneous IRE by performing histopathological analysis of tissue
samples from partial resection. MRI will be performed after 2, 7, and 28 days from
IRE procedure, and image analysis will be done to compare and evaluate any
changes (Wendler et al. 2015). Renal cell carcinoma tumors of 15–17 mm were
treated with IRE; results describe a new distribution of the ablation zone with
negative margins for all three tumors, complete tumor necrosis for two of them,
and residual tumor of unclear malignancy in the center of one of them. Results will
continue to be gathered as part of these clinical trials, which will help in the
optimization of IRE parameters to successfully treat renal tumors. If it is concluded
that IRE does not present significant advantage over other more commonly used
ablation techniques, a niche application of IRE could include central renal tumors in
close proximity to blood vessels and collecting system in which the nonthermal
mode of ablation can be exploited (Narayanan and Doshi 2016).
Pancreas
IRE has also been used to treat locally advanced pancreatic adenocarcinoma
(LAPC). A study from 2012 evaluated the efficacy of IRE for downstaging and
control of this disease (Narayanan et al. 2012). IRE was performed in 14 patients
where the mean tumor size was 3.3 cm. One patient received two treatments and
three patients presented with metastatic disease. Results showed only one case of
pancreatitis that was attributed to a complication from IRE treatment. Post-IRE scans
immediately after treatment and 24 h later that showed vascular patency was
preserved. Patients with metastatic disease eventually succumbed due to disease
progression, but two patients who were successfully treated did not show any sign of
disease for 11 and 14 months.
Two subsequent multicenter, prospective institutional review board (IRB)-
approved evaluations reported in 2014 and 2015 focused on the use of IRE to treat
locally advanced cancers. The study published in 2015 (Martin et al. 2015) presents
the case of 200 LAPC patients (stage III), comprising the largest evaluation to date,
who underwent either IRE alone (n = 150) or pancreatic resection combined with
IRE for treatment margin enhancement (n = 50). IRE was successfully completed in
all patients; they had initially undergone induction chemotherapy, and 52% were
additionally given chemoradiation therapy for a median of 6 months prior to IRE.
Six patients experienced local recurrence as reported with a median follow-up of
29 months. A median overall survival of 24.9 months ranging from 4.9 to 85 months
was calculated for this group of patients. This constitutes a substantial increment to
the median of 6–13-month survival for patients treated with alternate therapies and
demonstrates that IRE, with adjuvant chemotherapy, is the combination therapy that
induces the longest-term LAPC disease control currently reported in the literature
(Martin et al. 2015).
Pretreatment Planning
Computational modeling has revolutionized the level of precision and targeted

treatment that can be afforded by ablation therapies among many other surgical
procedures. IRE can be successfully delivered with a high degree of accuracy, thanks
to the use of algorithms that recreate the patient’s anatomy and simulate the chosen
set of parameters for the treatment with pulsed electric fields. A common pre-
treatment plan includes MRI or CT scans of the area of interest, 3D reconstruction
of the overall anatomy of this area, meshing (representation of the overall geometry
as a set of finite elements for computational analysis), iterative simulations using a
finite element model (FEM) that evaluates the total number of electrodes, electrode
geometry and placement, voltage, number of pulses, and delay between pulses that
will give the most comprehensive treatment of cancerous tissue while sparing as
much healthy tissue as possible.
Two of the main characteristics that are usually evaluated through computational
modeling of IRE treatments are the thermal and electric field distribution. This is
done in order to optimize all the parameters that would result in complete engulfment
of the cancerous tissue by an electric field above the threshold for cell death for the
particular cell type, while minimizing cell death through thermal damage and sparing
healthy tissues and critical structures.
In 2011, two separate studies from R.V. Davalos’ group reported on the first
clinical cases of the use of IRE to treat naturally occurring cancerous tissue in animal
patients. P.A. Garcia et al. (2011) used nonthermal irreversible electroporation and
adjuvant fractionated radiotherapy to treat a canine patient with an inoperable,
spontaneous malignant intracranial glioma. No adverse effects were reported after
an IRE protocol with 50-μs pulses delivered in 20 pulse trains with a voltage-to-
distance ratio of 1,000 and 1,250 V/cm that resulted in a ~ 75% reduction of tumor
volume after 48 h and allowed for subsequent fractionated radiotherapy which began
on the 16th day after IRE treatment. Complete remission was achieved for 149 days
at which point death ensued from radiation encephalopathy. R.E. Neal II et al. (2011)
reported on the treatment of a canine patient with a focal histiocytic sarcoma of the
coxofemoral joint that was causing sciatic neuropathy and bilateral pelvic limb
lameness. The original size of the neoplasm was greater than 136 cc located in
heterogeneous tissue composed of bone, muscle, arteries, and the sciatic nerve.
Complete remission was achieved 6 months after IRE treatment and chemotherapy.
Both relapsed and suspected chemoresistant tumor was completely ablated using
IRE alone with minimal damage to healthy tissues. The cancer has been in complete
remission for over 4 years which may suggest a synergistic immune response.
Kos et al. reported on the safe ablation of liver tumors in close proximity to major
blood vessels as a result of careful and successful treatment planning (Kos et al.
2015). A 48-year-old patient that had undergone a right hemihepatectomy (removal
of half the liver – the right side in this case) to remove the primary tumor presented
with metastatic disease near the remaining hepatic vein. The manufacturer of the IRE
pulse generator used in this study recommends a voltage-to-distance ratio of
1,500–1,700 V/cm for the treatment of tumors of the liver area; the authors used
this when developing the numerical simulation used as part of treatment planning
which gave an output of 19.9 cc of treated volume (tissue covered with at least
900 V/cm) and closely matched the actual clinical treatment. Complete tumor
regression and hepatic vein patency was achieved/retained after IRE treatment
with the patient being deemed recurrence-free at the 6-month time point post-IRE.
From the temperature rise observed in the electrodes during this study, the authors
recommend clinicians avoid placing the electrodes at a distance of less than 4 mm
away from critical structures (when using the settings recommended by the manu-
facturer) in order to prevent thermal damage to these from IRE procedure.
Thermal Damage Consideration
In electroporation-based therapies, there is a nonlinear increase in the bulk tissue

conductivity, as a result of the increased flow of ions through cells with
permeabilized membranes, after exposure to a high enough electric field. In 2014,
Garcia et al. evaluated the effects of the number of pulses and the electric field
strength on the percentage of cell death induced by irreversible electroporation
protocols (Garcia et al. 2014). The results showed that an increasing number of
pulses increase the probability of cell death induced by electroporation effects, but it
can also increase the probability of cell death induced by thermal damage as a result
of an increase in Joule heating. The study uses a statistical model of IRE cell death in
tissue previously reported by Golberg and Rubinsky in 2010 (Golberg and Rubinsky
2010). An interesting observation from the development and implementation of this
statistical model is the sharper delineation between electroporated and
non-electroporated tissue that can be afforded by an increase in the pulse number;
a sharp delineation between treated and untreated tissue is one of the main selling
points of this particular therapy since it allows for the treatment of malignant cells
and tissues in the vicinity of vessels, nerves, and bile ducts, which oftentimes
determine whether a tumor is deemed unresectable or untreatable by the more
commonly used thermal ablation techniques. These findings reiterate the importance
of parameter optimization during pretreatment planning in order to capitalize on the
great advantages of IRE while minimizing any deleterious effects to healthy tissues
and critical structures.
Most researchers cite necrosis due to excessive permeabilization and consequent
disruption of the osmotic balance as the killing mechanism of electroporation. In the
late 1990s, two independent papers were published in which it was shown in vitro
that electroporation not only caused necrosis but it also induced cell death with
features compatible with apoptosis (Hofmann et al. 1999; Piñero et al. 1997). In both
papers, it is reported that electroporation leads to chromosomal DNA fragmentation,
which is considered to be an explicit indication of late apoptosis. Another interesting
detail from the paper by J. Piñero et al. (1997) is a statement in the abstract where the
authors mention the possibility of killing tumor cells by electroporation, as opposed
to electrotherapy. This particular topic is not further developed in the manuscript, but
since in the introduction the authors refer to the use of electrochemotherapy for
treating solid tumors, it may indicate that the comment in the abstract was actually
proposing the use of irreversible electroporation for treating tumors. The mentality
within the scientific community was that irreversible electroporation would induce
tissue necrosis and not apoptosis; hence, it was considered a negative side effect of
reversible electroporation and something to be avoided in order to prevent instanta-
neous necrosis leading to massive tumor necrosis and possible ulceration (Miklavcic
et al. 2000).
B. Al-Sakere et al. performed in vivo IRE of tumors subcutaneously inoculated in
mice. The investigation evaluated multiple electroporation protocols to determine
the optimal parameters that led to complete regression of tumors inoculated in mice
(Al-Sakere et al. 2007). Optimal results were obtained by using protocols that
increased the number of pulses from the traditional 8 pulses delivered at 1 Hz for
ECT. Their best results consisted of 80 pulses of 100 μs in duration at 0.3 Hz
(an interval of 3.3 s between pulses with a delay between each set of 20 pulses)
with a voltage-to-distance ratio of 2,500 V/cm. With this protocol, complete regres-
sion was achieved in 12 out of 13 treated tumors, and no thermal effects were
observed. The results from this study demonstrated that it was attainable to increase
the number of pulses without inducing thermal damage and provided the foundation
for pulse parameters used for IRE in the clinic.
Although the importance to deliver protocols to mitigate traditional thermal
damage while maximizing the IRE zone has been described since the early literature,
the argument for a nonthermal mode of ablation for IRE has been previously
challenged. However, the consensus still favors that when the procedure is
conducted properly, the vast majority of cell or tissue death is induced by nonthermal
mechanisms. It has been observed that there may be some degree of thermal damage
to the tissue (necrotic tissue) directly in contact with the electrodes and in their
immediate vicinity; this is especially the case for the tissue in contact with the
charged electrode. From experimental results and computational simulations, it has
been observed that this necrotic area is then surrounded by cells undergoing apo-
ptosis or another form of cell death not caused by thermal damage. It is important for
clinicians and engineers to work together to establish the range of these zones such
that the critical structures that are being spared are not within the innermost region.
Therefore, as long as clinicians and researchers are aware of the different types of
cell death that can be induced by IRE procedures, tumors that were once considered
untreatable and unresectable may now find a legitimate contender in irreversible
electroporation procedures.
Tissue Heterogeneity and Implications for IRE
Despite the advantages from computational modeling during pretreatment planning,

the variability in the types of cells and tissues that comprise the tumor environment
provides an added challenge in simulating and successfully predicting the effects of
pulsed electric fields on each type of cell (Neal 2nd and Davalos 2009). Each cell and
tissue type has its own electrical properties and thus can experience a different effect
to the same applied electric pulses compared to its neighboring cells. One of the
contributing factors to tissue heterogeneity comes from differences in cell size and
geometry; IRE has been shown to be partially dependent on cell size where larger
cells might require lower electric field thresholds to undergo cell death. Moreover,
the electrical properties of cells and tissues do not remain constant throughout the
duration of an IRE treatment but instead vary nonlinearly (Ivorra and Rubinsky
2007). Attempting to take into account all of these changes, which are particular to
every type of cell or tissue, when developing numerical models for pretreatment
planning can become a very difficult task.
A second-generation platform for electric pulse delivery is investigating the use
of bipolar pulses delivered at higher frequencies than conventional IRE. Early results
suggest that the concerns with tissue heterogeneity may be circumvented with this
new platform. Experimental results show a closer resemblance of the electric field
distribution in the area of interest from the delivery of high-frequency irreversible
electroporation (H-FIRE) type of pulses to the solution of the Laplace’s equation
used in all computational simulations of these types of PEF ablations (Bhonsle et al.
2015). Bhonsle et al. showed a testament to this through experiments on vegetative
tissue, where IRE and H-FIRE types of pulses were evaluated and compared through
thermal imaging and electrical impedance measurements. Thermal imaging was
used as an indirect visualization of the electric field distribution, which enabled a
side-by-side comparison of the temperature and electric field distributions for both
types of PEF treatment. The authors show that the effects of impedance changes are
mitigated when the tissue is exposed to higher-frequency pulses, as in the case of
H-FIRE, compared to standard IRE. This may also explain why the electric field
distribution is more accurately predicted by the Laplace’s equation for H-FIRE than
it is for IRE.
Standard IRE pulses can induce muscle contractions that can cause an involuntary
shift in the electrodes’ location and lead to incomplete ablation of the target region or
damage to critical neighboring structures (e.g., blood vessels, nerves). General
anesthesia and neuroblockers are commonly used to counteract the effects of these
muscle spasms during pulse delivery. H-FIRE pulses have shown promising results
in reducing or completely eliminating muscle contractions – an attractive character-
istic in the adoption of this technique for clinical use (Arena et al. 2011).
Conclusions
IRE is a relatively new focal ablation technology. Due to its predominantly nonther-
mal mode of ablation, it has opened the door for the treatment of otherwise
inoperable tumors. It has already shown tremendous promise for the treatment of
tumors in the pancreas and prostate. The current state of IRE allows for successful
treatment of patients with a tumor located in a specific organ without systemic
metastases since it employs a localized or targeted mode of delivery. There are
also some constraints when it comes to tumor size; IRE works best for tumors
smaller than 3 or 4 cm, even though larger lesions have also been treated. There
are contraindications restricting the use of IRE to treat patients with pacemakers or
who have a history of cardiac arrhythmias and irregular heartbeats; this is due to the
possibility of inducing irregular heartbeats or dysrhythmias from the IRE procedure
even when cautionary measures, such as synchronizing pulse delivery to the
patient’s electrocardiogram (ECG), are taken in the clinical use of the technique.
As protocols refine to simplify the procedure and increase the ablation volume, the
use of IRE to treat renal and liver tumors will continue to expand. In particular, the
current IRENE (Wendler et al. 2015) and COLDFIRE-2 (Scheffer et al. 2015b) trials
will provide valuable insight as to optimize IRE for these indications. As it continues
to be established and refined by engineers, biologists, and clinicians working
together, its clinical utility has tremendous potential to expand the treatment of
other types of cancer and diseases.
Cross-References

Electrochemotherapy
Tissue Blood Flow
▶ Minimally Invasive Treatment by Electroporation: Remaining Challenges
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cct.2015.05.002
Diffusion Weighted Magnetic Resonance
Imaging for Detection of Tissue 41
Electroporation In Vivo
Faisal Mahmood and Rasmus Hvass Hansen
Abstract
Electroporation is advancing toward the treatment of non-superficial cancers.
With this an increased demand of noninvasive methods for verification of
electroporation-based treatments has emerged. Brownian motion of water is
present everywhere in the organism and, as such, indirectly reveals the micro-
scopic structure of the tissue, the main components of which are the intracellular
space, the cell membrane, and the extracellular space. Any change in the tissue
structure may inflict a change in the diffusivity of the water. Electroporation-
induced cell membrane permeabilization is believed to cause a number of mor-
phological changes of the tissue components, therefore potentially detectable
through water diffusion measurements. Diffusion-weighted magnetic resonance
imaging (DW-MRI) is a sophisticated MRI technique that utilizes the differences
in water diffusivity within the tissue to generate image contrast. From DW-MRI
so-called apparent diffusion coefficient (ADC) maps can be derived providing a
quantitative tool for water diffusivity assessment. Investigations of electroporated
tissue using DW-MRI in vivo show biomarker potential. Following irreversible
electroporation, the ADC shows a characteristic temporal evolution. Reversible
electroporation studies are limited but show that the ADC is increased during the
first 24 h after treatment. The complexity of biological tissue does not allow
DW-MRI to provide a detailed characterization of the electroporated tissue or the
membrane permeabilization itself. Nonetheless, bulk tissue features are accessi-
ble through quantitative DW-MRI, yet more advanced signal processing is
F. Mahmood (*)
Radiotherapy Research Unit (RRU), Center for Experimental Drug and Gene Electrotransfer
(CEDGE), Department of Oncology, Herlev Hospital, University of Copenhagen, Herlev, Denmark
e-mail: fama@regionh.dk
R.H. Hansen
Department of Radiology, Herlev Hospital, University of Copenhagen, Herlev, Denmark
e-mail: Rasmus.Hvass.Hansen@region.dk

DOI 10.1007/978-3-319-32886-7_167
724 F. Mahmood and R.H. Hansen
necessary if someday DW-MRI is to be used for “virtual histology” of electro-

porated tissue.
Keywords
Brownian motion • Water diffusion • Tissue structure • Response imaging •
Apparent diffusion coefficient • Diffusion-weighted MRI • Electroporation
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 724
What Is Diffusion? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 725
Diffusion Compartments in Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 725
Electroporation-Induced Diffusivity Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 726
Morphological Tissue Response to Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 726
Diffusion-Weighted MRI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 728
The Physics of Magnetic Resonance Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 728
The Standard Diffusion MRI Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 730
The Resolution of Diffusion-Weighted MRI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 732
Diffusion in Complex Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 734
Free and Restricted Diffusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 734
The Apparent Diffusion Coefficient (ADC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 736
Models for Water Diffusion in Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 736
Apparent Diffusion Coefficient Response to Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738
Apparent Diffusion Coefficient as Imaging Biomarker for Electroporation . . . . . . . . . . . . . . . 738
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 740
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 742
Introduction
Electroporation has proven to be an effective cancer treatment, for example when

used in combination with chemotherapeutics, mostly bleomycin. Targets such as
cutaneous metastasis from malignant melanoma, breast cancer, and head and neck
cancer are effectively treated with palliative intent (Part VIII, “Biomedical Applica-
tions of Electroporation,” ▶ Chap. 96, “Electrochemotherapy of Cutaneous Metas-
tases”). In recent years deep seated tumors have also been in focus for treatment with
electroporation (Part VIII, “Biomedical Applications of Electroporation,” ▶ Chap.
93, “Electrochemotherapy and Its Clinical Applications”). This advance in clinical
applicability increases demand for noninvasive verification of treatment delivery and
response assessment in electroporation-based treatments.
Diffusion-weighted magnetic resonance imaging (DW-MRI) is a well-known imag-
ing technique that produces images that reveal differences in water diffusivity within
the scanned tissue. The first study of water diffusion in the human brain was published
more than 30 years ago (Le Bihan et al. 1986). Since then many technical and
conceptual improvements have occurred, and DW-MRI has become an indispensable
clinical imaging modality especially for the investigation of neurological disorders.
In oncology, DW-MRI is becoming a standard for body imaging as well. Differ-
ent tissue types have different water diffusivities. One can imagine that pathological
41 Diffusion Weighted Magnetic Resonance Imaging for Detection of Tissue. . . 725
conditions of the tissue or tissue response induced by treatment may change the
water diffusivity. The ability of DW-MRI to noninvasively measure the diffusion of
water within the tissue is intriguing and potentially opens a window of possibilities
for investigation of the tissue structure essentially anywhere a change of the tissue
structure can be expected. In this chapter the role of DW-MRI in detection of
electroporated tissue is described. To understand why DW-MRI may be a good
choice of imaging modality for detection of tissue response in electroporation based
treatments, an introduction to the basic physics of diffusion is a good place to start.
What Is Diffusion?
Diffusion is a term used in various fields besides physics, and it can have different
meanings even in physics. In this text diffusion refers to the random thermal motion
of particles suspended in a fluid. A qualitative description of diffusion is often given
through examples, such as the spontaneous mixing of two or more types of particles
suspended in fluid and high concentration gradients related to the same particles
spontaneously disappearing over time. The quantitative description of diffusion that
follows will allow understanding the above phenomenon.
Diffusion is closely related to the discovery by the Scottish botanist Robert Brown
in 1827. While he was studying the pollen of the plant Clarkia pulchella immersed in
water, he noticed that particles ejected from the pollen moved randomly through the
water for no obvious reason. Later, in papers published independently by William
Sutherland in 1905, by Albert Einstein in 1905, and by Marian von Smoluchowski in
1906, a mathematical description of this Brownian motion was given. Diffusion
phenomenon may be seen as a macroscopic manifestation of the microscopic
Brownian motion.
Diffusion Compartments in Tissue
In biological tissue water is present in different compartments. Diffusion within the

intracellular fluid is represented by diffusion in the cytoplasm generally and in the
organelles. Diffusion in the extracellular fluid is represented by diffusion in the
interstitial fluid, intravascular fluid, lymphatic fluid, and various cavities, for exam-
ple, the ventricles of the brain. In normal brain tissue the extracellular space occupies
only about 20%, and the diffusion here is believed to be less free compared to that of
the intracellular space. In fact, the cerebral restricted intracellular diffusion has
sparked the creation of an entire scientific subfield, diffusion tensor imaging (DTI)
in which one of the goals is to track nerve fibers (Jones 2011).
How much each of the diffusion processes contributes to the DW-MRI measure-
ment depends on the tissue and its morphological (pathological) state. The first
clinical application of DW-MRI was in imaging the brain after stroke in adults
following the initial studies in cats (Moseley et al. 1990). The diffusivity of water
decreases in the area of the ischemic stroke. It is believed that it is a combined result
of water from the extracellular compartment moving into the intracellular (cytotoxic
edema), where the diffusivity of the water close to organelles and the cell membrane
is impeded, and the resultant swelling of the cells narrowing the extracellular
compartment.
Diffusion also takes places between the extracellular and intracellular compart-
ment. If the cell membrane permeability is increased, the water molecules will cross
the membrane more easily. However, this effect is accompanied by the more critical
loss of ionic equilibrium. So in electroporated tissue the membrane permeabilization
itself may result in increased diffusivity, but the loss of ionic equilibrium may result
in changes in cell morphology (e.g., cell swelling) which overshadow this effect.
This and other tissue responses due to membrane permeabilization are explained in
more detail in the next section.
Electroporation-Induced Diffusivity Changes
In order for DW-MRI to detect electroporated tissue, a change in water diffusivity of

the electroporated tissue is required. That such change actually occurs in electro-
porated tissue is implied by historical data.
Some of the first evidence of electrical breakdown of a cell membrane was based
on studies of a myelin sheath gap (Stämpfli 1958). Later in the 1960s and 1970s,
many studies showed that electrical fields were able to change the electrical proper-
ties of biological membranes. In 1972 the transient change of the membrane
permeability was demonstrated for the first time (Neumann and Rosenheck 1972).
The first in vivo electroporation study in which DW-MRI was used to investigate
treatment response was of laryngeal tumors implanted in mice (Dev et al. 2002). A
first phenomenological approach was attempted in a rat model of healthy brain tissue
electroporated with different electric field intensities (Mahmood et al. 2011). It was
hypothesized that since electroporation induces conductivity changes and since there
is proportionality between the electric conductivity and the diffusivity of the tissue,
diffusion-sensitive modalities such as DW-MRI may be able to detect electropora-
tion in vivo. That is, the assumption was that when electroporation breaks, the
membrane phospholipid bilayer ions and polar molecules such as water experience
less hindrance by the cell membrane, increasing the diffusivity of the water
molecules.
Morphological Tissue Response to Electroporation
The Maxwell relation relates the change in relative conductivity σ p/σ 0 = 1/λ of ions
in a suspension of impermeable (nonconductive) spheres, to the volume fraction of
the extracellular conductive medium (James Clerk Maxwell 1873; Barrande et al.
2007). This relation is written as
I.
II.
Normal cells Permeabilized cells
III.
Fig. 1 Schematic representation of a small section of tissue. Pink structures are cells and the black
background is the ECV. The length of the white arrows represents the diffusivity of water
molecules. Immediately after electroporation, the membrane becomes permeabilized increasing
water diffusivity. This is accompanied by one of several possible morphological changes shown in
the right panel. Examples: I extracellular edema, II cell swelling, III cell lysis/necrosis/apoptosis
[With permission of Springer, reprint from (Mahmood et al. 2011)]
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
λ¼ ð3 αÞ=2
where α is the extracellular volume fraction in the tissue and λ is the so-called
tortuosity of the extracellular volume (ECV). Tortuosity is generally used to char-
acterize the hindrance to diffusion seen in tissue, compared to diffusion in free
medium. The Maxwell relation implies that changes in the tissue structure is
detectable as a change in diffusional properties of the tissue. The tortuosity can be
seen as the fraction of free diffusion in a measurement. If λ equals 1 (i.e. α = 1) only
free diffusion is present.
The changes in tissue morphology described below represent different tissue
states that may result from electroporation. They could represent intermediate states
of the same overall process, e.g., apoptosis or necrosis, and the kinetics of any given
process is probably a function of the electroporation protocol (pulse parameter,
electrode design etc.) as well (Mahmood et al. 2015a). In general, it is believed
that adenosine triphosphate (ATP) depletion and generation of reactive oxygen
intermediates trigger many of the processes (Gabriel and Teissié 1994). As illus-
trated in Fig. 1 different tissue states can be represented by different ECV fractions,
which, according to the simple case of suspended spherical cells (Maxwell relation),
result in different tortuosities of the ECV, therefore different water diffusivities.
Morphological changes of the tissue include cell swelling (cytotoxic edema),
shrinkage, blebbing, lysis, and extracellular edema. In normal brain tissue, the ECV
fraction is about 0.2.
Electroporation can cause high influx of Na+ through the permeabilized mem-
brane. This hyperosmolarity state of the cell interior is believed to cause cell swelling
through water uptake by the cell (Abidor et al. 1994). If the ATP-fueled protein ionic
pumps fail to reestablish normal ion concentrations, the cell can progress to bio-
chemical arrest and eventually to necrosis (Kerr et al. 1972). Cell swelling reduces
the ECV fraction; hence the diffusivity of water is expected to decrease.
Both cell shrinkage and blebbing are morphological features of apoptosis. Shrink-
age is accompanied by blebbing of the plasma membrane which otherwise maintains
its integrity until the final stages of the process (Fackler and Grosse 2008). Blebbing
is the occurrence of irregular bulges in the plasma membrane caused by a localized
decoupling of the cytoskeleton from the plasma membrane (Kume-Kick et al. 2002).
This process is not necessarily associated with a change in the volume fraction of the
ECV, but diffusivity is probably decreased since the irregular curvature creates dead
ends for diffusing molecules.
Lysis refers to a complete fragmentation of the membrane. This eventually leads
to the disintegration of the cell and engulfment by phagocytes, consequently
augmenting ECV. Membrane lysis is usually only seen in dying cells, i.e., in the
process of necrosis and in the final stages of apoptosis (Sale and Hamilton 1967).
Lysis will increase diffusivity of water in the tissue.
Extracellular edema can result from different physiological processes including
increased capillary permeability (caused by bacterial infection, etc.). In the brain the
breakdown of the tight endothelial junctions which make up the blood-brain barrier
can cause extracellular edema, contributing to the enlargement of the ECV (see also
Part III, “Electroporation Phenomena on Tissue Level and Systemic Effects,”
▶ Chap. 35, “Effects of Reversible and Irreversible Electroporation on Endothelial
Cells and Tissue Blood Flow”). Locally the density of the cells in the edematous
tissue is reduced, and diffusivity of water is therefore expected to increase
(Mahmood et al. 2015a).
Diffusion-Weighted MRI
The Physics of Magnetic Resonance Imaging
To understand the basic principle of diffusion-weighted magnetic resonance imaging

(DW-MRI), a brief overview of the magnetic resonance principle is required,
although any attempt at completion is wholeheartedly abandoned, and the interested
reader is urged to consult the references (Brown et al. 2014; Nishimura 1996;
Westbrook et al. 2005).
Atoms with odd numbers of protons and/or odd numbers of neutrons possess a
nuclear spin angular momentum that gives rise to a magnetic moment smaller than
that of the electron by three orders of magnitude. Thus a considerable amount of
such moments usually has to be present and arranged in a somewhat coherent
fashion in order for any resulting magnetization to be measurable. The human
body consists largely of H2O (65% by weight, most of it contributed by oxygen),
and the proton 1H is its most abundant nucleus. The magnetic resonance (MR) signal
stems from the interaction of these nuclei with external magnetic fields. In an MR
scanner the specimen is exposed to the main (constant) magnetic field B0, the
radiofrequency (RF) field B1 which is turned on only at certain time points, and a
pulsed linear gradient field G that is used for the spatial labeling. In the absence of
external magnetic fields, the magnetic moments are randomly oriented yielding zero
net macroscopic magnetic moment (M = 0). In the following, we consider the
collective magnetic moments, denoted spins, of parcels of water molecules very
small on a macroscopic scale, but large enough to be ruled entirely by classical
electrodynamics. Something on the order of 10 microns cubed, which although tiny
still contains on the order of 1013 water molecules, is a good guiding size. At finite,
say, room or body temperature, and in the presence of the main magnetic field B0,
such spins will tend to align in the direction of B0 (by a few ppm) according to the
Boltzmann distribution, yielding a net macroscopic magnetic moment M0 along B0
in the z-direction. In addition, the spins precess about B0, with the Larmor frequency
ω = γB0, and if exposed to an RF field B1 along a different direction, this moment
M is rotated away from B0 around an axis and by an amount defined by the
polarization, magnitude, and duration of B1 and will now be precessing around a
new axis. This process is referred to as excitation of the spins. It might help the
reader in retaining sanity to imagine a coordinate system rotating along with the
magnetic moment with the Larmor frequency rendering the spin stationary in this
system. Notice that the wavelength of RF radiation is so large that within a good
approximation all spins in milliliter-sized samples experience the same B1. Typi-
cally, this could be a so-called 90 RF pulse that brings the spin into a plane
perpendicular to B0, i.e., into the xy plane (Fig. 2) or a 180 pulse reversing the
direction of the spins.
When the RF field is turned off, M will tend to return to its thermal equilibrium
state M0 through interaction with its surroundings (spin-lattice interaction). This
process is referred to as T1-relaxation of the spin and is essentially an exponential
decay (with a characteristic time T1) of the longitudinal component of M pointing in
the direction opposite to B0. Any transverse component of the spin will also decay
exponentially due to local interactions, field inhomogeneities, and fluctuations, and
given the usual wealth of imagination in naming physical processes, this is known as
T2 relaxation and characteristic time.
During the relaxation an RF receiver measures the signal induced by the change
in magnetization (the free induction decay). The mechanisms by which these
relaxation events occur are diverse and complex and depend on tissue type, implying
that the received signal contains information about the relaxation of spins from
different tissues.
Fig. 2 The magnetic moment

M precessing at the Larmor
frequency is reoriented by RF
field B1. Strength and duration
of B1 can be selected for 90
rotation, moving M into the xy
plane
To define the location of the signal, linear gradient magnetic fields are applied in
three orthogonal directions, slice selection, phase encoding direction, and frequency
encoding direction, changing the resonant energy and signals in a systematic manner
and dividing the body under investigation into plane slices, which again are sub-
divided into volume-pixels, or voxels.
The image contrast in a given MRI measurement is controlled by the choice of
so-called sequence parameters. Two main parameters are the echo time (TE), which
is the time between the excitation and the readout of the signal, and the repetition
time (TR), the time between each excitation. If, for example, an image has T2
contrast (is T2-weighted), the sequence parameters are chosen such that the image
mainly reflects T2 variations between tissues. As T2 differs between tissue types,
e.g. fat and muscle, the TE is selected to obtain the largest difference in signal from
these tissues.
The Standard Diffusion MRI Sequence
Refinement of the succession of magnetic RF fields and gradients used in MRI

(so-called MRI sequences) has led to special techniques such as diffusion-weighted
MRI (DW-MRI). DW-MRI utilizes the Brownian motion of water molecules to
generate image contrast.
In the standard DW-MRI procedure, pulsed magnetic gradients are utilized to
determine the mean-squared displacement hr2i of ensembles of water molecules over
a diffusion time Δ. The diffusion coefficient D is related to the displacement by
Einstein’s equation for diffusion (Eq. 1):
2
r ¼ 6DΔ (1)
de-phasing re-phasing
gradient gradient
δ
time
Diffusion Δ
No
Diffusion
Fig. 3 Illustration of the principle of a standard diffusion-weighted sequence: The upper panel
shows the motion-sensitizing gradients, and the lower panel shows the resulting spin phases. With
no diffusion the second magnetic gradient exactly cancels the phase difference induced by the first
magnetic gradient. In case of diffusion (illustrated as two spins switching position), the second
magnetic gradient fails to cancel the phase difference. This results in a reduction in net magneti-
zation and lower signal acquired by the receiver coil [With permission of Springer, reprint from
(Mahmood et al. 2011)]
The Stejskal-Tanner DW-MRI sequence (Stejskal and Tanner 1965) is basically a

spin echo sequence starting with a 90 RF excitation pulse and a 180 refocusing RF
pulse, with echo acquisition at time TE. Gradient pairs (usually in three orthogonal
directions) are applied symmetrically around the 180 RF pulse (Fig. 3). These
gradient pulses are what make this sequence motion sensitive and therefore are
also referred to as motion-sensitizing gradients. The time between the gradient pair is
the diffusion time Δ, the duration of each gradient pulse is denoted δ, and the
strength of each gradient is denoted G. Although there are many variations on the
theme of DW-MRI sequences (Jones 2011), this is a useful example to keep in mind.
How is the diffusion contrast achieved? If the spins in the volume being measured
are static, the dephasing of the spins introduced by the first gradient (dephasing
gradient) is exactly reversed by the second gradient (re-phasing gradient). On the
other hand, if the water molecules have diffused during the diffusion time Δ, a phase
difference will remain after application of the re-phasing gradient. This results in a
reduction of the net signal from the given volume. Generally, if the spins in the
volume being measured are limited in motion, the signal will be stronger, and if the
spins diffuse more freely, the signal will be accordingly weaker.
This means that in diffusion-weighted images, areas with low intensity represent
areas where water molecules diffuse more freely and areas with relatively high
intensity represent areas where the diffusion of the water molecules is less free. In
an experiment applying electroporation pulses in the rat brain (Fig. 4a), reduced
diffusivity in close proximity to the electrodes and increased diffusivity between
Fig. 4 In vivo electroporation of rat brain: (a) T2-weighted MRI with overlay of electrodes and the
electric field distribution (simulated with Comsol Multiphysics 3.5a; Comsol, Stockholm, Sweden).
(b) Diffusion-weighted image (b = 800 s/mm2) 2 h after electroporation (32 pulses of 200 V, 0.1 ms
each, at 1 Hz) delivered using an eight-pin conical electrode geometry (Mahmood et al. 2011) (c)
The ADC map
electrodes was seen (Fig. 4b). An ADC map provides quantitative estimates of
diffusivity pixel-by-pixel (Fig. 4c).
The Resolution of Diffusion-Weighted MRI
The image resolution of diffusion-weighted images is usually less than conventional

T1- or T2-weighted images as the remaining signal following the motion-sensitizing
gradients is quite low. However, the signal value of each image voxel in a diffusion-
weighted image is a representation of the microscopic events taking place in the
voxel. In a general clinical setting, the size of DW-MRI voxels is typically
(1–2) (1–2) mm in-plane times a 3 mm slice thickness.
Thermal motion on a molecular level is very complex. It occurs on a pico-second

time scale for water under normal conditions. In DW-MRI the diffusion time Δ is in
the order of milliseconds providing a measurement that is an average of a large number
of single collisions. In this case the central limit theorem shows that the distribution of
the distances covered by the molecules is Gaussian (Jones 2011). This is the case for
free diffusion in an unrestricted homogenous medium. The total variance for a
Gaussian distribution is proportional to the number of collisions, hence also to time:

σ 2 ¼ r 2 ¼ 6DΔ (2)
Equation (2) is of course again Einstein’s diffusion relation. Another crucial step
(cf. Peter Hänggi’s website (Hänggi 2016) for lots of details on the history of
Brownian motion) is now to posit that within the soup of molecular chaos a force
F giving rise to a molecular drift velocity, vd = μF where μ is the so-called mobility,
is balanced by the thermally induced diffusion at the absolute temperature T, leading
to the Einstein-Smoluchowski-Sutherland equation,
D ¼ μkB T (3)
where kB is the Boltzmann constant.

Combining Eqs. (2) and (3) shows that the mean-squared displacement of
particles increases with both temperature and the mobility of the molecules:
2
r ¼ 6μkB TΔ (4)
The diffusion coefficient of water in the temperature range between 10 C and
body temperature is approximately between 0.5∙103 mm2/s and 3∙103 mm2/s.
The diffusion time Δ is in practice set by the echo time TE, depending on the MR
sequence. It can be varied between a few milliseconds and a few seconds and is
limited by the duration of the RF pulse and motion-sensitizing gradients from
below and by the longitudinal relaxation time constant T1 from above. Applying
these numbers to Einstein’s equation, the diffusion length can be estimated as
lD(t) = < r2 >½ = (6DΔ)½ ~ 1–50 μm.
This range is in the general order of the size of the human cells, indicating that when
the diffusion time Δ is varied, the microscopic structure of the tissue can be probed. As
opposed to conventional MRI where the image contrast is basically relaxation time
dependent, hence essentially stationary over longer time intervals unless external con-
trast agents are introduced, DW-MRI holds the promise of measuring transient changes
induced on the cellular scale, or at least the macroscopic averages of such changes.
In an experiment using the Stejskal-Tanner sequence, the attenuation of the signal
is related to the gradient parameters and echo time by

δ
SðG, ΔÞ ¼ Sð0, ΔÞexp γ 2 G2 δ2 D Δ (5)
3
where γ is the gyromagnetic constant for hydrogen; G, Δ, and δ are the gradient
parameters as above; and S(G,Δ) and S(0,Δ) are the signal intensities at the mea-
surement echo for a motion-sensitizing gradient at strength G and zero, respectively.
In MRI, these parameters are usually lumped together in a single attenuation factor b
– or simply the b-value:

δ
b ¼ γ 2 G2 δ2 Δ (6)
3
measured in units of s/mm2 and giving Eq. (5) a somewhat more straightforward
form
SðbÞ ¼ Sð0Þexpðb DÞ (7)
By keeping the echo time constant and varying the gradient strength, the signal
attenuation can be measured at different values of b and the (apparent) self-diffusion
coefficient extracted.
Diffusion in Complex Tissue
Free and Restricted Diffusion
The self-diffusion of pure, bulk water is readily described by Eqs. (2, 3, 4) in the
previous section. It is assumed unimpeded, or free, as was also noted. In biological
tissue, water exists in many different compartments, more or less open to perfusion
or exchange. Here, water diffusion may very well be restricted in a space that is
crowded by cells and extracellular components, and the assumptions leading to
Eq. (2) have to be reconsidered. Whereas it can be intuitively grasped that introduc-
ing boundaries with special length scales and changing the number of collisions
renders the process decidedly non-Gaussian and invalidates the nice statistical
analysis leading to Eqs. (2, 3, 4), cf. Fig. 5, it is far from obvious how such
anomalous diffusion processes manifest themselves in DW-MRI. In fact, as was
already realized in nuclear magnetic resonance (NMR – the chemical analysis
progenitor of MRI) studies of porous materials in the early sixties, the diffusion
coefficient in Eq. (2) becomes a function of the diffusion (or echo) time in such
materials (Woessner 1963). Yet, from the beginning of the clinical use of DW-MRI,
there has been a lot of discussion of the source of echo attenuation, especially which
compartments of water generate the restriction. One could be excused for thinking
that the intracellular water in the cytosol would be an obvious candidate with the
great amount of organelles and macromolecules it has to negotiate its way around,
but this happens not to be the case, or at least not the whole story. The water
molecule in its supreme smallness deftly avoids most of the restrictions to its
diffusion inside the cell, and measurements by both neutron scattering (Jasnin
et al. 2008) and NMR (Persson and Halle 2008) find that most of the intracellular
Fig. 5 Gaussian diffusion is

free and restricted diffusion is
non-Gaussian
water behaves as in bulk. Indeed in the NMR study, it is estimated that 85% of the
water encounters very little or no restriction, whereas the remaining 15% “interacts
directly with biomolecular surfaces” seeing its motion reduced by more than an order
of magnitude. In other words, only the water close to membranes (or other obstacles)
is restricted in its diffusion. The very first observation of this kind in MRI was – to
our knowledge – in a very elegant experiment by Paul Callaghan et al. (1992), where
a systematic and reproducible signal enhancement at the walls of pentane-filled
micro-capillaries was clearly visible and quantifiable. This immediately lights the
thought that the reduction of the diffusion coefficient compared to that of bulk self-
diffusion should be proportional to the ratio of surface to volume of the water
container, and this is indeed confirmed in theoretical modeling of diffusion in
smooth pores (Mitra et al. 1993).
In essence, one is dealing with a set of three different time scales (Hurlimann et al.
1995; Grebenkov 2007), the diffusion length lD = (DΔ)½ in a given time Δ, the
dephasing length lG = (D/γG)1/3 which basically measures the diffusion length
required for a spin to acquire a 2π phase shift from the interaction with the pulsed
gradients, and a structural length scale lS indicating the typical distance between the
boundaries of the diffusion process. For very short lD(lD lS), the diffusion is
essentially free and measured as such in our typical DW-MRI if also lD lG,
resulting in a weaker attenuation; whereas in order to observe the diffusion restric-
tion of spins within lG of the boundaries, one needs lG lS and lG lD, such that the
phases of the spins further away from the boundaries through their less restricted
diffusion accumulate completely scrambled phases and do not contribute to the
image. This also links up nicely with the measure of tortuosity mentioned above in
relation to relative conductivities as one has λ = D(lD ! 0)/D(lD ! 1).
It all seems deceptively simple, as one would apparently merely need to design
the gradients properly in order to measure all kinds of restrictions. In practice large
diffusion times may result in poor signal-to-noise ratio (SNR) due to T2 relaxation,
and in clinical setting scanners operate at 1–3 Tesla, with SNR increasing less than
linearly as a function of field strength, and there are severe limits on the strengths and
rates of MRI gradients. In addition, the variety of water compartments means not all
signal attenuations are created equally and diffusively.
The Apparent Diffusion Coefficient (ADC)
Quantitative measurements developed on MRI scanners are notoriously difficult to

implement in a reproducible fashion which also holds a semblance of being machine
independent. Measures like diffusion coefficients, with its logarithmic relation to
signal strength and derived from several measurements, are among the most prom-
ising and robust candidates. From its inception as a clinical tool, it was realized that a
measurement of the diffusion coefficient using only small values of b (<200, say) is
prone to errors as the images are subjected to T2 contrast, so-called T2 shine-
through, and the echo attenuation with these weaker motion-sensitizing gradients
is dominated by signal loss from capillary perfusion and T2 inhomogeneities rather
than diffusion, a process usually referred to as pseudo-diffusion, or intravoxel
incoherent motion (IVIM) (Le Bihan et al. 1986). It is indeed striking to observe
how veins are clearly visible in low b images, but are suddenly “turned off” around
b = 50, which together with b = 0 is often used for quick venous mapping. To hedge,
the MRI community (Le Bihan et al. 1986) agreed to name the obtained results
apparent diffusion coefficients (ADC), and this is now the industry standard. Thus in
MRI, the echo attenuation (7) is written as
SðbÞ ¼ Sð0Þexpð-b ADCÞ (8)
with the b-value still determined by (6).

In reality hardware limitations as well as tissue properties and noise do not allow
b-values much above 1,000–3,000 s/mm2 on most clinical systems. At least two
measurements at different b-values are needed to calculate the ADC, and for a long
time (and still!) b = 0 and b = 1,000 s/mm2 were the preferred values, until it was
realized that using more b-values (and leaving b = 0 out altogether) yields better
estimates of ADC. In practice ADC is estimated as the slope of the linear fit of logS
(b), and in homogeneous media this poses no problems as the points line up nicely,
but heterogeneous media, like biological tissue, are more than likely to exhibit a
number of domains of b-values marking out separate ADCs, in good agreement with
the model of echo time-dependent diffusion coefficients. An attempt to illustrate this
has been made in Fig. 6.
Models for Water Diffusion in Tissue
It is possible to model the full b-value range with a better model than the mono-
exponential model. The bi-exponential IVIM model (Le Bihan et al. 1986) is a
popular alternative:
Fig. 6 Data points (blue dots)

from a highly idealized
DW-MRI measurement.
Straight fitted lines are
sketched to illustrate that the
data does not exhibit a simple
linear relationship over the
full range of b-values. The
lines indicate the three main
parts of the b-value interval,
where the endpoints of these
segments are variable and
tissue dependent
Sð bÞ
¼ ð1 f Þexpðb ADCÞ þ f expðb D Þ (7)
Sð 0Þ
This model takes into account the low b-values through the pseudo-diffusion
coefficient D*. The perfusion fraction f provides weighting of the two exponential
terms and can be interpreted as the fraction of signal originating from the blood pool
of the capillaries (tissue perfusion).
Some researchers have found kurtosis models useful (Jensen et al. 2005; Jensen
and Helpern 2010). The high b-values can be modeled using a kurtosis model. In
statistics, kurtosis is the fourth standardized moment of a distribution. For a Gaussian
distribution, the kurtosis is zero, and using this model the departure from the
Gaussian distribution can be probed:

Sð bÞ 1 2 2
¼ exp b ADC þ b ADC K (8)
Sð 0Þ 6
where K is the kurtosis. Such models usually require very high b-values (>3,000 s/
mm2) to fit properly, which is challenging on most clinical scanners and have yet had
little impact in studying non-cerebral tissues.
Many other diffusion models can be found in the literature (see, for example,
Jones (2011)) but are not covered here since none of these have been used to study
electroporation. The choice of model and b-value can have an effect on the outcome
in assessment of tissue response and should therefore be considered beforehand
(Mahmood et al. 2015c).
Apparent Diffusion Coefficient Response to Electroporation
The apparent diffusion coefficient (ADC) is the most intuitive of the quantitative
parameters that can be derived from DW-MRI. It is therefore also the most fre-
quently reported parameter in literature. In electroporation-based treatments, ADC is
the only parameter so far being used to study tissue response. In the paper
(Mahmood et al. 2015b), kurtosis was also reported, although no biological inter-
pretation of the results was attempted. The study did however show that ADC and
kurtosis provide complimentary information. In the following the focus will be on
ADC only, due to the dearth of studies using other parameters.
The morphological changes are described in electroporation-induced diffusivity
changes (section “Electroporation-Induced Diffusivity Changes”) and represent
different states of the tissue. Each state may be a function of the applied electric
field and time. Since in no practical situation the tissue is exposed to a perfectly
homogenous electric field (Part V, “Modeling of Electroporation and Related Phe-
nomena,” ▶ Chap. 56, “Electric Field Distribution and Electroporation Threshold”),
every sub-volume of the tissue (voxel) must be some weighted average of different
cell states. In addition, the tissue state is also a function of the tissue and cell type.
Apparent Diffusion Coefficient as Imaging Biomarker

for Electroporation
As explained above, the state of the tissue may be a function of the electric field. The
tissue may however not be entirely trichotomized into non-electroporated, reversibly
electroporated, and irreversible electroporated parts, but since electroporation is a
threshold phenomenon, it seems valid to apply this categorization. Nevertheless, as
stated in the previous section, each voxel may in reality represent an average of
slightly different tissue states.
Imagine that the temporal evolution of ADC following the delivery of electropo-
ration, ADC(t), has a unique feature. This would allow for an indirect and noninva-
sive identification of electroporation. Moreover, if the electroporation states exhibit
different ADC features, these tissue states may not only be detectable but also
differentiable.
In the field of oncology, Moffat et al. (2005) proposed a model of ADC change
following treatment in which it was suggested that the ADC decreases soon after
initiation of chemotherapy or radiation therapy due to cell swelling. This is followed
by cell death (lysis, necrosis) which results in an increase in the extracellular volume
and ADC. Treatment may also induce apoptosis which is associated with cell
shrinkage and increased ADC.
Figure 7 collects relative ADC data from the existing electroporation studies. The
scarce data of studies applying electric fields corresponding to reversible electropo-
ration suggest an initial increase in ADC till 24 h after electroporation (one
experimental condition at 500 V/cm showed no initial ADC change). In studies
where electric field pulses corresponding to irreversible electroporation were
Reversible electroporation
300 V/cm
ADC
Baseline ADC
500 V/cm
(Log axis)
4-30 m 1-3 m 24h 48h 1 mo
Irreversible electroporation
“Acute Apoptosis/
Swelling Normalization
effects” Necrosis
1250 V/cm 1305 V/cm
1000 V/cm >1200 V/cm

ADC
?
Baseline ADC
IRE Nano-knife
2500 V/cm 1200 V/cm
(Log axis)
4-30 m 1-3 h 24h 48h 1 mo
Fig. 7 The findings of existing studies of Apparent Diffusion Coefficient (ADC) change in
electroporated tissue are plotted. An arbitrary y-axis is used since numerical ADC data were not
reported in all studies. Few studies of reversible electroporation were available, all except one
(unchanged) suggesting an increased ADC within 24 h after electroporation. Studies of irreversible
electroporation showed consensus in the relative ADC change
applied, more data is available. ADC increases initially (4–30 min after electropo-
ration) followed by a decrease of ADC (1–3 h) and then an increase of ADC
(24–48 h) and a normalization of the ADC (1-month follow-up).
The ADC increase within the first 30 min after electroporation can result from cell
lysis which augments the ECV fraction of the tissue. Membrane permeabilization
itself could also explain this increase in ADC. Here it is simply referred to as acute
effects. The decrease of ADC 1–3 h after electroporation agrees nicely with the
swelling of cells. Further cell death (apoptosis, necrosis, lysis) explains the ADC
increase 24–48 h following electroporation. The normalization of ADC 1 month
after electroporation is due to the biological normalization of the tissue (including
formation of scar tissue).
Reversibly and irreversibly electroporated tissues are subdivided according to an

average electric field strength cutoff value of 1,000 V/cm. This number is not
completely settled although a recent study with histological validation confirmed
that electric field strength above 1,000 V/cm induced cell death in the brain
(Mahmood et al. 2015b). For other tissue types the cutoff value may be different.
Tissue types were indeed different in most of the studies including human laryngx
tumor xenograft in mice (Dev et al. 2002), normal brain tissue in mice (Mahmood
et al. 2011, 2015b), normal blood-brain barrier (Hjouj et al. 2012), fibro sarcom
xenograft in mice (Calmels et al. 2012), pancreatic carcinoma xenograft in mice
(Zhang et al. 2014), and hepato carcinoma in humans (Granata et al. 2015). Com-
parison between studies is further complicated by the fact that some studies report
voltage-to-distance ratios rather than electric field distributions and use different
electrode geometries. As can also be seen in Table 1, the electroporation protocols
(electric field strength or voltage-to-distance ratio (V/cm), duration, frequency, and
number) are different. It is well known that electroporation thresholds may depend
upon duration of the pulse as well as on the electric field strength. The DW-MRI
protocols were not directly comparable either, as different b-values and magnetic
field strengths were used and the DWI sequences are not reported in some of the
studies.
Conclusions
Diffusion-weighted magnetic resonance imaging (DW-MRI) has been used for

diagnostics of neurological disorders, especially acute ischemic stroke, since the
1990s. It is used because it works; the ADC is reduced within a few minutes after
arterial occlusion. In conventional MRI (T1/T2 weighted), a stroke can take up to
hours to become detectable. Nevertheless, it remains a matter of controversy what
actual causes the ADC to decrease and show up as intensity increase in the DW
image. The point is that biological tissues are extremely complicated, and it is too
optimistic to think that DW-MRI can characterize them in detail with the limited
number of parameters available.
There is no reason to believe that electroporated tissue is less complicated, and
thus a detailed characterization of electroporated tissue using DW-MRI in vivo is
not possible either at present. Despite the large complexities, a phenomenological
approach is nevertheless attempted. DW-MRI measures the average diffusive
displacement of water molecules within a macroscopic voxel, but it is not neces-
sary to know the details of individual cells if the aim is to detect the response of
bulk tissue. The water molecules probe the tissue structure in the microscopic
landscape made up by cells and extracellular components and provide an averaged
response measure.
All in vivo DW-MRI studies so far of electroporated tissue attempt to measure
diffusivity changes 4 min or more after application of pulses. Ivorra et al. (2009)
showed that the tissue conductivity is almost normalized after 4–5 min following
reversible electroporation, implying that membrane permeabilization is also
41
Table 1 Overview of studies of ADC change in reversibly and irreversibly electroporated tissue in vivo. Interstudy comparison is complicated by the variety of
electroporation protocols, target tissue, and DW-MRI protocols
V/cm Tissue Subject EP/chemo DW-MRI Timing Response
Dev et al. (2002) 1,305 Human Ex Six 99 μs, 1 Hz./ 14 T, b: 0.8, 7.4, 20.5, 24 h, 48 h ADC up at 48 h
larynx vivo bleomycin 40.3 s/mm2
tumor mice
Mahmood et al. 500,1,000 Normal In vivo Eight 100 μs, 3T, b:0, 1,000 s/mm2 4–20 min ADC up for high V
(2011) brain rats 1 Hz/no all time points
Hjouj et al. ? Normal In vivo 99, 50 μs, 4 Hz/no 1.5 T, b: ? 30 min Not quantitative, no
(2012) brain bbb rats visual effects seen
Calmels et al. RE: 1,250 Fibro- In vivo RE: Eight 100 μs, 4.7 T, b:0, 33, 134, pre – 48 h ADC down for IRE
(2012) IRE: 2,500 sarcoma mice 1 Hz/bleomycin. 300, 534, 835 s/mm2 at 1 and 3 h
xenograft IRE: 20 100 μs,
0.3 Hz
Zhang et al. 1,250 Pancreatic In vivo 64, 100 μs, 5 Hz 7 T, b:0, 500, 800 s/min2 pre, 30 min ADC up at 30 min
(2014) carcinoma mice
xenograft
Granata et al. IRE (nano-knife) Hepato- Human ? 1.5 T, b:0, 50, 100, 5 d pre, 1 mo Most ablated zones
(2015) cellular 200, 400, 600, 800 s/mm2 follow-up showed restricted
carcinoma diffusion. ADC
change
non-significant
Mahmood et al. 0, 300, 600 >1,200 Normal In vivo Eight 100 μs, 3 T, b:50, 100, 150, 5 min, 2 h, ADC and K up at
(2015b) brain rats 1 Hz/no 600, 800, 1,000, 800 s/mm2 24 h, 48 h 5 min, 2 h, 24 h (RE).
Diffusion Weighted Magnetic Resonance Imaging for Detection of Tissue. . .
ADC down at 2 h and

up at 48 h (RE)
741
normalized. The overall ADC response, however, lasts much longer. Studies with
reversibly electroporated tissue are limited but show that the ADC is increased 24 h
posttreatment. Studies of irreversible electroporated tissue on the other hand provide
more data and show a characteristic temporal change of the ADC. These promising
results indicate that DW-MRI is a serious candidate to become the standard clinical
tool for detection of electroporated tissue, as it is presently for detection of acute
stroke and certain cancers. Better studies are needed to find out whether DW-MRI is
capable of directly detecting membrane permeabilization, and more advanced pro-
cessing is necessary to allow a detailed characterization of the electroporated tissue.
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Effect of Electroporation on Blood-Brain
Barrier 42
Shirley Sharabi and Yael Mardor
Abstract
The blood–brain barrier (BBB) is a multicellular vascular structure that separates
the central nervous system (CNS) from the peripheral blood circulation. It forms
the major obstacle for effective drug penetration into the brain, thus limiting
extensively the efficacy of systemically administered therapeutic agents for
treating brain tumors as well as neurodegenerative diseases.
Electroporation (EP)-induced BBB disruption is a novel technique that can be
used for delivering high concentrations of therapeutic drugs to specific brain
regions.
Electroporation is a technique in which intense electric pulses are applied to
make the cell membrane transiently porous and increase permeability to ions and
macromolecules. When applied to the brain, electroporation can also induce
transient BBB disruption. The BBB can either be disrupted without causing
irreversible damage to the brain or in combination with irreversible electropora-
tion where BBB disruption surrounds a smaller irreversibly electroporated vol-
ume. The treatment can be performed in a minimally invasive manner using a
single intracranial electrode or require craniotomy if multiple electrodes are used.
It is possible to predict treatment outcome using statistical and computerized
models describing the electrical field distribution in the brain.
Electroporation-induced localized BBB disruption lasts in the order of
24–48 h. The relatively long duration of the disruption differs from reversible
electroporation which only lasts several minutes post treatment. The combination
of electroporation-induced BBB disruption with systemic administration of ther-
apeutic agent is suitable for treating primary or metastatic brain tumors as well as
localized neurodegenerative diseases.
S. Sharabi (*) • Y. Mardor (*)

Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel
e-mail: shirley.sharabi@sheba.health.gov.il; yael.mardor@sheba.health.gov.il

DOI 10.1007/978-3-319-32886-7_168
746 S. Sharabi and Y. Mardor
Keywords
Electroporation • Brain • Blood–brain barrier • Brain tumors • Chemotherapy
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 746
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748
Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748
Anesthesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748
Electroporation Treatment Location Within the Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748
Electrode Setups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748
Surgical Procedure for Electrode Implantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 749
Magnetic Resonance Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750
Treatment Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752
Typical Experimental Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 759
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 759
Introduction
The blood–brain barrier (BBB) is a multicellular vascular structure that separates

the central nervous system (CNS) from the peripheral blood circulation
(Obermeier et al. 2016). It is composed of several cell types including endothelial
cells, astrocytes, and pericytes in close association. The endothelium forms the
major interface between the blood and the CNS. Endothelial cells of the BBB are
different from those of extracranial tissues as they have continuous intercellular
tight junctions (TJs) and adherens junctions while they lack fenestrations. Tight
junction-associated proteins include occludin, claudin-1, claudin-5, and junc-
tional adhesion molecules (Abbott 2013; Abbott et al. 2006). Claudin-1 and
occludin are linked via zonula occludens (ZO) protein complexes to tight
junction-associated proteins that bind to the actin/myosin cytoskeletal system,
resulting in modification of the tight junction properties (van Tellingen et al. 2015;
Obermeier et al. 2013).
The endothelium acts as a “physical barrier” limiting paracellular movement of
molecules, and its main functions include control of molecular traffic and keeping
out toxins. The endothelium is also responsible for maintaining ion homeostasis and
low protein environment in the CNS in order to ensure optimal neuronal function
(Abbott 2013). Under pathological conditions, the function and organization of the
BBB can be altered. For example, brain pathologies including multiple sclerosis,
epilepsy, autoimmune deficiency syndrome, stroke, and some types of brain cancer
such as high-grade gliomas may cause BBB disruption. In glioblastomas (GBM), for
example, the alterations of the normal vascular function result in disrupted,
manifested as “leaky,” BBB, which can be depicted by contrast-enhanced MRI.
This is often used to evaluate tumor volume.
42 Effect of Electroporation on Blood-Brain Barrier 747
Even though the BBB is “leaky” enough for depicting the core part of GBMs, it
is insufficient for reaching therapeutic drug doses administered systemically.
Moreover, in large parts of the tumor, the BBB more closely resembles the intact
BBB and prevents efficient passage of cancer therapeutics (van Tellingen
et al. 2015). On top of that, the tumors are highly infiltrative. As such, residual
tumor, including the infiltrating zone surrounding the tumor, should be treated with
high efficacy as well, to prevent sublethal hits of tumor cells leading to the growth
of more malignant clonal cell populations. In low-grade gliomas, the normal
vascularization and the function of the BBB remain mostly intact. The BBB is
therefore a major obstacle for effective drug penetration into brain tumors
(Milojkovic Kerklaan et al. 2016). The treatment of neurodegenerative diseases
is also limited due to the BBB.
As direct drug delivery methods into brain tumors are restricted by the limited
diffusion of the drugs in the tissues, in the order of a few millimeters, strategies and
techniques to bypass the BBB are needed. Because of the important role of the BBB
in maintaining neuronal function, therapeutic BBB opening needs to be transient and
as localized as possible. This can be achieved by electroporation (EP) (Hjouj
et al. 2012; Sharabi et al. 2014, 2016)
During EP, electrical pulses are applied to the brain inducing an electrical field
that causes the creation of transient nanoscale pores in the cells, thus permeabilizing
the membranes to ions and macromolecules of membranes. Depending on the
function of the electrical field, the pulse parameters (strength, shape, duration,
number of pulses, and frequency), and various tissue-related parameters (such as
tissue type and cell size), EP can either lead to cell death, referred to as irreversible
EP (IRE), or to reversible permeabilization of the cell membrane, referred to as
reversible EP (RE) (Weaver et al. 2012; Yarmush et al. 2014). It was recently
discovered that EP applied to the brain can induce transient BBB disruption (Garcia
et al. 2012; Sharabi et al. 2014, 2016).
It is still unclear whether EP-induced BBB disruption is simply manifestation of
RE or whether a different mechanism is involved. Lopaz-Quintero et al. (Lopez-
Quintero et al. 2010) suggested that BBB disruption may occur independently of
membrane EP. They showed in vitro BBB disruption resulting from compromised
continuity of the ZO-1 proteins while the viability of the endothelial cells was not
affected and with no elevation in calcium uptake, suggesting BBB disruption
occurring below the threshold for EP, thus with no poration of the membrane.
Depending on the treatment parameters, the BBB can either be disrupted without
causing irreversible damage to the brain, or in combination with irreversible elec-
troporation (Garcia et al. 2012; Hjouj et al. 2012; Sharabi et al. 2014), where BBB
disruption surrounds a smaller irreversibly electroporated volume. This is most
useful for the treatment of brain tumors, where EP can be applied for inducing
IRE damage to the tumor core, surrounded by BBB disruption in regions of residual
tumor and surrounding infiltrating zone. The disruption can be used to provide
efficient uptake of systemically administered therapeutic agents.
Methods
Animals
The smallest animals recommended for experiments are rats, since the key issue is
breeching significant volumes of the BBB. For this purpose, it is ineffective to work
with mice, which are too small. Rats should preferably weigh 275–300 g. When
reaching this weight, the brain has reached its maximum volume of about 1.5 ml. In
addition, when using smaller rats, the skull is thin and more fragile and can therefore
break during perforation.
When working with naïve rats, the most common rat species is the
Sprague–Dawley rat. It is advisable to work with male rats to avoid menstrual
cycle effects. When working with intracranial brain tumor models, other species
may be required. For example, when working with the CNS-1 glioma tumor model,
it is best to work with Lewis rats; when working with the 9L gliosarcoma model, it is
best to work with Fischer rats.
Anesthesia
Intracranial electrode implantation, EP treatment, and MRI monitoring are all performed
under general anesthesia. The most common methodology is an intraperitoneal (IP) or
intramuscular (IM) injection of drugs such as Ketamine, Ketalar, Pental, and Rompun.
If imaging techniques such as MRI are used for monitoring EP effects
posttreatment, it is necessary to keep the animals anesthetized for relatively long
periods of time (hours), which requires more than one dose of anesthesia and control
of temperature using heating pads. In these cases, it is recommended to wait at least
15 min between anesthetic doses to avoid toxicity.
Electroporation Treatment Location Within the Brain
When applying Electroporation (EP) to the brain the most common treatment
location is the striatum. Nevertheless, it is possible to choose other brain areas.
Note that the conductivity of gray matter is assumed to be homogeneous and
isotropic while white matter is more anisotropic and inhomogeneous, where the
longitudinal conductivity is 5–10 times greater than the transverse conductivity
(Lozano and Hallett 2013). This might influence the shape and volume of the
induced BBB disruption significantly.
Electrode Setups
When applying EP to the brain there are two main possible electrode setups. One can
either use a multiple intracranial electrodes configuration (Miklavcic et al. 2012) or a
single intracranial electrode configuration which requires an external electrode as

well. The intracranial electrodes should not be thicker than 25 G, including insula-
tion (if used). This size ensures that the path of the electrode will cause little or
minimal undesired damage to brain tissue.
Single Intracranial Electrode Setup

This setup consists of a single insulated intracranial electrode and an external surface
flat electrode pressed against the rat chest. Electrically conducting gel should be
applied between the rat chest and the ground electrode for good electrical coupling.
This setup produces an electrical field that is strongest near the intracranial electrode
and decays with the distance. In order to produce a spherical effect surrounding the
tip of the electrode, insulate the electrode leaving only an exposed tip. For rats’
brains an exposed tip of 1 mm is recommended. The flat surface electrode should be
large enough to cover as much of the rat chest as possible and should be placed as
close as possible to the intracranial electrode in the horizontal plane. A small overlap
in the vertical plane is preferable, but if not possible, the horizontal distance between
the electrodes should not exceed 1 cm. If the electrodes are too far apart, the resulting
electric field might not be strong enough to induce EP. An example can be seen in
Fig. 4.
When using this setup with high numbers of treatment pulses or high treatment
voltages, irreversible EP is induced (followed by tissue necrosis) surrounding the
electrode tip, surrounded by a larger volume of BBB disruption. When applying low
numbers of pulses or low treatment voltages, this setup can induce small volumes of
localized BBB disruption, surrounding the electrode tip, without causing irreversible
damage.
Multiple Intracranial Electrodes Setup

This configuration most commonly consists of two or more intracranial electrodes,
where both the active electrode and the ground electrode are intracranial. The
electrical field with this setup will be strongest between the electrodes and can
produce, depending on the treatment parameters, either BBB disruption without
causing irreversible damage or irreversible damage surrounded by a thin volume of
BBB disruption (due to the rapid decay of the electric field).
Surgical Procedure for Electrode Implantation
Electrodes Implantation in Naïve Rats

For the implantation of a single intracranial electrode, a midline scalp incision can be
made to identify the bregma. For implantation in the striatum, a 1 mm burr hole
should be made 2 mm posterior and 3 mm lateral to the bregma. For implantation in
frontal white matter the burr hole should be made 2 mm anterior and 2 mm lateral to
the bregma. The electrode should then be placed stereotactically 5.5 mm deep into
the striatum or 3–3.5 mm deep into frontal white matter.
Note that the thin electrode will probably not be able to penetrate the thick dura;
therefore it is advisable to puncture a hole in the dura using a large-gage needle
before inserting the electrode.
For the ground electrode, a flat metal plate (preferably at least 2 4 cm) should
be pressed against the rat chest after applying electrical conducting gel for improved
electric coupling.
For two intracranial electrodes configuration, two burr holes may be made for the
electrodes with a 3–5 mm distance between them. Note that if burr holes are too
close, the skull might fracture. The remaining of the surgical procedure is the same as
for the one intracranial electrode setup. It is also possible to perform craniotomy for
the two-electrode setup. In the case of more than two electrodes, craniotomy is
always needed.
Electrodes Implantation in Tumor-Bearing Rats

As the main use of EP-based BBB disruption is for the disruption of BBB in tumors
and surrounding infiltrating zone, experiments using tumor-bearing rats are neces-
sary. Inoculation of intracranial tumors is typically performed in the striatum or in
the frontal white matter region of the rat’s brain.
Tumor inoculation is typically performed in a similar manner to the described EP
procedure. Under full anesthesia, a midline scalp incision is used to identify the
bregma, and a 1-mm burr hole is made 2 mm posterior and 2 mm lateral to the
bregma in order to target the striatum or 2 mm anterior and 2 mm lateral to the
bregma in order to target frontal white matter. A thin (up to 25 G) needle attached to
a syringe pump is placed stereotactically, typically 5–5.5 mm deep in the case of the
striatum and 3–3.5 mm deep in the case of frontal white matter. The cancer cells are
infused at a rate of 1–2 μl/min for 1–5 min, depending on the infusion volume. It is
recommended to seal the burr hole with bone wax post inoculation to prevent the
tumor from growing out of the skull.
The same burr hole can be reopened and used for EP treatment, although the
depth of the tumor needs to be evaluated by imaging prior to treatment to ensure the
tip of the electrode is inserted as close as possible to the center of the tumor.
Magnetic Resonance Imaging
Contrast-enhanced, T1-weighted MRI enables noninvasive evaluation of BBB dis-

ruption volumes. In combination with T2-weighted MRI for the evaluation of early
tissue response and late irreversible damage and gradient-echo MRI for the assess-
ment of bleeding, MRI provides a complete image of EP effects on the brain (Fig. 1).
For BBB disruption evaluation, a gadolinium (Gd)-based contrast agent (e.g.,
gadolinium-Dota) needs to be administered systemically prior to EP. The contrast
agent must be injected pretreatment due to the phenomenon of transient vessels
constriction post EP (Markelc et al. 2013). If intraperitoneal injections are used, a
dose of 0.02 mmol/kg can be injected 1 min prior to treatment, and the imaging
should be performed 20–30 min post treatment. At this time the concentration of the
Fig. 1 MR images of naïve rat brain post EP induced using a single intracranial electrode
configuration with 90 pulses of 50 μs duration at 600 V and 1 HZ. (a) BBB disruption as depicted
by contrast-enhanced, T1-weighted MRI obtained 30 min posttreatment. (b) Early tissue response
as depicted by T2-weighted MRI obtained 30 min posttreatment. (c) Irreversible damage as
depicted by T2-weighted MRI obtained 14 days posttreatment. (d) Gradient-echo MRI obtained
30 min posttreatment. Slight bleeding is observed along the electrode path
contrast in the blood system is at its peak. If the contrast agent is administered
intravenous, the rat should be scanned by T1-weighted MRI immediately
posttreatment. BBB disruption volumes can be calculated from the enhancing
volumes on the MR images. Although in other tissues it was reported that
Gd-based contrast agents can be entrapped in reversibly electroporated cells for a
long period of time (Kranjc et al, 2014), we found that in 24 h post treatment there
was no Gd enhancement left in the brain.
T2-weighted MRI should be acquired 30 min post treatment. Edema and early
tissue response can be calculated from hyperintense regions. Irreversible damage
that might be induced by the EP treatment can’t be evaluated at this time point. The
necrosis caused by the treatment can only be evaluated after full resolution of tissue
responses such as edema and inflammation. For this reason, irreversible effects can
be clearly depicted on T2-weighted MRIs obtained at least 8 days post treatment
(Hjouj et al. 2012) as hyperintense regions.
Possible procedure-related bleeding can be assessed by gradient-echo MRI.
Bleeding will be depicted as hyperintense regions. Note that bleeding is not a
desirable effect and if the procedure is executed correctly, bleeding in the treated
volume should not occur. Sometimes a small bleeding along the electrodes path can
be visible on gradient-echo MRIs obtained immediately posttreatment, but this
bleeding is later absorbed.
Treatment Parameters
EP-induced BBB disruption can be obtained alone or in combination with irrevers-

ible EP, as a spherical volume sounding EP-induced damage. The treatment param-
eters for both effects depend on the induced electrical field as well as on pulse
parameters such as the number of treatment pulses, pulse duration, and frequency.
Any change in one of the pulse parameters can influence the end result. As there are
numerous possibilities for each parameter, all the protocols discussed in this section
will consist of 50 μs pulses at a frequency of 1 Hz. The short pulse duration and
relatively long interval between the pulses can assist in preventing undesired thermal
effects. Nevertheless, it is advisable for each electrodes configuration, to ensure that
the temperature rise will be within the safe range either by measuring the temperature
or by computerized modeling (Garcia et al. 2011). More information regarding
temperature-related effects can be found in Temperature Increase and Thermal
Effects Due to Electroporation in Tissues.
Although discussing pulse amplitude is more straight forward, the resulting EP
effects depend on the electrode setup and therefore setting the pulse amplitude limits
the treatment possibilities to a given electrode configuration. For this reason, the
majority of this chapter refers to induced electrical fields rather than pulse ampli-
tudes. Examples of protocols applied to specific electrodes configurations are given
at the end of this section and in Fig. 3.
The electrical field threshold for inducing transient BBB disruption using a single
electrode setup was simulated by Sharabi et al. (2016) for various numbers of pulses
(50 μs pulses at 1 Hz) and is calculated using the following equation:
EFthresh ðBBBÞ ¼ 1040:1N0:169 (1)
where N is the number of applied pulses.

The electrical filed threshold for irreversible EP-induced damage to the brain
tissue can be calculated using:
EFthresh ðIREÞ ¼ 1462:3N0:181 (2)

Fig. 2 Electroporation
thresholds for inducing BBB
disruption and permanent
tissue damage as a function
of the number of treatment
pulses. The threshold for
inducing tissue damage
should be used as an upper
limit for inducing BBB
disruption without inducing
any damage
where N is the number of applied pulses.

The threshold in Eq. 2 represents the minimal electrical field for inducing brain
tissue damage. This equation should be used to determine the maximal electrical
field that can be used to achieve BBB disruption without causing any damage. If the
goal is to achieve complete ablation of tissue, much higher electrical fields are
needed (Eq. 3):
EFthresh ðIREÞ ¼ 1711N0:192 (3)
Figure 2 shows the electrical field thresholds for inducing BBB disruption and
damage. In order to avoid any tissue damage, the maximal electrical field applied
should be in the range between the two graphs.
In order to determine the pulse amplitude producing the desired electrical field, a
numerical model describing the electrodes configuration and tissue properties should
be constructed. Although it is possible to construct a 3D model, it is simpler to build
a 2D model of the rat’s head and chest and to assume symmetry. In a simple 2D
model, the head and chest can be modeled as a 30 15 mm ellipse with an initial
conductivity of 0.258 S/m to match the conductivity used by Sel et al. (Sel
et al. 2007). The electric field should be described by the Laplace equation for
electric potential distribution in a volume conductor:
∇ ðσðEÞ∇φÞ ¼ 0 (4)
where σ is the electric conductivity of the tissue, E is the applied electric field and φ is
the potential. When EP is applied to the tissue, a change in conductivity occurs within
the treated volume which is dependent on the electrical field. This change in conduc-
tivity can be described by a sigmoidal-shaped function in which 500 V/cm and 700 V/
cm are used as the inflection points (Corovic et al. 2013; Sel et al. 2007). These values
of course depend on the number of pulses and the pulse duration (Pucihar et al. 2011).
Dirichlet boundary condition should be applied to the surface of the active
electrodes:
φ ¼ φ0 (5)
and to the ground electrode:
φ¼0 (6)
where φ0 is the applied potential on the active electrode.

The boundaries where the analyzed domain was not in contact with an electrode
should be treated as electrically isolative, and Neumann boundary condition should
be set to zero on the outer border of the model:
@φ
¼0 (7)
@n
where n denotes the normal to the boundary.

Temperature change during the procedure can be evaluated by the modified
Pennes’ bioheat equation (Eq. 8) in the 2D numerical model with the inclusion of
the Joule heating source term. A duty-cycle approach can be used in order to reduce
model running time, in which a time-dependent solver for the duration of the
treatment is applied, and the thermal dissipation is multiplied by the pulse length:
00 @T
∇ðk∇TÞ þ wb cb ðT a TÞ þ Qmetþ q0 ¼ ρcp (8)
@t
Qmet ¼ σj∇φj2 (9)
where k is the thermal conductivity of the tissue, T is the temperature, wb is the blood
perfusion, cb is the heat capacity of the blood, Ta is the arterial temperature, q000 is the
metabolic heat generation, ρ is the tissue density, cp is the heat capacity of the tissue,
and φ is the local voltage amplitude. Qmet accounts for Joule heating, where ϕ is the
electrical potential and σ is the electrical conductivity of the tissue.
Examples of a numerical model built using COMSOL Multiphysics 4.3 are
shown in Fig. 3. More information regarding temperature-related effects can be
found in ▶ Part V, “Modeling of Electroporation and Related Phenomena.”
Typical Experimental Outline
Normal Brain
Typical experiments with naïve rats start with anesthesia followed by preparation for
treatment including scalp incision, skull perforation, dural perforation, and fixation in a
stereotactic frame. Once the rat is fixed in the frame, a Gd-based contrast agent is
administered IP, and the electrodes are inserted to the brain. One minute post admin-
istration of the contrast agent treatment by electrical pulses begins. When applying EP
to the brain, the treated animals experience an excitation wave of brain neurons.
Therefore, muscle contraction during pulses often occurs. In order to avoid electrode
Fig. 3 Example of a numerical model constructed using COMSOL Multiphysics 4.3. (a)
Geometrical model of head and chest with two 28 G, electrodes insulated except for 1 mm exposed
tips. (b) Geometrical model with one intracranial electrode insulated except for 1 mm exposed tip
and an external ground electrode (in blue). (c, d) Electric field distribution for two (c) intracranial
electrodes and one (d) electrode using an applied voltage of 250 V
movement during EP, which might cause undesired bleeding or dislocation of the
electrodes, the rats are placed in a stereotactic frame where the animal’s head is firmly
fixed to the frame. It is advisable to hold the hind part of the rats in place as well during
treatments. If the rats are kept for follow-up, the incision should be sutured following
pulses termination. It is also possible to use muscle relaxants, but since respiration
muscles may be affected, intubation and ventilation of the rat is required.
Twenty to thirty minutes post contrast administration, the rats are scanned by
gradient-echo, T1- and T2-weighted MRI for assessment of bleeding, BBB disrup-
tion, and early tissue response.
Fourteen days posttreatment, additional T2-weighted MRI is performed under
full anesthesia in order to evaluate irreversible damage to the brain tissue.
Tumor-Bearing Brain
Tumor inoculation is performed as described in section “Electrodes Implantation in
Tumor-Bearing Rats.” Post treatment the burr hole used for inoculation is sealed
with bone wax, and the surgical incision is sutured. EP treatment should be
performed only when the tumor is visible by MRI. Typically, in the order of
5 days posttreatment, it is advisable to perform treatment while tumors are less
than 30 mm3. Even though EP-induced BBB disruption volumes are significantly
larger than this volume, larger tumors may cause severe clinical deterioration and
even death prior to achieving the full effect of chemotherapy or irreversible EP.
On the treatment day, the rats are scanned by contrast-enhanced T1-weighted
MRI to evaluate the tumor volume as well as its depth. It is recommended to use ¾ of
the standard dose (typically 0.02 mmol/kg) in order to avoid accumulation of
contrast which might affect the results of the post treatment MRI. In any case,
minimum of 1 h should pass between the first contrast agent injection and the
second.
Following the MRI, the previous incision should be repeated. If a single intra-
cranial electrode is used, the burr hole should be reopened using a 25 G needle. If
several electrodes are used, craniotomy is advisable. Additional anesthesia should be
administered prior to the EP treatment or prior to surgical procedure, depending on
the depth of anesthesia.
Although the BBB is disrupted for over 24 h, if the experiment includes the
administration of chemotherapy or other therapeutic agents, the drugs must be
administered pretreatment since blood vessel vasoconstriction post treatment
(Markelc et al. 2013) may prevent penetration of the drugs into the brain parenchyma
for up to 24 h posttreatment. EP treatment should be timed to the peak serum
concentration of the drug. For IV cisplatin and bleomycin, it is 3 min prior to EP.
An additional ¾ dose of the contrast agent is administered IP 1 min prior to EP
treatment, and a second MRI scan for evaluation of the treatment effects is
performed 20 min posttreatment. Follow-up MRIs are performed periodically for
assessment of tumor volume changes. The first follow-up should be performed at
least 48 h post treatment in order to ensure that the contrast agent in the brain is a
result of tumor and not entrapment of the Gd due to reversible electroporation
(Kranjc et al. 2015)
Schematic representation of the experimental outline is shown in Scheme 1.
Examples of Treatment Protocols

All the examples of treatment protocols shown in this section were designed for an
experimental setup consisting of a single intracranial electrode (28 G). The electrode
was insulated except for a 1 mm exposed tip and placed in the rat striatum. An
external 5 4-cm flat ground stainless steel electrode was pressed against the rat’s
chest at a horizontal distance of 1 cm from the intracranial electrode (as shown in
Fig. 4).
With this setup, it was possible to obtain small BBB disruption without inducing
irreversible damage by applying 90 pulses of 250 V. Using these parameters
spherical BBB disruption with a radius of 1.4 0.2 mm was induced without
irreversible damage.
Using the same setup with a pulse amplitude of 600 V induced permanent tissue
damage with a radius of 1.3 0.3 mm surrounding the electrode tip, surrounded by
BBB disruption with a radius of 2.3 0.1 mm. Increasing the voltage to 1000 V
increased the tissue damage radius to 2.5 0.1 surrounded by BBB disruption with
a radius of 2.9 0.3 mm. As can be seen in Fig. 5, the ratio between tissue damage
volumes and BBB disruption volumes increases with the pulse amplitude. This
Scheme 1 Schematic representation of typical experimental outline for naive and tumor-
bearing rats
Fig. 4 Recommended setup

(demonstrated by a sagittal
T1-weighted MR image of a
naïve rat) for a single
intracranial electrode and an
external surface ground
electrode. The intracranial
electrode is placed in the
striatum, and the ground
electrode is pressed against
the rat’s chest with a
conductive gel
means that when higher electrical pulse amplitudes are applied, the radius of damage
increases by a larger extent than the radius of BBB disruption.
Figure 6 shows the radii of BBB disruption and damage that can be obtained with
different number of pulses applied at 600 V with the described electrodes setup. Note
that increasing the number of pulses does not increase the effect indefinitely because
of the plateau effect. Interesting to notice is that the ratio between BBB disruption
volumes and damage volumes remains constant. This means that when increasing
Fig. 5 Measurable parameters as a function of treatment pulse amplitude. (a) Radii of BBB
disruption and damage induced by 90 pulses at different pulse amplitudes. Setup consists of a single
intracranial electrode and a surface electrode as described above. (b) Ratio between damage and
BBB disruption volumes with the same setup
Fig. 6 BBB disruption and

irreversible tissue damage
radii as a function of the
number of treatment pulses
at 600 V. It can be seen that
the radii increase with the
number of pulses showing an
accumulative effect and
eventually reaching saturation
both the damage radius and the BBB disruption radius is desirable, increasing the
number of pulses is favorable over increasing the pulse amplitude. The ratio between
damage and BBB disruption volumes does not depend on the number of pulses.
These protocols are suitable for inducing EP in naïve rat brains. Treating tumors
requires further considerations as the electrical properties of tumors differ from those
of healthy brain tissues. Tumor cells are denser, and the electrical conductivity of the
tumor is generally higher than that of a normal brain. On top of this, gliomas are
often necrotic and inhomogeneous in nature. As such the electrical field distribution
is expected to differ, and the maximal electrical field in the tumor is expected to be
lower. Therefore, the treatment parameters should be adjusted accordingly, individ-
ually. For example, when treating rats bearing brain tumors of ~1.5 mm radius with
both 90 pulses and 180 pulses, applied with an amplitude of 600 V to the core, we
found no significant difference between the BBB disruption radii of the two pro-
tocols. The produced BBB disruption radius was on average 3.12 0.03 mm..
Conclusions
Electroporation (EP)-induced BBB disruption is a novel technique that can be used

for delivering high concentrations of therapeutic drugs to specific brain regions. EP
induces localized BBB disruption lasting 24–48 h. Systemic administration of
therapeutic drugs should be done pretreatment since vasoconstriction occurs imme-
diately posttreatment, preventing the penetration of drugs into the brain despite the
disrupted BBB (Jarm et al. 2010).
This time point is efficient as it uses both reversible EP effects, enhancing drug
uptake to the cells as well as the BBB disruption. The relatively long duration of the
disruption differs from reversible EP which only lasts several minutes post treat-
ment. The localized effect is suitable for treating brain tumors or metastases as well
as localized neurodegenerative diseases as the treatment can be performed in a
minimally invasive manner.
It is possible to disrupt the BBB using EP in a controlled manner either in a way
that would not cause any brain damage or in combination with irreversible EP. EP
can be performed using a single intracranial electrode and an external surface
electrode or using several intracranial electrodes. Using a single intracranial elec-
trode does not require craniotomy and is minimally invasive. It is mostly suitable for
the combined approach, as the electric field is strongest near the intracranial elec-
trode and tappers down to lower electrical field gradually, providing a relatively
small irreversible damage volume surrounded by larger BBB disruption. Several
intracranial electrodes are by nature more invasive, often requiring craniotomy, but
this type of setup enables disrupting larger volumes of tissue in a single treatment
without inducing damage. Single intracranial electrode produces a spherical-shaped
treatment volume while using several electrodes and can produce a more complex
treatment shape. Since the electrodes are close to each other in the multi-intracranial
electrodes setup, avoiding tissue heating is more challenging and should be taken
under consideration when planning the treatment, often requiring lower voltages. In
both cases, computerized models should be used for individual treatment planning.
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Medical Imaging of Electroporation
43
Eisuke Ueshima, Govindarajan Srimathveeravalli, and
Stephen B. Solomon
Abstract
Irreversible electroporation (IRE) is emerging as an important option for the
minimally invasive focal ablation of tumors in the liver, pancreas, prostate, and
kidney. Imaging plays a crucial role in selection of appropriate pulse parameters,
electrode placement, monitoring of pulse delivery, and evaluating outcomes of
IRE. The ability to clearly distinguish between regions of reversible and irrevers-
ible electroporation is crucial for the effective treatment of tumors. As electropo-
ration itself cannot be directly visualized in patients, the imaging of tissue
changes that arise following electroporation such as changes in perfusion, vascu-
lar permeability, diffusion, and functional or metabolic state of tissue is used to
monitor IRE in patients. Several imaging techniques, such as computed tomog-
raphy (CT), ultrasound (US), magnetic resonance imaging (MRI), and positron
emission tomography (PET) are now used for guiding and assessing IRE in vivo.
In this chapter, typical imaging features that manifest following IRE are discussed
and the physiologic effects that underlie these features as seen with different
imaging techniques in malignant and normal tissue in the liver, prostate, and other
major organs are described.
Keywords
Irreversible Electroporation (IRE) • Computed Tomography (CT) • Magnetic
Resonance Imaging (MRI) • Ultrasound (US) • Positron Emission Tomography
(PET)
E. Ueshima (*) • G. Srimathveeravalli • S.B. Solomon

Interventional Radiology Service, Department of Radiology, Memorial Sloan Kettering Cancer
Center, New York, NY, USA
e-mail: ueshimae@mskcc.org; srimathS@mskcc.org; SolomonS@mskcc.org

DOI 10.1007/978-3-319-32886-7_169
762 E. Ueshima et al.
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 762
Computed Tomography (CT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763
Ultrasonography (US) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766
Magnetic Resonance Imaging (MRI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 769
Positron Emission Tomography (PET) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 773
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 774
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 775
Introduction
Electroporation is a technique used to permeabilize cell membranes with external

electric fields. Exposure to an external electric field alters the transmembrane
potential of cells and causes formation of nanoscale pores in the bilipid layer.
Transient permeabilization of the cell membrane with recovery of membrane integ-
rity with limited or no cell injury is called reversible electroporation (RE) (see
▶ Chap. 99, “Electrochemotherapy of Primary Liver Tumors”). RE is an established
lab technique, used in the lab and with patients for the introduction of genes and
drugs into the cell. Extensive permeabilization of the cell membrane leading to loss
of homeostasis and cell death is termed irreversible electroporation (IRE – see
▶ Chap. 109, “Safety of Clinical Irreversible Electroporation”). IRE has been
developed as an ablation technique for the focal destruction of malignant tumors
in solid organs within the clinical setting.
Successful treatment is achieved when the entire tumor and tissue in the tumor
margin undergo IRE and not RE. Incomplete IRE of tumors can leave behind residual
viable tumor and cause recurrence. In the clinical setting, it is therefore important to
discriminate between reversible and irreversible forms of electroporation. Further,
IRE requires delivery of high voltage pulses in vivo, necessitating careful planning of
pulse delivery to targeted tissue and minimizing adverse effects on nontargeted tissue.
As permeabilization of cells due to IRE cannot be visualized directly, treatment effects
and success is determined by imaging physiological changes associated with success-
ful IRE (see ▶ Chap. 109, “Safety of Clinical Irreversible Electroporation”).
Imaging is therefore invaluable to address the needs that come with planning,
executing, and evaluating patients following IRE. Medical imaging can be used to
simulate treatment before it is even executed, and clinicians can use imaging to plan
electrode placement and optimize pulse parameters to ensure adequate tumor cov-
erage. During treatment, imaging can be used to guide the accurate placement of
electrodes according to plan and monitor treatment to ensure safety of the patient.
Following treatment, imaging can be used to determine treatment efficacy and
identify any residual or recurrent tumor. As complications may occur, imaging can
also be especially useful for diagnosing any rare complications such as vascular
stenoses or thrombus formation that may manifest following IRE.
43 Medical Imaging of Electroporation 763
Several imaging techniques that are used clinically with IRE include ultrasound
(US), magnetic resonance imaging (MRI), computed tomography (CT), and positron
emission tomography (PET) (Barabasch et al. 2016; Vroomen et al. 2016; Ryan et al.
2013). US and CT are most commonly used with patients to guide electrode
placement and monitor pulse delivery. Each imaging modality captures different
tissue or physiological changes that manifest from electroporation of tissue. The
changes are then interpreted to confirm successful IRE of the targeted tissue.
Techniques such as US, CT, MRI, and angiography can accurately capture
IRE-related direct tissue effects such as edema, hemorrhage, vascular congestion,
tissue necrosis, and devascularization. Techniques such as PET and some MRI
sequences can also capture functional tissue changes that arise following IRE such
as changes in metabolic changes and tissue perfusion/diffusion in tissue (see
▶ Chap. 42, “Effect of Electroporation on Blood-Brain Barrier”).
Established techniques for image guided ablation often rely on sustained and
large changes in tissue temperature for destruction of tumors and as such they are
commonly termed as thermal ablation techniques. On the other hand, while there are
transient increases in tissue temperature, cell death from IRE occurs largely from the
loss of homeostasis from electroporation of the plasma membrane and not from
temperature changes; hence, IRE is termed as a nonthermal ablation technique.
Therefore, owing to its nonthermal cell death mechanism, IRE treated tissue can
appear different from the findings that manifest following thermal ablation.
Imaging findings following thermal ablation are well documented under multiple
modalities; however, current understanding of imaging findings during or following
IRE in different organs and tumors is still developing. Thus, in this chapter, the
typical features that manifest following IRE, the underlying process captured by
different imaging techniques, and the clinical role of these techniques are discussed.
Imaging data presented from animal studies were acquired during Institutional
Animal Care and Use Committee approved experiments (Table 1).
Computed Tomography (CT)
Computed tomography is one of the most commonly used clinical imaging techniques
and utilized for image guided ablation as well. CT makes use of computer-processed
combinations of X-ray images acquired from different angles to produce cross-
sectional images of the scanned location. These images can be acquired using helical
or cone-beam techniques and reconstructed to axial, coronal, and sagittal images.
CT scans can be used for detecting the tumor to be treated with IRE, planning IRE
treatment, placing the needle electrodes into the tumor to deliver pulses, and
assessing the immediate posttreatment and long-term follow-up outcomes of treat-
ment. On CT scan, air density is defined as -1000 HU (Hounsfield Unit) and water
density as 0 HU, with tissue components such as organs, bone, or blood presented in
density relative to air and water. Generally, organs and solid tumors are observed to
Table 1 Useful imaging modality of IRE

Application Application
for for Application
treatment treatment for treatment
Advantage Shortcoming planning monitoring follow-up
CT Multi-planar Radiation Essential Essential for Commonly
imaging accurate used
electrode
High
placement
resolution
Ultrasound Portable Operator Not useful Realt-time Useful
dependent guidance
Rapid image
outcomes for
acquisition
electrode
No radiation Not suitable
placement
for imaging
deep tissue
MRI Multi-planar Imaging Useful Not useful Commonly
imaging susceptible used
Functional to artifacts
and metabolic Lengthy
assessment acquisition
No radiation time
PET (-CT) Metabolic Radiation Useful Useful Useful
imaging Lengthy
acquisition
time
be of almost the same density (called soft tissue density) and a contrast agent has to
be delivered to assist in differentiating tumor from normal tissue. The contrast agent
is typically delivered intravenously; the penetration and washout in normal and
tumor tissue vary substantially from differences in vascularization and the patency
of the vessels, allowing discrimination of tumor from normal tissue. Hemorrhage in
tissue/tumor and bone appear with high density while necrotic tissue and edema
appear with low density when compared to normal tissue. Therefore, with CT scans,
resultant changes after electroporation such as hemorrhage can be distinguished
easily from normal tissue component. Similarly, with contrast enhanced CT, ablation
related devascularization of tissue and subsequent necrosis of tissue can be readily
imaged. There are several reports describing the imaging features in CT during or
after IRE in both normal tissue and tumors. While some imaging findings following
IRE appear consistently, other findings do not appear consistently and are yet to be
validated as measures for evaluating the effects of IRE.
In the liver tissue ablated with IRE, noncontrast CT shows that ablated lesions are
isodense and cannot be clearly delineated immediately following treatment. The same
lesions appear hypodense on noncontrast CT at later time points. On dynamic contrast
enhanced CT (CECT), IRE lesions immediately after treatment demonstrate 3 zones
with different patterns of enhancement: (1) an inner nonenhancing zone, (2) a middle
Fig. 1 Contrast enhanced computed tomography (CT) images of liver before and after irreversible
electroporation (IRE) of metastatic tumors in a patient. (a) Arterial phase CT image before IRE
shows two well-enhanced lesions (*), consistent with metastatic tumors. (b) Noncontrast CT images
are used to navigate and place needle electrodes (arrowhead) into one of the lesions. (c) Arterial
phase CT image immediately after IRE of two lesions shows heterogeneous united low attenuation
of the ablated zones (dotted line) with ill-defined periablational hyperenhancing zones. (d) Portal
phase CT image immediately after IRE shows well-demarcated low attenuation of the ablated zones
with hyperenhancing zones (black arrows) at the peripheral portion of the ablated lesions. Gas
pooling (white arrow) is seen in ablated zone. Portal vein has been annotated with by «
zone with well-defined progressive internal enhancement, and (3) an outer ill-defined
enhancement zone with rapid wash out (Fig. 1c, d). The inner nonenhancing zone exists
around the electrode and is understood to demarcate the region of acute necrotic cell
death and is similar in appearance to thermal ablation. In the immediate vicinity of the
electrodes, cell death from IRE seems to be more immediate and severe than regions
that are further away. Also, any thermal effects during IRE are restricted to this region
and consequently the tissue may be devascularized, contributing to its appearance on
imaging. The middle zone with well-defined progressive internal enhancement is
unique to IRE and is not typically observed during imaging of thermal ablation. This
region corresponds to tissue where the effects of IRE lead to apoptosis of the tissue with
preservation of the vasculature. Preservation of the microvasculature and normal tissue
architecture allows penetration of contrast media to this zone, bringing its unique
imaging appearance which is best observed in portal phase CECT. The outermost zone
of ill-defined enhancement is consistent with the region of reversible electroporation
and vascular hemorrhage just outside the ablation zone (Chung et al. 2016; Lee et al.
2013).
Also in the liver, IRE creates uniform tissue ablation around vasculature and large
blood vessels, without perivascular tissue sparing. Although vessel narrowing has
sometimes been reported at early imaging follow-up, such imaging features are not
observed at later time points. Concurrently, heat sink effects or signs of vascular
damage are not observed following IRE (Lee et al. 2012).
IRE of pancreas appears different from IRE of liver. Immediately following
treatment, the ablation appears as a uniform hypodense zone and is often substan-
tially larger than the planned ablation. The apparent increase in size of the ablation
zone may be related to tissue edema and also because the pancreas is not an
encapsulated organ. In contrast to the appearance of IRE ablation in liver, the
ablation zone in the pancreas is ill defined, without properly delineated borders.
Further, internal enhancement (middle zone) or periablation enhancement (zone of
reversible electroporation) are not apparent. Compared to immediate postablation
imaging, the treatment region may demonstrate increased enhancement over longer
follow-up periods. This may be related to inflammation and the development of
granulation tissue and fibrosis (Akinwande et al. 2015).
The key benefits of CT imaging for guiding and follow-up post IRE ablation are
its superior spatial resolution, larger field of view, reduced distortion, and rapid
image acquisition (Table 2).
Ultrasonography (US)
Ultrasound imaging transmits pulses of high frequency sound waves and produces
pictures through patterns of tissue absorption and reflection of the waves. Many
different types of images can be formed using US imaging. The most well-known
type is a B-mode image, which displays the acoustic properties of a two-dimensional
cross section of tissue. In this mode of imaging, features can be strongly echoic (stone,
calcification, and gas), hyperechoic (fat, spongy, or necrotic tissue), isoechoic (solid
tumor or lesions, surrounded by uniform normal tissue), or echo-free (fluid collections,
cysts, and fibrosis). Other forms of US imaging can detect patterns of blood flow
(Doppler imaging) or changes in tissue stiffness (elastography). Specifically for IRE,
ultrasound is useful for rapid imaging of tumors that are not deeply situated (<10 cm),
needle placement with real time imaging, and monitoring of IRE progression. US can
also be used for immediate posttreatment assessment of the ablation zone. Several
reports have described US imaging features of IRE in the liver.
Thermal ablation of solid tissue, such as liver and kidney, typically causes the
treatment zone to appear hyperechoic on B-mode ultrasound. The hyperechoic
appearance is typically caused by microbubbles that form in the thermally injured
tissue and from tissue desiccation. In contrast, liver ablated IRE presents a hypo-
echoic zone in the early timing of energy deposition in IRE (Fig. 2a, b). At later acute
Table 2 Imaging features of computed tomography (CT) after IRE on liver and pancreas
Liver CT Timeline Immediately Later
after IRE
Features Isodense zone Hypodense zone (Well defined)
(ill defined)
Contrast Timeline Immediately ~ few minutes
enhanced after IRE
CT Features Inner Middle zone Outer
nonenhancing with well- ill-defined
zone defined enhancement
progressive zone with rapid
internal wash out
enhancement
Interpretation Region of Apoptosis of Reversible
acute necrotic the tissue with electroporation
cell death preservation of and vascular
the vasculature hemorrhage
(unique to IRE)
Pancreas CT Timeline Immediately Later
after IRE
Features Uniform Increase in size and less defined
hypodense
zone (ill
defined)
Contrast Timeline Immediately Later
enhanced after IRE
CT Features Hypodense Increased enhancement because of
zone (ill inflammation
defined)
imaging timepoints, this zone decreases in size and becomes more isoechoic
(Fig. 2b, c). At 60 min after IRE, the ablation zone appears as a hyperechoic area
with a peripheral hyperechoic rim (Fig. 2d). The initial hypoechoic region is caused
by water content (i.e., edema) in ablated area as a result of disruption of cellular
membrane by IRE. A delayed hyperechoic rim represents widened sinusoidal spaces
filled with red blood cells (Lee et al. 2007, 2010; Appelbaum et al. 2014). IRE can
sometimes cause microbubbles around electrodes in ablated tissue (Fig. 2a). This
may be the result of the formation of hydrogen microbubbles from electrochemical
reactions adjacent to the electrodes delivering the electric pulses.
Contrast enhanced ultrasound may provide further benefit for guiding and mon-
itoring treatments by improving depiction of vascularity of the target lesion. On
contrast enhanced ultrasound, the IRE ablation zone appears as an enhancing
hypovascular area in the arterial phase, in contrast to radiofrequency ablation
(RFA) induced thermally coagulated zones which never enhance over time. In the
portal phase, a clearly depicted hypovascular area is observed, showing the highest
correlation with gross pathologic findings of ablation. In the Kupffer phase (about
10 min after injection of contrast medium), the contrast between IRE ablation zones
Fig. 2 Typical US findings associated with IRE of normal porcine liver. (a) Scan obtained imme-
diately after IRE with the needle electrode still in place (arrowhead). Hyperechoic zone (white
arrows) seen around the electrode is consistent with “microbubbles.” The needle casts an acoustic
shadow (dark region) along the needle tract. (b) Scan obtained 1 min after pulse delivery demonstrates
a hypoechoic ablative zone (dotted circle), with well-demarcated margins. (c) Scan obtained 10 min
after ablation demonstrates the hypoechoic ablation zone, decreasing in size. (d) Scan obtained 60 min
after procedure shows an ablation zone that has lost the internal hypoechoic zone and with a
hyperechoic peripheral rim (black arrows). The portal vein is demarcated by an asterisk
and liver parenchyma is lower than in portal phase images as a result of faint liver
parenchymal enhancement (Sugimoto et al. 2015).
On ultrasound elastography, tissue stiffness can be predicted. Tissue stiffness in
the IRE ablation zones increases over time till 4 h posttreatment at which point it
plateaus. Tissue stiffness after IRE is less than after radiofrequency ablation and
microwave ablation, as the latter are thermal ablation techniques that lead to
denaturation of proteins and heat coagulation of ablated tissue while IRE is a
nonthermal ablation technique that causes less tissue denaturation (Au et al. 2013).
US imaging presents some unique advantages for guiding and assessing IRE
ablations. It can provide images in real-time without risk of ionizing radiation, and
the imaging devices are small and portable. However, US may be challenging to
perform in patients with cirrhotic livers as it can be difficult to differentiate tumors

from diseased liver (Table 3).
Magnetic Resonance Imaging (MRI)
The movement of protons and the innate magnetic fields found in different elements
are used to construct images during MRI. MRI uses magnetic fields and pulses of
radio wave energy to excite or perturb protons within the scanner to acquire different
types of images. MRI is also capable of performing nonanatomic functional imaging.
The movement of protons can be used to determine diffusion, perfusion, or spectro-
scopic characteristics of living tissue. MRI can employ different imaging sequences
such as T1 weighted image, T2 weighted image, fluid attenuation inversion recovery
(FLAIR), and diffusion weighted image. Some of these imaging sequences can be
combined with the use of intravenously administered MR imaging contrast to
enhance image acquisition. Therefore, MRI can provide superior anatomic detail
as well as contrast and functional status of IRE treated tissue.
Electroporation, IRE-related permeabilization of cell membrane, as well as sec-
ondary effects on microvasculature can cause fluid changes in the treated location.
These changes in turn cause edema, hemorrhage, or congestion, all of which can be
readily visualized with different MRI sequences. Generally, on T1 weighted image,
tissue hemorrhage appears as a region of high signal intensity. At the same time,
edema, congestion, and residual tumor all demonstrate high signal intensity on T2
weighted and FLAIR sequences. On diffusion weighted images, viable tumor and
infarction of tissue restrict the movement of protons and can be seen as regions with
high signal intensity. However, these typical intensities of MRI are not always seen
after electroporation and depend on the tissue (including its water and vascular flow)
and timing of image acquisition after electroporation.
MRI features after electroporation can vary substantially based on the tissue or
organ being treated and change considerably over time following electroporation. At
acute phase, images on MRI are usually influenced by the presence of edema and
hemorrhage. At later time points, presence of inflammation, fibrosis, and tissue
remodeling determines imaging findings.
In potato (vegetable) ablated with IRE, the ablated lesion can be seen as a high
intensity area. The lipids in potato tissue are in the intact cell membrane and are
organized in a tight structure, and protons in the molecule cannot vibrate freely.
When molecular defects are induced in the cell membrane, the lipids in the mem-
brane have additional degrees of freedom, resulting in a higher signal in T1 weighted
image and FLAIR sequence images, which suppress the effects of the liquid and
enhance the effects of lipids (Hjouj and Rubinsky 2010).
In liver ablated with IRE, ablation zones are immediately visible on MR images as
hypointense regions on T1 weighted images, and appear as homogeneously hyper-
intense regions when compared with normal liver parenchyma on T2 weighted images
and as hyper/iso-intense (relative to adjacent normal liver parenchyma) regions on
proton density weighted images (Zhang et al. 2010; Guo et al. 2010). Signal intensity
770
Table 3 Imaging features of ultrasound after IRE on liver

Ultrasound Timeline Immediately ~ Few minutes ~10 min Around 60 min
after IRE
Features Microbubbles around Hypoechoic zone Decreases in size and Hyperechoic area with a
electrode becomes more isoechoic peripheral hyperechoic rim
Interpretation Hydrogen microbubbles from Treatment related changes Widened sinusoidal spaces being filled with red blood
electrochemical reactions in tissue water content cells
Contrast Timeline Immediately ~ few minutes
enhanced after IRE Arterial phase Portal phase Kupffer phase
ultrasound (about 10 min after contrast injection)
Features Enhancing hypovascular area Clearly depicted Contrast between IRE ablation zones and liver
hypovascular area parenchyma is lower
Interpretation Preserved vasculature, not Clear demarcation of Result of faint liver parenchymal enhancement
observed in thermal ablation necrotic regions
E. Ueshima et al.
of the ablation zone on T2 weighted image becomes intermediately hypointense

compared with normal parenchyma and exhibits a hyperintense rim that is visible
around the ablation zone. Contrast-enhanced MRI visualizes the ablated lesion as a
hyperintensity area on T1 weighted images. This hyperintensity area, however, includes
both reversible and irreversible regions, resulting in overestimating of ablated region.
Penumbra-nulled IR images (inversion times set to null signal from peripheral penum-
bra surrounding ablation zone) seem to provide more accurate predictions of IRE
ablation zones than contrast enhanced T1 weighted image (Guo et al. 2011).
In hepatocellular carcinoma lesions ablated with IRE, a hyperintense core and a
hypointense rim are observed on T1 weighted images. On T2 weighted images, the
signal is heterogeneously hypointense. On diffusion weighted imaging (DWI), the
signal is not clearly discernible even for different b values (Granata et al. 2015).
Signal intensity of liver lesions immediately following IRE appears similar to signal
intensity on pretreatment imaging (Barabasch et al. 2016).
In prostate tissue 2–4 weeks after IRE, the ablated zone appears as a heterogeneous
hypointense zone on the follow-up T1 weighted images (Fig. 3b), but it is not clear
enough to separate the ablated region from surrounding normal tissue. On T2 weighted
images, the ablation zone appears as a hyperintense area interspersed with low intensity
area (Fig. 3c). On contrast-enhanced T1 weighted images, the ablated area clearly
appears as nonenhancing (Srimathveeravalli et al. 2016) (Fig. 3d). Although both T2
weighted images and contrast-enhanced T1 weighted images are useful to identify the
ablation zone, T2 weighted images are influenced by edema and inflammation. There-
fore, contrast enhanced T1 weighted images seem to provide a more accurate prediction
of IRE ablation zone than T2 weighted images (van den Bos et al. 2016).
Pancreas and pancreatic tumor treated with IRE can be seen as an ill-defined
region, as heterogenous hyperintense appearance onT2 weighted images, and as an
isointense region on T1 weighted images. These image findings can be associated
with limited healthy parenchyma surrounding the tumor and from treatment related
edema. The treated regions are not clearly discernible on diffusion weight images
and on contrast enhanced T1 weighted imaging (Vroomen et al. 2016).
In kidney ablated with IRE, the highest visual contrast between the treatment zone
and normal parenchyma can be obtained by fat-suppressed T2 weighted image. Treat-
ment region show inhomogeneous necrosis with small perifocal edema in the short-term
and sharply demarcated scars in the mid-term. The central necrotic regions appear
isointense when compared to the adjacent renal cortex on T2 weighted image and
diffusion weighted image and hypointense on T1 weighted image (Wendler et al. 2013).
In the brain of rats, diffusion weighted imaging is capable of detecting
electroporation-induced permeabilization of brain tissue. In the diffusion weighted
images, permeabilized area of brain tissue shows hyperintensity after electroporation
(Mahmood et al. 2015). On the other hand, in the brain of dogs, blood brain barrier
disruption appears as a zone of clear enhancement on T1 weighted images. Diffusion
weighted images shows no significant changes in blood brain barrier. Early treatment
effects and late irreversible damage are clearly depicted as hyperintensity on the T2
weighted images (Hjouj et al. 2012). IRE ablation zones are sharply demarcated and
iso- to hypointense on the T1 weighted image sequences, hyperintense on T2
Fig. 3 Representative MR images of patient prostate treated with IRE (a) and 2 weeks after (b, d)
IRE. (a) T2 weighted image before treatment shows hypointense tumor (*) in the left lobe of
prostate. (b) Ablated zone appears isointense and cannot be clearly separated from surrounded
normal prostate on T1 weighted images. (c) Ablated zone appears as a heterogeneous hyperintense
region (arrowheads) with an internal hypointense region (dotted area) on T2 weighted images.
Hypointense region shows an irreversible ablated area, whereas hyperintense region shows possibly
reversible area, associated with inflammation and edema. (d) Contrast enhanced T1 weighted
images show nonenhancing region (dotted area). This region demonstrates a completely ablated
area, matching the hypointense region on T2 weighted images ((c))
weighted images, and peripherally contrast enhancing following intravenous admin-

istration of Gadolinium contrast media (Ellis et al. 2011).
While CT, MRI, and US are used for assist in the setting of electroporation, they are
unable to monitor the process of pulse delivery or localize the electric field for more
accurate focal treatments. Magnetic resonance with electric impedance tomography
may reconstruct electric field distribution to images by measuring the electric current
density distribution and electric conductivity of the treated subject during application of
electric pulses. MR electric impedance tomography can thus be used for the prediction
of reversibly and irreversibly electroporated areas (Kranjc et al. 2015) (Table 4).
Table 4 Imaging features of magnetic resonance imaging (MRI) after IRE

Liver Timeline Immediately
after IRE
Sequences T1 weighted Contrast enhanced T2 weighted image
image T1 weighted image
Features Hypointense Hyperintense zone Hyperintense zone
zone
Interpretation - Includes both -
reversible and
irreversible areas
Prostate Timeline 2–4 weeks
after IRE
Sequences T1 weighted Contrast enhanced T2 weighted image
image T1 weighted image
Features Hypointense Nonenhancing zone Hyperintense zone with an
zone internal hypointense zone
(ill defined)
Interpretation - Irreversible zone Internal hypointense zone
matches with irreversible
zone
Positron Emission Tomography (PET)
Positron emission tomography (PET) uses small amounts of radioactive materials

called radiotracers. These radiotracers are taken up in relevant organs or tumors, and
a special radiation detector evaluates organ and tissue functions, such as blood flow,
oxygen use, and glucose metabolism. The most popular tracer is 2-deoxy-2-[fluo-
rine-18]fluoro- D-glucose (18F-FDG). 18F-FDG PET provides functional imaging
based on the increased glucose uptake of tissue and depicts metabolic abnormalities
before morphological alterations occur. Because cancer grows at a faster rate than
healthy tissue and inflammation needs more glucose, cancer cells and inflammation
absorb more 18F-FDG. PET scanner detects the radiation given off by the 18F-FDG
and produces color-coded images showing inflammation and cancer tissue. Cur-
rently, many PET scanners also include a conventional computed tomography
(CT) scanner. This allows images of both anatomy (CT) and function (PET) to be
taken during the same examination.
Tissue that has been destroyed with thermal ablation become metabolically
inactive and does not take up 18F-FDG. Recurrence and residual lesions then show
up as regions of intense 18F-FDG uptake.
PET scans of IRE ablation demonstrate a dynamic response to the IRE ablation.
At a few hours following IRE, completely ablated area has no uptake of 18F-FDG.
However, at a few days following IRE therapy, uptake of 18F-FDG surrounding the
ablated region can be seen. At one month or after following IRE, this peripheral
Fig. 4 Positron emission tomography (PET) CT of IRE ablation of metastatic liver tumor in a patient.
(a) Preablation image shows 18F-FDG uptake (*) in the right lobe of liver, demonstrating a metastatic
lesion from colorectal cancer. (b) The FDG avid lesion is used as an aid to guide electrode (arrowhead)
placement into the tumor. (c) At one month post-IRE, the treated region demonstrates faint 18F–FDG
uptake (white arrow) lower than surrounding liver indicating metabolically inactive tissue in the
ablated region (dotted area). (d) At three month post-IRE, contrast enhanced CT shows a hypodense
zone (dotted area) and no recurrence. Portal vein is identified with a black arrow
increase in FDG uptake is no longer present (Fig. 4). Increase of uptake at a few days
around the IRE region may be due to an inflammatory response increasing metabolic
activity. Therefore, an accurate assessment of IRE treatment with PET should be
performed after inflammation has resolved (Ryan et al. 2013; Neal et al. 2012).
Conclusions
In this chapter, typical imaging features that can be observed with different imaging
techniques following electroporation of normal and malignant tissue has been
described. Image guidance is crucial for planning and monitoring IRE treatment as
well as reviewing the efficacy of treatment and evaluating for recurrences after IRE.
Imaging features after electroporation can vary substantially based on the tissue or
organ being treated and changes considerably over time following electroporation.
While some of the imaging features are similar to what is observed following
thermal ablations, IRE demonstrates other unique features that are specific to this
ablation technique. As a relatively new clinical technique, our understanding of
tissue effects of IRE seen on imaging is still growing and our ability to accurately
monitor treatment with IRE will improve in the future.
Cross-References
▶ Effect of Electroporation on Blood-Brain Barrier

▶ Electrochemotherapy of Primary Liver Tumors
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Immunological Response to
Electroporation 44
Bashir Akhavan Tafti and Stephen T. Kee
Abstract
Minimally invasive energy-based ablation techniques such as radiofrequency
ablation, cryoablation, and irreversible electroporation are now widely used for
treatment of a variety of malignant lesions. An unexpected but interesting sequel
of such ablation methods has been regression of untreated distant metastases
following ablation of the primary lesion. This phenomenon has been fairly
studied following ablation with hypo- and hyperthermal modalities, especially
cryoablation, and is mostly ascribed to an adaptive antitumor immune response
induced by release of significant quantities of normally inaccessible tumor anti-
gens from the damaged tissue. Irreversible electroporation is a new, nonthermal
ablation technology that because of its unique characteristics is hypothesized to
be capable of inciting a strong tumor-specific adaptive immune response. How-
ever, very limited information regarding existence, extent, and efficacy of such
immune response is available in the literature. Therefore, based on literature
pertaining to other energy-based techniques, this chapter first provides a brief
summary of mechanisms proposed to be involved in the interaction between
immune system and tissue ablation methods. Then, data from studies investigat-
ing the local and systemic immune response following IRE will be presented and
analyzed.
Keywords
Irreversible electroporation (IRE) • Cryoablation • Adaptive immune response •
Antitumor immunity • Danger signal
B.A. Tafti (*) • S.T. Kee

Division of Interventional Radiology, Department of Radiological Sciences, University of
California, Los Angeles (UCLA) Medical Center, Los Angeles, CA, USA
e-mail: bakhavantafti@mednet.ucla.edu; SKee@mednet.ucla.edu

DOI 10.1007/978-3-319-32886-7_170
778 B.A. Tafti and S.T. Kee
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778
Immunomodulation by Thermal Ablation and Mechanisms Thereof . . . . . . . . . . . . . . . . . . . . . . . . . . 778
Irreversible Electroporation (IRE): A New Method for Tumor Ablation . . . . . . . . . . . . . . . . . . . . . . 780
Components of Cell-Mediated Antitumor Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782
Immunologic Response to Tumor Ablation with IRE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788
Introduction
Over the past few decades, advancements in cross-sectional imaging technologies

have resulted in rapid evolution and widespread use of minimally invasive energy-
based ablation techniques such as radiofrequency ablation (RFA), microwave abla-
tion (MWA), and cryoablation for the treatment of focal, otherwise unresectable
tumors, as well as treatment of patients who are not candidates for surgery or other
conventional therapies (Ahmed et al. 2011). While different in energy sources and
methods of delivery, the basic concept behind all these approaches is induction of
coagulative necrosis through transmission of destructive thermal energy to the target
tissue (Ahmed et al. 2011). Such thermoablative technologies offer several advan-
tages over surgical resection. Most notably, they are associated with reduced costs
and shorter hospitalization stays, better preservation of normal surrounding tissue,
and reduced morbidity (Pereira 2007). Therefore, energy-based ablation modalities
have become a rapidly growing area of research.
Immunomodulation by Thermal Ablation and Mechanisms

Thereof
Perhaps one of the most interesting and unexpected observations in this field has
been spontaneous regression of untreated distant metastases following thermal
ablation of the primary lesions (Bastianpillai et al. 2015; Haen et al. 2011). Although
the underlying mechanisms of this phenomenon have not yet been fully understood,
a growing body of evidence suggests that an adaptive antitumor immune response
(consisting of both cellular and humoral pathways) induced by release of significant
quantities of normally inaccessible tumor-specific antigens from the damaged tissue
plays a major role in this process. In addition, elimination of the inhibitory regulatory
T cells (Tregs), abundant in the tumor microenvironment, alleviates local immuno-
suppression and promotes the function of effector cytotoxic T cells. Tregs are a
subset of T cells which inhibit T-cell-mediated immunity, producing tolerance to
tumor antigens (Bastianpillai et al. 2015; Haen et al. 2011). They affect antigen
presentation and inhibit cytotoxic T cell and natural killer (NK) cell function. Except
as seen in cryoablation, the immune response induced by energy-based ablation
44 Immunological Response to Electroporation 779
modalities is generally insufficient for complete eradication of residual tumors and

maintaining a sustained antitumor immunological memory (Haen et al. 2011;
Machlenkin et al. 2005; Chu and Dupuy 2014).
A possible explanation for the disparity between cryoablation and hyperthermal
modalities such as RFA is that the latter methods cause significant protein denatur-
ation and loss of their three-dimensional configuration. This transformation can
destroy the structure of antigenic epitopes (i.e., antigen segments recognized by
the immune system), and, consequently, limited quantities of intact tumor antigens
remain available for stimulation of the immune system. In addition, coagulation of
the tumor bed vasculature prevents adequate release of tumor antigens into the
systemic circulation (Bastianpillai et al. 2015; Haen et al. 2011; Chu and Dupuy
2014). In contrast, while disrupting the cell membrane, the freezing process main-
tains the three-dimensional structure and integrity of epitopes and, thereby, results in
exposure of immune cells to intact native antigens (Bastianpillai et al. 2015; Haen
et al. 2011; Chu and Dupuy 2014). Ironically, cryoablation is not always immunosti-
mulatory and quite often triggers an immunosuppressive response. Diminished
antitumor response following cryoablation has been especially observed when larger
volumes of ablated tissue were left behind or when multiple lesions were ablated
simultaneously (Chu and Dupuy 2014). The latter observation has been subject to
extensive investigation and is probably best explained by a mechanism similar to
“danger theory” first described by Matzinger to describe the immune recognition of
“self” versus “nonself” in the context of the absence or presence of so-called danger
signals (Matzinger 1994; Fuchs and Matzinger 1996). The “danger theory” states
that three signals are required for initiating a cellular immune response against an
antigen. These include (1) presentation of an antigenic epitope by an antigen-
presenting cell (APC) in a format complementary to a T-cell receptor, (2) a
co-stimulatory signal between the APC and T cell, and (3) a danger signal to activate
the APC. Antigen recognition in the absence of danger signals prevents maturation
of APCs and suppresses cytokine release. This in turn leads to clonal deletion of the
responding T cells and can even result in peripheral tolerance and anergy (Curtsinger
et al. 2003). In tumor immunology, this third signal can come in the form of
(1) pro-inflammatory cytokines (e.g., interleukin 12 (IL-12)), (2) intracellular com-
pounds such as heat shock proteins (HSP), high-mobility group box chromosomal
protein 1 (HMGB1) or deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)
fragments recognized by Toll-like receptors, or (3) signals generated by tissue
structural changes including fibrinogen, oligosaccharides of hyaluronan, and
extradomain A (EDA)-containing fibronectin (Bastianpillai et al. 2015; Chu and
Dupuy 2014). During cryoablation, in situ cell death occurs via either necrosis or
apoptosis. While extra- and intracellular ice formation at the more central zones of
the ablation site cause irreversible membrane damage and cell death via coagulative
necrosis, sublethal temperatures at the more peripheral regions are not low enough to
induce necrosis, and, therefore, the cells undergo apoptosis. Necrosis results in
release of significant quantities of the aforementioned danger signals (e.g., HSP
70) and facilitates maturation of APCs. Heat shock proteins have also been shown to
be effective peptide chaperones, directing exogenous antigens toward major
histocompatibility complex 1 (MHC 1) molecules for presentation to T cells, and are

able to induce expression of co-stimulatory molecules on APCs. Chaperones are a
diverse group of proteins that oversee the correct intracellular folding and assembly
of polypeptides without being components of the final structure. In contrast, apo-
ptotic cells do not readily release their contents and, therefore, actively inhibit the
antitumor immune activity through the following mechanisms: (1) reducing the
availability of danger signals resulting in T-cell anergy, (2) recruitment of Tregs,
(3) decreased expression of co-stimulatory ligands on APCs or increased expression
of inhibitory T-cell coreceptors such as cytotoxic T-lymphocyte-associated protein
4 (CTLA-4), and (4) APC dysfunction. However, it should be noted that this
inhibitory effect applies mostly in the early post-ablation period and recent reports
suggest that in long term, apoptotic cells also hold considerable immunostimulatory
capacity (Scheffer et al. 2003). In addition, tumor cells are known to produce and
store immunosuppressive cytokines such as transforming growth factor beta
(TGF-β). Local and systemic release of such cytokines in their intact form following
apoptosis can further contribute to immune suppression and induction of anergy
against tumor antigens (Peng et al. 2007; Viorritto et al. 2007). Thus, the immuno-
logic impact of ablation may also vary depending on the tumor type and its innate
production of specific inhibitory cytokines. Based on the above information, and
knowing that both necrosis and apoptosis contribute to cell death following cryo-
therapy, the ratio between the extent of necrosis and apoptosis has been postulated as
the main determinant of the immune response type (i.e., stimulatory vs. inhibitory)
following cryoablation (Bastianpillai et al. 2015; Chu and Dupuy 2014). This
probably holds true for other tissue ablation modalities given the fact that variable
degrees of both necrosis and apoptosis are seen in the ablation zone. Figure 1
schematically summarizes the mechanisms discussed above.
Irreversible Electroporation (IRE): A New Method for Tumor

Ablation
IRE is a new, nonthermal ablation technology which is based on delivery of several

short pulses of electrical current to a target tissue. The ensuing electrical field
irreversibly damages cell membranes via creation of nanopores, thereby inducing
cell death by disrupting cellular homeostasis (Al-Sakere et al. 2007a; Lee et al.
2012). IRE has been tested successfully for ablation of normal and malignant cells in
the liver, heart, lung, prostate, kidney, and pancreas in small and large animal models
and very recently in human subjects without significant thermal damage (Wagstaff
et al. 2016; Thomson et al. 2011). Preclinical and clinical studies have also pointed
out several advantages for IRE over the more conventional thermal ablative tech-
niques. For example, the induced electrical fields only affect cell membranes and
spare the extracellular matrix and structures such as myelin sheaths, major vascula-
ture, and cutaneous connective tissue. It has also been demonstrated that
IRE-induced cell death can traverse these structures without causing any damage,
suggesting that IRE is a more prudent modality for treatment of tumors in close
Fig. 1 Mechanisms of cell death following thermal ablation and their effects on stimulation of
lymphocytes. Direct cell injury in the central part of the ablation bed results in necrosis. Intracel-
lular molecules such as heat shock protein 70 (HSP70) released by necrotic cells provide antigen-
presenting cells (APCs) with danger signals required for appropriate priming of T lymphocytes via
co-stimulatory molecules. Sublethal temperatures in the more peripheral zone cause apoptosis.
Stimulation of T cells in the absence of appropriate co-stimulatory signals results in anergy and
clonal deletion of the responding lymphocytes. In addition, apoptotic tumor cells may release
immunosuppressive cytokines (e.g., TGF-β) and further inhibit the immune response
proximity of such anatomical structures (Wagstaff et al. 2016). Moreover, preserva-

tion of the extracellular matrix and connective tissue scaffold facilitates rapid
resolution of the lesion with minimal to no scarring (Wagstaff et al. 2016). The
latter characteristic is of great importance as it makes the target volume more
amenable to follow up evaluation by conventional imaging modalities. Another
clinical advantage of IRE is the accuracy of imaging, particularly real-time ultra-
sound (US), in monitoring the extent of ablation as a result of immediate changes in
the treated tissue’s permeability to water. This feature is truly advantageous as US

could potentially be used during treatment to elicit repeat application of IRE pulses
or make adjustments in electrode positioning to ensure complete treatment of the
target lesions. Furthermore, IRE produces precisely delineated margins with cell-
scale resolution at the border of ablated and non-ablated tissues with good correla-
tion between microscopic and macroscopic findings (Wagstaff et al. 2016; Lee et al.
2010). Subsequently, the efficacy and outcome of IRE procedures can be evaluated
with higher accuracy. Another advantage of IRE is significantly shorter procedural
times (5–8 min vs. 30–60 min in comparison to RFA) (Lee et al. 2010). Finally, as
the elimination of tumor cells is not dependent on heat production, IRE remains
unaffected by the heat sink phenomenon and can be utilized reliably in highly
perfused tissues (Wagstaff et al. 2016; Lee et al. 2010).
There is solid evidence in the literature that IRE induces cell death through both
apoptosis, particularly at lower electric fields (Lee et al. 2012; Zhou et al. 2012), and
necrosis (Al-Sakere et al. 2007a, b; Li et al. 2012; Onik et al. 2007). Thus, and given
the results of studies documenting an immune reaction in the draining lymph nodes
of dogs undergoing IRE ablation of prostate (Onik et al. 2007), it was initially
hypothesized that IRE should be able to elicit robust local and systemic immune
responses against tumors because intact tumor-associated antigens and proteins
released from apoptotic cells as well as cytokines and danger signals unleashed by
necrotic cell death present the immune system with all three elements required to
incite a tumor-specific adaptive immune response. In addition, IRE ablation pro-
motes immediate edema and inflammation, rapidly attracting lymphocytes and
APCs to the treated region (Neal et al. 2013). It is known that antitumor immune
response mostly depends on the cell-mediated pathway wherein activation of both
CD4+ and CD8+ T lymphocytes is required. Therefore, it is worth summarizing the
interplay between the key components of this system (i.e., APCs, CD4+ lympho-
cytes, and CD8+ lymphocytes) before reviewing the available literature on immu-
nomodulatory effects of IRE. Figure 2 also demonstrates this interplay
schematically.
Components of Cell-Mediated Antitumor Immune Response
Professional APCs (also known as dendritic cells or DCs) are necessary for activation
of naïve T cells. APCs internalize antigens either through phagocytosis or receptor-
mediated endocytosis, process the antigens into peptide fragments, load those peptides
on class I or class II MHC molecules, and then display them on their membrane to
engage both helper CD4+ and cytotoxic CD8+ T cells. CD8+ and CD4+ cells
recognize and interact with classes I and II MHC molecule complexes, respectively.
As discussed previously, additional co-stimulatory “danger” signals are also required
for activation of the T cells. In addition, optimal activation of CD8+ cells and
generation of antigen-specific cytotoxic T cells depend on sufficient stimulatory input
from activated Th1-type CD4+ T cells. Insufficient generation of tumor-specific CD4+
T cells results in the host’s inability to eliminate malignant cells, and CD4+/CD8+ ratio
Fig. 2 Differentiation of T-cell subsets. CD4+ T cells are divided to “effector,” “memory,” and
“regulatory” subtypes, each playing a different role in adaptive immune response by secreting a
plethora of immunostimulatory or immunoinhibitory cytokines. The effector T cells further differ-
entiate into two major subtypes: Th1 and Th2
has been suggested as a key parameter for appropriate antitumor function. In practice,
CD4+/CD8+ ratio has been generally used as an indicator of cell-mediated antitumor
immunity and as a prognostic factor for cancer patients receiving immunomodulating
therapies (Bastianpillai et al. 2015; Li et al. 2012). A high CD4+/CD8+ T-cell ratio is
also associated with a reduced risk of tumor recurrence in certain cancers such as
hepatocellular carcinoma, whereas a relatively low number of Th1-type CD4+ T cells
with reversed CD4+/CD8+ ratios are associated with higher mortality rates (Unitt et al.
2006). This ratio has also been used in evaluation of antitumor immune response
induced by thermal ablation techniques including RFA and cryoablation (Li et al. 2010;
Zerbini et al. 2006).
CD4+ T cells are further divided to “effector,” “memory,” and “regulatory”
subtypes. As their names imply, effector cells secrete cytokines that interact with
and stimulate immune cells including other CD4+ T cells, while memory cells retain
their antigen affinity and act as late effector cells during repeat immune responses
(i.e., when the host is reexposed to the same antigen at a later time). In contrast,
regulatory T cells do not promote immune function but inhibit it instead. Tregs play
an essential role in controlling immune responses to self- and nonself antigens. The
effector T cells, in turn, differentiate into two major subtypes characterized by their
distinct cytokine expression patterns known as type 1 and type 2 helper T cells (Th1
and Th2 cells, respectively). Th1 effector cells are important in host immunity
against intracellular antigens such as bacteria, protozoa, and tumors. They are
triggered by IL-2 and IL-12 and secrete IL-2 and interferon gamma (IFN-γ) to
stimulate and activate downstream effector cells including cytotoxic CD8+ cells
and macrophages. In fact, experimental data showing a significantly higher
likelihood of developing certain types of cancers in IFN-γ knockout mice (IFN-γ / )

suggest a key role for CD4+ Th1 cells in tumor immunity. On the other hand, Th2 cells
play a major role in host immunity against extracellular antigens such as parasites and
modulate humoral immunity (e.g., antibody-producing B cells) through secretion of
cytokines such as IL-4, IL-5, and IL-10. On the contrary, high levels of tumor-
infiltrating Tregs associated with poor prognosis have been observed in many malig-
nancies. These cells produce high quantities of inhibitory cytokines IL-10 and TGF-β,
thereby suppressing the immune system and allowing for tumor evasion. IL-10 is a
multifunctional cytokine with immunosuppressive properties that has been found to be
a powerful inhibitor of Th1 cells. It also facilitates differentiation of naïve T cells
toward Tregs. Finally, it is worth mentioning that, very often, cancer cells obtain the
ability to produce copious volumes of IL-10 and TGF-β through mutation (Abbas et al.
2015).
Immunologic Response to Tumor Ablation with IRE
The very first study to investigate the immediate immune response following IRE
was performed in sarcoma-inoculated mice by Al-Sakere et al. (2007b). Although
many prior studies had reported infiltration of the ablation zone with inflammatory
cells after IRE, this was the first study to utilize immunohistochemistry to delineate
the post-ablation evolution of specific immune cell populations. Briefly, tissue
samples collected 0–72 h post-ablation were stained for macrophage 1 (Mac-1),
activated APC (CD86, CD80, and CD11c), and lymphocyte (CD4, CD8) cell surface
markers using respective specific antibodies and compared to control tumor samples
harvested before delivery of the electric pulses. Surprisingly, the results showed a
decline in all these cell types starting at 2 h which reached a nadir at 6 h and remained
unchanged up to 72 h after ablation. This was attributed to dissolution of injured
immune cells already residing within the tumor. In addition, no infiltration of
inflammatory cells into the ablation zone was noted during this time period. This
finding was ascribed to destruction of the tumor microvasculature architecture
precluding any new infiltrates, while larger blood vessels remained unperturbed
(Al-Sakere et al. 2007b). Although the authors did not provide any explanation for
the sharp contrast between these results and prior reports indicating notable
immunocyte infiltration into the ablation zone following IRE, variability in expres-
sion of cell surface markers by other inflammatory cells (e.g., neutrophils and plasma
cells vs. lymphocytes) and differences in intrinsic tumor features (e.g., hypo-
vs. hypervascularity) can be considered as potential reasons.
To shine further light on this matter and better characterize IRE-induced immune
response, Li et al. examined changes in peripheral blood T-lymphocyte subsets,
splenocyte cytokine expression patterns, and serum cytokine profile in an osteosar-
coma rat model over a 3-week period. The results were compared to experimental
groups undergoing sham or surgical tumor removal, as well as nontreated and non-
tumor-bearing animals (Li et al. 2012). The first parameters evaluated in this study
were the percentage of CD4+ and CD8+ T lymphocytes and the CD4+/CD8+ ratio.
The results showed a statistically significant increase in CD4+ T lymphocytes and

CD4+/CD8+ ratio in both surgical resection and IRE-treated groups 7 days after
operation in comparison to other groups. Moreover, the percentage of CD4+ T
lymphocytes and the CD4+/CD8+ ratio in IRE-treated animals were statistically
higher than surgical resection group 14 and 21 days postoperatively. In addition,
experimental subjects in the IRE group had similar percentage of CD4+ cells and
CD4+/CD8+ ratio compared to the non-tumor-bearing animals at the end of studies.
Ironically, the authors observed a significant decrease in the percentage of CD8+
cells in both surgical and IRE-treated groups 14 days after the operation compared to
control and sham procedure cohorts. However, the percentage of CD8+ cells in the
former groups did not show any significant differences in comparison to non-tumor-
bearing rats at post-op days 14 and 21. The authors also analyzed the percentage of
IFN-γ and IL-4-positive splenocytes as surrogates for Th1 and Th2 CD4+ cells,
respectively. While no statistically significant difference was observed between the
five experimental groups before operation, the percentage of IFN-γ-expressing
splenocytes distinctly increased in the IRE-treated group throughout the study.
However, the percentage of IL-4-secreting splenocytes remained unchanged
among all experimental groups at all time points. The authors also examined the
changes in serum levels of the immunosuppressive cytokine IL-10 and observed a
statistically significant decline in both IRE-treated and surgical resection cohorts
compared to sham and control groups. Further, IL-10 serum levels in the former two
groups approached values similar to that of non-tumor-bearing animals at postoper-
ative day 21. Collectively, these results implied that in contrast to surgical resection
which fails to evoke a sustained immune reaction despite having a transient immu-
nomodulatory effect, IRE ablation might be able to trigger an efficient systemic cell-
mediated antitumor immune response (Li et al. 2012). Finally, serum levels of
soluble interleukin-2 receptor (sIL-2R) were measured in all study cohorts at various
time points. Because binding of IL-2 receptor with its ligand (i.e., IL-2) results in
release of a segment of its alpha chain into the bloodstream, levels of serum sIL-2R
have been used as a surrogate marker for semiquantitation of the activated
T-lymphocyte population within tumor-infiltrating lymphocytes, in both primary
and metastatic malignant lesions. Thus, serum sIL-2R has been suggested as a useful
parameter for staging, treatment response monitoring, and restaging purposes in
cancer patients (Murakami 2004). The results showed a rapid decline in sIL-2R
levels in both IRE-treated and surgical resection but not sham or control groups
which reached statistical significance 7 days postoperatively. In addition, subjects in
the IRE-treated group demonstrated a more profound decrease in serum sIL-2R
levels at 14 and 21 days post operation compared to surgical resection group and
reached the same levels as non-tumor-bearing animals at the end of the experiments
(Li et al. 2012).
Although the latter study is perhaps the most comprehensive research on the
effects of IRE ablation on cellular antitumor immune response to date, some of its
findings should be interpreted with caution. For example, the ironic decrease in
percentage of CD8+ cells in IRE-treated and surgical resection groups might have
contributed to changes in CD4+/CD8+ ratio in these groups resulting in artificially
higher CD4+/CD8+ ratio. If this holds true, the increase in CD4+/CD8+ ratio cannot
be solely attributed to increase in tumor antigen-presenting CD4+ Th1 cells. As
another example, decline in serum sIL-2R levels might be predominantly caused by
elimination of tumor and tumor-infiltrating lymphocytes and, therefore, cannot be
ascribed to strengthening of the immune system by IRE ablation as originally
concluded by the investigators. Besides, as acknowledged by the authors, no exper-
iments were performed to further characterize the mechanisms underlying observed
changes in cellular immunity or to determine tumor specificity of the activated T
cells. Also, the efficacy of the elicited immune response in conferring resistance
against tumor recurrence, the sustainability of such presumed protective effect, and
the extent of IRE-induced antitumor response contribution to outcome improvement
were not investigated.
To determine if the IRE-elicited immune response truly enhances the outcome of
primary tumor ablation, and whether it can render the host with a systemic protective
capability against tumor rechallenge (e.g., reactivation of dormant metastatic
lesions), Neal et al. conducted a set of parallel experiments in immunodeficient
(ID) and immunocompetent (IC) mice inoculated with murine renal adenocarcinoma
(Renca) cells (Neal et al. 2013). Each cohort of animals was further divided into
treated and control (i.e., untreated) subgroups. Keeping all variables (e.g., tumor
size, electric field exposure, etc.) equal, tumors in treated subgroups were ablated
with an IRE pulsing protocol that used 60% of the predicted voltage-to-distance ratio
required for causing complete tumor regression in ID mice. The animals were
followed for up to 34 days post treatment. Surviving subjects with excessive disease
burden caused by tumor sizes greater than 18 mm in any dimension were euthanized
earlier. Eighteen days after ablation, treated mice were rechallenged by inoculation
of their contralateral flank using the same cell line. However, treated mice in the ID
cohort had to be sacrificed at up to 4 days post reinoculation based on primary tumor
size criteria, and, therefore, only rechallenge data from IC-treated subgroup were
analyzed. Figure 3 schematically summarizes the experimental setting. Primary
tumor size measurements showed a transient delay in tumor growth and a period
of progression-free disease in treated ID mice. However, tumor growth rate recov-
ered later and reached similar levels as both control groups around post-ablation day
20, indicating a suboptimal response. On the other hand, treated IC mice demon-
strated a significantly extended period of progression-free disease. In addition, mean
tumor volumes from each subgroup were measured every 5 days post-ablation and
compared to other cohorts. Although no significant difference was observed among
the four subgroups at 5 days, mice in treated IC cohort showed statistically signif-
icant lower tumor volumes at each time point after postoperative day 10. No primary
tumor recurrence was seen in 50% of the IRE-treated IC mice. Besides, the
rechallenged IC mice showed significantly slower, if any, tumor growth at the
secondary site of tumor inoculation. Differential growth rates between primary and
rechallenge tumors were also analyzed in IC mice which turned out to be signifi-
cantly lower in the latter lesions (Neal et al. 2013). The better outcome of treated IC
mice provided further evidence in support of the notion that IRE-induced immune
response can play a complementary role by eliminating residual malignant cells and
Fig. 3 Protective effect of systemic immune response following IRE ablation of primary
tumors. In comparison to immunodeficient (ID) mice, progression-free survival was significantly
longer in immunocompetent (IC) mice following IRE ablation of the primary tumors. In addition,
growth of the second tumors was substantially reduced or prevented entirely in IC mice. Animals
had to be sacrificed 16 days after tumor rechallenge due to the size of primary lesions
enhancing the antitumor efficacy of IRE. Further, the protective effect against tumor
rechallenge in treated IC mice supports the possibility of a systemic tumor-specific
immune reaction capable of attacking occult metastatic lesions, although long-term
sustainability of this response remains elusive.
Conclusions
In summary, IRE is a relatively new nonthermal tissue ablation technology that,

because of its nondestructive characteristics, holds great potential for focal ablation
of malignant lesions especially in niche applications such as pancreatic cancer and
tumors encasing critical structures (e.g., major vessels or nerves). The IRE-ablated
cells are eradicated through both necrosis and apoptosis. While apoptotic cells
maintain intact tumor-specific antigens that can be used by APCs for proper antigen
presentation to lymphocytes, necrotic cells release a myriad of co-stimulatory
molecules (i.e., danger signals) required for effective activation of effector and
cytotoxic T cells. In addition, preservation of large blood vessels and rapid repair
of endothelium in smaller vasculature after IRE provide an effective route for
immune cells to reach the ablation zone. Therefore, IRE provides the immune
system with all the necessary elements required for mounting an antitumor response
at local and systemic levels. Data generated by preliminary studies discussed above
provide evidence in support of this theory. However, IRE has only been investigated
for a relatively short period of time, and the limited body of evidence available in the
literature is not sufficient for full characterization of the observed immunological
response. Hence, robustness, tumor specificity, long-term sustainability, and
underlying mechanisms of the induced immune reaction are yet to be determined.

Besides, it is known that tissue response to IRE (e.g., presence or absence of thermal
damage, apoptotic vs. necrotic cell death, etc.) depends on several factors including
IRE pulse parameters (e.g., voltage-per-unit-distance ratio, pulse duration, pulse
interval, etc.) and intrinsic tissue characteristics (e.g., fat and water content). As
different tissues’ reactions to ablation can alter the invoked immune response, more
comprehensive studies are required for better understanding of the relationship
between ablation parameters and immune outcome. In addition, similar to what is
observed in cryoablation, volume and number of ablated lesions may play a role in
determining the type and extent of immune response incited by IRE. Moreover,
almost all of the investigations to date have been focused on cellular immunity, and
probable effects of IRE ablation on humoral immune response remain totally
unexplored. Finally, potential benefit of complementary interventions such as
administration of adjuvant immunostimulants which boost adaptive immunity
should be considered and further evaluated.
Cross-References
▶ Adjuvant Immunotherapy as a Tool to Boost Effectiveness of

Electrochemotherapy
▶ Combined Treatment of Electrochemotherapy with Immunomodulators
Tissue Blood Flow
Deep-Seated Tumors
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Part IV
Hardware Used in Electroporation Experiments
and Applications
Juergen F. Kolb
Marx-Generator Design and Development
for Biomass Electroporation 45
Martin Sack
Abstract
In 1923, Erwin Marx invented a pulse generator based on a capacitor discharge,
now known as Marx generator. Capacitors are charged in parallel connection and
discharged in series connection for voltage multiplication. The arrangement of
components enables a design, for which each element of the generator needs to be
designed for no higher voltage than the charging voltage per stage. Originally, the
Marx generator has been designed for high-voltage testing. In recent years, Marx
generators have been developed for the treatment of biological tissue by pulsed
electric fields (PEF). In contrast to Marx generators for high-voltage testing, these
generators are operated continuously at a repetition rate of at least 20 Hz.
Generators for high pulsed power applications might be set up using charging
coils and spark gap switches. Spark gap switches for extended lifetime have been
developed based on electrodes made of tungsten copper alloy and machined with
a homogeneous field profile. For triggered long-term operation, overvoltage
triggering in combination with artificial seed electron generation by means of a
corona discharge is applied. For another design, the use of semiconductor
switches enables the generation of rectangular or stepwise arbitrary waveforms.
Selected circuits for semiconductor-based Marx generators for generating posi-
tive, negative, and bipolar voltage shapes are described.
Keywords
Marx generator • Pulsed electric field treatment of biological tissue • Spark gap
switch • Overvoltage triggering • Semiconductor switch • Repetitive operation
M. Sack (*)
Institute for Pulsed Power and Microwave Technology, Karlsruhe Institute of Technology,
Eggenstein-Leopoldshafen, Germany
e-mail: martin.sack@kit.edu

DOI 10.1007/978-3-319-32886-7_203
794 M. Sack
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794
Principle of Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794
Ignition of Spark Gap Switches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795
Marx Generator Circuit for High-Voltage Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 799
Marx Generator for Treatment of Biological Material by Pulsed Electric Fields . . . . . . . . . . . . . . 801
Triggered Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 804
Semiconductor-Based Marx Generators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 807
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 811
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 811
Introduction
The Marx generator is a pulse generator for generating high-voltage pulses. It has
been invented by Erwin Marx in 1923 for the purpose of testing high-voltage
components intended for the use in the national grid against pulsed overvoltage
caused, e.g., by lightning strike or switching (Marx 1923). High-voltage testing is
still the main application of Marx generators. However, in recent years, Marx
generators for various industrial applications have become commercially available.
The development of those Marx generators was driven to some extend by the need of
pulse generators for the treatment of biological tissue by pulsed electric fields (PEF).
Such generators need to be capable of reliable continuous long-term operation at
pulse repetition rates of approximately 20 Hz and more. In comparison to other
pulsed power sources as, for example, transformer-coupled pulse generators or pulse
generators based on transmission lines, Marx generators combine a comparably low
inner impedance and, hence, a high-pulse current of up to more than 10 kA per
generator with a rather simple and cost-efficient construction. For large-scale appli-
cations with a high demand for pulsed power, Marx generators can be operated in
parallel configuration. Some design details of Marx generators are specific to the
application. Therefore, in this chapter, different basic designs concepts of Marx
generators are presented with the main focus on repetitively operated Marx gener-
ators for industrial applications. However, a small part of the chapter is also
dedicated to the Marx generator’s original application of high-voltage testing.
Principle of Operation
The Marx generator is based on the discharge of a capacitive energy storage. For
pulse generation, first, energy is stored to the capacitive energy storage during a
rather slow charging process and subsequently fast discharged to a load in a pulse.
Thereby, the pulse shape is adapted to the requirements by means of pulse-forming
circuit elements. For lightning impulse tests of high-voltage components depending
on the rated voltage level of the device under test, a testing voltage with a peak value
of more than a million volt is needed (Küchler 2009). The generation of such a high
45 Marx-Generator Design and Development for Biomass Electroporation 795
voltage with a generator based on a single capacitor only would be quite challenging,
because it would require all components of the pulse circuit to be designed for the
total charging voltage, including storage capacitor, discharge switch for pulse
generation, and capacitor charger.
Instead, according to the design by Erwin Marx, the pulse generator is set up from
separate stages. Each stage comprises a storage capacitor. For charging, the capac-
itors of all stages are arranged in parallel configuration. So all capacitors are charged
to an equal stage voltage. For pulsed generation, the capacitors are then connected in
series such that the pulse is driven by a total charging voltage equal to the sum of all
stage voltages. Apart from the capacitor, each stage comprises additional circuit
elements to establish current paths for charging and discharging, i.e., pulse genera-
tion. This design has the advantage that all circuit elements of the generator need to
be rated for the stage voltage only, which is a fraction of the total voltage required to
drive the pulse.
Figure 1 shows a simplified circuit diagram of a three-stage Marx circuit
connected to a resistive load. For charging the stage capacitors C1. . .C3 are connected
in parallel by means of the charging resistors RC. A charging device CD drives a
charging current until the desired charging voltage per stage is reached. The charging
resistors invoke a voltage drop along the charging path and delay charging of the
stage capacitors further away from the charging device. To allow all stages settling to
equal charging voltage, the charging current might be reduced towards the end of the
charging process. Closing the discharge switches per stage (SW1. . .SW3) establishes
series connection of the stage capacitors. The capacitors are discharged into the
resistive load RL. Depending on the design, the inductance of the leads or an
additional series inductance L may serve in combination with the stage capacitors
and load resistance as pulse-forming elements. A fraction of the capacitors’ dis-
charge current passes through the charging resistors. The value of the charging
resistors is usually chosen so large that the leakage current through the charging
path is small compared to the pulse current.
Ignition of Spark Gap Switches
Spark gap switches are robust and fast switching elements for high currents acting as
closing switches. Spark gap switches have been originally used by Erwin Marx for
his pulse generators, and they are still state of the art especially for Marx generators
designed for combined high-voltage and high-current applications. A spark gap
switch consists of a pair of electrodes with a gaseous dielectric in between. In
off-state the insulating dielectric prevents a current from flowing between the
electrodes. In on-state a dielectric breakdown has established a conductive path
between the electrodes and an arc of high conductivity bridges both electrodes.
In order to establish an electric breakdown in a spark gap switch, the following
two conditions must be met: (a) a number of seed electrons sufficient for initializing
the breakdown must be present, and (b) the electric field strength between the
electrodes needs to be sufficiently large to cause an initialization of the discharge
796 M. Sack
Fig. 1 Circuit diagram of a

Marx generator
by means of charge carrier multiplication. The rate of natural seed electron gener-
ation is in the order of 0.1. . .10 charges per cubic centimeter and microsecond (Frey
1996). The seed electron generation can be augmented artificially, e.g., by means of
irradiation by ultraviolet light. The static breakdown voltage of a spark gap switch is
determined according to Paschen’s law as function of the gas pressure p and the gap
distance d (Küchler 2009).
U D ¼ pd f ðpd Þ (1)
Moreover, the type of gas and the electrode material influence the breakdown
voltage. A detailed description of the processes can be found in Bluhm (2006).
If a comparably fast rising voltage is applied to a spark gap, the voltage across the
spark gap switch may rise above the static breakdown voltage temporarily due to
ignition delay. When the voltage applied to a spark gap switch exceeds the static
breakdown voltage, the initialization of the breakdown mechanism might be delayed
until sufficient seed electrons for breakdown initialization become available. In the
case of natural seed electron generation, this delay time may scatter due to the
statistical nature of the process. Subsequently, an electron avalanche is formed,
which may develop into a streamer discharge as soon as the critical number of
charge carriers for streamer inception is reached. After the conductive channel has
bridged the electrodes, it is heated up and forms a low resistive arc. Under the
assumption that the times for seed electron generation and arc formation are negli-
gibly short, the ignition time is dominated by the time required for channel forma-
tion. According to the area-time law by Kind (Eq. 2), the area between the curve
describing the voltage applied to the spark gap over time u(t), and the static
breakdown voltage U0 is constant for a given electrode geometry, gas, and pressure.
ð
tCF, End
ðuðtÞ U0 Þdt ¼ const: (2)

tCF, Start
Hence, a steeper voltage rise yields a shorter ignition delay but also a higher
dynamic breakdown voltage above the static breakdown voltage. The integration is
done for the time required for channel formation (CF), i.e., tCF,EndtCF-Start.
Due to the statistical nature of seed electron generation, the moment of break-
down after reaching the static breakdown voltage and, hence, the dynamic break-
down voltage scatter. As the rate of seed electron generation depends on the volume,
spark gaps with a larger volume of an electric field exceeding the critical field
strength for charge carrier multiplication exhibit less scattering. Spherical spark
gap electrodes, which are common for Marx generator designs used for high-voltage
testing, have to some extend an inhomogeneous electric field distribution in the area
between the electrodes. When using an electrode profile with a homogeneous field
distribution instead, scattering is lower (Frey et al. 2009).
Electrode profiles according to Borda and Rogowski (Prinz 1969) are both
designed for a homogeneous electric field distribution. The profile according to
Borda is more compact. It has been designed so that the electric field strength at
the surface of the electrode is constant. Rogowski’s profile has a homogeneous
electric field distribution due to a parallel-plate arrangement in its center. The border
is designed in a way that the shape is equal to an equipotential surface just without
electric field enhancement. In the Marx generator, the breakdown voltage is usually
adjusted either by a variation of the gap distance or the pressure. It should be noted
that the profiles according to Borda and Rogowski need to be designed for a fixed
electrode distance.
In a non-triggered Marx generator, the first spark gap switch ignites as soon as its
breakdown voltage is reached. The required seed electrons are present due to natural
irradiation. Usually, the distance of the first stage’s spark gap is reduced slightly to
guarantee that this spark gap switches first. The remaining spark gaps of the Marx
generator ignite due to the application of an overvoltage caused by a delayed
charging of the ground stray capacitances. Figure 2 shows the circuit of a three-
stage Marx generator including its stray capacitances.
Initially just before the ignition, the stray capacitances between the elements of
the ground side charging path to ground Cstr,G3 and Cstr,G5 are uncharged. The stray
capacitances between the elements of the charging path at high-voltage side to
ground are charged to the charging voltage per stage. After the spark gap of the
first stage has been ignited, the stray capacitance Cstr,G3 between the stage capacitor
C2 of the second stage and ground potential is charged immediately to the first
stage’s output voltage equal to the charging voltage per stage. The voltage across the
high-voltage side stray capacitance Cstr,G2 rises from stage voltage to twice of the
stage voltage. The sum of the stray capacitance between second and third stage
Cstr , st st3 + Cstr , st st4 and the capacitance of the second stage’s spark gap switch
CSG2 forms together with the high-voltage side and ground side stray capacitances of
798 M. Sack
Fig. 2 Marx generator including stray capacitances
the third stage a capacitive voltage divider. The voltage step due to switching the first
stage’s spark gap is transferred fast according to the ratio of this capacitive divider to
the ground stray capacitances of the third stage. Hence, the voltage across the second
stage’s spark gap SG2 is equal to twice the stage voltage diminished by the voltage
step due to capacitive charging of the third stage’s ground stray capacitances. If the
stray capacitance between both stages is small compared to the ground stray
capacitances of the third stage, the voltage across the second stage’s spark gap
may rise almost up to twice of the charging voltage per stage just after switching
of first stage’s spark gap switch. Subsequently, the ground stray capacitances of the
third stage are charged via the resistors of the charging paths between the second and
the third stage diminishing the overvoltage across the second stage’s spark gap
continuously. For proper operation, the Marx generator needs to be designed in a
way that the overvoltage across the second stage’s spark gap is sufficiently high and
lasts sufficiently long to cause its ignition. For fast ignition of the spark gap switches
without considerable delay, seed electrons need to be present immediately. However,
relying on the presence of naturally generated seed electrons only involves a
considerable uncertainty in the ignition delay. Hence, the spark gap switches are
usually arranged so that ultraviolet light originating from the already established
discharge of the preceding spark gap switch is able to generate seed electrons in its
neighboring switch. The ignition delay of the spark gaps in a seven-stage Marx
generator has been measured to be in the order of 100 ns between the ignition of the
first spark gap switch and the beginning rise of the load current (Sack and Bluhm
2008).
Marx Generator Circuit for High-Voltage Testing
In high-voltage testing applications, for which the Marx generator originally has
been designed, it is used to apply a well-defined voltage pulse to an in most cases
capacitive load. Voltage pulses due to lightning strike and switching of inductive
loads both are modelled as a superposition of two exponential functions according to
U0 τ1 τ2 h t=τ2 i
uðtÞ ¼ e et=τ1 : (3)
Rdmp CL τ2 τ1
Figure 3 shows a basic circuit, which is commonly used for pulse generation in
high-voltage testing applications, in its single-stage equivalent. The capacitor CG
represents the capacitance of the Marx generator and CL the capacitance of the
device under test as load. Initially, the capacitor CG is charged to a voltage U0. If the
spark gap switch closes first, CL is charged via the damping resistor Rdmp. This
process is described by the time constant τ1. Subsequently, both capacitors are
discharged via the discharge resistor Rdis. Under the assumption that charging of
CL occurs much faster than the discharge process, τ1 and τ2 can be approximated
according to
CG CL
τ1 ¼ Rdmp (4)
CG þ CL
τ2 ¼ Rdis ðCG þ CL Þ (5)
The ratio η between the peak value of the pulse voltage Û and the initial charging
voltage U0 can be calculated according to
^
U CS
η¼ ¼ (6)
U 0 CS þ CL
This single-stage circuit can be expanded to a Marx circuit according to Fig. 4
showing a three-stage generator. The damping and discharge resistors have been
distributed across the stages. With the number of stages n, the total charging voltage
U0 can be expressed as
U 0 ¼ n U stage (7)
800 M. Sack
Fig. 3 Single-stage equivalent of a basic circuit commonly used for pulse generation in high-
voltage testing applications
Fig. 4 Marx generator for

high-voltage testing
The effective capacitance for the pulse circuit CG is given by
Cstage
CG ¼ (8)
n
The Cstage and Ustage represent the stage capacitance and charging voltage per
stage, respectively.
In high-voltage testing applications, only a few pulses are applied to every device
under test. The pulses are applied as single pulses with up to several minutes time
between two pulses. Hence, Marx generators for testing applications are usually
designed for a lifetime of one to ten million pulses.
Marx Generator for Treatment of Biological Material by Pulsed

Electric Fields
In the recent years, Marx generators have been introduced to the pulsed electric field
treatment of biological material (electroporation) in industrial scale (Kotnik et al.
2015; Haberl et al. 2013; Reberšek et al. 2014; Mahnič-Kalamiza et al. 2014; Golberg
et al. 2016). For this application, the load has a comparably low resistance demanding
for a rather high-pulse current with a peak value in the order of approximately 4 kA . . .
12 kA, at a total charging voltage between approximately 300 and 500 kV, both
depending on the application (Mueller et al. 2007; Sack et al. 2005). The single-stage
equivalent circuit can be described as series resonance circuit comprising the pulse
capacitor CG the circuit inductance L and the load resistance RL.
The discharge is governed by the differential equation
uC ðtÞ þ RL Cu_ C ðtÞ þ LCüC ðtÞ ¼ 0 (9)
with the initial conditions uC(0) = U0
u_ C ð0Þ ¼ 0
For a series resonant circuit, a quality factor Q can be defined according to

rffiffiffiffi
1 L
Q¼ (10)
RL C
Depending on the quality factor, three different cases are distinguished:
Damped periodic case: Q >0.5:

δt δ
uC ð t Þ ¼ U 0 e cos ðωtÞ þ sin ðωtÞ (11)
ω
U 0 δt
uR ðtÞ ¼ RL e sin ðωtÞ (12)
Lω
Critically damped case: Q = 0.5:
uC ðtÞ ¼ U 0 ð1 þ δtÞeδt (13)

U0
uR ðtÞ ¼ RL t eδt (14)
L
802 M. Sack
Aperiodic case: Q < 0.5:
U 0 δt h 0 ω0 t 0 ω0 t
i
uC ðtÞ ¼ e ðω þ δÞe þ ð ω δ Þe (15)
2ω0
U 0 δt
uR ðtÞ ¼ RL e sinhðω0 tÞ (16)
Lω0
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi2 qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
RL 0 R2L
with ω ¼ 1
LC 4L 2 , ω ¼ 4L2
LC 1
, and δ ¼ R2LL .
For industrial applications, a compact design of Marx generator is desirable. The

effort for insulation can be reduced if a ground-symmetric voltage is applied to the
load (Sack and Mueller 2008). This is achieved by grounding the pulse circuit in the
center of the Marx generator as it is shown in Fig. 5. For the treatment of a flow of
material, the Marx generator is operated continuously. Marx generators with spark
gap switches have been designed for pulse repetition rates in the order of 20 Hz and a
lifetime in the order of 2∙109 pulses. For fast charging coils instead of charging
resistors are employed. Grounding the generator at its center actually splits the
generator into two halves. If the gap distance of the center spark gap SG1 is reduced
slightly, this spark gap switch ignited first. As a consequence, the negative half of the
generator comprising the spark gaps SG1 until SG4 ignites (Sack and Stängle 2007).
As there is actually no capacitive coupling between the first stage of the negative half
and the positive half comprising the spark gaps SG5 until SG7, the positive half does
not ignite immediately after spark gap switch SG1 has ignited. Instead spark gap
switch SG5 ignites after SG4, because the negative voltage of the generator’s
negative half is coupled via the load resistance to the positive half causing an
overvoltage across SG4. Subsequently, the positive half ignites in a top-down
order of the spark gaps with SG7 igniting last.
In a Marx generator designed for continuous operation, the spark gap switches are
operated in nitrogen gas rather than in the air. Without the presence of oxygen in the
plasma of the arcs, no nitric oxide is formed, as it would be the case when operating
the spark gaps switches in air. Moreover, the presence of oxygen causes a slightly
increased jitter of the ignition time of the spark gap switches, because the electro-
negative oxygen temporarily traps free electrons required for initialization of the
breakdown (Sack and Bluhm 2008).
The nitrogen gas is circulated through the gaps between the electrodes in a closed
loop. The gas movement removes debris from electrodes caused by erosion and
charged particles from the arc and, hence, fosters a fast reestablishment of the electric
breakdown strength of the gaseous dielectric after a breakdown. This is especially
important for operation at higher repetition rate of approximately 30 Hz and above.
In continuous operation due to the erosion of the electrodes, the electrode distance
increases, which may cause a continuous increase in the breakdown voltage without
countermeasures. A suitable electrode material exhibiting low burnup is tungsten-
copper alloy (WCu). It consists of a tungsten matrix with embedded copper. For the
Fig. 5 Marx generator for

repetitive operation operated
symmetrically to ground
potential
electrode material, the content of copper is in the order of 20 . . . 40%. Copper

exhibits good heat conduction. The melting point of copper with 1083 C is
considerably lower than the melting point of tungsten (3422 C). Thus, it can be
expected that the tungsten matrix stabilizes the structure of the material, and the
molten copper stays to some extend within the matrix. The evaporation temperature
of copper is with 2595 C below the melting point of tungsten, and the heat of
evaporation of copper is more than three times larger than the heat of melting of
tungsten. Hence, locally additional cooling of the electrode surface due to evapora-
tion may occur. Experiments showed that the pulse parameter set used in PEF
treatment devices the erosion of the spark gap electrodes is in proportion to the
Ðtp
load integral I ðtÞ2 dt . This corresponds well to a theoretical description of the
0
804 M. Sack
process based on the assumption that the copper melts rather than evaporates
(Zingerman 1960; Bluhm and Sack 2008). For electrodes made from tungsten-
copper alloy (WCu 80/20), which were machined with a Borda profile of 40 mm
diameter, for a load integral of 20 A2s a decrease in mass of 183 ng and for 81 A2s a
decrease of 824 ng per pulse and pair of electrodes has been determined. The
experiments have been performed at a current level of Î = 4.5 kA with a pulse length
of th = 1.4 μs and at Î = 4.5 kA with th = 1.4 μs, respectively, averaging over
500,000 pulses (Bluhm and Sack 2008). The increase in electrode distance with the
wear depends on the electrode diameter. Electrodes with larger diameter allow for
longer electrode lifetime. With an electrode diameter of 40 mm, the electrode
distance increases after 200,000,000 pulses by 1.9 mm at a burnup of 183 ng per
pulse and 8.7 mm at 824 ng per pulse, corresponding to about 15% and 73% of an
initial gap width of 12 mm, respectively. This example demonstrates that during
continuous operation, the wear must be compensated automatically either by a
continuous readjustment of the electrode distance or a decrease in pressure as long
as the spark gap switch is operated at a pressure above ambient pressure. The
operation above ambient pressure prevents air from getting into the nitrogen circuit.
The adjustment range of either pressure or electrode distance determines the service
interval for the spark gap switches depending on the expected burnup.
Triggered Operation
Triggering of the first spark gap allows to determine the moment of switching. By
triggering Marx generators for high-voltage testing, applications can be, for exam-
ple, synchronized to an AC voltage. For PEF treatment, triggering enables a parallel
operation of Marx generators for large-scale devices, if one Marx generator is not
able to deliver sufficient current. For a given pulse shape and total charging voltage,
the current is limited by the minimum circuit inductance, which is defined by the
stray inductance of the conductors. Although the circuit inductance can be reduced
to some extent by an antiparallel or also substantially coaxial arrangement of
conductors partly compensating the magnetic field, a parallel configuration of
generators allows for further reduction. However, in parallel operation, energy
oscillations between the generators may occur, which should avoided to prevent
additional losses and self-heating of components. Appropriate countermeasures are
damping the oscillations by additional damping resistors, which can be part of the
electrode system of a PEF treatment chamber, an exactly synchronized triggering of
the generators at the same time, or a combination of both (Sack et al. 2005).
Common trigger methods are the use of a three-electrode spark gap and the
initiation of a discharge by means of an igniting spark (trigatron). The three-
electrode spark gap has a ringlike trigger electrode between both main electrodes.
It is connected to a pulse generator via a capacitor. In off-state the potential of the
trigger electrode is controlled by means of two resistors of high impedance to a
potential of between half and one-third of the voltage between the main electrodes,
depending on the design. For triggering the potential is shifted by means of an
applied voltage pulse such that a breakdown between one main electrode and the
trigger electrode occurs. Subsequently, the potential of the trigger electrode becomes
equal to the potential of that main electrode. The breakdown of the remaining gap
occurs either due to overvoltage between the trigger electrode and the other main
electrode only or due to plasma, which enters the space between the trigger electrode
and the other main electrode though the center hole in the trigger electrode (Bluhm
2006). The plasma-initiated breakdown of the second part of the spark gap exhibits
less delay.
A trigatron trigger device has a trigger electrode embedded into one electrode of a
two-electrode spark gap. For triggering, a small spark is generated between this
trigger electrode and the surrounding main electrode. The breakdown of the main
spark gap is initiated by means of charged particles and UV light emitted by the
trigger discharge.
Both trigger methods have, apart from the main electrodes, a third electrode for
ignition, which is subjected to an increased wear. For Marx generators designed for
continuous long-term operation, overvoltage triggering may be applied (Sack et al.
2003, 2011). Figure 6 shows a simplified schematic. Either one or both charging
coils between the first and the second stage are replaced by a transformer-coupled
pulse generator. For triggering a pulse voltage is superimposed to the charging
voltage of the stage capacitor such that the voltage across the spark gap switch is
increased, until breakdown occurs. If a Marx generator is designed for delivering
pulses with a pulse length th in the microsecond range, its trigger pulse source needs
to be powerful enough to charge the second stage’s stray capacitances within some
100 ns to achieve a good synchronization of generators. After ignition, the trigger
device must provide transient insulation between both adjacent stages in the same
manner as a charging coil. If the trigger pulse generator would be a voltage source
and the transformer would have an excellent coupling factor resulting in a low stray
inductance, the combination of trigger pulse generator and transformer would be of
low impedance and, hence, would exhibit poor transient insulation. A transformer
with more stray inductance or an arrangement with an additional inductance in series
to the transformer’s high-voltage winding could improve transient insulation. How-
ever, charging the stray capacitances would be either slowed down or a higher pulse
voltage would be required. If the trigger pulse source instead is designed much like a
current source, it provides transient insulation between the stages and is able to
deliver sufficient current for fast charging of the stray capacitances.
Figure 7 shows a principal schematic of an overvoltage trigger device supplying a
current together with a part of the triggered Marx generator. It operates similar to an
ignition coil. An insulated-gate bipolar transistor (IGBT) has been employed as
switch. If the IGBT T closes, inductive energy is stored in the main inductance of the
transformer Tr. The pulse is generated after opening the IGBT. The current commu-
tates to the secondary winding, and the stray capacitances are charged until the
transient breakdown voltage of the spark gap switch SG is reached. After ignition of
the spark gap switch, the stage voltage is applied to the pulse transformer’s second-
ary winding. As it has reverse polarity compared to the trigger pulse, a diode in series
to the IGBT switch guarantees insulation of the low-voltage winding, independently
806 M. Sack
Fig. 6 Overvoltage triggered

Marx generator with two
trigger devices and
corona wire
Fig. 7 Overvoltage trigger device as current source connected to a Marx generator
whether the IGBT is conductive or not. The secondary winding of the pulse
transformer provides transient insulation of the stages.
If only one trigger generator in one charging path is employed, there is a leakage
current through the charging coil in the other charging path. After closing IGBT T
energy is transferred to the charging coil LC in the other charging path. The current
path comprises the stage capacitors C1 and C2 of the first and second stage, provided
that the trigger is situated between both stages. However, their capacitance can be
considered to be sufficiently large not to influence this current significantly. During
trigger pulse generation, the current driven by the charging coil supports charging of
the stray capacitances. Hence, the energy stored into the charging coil is not lost
(Sack and Mueller 2014). However, the trigger device needs to be powerful enough
to supply this energy additionally to the energy stored into the pulse transformer.
Depending on the available space for the trigger device instead of one more powerful
but bigger trigger device, two smaller and less powerful trigger devices can be
inserted into both charging paths as shown in Fig. 6. As both trigger devices are
operated synchronously, a leakage current though the neighboring charging path is
omitted.
For overvoltage triggering artificial seed electron generation is required for a
reliable ignition at low jitter. Seed electron generation can be easily achieved by
means of a corona discharge next to the triggered spark gap switch irradiating
ultraviolet light into the triggered spark gap switch. The corona discharge is
established either at the tip of a thin wire or along a conductive ring with a sharp
edge or made out of a thin wire around the gap between the electrodes of the switch
(CW in Figs. 5 and 6). In both cases, the corona electrode is electrically connected to
the cathode of the switch. The corona discharge is either supplied by the charging
voltage of the stage capacitor or by the trigger pulse only. The shape of the corona
electrode and its distance to the main electrodes are designed such that the corona
discharge stays in the high-field region around the corona electrode and is not able to
bridge the space to one main electrode of the switch.
The overvoltage trigger device can be designed as a two-terminal device, as a
direct replacement of one or both charging coils. It is supplied by the charging
current of the Marx generator.
Semiconductor-Based Marx Generators
Marx generators equipped with spark gap switches are limited in their repetition rate,
because of the time required for reestablishment of the electric breakdown strength.
Moreover, they can be operated as closing switches only, resulting in a complete
discharge of the capacitors after each pulse. Hence, the pulse shape is determined by
the passive circuit elements of the pulse circuit only. Marx generators equipped with
semiconductor switches may overcome these limitations. However, semiconductors
have less blocking voltage than spark gap switches. Hence, semiconductor-based
Marx generators are advantageous for applications demanding for lower output
voltage at higher repetition rate.
Semiconductor switches capable of closing and opening the circuit allow for only
partly discharging the stage capacitor during one pulse. If the stage capacitor is so
large that its voltage stays nearly constant during discharge, a substantially rectan-
gular pulse shape can be generated.
Figure 8 shows a Marx generator circuit for the generation of positive pulse
voltage equipped with IGBT switches. The high-voltage side charging path is
equipped with diodes, the ground-side charging path comprises IGBTs. For charging
the IGBTs TC1 and TC2 are conducting and the stage capacitors are charged in
parallel configuration. For pulse generation, the IGBTs for charging open and,
subsequently, IGBTs TP1. . .TP3 close and establish series configuration of the
capacitors. The diodes D2 and D4 together with the non-conductive IGBTs TC1and
808 M. Sack
Fig. 8 Semiconductor-based
Marx generator for positive
output voltage
TC2 provide insulation between the stages. Semiconductor switches must be pro-
tected against overvoltage. Diodes DCL1. . .DCL6 antiparallel to both IGBTs of one
stage provide voltage clamping. With both IGBTs in off-state, a current flowing in
the direction of the discharge current may bypass any stage via diode DCL1, DCL3, or
DCL5, respectively. A current in opposite direction charges the stage capacitor via
DCL2, DCL4, or DCL6. Thus, the voltage across the stage is limited to the momentary
voltage across the capacitor.
Individual control of each stage switch allows generating a stepwise arbitrary
voltage shape. The number of active stages determines the output voltage of the
generator. The discharge current bypasses inactive stages through DCL1, DCL3, or
DCL5. Individual control of the discharge switches may result in unequal discharge of
the stage capacitors C1. . .C3. If a capacitor of any upper stage has been discharged
further than a capacitor of a lower stage, without the diodes D1 and D3, reestablishing
the charging path again by closing the IGBTs TC1 and TC2 would result in a high
transient current. The diodes D1 and D3 prevent the stage capacitors from
discharging via the charging path. If the pulse switches are controlled in such a
way that the capacitors of the upper stages are always discharged to lower voltage
than the ones of lower stages, the diodes D1 and D3 can be omitted.
A negative pulse voltage can be generated by means of the circuit according to
Fig. 9. The ground-side charging path consists of diodes DCL2, DCL4, and DCL6 and
the high-voltage side charging path of the IGBT switches TC1 and TC2. The
Fig. 9 Semiconductor-based
Marx generator for negative
output voltage
capacitors C1. . .C3 are charged in parallel configuration to a positive voltage with
respect to ground. During discharge polarity reversal is performed by connecting the
positive pole of the first stage’s capacitor via TP1 to ground potential resulting in a
negative stage voltage at the output of the first stage. The other stages operate in the
same way. Due to negative output voltage, the diodes of the charging path are
blocking.
A series connection of bridge configurations each connected to a stage capacitor
allows generating bipolar pulses (Redondo et al. 2009; Sack et al. 2014). According
to Fig. 10, each switch of a bridge consists of an antiparallel configuration of an
IGBT and a diode. Charging of the stage capacitors is performed in parallel config-
uration via the charging path comprising series connections of IGBT switches TC1
and TC2 and diodes DC1 and DC2, respectively, and the low-side switches of the
bridges TPX3 and TPX4. The high-side switches of the bridges TPX1 and TPX2 are
isolating during charging. For pulse generation, the IGBTs of the high-voltage side
charging path are isolating. A positive pulse is generated by closing TPX2 and TPX3
of a bridge. At the first stage, the negative pole of the stage capacitor C1 is connected
to ground potential and the positive pole to the subsequent stage. For negative pulse
generation, TPX1 and TPX4 are conductive. Hence, the positive pole of the stage
capacitor is connected to ground potential and the negative pole to the subsequent
stage. The switches of the charging path need to be isolating for both polarities. If
either TPX1 and TPX2 or TPX3 and TPX4 are conductive, the stage is in idle mode
forming a bypass path for the current. If none of the IGBTs is conductive, the voltage
across a stage is clamped to the stage capacitor’s momentary voltage.
During arbitrary voltage generation, the stage capacitors are not discharged
equally. If the parallel configuration is reestablished for charging, transient currents
between the capacitors may occur. The diodes D1 and D2 prevent the stage capacitors
from discharging across the charging path. If the control scheme of the pulse
810 M. Sack
Fig. 10 Semiconductor-based Marx generator for bipolar output voltage
switches guarantees that switches nearer to the charging device are discharged
further, the diodes D1 and D2 can be omitted, because then the diodes DC1 and
DC2 of the charging path prevent transient currents in the charging path. In this case,
stages nearer to the power supply are charged first until each of them reaches the
voltage of its neighboring stage.
The semiconductor switches in the charging path cause losses and the voltage
drop across them progressively reduces the charging voltage of the stages. Thus, the
number of stages is limited, unless the voltage at the stage is not restored by means of
an on-stage voltage regulator.
Replacing the semiconductors in the charging paths by current compensated
chokes reduces charging losses significantly (Sack et al. 2016). However, the
achievable pulse length is limited by the size of the charging coils. Current com-
pensated chokes consist of two windings of equal turns, which are firmly magnet-
ically coupled. A current flow in opposite directions, which is the case during
charging, reduces the magnetic field resulting in low inductivity. Current flow in
equal directions as during discharge of the generator causes an increase in
inductivity. A Marx generator with current compensated chokes is similar to one
with common charging coils. It may form a series resonance circuit together with an
additional inductance and the load resistance. Switching the IGBTs on completely
before the load current is able to rise considerably and complete discharge of the
stage capacitors with each pulse enables zero current and zero voltage switching
allowing for a considerable reduction of switching losses. However, in this case, the
voltage shape is limited to that of a series resonance circuit.
In all mentioned circuits, IGBT switches can be replaced by field effect transis-
tors, especially metal-oxide-semiconductor field-effect transistor (MOSFET)
switches. MOSFETs made from silicon designed for a voltage level of several
100 V exhibit a larger on-state resistance than IGBTs of comparable voltage level.
However, MOSFETs might switch faster than IGBTs. So for applications requiring a
comparably low pulse current or fast switching MOSFETs might be preferable.
MOSFETs made from silicon carbide overcome the limitations of silicon MOSFETs
and even have the potential of replacing IGBTs. However, their price is still not
competitive compared to IGBTs.
Conclusions
Marx generators serve as pulse source for large pulse currents. Due to parallel
charging of the capacitors and discharging the capacitors in series configuration,
each component of a Marx generator needs to be designed for the stage voltage only.
For PEF treatment of biological material in large scale, Marx generators capable of
repetitive operation at long lifetime are employed. Depending on the design of the
device, Marx generators can be equipped with closing switches enabling complete
discharge of the capacitors only. Such switches might be either spark gap switches or
semiconductor switches, which can be operated in zero voltage and zero current
switching mode to reduce switching losses. The use of switches capable of both
closing and opening a circuit allows discharging the capacitors only partly to
generator rectangular pulses or pulses with stepwise arbitrary voltage shape.
Semiconductor-based Marx generators in bridge configuration enable to generate
bipolar pulses.
References
Bluhm H (2006) Pulsed power systems – principles and applications. Springer, Berlin/Heidelberg
Bluhm H, Sack M (2008) Industrial-scale treatment of biological tissues with pulsed electric fields.
In: Vorobiev E, Lebovka N (eds) Electrotechnologies for extraction from food plants and
biomaterial. Springer Science and Business Media LLC, Heidelberg
Frey W (1996) Lasergetriggerte Multikanalfunkenstrecken mit Magnesium-Aerosol-Schaltgas,
Dissertation, Universität Karlsruhe, 12.11.1996
Frey W, Sack M, Wüstner R, Müller G (2009) Gas-insulated self-breakdown spark gaps: aspects on
low-scattering and long-lifetime switching. Acta Phys Pol A 115:1016–1018
Golberg A, Sack M, Teissie J, Pataro G, Pliquett U, Saulis G, Töpfl S, Miklavčič D, Vorobiev E,
Frey W (2016) Energy-efficient biomass processing with pulsed electric fields for bioeconomy
and sustainable development. Biotechnol Biofuels 9:94
812 M. Sack
Haberl S, Miklavčič D, Serša G, Frey W, Rubinsky B (2013) Cell membrane electroporation – part
2: the applications. IEEE Electr Insul M 29(1):29–37
Kotnik T, Frey W, Sack M, Haberl Meglič S, Peterka M, Miklavčič D (2015) Electroporation-based
Küchler A (2009) Hochspannungstechnik, Grundlagen – Technologie -Anwendungen, 3rd edn.
Springer, Heidelberg/Dordrecht/London/New York
Marx E (1923) Verfahren zur Schlagprüfung von Isolatoren und anderen elektrischen
Vorrichtungen, German Patent 455 933
Müller G, Frey W, Sack M, Schultheiss C, Mayer H-G, Sigler J, Kern M, Günther U (2007)
Karlsruher Elektroporationsanlage KEA – Die Erfolgsgeschichte eines Technologietransfers in
die Industrie, Nachrichten des Forschungszentrums Karlsruhe, Jahrgang 39, 3/2007, pp
153–158
Prinz H (1969) Hochspannungsfelder. R. Oldenbourg Verlag, München
Reberšek M, Miklavčič D, Bertacchini G, Sack M (2014) Cell membrane electroporation –part 3:
the equipment. IEEE Electr Insul M 30(3):8–18
Redondo LM, Canacsinh H, Silva JF (2009) Generalized solid-state Marx modulator topology.
IEEE Trans Dielectr Electr Insul 16(4):1037–1042
Sack M, Bluhm H (2008) New measurement methods for an industrial scale electroporation facility
for sugar beets. IEEE Trans Ind Appl 44(4):1074–1083
Sack M, Mueller G (2008) Optimisation of an electroporation device for mash. In: Proceedings of
11th international conference on optimization of electrical and electronic equipment (OPTIM
‘08), Brasov, May 22–24, 2008
Sack M, Mueller G (2014) Testing of a modular over-voltage trigger device for Marx generators. In:
14th international conference on optimization of electrical and electronic equipment (OPTIM
2014), Brasov, May 22–24, 2014.
Sack M, Stängle R (2007) A bipolar Marx generator for a mobile electroporation device. In:
Proceedings of pulsed power and plasma science conference, Albuquerque, June 17–22,
2007, DVD-ROM
Sack M, Schultheiss C, Bluhm H (2003) Wear-less trigger method for Marx generators in repetitive
operation. In: 14th IEEE pulsed power conference, Dallas, June 15–18, 2003
Sack M, Schultheiss C, Bluhm H (2005) Triggered Marx generators for the industrial-scale
electroporation of sugar beets. IEEE Trans Ind Appl 41(3):725–733
Sack M, Stängle R, Mueller G (2011) Overvoltage trigger device for Marx generators. J Korean
Phys Soc 59(6):3602–3607
Sack M, Hochberg M, Mueller G (2014) Design of a semiconductor-based bipolar MARX
generator. In: IEEE international power modulator and high voltage conference (IPMHVC
2014), Santa Fe, June 1–5, 2014
Sack M, Hochberg M, Mueller G (2016) Synchronized switching and active clamping of IGBT
switches in a simple Marx generator. In: Proceedings of PCIM 2016, May 10–12, 2016,
Nuremberg
Zingerman AG (1960) Thermal theory of the electrical erosion of metals. Electrom 5(1):1427–1485
Measurement and Characterization
of Exposure Systems for High-Frequency, 46
Ultrashort Pulses
Philippe Leveque, Rodney O’Connor, and Delia Arnaud-Cormos
Abstract
This chapter summarizes the experimental dosimetry techniques developed for
exposures to high-intensity, ultrashort pulses in the nanosecond domain. The
dosimetry or characterization of the exposure conditions was performed by
experimental and numerical techniques. Typical frequency and time domain
techniques for characterization of exposure systems are described in this chapter.
In the frequency domain, classical microwave devices for low voltage are used
such as the vector network analyzer. Specific components and setups required for
time domain characterization of high-intensity electric field (>MV/m), high-
voltage (>kV), nanosecond duration pulses are presented. A new technique for
measuring temperature at the cellular and intracellular levels is also presented to
complete these macroscopic measurements at the level of the exposure system.
Numerical dosimetry that can serve to complete or to access quantities difficult to
measure was also briefly introduced.
Keywords
High voltage • Nanosecond and subnanosecond pulsed electric field • Dosimetry •
Characterization • Electro-optic probe • Microfluorimetric probe • Numerical
modeling
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 814
Experimental Macrodosimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 816
Frequency Domain Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 817
Time Domain Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 821
P. Leveque (*) • R. O’Connor • D. Arnaud-Cormos

University Limoges, CNRS, XLIM, UMR 7252, and LABEX “Sigma-LIM”, Limoges, France
e-mail: philippe.leveque@unilim.fr; philippe.leveque@xlim.fr; neurophoton@gmail.com; delia.
arnaud-cormos@ensil.unilim.fr

DOI 10.1007/978-3-319-32886-7_207
814 P. Leveque et al.
Numerical Dosimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 828

Macrodosimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 828
Microdosimetry and Nanodosimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 829
Experimental Microdosimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 830
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 831
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 834
Introduction
In order to study the response of biological samples exposed to high-voltage,

nanosecond pulsed electric fields (nsPEF), it is essential to understand physical
parameters such as electric field or temperature in those samples. Indeed, exposures
to intense electric fields at high repetition rates (Davalos et al. 2003; Pliquett 2003)
can lead, for example, to significant Joule heating that can cause damage to cells or
biological tissues.
Commonly, dosimetry consists of quantifying the energy absorbed by a target
exposed to an electric field, and it is a crucial factor for the control and validation of
biological investigations. In bioelectrics or bioelectromagnetics, the physical and
biological phenomena involved are complex and require the establishment of appro-
priate and well-characterized experimental technologies. Figure 1 shows an illustra-
tion of a typical exposure system for in vitro experiments. Developments in
experimental exposure systems typically focus on four main parts: pulse generation,
delivery system, dosimetry, and imaging and biological analysis. The generation part
is connected to the system for application of pulses, called “delivery system” or
“applicator.” The delivery system is the part of the exposure system that transforms
the generated voltage pulses into pulsed electric fields within the exposed biological
sample. An essential part of this relates to the quantification and the control of the
electric field and temperature in the sample. The dosimetry consists of experimental
and numerical quantification techniques of characteristic quantities. To observe and
analyze the sample behavior, ideally before, during, and after exposure, the exposure
system also may include developments on specific imaging systems. This chapter
focuses on measurement and characterization of exposure system for high-fre-
quency/ultrashort pulses, while another chapter of this book focuses on pulse
generation technologies (▶ Chap. 47, “Photoconductive Switching for Pulsed
High-Voltage Generators”).
Typically, the dosimetry or characterization of the exposure conditions is
performed by experimental and numerical methods. Dosimetry is multi-physical
involving both electromagnetism and thermodynamics but also multi-scale as the
magnitudes of characteristic quantities depend on the spatiotemporal scale consid-
ered. Three different spatial scales can be distinguished as illustrated in Fig. 2:
macroscopic, microscopic, and nanoscopic. When the characteristic quantities are
studied at the sample level as a whole, the scale is macroscopic, and the analysis is
46 Measurement and Characterization of Exposure Systems for High. . . 815
Fig. 1 Schematic illustration of typical experimental technologies for in vitro exposure systems of
biological cells to high-voltage pulsed electric fields. The exposure setup is composed of four main
parts: pulse generator, delivery system, dosimetric characterizations, and imaging systems
Fig. 2 Levels of experimental and numerical studies of pulsed electric field nanoseconds effects on
cells, from macroscopic to nanoscopic scale, from macrodosimetry to nanodosimetry: typical
macroscopic systems for nsPEF delivery (electroporation cuvette), experimental microscopic
observation, and numerical model of a single cell exposed to nsPEF, example of molecular
dynamics model of a nanoscale section of a biological membrane
called macrodosimetry. The microscopic scale is associated with the cellular level
and with microdosimetry analysis. When the magnitudes are considered at the
atomic level, the scale is nanoscopic with associated nanodosimetry analysis.
Within the general problem, biological mechanisms of pulsed electric fields
exposure like in the case of electroporation are nanoscopic and take place at the
atomic scale. Numerical studies have aimed at modeling electric field quantities
induced locally in simple spherical cell geometries placed between two flat elec-
trodes (Kotnik and Miklavcic 2006; Smith and Weaver 2008; Merla et al. 2011; Vu
et al. 2012). However, these studies did not fully consider the transient atomic
rearrangements observed in molecular dynamics simulations. In addition, experi-
mental electric fields are typically measured in aqueous suspensions composed of
electrolytes and living cells exposed to nsPEF using systems such as cuvettes (Kolb
et al. 2006; Walker Iii et al. 2006; Garon et al. 2007; Kirawanich et al. 2010; Kenaan
et al. 2011; Xiao et al. 2011; Kohler et al. 2012a), flat microchamber or wire
electrodes (Schoenbach et al. 2004; Sun et al. 2005; Frey et al. 2006; Pakhomov
et al. 2007; Rebersek et al. 2009; Arnaud-Cormos et al. 2011; Dalmay et al. 2011;
Wu et al. 2013), or transverse electromagnetic (TEM) cells (Kohler et al. 2012b,
2013). Based on these problems, one main question that emerges concerns the
relation between the macroscopically applied dose of electric field and the micro-
or corresponding ratio at the nanoscale.
Experimental and/or numerical dosimetry techniques are continuously being
developed to resolve this question. From the numerical standpoint, techniques on
the three previously mentioned scales exist. At the experimental level, however, the
developments decrease with diminishing scale; the vast majority of experimental
techniques concern the macrodosimetry, a smaller part the microdosimetry, whereas
nanodosimetry techniques are very limited due to the spatial and temporal scale
involved.
Section “Experimental Macrodosimetry” provides a brief overview of several
experimental and numerical dosimetry techniques. Experimental dosimetry tech-
niques at the macroscopic scale are first illustrated. With respect to macrodosimetry,
frequency and time characterizations of nsPEF exposure systems are presented,
particularly in terms of spectral bandwidth, temporal waveforms and pulse electric
field levels. Numerical dosimetry based on the finite-difference time-domain
(FDTD) method is briefly described, primarily at the macroscopic level. For micro-
dosimetry, a technique for measuring temperature at the cellular and intracellular
levels is presented in the last part of the chapter.
Experimental Macrodosimetry
Characterizations in the frequency and time domains can be carried out at both the
macroscopic level. Frequency domain characterizations can include measurements
of a delivery system’s S-parameters or a biological sample’s electromagnetic prop-
erties, as illustrated next.
Frequency Domain Characterization
One of the first basic macrodosimetric measurements illustrated in Fig. 3a consists of

the frequency characterization of nsPEF delivery system typically using a specific
microwave device called a vector network analyzer (VNA). The VNA is used to
determine the amplitude and phase of the reflection and transmission coefficients
commonly called S-parameters of a one-port or an active/passive two-port network,
depending on the frequency. The VNA compares the reflected wave to the incident
wave in a reference plane imposed during the calibration of the device. For example,
the VNA shown Fig. 3b and typically used in our measurements is the 8753E model
from Hewlett Packard, USA. It has a working frequency band from 30 kHz to
6 GHz, and its internal impedance is 50 Ω.
Prior to any measurement, the VNA is calibrated with a specific procedure.
During this procedure, the frequency band and a number of measurements points
are first set. Then, three known standards are successively measured: a short circuit,
an open circuit, and a matched load (50 Ω). Generally, the calibration plane is located
close to the characterized devices, i.e., at the coaxial cables output/input of the
device under test. A setup example for measuring the reflection coefficient of wire
electrodes in an nsPEF delivery system immersed in a biological sample is shown in
Fig. 3b. In this case, the calibration plane is located at the input connector of the
nsPEF system.
To determine the efficiency of energy transfer between the nsPEF generator and
the delivery system, the reflection coefficient (S11-parameter) at the input of the
studied system is frequently evaluated through measurement and simulation. The
lower the value of the reflection coefficient, the better the energy transfer and the
impedance matching between the generator and the delivery system. Typically, the
value of 10 dB for the reflection coefficient, i.e., 10% of the incident generated
power is reflected back by the delivery system, is considered acceptable and as a
reference for defining the system frequency bandwidth. To determine the resistive,
capacitive, or inductive characteristics versus the frequency, the equivalent imped-
ance of the system is also extracted from the reflection parameters. To determine the
intrinsic efficiency of a system, the transmission coefficient (S21-parameter) can be
studied. The transmission coefficient is considered between the output and input of a
two-port system. The higher the value of the transmission coefficient, the better the
energy transfer within the delivery system. Typically, the threshold at 1 dB for the
transmission coefficient, i.e., more than 90% of the input power is transmitted by the
delivery system, is considered as a reference.
Figure 3c, e illustrates an example of S-parameters for two nsPEF delivery
systems based on wire electrodes or a TEM cell containing a Petri dish with
biological solution. Several examples of the frequency characterization results for
different nsPEF delivery systems can also be found in Arnaud-Cormos et al. (2011),
Dalmay et al. (2011), Kenaan et al. (2011), and Wu et al. (2013).
A frequency characterization of the macroscopic electromagnetic properties of
dielectric media particularly of the biological samples can also be performed. For
determining the dielectric properties such as the relative permittivity and electrical
conductivity, a dielectric probe can be used, such the 85070E Dielectric Probe from
Agilent, USA, which is illustrated in Fig. 4a. These types of measurements are
commonly performed by immersing the probe in the center of the liquid or semisolid
sample to characterize. The measurements are nondestructive and may be performed
Vector Network Analyser

a
Port 1 Port 2
b1 b2
Forward return loss S11= a S22= a Reverse return loss
1 a2=0 a1 b2 2 a1=0
Device
b2 under test b1
Forward insertion loss S21= a S12= a Reverse insertion loss
1 a2=0 b1 a2 2 a1=0
c 0
–10
|S11| (dB)
–20
–30
–40
0 0.1 0.2 0.3 0.4 0.5
Frequency (GHz)
Fig. 3 (continued)
e 0 0
−5
−10 −1
|S11| (dB)
|S21| (dB)
−15
−20 −2
−25
−30 −3
0.5 1 1.5 2 2.5 3
Frequency (GHz)
Fig. 3 Measurements of delivery systems S-parameters. (a) Schematic of the experimental setup
for the frequency characterization of a delivery system under test. (b) Photo of the setup for
measuring the S-parameters of a wire electrode nsPEF delivery system. (c) Measured reflection
coefficient (S11-parameter) for a wire electrode delivery system. (d) Setup for measuring the
S-parameters of a TEM cell nsPEF delivery system. (e) Measured reflection coefficient (S11-
parameter) and transmission coefficient (S21-parameter) for a TEM cell delivery system
in real time. The complete setup is based on a vector network analyzer, which
measures the response of the material to the electromagnetic wave sent by the
previously calibrated dielectric probe (Fig. 4b). The calibration is performed at the
tip of the probe by connecting a specifically designed short circuit, by immersing the
probe in distilled water (equivalent of a matched load) and by leaving it in the open
air (equivalent of an open circuit). Complex permittivity, dielectric constant, dielec-
tric loss factor, and loss tangent are some of the parameters that can be obtained from
the electromagnetic wave reflection on the characterized medium. Generally char-
acterized biological solutions, e.g., Roswell Park Memorial Institute medium
(RPMI) or phosphate-buffered saline (PBS), exhibit dielectric permittivities around
75–78 and electrical conductivities ranging from 1 to 1.5 S/m, at low frequency and
at room temperature. Depending on the temperature, the permittivity slightly
a b Vector Network Analyser
ε(ω)
Port 1 Port 2
Control Unit
b1
+ S11= a
Software 1
Dielectric
=> ε(ω) dielectric
coaxial probe
characteristics b1 a1
Biological
solution
c
80
Relative permittivity
ε’ − measured
60
ε" − measured
ε’ − Debye’s fit
40 ε" − Debye’s fit
20
0
0 1 2 3 4 5 6
Frequency (GHz)
d 101
Conductivity (S/m)
100
10−1
σ − measured
σ − Debye’s fit
10−2
10−1 100 101
Frequency (GHz)
Fig. 4 Example of measured electromagnetic properties of a PBS buffer solution over

150 MHz–6 GHz frequency band. (a) Photo of the dielectric probe used for measuring dielectric
characteristics. (b) Schematic of the experimental setup for the frequency characterization of a PBS
buffer solution under test. (c) Complex relative permittivity measurements and fit with a Debye
model. (d) Equivalent electrical conductivity measurements and fit with a Debye model
decreases, whereas the conductivity increases. In certain conditions, it can be useful

to modify the conductivity of the biological solution to adapt the equivalent imped-
ance of a delivery system as described in Kenaan et al. (2011). An example of
measured electromagnetic properties versus the frequency of a PBS buffer solution is
shown in Fig. 4c, d. More details can be found in ▶ Chap. 16, “Single Cell Electrical
Characterization Techniques”.
Time Domain Characterization
Time domain characterizations are focused on determining the temporal character-

istics of nanosecond electric pulses. In the following part, temporal pulse profile
measurements by specific setups and components such as tap-offs, high-voltage
wideband probes, and high-intensity E-field probes are illustrated.
Tap-Off Measurements
Once the delivery systems and the biological samples have been characterized in the
frequency domain from an electromagnetic perspective, the next main step consists
of determining as precisely as possible the characteristics of the nsPEF applied to the
biological sample. Indeed, when assembling a generator with an nsPEF delivery
device through transmission lines and connectors, a fraction of the generated inci-
dent pulse is reflected at the transitions between the different components. The
fraction of the reflected pulse depends highly on the matching of the impedances
and the spectral frequency bandwidths of the pulse and the transmission chain
components especially those of nsPEF delivery system. Thus, it is important to be
able to dissociate the reflected pulse from the generated one in order to access the
applied pulse at the level of the biological sample. An example of a typical
configuration developed to apply and characterize nsPEF is shown in Fig. 5. This
particular setup is composed of a high-voltage nsPEF generator, coaxial transmission
lines, several high-voltage components, an oscilloscope for monitoring and mea-
surements, and a system that can deliver pulses to cells in suspension.
In this type of setup, the nsPEF generator is connected to the input of a component
called tap-off (model 245-NMFFP-100 from Barth Electronics Technology, USA)
via coaxial cables. The tap-off allows the real-time measurement of the incident
voltage pulse (Vi) and the reflected voltage pulse (Vr) propagating in the experimental
setup. A photo of this specific three-port component is shown in Fig. 5b. Port #3 is
also the measurement port which has a coaxial line of 4950 Ω input impedance. A
voltage pulse measured on port #3 is thus attenuated by a ratio of approximately
100 corresponding to 40 dB attenuation (50/(4950 + 50)). To measure incident and
reflected voltage pulses, electrical delays introduced by two transmission lines are
used. Based on propagation theory, an electrical delay (T = L/v) depends on the
transmission line length (L) and the wave propagation velocity (v = c/√er, where c is
a incident nsPEF
=> tap-off =>
nsPEF
generator
>=
4950 1:100
External 30 dB attenuator reflected nsPEF 50
trigger
Connector
Electrodes
Function generator
Trigger Monitoring Acquisition Oscilloscope
Petri dish Exposed cell
Fig. 5 (a) Typical experimental setup developed to apply and characterize nsPEF; in this example,
cells are exposed to nsPEF in a Petri dish. (b) Photo of the tap-off (245-NMFFP-100, Barth
Electronics Technology, USA) with annotation of direct and indirect line. (c) Photos of high-
voltage attenuator and 50 Ω loads
the light velocity and er corresponds to the transmission line relative dielectric
permittivity). As an example, consider a 1 m length of the transmission line of
er = 2.2 between the generator output and the tap-off and 3 m between the tap-off
and
the 8 delivery
pffiffiffiffiffiffiffi system. The corresponding electrical delay is 5 ns
1= 3:10 = 2:2 for the incident pulse. An electrical delay of 30 ns is then
obtained for a forward–backward pulse propagation between port #2 of the tap-off
and the delivery system. This will essentially allow one to display on an oscilloscope
the incident voltage pulse followed after a delay of 25 ns by the reflected voltage
pulse. The applied voltage (Vap) pulse to the input of the delivery system is obtained
by summing the incident (Vi) and reflected (Vr) pulses: Vap = Vi + Vr; the applied
pulse is calculated after compensation for the time offset due to the delay between
the forward and reflected pulses.
Pulses can be displayed through the measurement port of the tap-off on oscillo-
scopes. The latter are commonly limited in terms of maximum input voltage. To
avoid exceeding this limitation, attenuators (e.g., 20 dB, 10 dB, Barth Electronics
Technology, USA) are usually added at the tap-off output. High-voltage matched
loads (Fig. 5c) can also be used to characterize the generated pulses prior to
connection to the delivery system. Figure 6c, d illustrates an example of incident
and reflected pulses. Several examples of temporal characterization results of nsPEF
electrical pulses can be found in Merla et al. (2010), Arnaud-Cormos et al. (2011),
Dalmay et al. (2011), and Wu et al. (2013).
High-Voltage, Wideband Probe Measurements

A different method of measuring nanosecond electric pulses is based on high-voltage
probes as illustrated Fig. 6.
For the measurements, the two conductor pins of the probe are placed in direct
contact with the electrodes (Fig. 6a). The probe model 2440 from Barth Electronics
Technology, USA has a wide frequency band of 6 GHz and a risetime of 60 ps using
its supplied cable (1 m). The probe is designed to have the output terminated into a
50 Ω system, and it does not require any specific calibration procedure. Depending
on the output, the probe may be configured with two different resistances: a 950 Ω
input resistance will output a 1:20 voltage ratio, i.e., the probe output amplitude is
20 times less than its input given by the equivalent circuit resistances ratio 50:
(950 + 50); similarly, a 450 Ω input resistance will output a 1:10 voltage ratio. The
measurement principle and setup are described in Fig. 6b. A pulse example mea-
sured with this probe is illustrated (Fig. 6e) and in the article (Merla et al. 2010).
High-Intensity, E-Field Probe Measurements

From a more general perspective, to evaluate the potential effects of RF electromag-
netic fields on human health, electric field (E-field) and temperature probes are
widely used (Schmid et al. 1996; Pokovic et al. 2000; Schuderer et al. 2004). Indeed,
the interaction between electromagnetic fields that are variable in time and biological
systems such as tissues or cells can be quantified from the temperature rise or the
electric field induced in the exposed sample. The standard parameter for the assess-
ment of the interaction is the specific absorption rate (SAR) (Ahlbom et al. 1998;
Faraone et al. 2000; Ziegelberger 2009; Okano et al. 2010).
For experimental evaluation of the SAR by electric field measurements, metal
probes using diode-based sensors (Schmid et al. 1996; Faraone et al. 2000) and
electro-optical sensors (EO) (Cecelja and Balachandran 1999; Togo et al. 2008; Lee
et al. 2009, 2010) are mainly used. Because of their totally dielectric structure and
small size, the EO sensors are much less invasive than metal probes. To enable in situ
measurement in experimental bioelectromagnetic investigations, it is crucial to limit
the disturbances on the electromagnetic environment induced by the probe
(Schuderer et al. 2004). Metal probes are more sensitive than EO sensors. In
b Monitoring applied nsPEF

Oscilloscope
>=
Function
generator Acquisition 26 Attenuator
dB
External Biological HV probe

trigger solution 1:20
30 dB incident nsPEF 950 Ω
4950 1:100
=> =>
nsPEF
Cuv ette
generator
>=
tap-off
reflected nsPEF
Electrodes
c d 3
6
Incident pulse-Tapoff Reflected pulse- Tapoff
2
90%
4 1
Voltage (kV)
Voltage (kV)
2 11 ns
–1
10% –2
0
–3
Risetime = 0.5 ns
0 5 10 15 20 25 0 5 10 15 20 25
Time (ns) Time (ns)
Fig. 6 (continued)
e 6
Applied pulse - HV Probe
5
4 90%
Voltage (kV)
3
2 11 ns
1
10%
0
Risetime = 1.5 ns
–1
0 5 10 15 2 5
Time (ns)
Fig. 6 Probe for high-voltage nsPEF measurements. (a) Photo of a high-voltage measurement
probe, (b) Measurement principle and setup. (c, d) Incident and reflected pulses measured with the
tap-off. (e) Applied pulse measured with the high-voltage probe
addition, diode sensors are typically used to detect only the signal envelope, while
EO sensors are capable of measuring the electric field (amplitude and phase) in real
time over a wide frequency band. However, unlike metal probes, EO sensors are still
mostly prototypes developed for laboratory studies. Among them, very few are
developed and suitable for measuring high-intensity nsPEF.
Figure 7 shows an example of a prototype of an electro-optic probe for electric
field measurements developed by the start-up Kapteos. This millimeter-sized pig-
tailed wideband EO probe was developed for simultaneous measurement of one
component of the E-field and the temperature variation using a single EO crystal
(Duvillaret et al. 2002; Bernier et al. 2008). Both measurements are based on the
determination of the Pockels-effect-induced polarization state modulation of a laser
beam after a round trip across the EO crystal. The short-term polarization changes in
the optical signal from the probe yield the voltage measurement, while longer-term
changes yield changes in temperature. The EO probe crystal properties permit
wideband transient measurements (up to 10 GHz).
The active part of the probe is a 7.1 mm long, 1.8 mm diameter EO crystal of
congruent x-cut lithium tantalite (LiTaO3). LiTaO3 crystals are anisotropic and
sensitive to a single component of the electric field. The relative permittivity of the
EO crystal of 42 is well adapted to relatively high-dielectric-permittivity media, such
as biomedical or biological media. When immersed in an electric field, the optical
properties of the crystal change. The EO crystal is preceded by optical components
mounted at the output of an optical fiber. Its orientation relative to the optical fiber is
such that its axis of sensitivity to the electric field is perpendicular to the direction of
laser beam propagation. The EO probe is controlled by a servo control system
containing optical, electronic, and mechanical components (Duvillaret et al. 2002;
Bernier et al. 2008).
Prior to nsPEF measurements, the probe was characterized in terms of linearity,

sensitivity, selectivity, and dynamic in response to continuous-wave electromagnetic
fields (Jarrige et al. 2012). Measurements were performed in air and in a water
solution exposed to 1800 MHz CW signal inside a TEM cell. The probe had a
b Monitoring
Oscilloscope Monitoring
Voltage = f(E-field) T (°K)
Function Servo control

system
>=
generator Acquisition
Optic fiber
Cuvette
External
trigger
EO Crystal
30 dB incident nsPEF
Solution
4950 1:100
=> =>
nsPEF
generator
>= Electrodes
tap-off
reflected nsPEF
c 400
300
Electric field (kV/m)
200 11 ns
100
Tap−off
0 EO probe
−100
0 5 10 15 20
Time (ns)
Fig. 7 (continued)
d 600
Tap−off
500 EO probe
400
Electric field (kV/m) 300

FWHM = 3.9 ns
200
100
−100
0 5 10 15 20
Time (ns)
e
0.3 EO probe
Fitting
0.25
0.
80
ΔT (K)
0.15
Frequency
0.1 40
0.05
0
0 −20 0 20
Deviation Values (mK)
−0.05
0 30 60 90 120 150 180
Time (s)
Fig. 7 High-intensity, E-field probe measurements. (a) Photo of the EO probe inserted in an
electroporation cuvette. (b) Schematic of the experimental setup for the exposure and measurement
of high-intensity nsPEF. (c, d) Examples of electric field measurements using the EO probe and
compared with the tap-off measurements for 11 ns and 4 ns duration pulses. (e) Temperature
measurement with the EO probe and fitting; in the inset, distribution of the deviation and the
normal probability density function. In this configuration, a number of 120,000 pulses of 2.6 ns
duration at 1 kHz repetition rate were applied corresponding to an E-field intensity of 450 kV/m (#
[2012] IEEE. Reprinted with permission from Kohler et al. (2012a))
dynamic range reaching at least 60 dB, a linearity uncertainty within 0.6 dB, and
selectivity up to 25 dB was measured demonstrating the ability of the EO probe to
measure one unique component of the E-field. The sensitivity values of the EO probe
were 770 and 180 mV m1Hz1/2 in air and in water, respectively. The ability of the
EO probe to perform SAR assessments by simultaneous measurements of the
electric field and temperature was also demonstrated (Ticaud et al. 2012). The
measurements were made in a Petri dish filled with a biological solution placed in
a TEM cell and exposed to an RF signal. For the temperature measurements with the
EO probe, a standard deviation lower than 27 mK was attained. The SAR values
obtained via temperature and electric field measurements showed very good
agreement.
Studies were also conducted on the characterization of the probe response to
intense pulsed electric fields of nanosecond duration (Fig. 7) (Kohler et al. 2012a).
The measurements were carried out with the EO probe inserted in an electroporation
cuvette filled with a biological solution exposed to nsPEF (Fig. 7a). At the time, it
was to our knowledge the first report of high-intensity measurements made with an
EO probe in the literature (Fig. 7b). As shown in Fig. 7c, d, the electric fields
measured by the probe and deduced from voltage measurements via the tap-off
showed good agreement. Very good temporal measurement performance of the
probe was observed in terms of electric field levels reaching 1 MV/m. An example
of temperature measurement is also shown in Fig. 7e. In this configuration, a number
of 120,000 pulses of 2.6 ns duration at a 1 kHz repetition rate were applied
corresponding to an E-field intensity of 450 kV/m. This particular exposure led to
a total temperature rise of less than 0.3 K. The measurement uncertainty achieved
was 22 mK.
Numerical Dosimetry
For the knowledge of certain quantities such as the transmembrane potential at the
cellular level, the contribution of numerical modeling is essential and complemen-
tary to experimental techniques.
Macrodosimetry
The majority of numerical studies are primarily focused on modeling at the macro-
scopic scale of the nsPEF exposure systems. To perform a macrodosimetric study, a
numerical method such as the finite elements method (FEM), the finite-difference
time-domain (FDTD), or the method of moments (MOM) is required. For example,
once the structure is modeled, the FDTD numerical method typically solves Max-
well’s electromagnetic differential equations in the time domain within a spatially
meshed structure with elementary cells. We have advantageously used an FDTD-
based tool in a significant number of our dosimetric studies. Typical results for a
studied structure extracted from the FDTD simulations include reflection and trans-
mission coefficients, input impedance, and electric field intensity and voltage spa-
tiotemporal distributions. When modeling a structure, the dielectric materials are
considered with their electromagnetic macroscopic properties, i.e., relative permit-
tivity and electrical conductivity.
At high frequencies, specific absorption rate (SAR) is classically used to quantify
the exposure levels at a given frequency. The SAR can be obtained from temperature
measurements or electric field values. The dosimetric characterization can also be

assessed in terms of the specific absorption (SA).
SAR Assessment from Temperature Measurements

SAR values are computed from the temperature rise measurements using

@T
SAR ¼ C ðW=kgÞ (1)
@t t¼0
where C is the specific heat capacity of the considered medium (J.kg1.K1);

@T/@t corresponds to the initial slope of the temperature versus time (K.s1).
SAR Assessment from Electric Field

SAR values are calculated from the electric field as follows:
σE2
SAR ¼ ðW=kgÞ (2)
2ρ
where E is the local electric field (V/m), σ is the electrical conductivity (S/m), and
ρ is the sample density (kg/m3).
Specific Absorption (SA)

SA values are calculated as the time integral of the SAR. The specific absorption is
expressed in J/kg. For pulsed fields, the SA is a relevant parameter for characterizing
the energy absorbed per unit mass of biological tissue.
Several examples of modeling and macrodosimetry results of exposure systems
for nsPEF electrical pulses can be found in Arnaud-Cormos et al. (2011), Dalmay
et al. (2011), Kenaan et al. (2011), and Wu et al. (2013).
Microdosimetry and Nanodosimetry
At the macroscopic scale, the biological cells are not taken into account; only the
medium in which they are immersed is modeled. Analytical solutions are possible
only in specific configurations. Thus, it is necessary to use powerful numerical
methods to best approximate the response of the biological environment as a
whole but also locally through microdosimetric studies. Since the cell dimensions
(a few μm) are significantly smaller than the electromagnetic wavelengths contained
in typical nsPEF spectra (several cm), the wave propagation phenomena can be
disregarded. Thus, quasi-static approaches can be implemented to perform micro-
dosimetric studies. Classical approaches are based on solving the Laplace equation
by finite difference-based or finite elements-based methods. These techniques allow
analysis at the cell’s membrane scale (▶ Chaps. 55, “Different Approaches Used in
Modeling of Cell Membrane Electroporation”, ▶ 56, “Electric Field Distribution and
Electroporation Threshold”, and ▶ 59, “Transmembrane Voltage Induced by
Applied Electric Fields”).
The mechanisms for electroporation are nanoscopic, however, and take place at
the atomic scale. To bridge this gap between continuum electrical models and
discrete atomic interactions, molecular dynamics (MD) simulations can be used to
generate the time evolution of large sets of atoms under fundamental mechanical and
electric laws, with atomistic spatial resolution and sub-picosecond time resolution.
MD methods are intensively used in studies of the molecular mechanisms of
electroporation. MD simulations are run in order to generate atomic positions over
time as electroporation occurs. Then, distributions of electric charge densities can be
extracted from the resulting simulation trajectories (▶ Chap. 7, “Lipid Electropore
Lifetime in Molecular Models”).
Experimental Microdosimetry
We have so far seen dosimetry techniques at the macroscopic level of a system as a

whole. Via microdosimetry, we can go down to the cell level aiming to make the
junction between the characteristics (electric fields, temperature, etc.) obtained during
macroscopic scale experiments and those existing locally in the vicinity of a cell.
The effects observed with nsPEFs are often considered nonthermal as the total
energy of the pulses is low despite the high power. Some authors demonstrated by
analytical and numerical methods that an increase of the local temperature could occur
on the membrane of the cell, although no overall increase in temperature was observed
(Kotnik and Miklavcic 2000; Song et al. 2011). Measuring the temperature at the
subcellular level could therefore provide valuable information to understand how pulsed
electric fields interact with cells. Thus, rigorous macrodosimetric and microdosimetric
studies of the exposed biological sample are essential to establish connections between
electric fields and biological responses, to optimize nsPEF exposure protocols and
ultimately to enable reliable comparisons between experiments. In microdosimetry,
numerical implementations are more advanced (Kotnik and Miklavcic 2006; Smith
and Weaver 2008; Merla et al. 2009, 2010, 2011; Kotnik et al. 2012) as the lack of
appropriate measuring devices is a major obstacle to experimental investigations.
In this context, a microdosimetry experimental technique has been developed.
That allows microfluorimetry analysis of biological samples fluorescence at a micro-
scopic scale. More specifically, it consists of measuring the temperature at the
cellular level using the temperature-sensitive properties of the fluorophore rhoda-
mine B in response to nsPEF pulses. At the time of this study, no experimental
measurement of the temperature at the membrane or cellular scale was published
when the cells were exposed to high-intensity nsPEFs.
The technique has been implemented in two major stages. First, the fluorophores
were calibrated to determine their susceptibility to the temperature and the stability
with respect to the cell. To calibrate the variation of rhodamine B fluorescence as a
function of temperature, one method is to induce a fast temperature rise in the sample
and then compare changes in fluorescence measured by a reference probe (in this
case with a fluoroptic probe such as Luxtron XT-812, LumaSense Technologies, CA,
USA). A fast increase in temperature may be induced within a biological medium by
exposure to high-power continuous-wave (CW) electromagnetic fields.
The experimental setup for the fluorophore calibration is based on a compact open
transverse electromagnetic (TEM) cell containing a Petri dish with the cells labeled
with rhodamine B (Fig. 8). The applied fields are CW type at 930 MHz frequency with
50 W input power. The TEM cell is placed on the stage of an inverted microscope
(DMI6000, Leica) (Fig. 8a). The measurement setup is illustrated Fig. 8b for CW or
nsPEF exposures. For quantification of fluorescence, the 40 objective of the micro-
scope is positioned near the bottom of the Petri dish, through a hole in the TEM cell
bottom plate. The results obtained are shown in Fig. 8c, d. They indicate that a
temperature increase of 5 C was obtained in the sample after 11 min of exposure.
The decrease in fluorescence of rhodamine B was valued at 4.1 1.0% per degree of
temperature rise. The sensitivity of temperature measurement is less than 0.5 C.
Microscopic or macroscopic temperature changes were then investigated after
application of three million, 1.2 ns duration pulses of 2 kV amplitude in a TEM cell
(Fig. 8b). The nsPEFs were applied with a repetition rate of 3 kHz for a total of
17 min exposure time. The microfluorimetry measurements using rhodamine B in
cells showed no increase in the average temperature during the nsPEF exposure. This
was also the case for the measurements carried out macroscopically with the
fluoroptic probe. However, in both experiments the exposure to the nsPEF caused
interesting morphological changes associated with swelling and membrane bleb-
bing, similar to that previously reported in the literature.
Conclusions
This chapter summarizes recent developments in experimental dosimetry for high-

voltage, nsPEF exposure systems. The original developments focused on experimen-
tal measurement setups for high-voltage nanosecond pulses exposure. At the macro-
scopic level, complementary characterizations were carried out in the frequency and
time domain. Frequency domain characterizations include measurements of a deliv-
ery system’s S-parameters or a biological sample’s electromagnetic properties. Time
domain characterizations were focused on determining the temporal characteristics of
high-voltage nanosecond electric pulses. Measurements of the temporal pulse profile
by specific setups and components such as tap-offs, high-voltage wideband probes,
and high-intensity E-field probes were illustrated. At the microscopic level, a
b Temperature
Oscilloscope
Optical probe
30 dB
TEM Cell T° probe
Nanosecond
pulse
generator Tapoff
Petridish
Bidirectionnel
Circulator coupler
Signal Power microscope
generator amplifier objective
50
Powermeter
Fig. 8 (continued)
Fig. 8 Fluorescence temperature measurement technique for microdosimetry. (a) TEM cell placed
on the stage of an inverted microscope. (b) Measurement setup for CW or nsPEF exposures. (c)
When applying CW microwave power causing heating in the sample, the temperature-sensitive
fluorescence signal of RhodB is shown to decrease, while no changes are observed in the Rhod110
signal. (d) A normalized ratio of the RhodB/Rhod110 fluorescence signal fits well with the
macroscopic temperature measurements made by the Luxtron fluoroptic probe. (e) An example of
fluorescence variation observed in U87 cells exposed to a temperature increase of 3.5 C (# [2013]
IEEE. Reprinted with permission from Kohler et al. (2013))
microdosimetry technique for measuring temperature at the cellular and intracellular

levels was presented. Numerical dosimetry that can serve to complete or to access
quantities difficult to measure was also briefly introduced.
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▶ Photoconductive Switching for Pulsed High-Voltage Generators
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Photoconductive Switching for Pulsed
High-Voltage Generators 47
Delia Arnaud-Cormos, Vincent Couderc, and Philippe Leveque
Abstract
High-voltage (HV) ultrashort pulse technologies require adjustable pulse param-
eters such as duration, amplitude, shape, number of pulses, and the frequency
rate. Generators that can produce powerful electrical pulses with adjustable
characteristics are convenient but still not widely available. This chapter presents
a summary of key developments around HV pulse generators built on the frozen-
wave-generator concept. With this concept, a microstrip or coaxial transmission
line technology is used to store a bias voltage from a high-voltage source.
Ultrafast optoelectronic switches integrated on the line and triggered by laser
beam allow delivery of pulses into an output load. Due to their advantages,
photoconductive semiconductor switches (PCSS) are used in the linear running
regime. These generators can produce pulses of various shapes (rectangular,
bipolar, triangular), with durations ranging from a few hundred picoseconds to
a few tens of nanoseconds and with amplitudes up to tens of kV. Developed
generators include two types of transmission lines based on microstrip or coaxial
technology and two types of PCSS capable of switching up to 4 kV or 20 kV bias
voltages. This technology has enabled generation of pulses with peak amplitudes
of around 15 kV. It also allows the profiling of the pulses depending on the
conditions of PCSS commutations such as energy levels, optical delays, and
synchronous or asynchronous activation.
D. Arnaud-Cormos (*) • P. Leveque

University Limoges, CNRS, XLIM, UMR 7252, and LABEX “Sigma-LIM”, Limoges, France
e-mail: delia.arnaud-cormos@ensil.unilim.fr;
philippe.leveque@unilim.fr; philippe.leveque@xlim.fr
V. Couderc
CNRS, XLIM, UMR 7252, University of Limoges, Limoges, France
e-mail: vincent.couderc@xlim.fr

DOI 10.1007/978-3-319-32886-7_208
838 D. Arnaud-Cormos et al.
Keywords
High voltage • Nanosecond and subnanosecond pulse generator • Photoconduc-
tive switch • Optoelectronic switching • Pulse shaping
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 838
High-Voltage, Electric Pulse Generators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 842
Basic Operating Principle of PCSS-Based, High-Voltage, Electric Pulse Generators . . . . . 842
Photoconductive Switches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 844
Microstrip-Line Pulse Generators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 848
Coaxial-Line Pulse Generators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 852
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 854
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 856
Introduction
High-voltage (HV) ultrashort pulses have been increasingly used since the 1990s in
various industrial applications such as food processing, defense, environment, and
also biomedical science (Schoenbach et al. 2002; Kandušer and Miklavčič 2009). In
the latter case, many investigations have highlighted the effects of HV electric pulses
on biological cells (Weaver et al. 2012). More specifically, in the last decade many
studies have explored the effects of electric pulses with durations in the nanosecond
rage (3–300 ns) and intensities up to megavolt per meter (Schoenbach et al. 2004;
Garon et al. 2007; Napotnik et al. 2010). It has been shown that electric pulses with
durations of micro- and milliseconds impact external cell membranes and more
recently that nanosecond and subnanosecond pulses have effects also on the intra-
cellular structures and membranes (Weaver et al. 2012). However, the mechanisms
of interactions are not yet completely known or explained. In addition to the
multitude of biological parameters to investigate, the effects of specific parameters
of electric pulses are continuously being studied.
In bioelectrics or bioelectromagnetics, the physical and biological phenomena
involved are complex and require the establishment of appropriate experimental
technologies. Fig. 1 shows an illustration of a typical exposure system for in vitro
experiments. The developments on experimental exposure systems focus on four main
parts: pulse generation, delivery system, dosimetry, and imaging and biological
analysis. The generation part is connected to the system for application of pulses,
called “delivery system” or “applicator.” The delivery system is the part of the
exposure system that transforms the generated voltage pulses into pulsed electric fields
within the exposed biological sample. An essential part relates to the quantification and
the control of the electric field and temperature in the sample. The dosimetry consists in
experimental and numerical quantification techniques of characteristic quantities. To
observe and analyze the sample behavior, ideally before, during, and after exposure,
the exposure system also may include developments on specific imaging systems.
47 Photoconductive Switching for Pulsed High-Voltage Generators 839
Fig. 1 Schematic illustration of typical experimental technologies for in vitro exposure systems of
biological cells to high-voltage pulsed electric fields. Four main parts compose the exposure setup:
pulse generator, delivery system, dosimetric characterizations, and imaging systems
This chapter focuses on pulse generation technologies while another chapter of

this book focuses on measurement and characterization of exposure systems for high
frequency/ultrashort pulses.
For current bioelectric investigations, the generators must be capable of produc-
ing pulses with nanosecond and subnanosecond durations with HV pulse intensities.
Furthermore, implementations of generators those are able to output pulses with
adjustable characteristics, for example, amplitude, duration, or shape, are still rather
uncommon. The ability to generate pulses of variable parameters is highly requested
by biologists and medical doctors because it allows a detailed study of dose-effect
relations. It also allows the determination of the optimum parameters to maximize
the desired biological effects.
Typical parameters of an electrical pulse illustrated in Fig. 2 are:
– Duration commonly characterized by Full Width at Half Maximum (FWHM):

tens of nanoseconds to subnanosecond
– Amplitude: a few kilovolt (kV) up to a few tens of kV (20–30 kV)
– Temporal shapes: rectangular, bipolar, triangular, etc.
– Number of generated pulses and repetition rate
Fig. 2 Typical parameters of a

high-voltage electric pulse in
the nanosecond/
subnanosecond duration
range. (a) Illustration of pulse
repetition rate, peak voltage,
and energy. (b) One pulse can
be characterized by the
duration considered as the
Full Width at Half Maximum
(FWHM), maximum
amplitude, and rise time
considered between 10% and
90% of the pulse maximum
amplitude
The main characteristics of pulse generators are: variability of pulse parameters,

repeatability of the generated pulses, monitoring the number of pulses generated and
the frequency rate, coaxial output and impedance matching with the output devices
(cables, delivery systems, monitoring systems, etc.), synchronization with imaging
systems, friendliness of user interface, robustness, and compactness.
As discussed in Rebersek et al. (2014), the development of nano- and pico second
pulses require different techniques from micro- and millisecond generation and they
highly depend on the switching components turn-off times. Indeed, for generating
pulses of very short duration from a DC voltage source, the Pulse forming Line
(PFL) is considered as one of the simplest techniques. Consisting of a single
transmission line and one or more fast switches, the PFL can be used for high-
amplitude levels. One means of increasing the output voltage of the pulse generators
is the use of structures based on voltage multiplier circuits such as the Pulse Forming
Networks (PFN). PFN can amplify pulse output voltage while maintaining the
ability to vary their profiles.
Based on the PFL generation principle, “frozen-wave” generators allow the
shaping of the pulse using a transmission line. They are based on the storage of
Fig. 3 Electric scheme illustrating the frozen-wave generator components. Electric power, charged
by a high standard DC power source (bias voltage), is stored in a capacitor. The high-voltage pulse
is generated and output into a load using an ultrafast switch
electrical energy in a capacitor and its charging into a load in a very short time using
high-power switch (Fig. 3). Electric power, charged by a high standard DC power
source, is stored in a transmission line and output using an ultrafast switch.
In the literature, typical generation techniques of ultrashort electric pulses can be
found in Blumlein (Rebersek et al. 2009), Marx (Heeren et al. 2007), and magnetic
pulse compression generators (Tang et al. 2007; Akiyama et al. 2010). These
generators include one or several switche components that are usually power
semiconductor, spark gap, and optoelectronic. Among power semiconductors,
metal-oxide-semiconductor field-effect transistors (MOSFET) (Rebersek et al.
2009), thyristors (Akiyama et al. 2010), and diode opening switches (Tang et al.
2007) are commonly used for low breakdown voltage (several kilovolts or less) and
rise times of a few nanoseconds. For higher voltages, spark gap (Heeren et al. 2007)
or photoconductive semiconductor switches (Lee 1990; El Amari et al. 2010; El
Amari et al. 2011) are preferably implemented. Indeed, spark gap switches can
sustain up to several hundred kilovolts of voltage. However, the reproducibility of
the pulses generated is challenging because of the particular conditions necessary to
trigger these switches. Moreover, the jitter that corresponds to a fluctuation of the
emission time of the pulses relative to a clock is significant relative to the desired
pulse durations.
Among semiconductor switches, optoelectronic switches have found a large
variety of applications such as ultra-wideband radar, THz technology, and bioelec-
trics (Loubriel et al. 1997, Merla et al. 2010; Kohler et al. 2013). Optoelectronic
switches with controllable closing are particularly convenient for the generation of
pulses of kilovolt amplitudes, nanosecond and subnanosecond rise times, and
kilohertz repetition rates. The switches are referred in this chapter by their acronym
“PCSS” for “Photoconductive Semiconductor Switch.” A pulsed laser generally
provides the optical energy necessary for the control of the PCSSs.
PCSSs have two main running regimes, i.e., linear and nonlinear (or avalanche)
modes. In the linear mode, the PCSS semiconductor material creates an electron-
hole pair for each photon impinging on the switch. In this mode, for each photon an
extra electron will contribute to the conduction current (Renxi et al. 2001). Com-
pared to avalanche mode where a photon generates multiple electron-hole pairs by a
cascade effect (Zhao et al. 1993), the linear regime requires more energy for the same
optical switching efficiency. Indeed, the avalanche mode requires low optical energy
to switch the PCSS. But, the main limitations of this switching regime are the
maximum frequency rate and also the PCSS progressive destruction that reduces the
generator’s lifetime (Loubriel et al. 1997; Ma et al. 2004). To enable synchronization
with real-time imaging techniques for bioelectrics applications, PCSS linear regime
is recommended especially for low jitter. Photoconductive semiconductor compo-
nents are typically made from silicon (Si), gallium arsenide (GaAs), or silicon
carbide (SiC) substrates (Kelkar et al. 2008). PCSS enhanced performance in
terms of higher photoresponsivity, shorter carrier lifetime, and/or higher dark elec-
trical resistivity have been obtained using GaAs-grown alloys (Ospald et al. 2008).
Section “High-Voltage, Electric Pulse Generators” provides an overview of
frozen-wave pulse generators using PCSSs and their characteristics. Key results
and performance will also briefly be highlighted. More detailed in-depth descriptions
are available through the associated publications.
High-Voltage, Electric Pulse Generators
This section presents examples of PCSS-based, high-voltage, electric pulse genera-

tors. The section starts with the description of the generator’s working principle
followed by a subsection on photoconductive switch characteristics and perfor-
mance. Two types of generators implementing PCSSs on microstrip and coaxial
transmission lines are illustrated in the last two parts.
Basic Operating Principle of PCSS-Based, High-Voltage, Electric Pulse

Generators
Figure 4 illustrates the working principle and the main components of generators
built on the frozen-wave generator concept (Thaxter and Bell. 1995).
A high-voltage DC power supplies energy to a transmission line that acts as
capacitive energy storage element. Microstrip or coaxial transmission lines are
typically used. Two photoconductive semiconductor switches (PCSS) are integrated
in the transmission lines. A pulsed laser optically triggers the PCSSs. This allows the
delivery of the stored energy into the load (biological sample). The generated electric
pulse duration depends on the transmission line length and/or on the delay between
the triggering of the switches.
Figure 5 shows the general principle of generating an electric pulse using frozen-
wave generators. A lossless transmission line can be modeled by a set of capacitors
in parallel (C) separated by series inductances (L). The PCSSs integrated in the line
are in this case considered as ideal elements with zero resistance and switching time.
When the line is open and polarized at a bias voltage (V), the capacitors are charged
until the potential difference across them reaches the bias voltage. The voltage along
the line is then equal to V and 0 elsewhere. If an optical pulse illuminates PCSS2, the
switch is closing and it becomes the equivalent of a short circuit. The first capacitor
on the left of PCSS2 then begins to discharge into the first capacitor on the right of
PCSS2. The discharge ends when the potential difference reaches V/2 on both sides
Fig. 4 Schematic of the basic operating principle and main components of pulse generators. A
high-voltage DC power supply energy to a transmission line that acts as capacitive energy storage
element. Microstrip or coaxial transmission lines are typically used. Two photoconductive semi-
conductor switches (PCSS) are integrated in the transmission lines. A pulsed laser optically triggers
the PCSSs. This allows the delivery of the stored energy into the load (biological sample). The
generated electric pulse duration depends on the transmission line length and/or on the delay
between the triggering of the switches
Fig. 5 Modeling of an electric pulse generation using a transmission line embedding two ideal
PCSSs; Grey area: voltage along the line. (a) The line is first charged with a bias voltage V; PCSS1
and PCSS2 are open; PCSS2 is connected to a transmission line terminated with a matched load.
(b) PCSS2 triggered (closed) by an optical pulse – two waves propagating in opposite directions
from the position of PCSS2 are released
of PCSS2. The capacitors on the left of PCSS2 are then successively discharged
through the distributed inductors, while the right capacitor is charging. As illustrated
in Fig. 5, the charges/discharges result into two waves propagating in opposite
directions with a propagation velocity: v = 1/(LC)1/2, where L (H/m) is the induc-
tance per unit length and C (F/m) its capacitance per unit length of the line.
Figure 6 illustrates the generation of different shapes of electric pulses controlled
by the closing of PCSSs and by the termination load of port 1 near PCSS1. To
understand what happens to the wave propagating toward x when it reaches the
PCSS1, we define a reflection coefficient Γ as Γ = Er/Ei where Ei is the complex
amplitude of the incident electric field propagating toward –x and Er the reflected
electric field propagating toward x. It is known that for a short circuit Γ = 1 and for
an open circuit Γ = 1. This means that a wave incident on a short circuit is totally
reflected but the wave phase is shifted by π radians. In the case of an open circuit, the
reflection is total and its phase is preserved. In this configuration, the pulse obtained
on a matched load at the end of the line will be rectangular if the PCSS1 is not
illuminated, and bipolar if it is illuminated. Indeed, if the transmission line termi-
nates in an open circuit (PCSS1 open), there is no phase shift between the incident
and reflected pulses. A rectangular pulse is delivered to the load (Fig. 6a). If the line
is terminated in a short circuit on the left-hand side (PCSS1 closed), the wave
propagating backward undergoes complete reflection and is inverted upon reflection.
A bipolar pulse then propagates to the load (Fig. 6b). The total time T (s) of the pulse
generated depends on the distance L(m) separating the two PCSSs using T = 2 L/v
with v(m/s) of the wave propagation velocity in the transmission line.
Furthermore, controlling the power of the optical pulses illuminating the two
switches and the relative temporal delay between the two optical beams allows
control of the electrical pulse profile. Bipolar pulses with adjustable positive and
negative parts and pulses with adjustable frequency content between a few hundred
megahertz and 2 GHz can be generated as illustrated in Fig. 6c where PCSS1 is
triggered with a certain delay after PCSS2. Also, by charging the port 1 near PCSS1
with a 50-Ω matched load, rectangular pulses with adjustable duration can be
generated as Γ = 0, Er = 0 no reflected electric field (Fig. 6d).
Photoconductive Switches
Figure 7 shows two typically used photoconductive semiconductor switching com-

ponents that are high-voltage rectifier diodes sustaining up to 4 kV (RGP02-40E,
Vishay, USA) and up to 20 kV DC (X200FG, VMI, USA). The “open” switch state
is equivalent to a very high resistance (around 10 MΩ). This resistance can be
drastically decreased by illuminating the switch with a high-power laser beam,
transforming the switch from an insulator to a conductor. The latter case corresponds
to the “closed” switch state.
As mentioned previously, pulsed lasers provide the optical energy used to trigger
these generators’ PCSSs. Two Nd:YAG lasers operating at 1064 nm and generating
optical pulses of durations 2.4 ns and 35 ps have been typically used. The laser
a b
c d
Fig. 6 Generation of different shapes of electric pulses controlled by the closing of PCSSs and by
the termination load of port 1 near PCSS1; Γ denotes the reflection coefficient of the wave at the line
end. (a) Wave reflection at the level of “open” PCSS1, Γ = +1 – generation of a rectangular pulse.
(b) Wave reflection at the level of “closed” PCSS1 Γ = 1 – generation of a bipolar pulse. (c) Wave
reflection at the level of “closed” PCSS1; PCSS1 is triggered with a certain delay after PCSS2,
Γ = 1 – generation of a bipolar pulse with unbalanced positive and negative parts. (d) Wave at the
level of “closed” PCSS1, Γ = 0 as Er = 0 no reflected electric field – generation of a rectangular
pulse with adjustable duration
Fig. 7 Photoconductive semiconductor switches (PCSSs). (a) Photo of the 4 kV (with and without
packaging) and 20 kV maximum bias voltage PCSSs. (b) Electric diagram of the PCSS character-
ization setup, the switching performance are assessed as a function of the optical incident energy
and/or the bias voltage level
repetition rate is of 10 Hz or 20 Hz and can supply up to 30 mJ per optical pulse, an

energy level which is sufficient for PCSS switching.
Given the PCSS semiconductor absorption and depth of penetration, a laser
wavelength of 1064 nm is suitable for triggering these switches. PCSSs are packaged
in an axial semitranslucent glass that allows reducing the amount of optical power
required for complete switching. When triggering the PCSS in the linear mode, each
photon illuminating a switch is generating one electron-hole pair. When all gener-
ated electron-hole pairs have recombined, generally taking around 200 ns, the PCSS
returns to its “open” state.
PCSS switching performance has been characterized by assessing their resistance
and conversion efficiency versus their optical incident energy or the bias voltage
level (El Amari et al. 2011). The conversion efficiency (η) of the switch is defined as:
Vout
η¼ (1)
V=2
Fig. 8 Switching a
performance versus the
optical energy for the 20 kV
maximum bias voltage PCSS.
The optical pulse duration is
2.5 ns. When the optical
energy is varied, the bias
voltage is set to 8 kV. Without
illumination, a conventional
ohmmeter connected to the
PCSS indicates a resistance of
33 MΩ; the diode is then
effectively in the “open” state.
(a) PCSS equivalent
resistance variation versus the
optical energy. PCSS “closed” b
resistance as low as 6 ohms
was obtained. (b) PCSS
switching efficiency versus
the optical energy. PCSS
efficiency higher than 95%
was obtained
where V is the bias voltage and Vout is the maximum level of the pulse output
voltage, as defined in Fig. 7b. Note that V/2 is the maximum pulse voltage that can
be output from a frozen-wave-based generator and Vout is comprised between 0 and
V/2.
Figure 8 illustrates an example of PCSSs switching performance versus the
optical energy. The optical pulse duration is fixed to 2.5 ns. When the optical energy
is varied, the bias voltage is set to 8 kV. Without illumination, a conventional
ohmmeter connected to the PCSS indicates a resistance of 33 MΩ; the diode is
then effectively in the “open” state.
Figure 8a shows the PCSS equivalent resistance. As the optical energy increases,
the number of electron-hole pairs that can be created increases (until saturation) and
the PCSS exhibits better properties of electrical conductivity. In this configuration,
the PCSS equivalent resistance corresponding to the “closed” state was 6 ohms for a
10-mJ optical energy. Fig. 8b shows the PCSS switching efficiency. As the optical
energy received by the PCSS increases, excellent switching performance was
achieved with this PCSS. Efficiencies higher than 95% were obtained for 6 kV
bias voltage or 10 mJ optical energy, respectively. Compared to the 50-μJ optical
energy used for the 4-kV PCSS measurement performed in El Amari et al. (2011),
this component required higher optical energy. This can be explained by the higher
voltage that can be sustained by this component and by the less transparent packag-
ing materials of the 20-kV PCSS at the 1064 nm wavelength.
The equivalent resistance of the switch significantly increases with the bias
voltage (El Amari et al. 2011; Kohler et al. 2013). This can be explained by the
Franz-Keldysh effect, which translates in a shift in the optical absorption edge of the
PCSS toward longer wavelengths with increasing applied electric field (Lee 1990).
The residual resistance attained by the PCSS after optical illumination has a signif-
icant impact on the pulse shape; therefore, the generator design must consider the
evolution of resistance during the switching process. The knowledge of the PCSS’s
“closed” resistance value as a function of the bias voltage and the optical energy
allows one to identify configurations with higher performance and efficiency, for
example, by placing several PCSSs in parallel (to reduce the equivalent resistance)
or in series (to increase the bias and output voltage).
Depending on the transmission line types, nano- and picoseconds pulse genera-
tors based on microstrip or coaxial lines have been developed embedding PCSSs.
With microstrip lines, 4 kV maximum bias DC voltage PCSS have been
implemented, while coaxial lines allowed embedding PCSSs that could sustain up
to 20 kV maximum bias DC voltage.
Microstrip-Line Pulse Generators
With microstrip-lines-based generators, the energy is stored in a capacitive form in a

dielectric placed between two conductive elements of planar shape. As shown in
Fig. 9, one of the conductors serves as a ground plane and one is connected to the
high DC voltage source. The electric field generated when the upper conductor is
energized is mainly confined within the dielectric structure. This technology has
several advantages: The dimensions of the upper conductor can be easily modified
with inexpensive methods (photolithography); optoelectronic components can be
easily attached to this structure due to the planar configuration; and all the elements
incorporated into the structure are readily accessible. As far as drawbacks, these lines
can withstand relatively low voltages and radiation losses as the discontinuities
become important for high frequencies.
Figure 10 shows two examples of generators implemented on microstrip lines
with impedances of 50 Ω and 10 Ω, respectively. Depending on the prototypes, the
generators incorporate PCSSs sustaining up to 4 kV or 20 kV DC bias voltage. One
PCSS is grounded, while the one at the other end of the line is connected to the load.
In biological experiments, generated pulses are frequently delivered through coaxial
cables and/or exposure systems containing biological sample. These loads are
typically 50-Ω matched. For example, in the case of a standard electroporation
cuvette, containing the biological sample, its impedance is dependent on the sample
dielectric characteristics and the cuvette geometrical dimensions. For certain cuvette
geometry, samples with rather low conductivity (0.5 S/m) are required to match
50-Ω generators (Kenaan et al. 2011). However, biological experiments require more
Fig. 9 Microstrip a
transmission line.
(a) Composition: A dielectric
material placed between two
conductive elements of planar
shape. (b) Electric field lines
are mainly confined within the
dielectric structure between
the upper conductor and
ground plane
conductive solutions (1–1.5 S/m) that allow better maintenance of the cells’ wellness
and easier cell cultures treatments using standard protocols. Higher conductivity
values will decrease the equivalent impedance of the cuvette. For example, by
changing the conductivity from 0.3 S/m to 1.5 S/m, while keeping the same
geometry and gap distance between cuvette electrodes will decrease the equivalent
impedance from 50 Ω to 10 Ω. By increasing the conductivity, the matching
frequency bandwidth of the cuvette delivery system should extend allowing more
efficient pulse delivery within the biological sample. However, the optimization of
pulse transfer (energy and/or voltage) into the load conditions the development of
generators capable of presenting different output impedances. The impedance
matching is crucial for the control of applied pulses on biological samples and to
avoid multiple reflections between the generator and the delivery system.
Thus, the first illustrated generator (Fig. 10a) has a microstrip line of 50-Ω
impedance. The second generator (Fig. 10b) has a microstrip line of 10-Ω impedance
and a length of 130 mm between the two PCSSs. A gradual narrowing of the
microstrip line is used to transition between the microstrip-line widths (20 mm)
and the switches (about 2 mm wide). This later generator has also another innovative
feature that is the integration within the same compact structure of a cuvette delivery
system (Merla et al. 2010).
Figure 11 shows examples of measured temporal shapes of pulses obtained with
microstrip-line generators. The pulses are obtained with a 50-Ω impedance generator
(Fig. 10a) with PCSSs sustaining up to 20 kV DC bias voltage. The optical pulse
duration is 35 ps. Frozen-wave generators allow the generation of pulses with an
adjustable spectral content that depends on the activation conditions of the two
PCSS.
Examples of pulse shaping using delayed excitations of 0 ps, 250 ps, and 320 ps
between the activation of the two switches are shown in Fig. 11a. Asymmetrical
bipolar pulses with different durations of the positive and negative parts can be
Fig. 10 Examples of frozen-wave generators implemented on a microstrip transmission lines with

characteristic impedance of 50 Ω (a) and of 10 Ω (b). The dielectric substrate used for both
generators is 1.6 mm high and has a relative permittivity equal to 4.5. The generators integrate
PCSSs sustaining DC bias voltage up to 20 kV or 4 kV
obtained. In these conditions, pulse durations of 1.3 ns are achieved with a rise time
and fall time of 70 ps and 110 ps, respectively.
Figure 11b shows unbalanced bipolar pulses achieved using different optical
energies delivered to synchronously activated individual PCSSs. The optical energy
delivered to the PCSS connected to the output load were 500 μJ, 250 μJ, 110 μJ,
50 μJ, and 0 μJ, while the optical energy delivered to the PCSS connected to the
ground was 500 μJ.
A third example (Fig. 11c) illustrates the temporal pulse profiling when combin-
ing the two previous configurations, i.e., asynchronous activation and different
optical power levels delivery to the two PCSSs. When the grounded PCSS is
illuminated with 10 μJ and the PCSS connected to the load with 20 μJ, a monopolar
pulse with 1.7 ns duration was obtained for simultaneous activation. When the PCSS
Fig. 11 Examples of a 5
measured temporal shapes of 0 ps
pulses obtained with 250 ps
Voltage (kv)
microstrip-line generators. 320 ps
The pulses are obtained with
the 50-Ω impedance generator 0
shown in Fig. 8a with PCSSs
sustaining up to 20 kV DC
bias voltage. The optical pulse
duration is 35 ps. (a) Pulse –5
shaping using delayed 0 1 2 3 4
excitations between the Time (ns)
activation of the two switches.
(b) Pulse shaping using b 5 500 µJ
different optical energies
250 µJ
delivered to synchronously
110 µJ
Voltage (kv)
activated individual PCSSs.

50 µJ
(c) Pulse shaping 0 0 µJ
asynchronous activation and
different optical power levels
delivery to the two PCSSs. #
(2010) IEEE. Reprinted, with –5
permission, from El Amari 0 1 2 3 4
et al. (2010) Time (ns)
c 2 360 ps
1.7 ns
Voltage (kv)
3.3 ns
1
0
0 1 2 3 4 5 6
Time (ns)
connected to the load is activated before the grounded PCSS, a monopolar pulse with
3.3 ns duration was obtained. Conversely, when the PCSS connected to the load is
activated after the grounded PCSS, a 360 ps pulse duration was obtained.
An illustration of pulses generated with the 10-Ω impedance generator is shown
in Fig. 12. This generator integrates PCSSs sustaining up to 4 kV DC bias voltage.
Figure 12 presents the generated pulse and the applied voltage pulse across the
cuvette delivery system electrodes. Whereas the pulse shape is modified compared to
the generated one due to the cuvette equivalent impedance and frequency bandwidth,
a good consistency is obtained between applied measured and simulated pulses.
The numerical simulations have been carried out with an in-house solver based on
the finite-difference time-domain (FDTD) method.
A compact generator delivering pulses of a few nanoseconds based on the 4-kV
PCSS and a microchip laser has been developed. As mentioned previously,
Fig. 12 Examples of
measured temporal shapes of
pulses obtained with the 50-Ω
impedance generator shown in
Fig. 8b with PCSSs sustaining
up to 4 kV DC bias voltage:
generated (or incident) pulse,
applied pulse across the
cuvette delivery system
electrodes, FDTD numerical
simulation, and experimental
measurements
microstrip-based generators are limited by the relatively low voltages they can
sustain and by the radiation losses at high frequencies. Coaxial-based systems
have been thus developed as illustrated in the next subsection to overcome limita-
tions of microstrip lines.
Coaxial-Line Pulse Generators
As shown Fig. 13, a coaxial transmission line is a structure consisting of two

conducting materials, cylindrical and concentric, separated by a dielectric material.
The outer conductor serves as a reference potential and as a shield from external
electromagnetic fields. The inner conductor is connected to the source potential. For
cross-sectional dimensions and appropriate materials, it is shown that the generated
electric field is normal to the longitudinal axis of the conductors. This is true on a
continuous band of frequencies including the electrostatic fields, and the spectral
width is specific to the chosen structure.
Figure 13 also shows schematics and photographs of a coaxial generator example.
In this structure, each PCSS was integrated in separate coaxial boxes. Two types of
coaxial boxes have been developed with one-port and three-ports. The one-port box
is short-circuited at one end and connected to the coaxial line on the other end. The
three-port box was connected through port 2 to a HV DC power supply, through port
1 to a coaxial line, and through port 3 to the load. Apertures drilled in the outer
metallic architecture allow passing the optical energy required for the PCSS
switching.
The main benefit of coaxial-line generators consists in the possibility to readily
change the length of the coaxial line separating the two PCSS switching boxes.
Compared to the microstrip structure, the range of electric pulse durations that can be
generated is theoretically much larger. However, to achieve expected pulse genera-
tion performance, several specific parameters must be optimized. The connectors
serving as adapters between the different elements of the generation chain must be
chosen accordingly to prevent mismatching effects. Commercial connectors of THT
Fig. 13 Example of frozen-wave generators implemented on coaxial transmission lines with

characteristic impedance of 50 Ω. (a) Coaxial line: composition and electric field lines. (b) Pulse
generator example coaxial technology: axial cut view of coaxial boxes, connectors, and coaxial
cable (not to scale). (c) Photograph of the elements
type (THT .20, Radiall USA, Inc., CA, USA) specially designed to sustain DC
voltages up to 20 kV have been used. Their bandwidth at 3 dB reached 1 GHz. RG
214 coaxial cables served as energy storage element. For optimal energy transfer to
the 50-Ω impedance load, the characteristic impedance of the PCSS boxes, coaxial
cables, THT connectors should be equal to 50 Ω.
The internal part of the one-port and three-ports boxes was custom designed for
50-Ω impedance matching. To prevent unwanted and possibly damaging electrical
discharges, special care was taken to avoid air bubbles and apertures in the dielectric.
For the coaxial boxes, polytetrafluoroethylene (PTFE) dielectric material was chosen
for its good dielectric rigidity and temperature stability. The outer conductors are in
aluminum, while the inner conductors are made in copper; the later has a high
electrical conductivity. A 333 kΩ resistance was soldered between DC high-voltage
source and PCSS2 to limit both the current and the charging time constant of the
capacitive transmission line. This resistance and the PCSSs were welded so that the
length of their “wires” was kept the shortest possible. Indeed, connections through
single wires must be optimized as the inductance of single cylindrical wires increases
with increasing length and frequency. Moreover, to limit the electrical discharges
insulating oil can be injected at the PCSSs level.
Figure 14 illustrates the ability of coaxial-based generators to produce pulses with
different durations and shapes in the nanosecond range (Kohler et al. 2013). For the
examples showed, a 2.5-ns duration optical pulse was used with energy per optical
pulse of 6 mJ or 8 mJ; a DC bias voltage of 8 kV or 16 kV was charged on the line.
The presented pulses have durations of 8.3-ns, 9.5-ns, and 17.5-ns. Their
corresponding amplitudes are 4 kV for the 8.3-ns and 17.5-ns duration pulses and
7 kVor 13 kV for the 9.5-ns duration rectangular or bipolar pulses, respectively. Rise
times of 2.5–2.9 ns were measured for these pulses.
The generation of pulses with durations also in the subnanosecond range is
showed in Fig. 15. A 745-ps duration, 3.5-kV amplitude, 125-ps rise time pulse
was produced using 35-ps duration optical pulse with 1.05 mJ optical energy and
10 kV DC bias voltage.
The coaxial-based technology provides increased flexibility compared to micro-
strip lines, confers higher voltage-handling capability, and enables safer operation.
Indeed, pulses obtained with coaxial lines are generally smoother than those pro-
duced with microstrip lines. The coaxial implementation allows more flexibility in
pulse durations and higher intensities.
Conclusions
For bioelectric applications, high-voltage generators producing pulses in the nano-

second and subnanosecond duration ranges have been developed. Based on the
principle of frozen waves, the generators can deliver electric power stored in a
transmission line with an ultrafast switch called a PCSS into a load. Developed
Fig. 14 Typical coaxial lines a

generated pulses. For the
examples showed, a 2.5-ns
duration optical pulse was
used with energy per optical
pulse of 6 mJ or 8 mJ; a DC
bias voltage of 8 kV or 16 kV
was charged on the line.
(a) 9.5-ns duration rectangular
and bipolar pulses with 7-kV
and 13-kV amplitude,
respectively. (b) Rectangular
pulses with durations of 8.3-ns
and 17.5-ns, 4-kV amplitude.
# (2013) IEEE. Reprinted,
with permission, from Kohler b
et al. (2013)
Fig. 15 Typical coaxial lines generated pulse at the subnanosecond range. For the example
showed, a 35-ps duration optical pulse was used with energy per optical pulse of 1.05 mJ and a
DC bias voltage of 10 kV was charged on the line. A rectangular pulse with 745-ps duration, 3.5-kV
amplitude, 125-ps rise time pulse was generated. # (2013) IEEE. Reprinted, with permission, from
Kohler et al. (2013)
generators mainly include two types of transmission lines based on microstrip or

coaxial technology and two types of PCSS capable of switching up to 4 kV or 20 kV
bias voltages. Initially, the microstrip technology has been used for its advantages in
terms of compactness and ease of manufacture.
Pulses of monopolar and bipolar forms with durations between a few hundred
picoseconds to a few nanoseconds and maximum amplitudes of around 4 kV were
generated with this technology. To overcome the limitations of the latter concerning
the breakdown voltage and for more flexibility in terms of pulse length, the coaxial
technology was then implemented. This technology has enabled generation of pulses
with peak amplitudes of around 15 kV. It also allows the profiling of the pulses
depending on the conditions of PCSS commutations such as energy levels, optical
delays, and synchronous or asynchronous activation.
Cross-References

▶ Compact Nanosecond Pulse Generator Based on Printed Circuit Board Transmis-
sion Lines and Solid-State Switches
▶ Marx-Generator Design and Development for Biomass Electroporation
Ultrashort Pulses
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Basic Concepts of High-Voltage Pulse
Generation 48
Luís M. S. Redondo
Abstract
For electroporation experiments it is essential to generate, in a controlled way,
repetitive high-voltage pulses, with precise voltage amplitude and pulse widths,
in order to deliver well-defined energy packages to biologic loads. This transient
energy flow can be based on relatively simple circuits consisting of passive
discrete resistive-inductive-capacitive elements, transformers or transmission
lines, and switches, which convert the energy stored in the electric fields of
capacitors or magnetic fields of coils into well-defined voltage pulses. Here,
transient phenomena of single and stack circuits are described as a basis of the
generation and handling of high-voltage pulses, which are described elsewhere in
this handbook. These include direct capacitive discharge, comprising a single
switch or an association of circuits to circumvent the still voltage and current
limitation of semiconductors, which include adder and Marx generator circuits.
The use of a single step-up transformer or inductive adder circuit will be
described also. Also, the utilization of transmission lines to generate and format
pulses into well-defined loads is explained. In addition, the use of inductive
storage with normally closed switches is presented. Finally, an example of the
techniques that use resonant circuits with saturable inductor or transformer is
outlined. As the behavior of the load can influence greatly the shape of the voltage
pulse applied to the load as well as the current requirements of the switches,
during this chapter, the type of load will be evaluated in relation to the most
common type of biological type loads, with resistive or capacitive behavior,
respectively.
L.M.S. Redondo (*)

Lisbon Engineering Superior Institute, GIAAPP/ISEL, Lisbon, Portugal
EnergyPulse Systems, EPS, Lisbon, Portugal
e-mail: lmredondo@deea.isel.ipl.pt

DOI 10.1007/978-3-319-32886-7_209
860 L.M.S. Redondo
Keywords
Direct capacitive discharge • Association of circuits • Transformers • Transmis-
sion lines • Inductive storage discharge circuits • Resonant circuits with saturable
inductors
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 860
Capacitive Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 862
Direct Capacitive Discharge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 862
Utilization of Transformers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 866
Transmission Line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 869
Inductive Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 871
Resonant Circuit with Saturable Transformer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 873
Bipolar Pulse Generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 875
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 877
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 878
Introduction
Electroporation is associated with the permeabilization of biological cell membranes

induced by the application of electric fields, which are in general pulsed in order to
lower the power delivered to the cell medium and enhance energy efficiency (Kotnik
et al. 2012). These pulsed electric fields are generally created between two electrodes
by the application of high-voltage pulses to one electrode, being the other at a ground
potential. Hence, in order to make electroporation experiments, it is essential to be
able to generator pulsed high voltages, which must follow a number of requirements,
with the amplitude and the duration of the electric field applied to the cell medium
being exactly controlled (Kandušer and Miklavčič 2009; Reberšek and Miklavčič
2011; Reberšek et al. 2014). Hence, the voltage pulses are to be created with a
precise voltage amplitude and pulse width, with a set repetition rate or frequency.
These pulses are to be applied into a load with a considerable conductivity range,
meaning that the pulse generator must be capable of drawing a considerable amount
of current, or work as an open circuit, occasionally. In addition, the voltage pulse
amplitudes can usually range from hundreds of volts to tens of kilovolts. Where the
standard almost square voltage pulse can be characterized by several parameters
(Fig. 1):
• Risetime, tr: period of time from 0.1 to 0.9 U, where U is the pulse plateau
• Overshoot, ΔUs: voltage excursion, after the risetime, above the pulse plateau
U
• Width, Δt: pulse duration in the 0.5 U region
• Undershoot, ΔUd: voltage excursion, after the fall time, below zero volt
• Drop, ΔU: voltage fall during plateau
48 Basic Concepts of High-Voltage Pulse Generation 861
n
DUs
U
0.9U DU Uf
0.9Uf
U Dt
2
0.1U 0.1Uf
0
DUd t
tr tf
Fig. 1 Typical voltage pulse parameters
• Fall time, tf: period of time from 0.9 to 0.1Uf, where Uf=UΔU is the final pulse
voltage;
Considering the above requirements, there are several basic concepts that can be
used for high-voltage pulse generation. The key idea is that energy is collected from
some primary energy source of low voltage, stored temporarily in a relatively long
time and then rapidly released from storage and converted in high-voltage pulses of
the desirable pulsed power, as described in the block diagram of Fig. 2 (Pai and
Zhang 1995a). How is it possible to achieve such goals effectively and economically
is the heart of this technology.
From Fig. 2 one can see that, first of all, some kind of primary energy source must
be available, which can be electrical, chemical, mechanical, or other forms of energy.
However, in many electroporation applications, electrical energy, such that supplied
from mains, is found to be the most appropriate. Energy from the primary source is
usually slowly accumulated and stored, in a time scale greater than two to three
orders of magnitude in comparison with the high-voltage pulse widths (Pai and
Zhang 1995b).
Energy storage can be done in many different ways, where electrical energy
stored in capacitors and magnetic energy stored in inductors have been widely
employed. If one compares the energy storage density capabilities of electric and
magnetic fields, one finds, for electric energy
1
W E ¼ eE2 J=m3 (1)
2
and for magnetic energy
1 B2
WM ¼ J=m3 (2)
2 μ
862 L.M.S. Redondo
Fig. 2 High-voltage pulse

generation concept
where E and B are the electric and magnetic fields, respectively. Considering
acceptable parameters for nowadays material, as relative permittivity er = 10,
μ=μ0, E = 108V/m, B = 10T (Mankowski and Kristiansen 2000), results in
W E 4:4 105 J=m3
and
W M 4 107 J=m3
where it is seen that, usually, the energy density of inductive storage systems is, in
average, two orders of magnitude greater than that of capacitive systems. However,
inductive storage systems require opening switches instead of closing switches for
capacitive storage systems, being the last ones more common and with a higher
variety, performance, and more available with less price.
Considering electrical energy stored in capacitors, it will be described in this
chapter the key circuits for high-voltage pulse generation, which include direct
capacitive discharge using one circuit of a stack of them, transformers, and trans-
mission lines. These include the generation of monopolar and bipolar pulses.
Likewise, the basic circuit for high-voltage pulse generation, using magnetic energy
storage, will be described.
Capacitive Storage
Direct Capacitive Discharge
The most common topology for high-voltage pulse generation uses direct transfer
from a single or bank of capacitors C to the load, usually of resistive behavior R0, for
biological material, modulated by a normally open switch S, as shown in Fig. 3
(Gaudreau et al. 1998).
Considering, initially, the capacitor charged with voltage U, and then after the
closing of switch S, at t = 0, the voltage applied to the load v0 can be model as
v0 ¼ Uet=R0 C (3)
given an exponential decay-type voltage applied to the load, with time constant R0C.
If an almost square pulse needs to be applied to the load, it is necessary to open the
switch before most of the energy is discharged into the load. The time to open the
circuit and still have an almost square pulse depends on the parameters of the circuit
Fig. 3 Simplified direct

capacitor discharge topology
into a resistive load
and the operating conditions: the load resistance R0, the pulse width ton, and the
value of the capacitor C. The relation between them and the ratio between the final
and initial capacitor voltage, vCf/vCi, can be determined by equating the energy
balance as
Ei ¼ Ef þ E0 (4)
where Ei is the initial energy stored in capacitor C, with vCi = U,
1
Ei ¼ Cv2Ci (5)
2
and Ef is the energy stored in the capacitor C after the pulse, vCf,
1
Ef ¼ Cv2Cf (6)
2
and E0 is the pulse energy, with ton width,
U2
E0 ¼ ton (7)
R
resulting in
vCf 2ton
¼1 (8)
vCi CR0
From (8), it is possible to estimate that for a quasi-square pulse, the energy stored
in the capacitor has to be one or two order of magnitude higher than the energy
delivered to the load.
864 L.M.S. Redondo
In addition, the power supply that charges the capacitor must supply an energy
equal to the delivered pulse energy, E0, plus losses, during the charging, T-ton, period
(Redondo and Silva 2010).
E0
PU ¼ þ Ploss (9)
T ton
If the load is of capacitive nature C0, as shown in Fig. 4, then several operating
changes are imposed.
After the pulse the voltage is distributed between the two capacitors, which can be
equated by the charge balance as
CvCi ¼ CvCf þ C0 vCf (10)
resulting in
vCf C
¼ (11)
vCi C þ C0
However, the electrical efficiency of this discharge process is at best 50% when
C>>C0, meaning that half the energy is delivered to the load and half is dissipated in
heat in the circuit resistances. Hence, between pulses the power supply must
recharge capacitor C with at least two times the energy delivered to the load C0. In
order to increase the yield of such circuit, a different discharge approach should be
followed, for example, by introducing an inductance in between the two capacitors,
forming a capacitive-inductive-capacitive, CLC, circuit.
In addition, when the load is of capacitive nature, when the switch opens, the load
stays charged or is discharge via a longtime constant depending on the parasitic
resistance of the circuit. In order to circumvent this, another switch, Sa, must be

capacitor discharge topology,
with capacitive load

capacitor discharge topology,
with capacitive load, and
auxiliary switch to reset the
voltage after the pulse
connected in parallel with the load, as shown in Fig. 5, with an auxiliary resistor Ra,
which determines the fall time of the pulse, RaC0 (Redondo and Silva 2010).
There are very important additional characteristics of the direct capacitive dis-
charge circuit of Fig. 1; first the voltage rating of the switch S device must hold off
the capacitor voltage; second the primary power supply must have the same voltage
amplitude as the output pulse; third the energy stored in the circuit is all concentrated
in one capacitor bank, which can impose safety constraints. Finally, switch S has
floating trigger terminals, resulting in a trigger signal that must be transmitted with
galvanic isolation. When the voltage goes above a few kV, it is necessary to use more
complex topologies involving associations of single circuits, in order to distribute
the voltage by several switches.
A common approach to circumvent the voltage and current limitations of a single
switch is to stack in series and parallel multiple lower-voltage/ lower-current
switches. It is relatively easy to put in parallel switches due to their usually positive
temperature coefficient; their series assembly is much more complex. Considering
the tens of kilovolt range pulse amplitude that can be needed and the limitation of the
switch hold-off voltage, it is necessary to connect many switches in series to
assemble the circuit topology described above. In order that the stack operate
properly, it is necessary to add auxiliary circuits, such as resistive-capacitive
(RC) networks to balance the voltage in each switch both in steady and transient
operation (Gaudreau et al. 2001).
In addition, it is challenging to synchronously trigger many devices in series,
placed at different floating high-voltage potentials. Because of differences in switch
characteristics and their gate drive circuits, a complex monitoring and synchroniza-
tion of each switch gate are required, in order to maintain a good dynamic voltage
sharing of the switches and prevent their damage (Williams 2005).
Furthermore, the system has to be able to handle the failure of one of the switches
without destroying the whole assembly as well as an over-current emergency turnoff.
Hence, in order to prevent shutdown of a series stacked switch due to a defect arising
in one switch, redundant switches are, usually, included in the series, about 20%
more, such that the surviving switches share the voltage and the failed switch is still
able to carry the load current. This is true since the power switches used are usually
built to short circuit when failing (Welleman et al. 2007).
866 L.M.S. Redondo
Fig. 6 Simplified negative Marx generator topology
Hence, usually the best solution is to use more complex alternative topologies: the
Marx generator and adder circuit. Figure 6 shows a typical simplified Marx generator
topology for generating a negative pulse, where a number of n capacitors are charged
in series from a relatively low-voltage d.c. (direct current) power supply Udc and
subsequently connected in series with a load, applying v0 nUdc. In Fig. 6 circuit
each cell comprises an energy storing capacitor Ci, a diode Di and two switches, in
half-bridge configuration Spi for pulsing and Sci for charging, being i and integer
(Cassel 2004; Redondo and Silva 2009a).
For the adder circuit presented in Fig. 7, which generates a negative output
voltage v0 = nU, where U is the voltage in each capacitor, the main difference
from the Marx generator of Fig. 6 is that the Ci capacitors are charged independently,
in this case by isolated transformers through diodes Di, afterward they are connected
in series to the load by switches Si. Both Figs. 6 and 7 circuits are able to generate
multilevel pulse waveforms in the load, controlling the number of cells that are
applying voltage at each time, as each cell can be triggered independently (Redondo
et al. 2007; Liu et al. 2009).
For direct capacitive-type discharge, it is crucial to avoid that the energy stored in
the capacitor is transferred to the load in case of load short circuit and at the same
time limit the current that can destroy the switches. This is normally done by
measuring the current in the circuit in series with switches S, and when it reaches
a threshold value, the switches are put to off state.
Utilization of Transformers
A solution to avoid complex topologies and the need of high-voltage switches is the
use of a step-up pulse transformer to rise a voltage that does not exceed the voltage
rating of the switch to the desired output voltage, giving also galvanic insulation. In
this case the energy is stored in the primary capacitor C and transferred the load in
the secondary of the transformer. However, several issues must be taken into
consideration before designing the circuit (Smith 2002):
Fig. 7 Simplified negative

adder generator topology
• The parasitic elements (i.e., distributed capacitance and leakage inductance)

normally associated with the assembling of the transformer impose limitations
to the output pulse waveform, degrading the pulse risetime and voltage plateau.
• The transformer core volt-second product limits the transformer flexibility to
accommodate different operating conditions, such as duty ratio and frequency.
• After the pulse, it is mandatory to reset the transformer core magnetic flux back to
the initial status before the next pulse, which imposes an auxiliary circuit.
A common transformer topology is shown in Fig. 8 for generating output positive

voltage pulses in the load. For negative polarity, the ground should be placed in the
opposite terminal of the load resistor R0 (Redondo and Silva 2009b).
In Fig. 8, switch S modulates the voltages in the primary of the transformer, when
S on the voltage applied to the load is approximately
N2
v0 ¼ Udc (12)
N1
where N2/N1 is the transformer turns ratio, N. When S is off, the primary voltage is
approximately given by –Vz, which resets the transformer core. This is true if the
following equality exists:
U dc ton ¼ V z tr (13)
868 L.M.S. Redondo
Fig. 8 Transformer-based
topology for positive high-
voltage pulse generation
that expresses the volt-second (i.e., magnetic flux) apply to the core during ton pulse
that is reset during tr time, which must be less than the switch S off time. The
calculated volt-second swing can be calculated, from the Faraday’s law, using the
transformer physical parameters as
U dc ton ¼ N 1 BA (14)
where B is the maximum amplitude of the magnetic flux density (vs/m2 or T) and
A is the core area (m2). Finally, diode D2 on the secondary prevents that a negative
voltage is applied to the load.
In order to reduce the leakage inductance, a high number of primaries are
preferred, either a single switch can be used to control all the primaries or several
switches, each controlling one or a group of primaries. When several switches are
used, the current is shared between them. The fact that they drive different primaries
insures a better current sharing.
In addition, in order to reduce the parasitic effects of a high turns ratio trans-
former, it is normal to stack several pulse transformers with lower turns ratio, having
only a fraction of the total voltage in each one (Klein and Padberg 1995). With a
careful design, the leakage inductance and distributed capacitances of the equivalent
circuit are reduced as compared with one single equivalent transformer, leading to a
better risetime performance (Smith 2002).
Figure 9 shows an example of stack secondary windings with independent
primaries, or inductive adder topology, for generating negative voltage into the
load, where the capacitors in each cell are charged when the switches are off. Finally,
when the switches are on, the voltage in each primary is applied and added on the
series secondary (Holma and Barnes 2014; Cook 2000).
A useful feature of this topology is that by adding a diode in parallel with the
primaries, the gate control synchronization between each switch is no more critical;
each switch can be turned on or off independently. The output voltage will just be the
sum of the contribution from the closed switches, so one could use this opportunity
to adjust the output voltage of the pulse generator by just closing the right number of
switches.
Fig. 9 Inductive adder C1

topology for negative pulse
generation D1
L11 L12
S1
C2
D2
L21 L22
S2
Load
V0
Cn
Dn
Ln1 Ln2
Sn
In addition, the leakage inductance is lower when a transformer ratio of 1:1 is

chosen and is comparable to the one of a well-designed single transformer topology
with multiple primaries described earlier.
Transmission Line
The transmission line or pulse-forming line (PFL) is the most common type of circuit
topology for the generation of high-voltage pulses with less than 100 ns width. In
effect, a transmission line is a pair of conductors with distributed capacitor C and
inductor L with characteristic impedance:
rffiffiffiffi rffiffiffi
L μ
Z0 ¼ ¼ (15)
C e
where an input voltage wave injected in the line, with length l, propagates along the
line with a delay t0 given by
l
t0 ¼ (16)
v0
having a propagation wave velocity of
870 L.M.S. Redondo
Fig. 10 Basic pulse-forming line topology for positive pulse generation
c
v0 ¼ pffiffiffiffi (17)
er
where μ, e, er, and c are the medium permeability, permittivity, relative permittivity,
and speed of light, respectively.
A transmission line can be used as energy storage instead of a capacitor, being
charged slowly, after which the energy is released by means of a switch into the load,
as shown in Fig. 10, in order to generate high-voltage pulses (Mankowski and
Kristiansen 2000). When the switch is closed at t = 0, a pulse of amplitude U/2
propagates down the line toward its end, at length l; it then reflects and returns to the
switch at 2t0, which defines the pulse width. Upon return of the reflected pulse, the
load voltage becomes zero if the characteristic line impedance is Z0=ZL, i.e., the line
and load impedances are matched.
To get a complete transfer of energy from the line to the load, the impedance of
the load ZL has to match the characteristic impedance of the line Z0. In these
conditions the output voltage is approximately a square pulse with U/2 amplitude
and 2t0 width. Compared to a square pulse generator based on a capacitor discharge
(described in section “Capacitive Storage”), without other losses and in a matched
systems, the energy stored in the line is equal to the energy delivered to the load,
because the energy transfer is complete:
1 U2
CU 2 ¼ t0 (18)
2 2ZL
Another advantage is that the output current is limited, no matter what the load
impedance is, which inherently protects the switch against short circuits. However,
there is the requirement of load matching, which limits its use in dynamic loads and
the size of cable used. Considering the later, for 50 Ω coaxial cable, a 5 m length is
needed for 50 ns pulse width. For applications where larger pulse widths are required
and other load impedance, one can assemble a pulse-forming network (PFN) based
on discrete inductive-capacitive (LC) components, for different characteristic imped-
ance and longer pulses, >100 ns (Butcher 1989).
Fig. 11 Blumlein topology for positive pulse generation
Considering the pulse voltage amplitude limitation, of half the supply voltage, in
order to achieve pulse voltage amplitudes equal to the power supply voltage, is
necessary to couple two (or more) transmission lines, as shown in Fig. 11, called
Blumlein transmission line configuration (Mankowski and Kristiansen 2000).
In Fig. 11, fast closing of switch S causes voltage reversal in half the lines for a
time equal to the double transit time of the line, 2t0. The output pulse produced has
the same amplitude as the power supply voltage for ZL = 2Z0. It is also possible to
stack several of these circuits to achieve higher voltages.
Inductive Storage
A basic inductive energy discharge circuit is shown in Fig. 12, using capacitor C as
primary electric energy storage and a LC resonant circuit, where two types of
switches are used, normally open S1 and S3 and normally close S2 (Lindblom
2006). Initially, capacitor C is charged with Ui, switch S1 closes, and C is discharged
into the inductor producing an increasing sinusoidal current ii, while the energy in
the capacitor is transferred to the coil L as
ii ¼ I m sin ðωtÞ (19)
and angular oscillation frequency
1
ω ¼ pffiffiffiffiffiffi (20)
LC
After the first-quarter period of the sinusoidal LC-current, when current ii is

maximum
872 L.M.S. Redondo
Fig. 12 High-voltage pulse generation circuit based on inductive energy storage and opening
switch
Ui
I m ¼ rffiffiffiffi (21)
L
C
all the energy is stored in the inductor:
1
EL ¼ Li2i (22)
2
Then switch S2 opens and current ii is abruptly interrupted, producing a large
magnetic flux change in the inductor. The large change in magnetic flux induces a
large high voltage in the inductor v0:
dii
v0 ¼ L (23)
dt
Near the peak of v0, S3 closes and the load impedance sees the output pulse,
originating a discharge of the magnetic energy into the load. The main drawback
with this relatively simple but robust circuit is the serious demands this design puts
on the opening switch, as it has to carry the peak current and withstand the full-load
voltage.
In order to reduce the voltage over the opening switch, a transformer can be
introduced, as shown in Fig. 13 (Lindblom 2006). The opening switch S2 is located
on the primary side of the transformer and enables a reduction of the switch voltage
proportional to the winding ratio.
In relation to the inductive storage circuits above, the faster is the open switch, the
higher is the output voltage (Mankowski and Kristiansen 2000).
Fig. 13 High-voltage pulse

generation circuit based on
inductive energy storage with
opening switch and
transformer
Resonant Circuit with Saturable Transformer
A group of techniques for high-voltage pulse generation use the resonance between
capacitors and inductors, in series or parallel circuits, taking advantage of the
nonlinear properties of magnetic materials to achieve a large change in impedance
from ferromagnetic core inductors or transformers (Choi 2010). In effect, the
impedance of a magnetic core material varies from a large inductance Lu (i.e., high
permeability μru when magnetic core is unsaturated) to a small inductance Ls (i.e.,
low permeability μrs when magnetic core is saturated), such that μru> > μrs; hence
Lu> > Ls.
The inductance for unsaturated magnetic material can be calculated by
μ0 μru Am N 2
Lu ¼ (24)
l
where μ0 and μru are the free space and unsaturated relative permeability of the core,
respectively, Am is the magnetic material cross-sectional area, N is the number of
conductor turns, and l is the magnetic field path length. The inductance for saturated
magnetic material can be calculated by
μ0 μrs Am N 2
Ls ¼ (25)
l
where μrs is the saturated relative permeability of the core.
A common example of these techniques is presented in Fig. 14 circuit, based on a
saturable transformer (ST) with a 1:n primary to secondary turns ratio, where L1 and
874 L.M.S. Redondo
Fig. 14 High-voltage pulse generation circuit based on resonant circuit with saturable transformer
L2 are stray inductances; LS is the saturated inductance of the secondary winding of

ST, and the value relations between capacitors are C1 = n2C2 = n2C3, being C3 the
load (Choi 2010; Li et al. 2005). The circuit has a closing switch S in the primary and
a diode D in the secondary.
Supposing C1 initially charged to Udc, the energy in C1 is resonantly transferred to
C2, by closing switch S at t = 0, through L1 and the transformer ST, where diode
D provides the charging loop of C2, as seen in Fig. 14 (Choi 2010) (Li et al. 2005).
The maximum current in the primary circuit I1max and the charging time τ1 of C2 are,
respectively, as seen in Fig. 15:
rffiffiffiffiffiffiffi
C1
I 1max ¼ U dc (26)
2L1
and
rffiffiffiffiffiffiffiffiffiffi
L1 C1
τ1 ¼ π (27)
2
where, at time t=τ1, the maximum voltage of C2 is
V C2max ¼ nU dc (28)
The ST transformer is designed to saturate after the charging of C2, resulting in

the decoupling when the primary and secondary are decoupled; then the energy
stored in C2 is resonantly transferred to the load C3, as seen in Fig. 15 (Choi 2010; Li
et al. 2005), where the peak current I3max is given by
Fig. 15 Most significant I

voltage and current I3max
waveforms from Fig. 14
circuit operation
I3
I1max I1
t
I2
U
nUmax UC3
UC2
UC1
Umax
0 τ1 τ2 t
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
C2
I 2max ¼ V C2max (29)
2ðL1 þ LS Þ
and the charging time of C3, or the I2 pulse width, is given by

rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðL2 þ LS ÞC2
τ2 τ1 ¼ π ðL2 þ LS ÞC23 ¼ π (30)
2
where C2 = C3 and are in series.
After the pulse, and before the next one, it is necessary to reset the transformer
core in order to bring back the flux to the initial value. There are several ways of
doing that, being the most common one the utilization of a third winding that applies
a direct current, which generates an opposite flux (Barrett 1995).
Bipolar Pulse Generation
Whenever the use of bipolar pulses enhances the application of pulsed electric fields
into biological cell, the most common way to do that is by using a half and full-
bridge topology as shown in Figs. 16 and 17, respectively, where an output trans-
former can further increase the amplitude voltage by the addition of a step-up
876 L.M.S. Redondo
Fig. 16 Half-bridge topology

for high-voltage pulse
generation
Fig. 17 Full-bridge topology

for high-voltage pulse
generation
transformer, where the reset of the core is done by the alternating type of wave shape
(Shin et al. 2006; Wang et al. 2000).
Figure 16 shows a topology for generating positive and/or negative high-voltage
pulses, where switches Si must hold off the sum of the capacitor voltages, which is
2 U. However, for capacitive-type loads, it is not possible to discharge the load
capacitance after the pulse, since both S1 and S2 are off (Redondo and Silva 2010).
Figure 17 shows an alternative full-bridge topology for positive and/or negative
high-voltage pulses in comparison with Fig. 16 circuit, with only one power supply
is used and where switches Si hold off the capacitor C voltage U. The use of more
switches also increases the capability to deal with different load conditions. In fact,
with the full-bridge topology, positive and/or negative pulses can be applied to any
type of load. Nevertheless, depending on the flux of energy, not all the controlled
turn-off switches are always required (Redondo and Silva 2010).
It is also possible to stack several of Fig. 16 or Fig. 17 circuits in order to achieve
higher voltages. In alternative, bipolar Marx generator topologies are also available,
using from four to six switches per stage, which increases the operation complexity
of the circuits (Canacsinh et al. 2012).
Conclusions
The topological concepts presented in this chapter show the numerous techniques
that can be used for high-voltage pulse generation for generation of pulsed electric
fields in electroporation applications, depending on the requirements of the load and
on the type of pulse. The most important techniques described were:
• Based on electric energy stored in capacitors:

– Direct capacitive discharge, which manages very well conductivity variations
at the load, producing almost rectangular pulses, as long as the energy stored is
greater than ten times the pulse energy delivered to the load. The switch must
hold off the full voltage amplitude of the load, which is the power supply
voltage. Associations in a series of single circuits, as Marx generator or adder
circuit, makes possible the use of relatively lower-voltage amplitude power
supply and switches, but increases the system complexity.
– Pulse-forming line produces short square pulses in matched loads, transferring
all the stored energy to the load, with half the power supply voltage amplitude,
for single lines, of equal for Blumleins. Other pulse widths and load imped-
ances are possible for pulse-forming networks and stacks lines.
– Transformer, as a single device of inductive adders, can be used to further
increase the pulse amplitude; constraints are present mainly due to parasitic
that affect the voltage wave shape and core saturation and reset after pulse.
• Based on magnetic energy-stored inductors:
– Associated with resonant circuits, using opening switches and having more
energy density available;
• Based on resonant circuits:
– Associated with saturable inductor or transformer.
• Bipolar generation, using half and full bridges with or without transformers,
depending of the load type
Depending on the requirements of the load and the parameters of the application,
which impose the pulse protocol, the suitable technique for high-voltage pulse
generation is selected.
878 L.M.S. Redondo
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Industrial Pulsed Electric Field Systems
49
Michael A. Kempkes
Abstract
Pulsed electric field (PEF) systems have evolved considerably from the small,
hand-built, laboratory apparatus used for experiments in the 1990s. As this
technology has moved from the laboratory to commercial operations, the
demands on the systems themselves have grown substantially, along with sub-
stantially increased requirements for reliability and repeatability. Adding to these
challenges, the range of PEF applications has demanded higher levels of flexi-
bility from the PEF systems themselves.
In response to the growth of PEF applications, multiple companies are cur-
rently building PEF systems specifically for industrial use. These commercial
PEF systems are meeting the commercial demand and easing the transition to PEF
applications for new processes and companies. The key distinctions of industrial
PEF systems include their higher power/capacity, enhanced monitoring and
controls, and ability to meet commercial requirements for safety and operation
in a manufacturing environment. This chapter examines the requirements, unique
features, and current state-of-the-art in industrial PEF systems.
Keywords
Pulsed electric field • PEF • Food processing • Commercial • Industrial • Factory •
Extraction • Drying • Slicing • Peeling
Contents
Introduction/Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 882
Industrial Pulsed Electric Field Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 884
Nonthermal Pasteurization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 884
Tissue Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 885
Pulsed Electric Field System Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 887
M.A. Kempkes (*)

Diversified Technologies, Inc., Bedford, MA, USA

DOI 10.1007/978-3-319-32886-7_211
882 M.A. Kempkes
Industrial Pulsed Electric Field System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 888

Throughput/Capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 890
Automation/Data Logging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 894
Safety/Certification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 895
Industrial Pulsed Electric Field System Costs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 897
Pulsed Electric Field Processing and Commercialization Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 898
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 899
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 900
Introduction/Overview
Pulsed Electric Field (PEF) processing is a low temperature, nonthermal, non-

chemical, low impact process that induces electroporation of cell membranes in
microbes, plants, and animal cells (Fig. 1). Pulsed electric field processing involves
the application of short duration (1–20 μs), high voltage pulses that create an electric
field (from 1–50 kV/cm) across a liquid. The high voltage pulses are applied to the
liquid as it passes through a specially designed treatment chamber. Depending on the
desired effect, this electric field can create pores in the cell membranes (electropo-
ration). This modifies plant cells (at 1–10 kV/cm) and kills resident bacteria, molds,
and other microorganisms (at 15–50 kV/cm) (Barbosa-Canovas et al. 1999).
By using multiple treatment chambers to apply pulses to a stream of fluid in a
continuous flow process, kill ratios of 5–9 log reductions against bacteria in juices
and other liquid foods have been demonstrated, similar to those resulting from
thermal pasteurization. This nonthermal pasteurization ability has resulted in a
Fig. 1 PEF works through Cell membrane E

electroporation of the cell
membrane in bacterial, plant,
and even animal cells. + – + – +
Reversible electroporation is + – + – +
+ – + – +
used to introduce + –
chemotherapy drugs into
tumors and for genetic
engineering – the pores in the
cell walls close after the field
E < Ecnit Intact
is removed. Irreversible
cell
electroporation, the focus of
this investigation for food
processing, breaks the cell
membranes (Toepfl 2006)
E < Ecnit Reversible
E >> Ecnit Irreversible

49 Industrial Pulsed Electric Field Systems 883
Fig. 2 PEF-treated genesis

juice on sale in 2006 (Zhang
2008)
limited number of PEF-treated products around the world (Fig. 2, Clark 2006). More
recently, emphasis in industrial processing techniques has initiated the use of
electroporation of plant and animal cells as a pretreatment for slicing, drying,
extraction, etc., where PEF processing typically replaces heat treatments with
lower impact on the product and lower energy usage (Toepfl 2015).
The last 30 years have seen significant research into the use of pulsed electric field
(PEF) processing in the food industry. Two major, multi-party research consortiums
for PEF were initiated in the late-1990s in the USA and Europe, with the first
commercial-scale PEF system built by Diversified Technologies, Inc. (DTI) for
Ohio State University and the DUST Consortium in 2000 (Fig. 3). Although
adoption of PEF started slowly, by late 2015 over 100 commercially built PEF
systems were operating around the world. The pace of commercial adoption of PEF
systems appears to be accelerating as well.
Industrial PEF systems directly benefitted from the parallel development of
reliable solid state, high voltage pulsed power systems for the military and physics
world in this timeframe. Previous generation high voltage systems were built using
thyratrons, spark gaps, or vacuum tubes. These systems were expensive, difficult to
operate and maintain, and generally unsuitable for a food processing environment.
While there is no clear line between an R&D PEF system and an industrial
system, there are some distinct attributes which delineate them. The most obvious
is average power, which equates to throughput capability. Industrial PEF systems are
typically rated above ~30 kW average power, while most laboratory systems are
rated at a few kWs or less. Pilot plan systems can operate between these regimes and
be used either for large-scale R&D processes or small commercial operations. Other
884 M.A. Kempkes
Fig. 3 World’s first

commercial scale PEF
processing system, delivered
to Ohio State University
(OSU) by Diversified
Technologies, Inc. (DTI) in
2000. The system is capable
of treating 1,000–5,000 L/h
and is now operated at the
USDA ERRC in PA
(Gaudreau 2001)
unique features of industrial systems typically include higher levels of automation,

safety, and design for reliable, long-term operation.
In the last decade, PEF processing has made the leap from laboratory exploration to
a growing commercial processing market in several different industries. This commer-
cial demand, in turn, is leading to the rapid development of PEF systems for industrial
use. This transition has led to the development of larger, more automated PEF systems
which can meet the requirements of commercial food processing facilities.
Industrial Pulsed Electric Field Applications
Nonthermal Pasteurization
The original, and most studied, application of PEF in food processing has been
nonthermal pasteurization of liquids (Dunn 1987). Over a thousand peer-reviewed
PEF studies have been published over the last two decades, examining the impact of
PEF on microbial kill, taste, enzymes, proteins, and a host of other factors (e.g.,
Buckow 2013; Ganeva 2015; Grimi 2015; Jeyamkondan 1999; Min 2003; Yeom
2000). The overwhelming consensus from these studies is that PEF is an effective
approach to nonthermal pasteurization (Fig. 4, US FDA 2000), which has extremely
limited impact on the product’s taste and quality. This is only true within a limited
range of products, however. The primary limitation is that PEF only affects vegeta-
tive organisms (which have cell membranes that can be electroporated) and not
spores. This limits the use of PEF to primarily acidic foods, where spore outgrowth is
not a major concern. Other limitations have been reported for high protein foods
Inactivation of E.coli O157:H7 and E.coli 8739 by OSU-3 System. Treatment

conditions: flow rate: 1.33ml/s, pulse repetition rate 1000 Hz, pulse duration 3µs,
electric field strength 42.04 kv/cm for both E.coli O157:H7 and E.coli 8739
7
6
5 0 time (log cfu/ml)
4 62.38 micros (log cfu/ml)
3
recovery (logMPN/ml)
2
1
0
Ecoli O157:H7 Ecoli 8739
Fig. 4 Inactivation of E.coli by pulsed electric fields (Zhang et al. 2007)
(e.g., liquid eggs and dairy products), where denaturation can occur at high field
strengths. Future research and development is expected to increase the applicability
of PEF to these foods (Sperber 2011), as systems are developed which avoid micro-
heating of the product in the treatment chambers.
Within the nonthermal pasteurization market, commercialization has been slow
and scattered. Genesis Juice introduced the first commercial PEF-treated juices in the
USA in 2005 (Fig. 2), to very positive consumer response and sales (Clark 2006;
Zhang 2008). Unfortunately, Genesis was sold in 2007 for financial reasons
(unrelated to PEF), ending the initial commercial use of PEF. The worldwide
financial crisis in 2008 curtailed investment in PEF adoption for several years,
until several companies introduced mildly PEF-processed (not meeting the US
FDA required 5-log reduction of pathogens) juices and products in Europe around
2012 (Fig. 14, Toepfl 2015). In these cases, PEF served as a shelf life extension for
fresh juices, rather than being marketed as a food safety treatment.
The differing standards for pasteurization between the USA, Europe, and other
countries have also proved to be a hurdle for adoption of PEF systems. There is not a
common requirement for these systems, so each application (and installation) must
be validated independently for its target product and market. This adds significant
costs and time for companies desiring to adopt PEF pasteurization.
Tissue Modification
More recently, other applications of PEF processing have been developed and
introduced commercially. The most widespread of these has been potato processing,
to reduce breakage and cutting costs when making chips and French fries. This
process was pioneered by DIL/ELEA in Germany and accounts for the largest share
of industrial PEF equipment in operation in 2016 (Toepfl 2015).
Related applications for plant tissue modification via PEF such as drying or
extraction applications for a variety of crops have also been explored and proven
886 M.A. Kempkes
Fig. 5 Industrial PEF system

from DIL/ELEA, rated at
80 kW (Toepfl 2015)
Fig. 6 Tomato peeling with PEF. The first tomato is pulsed with 50 pulses, 1.7 kJ/kg, the second is
pulsed with 100 pulses, and the fourth is pulsed with 200 pulses. The third tomato is the control.
PEF can reduce the energy used in conventional steam blanching by nearly an order of magnitude
(Toepfl 2013)
in research trials, but are not known to be in commercial use at this time (Fig. 6).
Given the similarity of these applications to potato processing, both in terms of cost/
energy reduction and overall process improvement, it is expected that these appli-
cations will be adopted soon, and their use will accelerate in the near future.
Typically, all of these processes must be carried out at scales of tons per hour to be
commercially applicable. In response to these larger-scale PEF applications, indus-
trial PEF systems have emerged as well. At least four manufacturers have introduced
PEF systems, rated from 50–600 kW of average power (Figs. 5 and 8), with several
more selling PEF systems at lower powers. Several of these companies, including
Scandinova in Sweden and DTI in the USA, sell PEF systems as an extension of a
pulsed power business which initially focused on other applications (e.g., particle
accelerators, radar transmitters, etc.), while others focus exclusively on PEF systems.
In late 2015, an informal poll of manufacturers indicated that there were approx-
imately 100 PEF systems in operation around the world, split between commercial
and R&D applications (Kempkes 2015). This number appears to be growing at
~25% annually, but specific figures are difficult to track given the range of manu-
facturers and applications.
The benefits of these commercial PEF systems include solid-state switching,
higher average power/throughput, and a greater level of automation and control of
the PEF process itself. This automation simplifies the adoption of PEF in industry,
providing the highly consistent results, across a range of product attributes, required
for commercial food processing.
Pulsed Electric Field System Overview
There are three basic elements in a PEF system: (1) a DC power supply, which
transitions the AC power available from the utility into high voltage, DC power;
(2) the pulse modulator, which transforms that DC power into short, high peak power
pulses; and (3) the treatment chamber, where the high voltage pulses are applied to
the liquid itself (Gaudreau et al. 2004). Although there are many ways to build each
of these elements, they are common to all PEF systems.
The power supply converts the AC power available from the utility into high
voltage, DC power. DC power supplies are typically rated in terms of their average
power (in Watts), which must provide the power required for the desired PEF
protocol and throughput.
The second major element of a PEF system is the pulse modulator, which stores
and discharges short high voltage pulses from the DC power out of the power supply.
The design and construction of PEF pulse modulators builds on the development of
pulse modulators for other applications. Although it is possible to build a PEF
system using spark gaps, thyratrons, or other vacuum switches, and many early
PEF systems were built this way, virtually all modern industrial PEF systems use
solid-state devices (e.g., IGBTs) for generating the pulses. Industrial-scale PEF
systems with the reliability needed for commercial operation were not feasible
before these solid state systems were available.
There are several common modulator topologies, including hard switches (which
switch high voltage directly), Marx banks (in which multiple capacitors are charged
at low voltage in parallel, then erected to high voltage by switching them into a series
string during the pulse), and hybrid modulators (which are charged and switch at low
voltage and use a pulse transformer to reach high voltage pulses). In general, a hard
switch or Marx modulator will provide the most flexibility and pulse consistency
over a range of voltages and pulsewidths, as well as higher efficiency, so these
topologies are preferable for industrial systems.
888 M.A. Kempkes
The capabilities of the modulator are typically expressed in terms of peak power
(maximum pulse voltage and current delivered to the treatment chamber) and pulse
frequency. Additionally, the system must operate within the average power available
from the DC power supply, so:
peak power ðMW Þ pulse frequency ðHzÞ pulsewidth ðμsÞ < average power:
Typically, peak voltage and current are limited by the modulator design. There are
trade-offs between peak current capability and pulse frequency that must be taken into
account when designing the modulator, due to heating of the semiconductor switches.
The design of a specific modulator for a commercial application, therefore, is multi-
faceted and complex. Its specific capabilities and constraints will vary according to the
modulator topology and manufacturer. The goal in every case, however, is to reliably
achieve the desired PEF protocol on the targeted product(s), at the lowest cost.
The third major element of a PEF system is the treatment chamber, where the high
voltage pulses are applied to the product. The key attributes of the treatment chamber
are its ability to minimally impact the product flow, while ensuring that a consistent
electric field is applied to all elements being PEF treated. Unfortunately, these two
attributes are often in conflict, making design of the treatment chamber a critical
exercise in system optimization.
The two basic factors affecting treatment chamber design and operation are the
physical configuration of the chamber, and the electrode and insulator materials.
There are many chamber designs that have been developed and patented over the last
20 years, but the prevailing approach for pumpable fluids is the co-field flow
chamber design, developed and patented by OSU (Yin et al. 1997). This design
has been shown to provide a near-optimal balance between the flow and field
requirements (Fig. 7) in a simple, easy to manufacture design.
For the treatment of bulk foods (such as whole apples or potatoes), alternative
treatment chamber designs are required, such as parallel plates surrounding the
product being transported in a water bath (Fig. 10 shows one commercial example).
These are electrically feasible since the required field strengths are generally low for
these applications (<2 kV/cm.), (Toepfl 2006).
In all cases, there will be anodization (erosion) of the treatment chamber electrodes
during operation. The rate of this erosion depends on many factors, including the
electrode materials, the current density in the electrode material, and the average
current. Designing the electrode requires a balance between electrode costs
(for longer life) and downtime for electrode replacement. Electrode costs are typically
very, very small compared to the electricity required to run the PEF system, however.
Industrial Pulsed Electric Field System Requirements
One of the benefits of PEF processing is that it readily scales from small, laboratory
systems directly to industrial scale systems, as long as the same treatment conditions
(field strength and treatment time) are maintained. This significantly simplifies the
Fig. 7 Cutaway view of OSU

co-field flow chamber built
under license by DTI. The
liquid food flows through the
small hole in the center of the
chamber diameter, and the
high voltage pulses are
applied to the center
conductor, establishing two
treatment areas within the
chamber itself (Kempkes et al.
2012)
Fig. 8 Industrial PEF system

from DTI. This system
provides 150–300 kW of
average power, sufficient to
pasteurize 5–20,000 liters per
hour of solution at up to
40 kV/cm (DTI)
transition of PEF processes from the laboratory to the processing plant. There are
several attributes, however, that divide commercial/industrial PEF systems from the
systems typically used in universities and corporate R&D centers. These include:
• Throughput/Capacity
• Automation/Data Logging
• Safety/Certification
890 M.A. Kempkes
Throughput/Capacity
The PEF process itself operates at any scale – as long as the field strength and dose
(total treatment time) applied to the product are the same. This means that treatment
protocols can be developed in laboratory and pilot scale systems, and these protocols
can directly transition to commercial scale PEF operations (Zhang et al. 2007). Any
discussion of PEF system design must be based upon the development of an
effective PEF treatment protocol (i.e., field strength and treatment time) for the
desired application. In designing a PEF system, this protocol must be known. The
PEF treatment protocol can vary significantly across different applications and
products (Mahnič-Kalamiza et al. 2014). Development of the optimal treatment
protocol is a critical step to commercial PEF processing.
Adapting to high flow rate is the key to commercial scale-up of PEF systems. This
typically requires three major parameters: peak voltage (which determines treatment
chamber size), peak current (determined by the field strength/liquid conductivity),
and average power, which is directly proportional to flow rate/capacity.
From that point, the characteristics of the product, and the desired processing
capacity, are the two major remaining considerations to be accounted for in the
system design. These are typically expressed as the liquid conductivity and flow rate.
These two factors, when combined with the desired protocol, form the basis for the
electrical specifications of the industrial PEF system.
Conductivity and Flow Rate

A laboratory PEF system typically processes a few liters per hour, and the flow can
be reduced to stay within the system’s electrical capabilities, so throughput is not a
critical factor. Scaling to commercial volumes, however, requires processing thou-
sands of liters per hour – orders of magnitude higher throughput (and average power)
than a lab system.
The power required to apply a given PEF protocol (field strength and treatment
time) is determined by the conductivity of the fluid and the desired flow rate.
Conductivity is readily measured with inexpensive meters and allows direct calcu-
lation of the current that will pass through a volume of fluid, through application of
Ohm’s law. Power can be calculated as:
P ¼ E2 σV t:
Where P = Average Power (kW); E = Electric Field (kV/cm);σ = conductivity

(mS/cm); V = Volume (liters/second); and t = Treatment time (μs).
Power increases by the square of the required field strength – making field
strength the most critical parameter to establish and maintain, in order to minimize
electrical power requirements in the PEF process. Since electroporation is a thresh-
old effect, small changes in the field strength can result in significant changes in the
electroporation levels achieved, creating a very high need for balance between
effectiveness (at higher than threshold field strengths) and cost (Gaudreau et al.
2004). Power also increases linearly with flow rate or conductivity (for a given
treatment protocol). A 100 kW average power system can, by definition, process five
times the volume of product per hour as a 20 kW system, at the same conductivity
and treatment protocol.
Another way to look at this relationship is that power is energy per unit time, so
the energy delivered per liter, multiplied by the number of liters to be processed in a
given time, gives the total average power required in the PEF system. The energy per
liter also determines the temperature rise in the product, since all of the PEF energy
becomes heat.
Finally, the flow rate impacts several other factors, which in turn affect the size
and cost of the PEF system. These include the maximum allowable pressure in the
pumps and treatment chambers, the minimum chamber dimensions (to allow partic-
ulates or solids), and the pattern of the fluid flow in the treatment chamber (where
turbulent flow is highly desirable). These constraints, in turn, impact the electrical
specifications and design of the PEF system.
Pulse Shape/Frequency
A critical issue in PEF system performance is the electrical pulse shape delivered by
the PEF system. PEF is believed to work on a voltage threshold, and only exposure
time above that critical field strength will achieve the desired electroporation result
(Buckow et al. 2013). In an ideal PEF pulse, there would be zero rise- and fall-time,
and the flattop of the pulse would be at a constant voltage – and applied in a perfectly
consistent treatment chamber. All of the pulse energy, in this case, would be at the
desired field strength, contributing to electroporation.
In practice, however, this ideal pulse shape is not realizable. All high voltage
pulses have finite rise and fall times, where the full voltage is not present (Fig. 9).
Furthermore, all treatment chambers have variations in field strength within them.
Characterizing the actual field applied, and its duration, is therefore subject to
Fig. 9 High voltage pulse from the industrial PEF system at 35 kV, 150 A, 10 μs, and 2 kHz. Trace
1: Trigger; Trace 2: Voltage; Trace 4: Current (DTI)
892 M.A. Kempkes
Fig. 10 Parallel plate conveyor for potato treatment (DIL)
general estimates or, more commonly, ignored all together by simply increasing the
field strength and treatment time until the desired impact is achieved.
Local heating and electrolysis also combine to limit the pulsewidths to a few
microseconds (<10–20 μs at 30 kV/cm, for example) for high field PEF applications
– at longer pulsewidths, arcing will occur. At these short pulsewidths, the disparity
between the ideal and actual pulse shapes is critical, since more of the total
pulsewidth consists of the rise- and fall-times. This translates into wasted power,
which does not contribute to the PEF treatment. Longer pulsewidths (milliseconds,
rather than microseconds) are possible at lower field strengths, which simplifies the
pulse requirements at low field strengths, for plant tissue permeabilization.
For industrial systems, therefore, both PEF effectiveness and efficiency drive us
to try to achieve the closest possible approximation of a perfect square wave pulse
(Fig. 11). At high average power, the wasted costs of higher field strengths and
treatment times than absolutely required can be an appreciable percentage of the total
PEF cost. There is substantial incentive, therefore, to try to optimize both the pulse
shape and the treatment chamber design in industrial systems – even if this requires
higher capital cost at the outset.
Hard switch or Marx modulators are preferable for PEF applications, because
they deliver consistent pulses over a range of conductivities (Toepfl 2015). In a
hybrid modulator, the inductance of its pulse transformer slows the pulse rise and
fall-times and typically presents a 5–10% power loss to the system. More impor-
tantly, because of the transformer impedance, the pulse shape will change when the
conductivity of the product changes, which is typical for virtually every PEF-treated
product as a result of variations resulting from different seasons, sources, or even
Fig. 11 This nearly ideal PEF pulse, at 35 kV, 150 A, 10 μs is the same one shown in Fig. 10. The
rise- and fall-times of the pulse are <2 μs, and the voltage pulse (Channel 2) is nearly perfectly flat.
Depending on how the pulsewidth is measured (pulse command, 70% peak, or 100% peak), the
reported pulsewidth for this one pulse could vary from 10–14 μs. This 40% variation can signif-
icantly skew the reported results from different researchers, even for the same pulse. If the pulse
were not flat, even greater variation in reporting would be likely
weather at harvest. This means that the treatment protocol will vary – sometimes
significantly – as the product attributes change.
The final parameter to be assessed in the design of a PEF system is the required pulse
frequency. The total energy delivered to a volume of fluid is known from the treatment
protocol. As more liquid is processed, the average power (which is energy per unit time)
goes up linearly. In conventional pulsed power systems, such as radar transmitters,
increasing the average power is typically achieved by increasing the pulsewidth,
allowing the pulse frequency to remain at reasonable levels for traditional modulator
technologies (i.e., less than 1 kHz). It is also more efficient to run at longer pulsewidths,
since the rise and fall times of the pulse account for a lower percentage of total pulse
energy. It is not always possible, however, to run at high fields and long pulsewidths
before arcing occurs. Without the ability to increase the pulsewidth, the only remaining
options are to increase the peak power in each pulse (which directly increases the cost of
the PEF system) or to operate with more pulses per second (increased pulse frequency).
Operating at several kilohertz pulse frequency is common in industrial PEF systems.
For industrial PEF systems, operating at high average power and high capacity,
the trade-offs between peak power and pulse frequency also impact the treatment
chamber sizing (and the number of treatment chambers). The specific designs to
balance these trade-offs will vary according to the pulsed power capabilities of the
PEF system manufacturer, whose objective is to optimize equipment cost, perfor-
mance, and reliability.
894 M.A. Kempkes
Automation/Data Logging
The second major distinction between R&D and industrial PEF systems is their
method of operation. To conduct PEF research, the researcher needs control of all the
PEF parameters, in order to vary the treatment over a range of possible conditions,
and observe the resulting impact. The researcher will closely monitor the operation
of the PEF system while the tests are being conducted, and the PEF system is
typically operated by itself, independent of any other systems.
For industrial systems, however, the goal is to minimize both labor costs and the
variation in the PEF treatment applied. This means automating the settings and
operational parameters of the PEF system, as well as coordinating the operation of
the PEF system with both upstream (pumps, conveyors, etc.) and downstream
(slicers, bottling systems) equipment. This requires both internal controls for oper-
ation and fault detection/handling, as well as interfaces to a plant level control
system for coordination with other systems on the processing line. An industrial
computer (known as a Programmable Logic Controller or PLC) is the standard
approach to these control systems. The PLC can monitor and control all of the key
PEF system parameters, allowing (for example) the pulse frequency of the PEF
system to be varied in accordance with the product flow rate in the process line
(Fig. 12). The control system must also be capable of handling any faults in the PEF
Fig. 12 Sample PLC display screen for the lab PEF system (DTI)
equipment or product treatment (arcing, clogging, etc.) in coordination with other

elements of process line. Monitoring the product temperature change, conductivity,
and pulse current provides the ability to validate the consistency of the PEF
treatment and even indicate when replacement of treatment chambers is required.
The one unique feature of the PEF system in a food processing plant is the short,
high voltage pulse itself. This pulse occurs on a time scale that is incompatible with
industrial PLCs, so there is a trade-off between collecting and storing the pulse itself,
through a fast digitizing oscilloscope, or measuring only secondary characteristics
(power supply voltage, average power, product temperature, etc.) within the PLC
system. Measuring and storing each pulse can result in terabytes of data very quickly,
so actual pulse data is typically collected either intermittently (once every several
minutes) or in the event of a fault. In most other respects, the PEF system can be
integrated into the food processing plant like any other piece of equipment – it has
operating parameters that must be set and monitored, as well as diagnostic param-
eters (average power, temperature, etc.) to monitor.
Finally, industrial systems must record (or log) their performance, faults, etc.,
during operation. For food safety processes (i.e., nonthermal pasteurization), this
information is critical and mandatory for HACCP monitoring and regulatory com-
pliance. This data logging can be accommodated within the PEF system itself, or the
PEF system can report its operating and monitored parameters to a plant-level
recording system.
Safety/Certification
The final major difference between industrial and R&D PEF systems relates to safety
and certifications. While all PEF systems must be built to allow safe operation and
prevent injury, the level of safety required in an industrial environment is much
higher than in a laboratory. R&D systems are typically operated by trained person-
nel, highly familiar with the equipment (Fig. 13). Industrial systems may be operated
and maintained by a range of personnel, much less aware of the hazards of the
system.
In general, industrial systems are governed by three levels of standards: Interna-
tional (CE, UL, IEEE, NEMA, etc.), Local/National (OSHA, local building codes,
etc.), and Corporate (which may incorporate these other standards, as well as specific
plant standards). Installing an industrial PEF system in a commercial plant requires
compliance with all three levels. In many cases, there are no specific standards for
high voltage equipment, so the judgment of the certifying authority or inspector is
crucial.
The treatment chambers, where the high voltage pulse interacts with the product,
present the most complex safety issues – combining high voltage, the product, and
potentially mechanical processes (pumps, conveyors, etc.) into one piece of equip-
ment. The treatment chamber design must also accommodate both cleaning/
unclogging when necessary, as well as replacement of the electrodes in a simple
and safe manner.
896 M.A. Kempkes
DANGER
EQUIPMENT DESCRIBED WITHIN THIS DOCUMENT PRESENTS LETHAL VOLTAGES TO ANY
APPARATUS CONNECTED TO IT.
IMPROPER INSTALLATION, OPERATION, AND MAINTENANCE OF THIS EQUIPMENT CAN
RESULT IN DEATH OR SERIOUS INJURY.
ASSUME HIGH VOLTAGE IS PRESENT UNLESS SAFETY GROUNDS ARE ATTACHED.
ALWAYS DE-ENERGIZE THE POWER SUPPLY AND HARD GROUND THE HIGH VOLTAGE
OUTPUT AND RETURN PRIOR TO PERFORMING ANY WORK ON THE SYSTEM OR EQUIPMENT
CONNECTED TO IT.
IN THE OFF STATE, A CURRENT OF UP TO A FEW MILLIAMPERES CAN FLOW CREATING A
SHOCK HAZARD, PARTICULARLY IF OUTPUT IS OPEN CIRCUITED AND EXCESSIVE
CAPACITANCE IS PRESENT AT OUTPUT.
WARNING
ONLY PERSONNEL PROPERLY TRAINED IN WORKING WITH THESE TYPES OF
HAZARDOUS ENERGY SOURCES SHALL OPERATE THIS SYSTEM.
COMPLETE ALL OPERATIONS IN ACCORDANCE WITH APPLICABLE SAFETY
REGULATIONS AND STANDARD HIGH VOLTAGE ENGINEERING PRACTICES.
USE PERSONAL PROTECTIVE EQUIPMENT AND HIGH VOLTAGE GROUNDING STICKS
WHERE APPLICABLE.
CAUTION
BEFORE ATTEMPTING ANY INSTALLATION OR OPERATION OF THIS SYSTEM, READ
AND UNDERSTAND ALL INSTRUCTIONAL MATERIALS THAT RELATE TO THIS
EQUIPMENT.
IF ADDITIONAL INFORMATION IS REQUIRED, CONSULT WITH DIVERSIFIED
TECHNOLOGIES, INC. BEFORE PROCEDING ANY FURTHER.
THE OPERATOR OF THIS EQUIPMENT SHALL ENSURE THAT UNDER ALL
CIRCUMSTANCES ONLY HIGH VOLTAGE QUALIFIED PERSONNEL SHALL INSTALL,
OPERATE, AND MAINTAIN THIS EQUIPMENT USING ALL OF THE APPLICABLE
STANDARD HIGH VOLTAGE SAFETY PRACTICES.
Fig. 13 Warning label for DTI PEF system
In the initial generation of PEF systems, safety features included shielding/

covers/interlocks around the high voltage equipment and treatment chambers
temselves, up to personnel exclusion zones with fences/interlocks at a distance
from the equipment itself during operation.
Given the need for an industrial PEF system, with high power electrical connec-
tions, water/plumbing, mechanical assemblies (pumps and motors), operating in a
food processing environment, safety and regulatory compliance can be a significant
cost in the development and fabrication of the PEF system. Fortunately, industrial
standards are relatively uniform around the world, and once a system is approved, it
typically requires few modifications for other facilities/locations.
Industrial Pulsed Electric Field System Costs
The cost of PEF processing is made up of two major elements: the capital cost for a
level of capacity (in $ /ton/hour) and the cost of the electricity needed to provide the
PEF treatment itself. PEF systems are typically similar or lower in labor utilization
than similar, conventional processes, so labor costs are not included here. They are
also very compact compared to thermal pasteurizers, so facility costs are not a
significant issue.
Capital cost for the PEF system is the largest element in the cost calculation, but
typically not the largest overall cost. PEF system capacity directly affects the capital
cost. Higher capacity/throughput means increasing the peak and average power in
the system, which directly impact the power supply and modulator costs in the PEF
hardware. Larger PEF systems are, not surprisingly, more expensive than
smaller ones.
PEF system costs do not increase linearly with power, however. Larger systems
are generally more economical than smaller systems on a volume adjusted basis. In
addition, the operating range of an industrial PEF system is typically very narrow
compared to an R&D system, since the treatment protocol for a given process has
already been developed and optimized. A (very) general rule for assessing the size
and cost of commercial PEF systems is that the cost increases by the square root of
the capacity increase, so an order of magnitude increase in capacity system will only
cost 3–3.5X more than the smaller system (All of the cost estimates provided in this
section are very general starting points. While they are consistent with PEF systems
sold by DTI and other manufacturers recently (2016), they do not reflect specific
systems. The actual cost of a PEF system will depend on a number of factors beyond
its average power.).
Beyond a certain power level, however, the required size of the PEF equipment
(in terms of voltage, peak power, and average power) exceeds commercial pulse
power system capabilities. This crossover point can be as low as 50 kW or as high as
500 kW–1 MW of average power, depending on the manufacturer. Above this
crossover point, the only way to increase capacity is by operating multiple systems
in parallel. Improvements in system efficiency (even at the expense of higher initial
system costs) will also provide the lowest overall processing cost, and the closer the
pulse shape of the PEF system is to the ideal pulse (zero rise and fall-time, perfect
flattop), the higher the system efficiency.
The capital cost of a large industrial PEF system is generally smaller than its
electricity bill over its lifetime. For example, a 200 kW PEF system might
cost ~ $300–$500 k (excluding ancillary equipment). Over 5 years of depreciation,
this capital cost is $7–$10 per hour of operation. Its electricity cost would be
898 M.A. Kempkes
approximately twice this cost (per hour), depending on local electricity rates (Elec-
tricity costs are assumed to be $0.08–0.12 USD/kWh.). This total cost of $20–30 per
operating hour, in turn, is divided over the amount of product processed in that time,
which could range from 1 to 100 tons, depending on the PEF application. The
resulting cost is typically fractions of a cent per liter or kg of processed product. A
solid-state PEF system should have a lifetime of 10–20 years, so the cost will
decrease significantly after the depreciation period.
With all of these costs taken into account, PEF for nonthermal pasteurization is
typically around twice the cost per liter of thermal pasteurization, but only a fraction
(10–20%) of the cost of high pressure processing (HPP), which is the common
alternative for nonthermal pasteurization (Sampedro et al. 2013; Toepfl 2013). For
plant tissue modification applications, the cost of PEF and its conventional alterna-
tives span a much wider, but overall lower, range. PEF costs are generally a fraction
of the cost of the heat treatment commonly used in applications such as peeling or
extraction, with reported cost reductions of 30–90% for PEF. Given the extremely
wide range of possible applications and conventional processes, these applications
must be assessed on a case-by-case basis.
Pulsed Electric Field Processing and Commercialization Status
Modern PEF processing has been the subject of focused R&D for over 20 years.
Initially, the emphasis was on validating and defining the ability of high voltage
pulses to kill bacteria, molds, and other potential pathogens, as a nonthermal form of
pasteurization. In the early 2000’s, consortia in both the USA and Europe focused on
scaling the results discovered in the laboratory to commercially viable processes and
systems (Buckow et al. 2013). Since that time, several key developments have
brought PEF processing to the brink of wide-scale commercial adoption. Genesis
Juice Corporation introduced the first FDA approved, PEF processed juices to the
US market in August, 2005 (Fig. 2). Market acceptance of their PEF processed juice
in the USA, although small, was strong and growing. At approximately the same
time, DTI introduced a 25 kW pilot-scale PEF system, capable of treating 100–500
liters/hour of juices or other products, allowing researchers to process a variety of
products for both R&D and preproduction process definition (Gaudreau et al. 2006).
As a result, Genesis and DTI were awarded the Institute of Food Technologists’
Food Technology Industrial Achievement Award in 2007 (Zhang 2008).
The first known commercial deployment of an industrial scale PEF system
occurred in 2006 in Mesa, Arizona.This system, rated at 150 kW and 10,000 L/h,
was deployed for wastewater treatment, rather than food processing. In over 6 years
of operation, this system demonstrated the ability to meet stringent operational
requirements for efficiency, reliability, electrode life, and unattended operation
(Kempkes et al. 2012). Since that time, DTI and others have developed and sold a
number of lower cost industrial PEF systems. The system shown in Fig. 2, for
Fig. 14 PEF processed juice

on sale in Europe. PEF
processing is applied for
extended shelf-life, not
nonthermal pasteurization,
and the juice is labeled as
“fresh”
example, has the same basic performance as the system shown in Fig. 8, but the
system cost dropped by more than 50% between 2000 to 2012.
In parallel, a number of lab and pilot scale systems have been deployed to
research facilities around the world, including the USA, Europe, and Australia.
These systems have supported PEF process development and research on basic
PEF effectiveness for a range of applications and products.
In the last decade, several companies have entered the PEF systems market, either
from the pulsed power industry or from the food industry. Multiple companies are
now selling industrial-scale PEF systems, focused on a range of applications
(Kempkes 2015). Approximately 100 PEF systems were operating around the
world in 2015, with over half of them in industrial applications. Most of these
commercial systems are being used for tissue modification (tissue softening, extrac-
tion, etc.), in addition to a limited number of liquid food products introduced in
Europe and Asia (Fig. 14, Buckow et al. 2013).
Conclusions
The application of PEF to industrial food processes builds directly on two pillars: the
development of reliable, solid state high voltage pulsed power systems for the
military and high energy physics and the extensive research base in PEF for
nonthermal pasteurization. Both pillars arose independently in the 1990’s, and
their merger has led directly to the emergence of industrial PEF systems in the last
decade. Today, new applications of PEF processing are emerging at a rapid pace,
extending the application of PEF into a range of other areas dependent on electro-
poration of cells, including drying, extraction, and preprocessing of both foods and
900 M.A. Kempkes
other streams (e.g., algae and wastewater). Commercial markets for PEF systems are
growing, and this is expected to accelerate as the benefits of PEF processing become
more widely known.
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bioprocessing – applications, process and equipment design and cost analysis, PhD thesis,
Technical University of Berlin
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fields. Presented at the institute of food technologists annual meeting, Chicago, IL
Compact Nanosecond Pulse Generator
Based on Printed Circuit Board 50
Transmission Lines and Solid-State
Switches
Yan Mi, Jialun Wan, and Yanyuan Zhang
Abstract
In order to explore the biomedical application of nanosecond pulsed electric fields
(nsPEFs), researchers have used numerous methods to develop nanosecond pulse
generators. Transmission line-based generators are widely used for their good
reliability and simplicity. With traditional method, strip lines and spark gap
switches are almost homemade. At the same time, manufacture techniques of
PCB (printed circuit board) strip line and solid-state switch with universal
manufacture standards will benefit the generators to be compact, portable, and
reliable. This chapter provides guides to develop a compact nanosecond pulse
generator for biomedical application based on PCB transmission lines and solid-
state switches. Firstly, the principle of Blumlein strip line pulse generator includ-
ing traditional balanced, unbalanced, and stacked Blumlein strip line is presented.
Unbalanced Blumlein strip line could be used to generate output voltage with
variable duration and variable amplitude. Stacked Blumlein strip line could be
regarded as a voltage multiplier or a current amplifier. Then, as a main part, this
chapter proposes the detailed design key points of this kind of compact generator
including PCB strip line, PCB microstrip line, stacked PCB transmission lines,
and stacked solid-state switches. Characteristic parameters, attenuation and
design procedure with detailed equations of different strip lines, and PCB design
techniques with parameters of different dielectric materials are also provided.
Y. Mi (*) • J. Wan
State Key Laboratory of Power Transmission Equipment and System Security and New
Technology, College of Electrical Engineering, Chongqing University, Chongqing, China
e-mail: miyan@cqu.edu.cn; wanjialun@cqu.edu.cn
Y. Zhang
The State Grid Chongqing, Changshou Power Supply Company, Chongqing, China
e-mail: 931286429@qq.com

DOI 10.1007/978-3-319-32886-7_212
904 Y. Mi et al.
Keywords
Nanosecond pulse generator • Nanosecond pulsed electric fields (nsPEFs) • PCB
(printed circuit board) • Blumlein transmission line • Solid-state switch
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 904
Theory of Blumlein Strip Line Pulse Generator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 906
Traditional Blumlein Strip Line Pulse Generator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 906
Unbalanced Blumlein Strip Line Pulse Generator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 907
Stacked Blumlein Strip Line Pulse Generator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 910
Design of The Nanosecond Pulse Generator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 912
Strip Line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 912
Microstrip Line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 916
Design of a Balanced and Unbalanced Transmission Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 919
Combination Design of PCB Transmission Line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 922
Design of Solid-State Switch in Planar Transmission Line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 922
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 924
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 924
Introduction
Nanosecond pulsed electric fields (nsPEFs) have been shown to be effective to

penetrate into biological cells and permeabilize both organelle membranes and
plasma membrane (Schoenbach et al. 2002; Schoenbach and Joshi 2004; Vernier
et al. 2006; Pakhomov et al. 2007; Batista Napotnik et al. 2016). Programmed cell
death (PCD, i.e., apoptosis) in cultured cells and tumor-growth inhibition in mice
models were observed (Beebe et al. 2002; Chen et al. 2010; Nuccitelli et al. 2006,
2009, 2013). Recently, Nuccitelli et al. (2014) reported the first successful human
trial for basal cell carcinoma (BCC) tumors subjected to 100–1000 electric pulses
with 100 ns in duration, 30 kV/cm in amplitude, and 2 Hz in repetition rate. These
studies indicate that this highly localized and drug-free physical technique offers a
promising new therapy for tumor treatment.
To explore the biomedical mechanism and clinical application of tumor treatment
with nsPEFs, researchers have used numerous methods to develop nanosecond pulse
generators. Nanosecond pulse generators based on the transmission line principle are
widely used in biomedical applications because of their good reliability and sim-
plicity. There are different types of transmission line such as coaxial line, parallel
cylindrical line, strip line (or parallel plate line), radial line (or disk line), helical line,
and taper line (Pai and Zhang 1995). Coaxial transmission line is mostly used to
generate nanosecond square wave pulse for biomedical applications. Behrend et al.
(2003) developed a single-ended transmission line pulse generator based on four
50 Ω coaxial cables in parallel and spark gap switch. Kolb et al. (2006) designed a
Blumlein transmission line pulse generator with two 2 m long coaxial cables and a
solid-state switch for real-time observation of individual cells exposed to nsPEFs.
50 Compact Nanosecond Pulse Generator Based on Printed Circuit Board. . . 905
However, coaxial transmission line has the disadvantage of occupying a large space.
For example, to match a 10 Ω biological cell cuvette in medical experiments, two
sets of coaxial parallel groups are needed to constitute a Blumlein transmission line
system to generate a pulse width of 100 ns square pulse. As a result, a total of 200 m
length of 50 Ω coaxial is required. Apparently, the volume of the nanosecond pulse
generator will be extremely large.
Therefore, many researchers introduced the planar transmission line as an alter-
native to achieve the characteristics of compactness and portability. The first gener-
ation of the planar transmission line is the strip line. The second generation of the
planar transmission line is the microstrip transmission line, which was developed in
the 1950s. Deng et al. (2000) developed a nanosecond pulse generator to investigate
intracellular electromanipulation (IEM) of nsPEFs. A Teflon-based Blumlein strip
line, with a volume of 2 800 mm (length) 25 mm (width), and a pressurized
spark gap as switch were designed to produce an 8 ns pulse with voltages between
2 and 30 kV across a 10 Ω cuvette load. Katsuki et al. (2002) presented a nanosecond
pulse generator based on a 30 Ω, 480 mm long, Teflon-based Blumlein strip line with
a low inductance spark gap switch to deliver square wave voltage pulses of 45 ns
duration to bacterial treatment chamber. Behrend et al. (2003) proposed a compact
nanosecond pulse generator with water-based Blumlein strip line and an overvolted
air spark gap switch to deliver 3–5 ns long pulses to 10 Ω cuvette load. Kolb et al.
(2006) designed a nanosecond pulse generator based on a 10 Ω, 2000 mm long,
U-shape, Teflon-based Blumlein strip line with a spark gap switch which generates a
negative square wave voltage of 40 kV and 10 ns. Ma (2007) developed a high-
voltage nanosecond pulse generator based on the Teflon-based spiral-shaped strip
line and discussed the double voltage principle of the spiral-shaped strip line system.
The output pulse amplitude of the device reached 2n U0 (U0 is the DC charging
power supply voltage, and n is the layers of the spiral strip line). Li et al. (2011)
designed a nanosecond pulse generator with a DMD (Dacron/Mylar/Dacron)-based
spiral-shaped strip lines, with volume of 290 220 mm, and spark gap switch. A
quasi-square wave of 12 kV and a pulse width of 200 ns were obtained at a load of
1.1 Ω. Thus, a reasonable choice for the rolled strip line can be made depending on
the application such that the output characteristics can be improved.
The pulse generator will be compact, portable, and reliable, which seems more and
more important for biomedical applications, if the PCB (printed circuit board) trans-
mission line and solid-state switches are introduced into the pulsed power technology.
Strip lines and spark gap switches mentioned above are almost homemade by
different researchers themselves without any universal manufacture standard. The
performances of these devices with the same parameters by different researchers may
change a lot according to different manufacture techniques. At the same time, the
manufacture techniques of PCB and solid-state switch have been developed for a long
time with universal manufacture standards, which satisfies the demand of planar strip
line and discharge switch with better reliability and reproducibility. Furthermore, PCB
strip line and solid-state switch may occupy a smaller volume than traditional devices,
which will benefit the generator to be compact and portable.
906 Y. Mi et al.
Romeo et al. (2013) designed a nanosecond pulse generator based on the PCB
Blumlein microstrip transmission line and solid-state switches. The idea of inter-
changeable transmission line with three FR-4 (FR-4 epoxy glass cloth laminate,
er = 4.4)-based PCB microstrip transmission lines was realized to match a 10 Ω,
50 Ω, and an 133 Ω load with maximum achievable pulse length of 40 ns, 40 ns, and
100 ns, respectively. Two identical solid-state switches, which can be independently
triggered with a certain delay time among each other, were used to generate pulses
with variable duration and polarity. Mi et al. (2016) proposed a nanosecond pulse
generator based on an unbalanced Blumlein multilayered microstrip transmission
line and solid-state switches. A multilayered microstrip transmission line is stacked
onto the four-layered FR4-based PCB to minimize the system volume to 400 mm
(length) 400 mm (width) 2.6 mm (thickness). A double-switch synergy with
stacked solid-state switches was applied to achieve variable pulse width and to
match different load impedance.
This chapter aims to provide guides to develop a compact nanosecond pulse
generator for biomedical application based on PCB transmission lines and solid-state
switches. Firstly, the principle of Blumlein strip line pulse generator including wave
propagation analysis is presented. Then, as a main part, this chapter provides the
detailed design key points of this kind of compact generator including PCB strip line,
PCB microstrip line, stacked PCB transmission, lines and solid-state switches.
Theory of Blumlein Strip Line Pulse Generator
In order to explore the biomedical effects of nsPEFs, square wave waveform is

commonly used for its simpler and better understanding of frequency characteristics
than other waveforms such as exponential decay waveform. For this purpose, trans-
mission lines are widely used. In general, there are two main types of transmission
line, i.e., single transmission line and Blumlein transmission line (Martin et al. 1996).
The main disadvantage of single transmission line is that the voltage amplitude on the
matched load is just half of the charging voltage. In addition, the output switch is located
before the load, and therefore, if dynamically charged, a prepulse could be coupled to
the load (Bluhm 2006). These shortcomings are avoided by the Blumlein transmission
line. As a result, almost all transmission line pulse generators developed for biomedical
effects of nsPEFs are based on Blumlein transmission line. As a consequence, this
section only analyzed the theory of Blumlein strip line pulse generator. However, three
types Blumlein strip lines including traditional balanced Blumlein strip line, unbalanced
Blumlein strip line, and stacked Blumlein strip line are taken into account.
Traditional Blumlein Strip Line Pulse Generator
The basic structure of a Blumlein transmission line may be conceptually considered

as two simple transmission lines connected in a way that they are charged in parallel
Fig. 1 Diagram of traditional R1 R2

Blumlein strip line pulse T-1 T-2
generator
DC S
Fig. 2 Voltage waveform on U

the load of traditional
Blumlein strip line pulse U0
generator
0 t 3t t
configuration and discharged in series. Figure 1 shows the diagram of traditional

Blumlein strip line pulse generator. Traditionally, the characteristic impedances, Z1
and Z2, of two simple transmission lines, T-1 and T-2, are the same and equal to Z0,
and the matched load R2 = 2Z0. At the beginning, the distributed capacitance of T-1
and T-2 is fully charged by the DC power supply, and then S is turned on. Because
the conductor of T-1 is grounded, resulting in an incident wave (U=U0) propagat-
ing from left to right of T-1. After elapsed time τ, where τ is time required for an
electromagnetic wave to propagate through the entire T-1, the negative voltage wave
reaches the matched load, and the reflected and transmitted waves appear. Given the
equivalence of Z1, Z2, and load 1/2R2, the reflected and transmitted waves are equal
to 1/2U0. Accordingly, a voltage wave with the amplitude of U0 is formed on the
load. Eventually, after several reflection and transmission of the waves, a pulse
waveform is formed on the load at time τ3τ, as shown in Fig. 2.
Unbalanced Blumlein Strip Line Pulse Generator
In some other cases, the characteristic impedances, Z1 and Z2, of two simple
transmission lines, T-1 and T-2, are not the same. As shown in Fig. 3, an additional
switch S2 is used to generate voltage with variable duration and variable amplitude.
Unlike the traditional transmission line system analyzed above, the impedance of
the unbalanced Blumlein strip line system used in this section is Z1>Z2. When S is
opened, an incident voltage wave (U1=U0) travels from the left to the right end of
T-1 and is accompanied by the incident current flowing through S1. The incident
current is given by:
i1 ¼ U 0 =Z 1 , (1)
908 Y. Mi et al.
Fig. 3 Diagram of Z1=30 W Z2=20 W

unbalanced Blumlein strip R1 R2 = 50 W
line pulse generator T-1 T-2
S1
DC
S2
t0 + t
t0
where U0 is the charging voltage of DC power supply and Z1 is the characteristic

impedance of T-1.
With certain values of U0, the larger the Z1, the smaller the i1, as shown
through Eq. 1. A smaller current flowing through S helps to reduce the number of
solid-state switches needed to be connected in parallel to maintain the flow of
current. A value of Z1 that was as large as possible was needed, which will
inevitably lead to a smaller Z2 under a certain R2. However, because of the
precision error during manufacturing the multilayered microstrip transmission
line, it is hard to ensure that the impedance of Z2 is small and is only several
ohms. Taking into account economic factors and feasibility, we set Z1 = 30 Ω
and Z2 = 20 Ω, respectively.
Wave Propagation Analysis

Here, the wave propagation was analyzed with the assumption that R2 = Z1 + Z2 =
50Ω (i.e., Z1 = 30 Ω and Z2 = 20 Ω) as shown in Fig. 4. The analysis is as follows:
(a) Using MSL to denote microstrip line, the distributed capacitance of MSL-1 and
MSL-2 is fully charged by the DC power supply at t = 0, and then S1 is
turned on.
(b) When S1 is turned on at t = 0, the conductor of MSL-1 is grounded, resulting in
an incident wave (U1 = U0) propagating from left to right of MSL-1 during
0<t<τ, where τ is time required for an electromagnetic wave to propagate
through the entire MSL-1.
(c) During τ<t < 2τ, refraction and reflection of the incident wave, U1, occurs at t
=τ, when 2τ is the time when U1 reaches the right side of MSL-1, as shown in
Fig. 4c. At the same time, a rectangular pulse begins to emerge on R2.
(d) When 2τ<t<3τ, there is voltage wave propagation as shown in Fig. 4d, and the
rectangular pulse on R2 remains.
(e) When 3τ<t<4τ, voltage wave propagation occurs as shown in Fig. 4e. The
absolute value of the potential difference at R2 at both ends drops dramatically to
0.2U0, leading to a tail pulse after the main pulse. The pulse amplitude formed on
R2 is small because the refraction and reflection amplitudes decline to 0.08U0
after 4τ, and thus there is no wave propagation. The overall voltage waveform is
shown in the black line in Fig. 5.
Fig. 4 Analysis of wave

propagation of unbalanced
Blumlein strip line Pulse
Generator (Mi et al. 2016)
Fig. 5 Voltage waveform on

R2 (R2= Z1+ Z2) of
unbalanced Blumlein strip UR / U0
line pulse generator 1
Dt
0 1 3 5 7 9 t/t
“Clipping” Strategy
Figure 5 shows that the overall voltage waveform is a stepped wave. In other words,
tail pulses exist after the main pulse. These pulses should be eliminated. For this
purpose, another switch, S2, is turned on after the main pulse is introduced to short
R2. In this manner, the voltage waveform of R2 becomes a square wave without tail
pulses. Also, when S2 is turned on at different times, 2τ–3τ, a number of pulses with
different widths will be obtained. If Δt=τ (when S2 is closed at 2τ), the output of the
910 Y. Mi et al.
Fig. 6 Voltage waveform on

R2 (R2Z1+ Z2) of
U R / U0
unbalanced Blumlein strip
line pulse generator 1+k
Dt
0 1 3 5 7 9 t/t
Fig. 7 Timing sequence of

the switches and the S1
waveform of R2
S2
VR2
time
0 t t + Dt
system is a rectangular pulse with width τ, where τ is 50 ns. It is obvious that when
the red dotted line coincides with 3τ (when S2 is closed at 3τ), a rectangular pulse
with the maximum width of 2τ (i.e., 100 ns) will be obtained.
Also, a series of pulse width (i.e., a lot of pulses with different widths) is obtained
because of the introduction of S2 close to the tail pulses. At the same time, the
system is no longer sensitive to the impedance of R2 because of the short circuit
clipping strategy. Under the condition R2Z1+ Z2, the peak voltage of R2 will be
almost two times that of the charging voltage U0 in the main pulse. The overall
voltage waveform is shown in Fig. 6 (where k = R2 Z1 Z2/R2 + Z1 + Z2). The
value of Δt determines the pulse width of the output pulse. The longer Δt is, the
wider the width of the rectangular output pulse will be.
Timing Sequence of S1 and S2

The control sequence of solid-state switches S1 and S2 is shown in Fig. 7. When S1
is turned on at t = 0 and S2 is turned on at t = τ + Δt, the pulse width on R2 is
determined by Δt(2τ < Δt < 3τ). To obtain a certain pulse width t1, Δt=t1. Finally,
the waveform of R2 is given by VR2.
Stacked Blumlein Strip Line Pulse Generator
When several Blumlein strip lines are connected together, the system is called a
stacked Blumlein strip line pulse generator. Traditional Blumlein strip line discussed
SPL-S11P1 SPL-S21P1 SPL-S51P1
SPL-S11P5 SPL-S21P5 SPL-S51P5

R2
R1 SPL-S12P1 SPL-S52P1
U0 SPL-S12P5 SPL-S52P5
Fig. 8 Diagram of five-stage stacked Blumlein strip line pulse generator
in above sections can be stacked to yield either high voltage or current depending on
the way they are structured. In other words, this stacked Blumlein strip line could be
regarded as a voltage multiplier or a current amplifier.
The schematic block diagram of a high-voltage nanosecond pulse generator based
on stacked series–parallel PCB transmission line is shown in Fig. 8. The generator
consists of a DC power supply, the current limiting resistor R1, the solid-state switch
series–parallel group MOS (metal–oxide–semiconductor), and the Blumlein trans-
mission line. In the picture, a S*&-stage series, P*-stage parallel Blumlein transmis-
sion line is displayed, where the upper case S represents series connection, P stands
for parallel connection, * represents the number of stage, and subscript & stands for
the number of strip line in each stage of Blumlein transmission line. In order to
simplify the description, a five-stage series, five-stage parallel Blumlein transmission
line is described. The five-stage series Blumlein transmission line is composed of
strip lines numbered from SPL-S11P1 to SPL-S51P1 and from SPL-S12P1 to
SPL-S52P1. Each strip line is connected to the same parallel four-stage transmission
line. Then, the five-stage series, five-stage parallel Blumlein transmission line can be
obtained. In Fig. 8, the characteristic impedance of each single transmission line is
Z0, and the propagation delay of the electromagnetic wave is τ, where τ is time
required for an electromagnetic wave to propagate a single transmission line. The
series–parallel line can be equivalent to a Blumlein transmission line, which has
1/5Z0 impedance and 5τ transmission delay. Thus, the matched load R2 is 2/5Z0. The
final output waveform is shown in Fig. 9. Different waveforms can be obtained by
changing the number of series and parallel connections.
As shown in Fig. 8, if the load is connected between the surface layer of two
transmission lines and the two high-voltage wires are connected directly, then the
waveform polarity on the load will be changed, as compared with Fig. 1.
912 Y. Mi et al.
Fig. 9 Voltage waveform on U

the load of five-stage stacked
Blumlein strip line pulse U0
generator
0 5t 15t t
Design of The Nanosecond Pulse Generator
The planar transmission line is a kind of microwave transmission line developed in

1960s. It has the advantages of wide frequency band, low cost, and high reliability,
but the disadvantage is the large loss, low-power capacity. The plane transmission
line has many kinds of structure, such as strip line, microstrip line, coplanar
waveguide, and dielectric waveguide, and the transmission characteristics can be
controlled by adjusting the two-dimensional plane dimension (Collin 2000). This
section will introduce features and design methods of the strip line and the microstrip
line and the combination design of the transmission line based on PCB.
Strip Line
Strip line is a kind of three conductor TEM (transverse electric and magnetic field)
transmission lines; its structure is shown in Fig. 10. The outermost two conductor
plates are connected to the ground, and the conductor strip is located in the symmetry
center of the ground floor. Usually, the dielectric is filled between these conductors,
its relative permeability μr = 1, and relative permittivity is er. The distance between
the upper and lower conductor plates is b, conductor strip width and thickness are
W and t.
The strip line transmission line can be seen as an evolution from the coaxial line,
as shown in Fig. 11. The structure of the strip line makes the electromagnetic wave
propagate in the medium, so its loss is relatively low.
Characteristic Parameters of Strip Line

The main mode of strip line is TEM. The phase velocity is:
1
V p ¼ pffiffiffiffiffiffiffiffiffiffi (2)
L0 C0
where C0, L0 is the distributed inductance and distributed capacitance of the strip
line in per unit length, respectively. So the characteristic impedance of the strip line is:
rffiffiffiffiffiffi pffiffiffiffi
1 L0 er
Zc ¼ ¼ ¼ (3)
V p C0 C0 cC0
Dielectric
Substrate
b t Ground Plate
w
Conductor Strip
Fig. 10 Structure of strip line
Fig. 11 Evolution and a

structure of the strip line. (a)
The evolution of the coaxial
line to the strip line. (b) Field
structure of strip line
b
E
R
The accurate calculation of the characteristic impedance of the strip line is much
more complicated than that of the coaxial line. A simple method, considering the
influence of edge effect, is equivalent to widen the conductor strip width. The effect
is equivalent to adding an arc at both ends of the conductor, with the radius of
R = 0.2205b, so the conductor strip width is We+2R (We is equivalent width). So the
unit length capacitance between the conductor strip and the ground plate is
eðW e þ 2RÞ 2eðW e þ 0:441bÞ

C0 0 ¼ ¼ (4)
b=2 b
So the capacitance of the strip line in per unit length is:
4eðW e þ 0:441bÞ
C0 ¼ 2C0 0 ¼ (5)
b
914 Y. Mi et al.
And the characteristic impedance of the strip line is:
30π b
Z 0 ¼ pffiffiffiffi , (6a)
er W e þ 0:441b
8
> W
We W< 0 ,
b
> 0:35
¼
W 2 : (6b)
b b>
: 0:35 b ,W
< 0:35
b
According to the Eq. 6, the characteristic impedance of the strip line decreases with
the increase of the conductor strip width W. At the same time, the equation
was established with the assumption that the conductor strip thickness is 0, of which
the accuracy is about 1%. And the expression, derived by Wheeler (1965) is as follows:
2 sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 39
=
30 4 1 48 1 8 1 2
Z0 ¼ pffiffiffiffi ln 1 þ þ þ 6:275 (7)
er π m π m m m ;
and
w Δw
m¼ þ ,
bt b t h x 2 0:0796x n

Δw x
¼ 1 0:5ln þ ,
b t π ð1 xÞ 2x w=b þ 1:1x
2 t
n¼ ,x ¼ ,
2 x b
1þ
3 1x
where t is the thickness of the conductor strip. When w/(b–t) <10, the accuracy is
lower than 0.5%.
Power of Strip Line

The power of the strip line is mainly affected by two factors: the breakdown field and
the maximum temperature rise of the dielectric. It is easy to break down at the edges
and corners of the conductor strip, so it is difficult for the strip line to transmit high
power. The power capacity can be improved when the edge is changed to a smooth
circle.
Attenuation Coefficient
The attenuation coefficient α of the strip line includes three parts: the conductor
attenuation coefficient αc, the attenuation coefficient of the dielectric αd, and the
radiation attenuation coefficient αr. However, the conductor strip width is
much smaller than the width of the ground plate, and the distance b between the
ground plates is much smaller than the transmission wavelength, so the αr can be
neglected. The approximate results of the conductor attenuation coefficient αc (unit:

Np/m) is:
8
>
> 2:7 103 Rs er Z0 pffiffiffiffi
< A, er Z 0 > 120Ω
αc ¼ 30π ðb tÞ , (8)
> 0:16Rs B,
> pffiffiffiffi
: er Z0 > 120Ω
Z0 b
and

2W 1bþt 2b t
A¼1þ þ ln ,
b t π b t t
b 0:414t 1 4πW
B¼1þ 0:5 þ þ ln :
0:5W þ 0:7t W 2π t
The attenuation coefficient of the dielectric is (unit dB/m):

pffiffiffiffi
27:3 er
αd ¼ tan δ (9)
λ0
where λ0 is the wavelength in free space and tanδ is tangent value of the dielectric
loss angle.
Design of Strip Line

However, the known conditions are only the strip line characteristic impedance Zc,
the relative permittivity er of the dielectric material, and the conductor thickness t.
Thus, the following formulas are needed to obtain the value of w/b:
w we Δw
¼ , (10)
b b b
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
we 8ð1 t=bÞ eA þ 0:568
¼ , (11)
b π eA 1
( " 2 m

Δw t=b t=b 0:0796t=b
¼ 1 0:5ln þ , (12)
b π 2 t=b we =b 0:26t=b
pffiffiffiffi
2 Z 0 er
m¼ ,A ¼ : (13)
2t=b 30
1þ
31 t=b
The strip transmission line phase velocity can be expressed as follows:
c
vp ¼ pffiffiffiffiffiffiffi : (14)
eeff
916 Y. Mi et al.
where eeff is the effective permittivity.

The length of the transmission line can be calculated as follows:
τ c τ
L ¼ vp ¼ pffiffiffiffiffiffiffi , (15)
2 eeff 2
where c is the speed of light and τ/2 is the time for electromagnetic propagation in
a single transmission line.
In the design process, if the size is improper, or the precision of the process is low,
it will produce higher mode TE (transverse electric) and TM (transverse magnetic).
The size of the strip line is chosen to follow the following rules:
8 pffiffiffiffi
>
> λmin > 2W er
< pffiffiffiffi
λmin > 2b er
(16)
>
> W ¼ ð3 6ÞW
: g
b λ0 =2
Microstrip Line
Microstrip line, a kind of nonsymmetrical planar transmission system, is the most

widely used transmission line, and its geometric structure is shown in Fig. 12. W is
the width of the conductor strip, t is the thickness, er and h are the relative
permittivity and the thickness of the dielectric substrate, and the ground plate
width is D.
The microstrip line can be seen as a double transmission line, as shown in Fig. 13.
A thin conducting plate is inserted between the two conductors, which does not affect
the original field distribution, and then the conductor under the plate is removed. The
left transmission line becomes a strip, that is, a microstrip line is formed.
Because the conductor strip is in the mixing medium between the air and the
dielectric substrate, component of the field should not only meet the metal boundary
Fig. 12 Structure of
microstrip line
Conductor Strip
Dielectric
Substrate
t
h w
Ground Plate
D
Fig. 13 Evolution of a
double conductor
conditions but also satisfy the boundary conditions on the dielectric surface. Thus
the electromagnetic field may have a longitudinal component of Ez and Hz, so it may
also transmit TE and TM modes. However, it can be proved that when the thickness
h is much smaller than the wavelength, the longitudinal component of the field is
very small, which can be approximately regarded as the TEM mode, so the main
mode in the microstrip line is called “quasi-TEM.”
Characteristic Parameters of Microstrip Line

The medium of the microstrip line includes an air and a dielectric substrate;
therefore, the relative permittivity cannot be considered only in the air or the
substrate. The equivalent relative permittivity is expressed by ere, which is defined
as the ratio of the real capacitance in per unit length C0 and equivalent capacitance
per unit length in air medium C1:
C1
ere ¼ (17)
C0
Therefore, the actual microstrip line can be considered as a conductor strip filled
with a kind of single and uniform dielectric. The phase velocity is:
c
V p ¼ pffiffiffiffiffi (18)
ere
Wavelength of the waveguide λg is:
λ0
λg ¼ pffiffiffiffiffi (19)
ere
where the λ0 is the wavelength in free space.

The characteristic impedance of the microstrip line with zero thickness is derived
by Wheeler. H. A (the accuracy is better than 1%):
8
> 60 8h w 1 w
> ln þ , < 1
< pffiffiffiffiffiffi
>
eeff
ffi
w 4h 2 h
Zc ¼ 120π w , (20)
>
> h i , 1 < < 20
> p ffiffiffiffiffiffi
ffi w w
: eeff þ 1:393 þ 0:667ln þ 1:444 h
h h
918 Y. Mi et al.
and
er þ 1 er 1 1
eeff ¼ þ rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ,
2 2 h
1 þ 12
w
where W is conductor width; h is thickness of the dielectric substrate; and er, less than
16, is relative permittivity of the dielectric substrate. When the thickness t ¼
6 0, W in the
formula can be replaced by the equivalent width of conductor We = W + ΔW:
8
> t 2h 1
>
> 1 þ ln , W
>
< π t
h
2π
ΔW ¼ (21)
>
> t 4πW 1
>
: 1 þ ln , W
<
h
π t 2π
Attenuation Coefficient
The attenuation coefficient α of the microstrip line includes three parts: the conduc-
tor attenuation coefficient αc, the attenuation coefficient of the dielectric αd, and the
radiation attenuation coefficient αr. When the size selection of the microstrip line is
appropriate and the frequency is not high, the radiation loss is very small, generally
negligible. So the attenuation coefficient of the microstrip line can be written as:
α ¼ αc þ αd (22)
Because the current distribution in the cross section of the conductor and the
ground plate is not uniform, the calculation of the αc is very complicated. Its final
expression can refer to the relevant books.
The expression of the dielectric loss coefficient of microstrip transmission line is
as follows:
pffiffiffiffiffi
π ere qer tan δ
αd ¼ (23)
λ0 ere λg
Design of Microstrip Line

In general, to obtain the specific impedance of the microstrip transmission line Zc,
physical dimension w/h of the microstrip transmission line is needed, which is
calculated as:
8
>
> 8eA w
w < 2A
, 2
¼ 2
e 2 h
(24)
h >> er 1 w
: B 1 lnð2B 1Þ þ C ,2 <
π 2er h
and
rffiffiffiffiffiffiffiffiffiffiffiffi
Z0 er 1 er 1 0:11
A¼ þ 0:23 þ
60 2 er þ 1 er
377π
B¼ pffiffiffiffi
2Z0 er
0:61
C ¼ lnðB 1Þ þ 0:39
er
In addition, the phase velocity of the microstrip transmission line is:
c
vp ¼ pffiffiffiffiffiffiffi (25)
eeff
So the length of the microstrip transmission line is:
τ τ c
L ¼ vp ¼ pffiffiffiffiffiffiffi (26)
2 2 eeff
where c is the speed of light and τ/2 is the time for electromagnetic propagation in
a single transmission line.
Design of a Balanced and Unbalanced Transmission Lines
Using a single strip or microstrip line as high-voltage pulse forming network, the
voltage amplitude on the load is only half of the charging voltage. When the
Blumlein transmission line is adopted, the voltage amplitude of the load is equal
to the charging voltage, thereby reducing the requirement of the switch voltage
tolerance. This section will introduce the design of two kinds of planar transmission
lines on printed circuit board (PCB).
Design of Balanced Transmission Line

The balanced transmission line means that the impedance of the two sections in the
Blumlein transmission line is equal to each other. Compared with the coaxial
transmission line, the length of the strip or microstrip line has been greatly reduced.
However, when the pulse width of the output waveform is several hundred nano-
seconds, the length of the strip or microstrip line should be a few meters. In order to
use the advantages of planar transmission line, the winding conductor strip can be
arranged on the PCB, which can greatly reduce space occupation. Furthermore, if the
length of the conductor is up to tens of meters, the conductor can be arranged in a
multilayer PCB. The cross section of the strip line and the microstrip line in PCB and
the load connection diagram are shown in Fig. 14.
920 Y. Mi et al.
a Via Hole b
Via Hole Via Hole
Top Layer
Strip1
Strip1
GND GND
GND
Strip2
Bottom Layer Strip2

R
c R d
High
Voltage
Connection
High Voltage
Via Hole Connection
Fig. 14 Cross-sectional diagram of planar transmission line and the load connection diagram.
(a) Cross section of the strip line with five-layer PCB. (b) Cross section of the microstrip line with
three-layer PCB. (c) Arrangement of Blumlein transmission line. (d) Arrangement of unbalanced
transmission lines
Generally, PCB materials can be divided into two categories: rigid substrate
materials and flexible substrate materials. And the important variety of rigid sub-
strate material is copper clad laminate (CCL). It is made through the using of
reinforcing material, impregnated with resin adhesive. Covering with copper foil,
products can be made under high-temperature and high-pressure environment by
using steel plate as mold. There are many kinds of classification method for CCL.
According to the resin adhesive of PCB, part of the results are shown in Table 1.
Among them, the most common use in circuit board is FR-4. The main technical
features and applications of the FR4 are electrical insulation performance stability,
good flatness, smooth surface, and thickness tolerance standard. Figure 14c and 14d
show the distribution of the Blumlein transmission line, which are divided into the
right and left groups. If the strip line is arranged on the upper and lower sides of the
PCB, the total layer is five. The Toplayer, Ground (GND), and Bottomlayer are
ground plates, which are connected together through via holes, and Strip1 and Strip2
are connected in series through the same way. It is shown by the figure, Toplayer,
Strip1, and GND constitute the upper layer strip line, and GND, Strip2, and
Bottomlayer constitute the lower layer strip line. Similarly, if the microstrip line is
arranged on the upper and lower sides of the PCB, the total layer is three. The Strip1
and Strip2 are the conductor strips, which are arranged in the upper and lower
50
Table 1 Type of resin and use of substrate materials

Relative
Breakdown permittivity Dielectric loss
Specifications Resin Reinforcing material Properties and uses voltage (1 MHz) factor(1 MHz)
XXXP Phenolic Insulating paper Suitable for household appliances 12.3 kV/ 5.5 0.05
resin manufacturing and hot punching mm
XXXP-C Phenolic Insulating paper Punching at lower temperatures than 12.3 kV/ 5.5 0.05
resin XXXP grade mm
FR-4 Epoxy Glass fiber cloth Computer, instrumentation, and other 14.2 kV/ 5.5 0.04
resin electronic products mm
CEM-3 Epoxy Glass fiber cloth, Electronic product 13 kV/ 5.4 0.035
resin insulating paper mm
Compact Nanosecond Pulse Generator Based on Printed Circuit Board. . .
921
922 Y. Mi et al.
surfaces. They are also connected in series through via hole, and the middle layer
GND is actually only one layer.
Design of Unbalanced Transmission Line

The design idea of the unbalanced transmission lines is the same as that of the
balanced transmission line. However, it is needed to calculate the parameters of two
transmission lines in the unbalanced Blumlein transmission line, respectively. At the
same time, according to the analysis of unbalanced transmission line wave process, a
stepped wave line is formed on the load. In order to get a pulse square wave, the
wave needs to be cut off.
Combination Design of PCB Transmission Line
When the PCB transmission line is completed, the characteristic impedance and
length of the transmission line cannot be changed. For a single piece of PCB
transmission line, its characteristic impedance and length are generally limited to a
few hundred and decade meters. To obtain a variable characteristic impedance and
transmission line length, a few pieces of PCB can be connected in series, parallel,
and series–parallel. Because the thickness of the PCB is only a few millimeters, the
PCB can be stacked with the special design method, so as to achieve the purpose of
changing the parameters. Schematic diagram of stacked PCB is shown in Fig. 15,
where the orange parts are copper pillars, used for electrical connection, and the
green parts are insulated pillars, used for mechanical connection. When matching
different impedance load, it needs to change the characteristic impedance of the
Blumlein transmission line. Therefore, the ground plate and the conduction strip of
each piece of PCB are connected in parallel, which makes the impedance of the
entire Blumlein transmission line into 1/n Zc. When the width of high-voltage pulse
needs to be adjusted, it needs to change the length of the transmission line. The
specific method is to link the ground of the first PCB with the ground of the second
and the end of the first conductor strip and the starting point of the second conductor
strip. If the impedance and length of the transmission line both need to be adjusted,
you can use a similar method to cascade parallel, a similar method can be used with
series–parallel connection.
Design of Solid-State Switch in Planar Transmission Line
Design of Solid-State Switch

The switch unit, which is involved with the voltage amplitude, rise/fall time, pulse
width, and repetition rate, is a key part of the pulse power technology field. A
traditional gas switch has a large volume, low reliability, low repetition frequency,
and large dispersion. In contrast, a solid-state switch has the advantages of small
volume, long service life, high repetition frequency, and high reliability.
a High Voltage Connection b High Voltage Connection
Strip 1 Strip1
GND1 GND1
GND1 GND1
Strip2 Strip2
Copper Pillar Load Copper Pillar
Connection
Strip3 Strip3
GND2 GND2
GND2 GND2
Strip3 Strip3
Via Hole Load

Via Hole High Voltage
Connection Connection
Fig. 15 Schematic diagram of stacked PCB. (a) Schematic diagram of parallel connection. (b)
Schematic diagram of series connection
The solid-state switch unit typically includes control circuit, drive circuit, and
solid-state switch. Pulse width should be taken into account when selecting
switch, because the rise time of a commercial insulated-gate bipolar transistor
(IGBT) is usually lower than dozens of nanoseconds; it is too slow to obtain
rectangular pulse with a width of less than 100 ns. Compared with an IGBT, a
metal–oxide–semiconductor field-effect transistor (MOSFET) has a faster rise time,
which makes it the perfect switch for pulse duration from dozens to several hundred
nanoseconds.
Combination of Solid-State Switch and PCB

Normally, the PCB transmission line and the solid-state switch are designed sepa-
rately, which is beneficial to the test. In this case, it needs to design the corresponding
connection device on the PCB and the solid-state switch, such as via holes and solder
pads, then connecting directly or through short wire or copper strip. However, the
presence of wire and contact points, it will increase the stray inductance and contact
resistance. In order to reduce the influence of the stray parameters on the pulse
waveform, the transmission line and the solid-state switch can also be designed on
924 Y. Mi et al.
the same piece of PCB. In this case, their connection line is the shortest, and the flat
wire is adopted, which is conducive to improving the shape of the waveform and the
efficiency of generator.
Conclusions
In order to improve the nanosecond pulse generator for biomedical application to be

compact, portable, and reliable, this chapter provides guides to develop a compact
generator based on PCB transmission lines and solid-state switches. The principle of
Blumlein strip line pulse generator was presented. Unbalanced Blumlein strip line
could be used to generate output voltage with variable duration and variable ampli-
tude. Stacked Blumlein strip line could be regarded as a voltage multiplier or a
current amplifier. This chapter proposed the detailed design key points of this kind of
compact generator including different strip lines and stacked solid-state switches.
Characteristic parameters, attenuation and design procedure with detailed equations
of different strip lines, and PCB design techniques with parameters of different
dielectric materials were also provided. Multilayered PCB strip line occupies a
smaller volume than PCB microstrip line. However, the attenuation of stacked
PCB transmission lines (more than three stages) should be considered.
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Cliniporator: Medical Electroporation
of Tumors 51
Claudio Bertacchini
Abstract
The development of the medical device Cliniporator™ started in 2000 with the
homonymous European project, and, at its end, the prototype of the device was
ready to be certified. The work done up to that moment has been used for a new
European project called European Standard Operating Procedures for the Electro-
chemotherapy and Electrogenetherapy (ESOPE). During the ESOPE project,
standard operating procedures (SOP) have been established, and the certified
device has been used for clinical trials. Some years after the end of this project, a
technological redesign of the device enhanced its capability giving the possibility
to produce two different models of the device, one for gene therapy and small
internal or superficial tumors and the other for big, deep, solid tumors. Both the
devices are driven using the same type of pulses. What is mainly changed from
the beginning is the technology used to control the device, the user interface
technology, and the new types of electrodes allowing to approach different type of
tumors in different locations of the body. The ability to operate during open
surgery has required the capability to stay synchronized with the absolute refrac-
tory period of the heart. On the other side, the need to treat big deep tumors also
required the development of accessory tools to help the surgeon to plan the
intervention optimizing the number of electrodes to be used and their placement.
Keywords
Electroporation device • Electrochemotherapy • Electrogenetherapy • Predefined
treatment parameters • High voltage • Low voltage • Graphic user interface •
Alternative pulse protocol • Preoperative planning • Electrodes
C. Bertacchini (*)
Igea S.p.A., Carpi, Italy
e-mail: c.bertacchini@igeamedical.com

DOI 10.1007/978-3-319-32886-7_214
928 C. Bertacchini
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 928
Fields of Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 929
Cliniporator: An Electroporation Platform . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 930
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 930
Hardware Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 930
The New Cliniporator™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 931
Cliniporator VITAE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 933
Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 935
Software Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 937
Graphic User Interface Wizard Architecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 937
Working Parameters Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 943
Treatment Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 945
Electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 945
Types of Electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 945
Preset geometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 946
Custom Geometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 948
Electrode Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 950
Device Certification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 950
Council Directive 93/42/EEC 1993 and Amending Directive 2007/47/CE . . . . . . . . . . . . . . . . 950
Main Standards Applicable to the Device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 953
Cliniporator™ use strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 954
The Electric Field as Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 954
Electrode Parallelism to Ensure Electric Field Uniformity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 954
Needles Geometry and Treatment Area Coverage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 955
The Alternative Pulse Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 955
Preoperative Planning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 956
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 958
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 958
Introduction
Everything started in the far 2000 when Igea has been involved in the European
project named “Cliniporator” (QLK3-1999-00484, Cliniporator: a new adaptive
generator for DNA electrotransfer in vivo for gene therapy) (http://cordis.europa.
eu/project/rcn/51516_en.html). The aim of that project, coordinated by Dr. Lluis
M. Mir of the Institute Gustave-Roussy-France, was to demonstrate the basis of
the efficacy of the electroporation used to enhance the gene transfer. The research
work done on this project brought to design a prototype of a device (the
Cliniporator™) able to generate high-voltage and low-voltage pulses to be used
for this purpose. In fact the High Voltage pulses have the effect to permeabilize the
cell membrane and the Low Voltage pulses have an electrophoretic effect used to
transport the DNA inside the cell (Miklavčič et al. 2000; Šatkauskas et al. 2002,
2005; André and Mir 2004; Mir et al. 1991a, 1998). The approach selected for this
project was based on the reversible electroporation because the cells must stay
alive after the treatment.
51 Cliniporator: Medical Electroporation of Tumors 929
Into the 2002 the work done in the Cliniporator project has been used as basis for
the ESOPE project (QLK3-2002-02003: ESOPE: European Standard Operating
Procedures for the Electrochemotherapy and Electrogenetherapy) (http://cordis.
europa.eu/project/rcn/67434_en.html). During this project, the Electrochemotherapy
(ECT) approach to the tumors has been tested and verified. The base of this approach
is that opening pores on the cell membrane, enhancing its permeabilization, we can
improve the effect of drugs that usually cannot enter, or have some difficulty to enter,
into the cells due to the big dimension of their molecules (Mir et al. 1991; Serša et al.
1995). During the project drugs like bleomycin and cis-platinum have been tested
and their toxic effect evaluated in clinical trials (Marty et al. 2006). Their selection
and usage, according to the number of nodules and their dimension, has been defined
into the standard operating procedures (SOP) document (Mir et al. 2006). This
document should be used as guideline for each electrochemotherapy treatment in
order to select not only the drug but also the electrodes and all the other aspects of the
treatment.
Fields of Application
One of the goals of the ESOPE project was to have the new Cliniporator™ device
certified as medical device for clinical trials and compliant with the European
Medical Device Directive to be used for clinical applications. Its intended use is
the electroporation of the tissues, and its main indications for use are:
• Local treatment of subcutaneous and cutaneous metastasis independently from

their source and from previous therapies
• Treatment of local skin tumors relapse
• Electroporation-based gene therapy
• Electroporation of the human tissue
• Electroporation of cell cultures and in animal models
Following these indications for use and the ESOPE procedures, the
Cliniporator™ has been used to treat superficial tumors and to do research on gene
therapy since 2005.
After several years of research and clinical use, Igea redesigned the first model of
Cliniporator™. The result of this work is a new generation of devices with different
capabilities and field of use:
• The new Cliniporator™, used not only for superficial tumors and gene therapy
but also for internal tumors
• The Cliniporator Vitae, used for superficial tumors, internal tumors, solid (includ-
ing bones) tumors, and for deep or big tumors
930 C. Bertacchini
Cliniporator: An Electroporation Platform
Introduction
After the big effort done during the projects Cliniporator and ESOPE, Igea started
with the commercialization of the first device having model EPS01. The device has
been initially located into reference centers in different countries starting from the
ones directly involved in the projects. For several years, the manufacturer worked to
disseminate the technology and teach it to doctors like oncologists, surgeons,
intervention radiologists, and others involved in the cancer treatment. During this
work, considering the feedback from the field, the manufacturer started to improve
the device and the electrode models and to expand the fields of application. The
result of this work can be seen as the birth of an electroporation platform based on
two different models of devices and new types of electrodes for specific application
or electrodes that can be used with all the devices like the finger electrodes, the
ElectroPoration Series Adjustable (EPSA) electrodes for ECT, and the variable
geometry disposable (VGD series) electrodes.
Hardware Structure
The Cliniporator™ model EPS01 (see Fig. 1) is the first device obtained from
research. It has been developed in collaboration with the universities of Ljubljana
(Slovenia) and Bielefeld (Germany). For this reason a large number of boards,
having a specific behavior, composed it. A microprocessor system connects together
and coordinates all these boards.
The microprocessor card exchanges its data and the measured treatment data with
the console through a RS232 serial communication. The user interface software,
Fig. 1 Cliniporator™ model EPS01

used to control the device, is a graphical software developed under the National
Instruments Labview framework, running on a medical grade personal computer,
having Windows XP as operative system and the Labview run-time engine.
The New Cliniporator™
The redesign activity made on the Cliniporator™ was specifically directed to

improve its technology replacing obsolete components, improving the intrinsic
safety of the device, reducing the number of boards, and simplifying the user
interface using a more stable operative system and a faster specific language for
the application. A new feature has also been added to the device: the possibility to be
used even during open surgery to treat internal tumors.
From the electronic hardware point of view, the implemented improvement can
be resumed with the following points:
(a) All the programmable and control logic has been redesigned and controlled with
an unique Xilinx Spartan3E fpga (field of programmable gate array: a type of
programmable logic). The usage of a single fpga improves the deterministic
control of the whole hardware and adds a faster parallel control of the different
sections of the device. The fpga is divided into several sections working in
parallel and exchanging synchronization data at a 20MHz rate:
• The communication section
• The pulse generation section
• The treatment control section
• The charge/discharge section
• The output relays control section
• The measure section
With this approach, the power unit is seen as a group of memory register from the
console. These registers are used to show the status and the alarms of the hardware
and to store all the treatment parameters. In this way, the fpga has the complete
control of the treatment delivery ensuring the maximum safety for the patient due to
its deterministic behavior.
(b) The communication with the console has been improved and sped up with the
introduction of the Universal Serial Bus (USB) communication interface over
optical fiber passing from 56,000 bit/s to more than 1 Mbit/s.
(c) The cards controlling the high-voltage section and low-voltage sections have
been redesigned using more efficient high-voltage power supplies able to gen-
erate voltages up to 1200 V for the high-voltage section and 250V for the
low-voltage section. The original two cards have been compacted into one single
card for each section having the functionality of power supply generation,
voltage amplification, and energy storage.
(d) The measure cards has been compacted with the output card moving also into the
new card the feature to generate the pulses. In this card, the sampling time has
932 C. Bertacchini
been reduced to a 1MSPS (mega sample per second) introducing serial analog-
to-digital converters and simplifying the isolation of the sampled data. At the
same time, programmable amplifiers have been used to improve the precision of
the sampled data at low voltages.
(e) The console has been replaced by a certified medical grade panel PC (all-in-one
personal computer) with touch screen. This simplified the console eliminating the
needs of mouse and keyboard. The use of a certified medical panel PC simplifies
the certification procedures ensuring also a more stable life of this component.
(f) The requirement to use the device during open surgery has been fulfilled
implementing the synchronization with the absolute refractory period of the
heart to avoid the risk to induce atrial or ventricular fibrillation during the pulses.
With this feature, the device delivers a group of pulses 50 ms after the R wave,
i.e., during each absolute refractory period of the heart. To do this the device
requires to work in conjunction with an R-wave trigger device. The R-wave
trigger device analyzes the heartbeat and detects the R-wave that is the starting
moment of the absolute refractory period of the heart. To make the device safer,
an algorithm analyzing the frequency of the R-wave trigger has been
implemented. This algorithm accepts as valid the triggers having a period longer
than 500 ms (<120 heartbeat per minute) and can detect when the trigger is lost
and if the trigger signal has wrong characteristics. In particular the trigger signal
must be a signal with active high logic level and length of 150 ms.
From the software point of view, the main change has been the replacement of the
operative system with Linux. The use of the Gentoo distribution, which is always
compiled on the hardware, ensures to have an operative system that is efficient, fast,
and stable. Another change has been the complete new design of the application
using the C/C++ language and the GTK+ libraries for the graphic interface. The use
of the C/C++ language ensures to have a compiled software without the need of a
run-time engine or a framework like in Labview or dotnet under Windows. Without
the interpreter and using a machine language, the software execution is the fastest,
and it is not necessary to have an expensive PC with high characteristics to let run
adequately the software. With this solution, we had also the possibility to design a
user-friendly software interface with custom icons and simple and efficient screens.
A third change, done considering the field of application, has been the removal of the
patient database introducing the concept of exportable treatment file. This change
takes its origin from the evidence that the doctor needs to prepare the treatment
before going into the operating room where usually the device is located. This means
the database into the device is useless, and on the contrary, it is more useful to have
the treatment data on the patient file where they can be easily evaluated.
Finally also the mechanical hardware has been redesigned having in mind new
requirements that can be resumed into the need to have a new user-friendly device,
movable, but not portable, adequate to work in the operating theater without the
needs to have an external trolley, and having a limited number of components.
The result is the device model EPS02 shown in Fig. 2 having a power unit with a
simpler electric/electronic architecture (see Fig. 3) composed of only six cards.
Fig. 2 Cliniporator™ model

EPS02
Mechanically, it incorporates a cart that joins the console and the power unit into a
unique structure with four wheels allowing to move the device with a minimum
effort. Thermoformed external plastics, designed according with the console shape,
give to the device the final look. The redesign of the Cliniporator™ has been done
having in mind also new possible applications, thus hardware and software have
been redesigned considering the possibility to change easily the configuration of the
device. This change of configuration has been implemented in the new
Cliniporator™ with the possibility to insert or remove easily the card for the
low-voltage section implementing with a small change a device specific for electro-
genetherapy (EGT) or specific for the electrochemotherapy (ECT). The hardware
automatically identifies the presence of the card for the low-voltage section, and,
sending this information to the software, the features for the gene therapy, normally
disabled, are automatically enabled.
Cliniporator VITAE
The need to use the device in situations where the electric field has to be generated
between needles having a distance longer than 1 cm and up to 3 cm with custom
configurations required the design of a specific set of cards for this purpose. This
activity brought to the development of the Cliniporator VITAE (see Fig. 4). In this
device, the high-voltage power supply is able to generate voltages up to 3500V, but
due to this high voltage, the amplifier has not been developed preferring the approx-
imation of a free short discharge of the capacitors to generate square pulses. The use of
934
LVC-AMP
3
High Voltage
1 2
Front End
Transformer
8 HVC-AMP 4 PGM 5 Switching 6 7
High High
High Voltage Voltage Voltage
AC-DC Patient Interface

9 10
USB2FPGA 19
11 12
15 Data Lines
17 LV Power Supplies
Medical Grade
Swit ching Panel PC HV Lines
RFID PS 18 Protective Earth
13
Card Mains
16
Optical fiber
14 Double Pedal
Swit ching
PS
C. Bertacchini
Fig. 3 New Cliniporator™ block diagram

Fig. 4 Cliniporator VITAE

model VGP02
such a high voltage required to meet more stringent safety requirements ruled by the
European Standard EN60601-1. Another characteristic of this device is that it can be
used only for the electrochemotherapy having also the electrochemotherapy part of the
new Cliniporator™ inside. In this way the device can be used independently with the
two different modalities, the “preset mode” and the “custom mode,” switching freely
from one to the other. The hardware architecture of the device (see Fig. 5) shows that
this device is composed by two independently controlled devices.
The software enables the use of each part according with the choice of the user.
With these characteristics, the Cliniporator Vitae can be used safely with custom
electrodes configurations.
Since the two power hardware are similar but not completely compatible, a new
mechanical part has been developed to let them integrate. The software, developed
as graphic user interface (GUI), runs on the same console type of the new
CliniporatorTM, and even for this device, the operative system is Linux.
The GUI has been derived from the one of the new Cliniporator™ integrating the
two working modes.
Software
The software of the Cliniporator™ devices has been improved during the life of the
device. During the Cliniporator project, the software has been developed using
Labview because it is the most powerful development tool able to enhance the
realization of a software specific for instrumentation control and test tool
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Fig. 5 Cliniporator VITAE block diagram

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development. The effort done during the development phase allowed the realization
of a nice software on a Windows-based personal computer allowing a good control
of the device. With the mass production of the device, the problems due to the
operative system and the continuous upgrades required by hardware and software,
together with the new requirements coming from the users, brought to a redesign of
the device. The software redesign started from a new baseline: the use of a stable
operative system together with a development language more efficient and specific
for the applications or, in other words, a compiled language not requiring a run-time
engine, an interpreter for meta-languages, makes the system slow.
Software Structure
Since the structure of the software during the first development was a little bit
complex, during the redesign a wizard approach has been preferred to guide the
user during the treatment. This approach is common for both the devices: the new
Cliniporator™ and the Cliniporator VITAE. The new software, running into as
Linux environment, has been developed to take the advantages of the client-server
approach. Following this approach the software has been divided into two separate
programs running at the same time. One program is a server, named epcore, having
the functionality to speak directly with the hardware, through the USB, the GUI, and
the radio-frequency identification (RFID) peripheral. The other is the GUI, having
the functionality to interact with user through the touch screen and the display, to
export the data through a USB pen drive, and to exchange the informations with the
server. Figure 6 shows this structure for the new Cliniporator™, and the
corresponding structure for the Cliniporator VITAE is very similar to this one.
Graphic User Interface Wizard Architecture
The GUI wizard architecture (see Fig. 7) is composed of several pages helping and
guiding the user into the treatment delivery. The wizard allows always the user to go
back from a screen to the previous. The moving to the next screen has instead to be
enabled doing the actions required by the software.
The first page (see Fig. 8), common to both the new Cliniporator™ and the
Cliniporator VITAE, shows the self-test of the device done just after the power on. In
this page the device performs a series of tests to check its functionality. Only if the
device passes all the tests, the user can continue to use it for the treatment
The second page (see Fig. 9) is the login page. Here the software asks the user to
enter username and password to enable the use of the device. This feature is
important about privacy because the device contains sensible data about the patients,
and only authorized people can have access to them.
The next page (see Fig. 10), the third, is the mode selection screen. It allows the
user to select between the working modes and the export of the treatment data. On
the new Cliniporator™, the choice is between the electrochemotherapy (ECT
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CONSOLE APPLICATION Touch Screen
USB Pendrive GUI Application Display
TCP/IP protocol
SERVER
RFID Card USB Port
LINUX OPERATIVE SYSTEM
Fig. 6 Software architecture
Parameters
Boot SelfTest Edit or
(EGT only)
Esope Edit
ECT/
Shutdown EGT Patient Electrodes
Login Treatment
Data Selection
Login
Mode View
Export
Selection
Fig. 7 GUI architecture

Fig. 8 Self-test screen
Fig. 9 Login screen

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Fig. 10 Mode selection screen
mode) or the electrogenetherapy (EGT mode), but on the Cliniporator VITAE,

where only the Electrochemotherapy can be done, the choice is between the preset
geometry, the same of the new CliniporatorTM, and the custom geometry.
The fourth page (Fig. 11), the patient data screen, asks to the user to insert the
patient data and eventually to add some notes (e.g., type of disease and quantities of
drug used) about the treatment.
At this point, the user can proceed to the next page, the electrode selection screen
(Fig. 12). The actions that can be taken are different according to the device and the
use mode selected:
(a) In the ECT mode of the new Cliniporator™ or in the preset mode of the
Cliniporator VITAE, the user can select one or more electrodes, having pre-
defined treatment parameters (see the Table 4), to be used during the treatment.
(b) In the EGT mode of the new Cliniporator™, the user can select only one
electrode and then it has to open the edit screen to create or reload a treatment
protocol.
(c) In the custom mode of the Cliniporator VITAE, the user can select the type, the
number, and the configuration of the electrodes. From this page the user can
proceed into the next page, the parameters screen, where he has to approve the
treatment protocol. In case of a custom electrode configuration, the user is asked to
place the electrodes and then to define the distances from each other (Fig. 13a–b).
In the electrode selection screen, or in the parameters screen for the Cliniporator
VITAE, the user has the possibility to enable the synchronization with the heartbeat.
Fig. 11 Patient data screen
Fig. 12 Electrode selection screen

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Fig. 13 (a–b) Parameter screens
This operation must be done after the connection of an approved R-wave trigger
device to the Cliniporator™.
When the electrodes and the protocols have been selected and approved, the
wizard architecture lets the user to proceed into the treatment screen (Fig. 14).
Here the user can charge the capacitors, start the treatment, and check the final
Fig. 14 Treatment screen
result looking to the measured data. In the Cliniporator VITAE, it is also

possible to see the electrocardiographic track, if the R-wave detector has been
enabled and connected, and to have a zoom on the measured data to have a look
to a specific group of pulses. Always in the treatment screen, the user can use
two buttons working as a shortcut to go directly in the patient screen to do a
new treatment with a new patient or to go in the login screen to exit and shut
down the device.
Working Parameters Selection
As explained in the previous chapter, the working parameters selection is different

according to the device and the working mode. Each device has its specific
working range defined from the amount of energy that can be stored into the
capacitors. The new Cliniporator™ has the parameters range defined by the follow-
ing Tables 1 and 2.
The Cliniporator VGP02 has the parameters of the preset geometry equals to the
ones of the ECT working mode of the new Cliniporator™. The custom geometry
instead has the following parameters:
The Table 3 below shows maximum and minimum values for each parameter
considered alone.
In any case, for both the devices, the parameter selection is driven by the
following rules:
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Table 1 Cliniporator™ high-voltage pulse ranges

Working mode ECT EGT
Output channels 7 7
Number of pulses 4–8 1–10
Pulse amplitude 400/730/960 V 100–1000 V
Pulse length 100 μs 50–1000 μs
Rise time <2 μs at 1000 V <2 μs at 1000 V
Pulses repetition frequency 5000 Hz 1–5000 Hz
Maximum deliverable current 20 A 20 A
Pulse amplitude accuracy 5% 5%
Pulse length accuracy 2 μs 2 μs
The product of number of pulses times pulse duration must be less than 1000 μs
Table 2 Cliniporator™ low-voltage pulse ranges

Working mode EGT
Number of pulses 1–10
Pulse amplitude 20–200 V
Pulse length 1–1000 ms
Pause between pulses 1–2000 ms
Maximum deliverable current 5A
Pulse amplitude accuracy 5%
Pulse length accuracy 1%
Pause between high-voltage and low-voltage pulses 1–2000 ms
Table 3 Cliniporator VITAE: variable geometry pulse ranges

Output channels 6
Number of pulses 8–320 in groups of 4 with group inversion of
polarity
Switching interval between electrode pairs 3.1 s
Pulse amplitude 500–3000 V
Minimum distance between electrodes 5 mm
Pulse length 100 μs
Rise time <4 μs at 1000 V
Pulse repetition frequency 1/1000 Hz
Maximum deliverable current 50 A
Max. pulse energy for each pulse (nominal) 15 J
Pulse amplitude accuracy 5%
Pulse length accuracy 2 μs
(a) In the ECT mode, the device always tries to generate an electric field starting
from a voltage to distance ratio of 1000 V/cm between each couple of electrodes.
Therefore, it can have a predefined voltage value or can adapt it to the distance
between the needles or the plates.
(b) In the EGT mode, on the new Cliniporator™, the user has the possibility to
generate bigger or smaller electric fields into the tissue always starting from a
voltage to distance ratio derived from the treatment parameters. Thus, the
parameters selection must be done carefully.
(c) On the Cliniporator VITAE in custom mode, the user is asked to introduce, as
parameter, the distance between the electrodes. Since the minimum distance
between the electrodes is 5 mm, the software forbids to set a smaller distance. In
case of a distance setting bigger than 3 cm, a warning is shown to the user
because the voltage to distance ratio is lower than 1000 V/cm.
Treatment Management
The treatment management is very simple. Once the treatment has been planned, the
surgeon has, first to select and activate the electrodes; then he/she can insert the
electrodes into the patient body. Only at this moment, the capacitors should be
charged in the treatment page of the graphic user interface. When the capacitors
are charged, pressing the arm pedal, the system transfers the treatment parameters
from the console to the power unit of the device. After that a countdown starts, and
the user has 10 s to press the pulse pedal in order to start the treatment. If this time-
out elapses, the system automatically aborts the treatment. During the treatment,
sounds are generated to signal the status of the device: a single beep at the beginning
and double beep at the end. Then the data are uploaded from the memory and shown
on the graphs. At the same time, the Cliniporator™ recharges the capacitors to be
ready, within few seconds, to deliver a new treatment.
Electrodes
The electrodes for the Cliniporator™ can be divided in different types according
with their usage. In any case, being the applied part of the device, they are single use
and are provided sterile to the final user.
Types of Electrodes
A first level of classification that can be applied to the electrodes is related to the
geometry. Starting from the beginning, the electrodes of the Cliniporator™ have
been characterized from a preset geometry and from the presence of a couple of
handlers where to connect different types of applied part. All these electrodes have
been grouped into a series named EPS. All the needles of this series are made of
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American Iron and Steel Institute type 304 (AISI 304) stainless steel with a pencil tip
and a diameter of 0.7 mm. The tip and the diameter are the result of some test done,
during the development of the device, to evaluate the best shape ensuring a good
strength to the bending, the minimum insertion effort, and the minimum damage to
the tissues. Next evolution brought to the birth of new electrodes for preset geometry
and electrodes for variable geometry, specifically done to be used with big or internal
tumors.
Preset geometry
The choice to have electrodes with different predefined geometries has been selected
to help the surgeon to use the device and learn the usage technique. Another
advantage of the preset geometry is that all the treatment parameters are already
defined and are automatically loaded into the device when the electrodes is selected.
With this geometry the user has simply to ensure that the tumor nodules stay into the
volume defined by the needles. Doing this, the treatment is very simple and fast, and
the user can stay focused on the patient. The electrodes developed during the project
Cliniporator™ have been collected under the name of “EPS series.”(Fig. 15a–c).
The figures above show the different types of electrodes of the EPS series.
Linear
Linear electrodes (see Fig. 15b) are composed by two rows of four needles. The
needles of each rows are connected together so the electrical connection of this
electrode is basically bipolar. The needle rows have a distance of 4 mm, and their
height can be from 10 mm up to 40 mm. Following the SOP, the treatment
parameters are eight pulses 100 μs long at 400 V and with a repetition frequency
of 5 kHz.
Fig. 15 (a) Hexagonal electrode. (b) Linear electrode. (c) Plate electrode
Hexagonal
Hexagonal electrodes (see Fig. 15a) have seven needles, six placed with a hexagonal
shape and one in the central position. The distance between the needles is 7.3 mm, and
in this case, their height can be from 10 mm up to 40 mm. With this type of electrode, it
is possible to treat tumor nodules of bigger dimensions with respect to the linear
electrodes. Following the SOP, the treatment parameters are four pulses 100 μs long at
730 V and with a repetition frequency of 5 kHz applied to each couple of needles with
the polarity exchange. Since the couples of needles to be used without any repetition
are 12, this treatment applies totally 96 pulses: 8 pulses to each couple.
Plate
Plate electrodes (see Fig. 15c) have been specifically designed for usage in specific
parts like the ears and nose or when the tumor nodules has an extruded part that can
be treated without the insertion of needles into the skin. These electrodes have two
plates made of AISI 316L stainless steel with a distance of 8 mm. Following the
SOP, the treatment parameters are eight pulses 100 μs long at 960 V and with a
repetition frequency of 5 kHz. The pulse amplitude, suggested by the SOP for this
electrode, is related to the difficulty to electroporate the skin due to the tissue
properties, anatomy, and geometry. A bigger voltage helps to do this.
EPSA Series
The EPS series of electrodes for preset geometry has been redesigned with the
following goals:
(a) Eliminate the handles doing a complete single-use applied part.

(b) Create an insertion guide to help the user during the electrode insertion to
maintain the parallelism between the needles.
(c) Create a mobile part that can cover the needles at the end of each application
without the risk of puncture for the user.
In this way also, the disposal of the electrode is safer. The new series has been
named ElectroPoration Series Adjustable or EPSA. The electrode models of this series
are the following (see Fig. 16a, b): (a) linear electrodes and (b) hexagonal electrodes.
Finger
This model of electrode has been developed to be used into the human orifices, e.g.,
the oral cavity. Its name is due to the characteristic to be wearable and in particular
that it has to be inserted into a finger to be used. It is a linear electrode composed by
two rows of three needles each. The distance between the rows is always 4 mm, but
the height of the needles can be 5 or 10 mm. Two submodels (see Fig. 17a, b) have
been done according to the position of the needles: (a) the LG (longitudinal) model
having the needles exiting from the tip and (b) the OR (orthogonal) model having the
needles exiting from the down side of the finger. Following the SOP, the treatment
parameters are eight pulses 100μs long at 40 V and with a repetition frequency of
5 kHz.
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Fig. 16 (a) Hexagonal EPSA electrode. (b) Linear electrode
Fig. 17 (a) Orthogonal finger. (b) Longitudinal finger
Custom Geometry
Starting from the superficial tumors, the feedback from the field has shown the need
to treat also other type of tumors. Thinking to expand the application field of the first
Cliniporator™ device, a new specific and more powerful device has been developed
to be used with big nodules, deep tumors, or bone tumors. These tumors can have
specific insertion constraints: tissues that cannot be treated or that have no available
access path could require distances between the electrodes longer than 1 cm. Such long
distances bring to the delivery of pulses with a larger voltage and current causing the
need to have an energy amount bigger than the one stored into the Cliniporator™. In
these cases, the preset geometry could not be applicable. Therefore, a possible solution
is to have single needles, with different length and with an active part from 10 mm up
to 40 mm. These electrodes have to be inserted independently, but maintaining the
parallelism one from the others, to define the treatment volume. To create a treatment
volume, deep into the body, it is also necessary to insulate the electrode except its
distal part (the active part) to protect healthy or sensitive tissues and treat only the
tumor nodule. Electrodes for bone application must also have a trocar tip, otherwise it
will be difficult to penetrate them; electrodes for soft tissues, instead, can have the
standard pencil tip. The first needles having these characteristics have been grouped
into the variable geometry (VG) series. The connection with the device is done with a
specific re-sterilizable cable. Now, after their first period of usage, the VG series has
been updated into the variable geometry disposable (VGD) series.
VGD Series
The VGD series (Fig. 18) is characterized again by needles made of AISI 304 stain-
less steel. The changes made to improve the VG series are the following: (a) all the
needles have a trocar tip; (b) each needle is insulated with a polyimide tube with a
thickness of few tenth of microns; (c) each needle is equipped with laser-made
measuring notches; (d) the tip has been sandblasted to increase the echogenicity;
(e) each needle has a grip with integrated connector for the connection cable; (f) each
needle has its single-use cable to be connected with the device. Following the SOP,
the treatment parameters are similar to the ones of the hexagonal electrodes: 4+4
pulses (due to the polarity change) 100 μs long at a voltage adequate to generate a
voltage to distance ratio of 1000 V/cm. In this case, the repetition frequency is
1000 Hz to limit the risk of local accumulation of energy.
RFID Identification of Electrodes

Since all the electrodes of the Cliniporator™ has to be single use as measure of risk
control, it has implemented their identification through an RFID system based on the
Fig. 18 Variable Geometry Disposable (VGD) series electrode with its connection cable
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standard ISO 15693 (International Standard Organization) working at a frequency of

13.56 MHz. Each electrode has a tag containing a specific identification code, and,
when activated, the electrode and the patient are linked together. Each electrode stays
also active only up to 6 h from the first activation. After that time the electrode is no
be longer accepted by the device. In case of identification issues, the device can
perform a manual emergency identification of the electrodes allowing the user to
complete the treatment.
This type of identification is full operative on the electrodes for the preset
geometry and will be available soon on the electrodes for variable geometry.
Electrode Parameters
Table 4 shows the treatment parameters for each type of electrodes that can be used
with the Cliniporator™ devices.
Device Certification
When a company decides to produce a device, the first thing to do is to define the
type of the device and its field of application. To help the manufacturers, the
European Commission released a series of laws called directives that must be used
to classify and to drive the design and the manufacturing of each device. The
essential requirements defined for each directive are mandatory for the manufac-
turers, but they do not say anything about technical specifications of each product.
The European Commission can decide to ask a recognized European Standard
organization (CEN, CENELEC, or ETSI) to deliver a specific standard or the
transposition of an international standard under the form of a European Standard
(EN) so that the manufacturers can follow these standards to demonstrate that
products, services, or processes comply with relevant European Union legislation.
The compliance with these harmonized standards ensures a high-quality level of the
devices and the conformity with one or more essential requirements of the European
directives.
Council Directive 93/42/EEC 1993 and Amending Directive 2007/

47/CE
For a medical device, the directive to be followed is the Council Directive 93/42/
EEC 1993, usually called Medical Device Directive or MDD 93/42, with amending
Directive 2007/47/CE. This directive, in the article 1, specifies when a device is a
medical device.
A fundamental article of the medical device directive is the article 3 saying that
a medical device must meet the essential requirements, specified in the Annex I,
51
Table 4 Electrode parameters

Number Number Pulses
Cliniporator Working Model of of output of Electrode geometric Pulse Pulses Pulses repetition Pulse Maximum
device mode electrodes channels electrodes description number amplitude duration frequency sequences current
EPS02/ ECT /preset N-xx-HG 7 7 Hexagonal HV: 4 HV: 730V HV: 100 μs HV: (1 or 24 HV: 730V HV: 20 A
VITAE geometry configuration 5000) Hz configurations
Diam:0.7 mm/length:
10 mm /20 mm/
30 mm (xx)
N-xx-4B 2 8 (2 4) Linear configuration HV: 8 HV: 40V HV: 100 μs HV: (1 or No HV: 40V HV: 20 A
Diam: 0.7 mm/ 5000) Hz
length: 10 mm/
20 mm/30 mm (xx)
P-30-8B 2 2 Linear configuration HV: 8 HV: 960V HV: 100 μs HV: (1 or No HV: 960V HV: 20 A
(10 mm 30 mm 5000) Hz
0.8 mm)
L-xx-OR 2 6 (2 3) Finger configuration HV: 8 HV: 400V HV: 100 μs HV: (1 or No HV: 400V HV: 20 A
with orthogonal 5000) Hz
Cliniporator: Medical Electroporation of Tumors
linear needles Diam:

0.7 mm/length:
5 mm/10 mm (xx)
L-xx-LG 2 6 (2 3) Finger configuration HV: 8 HV: 400V HV: 100 μs HV: (1 or No HV: 400V HV: 20 A
with longitudinal 5000) Hz
linear needles Diam:
0.7 mm/length:
5 mm/10 mm (xx)
Diam: 0.7 mm/
length: 5 mm/10 mm
(xx)
N-xx-HG 7 7 Hexagonal HV: 4 HV: 730V HV: 100 μs HV: (1 or 24 HV: 730V HV: 20 A
configuration Diam: 5000) Hz configurations
0.7 mm/length:
40 mm (xx)
(continued)
951
952
Table 4 (continued)
Number Number Pulses
Cliniporator Working Model of of output of Electrode geometric Pulse Pulses Pulses repetition Pulse Maximum
device mode electrodes channels electrodes description number amplitude duration frequency sequences current
EPS02 EGT N-xx-4B 2 8 Linear configuration HV: HV: HV: HV: No HV: HV: 20 A
Diam: 0.7 mm/ 1–10LV: (100–1000) (50–1000) (1–5000) 100–1000V LV: 5 A
10 mm/20 mm (xx) 1–10 VLV: μs L: HzLV: LV:
(20–200) V (1–1000) (0.45–500) 20–200V
ms Hz
2 2 Linear configuration HV: 1–10 HV : HV: HV: No HV: HV: 20 A
(10 mm 30 mm (100–1000) (50–1000) (1–5000) Hz 100–1000V
0.8 mm) V μs
LV: 1–10 LV: LV: LV: LV: LV: 5 A
(20–200) V (1–1000 ) (0.45–500) 20–200V
ms Hz
VITAE Soft tissue VGD- 2–6 2–6 Single long needle HV: 4+4 HV: HV: 100 μs Custom 500–3000V 50 A
custom 12xxTyy Diam: 1.2 mm/active (polarity (500–3000) (500–3000) configuration
geometry part: 10–40 mm (xx); exchange) V V
ECT total length:
12–24 cm
Bones VGD- 2–6 2–6 Single long needle HV: 4+4 HV: HV: 100 μs Custom 500–3000V 50 A
custom 18xx-Tyy Diam: 1.8 mm/active (polarity (500–3000) (500–3000) configuration
geometry part 10–40 mm (xx); exchange) V
ECT total length:
12–24 cm (yy)
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taking in account of its intended purpose. The essential requirements are a series of
rules defined to ensure the safety, the efficacy, and reliability of the device. For this
reason, an important essential requirement is the need of a clinical validation of the
device.
Another main point of the medical device directive is the classification of the
devices defined into the article 9 in accordance with Annex IX of the same directive
(Council Directive 93/42/EEC 1993). The directive defines four main classification
levels: class I, class IIa, class IIb, and class III. The Annex IX provides the rules
to define the class of each device. According with these rules, the different
Cliniporator’s models have a different classification. The new CliniporatorTM is a
class IIa device because, following the rule 9, it is an active therapeutic medical
devices intended to exchange energy with the patient. The Cliniporator Vitae instead
is a class IIb device because not only it can be classified as the new Cliniporator™
but it delivers energy in a potentially hazardous way. In fact, the classification of the
Cliniporator Vitae is due mainly to the amount of energy stored into the capacitors,
allowing treating tumors of big dimensions and delivering pulses with amplitude up
to 3000 V.
Since the manufacturer has its quality system certified ISO 13485 and since the
classification requires that the conformity to the medical directive is released by a
notified body, the conformity assessment procedures follow the Annex II of the
directive, based on a full quality assurance, using the Italian Institute of Quality
Mark (IMQ) as notified body. Therefore, all the Cliniporator™ devices have a
CE0051 marking.
Main Standards Applicable to the Device
To be compliant with the essential requirements of the MDD 93/42 (Council

Directive 93/42/EEC 1993), the main applicable harmonized standards are the
following:
• Safety: EN 60601-1-1:2006/A1:2013 (EN 60601-1:2006/A1:2013 Medical elec-

trical equipment Part 1) and the related applicable collateral and particular
standards. In particular for the CliniporatorTM device, the collateral standards
are:
– EN 60601-1-1:2001 General requirements for safety – Collateral Standard:
Safety requirements for medical electrical systems
– EN 60601-1-2:2007 General requirements for basic safety and essential per-
formance – Collateral Standard: Electromagnetic disturbances, Requirements
and tests
– EN 60601-1-6:2010 General requirements for basic safety and essential per-
formance – Collateral standard: Usability
• Usability: EN 60601-1-6 (EN 60601-1-6:2010 Medical electrical equipment –
Part 1-6). This standard basically asks to apply the European Standard:
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– EN 62366:2008 Medical devices – Application of usability engineering to

medical devices that has to be applied in any case being an harmonized
standard of the Medical Device directive 93/42
• Risk Management: EN 14971:2012 Medical devices – Application of risk man-
agement to medical devices (ISO 14971:2007, Corrected version 2007-10-01).
Cliniporator™ use strategies
The correct execution of a treatment with the Cliniporator™ is related to several

aspects that have to be considered by the user before and during the treatment. These
aspects are resumed under the following topics:
• The evaluation of the electric field to be used as reference for the treatment
parameters
• The evaluation of the geometry to be used during the treatment to ensure the
coverage of the treatment volume
• The check of the parallelism between the needles to ensure an uniform electric
field
• The evaluation of the current delivered during the treatment in case of problem to
apply the alternative pulse protocol
The first two topics, when the user works with custom geometries, brought to the
need to have a tool to help the planning of the treatment.
The Electric Field as Reference
The theory of the electroporation says that this process is a reaction of the cell
membrane to the presence of an electric field and that each type of cell seems to
have a specific value of electric field triggering the electroporation (Neumann and
Rosenheck 1972; Miklavčič et al. 2006a). For this reason and since the device is
voltage controlled and cannot take into account the real characteristics of the tissue, an
electric field (E) between two electrodes can be controlled through the ratio between
the potential U applied to an electrode and the distance d from it (E = U/d). Therefore, it
is not only important to evaluate the electrodes placement and distance defining the
correct voltage to be applied, but, since the electrodes can be placed around and into
the tumor, it is also important to define a volume around the tumor having a local
value of electric field adequate to trigger the electroporation of the cells.
Electrode Parallelism to Ensure Electric Field Uniformity
One of the more important conditions to have a good electroporation is the unifor-
mity of the electric field all around the treatment volume. This uniformity can be
easily obtained using parallel electrodes and applying a correct difference of poten-
tial between them. This brought to create specific applied parts characterized by
parallel needles (or plates) having different geometrical configurations. Typically
electrodes have linear (dual in line electrodes) or hexagonal geometry. The experi-
ence shows that sometimes it could be difficult to maintain the required parallelism.
When it is not possible to ensure the parallelism between the electrodes, it is
necessary to have another parameter ensuring the goodness of the electroporation.
This parameter is the local value of the electric field. Considering that our body has
different types of cells and that it is almost impossible to have a homogeneous tissue
during a treatment, studies have defined a minimum value of electric field able to
trigger the electroporation for different types of cells. This value is bigger than
250 V/cm and has to be used as the minimum value of electric field triggering the
electroporation (Miklavčič et al. 2006a). The maximum value of the electric field,
instead, should not need to exceed 1000 V/cm (Sersa et al. 2008).
Needles Geometry and Treatment Area Coverage
Another aspect linked with the estimation of a volume having an adequate value of
electric field is the coverage (Miklavčič et al. 2006b). Using this third aspect together
with the others to have a good electroporation, the treatment planning will give to the
user a complete information about how to ensure the coverage of the treatment zone
with an electric field having theoretically the best value, but in the worst case an
adequate value. Thus, it is very important for a successful electroporation to verify
that an electric field having a gradient value not lower than 400 V/cm (Cadossi et al.
2014; Kranjc et al. 2015) using applied parts with a high grade of parallelism
between the electrodes completely covers the treatment volume.
The Alternative Pulse Protocol
The evaluation of the current can help the user to evaluate how the treatment has
been delivered. Studies have shown that the changes in the current and in the
dynamic conductivity could be used to detect the electroporation (Cukjati et al.
2007), but this approach is still under study. The Cliniporator™ instead uses the
result of an analysis about the correlation between the current flowing and the
effectiveness of the electroporation. The result of this study shown that if the current
is above a certain threshold and all the pulses have been applied properly, the
treatment efficacy is good. Otherwise, it is recommended as an evaluation about
the possibility to apply another treatment.
The Cliniporator Vitae has a new feature that can help the surgeon when a
current alarm is present during the treatment delivery. This feature has been
called alternative pulse protocol (APP). The APP has been developed following
the result of a study (Fini et al. 2010; Ongaro et al. 2011) showing that the same
electroporation effects can be obtained with a reduced value of potential applied
956 C. Bertacchini
to the needles and with an increased number of pulses. A table resuming the
results of the study has been implemented into the software. There are different
values of electric field, associated with bigger number of pulses, which can
generate the same electroporation effect of the standard treatment. The APP
starts automatically, at the end of a treatment with current problems. The system
analyses the couples of electrodes experiencing the problem, and, in case of an
overload alarm, it performs a post pulse to evaluate the impedance of the tissue.
With this information and considering the maximum allowed current, it is
selected, from the table, the correct value of pulse amplitude and number of
pulses to be applied to that specific couple of electrodes. In case of a low current
alarm, the APP asks simply to check the connections and the electrode placement
before to apply again the same treatment.
Preoperative Planning
The difficulty to operate with the Cliniporator VITAE and in particular the problems
related to the electrodes placement brought the manufacturer to work on an external
tool helping the surgeon during the treatment planning. The tool has the functionality
to do an automatic placement of the needles within or around a predefined area
segmented by user based on an estimation of the coverage of the electric field and a
minimization of the number of electrodes required to get it. The calculations are done
considering the 2D image of the electrode insertion plane under the approximation of
homogeneous tissues and conductivity. The result of this work made with the effort
of a Slovenian company has been a software named Pulsar. This software, that is not
predictive, can now be provided with the Cliniporator VITAE, to help the surgeon to
do the preoperative planning of the electroporation intervention starting from a
Digital Imaging and Communication in Medicine (DICOM) image representative
of the target lesion.
The surgeon starts the planning loading a DICOM image that shows the target
area from the electrode insertion view. On this picture, the surgeon has to draw the
boundaries of the lesion and, if present, the forbidden zones. The forbidden zones are
parts of the body, where the electrodes cannot be inserted, like vessels or nerves or
other critical tissues or structures. In case an image is not available, the surgeon can
draw the treatment area from scratch. Starting from the obtained picture, the software
optimizes automatically the electrode placement to get the complete coverage of the
treatment area with the required electric field.
The result of this optimization (Fig. 19) is a picture showing the estimation of the
electric field coverage. This estimation has to be evaluated by the surgeon and
accepted. When accepted, the software creates the protocol and asks the user, if
needed, to divide the treatment area into subdomains to simplify the treatment
considering that the device has only six output channels. The final step is the export
on an USB pen drive of the planning, with the image of the tumor and the forbidden
zones, to be used with the Cliniporator Vitae.
Fig. 19 Pulsar screens: (a) DICOM image; (b) tumor boundaries; (c) optimization; (d–e)
subdomains
958 C. Bertacchini
Conclusions
This chapter has shown the medical device Cliniporator™ from the origins to the
actual devices composing an electroporation platform. This device has been the first
clinical device using the electroporation to enhance the effect of drugs for tumor
treatments. The author has provided an overview not only of hardware, software, and
applied parts but also of the effort done to get the CE0051 marking. A small part of
the chapter has provided also an overview of the main treatment strategies needed to
have an effective electroporation treatment. The big effort made to bring the elec-
troporation in the clinic makes the Cliniporator™ as a milestone. With this medical
device, the application of the electroporation to the tumor treatment has become a
reality creating a methodology with a minimal invasivity, a very fast application
time, and a simple and clear usability.
References
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applications. Gene Ther 11(1):S33–S42
Cadossi R, Ronchetti M, Cadossi M (2014) Locally enhanced chemotherapy by electroporation:
clinical experiences and perspective of use of electrochemotherapy. Futur Oncol 10:877–890
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Cukjati D, Batiuskaite D, André F et al (2007) Real time electroporation control for accurate and
safe in vivo nonviral gene therapy. Bioelectrochemistry 70(2):501–507
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safety and essential performance
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and essential performance – Collateral standard: Usability
Fini M, Tschon M, Ronchetti M et al (2010) Ablation of bone cells by electroporation. J Bone Joint
Surg Br 92(11):1614–1620
of electric field distribution in mouse tumor during electroporation. Radiology 274:115–123
Marty M, Sersa G, Garbaya JR et al (2006) Electrochemotherapy – an easy, highly effective and
safe treatment of cutaneous and subcutaneous metastases: results of ESOPE (European Standard
Miklavčič D, Semrov D, Mekid H et al (2000) A validated model of in vivo electric field distribution
in tissues for electrochemotherapy and for DNA electrotransfer for gene therapy. Biochim
Biophys Acta 1523(1):73–83
Miklavčič D, Pavšelj N, Hart FX (2006a) Electric properties of tissues. Wiley encyclopedia of
biomedical engineering. Wiley, New York, pp 3578–3589
Miklavčič D, Corovic S, Pucihar G et al (2006b) Importance of tumour coverage by sufficiently
high local electric field for effective electrochemotherapy. Eur J Cancer Suppl 4(11):45–51
Mir LM, Orlowski S, Belehradek J Jr et al (1991a) Electrochemotherapy potentiation of antitumour
effect of bleomycin by local electric pulses. Eur J Cancer 27(1):68–72
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malignant tumours by electrochemotherapy. Br J Cancer 77:2336–2342
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Ongaro A et al (2011) Identification of in vitro electropermeabilization equivalent pulse protocols.
Project reference: QLK3-CT-1999-00484QLK3-CT-1999-00484. Cliniporator: a new adaptative
generator for dna electrotransfer in vivo for gene Therapy. Available via DIALOG. http://cordis.
europa.eu/project/rcn/51516_en.html
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Mechanisms of in vivo DNA electrotransfer: respective contributions of cell electro-
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Surg Oncol 34:232–240
Development of Pulsed Antennas for
Noninvasive Exposures 52
Shu Xiao
Abstract
Electric pulses in the subnanosecond range can be radiated with pulsed antennas.
Large antennas, such as prolate spheroidal antennas, allow the pulses to be
focused in biological tissues, but the field intensity is generally low as the pulses
suffer the losses including the one proportional to the distance of the targeted
tissue, the reflection loss at the air-tissue interface, and the attenuation loss in the
tissue. Thus, the field intensity may not be sufficient to cause significant biolog-
ical effects such as stimulation or electropermeabilization. Small antennas, such
as a dielectric rod antenna, can be used to improve the field intensity. The
dielectric antenna has a conical wave launcher, a rod wave guide, and a conical
emitter. It can make direct contact with tissues, eliminating the propagation in free
space and reducing the reflection loss occurring at the air-tissue interface. Since
the antenna is physically small and the distance to the targeted tissue is short, the
propagation loss (which increases with distance) can be lowered. From the
antenna perspective, small antennas are not as efficient as large antennas as
their sizes are typically smaller than the spatial distribution of the pulse, and
therefore, a high-voltage source needs to be employed to drive the antenna. But
small antennas have the advantages of higher coupling efficiency to tissue and
smaller footprint on the tissue surface. In a case that a subnanosecond pulse
(100 ps) was delivered for transcranial stimulation, the simulation results showed
that the penetration depth is approximately 2 cm and the field is concentrated in
an area of 1 1 cm. A pulsed power system capable of generating a few
hundreds of kilovolts is expected to be sufficient to drive the antenna in order
to generate electric fields sufficient for biological responses.
S. Xiao (*)
Department of Electrical and Computer Engineering, Frank Reidy Research Center for Bioelectrics,
Old Dominion University, Norfolk, VA, USA
e-mail: sxiao@odu.edu

DOI 10.1007/978-3-319-32886-7_221
962 S. Xiao
Keywords
Subnanosecond pulses • Picoseconds pulses • Pulsed antenna • Dielectric antenna
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 962
General Consideration of Wave Propagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 963
A Dielectric Rod Antenna . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 966
Radiated Electric Field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 968
Pulsed Power to Drive the Antenna . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 970
Application of Antenna to Stimulate Neurological Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 972
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 974
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 975
Introduction
Ultrashort electric pulses in the subnanosecond range promise a breakthrough in

bioelectrics, i.e., antennas can be used as the delivery system rather than electrodes,
thus allowing for noninvasive access to deep tissues. Recent in vitro bioelectric
studies have created better understanding of the responses of biological cells
exposed to subnanosecond pulses, and the approximate electric field thresholds
were determined. The first experiment of neuronal stimulation was reported (Xiao
et al. 2013) in which the electric field was approximately 10–50 kV/cm when the
pulses were repetitively applied. Other effects, such as cell permeabilization or cell
death, require prolonged exposure accompanied by additional heat (Xiao et al. 2011;
Camp et al. 2012). For a single pulse, a much higher electric field is required. An
electric field of 200 kV/cm for a 500 ps pulse was needed in order to depolarize a cell
membrane or to open voltage-gated channels (Semenov et al. 2015; Xiao et al.
2016). It can be thus assumed that cell death by subnanosecond pulses is generally
hard to achieve, but stimulating cell signaling or modulating cell functions is
attainable with moderate exposure conditions in terms of electric field magnitude,
pulse number, and pulse repetition rate at moderate heating.
Coupling electric fields to biological tissues was extensively investigated in the
1950s and most of the research focused on narrow band, continuous waves (Schwan
and Li 1956). Ultra-wideband electric fields were shown to create fewer hot spots
than narrow band fields and thus can be a better option in therapy (Converse et al.
2006). To deliver wideband pulses, travelling wave antennas, resistively loaded
antennas, and multi-resonance antennas can be used (Kanda 1986; Chen 2007).
With new materials and signal processing techniques, more broadband antennas are
now emerging. While there can be various types of antennas suitable for wideband
operation (Wiesbeck et al. 2009), their power capability in general is low and
prevents their use in bioelectrics. The high-field threshold calls for high-power
antennas. The prolate spheroid impulse radiating antenna (PSIRA), a reflector
type antenna for near-field targeting designed by Baum et al. (Baum 2007; Kumar
et al. 2011), has the potential to generate large electric fields in tissue when it is
52 Development of Pulsed Antennas for Noninvasive Exposures 963
driven by a pulsed power system. The electric fields produced by the reflector type
antenna can be sufficient to induce biological effects. Previously, a parabolic
impulse radiating antenna (IRA) designed for long distance targeting was shown
to generate a peak electric field of 68 kV/m at 10 m when the pulse fed into the
antenna was 1 MV and had a rise time of 200 ps and pulse width [full width at half
maximum (FWHM)] of 400 ps (Prather et al. 2003). At a shorter distance, the
electrical field in air could be much higher and close to the required field strength
for stimulation of cells or tissues (approximately 20 kV/cm). Yet, the electric field in
a biological target is strongly reduced compared to that in air because of the
reflection at the interface of air and tissue, which is due to the large difference in
dielectric permittivity. To reduce the reflection, it is important to keep the wave
propagating in a dielectric rather than air until it reaches the tissue. A dielectric
antenna provides such an option and can also emit high-intensity electric fields. The
dielectric constant can be chosen to approximate the tissue, allowing one to increase
the transmittance of radiation by making direct contact between the antenna and
tissue. Also, because the antenna is dielectrically loaded, it can be constructed in a
much smaller size than a prolate-spheroidal antenna, which typically is constructed
in air.
General Consideration of Wave Propagation
Tissues that are under consideration usually have a high water content, which gives
them a high dielectric constant. The skin, for example, has an average dielectric
constant of approximately 40 despite that it has multiple layers (epidermis, dermis,
and hypodermis) (Sasaki et al. 2014). This high constant stays relatively stable
throughout a wide band (from 0.9 to 2.45 GHz), and therefore, the pulse broadening
due to dispersion is insignificant (Fig. 1). For a pulse from medium 1 (e1) entering
medium 2 (e2), the transmission coefficient for a planar wave is
" 12 #1
Etransmitted e2
T¼ ¼2 1þ (1)
Eincident e1
For a pulse in air (e1 = 1) penetrating the skin (e2 = 40), T = 0.3. This is a
significant reduction of the field. But if the pulse is in a dielectric that has a dielectric
constant of about (e1 = 10), T can be greatly increased, e.g., to 0.9. One needs to be
cautious though in using this transmission coefficient, as the wave emitted from the
antenna in general is not planar in nature. Nonetheless, reducing the difference
between dielectrics becomes critical for increasing transmission.
An electromagnetic pulse can be confined in a dielectric rod and propagates over
a distance. The dielectric rod is widely used as optic fibers in which total reflection
occurs and the associated energy loss is negligible. While many modes of waves can
propagate in the rod, the fundamental mode (lowest) is HE11 mode, for which there is
964 S. Xiao
Fig. 1 The dielectric

constants for typical
biological tissues in the range
of 0.9–2.45 GHz (Data was
taken from the HUGO model
(CST 2016))
no cutoff frequency. As such, wideband signals are favored. Outside the waveguide,
an evanescent wave propagates with the speed of light in free space, but the field
intensity decreases exponentially in the radial direction from the waveguide surface.
So the energy is mostly confined in the center of the waveguide. When determining
the diameter of the rod, a rule of thumb is that the radius R should be greater than the
spatial width of the pulse:
tw c0
R > pffiffiffiffi (2)
er
where tw is the pulse duration and er is the rod’s dielectric constant. This means
that the pulse reflected from the rod-air interface will be temporally separated from
the main pulse on the axis as it progresses. The axis is the location of the highest
power density, and consequently, the pulse is defined mainly by the waves on the
axis. For small-radius rods, the reflection adds to the existing pulse in time, creating
distorted waveforms. This distortion is pronounced for low-frequency waves or long
pulses. Therefore, the radius essentially determines the low-frequency limit of the
antenna.
Once the pulse enters a homogeneous tissue, the field is usually confined in a
single spot and the size is determined by the pulse width. This differs from the field
distribution of a standing wave pattern created by narrowband radiation. In a target
that comprises multiple tissues, the pulse may experience reflections and refractions,
forming a standing-wavelike field distribution, but the field distribution is still
largely dictated by the pulse width. Very thin or small structures may slightly
broaden the pulses, but will not change the overall field distribution. As shown in
a simulation study (Guo et al. 2013), when a pulse radiated by a PSIRA was sent to
the brain, it was focused in the deep region (7–8 cm from head apex) despite the
inhomogeneous composition of the brain. This can be understood in a simple
scenario in which a planar wave propagates in a 1-D stratified target (Fig. 2)
where there is a thin layer of heterogeneous tissue. Suppose there is an interest
Fig. 2 The electric pulse shape observed in multiple layers in the presence of a heterogeneity. The
circle indicates an observer in a medium observing the original transmitting pulse and the reflected
pulse by a heterogeneity (dark color). The pulse that has two peaks in time corresponds to two spots
in the space, whereas one peak corresponds to a single spot
point where one needs to observe the field (indicated by the circle in Fig. 2). When
the original transmitting pulse encounters a heterogeneity that has a different dielec-
tric constant (ed) and a thickness of l, scattering occurs. The backward-scattered
pulse will be seen by the observer along with the original pulse. Inside the hetero-
geneity, the transmitted pulse will continue to propagate until it hits the second
discontinuity, which is the first dielectric. A second back scattering will occur. After
a round-trip time (2Tp) of the pulse in the heterogeneity, the observer will detect a
second signal. The delay between the first peak and the second peak is determined by
the dielectric property and the thickness of the dielectric.
Under the condition that the round-trip time is longer than the EM pulse width,
i.e., 2Tp > tw, the two pulses can be differentiated. The manifestation of these pulses
is that they form two adjacent but discreet spots in space. But when 2Tp < = tw,
these two pulses cannot be resolved and they all appear as a single spot. In
microwave imaging, the pulse width determines the minimum resolvable dimension
of the tissue heterogeneity, known as the “diffraction limit.”
pffiffiffiffiffi
l ¼ tw c0 = ϵ d (3)
In a treatment where one wishes to confine the electric pulse in a single spot, Eq. 3
shows the largest size of an object that will not cause extra spots. Evidently, the
diffraction limit is determined by the pulse width and the dielectric constant of the
tissue heterogeneity. Shorter pulses can be confined rather well, but their spot size
can easily be broadened in space by small heterogeneities with higher dielectric
constants. For example, for a pulse of 120 ps, if the heterogeneity has a dielectric
constant close to water (ed = 80), the diffraction limit l is 2 mm, which means any
heterogeneities greater than 2 mm will spread the pulse. Generally, an EM pulse
could experience multiple reflections at dielectric interfaces, resulting in resonance
966 S. Xiao
phenomenon. In this case, if the medium is lossy, the resonance is damped and will
not affect the size of the spot that has the largest field.
A Dielectric Rod Antenna
Large antennas such as IRAs will encounter several issues when the pulse is directed
to a tissue. First, launching electric pulses from pulse antennas is usually done in air,
which is a common approach in IRAs. After being emitted as a spherical wave, the
pulse’s intensity drops and is proportional to the inverse of the distance from the
focal point, although the field will eventually converge in the vicinity of the second
focal point. Antennas with a large reflector area may slow this field drop. Second, the
radiated pulse faces the reflection loss once it encounters tissue. Finally, the trans-
mitted pulse in the tissue will be attenuated due to tissue’s finite resistivity. All these
factors suggested that delivery of a pulse from air to a deep target may not be as easy
as generally conceived. One should instead look into shallow targets, for example,
those that are a few centimeters deep. In this case, the pulse can be confined in a
dielectric antenna of manageable size, and the reflection loss can be greatly reduced.
As the propagation distance is reduced, the field at the target may not drop too fast
even in the presence of tissue attenuation and should still be sufficient to meet the
threshold. Such a dielectric antenna was studied in Petrella et al. (2016a) and consists
of three sections:
1. A wave launching section that is a hollow dielectric conical TEM wave guide.
This structure is commonly used for launching waves to a dielectric lens (Baum
1976). In principle it is an aperture antenna but has a small aperture area.
2. A dielectric rod, i.e., a cylindrical wave guide, which is used to confine the
electromagnetic waves and guide them to the emitting section
3. A dielectric cone as the wave emitting section. The conical segment acts like a
convex lens, confining the electric field to a small area, and provides the coupling
between antenna and tissue, reducing reflections.
In section 1, two triangular metal plates are bent in a conical shape to symmet-
rically cover part of the dielectric cone. This part of the antenna is defined by two
angles: the apex angle θ0 and the azimuth angle ϕ0. Such an antenna was studied
(Shen et al. 1988). The important features of an infinitely long version of this antenna
(consisting of only the wave launching section) are (1) frequency independence
(important for wide-band pulses), (2) uniform azimuthal directivity, and (3) TEM
(transverse electromagnetic) mode excitation. However, one has to deal with an
antenna that has a finite length, which can be as short as a few centimeters. The
scattering of the electromagnetic waves at the end of the conical transmission line
will cause pulse ringing or pulse broadening at the target. This corresponds to the
increase in pulse duration at the target. This may be unfavorable for radar or
communication applications, but for biological applications, it may be useful since

the threshold electric fields for biological effects generally are reduced as the pulse
duration increases, thus easing the requirement of the pulsed power system.
The radiated field from a dielectric cone can be obtained from antenna aperture
theory. The dominant field is in the y direction for a cone that has the z axis, and it
can be estimated (Baum 1987) as
!
ð1Þ a
2
@ R0 ð2Þ a
2
R0
E y R 0 , t ¼ E 0 α y, y f t þ αy, y 2 f t (4)
R0 c @t c R0 c
where R0 is the distance of an observer on the axis from the aperture center. The
input pulse f(t) and its time derivative term @f(t)/@t represent the speed of the charges
(or current) and the acceleration, respectively. The coefficients α(1 or 2)y,y, depicting
the integral of the tangent electric fields over the aperture plane, indicate that
homogeneous unidirectionally aligned “currents” resulting from a uniform illumi-
nation produce larger coefficients and hence larger electric fields. In this case, a low
impedance conical wave launcher with a large covering angle (ϕ0) is preferable for
radiating waves at high intensity. The aperture size preferably is comparable to the
observing distance, so the ratios of α(1or2)y , y over R0 or R02 are not small. Seemingly
it would be a good practice just to have section 1 as an antenna that makes contact
with the target. However, doing so will result in a field pattern on the target that is
identical to the static field established by the two curved plates. Dominated by the
second term in Eq. 4, the field would be concentrated near the two projected regions
from the gaps of the curved metal plates. This is undesirable if one wishes to confine
the field in the centered region. To resolve this problem, the static electric field,
governed by the antenna structure, needs to be suppressed by increasing the distance
from the target. As indicated by Eq. 4, one needs to increase R0 and reduce the field
dependence of the 1/R02 term. A dielectric rod in this case provides such separation
and in the meantime confines the electric field.
At the intersection of the conical wave launching section with the cylindrical
wave guide (section 2), and in order to preserve the pulse shape of the incident pulse
in the cylindrical wave guide, the incident waves (section 2) should ideally be planar
waves. However, this can only be achieved by using a small slope launcher (large
β0 and small θ0), which resembles to two parallel plates in some degree. It seems that
keeping the wave launching section short (large θ0) has certain advantages such as
low losses due to scattering and high axial electric fields in the rod. However, that
needs to be balanced against the distortion of the waves. Considering the importance
of preserving the pulse shape in the antenna, it might be still preferable to use a wave
launcher with a small apex angle.
As far as the length of the dielectric wave guide L is concerned, it should satisfy
the condition (Eq. 5):
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi pffiffiffiffi
R R er
þ R þL
2 2
L > tw (5)
sin θ0 tan θ0 c0
968 S. Xiao
Fig. 3 A dielectric rod antenna used for delivering electrical pulses to shallow targets
This condition ensures the pulse will not be broadened once it reaches the end of
the waveguide. The waves scattered at the discontinuity at the interface of the
dielectric rod and the air also contribute to the pulse duration if they reach the end
of the wave guide at the same time and cause a broadening of the pulse.
The purpose of the third section of the antenna is to radiate the electric field into
the tissue. Applying the ray theory, the dielectric taper refracts the incident waves
and directs them toward the apex. In this way the terminal taper acts like an axicon
with a majority of the energy entering the tissue near the apex. The impinging ray
should be reflected at the surface of the cone and then exit from the cone apex. This
requires the angle of the cone to be α0, the critical angle for total internal reflection
(Fig. 3):

1 1
α0 ¼ sin pffiffiffiffi (6)
er
This ensures the rays that are in parallel with the axis reach the cone apex and
radiate into the tissue. For fields scattered away at the end of the wave launcher, the
incident angle, α, can be larger than α0. They are still totally reflected, but the
reflected waves create focal points inside the cone, rather than reaching the cone
apex. When α is less than α0, refraction occurs, and the transmitted wave departs the
cone and becomes spread around the cone apex. This causes a widening of the focal
spot in the tissue.
Radiated Electric Field
An antenna that has taken into account the preceding discussions was designed and
optimized through simulations (Petrella et al. 2016a). The geometry is shown in
Fig. 4a. The dielectric material for the antenna has a dielectric constant of 7.3 and
was made of a proprietary nanocomposite material by TPL (Albuquerque, New
Mexico). The antenna was tested in the free space (Fig. 4b) with subnanosecond
pulses (5-P-V, FID, Germany) and a Prodyn AD-80(R) D-dot sensor. The electric
Fig. 4 The experimental setup for characterizing the antenna in the free space. (a) The dielectric
rod antenna. (b) A Prodyn D-dot sensor was used
field on the axis at 3 cm from the apex of the antenna is shown in Fig. 5. As indicated
in Eq. 4, the electric field has temporal dependences on the original pulse and its time
derivative. But even for such a short distance, the electric field already exhibits
almost identical temporal dependence as the time derivative (comparing Fig. 5a, b).
It becomes obvious that having a fast rise time is beneficial to the radiation. Usually,
if a pulse is measured in a far zone, the area of the pulse should be zero in order to
satisfy the charge neutral condition. This is the case for an IRA, whose pulse
waveform has a prepulse and impulse. Their areas are equal but opposite in sign,
leaving the total algebraic area summation to be zero (no dc in the radiated field). In
this antenna, because the measurement was done in the near-field region and the
coupling of signals can occur through stray capacitances, it is not clear that the area
under the pulse should be zero and the exact match of pulse measurement and
simulation result may not be realizable.
It is noted that a typical symmetrical (balanced) antenna like the one shown in
Fig. 4 should be connected through a balun transformer to an unbalanced transmis-
sion line, such as a coaxial cable that has conductors of different dimensions so that
the current distributions on the antenna conductors are identical in order to suppress
common mode radiation. This is particularly important if one is interested in the far
field. But if the near field is of interest, one can simply use a coaxial cable to feed the
pulse, and a balun becomes unnecessary, as the differential-mode wave from the
symmetrical curved plates arrives at the target much earlier than the common mode
radiated from the outer shielding of the cable. Temporally, they are separated. In
970 S. Xiao
Fig. 5 (a) The voltage fed to

the antenna and its time
derivative. (b) The electric
field measured on the axis of
the antenna along with the
simulation result
biological applications, the role of the common mode may not be as detrimental as in
communications or radar, and they may even be helpful in lowering the electric field
thresholds for some biological effects.
Pulsed Power to Drive the Antenna
The electrical pulses that drive a pulsed antenna should have a transient phase in the
subnanosecond range as the flat part does not contribute to the field. In a simple case,
a waveform generated by discharging a capacitor in fast time should suffice.
Considering the high electric field thresholds for biomedical applications, the driving
pulses should be high voltage. Usually, they are produced by a Marx generator or a
Tesla transformer. The schematic of a complete system that has a Tesla transformer is
shown in Fig. 6. The antenna can be treated as a capacitive load that is driven by a
rapid charger. The Tesla transformer operates in the dual resonant condition and has
an air core (DRPT, Dual Resonance Pulse Transformer) as it has a linear current-flux
density relationship. It allows for a near 100% efficiency when the circuit is in
Fig. 6 The high-power dielectric antenna can be driven by a dual resonant air core transformer
through a peaking switch, which produces fast rise time of the pulse
resonance condition. The DRPTs are low cost, compact, and simple to build and can
be constructed to function in the megavolt range. For these reasons, DRPTs are
considered viable alternatives to Marx generators which require a network of
switches and capacitors to operate. The discrete circuit model is accurate for
describing the operation of the DRPT. An ideal dual resonant condition can be
used to describe the temporal evolution of the pulse voltage and current. The
capacitors in the primary have no equivalent series inductance and are connected
by ideal wires. The only inductance present in the circuit is contained in L1 and L2.
The gain or the maximum value of the second voltage excursion is described by
the square root of the ratio of the inductance or capacitor values (Korioth et al. 1999):
rffiffiffiffiffi rffiffiffiffiffiffi
Vo L2 C1
¼ ¼ (7)
V L1 C2
For DRPTs this is considered to be an estimate. The gain is for the ideal case
where the transformer is both perfectly resonant and has no resistive losses or losses
in magnetic flux. In a slow charging
qffiffiffiffi resonance frequency system, it is possible to
L2
achieve a voltage gain of 0:9 L1 (Lim et al. 2011). However, it is unlikely to have a
qffiffiffiffi qffiffiffiffi
gain of 0:9 LL21 in an ultrafast low inductance system. A gain of ð0:6 0:7Þ LL21 was
considered realistic for this setup.
Figure 7 shows typical waveforms for the primary voltage, secondary voltage,
and secondary current generated in a DRPT charger (Petrella et al. 2016b). It also
shows the relation between the charging voltage of the primary capacitor and the
peak of the secondary voltage. When the transformer was operated in air without an
insulating medium, the primary voltage was 11.8–41.2 kV (which is the maximum
capacitor rating). This sweep of primary voltages produced linearly increasing
secondary voltages from 47 to 124 kV. This represents a range of voltage gains
between 3 and 3.98.
With the fast-ringing voltage, one can use a peaking switch to generate a fast rise
time pulse and feed it to the pulse antenna, like the one shown in Fig. 6. This concept
was implemented in JOLT, an impulse-like radiator (Baum et al. 2004). The peaking
switch breaks down at the second peak of the voltage across the second transformer
winding, where the energy transfer reaches maximum. The closing speed of peaking
972 S. Xiao
40 150 1500
Secondary Voltage (kV)
Secondary Current (A)

Primary Voltage (kV)
100 1000
20
50 500
0 0 0
-50 -500
Primary Voltage
-20
Secondary Voltage
Secondary Current -100 -1000
-40 -150 -1500

-600 -400 -200 0 200
Time (ns)
Fig. 7 The voltage and current waveforms measured in the transformer (Petrella et al. 2016b)
switches can be hundreds of picoseconds. They are usually spark gap switches with
liquid or gas as switching medium. Oil, for example, was used in JOLT. For a gap
distance of 0.76 mm, the rise time of the pulse can be as short as 130 ps when a
charging pulse of 1 MV was applied. To remove the debris generated in the
breakdown, a high flow rate was maintained when the system was operated at
200 Hz. To have a higher operation rate, one can also use high-pressure gas switches.
At 10 atmospheres, an air spark gap exhibited a hold-off strength of 1 MV/cm
(Cravey et al. 1992).
Application of Antenna to Stimulate Neurological Tissue
The classic applications of electric pulses mostly include electroporation and electro-
stimulation, which are the result of a change in transmembrane potential. Ultrashort
electrical pulses radiated from antennas have not been used in these applications.
Previous in vitro data showed that electroporation by subnanosecond pulses is hard
to achieve, especially if one wants to introduce large molecules to cells through
membranes (Xiao et al. 2011). Stimulation of excitable tissues, on the other hand,
seems more achievable (Xiao et al. 2013). The research of biomedical application of
antenna pulses in general is scarce, although the promise of noninvasive delivery
could change the current status. As an engineering effort in advance of the biological
research, the dielectric rod antenna was studied in a simulation aiming to stimulate
neurological tissue (Petrella et al. 2016a). The antenna was radiated to the brain in a
HUGO model (Computer Simulation Technology, Microwave Studio ®). It is a
partial human head voxel model and consists of seven dielectrics with dielectric
properties of skin, fat, bone, blood, optic nerve, and brain (gray and white matter). A
dielectric that has er = 7.3 was selected as the antenna dielectric material.
Fig. 8 Cross-sectional view of the brain tissue at three depths shows the field decreasing in
magnitude and size with increasing depth (Petrella et al. 2016a)
The performance of the antenna was examined with respect to the electric field
gradient, as the electric field decreases as the wave penetrates, but the field at the skin
covering the skull should not be excessively large compared to that in the target
zone, so that damage can be avoided; the spot size of the field confinement as well as
the magnitude of the electric field in the brain tissue was also looked upon. If one
defines the stimulation spot size as the area that has an electric field value greater
than 90% of the peak value, the thickness and width of the stimulation spot can be
estimated to be 5 mm by 11 mm (Fig. 8). At the surface, the spot size is on the order
of the diameter of the dielectric rod and shrinks until reaching 11 mm in width in the
brain tissue (2 cm deep). This field concentration is caused by tissue attenuation. The
electric field is highest at the center and remains so at deeper regions in the brain, but
as the depth increases, the peak electric field decreases, and the electric field in the
brain tissue is approximately 42% of that at the skin surface (Fig. 9). One should
notice that 42% is higher than 36.8% (1/e), a number that is typically used to define
the penetration depth, which means the penetration depth could be deeper than 2 cm
for the pulse that was considered (100 ps Gaussian pulse). For a narrow band signal,
which is defined by one frequency, the penetration depth can be determined directly
from a known formula (Petrella et al. 2016a). But for wideband pulse, the
974 S. Xiao
Fig. 9 The electric field

distribution from the skin to
the brain. The electric field
magnitude decreases from
27.6 V/m at the skin surface
and reaches 11.5 V/m inside
the brain tissue
penetration depth is not obvious, especially if the target consists of several tissues.
One can only determine it from the simulation. In terms of absolute field, the peak
field in the brain is 11.5 V/m for a 1 V input. This implies that skin tissue may need to
tolerate a field level two to three times that of brain tissue. Using 20 kV/cm as a
threshold for a biological effect, a 175 kV pulse power supply would be required.
Conclusions
The research of biological applications and delivery of subnanosecond pulses is an

emerging area. In one way, the biological effects have not been studied as exten-
sively as those caused by longer pulses. On the other hand, the high-power antennas
for delivery of subnanosecond pulses have not been developed. Nonetheless, the
pulsed power technology to generate subnanosecond pulses has made significant
progress in that 1 Megavolt sources are commercially available. This chapter
attempts to provide an example of how a high-power antenna can be designed.
Historically, the delivery of these ultrashort pulses has focused on long range, far
field. It was found that the electric field is less likely to meet the biological
thresholds. Thus, some effort was turned to the near-field application. Even so,
one still faces two major challenges: the delivery of pulses from air to tissue and
tissue attenuation. A step to reduce the severity of these challenges is to aim at
subcutaneous targets at a depth of several centimeters. A dielectric antenna in this
case can be a good option, at least better than needle electrodes. In a simulation,
electric pulses in the hundred picosecond range were successfully delivered to
subcutaneous regions of the brain. The antenna makes direct contact with tissue,
with no air gap, thus reducing the large reflection loss due to the difference in the
permittivity of air and tissue. For the dielectrically loaded antenna er = 7.3, the
electric field at a depth of 20 mm (inside brain tissue) was shown to be 11.5 V/m for a
1V input, and it is approximately 42% of that at the skin surface. In order to stimulate
the tissue, the critical electric field needs to be on the order of 20 kV/cm, as found in
the previous study. This critical field corresponds to an input voltage of 175 kV. This
voltage indeed is achievable with currently available pulsed-power systems. How-
ever, one should keep in mind that the targeted tissue type is not limited to
neurological tissue but can also be used for superficial targets such as skin or tissues
in culture.
Acknowledgment This work was sponsored by an R21 grant (1R21EB016912-01A1) from the
National Institute of Biomedical Imaging and Bioengineering, NIH, and a MURI project, “Nano-
electropulse-induced electromechanical signaling and control of biological systems” from AFOSR.
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1516-7
Beyond Electroporation Pulse Parameters:
From Application to Evaluation 53
Matej Reberšek
Abstract
Electroporation is a phenomenon that occurs in the membranes of biological cells
exposed to strong electric fields. The exposure of cells to strong electric fields
leads to temporary permeabilization of biological cells, thus allowing easier
transport of ions and molecules across the cell membrane. Electroporation is
already widely used in several biomedical and biotechnological applications. The
efficacy of electroporation strongly depends on the electric field parameters and
also other biophysical parameters. Strong electric fields are generated between the
electrodes by the delivery of high-voltage electric pulses to the electrodes close to
each other. Results obtained in different electroporation studies may vary signif-
icantly. This may be because the determination and description of the electric
fields to which cells are exposed during experimental work were not adequately
addressed or at least were not reported in sufficient details. To enable the
reproduction of electroporation experiments, researchers should adequately mea-
sure the electroporation pulses, evaluate the generated electric field, and provide
adequate description of experimental methods. In this chapter, parametrization
and parameters of electroporation pulses are described, techniques of controlling
the generation sequence of electroporation pulses are specified, and recommen-
dations for electroporation pulse measurement and reporting in electroporation
studies are suggested.
Keywords
Applications • Cells • Electric field • Electroporation • Evaluation • Generation •
Measurement • Parameters • Pulses • Recommendations
M. Reberšek (*)
e-mail: matej.rebersek@fe.uni-lj.si

DOI 10.1007/978-3-319-32886-7_222
978 M. Reberšek
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 978
Parameters of Electroporation Pulses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 979
Pulse Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 980
Series of Pulses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 985
Generation of Electroporation Pulses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 989
Electroporation Applications and Effects of Electroporation Pulse Parameters on Cells . . . . . 990
Measurement of Electroporation Pulses and Evaluation of Pulsed Electric Fields . . . . . . . . . . . . 993
Recommendations for Reporting in Electroporation-Based Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . 994
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 995
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 996
Introduction
Biological cells are separated from its surrounding environment by a cell mem-
brane. The cell membrane is selectively permeable to ions and organic molecules
and thus controls the transport of essential and redundant or even harmful sub-
stances in and out of the biological cells. This separation and controlled transport
of substances is vital for the cell and organism survival; however, it sometimes
prevents or reduces the effectiveness of certain biomedical or biotechnological
treatments. Ineffectiveness of certain treatments can be improved by increasing the
permeability of the cell membrane. The permeability of the cell membrane can be
temporarily increased by several methods. One of such method is electroporation
(Kotnik et al. 2012).
Electroporation is already widely used in several biomedical and biotechnological
applications. Thus the electroporation is already used for treating tumor cells with
electrochemotherapy (ECT, ▶ Chap. 93, “Electrochemotherapy and Its Clinical
Applications” by Gehl and Serša), for ablation of normal and tumor tissue with
irreversible electroporation (IRE), for delivery of DNA molecules into cells with
gene electrotransfer (GET), for microbial inactivation in liquid foods and waste-
waters), for extraction of molecules from the cells), etc. (Haberl et al. 2013).
Electroporation is achieved by the delivery of electroporation pulses to biological
cells. The electroporation pulses are electrical pulses with a particular time course to
achieve electroporation. The electroporation pulses are generated by pulsed power
generators known as electroporators. Pulsed power generators operate in two phases,
a charge and a discharge phase. During the charge phase, electrical energy is
accumulated over a long period of time in an energy storage element such as
capacitor. In the discharge phase, stored electrical energy is quickly released into
the load, thus generating an electrical pulse. In electroporation, the load is a cell
suspension or a tissue. Electrically, cell suspension or tissue can be modeled as a
capacitance and variable resistance in parallel where resistance in principle decreases
with the level of cell permeability. Electroporation pulses are delivered to the cells
53 Beyond Electroporation Pulse Parameters: From Application to Evaluation 979
through electrodes between which a pulsed electric field (PEF) is generated, thus
exposing cells to PEF (Reberšek et al. 2014).
During the process of electroporation, series of electroporation pulses are usually
delivered to the electrodes. Each series has a specific voltage and current time course
which can be measured by oscilloscope using voltage and current probes. However,
the cells are exposed to the PEF between the electrodes, and it is currently not
possible to measure the time course and distribution of the electric field during the
delivery of the pulses. Therefore, the time course and distribution of the PEF
between the electrodes to which the cells are exposed to must be calculated from
the electrode geometry and measured electroporation pulse time course (Batista
Napotnik et al. 2016). Time course of the electroporation pulses can be displayed
in a figure. However, to quantitatively compare different electroporation pulses, a
parametric description of electroporation pulses is needed which should be made as
accurate as possible.
The process of the electroporation is strongly dependent on the pulse parameters
of the delivered electrical pulses and also other biophysical parameters such as
temperature, osmotic pressure, size and shape of the cell, etc. (▶ Chap. 75, “Param-
eters Affecting Cell Viability Following Electroporation In Vitro” by Rols). In order to
control the process of the electroporation and to exactly specify the experimental
method, and thus enable the reproduction of experiments under the same conditions,
researchers should determine and describe these electrical parameters precisely in their
electroporation-based studies (Campana et al. 2016; Raso et al. 2016).
Results obtained in different electroporation studies vary significantly (Batista
Napotnik et al. 2016; Raso et al. 2016). This may be because in some studies the
determination and description of the electric fields to which the cells were exposed
during experimental work were not adequately reported or at least were not reported
in sufficient detail.
The focus of this chapter is on parametric description of electroporation pulses,
methods for controlling the protocol of generation of electroporation pulses, the use
of specific waveforms and series of pulses, measurement and evaluation of electro-
poration pulse delivery, and recommendations for reporting in electroporation-based
studies.
Parameters of Electroporation Pulses
Parametric description of electroporation pulses is used for quantitative presentation

and comparison of the electroporation pulses used in different studies. Parameters
are determined from the time course of pulse voltage or current. A series of several
electroporation pulses are usually used in electroporation. If all the pulses are similar,
it is enough to define one and the way of their repetition. Parameters of electropo-
ration pulses are therefore divided into parameters of a pulse and parameters of a
series of pulses.
980 M. Reberšek
Pulse Parameters
Electrical pulses may vary in their shape, amplitude, duration, and polarity. Ampli-
tude and duration of the pulse are a part of a pulse shape and are the most influential
parameters in electroporation. During the transmission of extremely short pulses
from the generator to the load, a reflection of the pulse may occur on the transmission
path due to impedance mismatch which will affect the pulse shape, amplitude,
duration, and polarity of the pulse. Electroporation pulses that are delivered to the
electrodes generate pulsed electric field (PEF) between the electrodes to which the
cells are exposed to. Therefore, in order to specify precisely the experimental
conditions to which the cells were exposed to, a distribution of electric field between
the electrodes has to be determined or calculated from the pulse amplitude.
Pulse Shape, Amplitude, and Duration

Predominantly, two main pulse shapes are used in electroporation studies: square
wave pulses and exponentially decaying pulses. Each pulse shape has its own
descriptive parameters.
Ideal square wave pulse has a sharp turn on and off times and stable amplitude
during the pulse (Fig. 1a). Ideal square wave pulse can be characterized by standard
pulse parameters, i.e., amplitude (U) and pulse duration (tFWHM) where U is a plateau
amplitude calculated as a mean value of the pulse plateau amplitude and tFWHM is a
duration of the pulse at half amplitude (FWHM – full with at half maximum).
In reality, the amplitude of the pulse requires some time to rise and fall on the
capacitive load, as is the biological load. Rise (tR) and fall time (tF) parameters
are used for quantification of the duration of this transition (Fig. 1b). Rise time
equals the time in which the amplitude rises from 10% to 90% of its plateau
amplitude (U ) and vice versa for the fall time. During the on/off switching of
electroporation pulses, some overshooting or ringing may occur (Fig. 1c) which
can be quantified by maximal amplitude (UMAX) of the overshoot and by the time of
ringing (tRING).
Some pulse generation techniques, such as square wave pulse generator
(Reberšek et al. 2014), deliver the energy to the load directly from the energy storage
capacitor. In this case, the amplitude (U ) of the pulse will drop (ΔU ) during the pulse
delivery (Fig. 1d), and if the capacitance (C) of the energy storage capacitor is too
low in comparison to the load impedance (ZL), this drop will be seen on the pulse
amplitude:
ΔU tP
ffi (1)
U C ZL
When square wave voltage pulses with short rise time are applied to the cell
suspension or tissue, a very distinguished current time course can be observed. As
the load is capacitive, a spiky capacitive current at the beginning of the pulse is
observed (Fig. 2a) also referred to as displacement current. The current in time settles
and becomes only resistive.
Fig. 1 Time course and electrical parameters of square wave electroporation pulse. (a) Ideal square
wave pulse, (b) square wave pulse with slower rise and fall times, (c) overshooting and ringing
during voltage switching, and (d) amplitude drop during the pulse delivery. Square wave electro-
poration pulse can be characterized by: amplitude (U ) which is the mean value of the pulse plateau;
pulse duration (tFWHM) which is the width of the pulse at 50% of the amplitude (U ); rise time (tR) in
which the amplitude rises from 10% to 90% of its amplitude (U ) and vice versa for the fall time (tF);
maximal amplitude (UMAX); ringing time (tRING) which is the time of amplitude oscillation; and
amplitude drop (ΔU ) which is the drop of the pulse amplitude during the pulse delivery
During the delivery of the pulses, the cells may become electroporated (mem-
brane conductivity is increased) and the cell suspension or tissue consequently less
resistant (more conductive). Moreover, the cell suspension or tissue may become less
resistant also due to Joule heating during the delivery of the pulses. In both cases, the
current will rise (ΔI ) during the pulse delivery (Fig. 1b). Both effects, capacitive
current and current rise, are usually observed together on typical electroporation
current time course (Fig. 1c). Rise of the current after the spike transition was used to
monitor the electroporation process during the pulse delivery (Cukjati et al. 2007).
Ideal exponential pulse can be characterized by maximal amplitude (UMAX) and
time constant (τ) of the pulse decay:
U ðtÞ ¼ Umax eτ ;

t
t0 (2)
Time constant can be derived by extrapolating decay rate to timeline; however, it

is usually much easier to find pulse amplitude equal to 37% of UMAX whose time
equals to τ (Fig. 3a).
982 M. Reberšek
Fig. 2 Time course and electrical parameters of the current through the load during the delivery of
square wave voltage pulse with short rise time to the load. (a) Capacitive current, (b) rise of current
due to increase of conductivity, and (c) typical electroporation current which includes both effects.
Current square wave electroporation pulse can be characterized by amplitude (I ) which is the
middle value of the pulse plateau, pulse duration (tFWHM) which is the width of the pulse at 50% of
the amplitude (I ), maximal amplitude (IMAX), and current rise (ΔI ) which is the rise of the pulse
current during the pulse delivery
Fig. 3 Time course and electrical parameters of exponential electroporation pulses. (a) Ideal
exponential pulse and (b) exponential pulse with slower rise time. Ideal exponential pulses can
be characterized by maximal amplitude (UMAX) and time constant (τ). Exponential pulses with
slower rise times are preferable characterized by standard pulse parameters such as rise (tR) and fall
time (tF), pulse duration (tFWHM), and maximal amplitude (UMAX)
However, as with the square wave pulses, also at the exponential pulses amplitude
cannot be switched instantaneously from zero to UMAX but has a certain turn on or
rise time (tR). In this case, the time constant is more difficult to be derived from the
time course of the voltage; therefore, it is preferred to characterize such pulses with
standard pulse parameters (Fig. 3b) such as rise (tR) and fall time (tF), pulse duration
(tFWHM), and maximal amplitude (UMAX).
Polarity
Electroporation pulses may also vary in polarity and can be either unipolar or bipolar
(Fig. 4). Unipolar pulses have only positive or only negative amplitude (Fig. 4a),
while bipolar have both. Ideal bipolar pulses have the same positive and negative
amplitude and positive and negative pulse duration and have no delay between the
positive and negative pulse (Fig. 4b). However, intentionally or because of pulse
Fig. 4 Polarity of a single electroporation pulse: (a) unipolar pulse, (b) ideal bipolar pulse, and (c)
bipolar pulse with delay. Bipolar pulses can be characterized by positive amplitude (UP), negative
amplitude (UN), duration of positive pulse (tP), duration of negative pulse (tN), and delay between
the positive and negative pulse (tDELAY)
generation method, positive and negative pulses may be different in amplitude or

duration, or delay may occur between the positive and negative pulse (Fig. 4c).
Pulse Reflections
Pulse reflections may be significant when short (nanosecond) electroporation pulses are
used. Electric pulses propagate through the cables with the speed of light which in
coaxial cables made from dielectric materials is a bit slower than in the vacuum. For
example, electrical pulses propagate through typical coaxial cable with approximately
20 cm/ns, while light in vacuum propagates with 30 cm/ns. Because of finite pulse
propagation speed, pulses when generated do not have the information about the loads
impedance, and its voltage current ratio is not yet adjusted to the load but to the
characteristic impedance of the generator. If there is no impedance, discontinuity from
the generator to the load the voltage current ratio remains unchanged, and the reflections
do not occur. Otherwise, when there are impedance mismatches on the transmission path
of the pulse from the generator to the load, the pulse will be reflected on these mismatches
(Fig. 5). Usually, the impedance mismatch occurs at the generator and the transmission
line connection or at the transmission line and the load connection. The ratio between the
reflected (U1) and the incident wave (U) is defined by reflection coefficient (Γ):
U1 ZL ZS
Γ¼ ¼ (3)
U ZL þ ZS
where ZL equals to the impedance toward the load and ZS equals to the impedance
toward the source (Smith 2002). If the rise time of the pulse is much longer than the
electrical length of the transmission path, which equals to propagation time of the
pulse from the pulse generator to the load, the voltage current ratio will be adjusted
within the rise time, and reflections will not be observed. Otherwise, non-inverted
reflections (Fig. 5a) can be observed if the impedance of the load is higher than the
impedance of the source or transmission line, and inverted reflections (Fig. 5b) can
984 M. Reberšek
Fig. 5 Reflections of short (nanosecond) electroporation pulses. (a) Not-inverted reflections occur
when impedance of the load is higher than the impedance of the transmission line, and (b) inverted
reflections occur when impedance of the load is lower than the impedance of the transmission line.
Pulse reflections can be characterized by reflection coefficient (U1/U ) and delay of the reflection
(tDELAY). If the reflection is inverted, reflection coefficient is negative
be observed if the impedance of the load is lower than the impedance of the source or
transmission line (Kolb 2010).
Pulse reflections can be characterized by the amplitude of the reflected wave (U1)
and the delay between the incident and reflected wave (tDELAY). With these param-
eters, also reflections of the reflected wave can be estimated.
Electric Field
The cells are exposed to pulsed electric field which is generated by the delivery of the
electric pulses to the electrodes. Therefore, to fully characterize the conditions to
which cells are exposed, electric field should also be estimated or calculated from
the pulse amplitude on the electrodes. Electric field is a field of vectors that are
assigned to each point in space where each vector represents Coulomb force
per electric charge. Electric field can be homogeneous or inhomogeneous where
homogeneous electric field is a field of equal vectors. The homogeneity of the
electric field depends on the geometry of the electrodes and the homogeneity of
the load conductivity (▶ Chap. 56, “Electric Field Distribution and Electroporation
Threshold” by Kranjc and Miklavčič). Plate electrodes (Fig. 6a) with short distance
(d) between the electrodes in comparison to the width of the electrodes have
relatively homogeneous electric field (E) which can then be estimated by
U
E¼ (4)
d
where U is the amplitude of the pulse. For needle electrodes (Čorović et al. 2007),
microelectrodes (Kolb et al. 2006; Arnaud-Cormos et al. 2011), or collinear elec-
trodes, the electric field must be calculated by numerical methods (Fig. 6).
a b
450 250
400
200
350
300
150
V/cm
V/cm
250
200
100
150
100 50
1 cm 50 1 cm
0 0
c 200
d
200
180 180
160 160
140 140
120 120
V/cm
V/cm
100 100
80 80
100 mm 1 cm
60 60
40 40
20 20
0 0
Fig. 6 Numerically calculated (Comsol Multiphysics 5.2, USA) static electric field between:
(a) plate electrodes, (b) needle electrodes, (c) microelectrodes, and (d) collinear electrodes in
flow-through chamber. Voltages applied to the electrodes are 1 V on the microelectrodes (c) and
100 V on other electrodes (a, b, d). The material used in the model is iron for the electrodes and
more conductive water (1 S/m) for the cell suspension. Colors are proportional to the electric field
strength. White area in microelectrodes represent cover glass, and in flow-through chamber, a tube
made of electrically insulating material. Electric field between plate electrodes is homogeneous with
the exception of the edges. Electric field between other types of electrodes is much stronger near the
electrodes in comparison to the middle. Around sharp edges of the electrodes high electric field is
present
Series of Pulses
Electroporation pulses are mostly used in series, i.e., several pulses are delivered in
sequence. There are many different ways how to combine electroporation pulses in
series. Researchers may deliver different number of pulses, vary pulse repetition
rates, deliver pulses in bursts, use combination of different pulses, or deliver
electroporation pulses in different directions relative to the cell. During the delivery
of series of pulses, electroporation pulses may unintentionally differ from pulse to
pulse. One of such occurrence which frequently occurs is an amplitude drop from
pulse to pulse. Depending on the method of combining the electroporation pulses in
series, the series of electroporation pulses are parametrically described in several
different ways.
986 M. Reberšek
Number of Pulses and Pulse Repetition Rate

The simplest and most commonly used way of combining electroporation pulses in
series (Fig. 7) is to uniformly repeat several (N) pulses with a specific pulse
repetition rate (PRR):
1
PRR ¼ ; tPERIODE ¼ tPULSE þ tPAUSE (5)
tPERIODE
where tPULSE is the duration of the pulse, tPAUSE is the time between the pulses, and
tPERIODE is the time from the beginning of one to the beginning of the next pulse.
Bursting
Electroporation pulses are sometimes delivered in bursts which means that the
electroporation pulses are per partes generated with high pulse repetition rate
(PRR) in several blocks/bursts which are separated by the burst delay (Fig. 8). The
complete number of delivered pulses (N ) in such treatment equals to
N ¼ NB NP (6)
where NB equals to the number of bursts and NP equals to the number of pulses in
each burst.
The bursts are uniformly repeated with a specific burst repetition rate (BRR):
1
BRR ¼ ; tBURST_PERIODE ¼ tBURST þ tBURST_DELAY (7)
tBURST_PERIODE
where tBURST is the duration of the burst, tBURST_DELAY is the time between the
bursts, and tBURST_PERIODE is the time from the beginning of one to the beginning of
the next burst.
Fig. 7 Series of uniformly repeated electroporation pulses. Series of pulses can be characterized by
number of pulses (N ) and pulse repetition rate (PRR). PRR is the inverse value of the pulse period
(tPERIOD) which equals to the time between the beginnings of two pulses or the sum of pulse (tPULSE)
and pause duration (tPAUSE)
Fig. 8 Electroporation pulses generated in bursts. Bursts can be characterized by the number of
bursts (NB), number of pulses in bursts (NP), burst repetition rate (BRR), and pulse repetition rate
(PRR) in each burst. BRR is the inverse value of the burst period (tBURST_PERIOD) which equals to
the time between the beginnings of two bursts or the sum of burst (tBURST) and burst delay duration
(tBURST_DELAY)
Fig. 9 Combination of
different types of
electroporation pulses
separated by the delay
(tDELAY)
Combining Different Pulses

Short and long electroporation pulses have different effects on biological cells. To
combine different effects or to achieve synergistic effect between different types of
pulses, they can be combined together in series (Fig. 9). Each type of electroporation
pulses are in this case usually generated in a particular block, which are combined
together in a sequence and separated from other blocks by the delay (tDELAY). The
sequence of generating different types of electroporation pulses may in this case be
of great importance.
Different Pulse Directions

Electroporation pulses can also be delivered to the cells so that the electric field
direction with respect to the cell is different in each block of pulses (Fig. 10).
Between two electrodes (Fig. 10; 1,2), electrical pulses can only be delivered in
one (single polarity, SP, Fig. 10a) or in two directions (both polarities, BP, Fig. 10b).
However, with multiple electrodes or by changing the position of the electrodes with
respect to the cell, the electroporation pulses can be delivered to cells in multiple
directions relative to the cell. Between four electrodes (Fig. 10; 1,2,3,4) positioned in
988 M. Reberšek
a square configuration, electrical pulses can be delivered also in two orthogonal

directions (orthogonal single polarity, OSP, Fig. 10c) or in four directions (orthog-
onal both polarities, O BP, Fig. 10d). Hexagonal arrangement of electrodes is also
used to deliver the electroporation pulses in multiple directions relative to the cell
(▶ Chap. 51, “Cliniporator: Medical Electroporation of Tumors” by Bertacchini).
Amplitude Drop in a Series

During pulse delivery, the energy is transferred from the generator to the load and the
voltage on the energy storage capacitor in the pulse generator drops. If the capaci-
tance of the capacitor is too low in comparison to the load resistance or the high-
voltage power supply cannot recharge the capacitor in the pause between the pulses,
this drop can be seen on the pulse amplitude (Fig. 11). During the delivery of series
of electroporation pulses, amplitude of pulses may drop (ΔUTOTAL) much more
significantly than at single pulse delivery (ΔU ).
Fig. 10 Delivery of
electroporation pulses in
different directions with
respect to the cell: (a) single
polarity (SP), (b) both
polarities (BP), (c) orthogonal
single polarity (OSP), and
(d) orthogonal both polarities
(OBP). Electrodes in square
configuration are numbered
from 1 to 4
Fig. 11 Amplitude drop

during the generation of
several square wave pulses
Generation of Electroporation Pulses
Electroporation pulses are generated by pulsed power generators known as electro-

porators. They are usually composed of user interface, control unit, high-voltage
(HV) power supplies, pulse amplifiers, and output module (Fig. 12). User interface is
used to monitor and to enter the parameters of electroporation pulses into the device
by the user. Control unit controls the HV power supplies and output module and
generates control signals for the pulse amplifiers. Control signals for the pulse
amplifiers are usually digital signals that define the timing of the electroporation
pulse generation sequence. HV power supplies provide the energy for the pulse
amplifier. Pulse amplifiers amplify or transform the control signals into high-voltage
pulses. Electroporators may have more than one HV power supply and pulse
amplifier depending on how many different combinations of electroporation pulses
they generate. Internal pulse measuring probes and commutator for several elec-
trodes are optional in electroporators and are located in the output module. Herein-
after the generation of control signals in electroporators will be described, whereas
the pulse amplifiers are thoroughly described in other chapters.
A general purpose function generator is very often used to generate control
signals for laboratory or prototype electroporators because of its simple user inter-
face and capability of generating wide range of signals. Function generators usually
have a pulse function where amplitude, offset, duration, and period of the pulse can
be set. On the 50 Ω input of the amplifier amplitude and offset of the pulse are
usually set to 5 V and 2.5 V, respectively, and on high impedance input 2.5 V and
1.25 V, respectively, to meet the digital levels of the pulse amplifier. Duration should
equal to pulse duration, or some adjustment can be made if the amplifier has unequal
turn on and off times. Period should be set according to pulse repetition rate. After
setting the pulse, burst function is usually used on function generator to define the
number of pulses. External triggering can be used on function generator for syn-
chronization with other devices. To generate more complex control signals as
bipolar, bursts, or combination of different pulses, several function generators can
be combined so that each generates particular time sequence and then triggers the
next function generator.
Fig. 12 General block

diagram of an electroporator
990 M. Reberšek
For electroporators that are more commonly or generally used, special user
interfaces and control units are usually designed. For simple electroporators with
slow control pulses, microprocessors can be used to generate control signals. In
order to achieve precise timing of electroporation pulses, a simple real-time operat-
ing system (RTOS) is advisable to be implemented in microprocessor. Simple RTOS
can be implemented by setting up the interrupt handler and timer interrupt on
microprocessor. The sequence of the electroporation pulses is then generated by
counting the interrupts and thus defining the real-time, setting the flags for each state
of pulse generation, counting the flags and thus defining the number of pulses/bursts/
sequences, and changing the level of the control signal according to the state of the
pulse generation.
For complex electroporators with fast control pulses, a custom made electronics
must be used which can be most easily implemented on field-programmable gate
array (FPGA). In contrast to microprocessors which process the data serially, custom
made electronics can process the data in parallel. Thus, the custom electronics may
generate much shorter and much more complex control signals than the micropro-
cessors. The control electroporation signals are generated in FPGA by using state
machine in which each state represents the state of pulse generation (pulse,
pause. . .). High-frequency oscillator is used to define the real time of the state
machine. Electroporation pulse parameters are stored in several registers which are
during the operation of the state machine continuously compared to counters that
measure the duration of each state. The level of the control signals are changed
according to the state of pulse generation.
Electroporation Applications and Effects of Electroporation Pulse

Parameters on Cells
Pulse amplitude and duration are the most influential parameters of electroporation
pulses, because their value has the greatest influence on the course of electropora-
tion. The effects of pulse amplitude and duration are interdependent (Fig. 13).
Namely, the longer are the pulses, the lower amplitude is needed to achieve equiv-
alent level of plasma membrane permeabilization (Pucihar et al. 2011). To achieve
reversible electroporation, voltage of the pulses must be higher than the reversible
Fig. 13 Areas of reversible

and irreversible
electroporation depending on
the pulse amplitude and pulse
duration (Adapted from
Pucihar et al. 2011)
Fig. 14 Applications of electroporation pulses
voltage threshold which depends on the pulse duration. If the voltage is further
increased over the irreversible voltage threshold, the irreversible electroporation is
achieved. If the amplitude drops during the pulse delivery, the permeabilization of
the cells will be lower, and if the voltage of the pulses drops below the reversible
threshold, the contribution from that pulses to the permeabilization of the cells will
be zero.
Outcome of an application of electroporation pulses to biological cells are diverse
and are controlled mainly by electroporation pulse parameters (Fig. 14). Two main
pulse durations are used in electroporation, long pulses (μs and ms) where duration
of the pulses is longer than the time constant of the plasma membrane charging and
short pulses (ns) where duration of the pulses is shorter than the time constant of the
plasma membrane charging. Long pulses affect mainly cell plasma membrane while
short pulses affect also intracellular membranes of organelles. Combination of this
two pulse duration is sometimes used for achieving combined or synergistic effect.
When the voltage is higher than the irreversible threshold the process leads to cell
death which is at long pulses mainly necrotic while at short pulses mainly apoptotic.
Reversible electroporation with the voltage between the reversible and irreversible
threshold may lead to introduction of small molecules or extraction of molecules,
with electrophoretic pulses also to introduction of large molecules, and with
dielectrophoretic pulses also to cell fusion. Electrophoretic pulses are used for
electrophoretic drag of charged molecules. Electrophoretic pulses are usually long
992 M. Reberšek
pulses (ms). Dielectrophoretic pulses are used for bringing the cells together to
achieve better cell fusion. Dielectrophoretic pulses are around 1 MHz, several
seconds long, sinusoidal pulses with amplitude below irreversible threshold.
Electroporation induced cell death can be used for irreversible electroporation of
tumor cells (IRE) and non-thermal pasteurization). Introduction of small molecules
can be used for electrochemotherapy (ECT; ▶ Chap. 93, “Electrochemotherapy and
Its Clinical Applications” by Gehl and Serša). Extraction of molecules can be used
for production of biological substances (Haberl et al. 2013). Introduction of large
molecules can be used for gene electrotransfer (GET). Cell fusion can be used for
production of heterohybridoma cells (Rems et al. 2013).
It is easier to generate the exponential pulses than the square wave pulses.
However, square wave pulses are more often used in electroporation applications
because their relevant electrical parameters are much less dependent on the load and
square wave pulses offer better survival rate thus offering good control over elec-
troporation process and reproducibility of experiments.
At long electroporation pulses the rise and fall times have no significant effect on
electroporation (Kotnik et al. 2003). However, at short electroporation pulses the rise
time has theoretically an effect on electroporation as induced transmembrane voltage
is higher with shorter rise time (Kotnik and Miklavčič 2006).
Single pulse electroporation is rarely used, mostly in electroporation phenomenon
research. The effect of pulses is much stronger if several pulses are used instead of
one. However, the effect reaches a plateau at a certain number of pulses. Very large
numbers of pulses are used at nanosecond electroporation and irreversible electro-
poration where one pulse has very low effect.
Unipolar pulses release metal from the electrodes into the biological load which
can be reduced by using bipolar pulses (Kotnik et al. 2001). However, if the bipolar
pulses are very short and with a very small delay, the second pulse of reversed
polarity may not have any additional effect or may even (to some extent) cancel the
effect of the previous pulse of forward polarity (Pakhomov et al. 2014).
The impact of the pulse repetition rate (PRR) is the least understood, resulting in
controversial findings and treatment recommendations. Aside from the trivial
heating effect that increases with increased PRR (less time for heat dissipation
between the pulses), experimental and theoretical studies using different endpoints
reported significantly greater bioeffects at higher PRR, significantly greater effects at
lower PRR, biphasic or more complex dependences, or relatively little role of PRR
within studied limits (Pakhomova et al. 2011).
Higher PRR leads to less stimulation of nerves and consequently less pain
(Županič et al. 2007), and less muscle contraction (Arena et al. 2011).
Combination of different types of electroporation pulses can also be used to
improve electroporation applications, inactivation of bacteria can be improved by
using combination of nanosecond and microsecond pulses (Žgalin et al. 2012),
and GET can be improved by using nanosecond first and microsecond electro-
poration pulses (Guo et al. 2014) or combination of microsecond and millisecond
pulses (Šatkauskas et al. 2002). The sequence of generating different types of
electroporation pulses is for GET of great importance; especially in vivo. Delivery of

pulsed electric field in different directions with respect to the cell can also be used to
improve GET and cell fusion (Reberšek et al. 2007).
Measurement of Electroporation Pulses and Evaluation of Pulsed

Electric Fields
Electroporation pulses are usually measured by multi-channel oscilloscope, and

voltage and current probes. All the measurement equipment should be calibrated.
Bandwidth of the probes and oscilloscope should be higher than the bandwidth of
the pulse. The bandwidth of the pulse (BW) can be estimated from the rise (tR) or fall
time (tF) of the pulse, whichever is shorter:
0:35
BW ffi (8)
tR
Also, maximal non-destructive amplitude of the voltage/current probes should be
higher than the maximal amplitude of the pulse.
Measurements of the pulse should be conducted on the electrodes, or, if that is
not possible, the voltage on the electrodes should be determined by evaluating
losses and reflections of the pulse between the measuring point and the electrodes
(Kenaan et al. 2011). Losses in the pulse transmission line may be considerable
when short/high-frequency pulses are used or when the current of the pulse is very
high and thin or long cables are used to deliver pulses. Reflections may occur if the
rise or fall time is comparable to the electrical length of the pulse transmission path.
By measuring the current at long electroporation pulses and observing the reflec-
tions at short electroporation pulses, it is possible to detect bad connection with
the load.
Measurement of the pulse amplitude should be made and checked in each
experiment where pulses are delivered. During the measurement, all the electropo-
ration pulses should be acquired including the reflections of the pulses. Sequencing
(Sequence mode on oscilloscope) can be used to acquire series of pulses with long
pauses; however, the number of pulses must be preset on oscilloscope in this case.
Zooming can then be used for observing each pulse when large numbers of pulses
are acquired. For good resolution of zoomed pulses, an oscilloscope with sufficient
acquisition points or memory has to be used.
Some electroporators enable internal measurement of electroporation pulses.
In this case, the user should verify if the internal measurement system is adequate
for electroporation pulse measurement, as commonly this is not the case. Man-
ufacturer has to specify accuracy of measurements and provide periodic
calibrations.
After acquiring the amplitude of the electroporation pulses on the electrodes,
electric field within the biological load induced by the delivered electroporation
994 M. Reberšek
pulses should be calculated or otherwise determined. Electric field can be calculated

analytically if the electrode geometry is simple or by numerical methods for more
complex geometry of electrodes (Čorović et al. 2007). For adequate calculation of
the electric field, the electrical properties or the name of the material and dimensions
of the chamber/electrodes and the biological load are required.
Recommendations for Reporting in Electroporation-Based

Studies
Electroporation pulses are generated by electroporators and delivered to the cell

suspension or tissue through the electrodes. Therefore, it is highly recommended that
an appropriate description of the electroporators and electrodes is provided. For
commercial equipment, the name of the model and the manufacturer should be
specified. If the equipment is a laboratory prototype or specially fabricated device,
an adequate description of electrical configuration and components should be pro-
vided in order to reproduce the sample exposure to pulsed electric field (Raso et al.
2016). However, a device may malfunction unnoticeably, aging of electrical com-
ponents may change the operation of the device, manufacturer may introduce
improvements or produce different versions of the device which operate differently,
time course of delivered electroporation pulses depend on the load, and other
researchers may not have specified device. Therefore, it is crucial to determine the
conditions to which the cells were exposed to and to represent/report them ade-
quately. To do that, the electrical pulses must be measured and the electrical field to
which the cells where exposed to has to be evaluated. To ensure that the reports on
electroporation methods are unbiased, researchers should (1) describe exactly how
the electric pulses were measured, (2) provide complete time-domain waveforms and
parameters of the electric pulses at the electrodes, and (3) define the electric field to
which the cells were exposed to.
To describe exactly how the electric pulses were measured:
• The name of the model and the manufacturer of the oscilloscope and probes
should be specified if the measurement equipment is commercial. If probes are
specially fabricated or a laboratory prototype, an adequate description should be
provided in order to reproduce the probes and maximal nondestructive amplitude,
and bandwidth of the probes should be specified;
• The point of the measurement should be specified. The point of the measurement
should be conducted on the electrodes. If that is not possible, the losses and
reflections of the pulse between the measuring point and the electrodes should be
evaluated to estimate the amplitude on the electrodes;
• It should be specified if the measurements of the pulses were made and checked in
each experiment where pulses were delivered.
To provide complete time-domain waveforms and parameters of the electric

pulses at the electrodes:
• The time course of the electroporation pulse and the time course of complete
series of pulses measured or estimated on the electrodes should be displayed. If
more than one combination of electroporation pulses are used, the time course of
the electroporation pulse from each combination should be displayed. Individual
pulse should be displayed in such a manner that the amplitude and polarity of the
reflected waves are visible, even if they are very small. Therefore, at least double
pulse traveling time through the system should be displayed.
• The parameters of the electroporation pulse and series of pulses should be
described. If more than one combination of electroporation pulses are used,
parameters of the electroporation pulse from each combination should be
described.
To define the electric field to which the cells were exposed to:
• The results of calculated or otherwise determined electric field within the biolog-
ical load induced by the delivered electric pulse should be displayed. Electric field
can be calculated analytically or by numerical methods.
• All the necessary data to determine the electric field within the biological load
should be specified or cited, e.g., the electrical properties or the name of the material
used and the dimensions of the chamber/electrodes and the biological load.
Conclusions
The scientific studies and reports should be unbiased and comprehensive for others to
be able to replicate results. In electroporation-based studies, it is therefore very
important to adequately determine the electric field to which the cells were exposed
during the study and to report the methods and results of electric field determination in
sufficient detail to allow their replication. In this chapter main features that should be
described in every paper on electroporation are identified: the measurement protocol,
time course of the pulse, and electric field determination. The parameters, the methods
of generation, and applications of electroporation pulses are described. Moreover,
recommendations for appropriate measurement of electric pulses and evaluation of
electric field to which the cells are exposed are outlined. Consider also recommenda-
tions published recently for clinical (Campana et al. 2016) and food and biotechno-
logical (Raso et al. 2016) electroporation experiments. With these in mind, researchers
should conduct and present their results in a more comprehensive way which will
allow faster progress in this interesting and expanding field of research.
Cross-References

▶ Cliniporator: Medical Electroporation of Tumors
996 M. Reberšek

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Part V
Modeling of Electroporation and Related
Phenomena
Damijan Miklavčič
Treatment Planning for
Electrochemotherapy and Irreversible 54
Electroporation of Deep-Seated Tumors
Bor Kos
Abstract
Electrochemotherapy and irreversible electroporation can be used to treat deep-
seated tumors. Key to treatment success is ensuring that the entirety of the target
tumor is covered with electric fields of sufficient strength during the treatment.
Treatment planning using numerical methods has long been established in radio-
therapy, and this chapter presents the necessary tools to realize a similar treatment
planning framework also for electrochemotherapy and irreversible electropora-
tion. Treatment planning consists of identifying the target tumor and surrounding
tissues on tomographic medical images. This reconstruction can be used to build a
numerical model of the region of interest with by assigning correct conductivities
to each tissue in the treatment zone. Using the finite element method, electric
fields for a given electrode configuration can be determined. By coupling the
numerical model of electroporation with appropriate optimization algorithms, the
voltage to be delivered to each electrode pair can be determined and the positions
of electrodes can be adjusted to ensure successful coverage of the target volume.
Possible approaches to execute the treatment according to the prepared treatment
plan are also discussed at the end of the chapter.
Keywords
Irreversible electroporation • Electrochemotherapy • Deep-seated tumors • Treat-
ment planning • Radiotherapy • Optimization
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1002
Electric Field in Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1003
Numerical Methods for Computing Electric Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1005
B. Kos (*)
e-mail: bor.kos@fe.uni-lj.si

DOI 10.1007/978-3-319-32886-7_2
1002 B. Kos
Predicting Response to Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1008

Predicting Response Based on Electric Field Thresholds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1009
Modeling Probability of Cell Survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1011
Optimization and Treatment Plan Execution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1013
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1015
Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1016
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1016
Introduction
Electroporation is a physical mechanism in which externally applied electric field

pulses cause changes in the cellular membrane, which increase the membrane’s
permeability. Depending on the strength, duration, and number of the applied
electric field pulses, these changes in the cellular membrane can either be reversible
or irreversible, with irreversible electroporation requiring a higher electric field
strength, a greater number of pulses, or both. Both principal types of effect
can be used to treat deep-seated tumors in various organs with good success rates,
more so, when we consider that many of these studies treated patients with no further
therapeutic options: liver (Thomson et al. 2011; Edhemovic et al. 2014), kidney
(Thomson et al. 2011; Narayanan and Doshi 2016), pancreas (Martin 2013),
prostate (Onik et al. 2007), and metastases in bones (Gasbarrini et al. 2015; Bianchi
et al. 2016). Reversible electroporation is used in conjunction with certain chemo-
therapeutic drugs (bleomycin and cisplatin), which greatly potentiates their
antitumor effectiveness and has a number of other beneficial effects; this kind
of treatment is termed electrochemotherapy (ECT) (Mir et al. 1991; Miklavčič
et al. 2014). Irreversible electroporation (IRE), on the other hand, can be used to
directly ablate tissue, and directly causes either apoptotic or necrotic cell death;
irreversible electroporation and nonthermal irreversible electroporation ablation are
terms used in the literature (Davalos et al. 2005; Jiang et al. 2015).
For a treatment of a deep-seated tumor to be successful, it is necessary to ensure
that the whole tumor is covered with a sufficiently strong electric field, exceeding the
reversible or irreversible electroporation threshold, depending on the type of treat-
ment that is being performed, i.e., electrochemotherapy or tissue ablation by
irreversible electroporation, respectively. For deep-seated tumors, the electric field
pulses are delivered to the target area by needle electrodes, which can be either in a
fixed array or as individual electrodes. These electrodes need to be introduced in the
immediate vicinity of the tumor or inside the tumor, because the strongest intensity
of electric fields is found at the surface of the electrodes. Further away from the
electrode surface, the electric field decreases rapidly, but the speed of this decrease is
dependent on many factors, such as the shape of the electrode, diameter of the
electrode needle, and on the proximity of the counter electrode. If we consider an
imaginary point and move it from the surface of one electrode towards the surface of
the second electrode, the electric field will first decrease to a certain point and then
start increasing again. The values of electric field strength along this path are
54 Treatment Planning for Electrochemotherapy and Irreversible. . . 1003
dependent on the path itself, the distance between electrodes, the shape of the
electrodes, the applied voltage, and the electrical properties of the tissue(s) in
which the electrodes are inserted. Additionally, given the limitations of available
electroporation pulse generators in terms of maximum voltage and maximum current
also typically requires the use of more than one electrode pair, which further
complicates predicting the response of the treatment as a whole. As can be inferred
from the number of variables affecting the electric field distribution, the distribution
of electric fields in tissue is not necessarily intuitive and easy to determine, therefore
it is advisable to use some kind of treatment planning to determine the best way to do
a procedure of this kind.
Treatment planning is a term that originated in radiotherapy. With the spread
of 3D imaging modalities such as ultrasound, computed tomography (CT), and
magnetic resonance imaging (MRI) in 1980s, these imaging modalities became
ever more commonly used to plan therapeutic radiation therapy (Jaffray et al. 2007).
In this approach, medical imaging is first performed on a patient to determine the
anatomy of the patient, the location of the tumor to be treated, and its surrounding
regions. This allows the physician in charge of the treatment to delineate the clinical
target volume (CTV), that is, the gross tumor volume (GTV) surrounded by a
clinically relevant safety margin. This safety margin is determined based on the
histology and aggressiveness of the tumor. Surrounding critical structures are also
defined and then used in the treatment planning step to design the irradiation pattern
in such a way that a sufficient dose is delivered to the CTV while limiting the
irradiation of the surrounding healthy tissue and especially to any critical tissues
present. The treatment is then delivered in multiple fraction over several weeks,
while controlling the patient positioning and in image-guided radiation therapy also
following the positioning of the CTV itself due to breathing, tissue deformation, etc.
(Jaffray et al. 2007; Bujold et al. 2012). While dedicated off-the-shelf treatment
planning software for radiotherapy planning is available on the market with appro-
priate certification as medical devices, this is unfortunately not yet the case for
electrochemotherapy and irreversible electroporation. A proof-of-concept tool
Visifield has been developed at the University of Ljubljana (www.visifield.com,
University of Ljubljana, Slovenia) (Marčan et al. 2015), but this cannot yet be used
in the clinical setting. The aim of this chapter is therefore to present the methods and
procedures that can be used to make individualized treatment plans for ECT or IRE.
Electric Field in Tissue
In this subsection, the effects that different electrode geometry has on electric field
distribution in the tissue will be illustrated. Treatment planning in electroporation-
based treatments can follow a similar paradigm as for radiation therapy, with some
steps modified to take into account the different physics involved in electroporation
(Pavliha et al. 2012). One of the essential in-between steps between imaging and
computation of electric fields in tissue is image segmentation into different tissues.
1004 B. Kos
This allows us to take into account the markedly different electrical properties that
different tissues have, and at the same time also serves as the delineation of the GTV
for the treatment planning process.
Plate electrodes were the most used electrode types in the initial studies of
electrochemotherapy. They have a simple geometry, where the tissue to be treated
is compressed between two parallel plates. This makes the setup very repeatable, and
the electric field delivered to the target tissue is roughly approximated as the ratio
V/d, where V is delivered voltage and d is the distance between the internal faces of
the electrode. However, this simplification is only true in homogeneous tissues,
when the distance between the electrodes is small in comparison to their width and
length and if the conductivity of tissue remains constant irrespective of the applied
electric fields. The last issue with plate electrodes is that they can only be used on
relatively small target areas and cannot be inserted into tissue.
Further increasing the complexity of planning ECT and IRE treatments of deep-
seated tumors is the fact that different tissues in the human body have markedly
different electrical properties. Examples of tissues with the lowest conductivity
include the outer layer of the skin (stratum corneum), bone, bone marrow, and fat
tissues, while tissues with the highest conductivity include various glands, small
intestines, and muscle (Gabriel et al. 1996). Tumor tissues generally have a higher
conductivity than surrounding tissues as well (O’Rourke et al. 2007; Haemmerich
et al. 2009; Peyman et al. 2015). Additionally, tissue conductivity also increases as a
function of electric fields, because electroporation causes additional conductive
pathways to form through otherwise insulating cell membranes. This is shown in
Fig. 1, where the conductivity of liver and tumor tissue is presented as a function of
electric field (Corovic et al. 2013).
For a situation with plate electrodes and a nonhomogeneous tissue, the electric
field strength is dependent on the thickness and the conductivity of each different
tissue, but generally the tissue with higher conductivity “sees” a lower electric field
than the tissue with a lower conductivity. The exact electric field in these non-
homogeneous tissues quickly gets impossible to calculate analytically, so computa-
tional methods have to be used.
Figure 2 shows an illustration of the influence of tissue conductivity and electrode
shape on the electric field in tissue. For better clarity, only electric fields above
400 V/cm are shown. An illustration of electric fields between plate and needle
electrodes is shown. The electrodes are 1 cm apart, and 1000 V is applied between
them. The upper row (panels A and B) shows the electric field between plate
electrodes. For example with homogeneous tissue, the electric field is the exact
value of voltage-to-distance ratio only in the point exactly between the electrodes
(coordinates 0,0 on panel A). This is clearly not the case in panel B, where the tissue
to the left of the slanted line has lower conductivity by a factor of 3 than the tissue to
the right of the dividing line. This conductivity ratio is similar to the ratio between,
e.g., liver and muscle (Gabriel et al. 2009); however the difference between tumors
and surrounding tissue can be even higher than 5 (Haemmerich et al. 2009), which
amplifies this effect even further. The panels in the bottom row (C and D) show the
electric field between needle electrodes. The difference between needle electrodes
Fig. 1 The effect of electroporation on tissue conductivity. The initial conductivity of liver and
liver tumors is based on the measurements of ex-vivo human liver and liver tumors (Haemmerich
et al. 2009). At sufficiently strong electric fields, the conductivity starts to increase and the final
factor of conductivity increase in this graph is 3.5 for liver and 3 for tumor (Corovic et al. 2013)
and plate electrodes is that needle electrodes have an even more inhomogeneous
electric field distribution, and the electric fields exactly between electrodes is
actually lower than the voltage-to-distance ratio. This effect is even more
pronounced in the case with nonhomogeneous tissue (panel D). In tissues, this
kind of conductivity change is expected on any interface between two different
tissues, such as liver tumor, kidney tumor, or muscle tumor. While the conductivity
transition is immediate in Fig. 2, in actual tissue, it would be more gradual across
a millimeter or two, therefore the electric field would have a more gradual transition,
but it would still be present. Tissue inhomogeneity thus renders electric field in tissue
very difficult to predict without computer simulations.
Numerical Methods for Computing Electric Fields
Previously the reasons for using numerical methods when planning any kind of
electroporation treatment were demonstrated. One of the numerical methods, which
has gained the most traction in electroporation research, is finite element method
(Sel et al. 2007; Županič and Miklavčič 2010; Zupanic et al. 2012). The finite
element method is based on discretizing the model geometry into a mesh of finite
1006
Fig. 2 The influence of electrode shape and tissue inhomogeneity on electric field distribution. The figure shows: (a) plate electrodes inside homogeneous
tissue; (b) plate electrodes inside a nonhomogeneous tissue; (c) needle electrodes in homogeneous tissue; and (d) needle electrodes in nonhomogeneous tissue.
B. Kos
In examples (b) and (d), the following relation holds for conductivities: σ2=3 σ1
elements, on which the unknown quantity (in the case of treatment planning for
electroporation this is the electric potential) is presumed to change according to some
simple function (usually a polynomial). The solution of the whole problem is then
translated into solving a set of algebraic equations for steady state problems. The
computation of the electric field distribution during an electroporation pulse is an
example problem, which can be simplified by assuming that it is a steady state
problem.
This simplification of electric field pulses to a static electrical conductive media
problem can be justified as follows. Most electroporation pulses for electro-
chemotherapy and irreversible electroporation use 100 μs long pulses. These pulses
are long compared to the cell membrane charging constant, which means that the
induced transmembrane voltage will reach its maximum value long before the end of
the pulse (Kotnik et al. 2012). Therefore, it is possible to assume that all the transient
phenomena of electroporation occur in the beginning of the pulse and have settled
out before the end of the pulse. With this simplification, it is possible to greatly
shorten simulation times for electroporation models (Ivorra 2010). The equation to
solve then becomes the Laplace equation for electric potential:
∇ ðσ∇V Þ ¼ 0, (1)
where σ is tissue electric conductivity, ∇ is the gradient operator, and V is the electric
potential. In order to take into account the increase in conductivity due to electro-
poration, the conductivity σ should also be a function of the electric field, which is
defined as:
E ¼ ∇V: (2)
The following text will assume that Comsol Multiphysics (COMSOL AB,
Stockholm, Sweden) is used for the finite element modeling since it has a relatively
accessible user interface, and academic discounts are available for the software, but
the same equations and equivalent solvers can also be implemented for example in
free open-source finite element method packages FEniCS (https://fenicsproject.org/)
(Alnæs et al. 2015) and FreeFem++ (http://www.freefem.org/) (Hecht 2012).
The Comsol Multiphysics AC-DC model provides a physics interface for modeling
the electric fields and currents in tissue, when pulses are applied through electrodes.
The most appropriate mode for modeling electroporation is called electric currents.
The first important choice that needs to be made is whether to model in three
dimensions or to use a two-dimensional approximation. The former is much more
computationally intensive and requires more computer resources, but gives a more
representative result in the whole computational domain. On the other hand,
a two-dimensional approximation is much faster to compute and less likely to run
into issues with computational stability and therefore also allows the user to easily
make more experiments.
The initial step in numerical treatment planning is the creation of a representative
patient geometry. The easiest approach is to use a geometrical representation using a
1008 B. Kos
sphere or spheroid to represent the target tumor. This can be done directly in the
graphical user interface of the Comsol Multiphysics software. The next step is the
inclusion of electrodes – for deep-seated tumors these are typically needles of some
kind, which can be adequately modeled by a cylinder. The tissue surrounding
electrodes can be simply defined as a box or cylinder with sufficiently large
dimensions. This kind of schematic geometry can serve as an initial step or a learning
tool for treatment planning, but is still capable of giving valuable results (Garcia
et al. 2011, 2014; Denzi et al. 2015).
Once the geometry is defined, voltage can be applied to the electrode parts of the
geometry via boundary conditions. The most basic way to define that is the Dirichlet
boundary condition, which is essentially a fixed potential of the boundary of
the electrode. In Comsol Multiphysics AC/DC module, this can be defined as an
electric potential boundary condition, which allows the user to specify the fixed
voltage at the electrode boundary. An even better option is using the Terminal
boundary condition, which also provides a user-friendly interface for the computa-
tion of the total current delivered during the pulse. The interface for including
electric field-dependent conductivity can be found by defining a smoothed step
function (smoothed Heaviside function) in the material properties of the tissue
(Garcia et al. 2014).
In order to realize a more detailed patient-specific geometry some more steps
need to be performed. One approach entails using the segmented medical images to
extract the surface of different tissues and combining them with a detailed model of
the electrodes. This approach, however, can quickly run into difficulties with
generating the mesh for finite element method when attempting to insert the elec-
trodes into the detailed geometry model with complicated geometry. Specialized
software tools exist, which can help with that, but the process still involves a
considerable amount of manual work. Another approach represents using Comsol
Multiphysics in conjunction with Matlab (Mathworks, Natick, MA). This allows the
generation of the mesh by taking into account as a solid geometry only the electrodes
and a suitably large bounding box. The conductivities of different tissues can then be
assigned directly to the free mesh elements by using an interpolation function for
conductivity in Comsol Multiphysics (Aström et al. 2009). This method was used to
generate the example figures in the continuation of the chapter.
Predicting Response to Treatment
The computation of electric field is the first prerequisite of treatment planning which
then needs to be evaluated in terms of expected biological outcome. In the first
studies of ECT, electric field thresholds have been determined for trains of eight
100 μs pulses, delivered at 1 Hz pulse repetition frequency (Mir et al. 1991). This
kind of pulse protocol has also been the most widely used electroporation protocol in
clinical practice, but different number of pulses can also achieve the same effect,
albeit at different electric field thresholds (Pucihar et al. 2011). For IRE, a higher
number of pulses is typically used (Jiang et al. 2015), which has the benefit of
reducing the electric field strength needed to achieve ablation, but also increases the
thermal load of the treatment and increases the treatment duration.
Predicting Response Based on Electric Field Thresholds
The total volume of tissue that can be covered with electric field above the electro-
poration threshold with a single pair of electrodes is limited by the maximum voltage
and current that a pulse generator can deliver. Therefore, it is necessary to develop a
method of accounting for successivle coverage from more pairs of electrodes. The
number of possible electrode pairs is given by Eq. 3, where n is the number of
electrodes:
nðn 1Þ
Possible pairs ¼ : (3)
2
Currently, the two most widely available pulse generators for ECT and IRE have
connections for six individual electrodes, which yield a total of 15 possible pairs.
Depending on the positioning of the electrodes, some of the possible pairs might not
make sense, such as when given two electrodes are too far apart to produce
therapeutically effective electric fields between them.
The most conservative method for evaluating cumulative coverage of multiple
electrode pairs is considering each electrode pair and the pulses delivered to it as a
separate instance, by defining the volume of tissue above the electroporation thresh-
old appropriate for the application as completely treated. The coverage of each
successive electrode pair can then be superimposed and the total coverage deter-
mined from this superposition. This approach is illustrated on Fig. 3. Another reason
that this approach is conservative is that it slightly underestimates the volume of
treated tissue, since the conductivity of tissue is increased due to delivery of pulses to
one electrode pair and the increase in conductivity persists for a short duration after
the pulse delivery switches to the next electrode pair (Ivorra et al. 2009).
More insight into the coverage of the target tissue with electric field can be
determined by an approach analogous to dose-volume histograms, which are typi-
cally used in radiotherapy treatment planning (Zupanic et al. 2012). This kind of
graph is shown in Fig. 4 and gives information what volume fraction of the GTV is
covered by electric fields of at least the strength indicated on the x-axis. Here the
treatment equivalent electric field after the application of pulses to n-th electrode pair
(from the total p pairs) is determined as follows:

max EEQ n1 , En ; n > 1
EEQn ¼ , (4)
En ; n¼1
where EEQ n1 is the treatment equivalent field from the electrode pairs 1 through
n1. This approach is conservative in the sense that the whole CTV is being
1010 B. Kos
Fig. 3 Illustration of the successive coverage of a larger volume by utilizing more electrode pairs.
By delivering pulses between more than one electrode pairs, a larger CTV can be covered with
sufficiently strong electric fields. Homogeneous tissue is presented in this figure for clarity, and
applied voltage is 1000 V between each electrode pair. Area covered by each electrode pair is
indicated by different colors; each treatment field represents electric field strength above 400 V/cm
subjected by more than just one train of pulses. And to ensure the complete coverage
of the CTV, some overlap between volumes treated by each electrode pair is
inevitable, as can be seen in Fig. 3. The consequence of this is that some parts of
the CTV are in fact experiencing two, three, or even more pulse trains of comparable
electric field strength. The effective electroporation thresholds would therefore
be actually even lower, since the electroporation thresholds are reducing with
increasing number of pulses (Pucihar et al. 2011). The overlap of coverage can
also be seen in Fig. 4, where, e.g., the volume fraction of the tumor after the third
electrode pair (between electrodes 2 and 5) is the same as after the second electrode
pair at 400 V/cm. At higher electric field strengths, it can be seen that the third pulse
sequence increases the volume fraction by about 0.1. Due to the positioning of the
electrodes and pair selection, a larger jump in coverage can be seen again for pair
number 4 (between electrodes 2 and 6).
However, looking only at the integrative coverage of the CTV provides only
a descriptive statistic of the total coverage, without indicating where the coverage is
lacking. Another approach is to present the results of the model directly on the
original patient medical images, which have been used to generate the patient-
specific model. This kind of visualization is shown in Fig. 5, and is based on the
same case as Fig. 4.
Fig. 4 Electric field histogram. The cumulative volume fraction which is covered by electric fields
of at least the strength indicated on the x-axis is shown after each electrode pair application. The
data of electric field coverage was taken from the treatment plan of a patient with colorectal
carcinoma metastases in the liver treated with ECT (Edhemovic et al. 2014)
Modeling Probability of Cell Survival
The approach based on electric field thresholds is a good approach based on

its simplicity and ease of understanding; however it has drawbacks for the task
of treatment planning. The first is that the electric field threshold presents a very
sharp delineation between “treated” and “untreated” tissue – namely if 400 V/cm
is considered as a threshold for reversible electroporation in tumor tissue, and parts
of the tumor, where the electric field strength determined by treatment planning
would be considered fully treated, while any parts that fall even a little below
the threshold would be considered completely untreated. This kind of result is overly
simplistic in the fact that actual cells which are exposed to either 399 V/cm or
401 V/cm have a very similar probability of survival.
The possible solution to this drawback of the electric field threshold for predicting
the tissue response to ECT or IRE is therefore statistical modeling of the cell
survival. The cell survival model called Peleg-Fermi model based on the original
author of the model used for predicting microbial inactivation after exposure to
1012 B. Kos
Fig. 5 Electric fields in tissue after all electroporation pulses have been delivered. The maximum
value of electric field is shown in all tissues. Since the electrodes are located in the same axial plane
as the MRI scan, the electrode track can be seen in the image. The electrode used in the treatment
plan has a diameter of 1.2 mm and a length of the active part of 4 cm
pulsed electric field has already been applied to IRE treatment planning (Garcia
et al. 2014) in the following way (Fig. 6). A survival function is defined as follows:
1
S¼ (5)
E Ec ð nÞ
1 þ exp
Að n Þ
where S is the probability of cell survival (ranging from 0 to 1), E is the local electric
field determined by numerical modeling, Ec(n) is the critical electric field at which
50% of the cells are expected to die, and A(n) is the shape factor, which determines
the width of transition zone between fully treated and untreated regions. A larger
value of A results in a wider transition area and a lower value of A results in a
narrower transition area. The benefit of using this approach is that both Ec and A are
a function of the number of pulses delivered to the treated area, and are defined as
follows:
Ec ðnÞ ¼ Ec0 ek1 n , (6)
AðnÞ ¼ A0 ek2 n , (7)

Fig. 6 The landscape of the probability of survival according to the Peleg-Fermi model depending
on the number of pulses and electric field strength (for values of the parameters: Ec0 = 750 V/cm,
A0 = 15 V/cm, k1 = 0.015, k2 = 0.003). The figure shows that the transition region between fully
electroporated and nonelectroporated tissue spans about 200 V/cm of electric field strength at
10 pulses; however at 90 pulses this transition region becomes narrower at 100 pulses
Ec0 and A0 are initial values for the parameter, which are theoretical values at
0 pulses, while the k parameters define the rate at which the values decrease with
increasing number of pulses. At this point it is important to stress that the values of
the four parameters need to be experimentally determined and validated for different
tissues and that they have a limited interval in which they can be used. By increasing
the number of pulses above a reasonable number, this equation has the possibility
to return clearly erroneous results since the value of S when n approaches infinity
is 0. With this limitation in mind however it is possible to apply this equation to the
treatment planning problem successfully.
Optimization and Treatment Plan Execution
When performing treatment planning, the goal is to generate technically feasible

electrode positioning, which will ensure the complete coverage of the CTV, at the
same time limit damage to potential critical structures in the vicinity of the CTV, all
while maintaining compliance with the hardware limitations of the available pulse
1014 B. Kos
generators. Due to these considerations, it is not always the best approach to increase
the voltage to electrode pairs, since that can run the risk of exceeding the maximum
current limitation of the pulse generator. Additionally, if the electrodes are too far
apart, or if the contrast between conductivities of the GTV and the surrounding
healthy tissue is too large, it can be impossible to achieve sufficiently strong electric
fields to successfully treat the target. The use of optimization algorithms can simplify
the search for appropriate electrode positions and voltages. The considerations above
need to be formalized into a fitness function, which allows the optimization algo-
rithm to compare candidate solutions.
The fitness function has to be designed differently for ECT and IRE, since the two
different approaches require different degrees of coverage. ECT requires the whole
CTV to be covered with at least with electric fields above the reversible electropo-
ration threshold. Fields above the irreversible electroporation threshold in the CTV
are not required, but are not detrimental to the success of the treatment. For IRE
however, the whole CTV needs to be covered with fields above the irreversible
electroporation threshold. Additionally, for IRE, temperature rise is an important
consideration, especially when electrodes are located near sensitive critical struc-
tures (Kos et al. 2015). Example fitness functions would be based on the approach
described in (Zupanic et al. 2012):
FECT ¼ 100 V CTV sur

rev 10 V irr , (8)
FIRE ¼ 100 V CTV sur

irr 10 V irr , (9)
In Eqs. 8 and 9, the values of VCTV indicate the volume fraction of the CTV, which
is covered by fields above the reversible or irreversible electroporation threshold
indicated by the subscripts rev and ire, respectively. The volume of surrounding
tissue above the irreversible electroporation threshold is intended to minimize
damage to surrounding critical structures.
For treatment planning optimization, two general situations can be anticipated when performing
optimization. In the first situation, the electrode positions are fixed by anatomical, surgical, or some other
constraints and only the voltages can be varied. In the second situation, the optimization algorithm can
have the full freedom of electrode positioning and voltages between each electrode pair, therefore the
position and direction of each electrode can be varied, as well as the voltage between each electrode pair.
The first situation is less complex, since the smaller number of parameters limits
the possible number of solutions to a more manageable number. In this case, it is
feasible to compute the solution of electric field between each electrode pair for most
feasible voltages and then use the optimization algorithm to select the best combi-
nation of voltages, which achieve sufficient CTV coverage with electric field and stay
within the constraints of the pulse generator. A gradient optimization algorithm can
be used in this case. This requires the computation of the partial derivatives of the
fitness function with respect to all parameters. The algorithm then changes the
parameter, which results in the largest increase of the fitness function. In the next
iteration, the gradient needs to be determined again, so that the appropriate parameter
can be adjusted, until the solution converges to a maximum. With a gradient
algorithm it is not possible to guarantee that this maximum is not a small local
maximum instead of a true global maximum.
When the electrode positions also have to be determined by the optimization
algorithm, the best approach is to use a genetic algorithm, which attempts to mimic
natural selection (Zupanic et al. 2012). For optimization with a genetic algorithm, all
parameters of the treatment need to be encoded in a “chromosome,” which is a
numerical variable in the optimization algorithm, which contains the position and
direction of all electrodes and the voltage delivered between each electrode pair.
Initially a population of candidate solutions is generated at random and the fitness
function is evaluated for each member of the population of solutions. A number of
best performing solutions are selected to “breed” in order to generate the next
generation. The chromosomes of the next population are generated by crossing
over the chromosomes of random pairs from the breeding individuals. Finally,
mutation is introduced, which is a random change of a single parameter, can occur
after the cross-over phase and generate new values for parameters, which have not
previously been present in the population. This process is repeated, until a solution
which satisfies the criteria is found. This approach is computationally much more
intensive, because with changing electrode positions, the whole treatment (all
electrode pairs) needs to be computed for every new candidate solution. But the
benefit of using the genetic algorithm is also that it is much less likely to get stuck in
a local maximum in comparison to a gradient-based algorithm. Because the genetic
algorithm changes the parameters randomly (but based on previous successful
solutions), it can also find a successful solution faster than a gradient-based algo-
rithm, which has to make small improvements on existing solutions.
Conclusions
Treatment of deep-seated tumors with electrochemotherapy (ECT) or irreversible

electroporation (IRE) is a challenging procedure, which requires the careful design
of the procedure and good control of positioning of the electrodes and careful
selection of voltage for each electrode pair. Analogously to radiotherapy, where a
physical mechanism (ionizing radiation) is used to elicit a biological response, in
electroporation a different physical mechanism (nonionizing pulsed electric fields) is
used to elicit a biological response (uptake of cytotoxic drugs in electro-
chemotherapy, or apoptotic cell death in IRE). The physical laws which govern the
distribution of electric fields in tissue are well understood and lend themselves to
efficient numerical solutions. The numerical methods can be used in conjunction
with tissue segmentation to predict the response to the treatment and to tailor the
treatment parameters in a way that will maximize the probability of a successful
treatment outcome. Optimization algorithms provide the means to automatically
adjust voltages and electrode positions until a treatment plan with sufficient quality
and robustness is generated. This can then be translated into practice by following
1016 B. Kos
instructions in the treatment plan or by using some kind of navigational assistance

device, which guides the performing physician in positioning the electrodes
according to the treatment plan.
Cross-References
▶ Cliniporator: Medical Electroporation of Tumors

▶ Mathematical Models Describing Cell Death due to Electroporation
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Different Approaches Used in Modeling
of Cell Membrane Electroporation 55
Clair Poignard, Aude Silve, and Lars H. Wegner
Abstract
Cell electroporation is a complex phenomenon, which consists in the emergence
of defects in cell membranes subjected to electric pulses. Since the end of the
1990s, biophysical models have been developed to explain and predict the
conditions for cell electroporation. However the recent biological data, in partic-
ular those dealing with the influence of the repetition rate of the pulses, challenge
these biophysical models. In this chapter, different approaches to model electro-
pore formation are presented. The simplest equivalent circuit model is first
presented. Biophysical approaches are rapidly overviewed. For each approach,
advantages and disadvantages are also discussed, in terms of physical meaning
and validation with the experimental data. Then phenomenological approach is
introduced. Such approaches consist in designing the model on an empirical basis
thanks to the experience. Even though the physical bases of such models are still
lacking, they provide new interesting views on the electroporation processes, as
described by the experiments. The aim of the chapter is to introduce the reader to
different ways of modeling cell membrane electroporation and to provide some
possible directions to obtain a more reliable theory of electroporation in accor-
dance with the experiments and with a justified theoretical basis.
C. Poignard (*)
Team MONC, INRIA Bordeaux-Sud-Ouest, Institut de Mathématiques de Bordeaux, CNRS UMR
5251 and Université de Bordeaux, Bordeaux, France
e-mail: clair.poignard@inria.fr
A. Silve • L.H. Wegner
Karlsruhe Institute of Technology, Institute for Pulse Power and Microwave Technology (IHM),
e-mail: aude.silve@kit.edu; lars.wegner@gmx.net

DOI 10.1007/978-3-319-32886-7_3
1020 C. Poignard et al.
Keywords
Cell electroporation modeling • Biophysical and mathematical models for pore
formation
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1020
Cell Electrical Modeling Before Electroporation: The Linear Regime . . . . . . . . . . . . . . . . . . . . . . . 1021
Equivalent Circuit for Cell: The 0D Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1022
3D Maxwell Equations and Their Simplication to the 3D Model . . . . . . . . . . . . . . . . . . . . . . . . . 1023
Spherical Cell in a Unidirectional Constant Field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1024
Basic Concepts in Biophysics for Pore Formation in Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1027
Single-Pore Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1027
From One Pore to the Total Pore Density Equation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1029
Pore Radii Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1029
The Krassowska-Neu Model for Electroporation Current . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1031
Back to the Electric Equation and Drawbacks of the Krassowska-Neu Model . . . . . . . . . . . 1032
Phenomenological Models for Membrane Conductivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1034
Biological Evidences of Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1034
The Two-Step Process of Membrane Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1035
Calibration of the Model with Patch-Clamp Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1035
Biophysical Basis of the Phenomenological and Link with Pore Radius Evolution . . . . . . 1036
The Role of the Surface Diffusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1038
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1039
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1041
Introduction
Eukaryotic cell is a complex biological entity, which is the main constituent of any
biological tissues: it is somehow the base unit of any living organism. These cells are
generically composed of cytoplasm, which includes the nucleus, mitochondria, and
other organelles that are necessary for life. This cytoplasm is protected from the
extracellular stress by the plasma membrane, which is a phospholipid bilayer. This
barrier plays a double role of protecting the cell and controlling exchanges between
the cytoplasm and the extracellular medium. In the 1970s, it has been observed that
electric shock may change transiently the membrane, allowing the entrance of
usually non-permeant molecules into the cytoplasm. This phenomenon, called
electroporation or electropermeabilization, has then been studied for cancer treat-
ments, by treating cells with cytototoxic drugs – such as bleomycin or cisplatin –
while exposing them to pulsed electric fields. This treatment called electro-
chemotherapy (see the chapters by L.M Mir, Gehl and Serša, and also Cadossi
et al.) is now used standardly in more than 40 cancer institutes in Europe for
cutaneous tumors, and several clinical studies are driven for deep located tumors.
Even though the bases of cell electroporation are well known, several experimental
observations are still unexplained, and the modeling of the phenomenon suffers from
a lack of accuracy. The aim of this chapter consists in presenting different
55 Different Approaches Used in Modeling of Cell Membrane Electroporation 1021
σm
Ci
Eext Electrical Vm εm
Permeabilisation Transport
description Cout
ρm
Fig. 1 Schematic process of electroporation
approaches to model cell membrane electroporation and in discussing the pros and
cons of each approach.
Generally speaking, the process of electroporation can be schematized by Fig. 1:
Exposure to applied external electrical field leads to an increase of the transmembrane
voltage Vm, which induces an increase of membrane conductivity σ m, permittivity em,
and permeability pm. Increased membrane conductivity and permittivity, in turn, affect
membrane voltage til the end of the pulse. Membrane permeability is not intrinsic: it
depends on the considered molecule and is detected thanks to transport of molecules
(PI, DNA plasmids, etc.). The aim of the chapter is to focus on the electrical
description of the cell membrane, without accounting for the transport process,
which is still unclear. The chapter is organized into five sections. First, the linear
electric model of Schwan et al. (Foster and Schwan 1989) is presented. The cell is
composed of a conducting cytoplasm surrounded by a resistive thin layer. Equivalent
circuit model is described and complexified by the generic model in which the thin
membrane is accounted for by imposing equivalent transmission conditions across the
interface between the cytoplasm and the outer medium. Electroporation phenomenon
modeling is then addressed. In section “Basic Concepts in Biophysics for Pore
Formation in Liposomes,” the biophysical models for electropore formation are briefly
introduced, and the model of Krassowska and Neu referred to as the KN model,
considered as the most advanced description of electroporation, is described. In section
“Phenomenological Models for Membrane Conductivity,” phenomenological
approaches which consist in designing a model thanks to the experimental observa-
tions are proposed. Phenomenological models avoid the drawbacks of the other
models, in particular when considering the effect of pulse repetition. In conclusion,
current challenges in cell electroporation modeling are given, in order to obtain models
reliable and in complete accordance with the experiments.
Cell Electrical Modeling Before Electroporation: The Linear

Regime
In the Schwan model (Foster and Schwan 1989), the cell is composed of a homo-
geneous conducting cytoplasm, which is roughly tens of micrometers in diameter,
surrounded by a few nanometers thick, insulating membrane (see Fig. 2).
Fig. 2 Electrical model of biological cell by Schwan et al. (Foster and Schwan 1989) and its
equivalent electric circuit. The cytoplasm Oc is protected thanks to the thin membrane Om, whose
thickness h is about a few nanometers. The cell is embedded in an extracellular medium denoted by
Oe. The entire domain is denoted by Ω. (a) Schematic cell and dielectric parameters. (b) Equivalent
electric cell model
Equivalent Circuit for Cell: The 0D Model
The simplest way to model the cell is to derive an electric circuit model in which the
cell cytoplasm is described by a resistivity Rc, the cell membrane is identified by a
capacitor whose capacitance equals Cm, and the ambient medium is described by a
resistivity Re as given by Fig. 2b. Kirchhoff’s circuit law writes then
dVm
V cell ¼ V m þ Rc Cm : (1)
dt
If a static electric field of magnitude E is applied to the cell of radius rc, one infers
dVm
2r c E ¼ V m þ Rc Cm : (2)
dt
Assuming that Vm is zero at the initial time and that the electric field E is constant,
then Vm is given by

V m ðtÞ ¼ 2r c E 1 et=τ , with τ ¼ Rc Cm : (3)
Interestingly, this very simple model exhibits a linear dependency of the trans-
membrane voltage on the radius. Such a linear dependency is intrinsic, and it will be
recovered in the 3D model, as explained below. However, this equivalent model is
too rough, and in particular it cannot describe the influence of the cell shape or the
effect of the direction of the electric field on the membrane voltage. Therefore, it is
necessary to use partial differential equations (PDEs), which describe the electric
field in the whole cell.
3D Maxwell Equations and Their Simplication to the 3D Model
Maxwell equations and the standard constitutive laws for a dielectric material with
permittivity e and conductivity σ read
curlH ¼ e@t E þ σE, curlE ¼ curlH: (4)
This 3D vector system is quite complex to solve, especially for high contrast
material as biological cells. However, neglecting the time variation of the magnetic
field leads to a curl-free electric field E, which then implies that E derives from a
scalar potential E ¼ ∇V. Then, taking the divergence of the left equation leads to
the following PDE on V called the electroquasistatic formulation:
∇ ðe∇@t V Þ þ ∇ ðσ∇V Þ ¼ 0; (5)
to which initial and boundary conditions are imposed, for instance,
V j@Ω ¼ g, V jt¼0 ¼ V0: (6)
For the cell cytoplasm and the extracellular medium, the ratio e/σ is about 109s
(see the parameters of Fig. 2a), meaning that up to several megahertz, the displace-
ment currents can be neglected. However, due to the high resistivity of the mem-
brane, these currents have to be accounted for in the thin layer (Poignard 2009);
hence V is the continuous solution to
∇ ðσ∇V Þ ¼ 0, in Ohc [ Ohm [ Oe ; (7)

σ@n V Γ þ , Γh ¼ em @t @n V Γ , Γþh þ σ m @n V Γ , Γþh , on Γ and Γ h respectively; (8)
V j@Ω ¼ g, V jt¼0 ¼ V 0 ; (9)
where Γ h and Γ are the respective outer and inner boundaries of the cell membrane, and
the normal vectors are taken from the inner to the outer part of the cell. Even though this
equation is a rough simplification of the Maxwell vector equations, ∇V describes quite
precisely the electric field at low frequency, and thus it is widely used in the electrical
bioengineering community. However, due to the high resistivity and the small thickness
of the membrane, it is still complex to solve accurately the above equation on V.
To perform computations on realistic cell shapes without meshing the cell
membrane, Pucihar et al. (2006) propose to replace the membrane by an equivalent
condition on the boundary of the cytoplasm (see Fig. 3). Denoting by S0 the surface
conductance and by Cm the capacitance of the membrane defined as
Cm ¼ em =h, S0 ¼ σ m =h; (10)
and denoting by Oc the whole cell
Oc ¼ Ohc [ Om ; (11)
the electric potential V is approached by U, the solution to
ΔU ¼ 0, in Oc [ Oe ; (12a)
σ e @n U jΓ þ ¼ σ c @n U jΓ ; (12b)
Cm @t V m þ S0 V m ¼ σ c @n U jΓ , where V m ¼ U jΓþ U jΓ ; (12c)
U j@Ω ¼ g, Ujt¼0 ¼ V 0 : (12d)
It is worth noting that unlike V, the approximate potential U is discontinuous

across the interface: this is the effect of the high resistivity and the small thickness of
the membrane. Equation (12c) corresponds to a contact resistance model, rigorously
justified by Poignard and Perrussel (Perrussel and Poignard 2013). It is a generali-
zation of (2), accounting the cell geometry and the electric field orientation thanks to
the Neumann derivative.
Spherical Cell in a Unidirectional Constant Field
For simple shapes and simple electric field, it is possible to compute analytically the
transmembrane voltage. For instance, let assume that the cell is a sphere of radius rc
Fig. 3 Cell model with a

zero-thickness membrane.
The influence of the
membrane is described
through its capacitance Cm
and its surface conductance S0
embedded in the whole space ℝ3 and submitted to the unidirectional electric field
E ¼ EðtÞez . Using polar coordinates x ¼ r cos θ sin φ, y ¼ r sin θ sin φ, z ¼ r
cos φ – the Kirchhoff law (12c) reads

2σ e σ c 3σ e σ c
Cm @t V m þ S0 þ Vm ¼ EðtÞ cos φ: (13)
r c ð2σ e þ σ c Þ 2σ e þ σ c
Therefore, for a time-constant electric field, one obtains the explicit expression of Vm:
3 1 rc Cm
V m ðt, φÞ ¼ r c E 1 et=τm cos φ, with τm ¼ :
2 σ c þ 2σ e 2σ c σ e
1þ r c S0 þ r c S0
2σ c σ e σ c þ 2σ e
(14)
In the linear regime, the membrane conductance is of order 1, and since the cell
radius is of the order tens of microns, one has
1 r c Cm ðσ c þ 2σ e Þ
1, and τm : (15)
σ c þ 2σ e 2σ c σ e
1þ r c S0
2σ c σ e
Thus one recovers the linear dependency of the membrane voltage with respect to
the cell radius but with a different constant compared with the equivalent circuit
model.
One interesting feature of formula (14) lies in the fact that the membrane conduc-
tance has to increase dramatically to impact the membrane voltage. Roughly, for
values of S0 increasing from 1 to 104 S/m2, the changes on the potential is only
about 10%. Therefore, direct measurements of the membrane potential cannot detect
TMV Depolarization
Hyperpolarization
Fig. 4 Left: Schematic angular variation of the transmembrane voltage according to formula (14).
Right: Fluorescent measurements and relative fluorescent with respect to the angle (From Poignard
and Silve 2014)
any increase of membrane conductance below 1,000 S/m2. Another interesting feature
is that the constant time τm is bounded by below by r c Cm ðσ c þ 2σ e Þ=ð2σ c σ e Þ, which is
about several tenth of microseconds for cells, and thus the membrane voltage stabilizes
within a few microseconds.
Formula (14) predicts the sinusoidal behavior of the transmembrane voltage in
the linear regime as observed by the experimental data (Fig. 4).
Remark 1 (On the Schwann equation and short pulses)

As mention above, for square pulses longer than the intrinsic membrane charging
time τm, equality (14) leads to the so-called Schwann equation, which gives the
transmembrane voltage value at the end of the pulse: V m t τm ð3=2Þr c E cos ϕ,

since 1 et=τm rm 1. It is worth noting that such formula does not hold for short
pulses, which lasts a few nanoseconds. For such pulses, the membrane voltage is
approximated by
3 t 3σ e σ c t
V m ðt, φÞ t τm r c MðEÞ cos φ MðEÞ cos φ (16)
2 τm σ c þ 2σ e Cm
where M(E) is the mean value of E along the pulse:
Z t
1
MðEÞ ¼ EðsÞds: (17)
t 0
The important fact is that the transmembrane voltage for short pulses does not
depend on the cell radius but on the time of pulse application, and the mean value of
the field amplitude is involved.
Basic Concepts in Biophysics for Pore Formation in Liposomes
The creation and growth of single pores in vesicles have been studied for many years
(Kroeger et al. 2009; Ryham et al. 2011; Sandre et al. 1999; Weaver and
Chimazdzhev 1996). At the end of the 1990s, Sandre et al. (1999) have studied the
pore creation in vesicles embedded in a highly viscous fluid. The high viscosity of
the ambient medium made possible to visualize pore formation in stretched vesicles
in real time and thus the comparison of the theory with the experiments. Unfortu-
nately, such experiments have not been performed in electroporation research. Note
that changes in the medium viscosity would imply dramatic changes in the medium
conductivity and in the lipid properties, which thus would complexify the modeling.
However since the end of the 1990s, biophysicists proposed different model for pore
formation in membranes subjected to high transmembrane voltage. An extensive and
physical presentation of such approaches is given in the first section.
Single-Pore Models
Pore radius models are based on the description of the free energy of membranes,
thanks to which Langevin-type equation provides the pore radius evolution. More
precisely, given the membrane energy Em as a function of the pore radius r, the time
evolution of the single-pore radius r behaves as follows accordingly to Weaver and
Chizmadzhev (1996):
dr D
¼ @ r Em ; (18)
dt kB T
where D, kB, and T hold, respectively, for the diffusion coefficient, the Boltzmann
constant, and the temperature. The two next subsections deal with the two main
models that describe the single-pore radius that referred to, respectively, as
Brochart–Wyart, de Gennes, Sandre - Chizmadzhev and Weaver (BGS-CW)
model and dominant aqueous viscosity (DAV) model.
The Classical Brochart–Wyart, de Gennes, Sandre - Chizmadzhev and

Weaver Model
Brochart–Wyart, de Gennes, and Sandre (Sandre et al. 1999) on one side and
Chizmadzhev and Weaver (Weaver and Chimazdzhev 1996) on the other side
proposed the membrane free energy Em given by
Cs
Em ðr Þ ¼ Ef ðr Þ þ Ep ðr Þ ¼ 2πγr πr 2 σ 0 þ ap V 2m þ 4 : (19)
r
In the above equation, γ is the line tension, which tends to shrink the pore, while
σ 0 is the surface tension of the stretched vesicles. The term Cs holds for the steric
repulsion of the lipids. It ensures that a small space r0 between the phospholipids
remains at rest and has a very low influence for pore radii above r0. The term ap is
the membrane capacitance and Vm is the transmembrane voltage. The pore radius
evolution, which is called here BGS-CW model, is then derived thanks to (18) and
Stokes–Einstein – that is, kB T=D ¼ 2πηm δ where δ is the membrane thickness –
such that

dr 1 Cs
¼ γ r σ 0 þ ap V 2m 5 : (20)
dt ηm δ r
One can point out a few drawbacks of the model. The first one lies in the fact that
pores are assumed cylindrical all along their lives. Such assumption seems justified
during the formation, according to the very recent study of Sengel and Wallace
(2016). However there is no physical justification of circular shrinkage, in particular
the lateral diffusion of membrane lipids is important (a few nm2 per ns) and should
change the shape of the defects. In addition pore expansion is exponentially fast
above the critical radius rc given by
γ
rc ; (21)
σ 0 þ ap V 2m
which is hardly defensible since the pore radius should be bounded at least by half of
the membrane circumference. On the other hand, if the membrane voltage is stopped,
the pore shrinks, but the shrinkage is linear as soon as the radius is smaller than γ/σ0,
whereas Sandre et al. have reported an acceleration of the shrinkage for small radii
(Sandre et al. 1999). More recently, Kroeger et al. (2009 and Ryham et al. (2011)
have pointed out that experiments are at odds with the linear closure, and they
derived in two different ways a curvature-driven pore model.
Curvature-Driven Pore Closure: The Role of Aqueous Viscosity

In Ryham et al. (2011), Ryham et al. suggested that the aqueous viscosity of the
ambient medium impacts the pore dynamics. They derived the dominant aqueous
viscosity (DAV) model by adding a force Fs, which accounts for the lateral stresses
generated on the bilayer:
dr
Fs ¼ Cηs r ; (22)
dr
where C is a non-dimension constant, whose value is around C 8 according to
(Ryham et al. 2011) and ηs is the viscosity of the solution. Summing up all the forces,
the following equation on the pore radius evolution in membrane submitted to a
transmembrane voltage holds
dr dr Cs
Cηs r þ ηm δ ¼ γ þ r σ 0 þ ap V 2m þ 5 : (23)
dt dt r
Note that the steric repulsion Cs/r5 is not given in the DAV model as written in
Ryham et al. (2011); however it is necessary to prevent negative radii. The main
insight of this model, which fits very well the experiments of Portet and Dimova
(2010), as shown by Fig. 5 of Ryham et al. (2011), is the predominance of the
aqueous viscosity. Actually even if ηs is much smaller than the lipid viscosity, for
pore radii bigger than 1 μm, the term Cηsr plays a crucial role in the pore closure
since the membrane thickness h is very small of order 10 nm. The term
ηeff ðr Þ ¼ ηm h þ Crηs ; (24)
can be seen as the effective membrane viscosity, which increases linearly with
respect to the pore radius. Ryham et al. pointed out that due to the membrane
thinness, the aqueous viscosity cannot be neglected, explaining the curvature-driven
pore closure. Kroeger et al. used another reasoning based on the electrochemical
potential to obtain a similar equation accounting for membrane viscosity (see Eq. 7
of Kroeger et al. 2009), keeping the hypothesis of cylindrical pores.
From One Pore to the Total Pore Density Equation
From these models, Smoluchowski equation, which is a drift–diffusion equation on

the distribution function of pores of radius r, gives the pore distribution in the space
of radii. The very important insight of Krassowska and Neu in the late 1990s was to
approach Smoluchowski equation by an ordinary differential equation on the total
pore density Nep, thanks to a subtle asymptotic analysis (Neu and Krassowska 1999).
Interestingly Kroeger et al. (2009) pointed out that both BGS-CW model and DAV
model of pore radius lead to the same ordinary differential equation for the pore
density

dNep V 2m =V 2ep N ep qV2m =V 2ep
¼ αe 1 e ; (25)
dt N0
where Vep is the threshold membrane voltage above which electroporation occurs
and No is the pore density at rest, when Vm equals 0 and α and q > 1 are ad hoc
parameters.
Pore Radii Evolution
In the pore density model, all the pores are created with the same radius, rm. This is
due to the averaging and the asymptotic analysis performed to pass from the
Smoluchowski equation to the simple differential equation on Nep. In Krassowska
and Filev (2007 and Smith et al. (2004), Krassowska et al. add another equation for
the pore radius evolution rj, for j = 1, . . ., K, which has the same basis of Eq. 20:
Fig. 5 Numerical simulations of Krassowska-Neu model from Fig. 3 of (Smith et al. 2014)
(Courtesy of J.C. Weaver). “Electrical and poration response to a 1 kV/cm, 100 μs trapezoidal
pulse with 1 μs rise and fall times. (a) Transmembrane voltage spatially averaged over the polar
quadrants, during the initial 5.5 μs and at the end of the pulse from 97 to 103 μs. Red “+” indicates
the hyperpolarizing anodic side, and black “” side and black “” indicate the depolarizing
cathodic side. (b) Angular profile of transmembrane voltage at 99 μs. Background-shaded regions
indicate the angular extent of EP around the anodic and cathodic sides of the membrane. Red and
black dots mark the location of the anodic and cathodic poles on the membrane, respectively.
c Equipotentials (black) and pore density, n(θ) (white), at 99 μs”
!
dr j D 2 Cs
¼ ap r j V m 2πγ þ πr j σ eff r j þ 5 ; (26)
dt kB T rj
where ap and σ eff are defined as

Fmax 2σ 0 σ 0
ap r j ¼ , σ eff r j ¼ 2σ 0 !2 : (27)
1 þ r h = r j þ rT XK r 2j
1 1
4 j¼1 R2cell
In Krassowska and Filev (2007), the link between K and Nep is not precisely
stated, but it seems that the following equality holds:
K ðtÞ ¼ 4πR2cell N ep ðtÞ: (28)
The Krassowska-Neu Model for Electroporation Current
From the total pore density Nep and from the description of the pore radii evolution,
Krassowska and Neu et al. (Krassowska and Filev 2007; Neu and Krassowska 1999;
Smith et al. 2004) propose the following nonlinear electroporation current Iep:
X
K ðtÞ

I ep ðt, V m Þ ¼ iep r j , V m ; (29)
j¼1
where iep(rj, Vm) is the current through a single pore of size rj. In Krassowska and
Filev’s paper (Krassowska and Filev 2007), the current is given by
2σr j
iep r j , V m ¼ Vm; (30)
1 þ δ= 2πr j
which is used in recent works by Weaver et al. (Son et al. 2016) and Li and
Lin (2011).
Note that the way K(t) is computed is still unclear. For numerical purpose, Filev
and Krassowska proposed to consider only two kinds of pores: Ksmall for small pores
and Klarge large pores. At the beginning, all the pores are created at the same radius,
with a density Nep. Since each node is associated to a surface area δA, the number of
small pores Ksmall is given by K small ¼ δANep on the nodes of the mesh. Then at any
point of the mesh on the membrane, the pore radii evolve, and if the radius increases
locally, then the Ksmall is decreased and Klarge increases. One of the drawbacks lies in
the fact that the number of pores depends on the mesh, which means that the
numerical method is not mathematically consistent: changing the mesh changes
the number of pores passing from Ksmall to Klarge. However since the transmembrane
voltage value is quite robust with respect to the membrane conductance, as shown in
subsection “Spherical Cell in a Unidirectional Constant Field” of section “Cell
Electrical Modeling Before Electroporation: The Linear Regime,” such a variability
should not be troublesome, even though it raises modeling issues.
Back to the Electric Equation and Drawbacks of the Krassowska-Neu

Model
In order to link the biophysical model of electroporation to the electric cell model,
the Kirchhoff Eq. 12c is changed into
Cm @t V m þ S0 V m þ I ep ðt, V m Þ ¼ σ c @n U jΓ : (31)
Krassowska-Neu model provides a biophysical explanation of pore formation.

The grounds of the model consist of cylindrical pore formed by transmembrane
voltage. Such hypothesis has been recently confirmed, at least during the formation
process, by optical signal-channel recording in Sengel and Wallace’s paper (2016),
even though more complex phenomena are pointed out such as electropore diffusion
and the resealing has not been addressed in this study. Interestingly, KN model
provides a link between pores of the membrane and global electric potential in the
whole cell. From the numerical point of view, the model is not easy to solve because
it is nonlinear and stiff (the dynamic of pore creation is very rapid); therefore,
accurate numerical methods combined with very fine mesh is needed. The compu-
tational cost is quite high, especially for finite element methods; therefore most of the
numerical studies focus on bidimensional simulations.
The typical behavior of transmembrane voltage Vm is illustrated by Fig. 5, which
comes from Smith et al. (2014). During the first microsecond, the membrane is being
charged as a capacitor, but once the transmembrane voltage reaches a threshold
value, pores are created, leading to a decrease and then a stabilization of the
transmembrane voltage within 2 μs. At the equator, the electric field is tangent to
the membrane, and thus the transmembrane voltage does not increase: no electro-
poration occurs. At the pole, the electric field is normal, and the transmembrane
voltage is the highest before electroporation; therefore, this is the privileged location
for pore formation, as shown in Fig. C. 5. Such privileged locations near the poles
facing the electrodes have been corroborated qualitatively by the experiments in
many studies, in particular from Teissié and Rols’ group (see, for instance, Escoffre
et al. 2009; Rols et al. 1998 and reference therein), Miklavčič’s group (Kotnik et al.
2010, 2012), and also Vernier’s team (2003).
KN Model vs Experiments
One of the main drawbacks of the KN model lies in the fact that it barely corrob-
orates quantitatively the experiments. One can point out here a few experimental
observations non-accounted for by the KN model.
• According to Son et al. (2016), the maximum electroporation should be achieved

after the first pulse. Patch-clamp experiments on Chinese hamster ovary DC3F
performed by Wegner et al. exhibit a cumulative effect of the pulses (Wegner et al.
2015) leading to a persistent permeabilization, in addition to the transient permeabi-
lization accounted for by the models (see Fig. 6). As mentioned by Wegner et al.,
such a persistent effect could result from a change in lipid property (such as
a b
1 ms 1 ms
10 2 nA
5 nA 4 1 nA
5 nA 16
3 10 15
4
1
1
5 ms
10 ms
Specific membrane conductance (nS/pF)
Specific membrane conductance (nS/pF)

3.0
2.5
2.5
2.0
2.0
1.5
1.5
1.0
1.0
0.5 0.5
0.0 0.0
2 4 6 8 10 12 14 16 18 20 2 4 6 8 10 12 14 16 18 20
Pulse number Pulse number
Fig. 6 Experimental patch-clamp measurements on DC3F (Wegner et al. 2015). “Two examples of
the current response to repetitive application of double-pulse protocol, consisting of a depolarization
to 320 mV command voltage for 10 ms and successive clamp at 80 mV for 25 or 100 ms (a and b,
upper; note that part of the trace is omitted in b). Subsequently, the membrane was stepped back to
0 mV for 5 s before the next pulse of the same type was applied. Individual repetitions are
superimposed as indicated by numbers. The membrane conductance at the physiological voltage
range was calculated from the difference in steady-state current level induced by a voltage step from
80 to 0 mV (redrawn at an enlarged scale in a, b, as indicated by the arrows). Bar graphs (a, b,
lower) show the conductance levels for the applied sequence of pulses. The conductance increased
stepwise (dotted lines), either in several steps (a, pulses 3, 4, and 10) or in one step (b, pulse 15).
These two examples were selected to reflect the variability among individual cells”
oxidation), which is not accounted for in the KN model. Interestingly, patch-clamp

measurements of membrane intensity do no exhibit systematically a stabilization
during the pulse unlike KN model. According to Wegner et al. (2015), for some
patch-clamped cells, the current response does not stabilize during the pulse, and
moreover the membrane conductance increases with the number of pulses.
• Such cumulative effect is also reported in experiments performed by Pakhomova
et al. (2011) and Dermol et al. (2016). Whether the pulse repetition rate induces a
sensitization or a desensitization is still controversial. Recently Silve et al.
reported that high repetition rate of similar pulses is less efficient than the same
pulses repeated at a lower rate (Silve et al. 2014). These two studies have different
explanations of the same phenomenon: the pulse repetition rate has an impact on
the electroporation.
• From the modeling point of view, since membrane is a dynamic soft matter (lipids
are constantly in motion), it is hard to imagine that no diffusion or motion of pores
is involved in the description of pore density. Another important drawback is that
saturation of the membrane conductance and pore formation is not accounted for
as described, e.g., in Wegner et al. (2015).
The KN model has been extensively studied in silico during the past decade,
providing interesting explanations of electroporation. However the modeling has
several drawbacks making the model hardly predictive. The main drawbacks of such
modeling lie in the number of non-measurable parameters and the sensitivity of the
results to the parameters which makes the model calibration hardly obtainable. Even
though biophysics gives a range for their values, the model is too sensitive to slight
modification of many parameters, making hard the parameter calibration with
biological data.
Phenomenological Models for Membrane Conductivity
In order to compare theoretical models and experimental measurements, an alterna-

tive to biophysical models is to develop phenomenological models with the fewest
parameters to describe the main features of the experiments. The fact that a few
parameters are involved facilitates the parameter identification to obtain results that
are quantitatively in accordance with the experiments, the drawbacks being the
physical justification of the modeling.
Biological Evidences of Electroporation
The phenomenological model of membrane permeabilization proposed by Leguébe

et al. in (Leguébe et al. 2014) is based on the following assumptions, which come
from experimental observations:
• Permeabilization results from a long-term effect of defects in the membrane

related to an alteration of phospholipids in the membrane. Such alterations may
be due to the combination of high electric field and water molecules inside the
membrane. Actually it has been reported by Harakawa et al. (2005) that electric
field changes the phospholipid composition, by altering the lipid property.
• The dynamics of alteration and reconstruction of the membrane are much longer
as observed by the experiments (Rols et al. 1998). The alteration of the lipids is a
physical phenomenon, which occurs as long as pores are present on the mem-
brane and which is persistent after the end of the field pulse. On the contrary, the
membrane recovery is a biological phenomenon, which takes time: it happens for
minutes after the electric shock.
• Lipids diffuse along the membrane at a speed dL around 1 μm2/s (Fahey and Webb
1978), which is non-negligible compared to the lapse of time between two pulses
(usually of the order of 1 s), and therefore this surface diffusion has to be
accounted for.
The Two-Step Process of Membrane Electroporation
Leguébe et al. proposed in 2014 a two-step model for membrane electroporation,

describing the membrane surface conductance Sm as
Sm ðt,Þ ¼ S0 þ S1 X1 ðt,Þ þ S2 X2 ðt,Þ; (32)
where S0 is the membrane conductance at rest, S1 is the surface conductance

of the fully porated membrane, and S2 is membrane conductance due to the
long-term effect of electroporation. The non-dimension variables X1 and X2
refer to as the degree of poration during the pulse and the degree of long-term
changes in the membrane, respectively, and they satisfy the following
equations:
@X1 βðV m ðt,ÞÞ X1 ðt, V m ðt,ÞÞ

¼ , t > 0; (33a)
@t τ1
@X2 h iþ
dΓ ΔΓ X2 ¼ ðX1 X2 Þ=τ2 , t > 0; (33b)
@t
X1 jt¼0 ¼ X01 , X2 jt¼0 ¼ X02 (33c)
where []+ denotes the positive part, while τ1 and τ2 are the characteristic times of
“pore” creation and changes in the membrane. The function β is a sigmoidal
function, for instance,

ðV ep =ðλþυ0 ÞÞ
2 1 þ tan kep jλ þ υ0 j=V ep 1
βðλÞ ¼ e , or βðλÞ ¼ (34)
2
The Kirchhoff law is then given by
Cm @t V m þ Sm ðt,ÞV m ¼ σ c @n UjΓ : (35)
Such model involves six parameters: Vep, the two characteristic times τ1, τ2, the
diffusion coefficient dΓ, and the two conductances S1 and S2.
Calibration of the Model with Patch-Clamp Experiments
Patch-clamp experiments provide interesting direct measurements of the current that

flows across the membrane, for different pulses. Considering patch-clamp experi-
ments on DC3F of (Wegner et al. 2015), it is possible to fit the model parameters
with the data. The idea is to use the angular invariance of the command voltage g.
Assuming that the source potential is applied at the center of the cell – assumed
spherical – on a ball of radius Rs, the flux σ c @r Ujr¼Rc reads

Rc Rc Rc
σ c @r Ur¼Rc ¼ ðV m þ gÞ= þ 1 ; (36)
σ e σ c Rs
and thus the membrane potential Vm satisfies
I ðtÞ :¼ Cm @t V m þ Sm ðt, V m Þ ðV m þ υ0 Þ

Rc Rc Rc
¼ ðV m þ gÞ= þ 1 : (37)
σ e σ c Rs
The feature of the above equation is twofold: on one hand, the patch-clamp
technique measures directly the current I, and thus one can compare measured and
computed intensities. On the other hand the right-hand side of the formula makes it
possible to obtain the transmembrane voltage from the measured intensity. The value
of the parameters to obtain the currents of Fig. 8 is given in Table 1 of Leguébe
et al. (2014).
Phenomenological models being designed to be calibrated with the experiments
(see Fig. 7), it is natural to try to identify parameters that match the data, the key
point being to find a set of parameters such that numerical results fit with as many
experiments as possible. Indeed, if different set of parameters are needed for each
experiment, it means that the model is not reliable. Interestingly, Fig. 8 shows that it
is possible to find a set of parameters so that the numerical simulations fit quite well
with the data. The two functions X1 and X2 seem necessary to describe the data: the
first X1 increases very fastly during the pulse and vanishes at the end of the pulse,
while X2 increases more slowly but remains after the pulse. Fitting the KN model
with these patch-clamp data is proved to be difficult. One reason is the huge number
of parameters, which prevents a systematic sensitivity analysis of the model. The
two-step process exhibited by the data is hard to obtain with the variable Nep.
Biophysical Basis of the Phenomenological and Link with Pore

Radius Evolution
Equation (33) of the phenomenological model of Leguébe et al. is very different

from the approach of Krassowska et al. More precisely, Eq. 33a can be linked to the
pore density Eq. 25, but the reaction–diffusion (33b) involves surface diffusion of
lipids which is not accounted for in Krassowska et al. approach. Even though the
physical bases of such reaction–diffusion model is still unclear, it can be related to
the phase-ordering kinetics approach, as described in the seminal book of Bray
(2002). The concept being to look at the membrane as a two-phase medium
described thanks to the order-parameter field ϕ ¼ 0: the lipid phase, corresponds
Fig. 7 Comparison between simulations and patch-clamp data. The applied voltage is a rectan-
gular function (as described in Wegner et al. 2015) with a voltage amplitude of 820 mV during
10 ms. Thanks to the model, one can compare the currents but also the transmembrane voltage
(TMV). The membrane conductance is increasing during the pulse delivery
to the state ϕ and the water phase to ϕ ¼ 1. The Landau free energy of the membrane
subjected to transmembrane voltage Vm is given by the functional H(ϕ):
Z
1 1
H ð ϕÞ ¼ j∇ϕj þ V ðϕÞ þ Cm ðϕÞV m ds;
2 2
(38)
Γ 2 2
where V is a double-well potential with two minima 0 and 1 and Cm(ϕ) is the
membrane capacitance depending on ϕ. For instance, Weaver and Chizmadzev
proposed Cm ¼ CW ϕ þ CL ð1 ϕÞ in Weaver and Chimazdzhev (1996). From this
Landau free energy, the so-called model A (Bray 2002) describes the evolution of the
order parameter:
@H
@t ϕ ¼ d ðϕÞ; (39)
@ϕ
leading to the following reaction–diffusion on ϕ:
Fig. 8 Comparison of the computed and measured currents for different applied voltages to DC3F
for a well-chosen set of parameters. Experimental setup is described in (Wegner et al. 2015).
Interestingly, the measured current exhibits a two-step increase, as in the model. Such results
cannot be obtained easily with the KN model. a Applied voltage amplitude: b applied voltage
amplitude: c applied voltage amplitude: 860 mV, 460 mV, and 140 mV
1
@t ϕ ¼ dΔΓ ϕ V 0 ðϕÞ þ ðCW CL ÞV 2m : (40)
2
The right-hand side of phenomenological model can be seen as a specific model
of the term V 0 ðϕÞ 12 ðCW CL ÞV 2m in the above equation. The main insight of such
approach is to avoid the simplistic assumption of cylindrical pores. It is more
general, but the equation on single pore can be derived from it. More precisely,
making the assumption that only one cylindrical pore of radius r is present in the
membrane supposed to be flat and infinite, then the last equation leads to an equation
similar to Eq. 20, as described in Bray’s book (2002).
The Role of the Surface Diffusion
The main novelty of Leguébe et al. model lies in the surface diffusion, which is
numerically difficult to tackle. It is thus important to determine whether or not such
surface diffusion plays an important role. Theoretically, the diffusion induces a delay
in the spatial response of the membrane; therefore, diffusion should appear when
several identical pulses at different frequency are applied. In model without surface
diffusion, the maximal electroporation occurs after the first pulse, and then the pulse
efficiency decreases as reported by Weaver et al. in Son et al. (2016). In particular the
influence of pulse frequency is not accounted for. Surface diffusion induces a
complex response, which is a balance between the characteristic time of lipid
diffusion and pulse frequency.
In order to investigate the role of the surface diffusion, 3D simulations of a
spherical cell submitted to ten permeabilizing micropulses (10 μs, 40 kV/m), with
various repetition rates from 1 to 1,000 Hz, are presented. The lateral diffusion of the
lipids on the membrane is set to
dL ¼ 1012 m2 s1 ;
which is in the range of the measured lateral diffusion of the lipids in cell membranes
(Fahey and Webb 1978).
The average permeabilization X2 of the membrane is computed at any simulation
time. Figure 9 shows the distribution of X2 on the surface of the cell at different
instants of the 1 Hz and 1,000 Hz simulations.
For the case of fast repetition rate, the altered lipids do not have time to be evenly
spread on the membrane. Since the next pulse alters the same region as the previous
one, therefore the total quantity of altered lipids is lower than for the 1 Hz case.
Figure 10 presents the average of X2 after each pulse. As expected, the permeabi-
lization is more efficient if enough time is left between pulses to let the lipids diffuse.
These simulations corroborate qualitatively the results of high voltage/low volt-
age experiments (Pucihar et al. 2002; Šatkauskas et al. 2005) that, within the first
seconds after the pulses, show a better permeabilization when the lapse of time
between two consecutive pulses is longer.
Conclusions
In this chapter, biophysical and phenomenological ways to model cell electro-

permeabilization have been presented, and pros and cons for each approach are
discussed. To summarize, biophysical models are more reliable due to their theoret-
ical grounds, but they generally involve too many parameters that prevent quantita-
tive comparisons with the biological data. On the other hand, phenomenological
models are designed to account for the biological observations, but they suffer from
a lack of mechanistic basis. An interesting challenge in cell electroporation modeling
would be to enrich the phenomenological modeling with theoretical grounds, and the
phase-ordering kinetics theory could be the appropriate way to tackle this
challenges.
The long-term goal would consist in providing a model that can predict quanti-
tatively the degree of cell permeabilization and the amount of molecules taken up by
electroporation. The fitting of the parameters is thus a crucial point, which is
common to these perspectives.
0.8
0.6 After pulse 1 Before pulse 2 After pulse 2 After pulse 5 2 s after pulse 10
0.2
0
X2 (A.U.)
Fig. 9 Numerical illustration of the lateral diffusion effect (Leguébe et al. 2014). Influence of the
pulse frequency on the membrane permeabilization X2. The magnitude of each pulse is 40 kV/m
during 10 μs. Ten pulses are applied on both cells, but the time between pulses is different: 1 s for
the top line, 1 ms for the bottom line. After ten pulses, the average of X2 is around 8 108 for the
1 Hz case and half for the 1,000 Hz case
0.8
0.6 1 Hz
X2 (A.U.)
10 Hz
100 Hz
0.4 1000 Hz
0.2
0
1 2 3 4 5 6 7 8 9 10
Pulse number
Fig. 10 Numerical illustration of the increase of the variable X2 (Leguébe et al. 2014). Average
permeabilization X2 on the cell after each of the ten pulses of Fig. 9 for different pulse repetition
rates
Acknowledgments This study has been carried out with financial support from the French State,
managed by the French National Research Agency (ANR) in the frame of the “Investments for the
future,” Programme IdEx Bordeaux, CPU (ANR-10-IDEX-03-02).
Numerical simulations presented in this paper were carried out using the PLAFRIM experi-
mental testbed, being developed under the Inria PlaFRIM development action with support from
LABRI and IMB and other entities: Conseil Régional d’Aquitaine, FeDER, Université de Bor-
deaux, and CNRS (see https://plafrim.bordeaux.inria.fr/).
C.P. is partly granted by the Plan Cancer project DYNAMO (9749 Inserm), and the Plan Cancer
project NUMEP (11099 Inserm). C.P. is also member of the European lab of CNRS, LEA EBAM,
on electroporation.
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Electric Field Distribution and
Electroporation Threshold 56
Matej Kranjc and Damijan Miklavčič
Abstract
This chapter is dedicated to fundamentals of interaction between cells and tissues
exposed to the externally applied electric field. Through experimental work and
mathematical modeling, it has been shown that an accurate coverage of tissue
with sufficiently large electric field presents one of the most important conditions
for successful outcome of electroporation-based applications. The electroporation
process as well as cell viability is also governed by other parameters of applied
electric pulses and characteristics of targeted tissue; thus, different electroporation
threshold values of the electric field for reversible and irreversible electroporation
are being reported. Electric pulses and tissue structure also define established
electric field distribution which is difficult to predict. Still, numerical modeling
has proven to be very efficient in providing simulated maps of electric field
distributions for various electrode geometries and tissue structures, especially if
they incorporate nonlinear behavior of tissue electrical properties. Electric field
distribution in tissues can also be indirectly monitored using magnetic resonance
techniques that enable determination of electric field distribution in situ while
taking into account nonlinear changes that occur in the tissue due to electropo-
ration. Brief introduction to the monitoring method together with maps of electric
field distributions in animal and vegetable tissues obtained by means of magnetic
resonance techniques is presented in the last part of the chapter.
Keywords
Reversible electroporation • Irreversible electroporation • Electrical conductivity •
Numerical modeling • Magnetic resonance electrical impedance tomography
M. Kranjc (*) • D. Miklavčič (*)

e-mail: matej.kranjc@fe.uni-lj.si; damijan.miklavcic@fe.uni-lj.si

DOI 10.1007/978-3-319-32886-7_4
1044 M. Kranjc and D. Miklavčič
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1044
Electroporation Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1044
Values of Thresholds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1047
Electric Field Distribution in Biological Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1048
Mathematical Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1049
Numerical Modeling of Electric Field Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1050
Monitoring of Electric Field Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1051
Magnetic Resonance Electrical Impedance Tomography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1051
In Vivo Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1052
Plant Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1054
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1057
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1058
Introduction
Most theoretical studies predict that the application of electric pulses to the cell results
in structural changes which occur in the membrane in the form of hydrophilic pores.
This phenomenon, termed electroporation (sometimes also electropermeabilization),
allows various normally nonpermeant molecules to enter the cell by crossing the
membrane. When electric parameters (number, shape, duration, and repetition fre-
quency of electric pulses, direction of electric field), electrode geometry, and electrode
positions are appropriately chosen and consequently the cell is exposed to adequate
electric field, transient structural changes can be attained. After a certain period of
time, the membrane reseals and the cell survives. This is termed reversible electropo-
ration as cell preserves its viability. On the contrary, irreversible electroporation leads
to cell death as the applied electric field is too strong and the cell does not regain its
homeostasis after electroporation. Through experimental work and mathematical
modeling, it has been shown that an accurate coverage of tissue with sufficiently
large electric field presents one of the most important conditions for successful
outcome of electroporation applications such as electrochemotherapy, irreversible
electroporation tissue ablation, and pulsed electric field processing, to name a few.
In the first part of chapter, relation between transmembrane voltage and electric
field is briefly explained, followed by an overview of electroporation outcomes in
relation to applied electric field strength. The second part of the chapter is dedicated
to electric field distribution, how it is established, and how it depends on tissue
properties. In addition, monitoring of electric field distribution is presented together
with results in biological tissues.
Electroporation Threshold
Cell electroporation occurs when the externally applied electric field is above the
electroporation threshold value. The cell can be exposed to the electric field by an
application of electric pulses which establish an induced potential difference across the
56 Electric Field Distribution and Electroporation Threshold 1045
cell membrane, i.e., transmembrane voltage (see ▶ Chap. 59, “Transmembrane Voltage
Induced by Applied Electric Fields”), which is added to the resting membrane potential.
The cell membrane becomes permeable when transmembrane voltage, a sum of
induced potential difference and resting membrane potential, exceeds certain critical
value. The standard model of electroporation or model of pore formation describes
electroporation phenomenon as a formation of aqueous pores due to applied trans-
membrane voltage. Aqueous pores are established after transition from the hydrophobic
to the hydrophilic state which are then stable due to a local minimum of free energy.
This state is reversible until applied voltage remains under critical value, whereas when
this value is exceeded, an irreversible breakdown of the membrane occurs.
Relation between applied electric field E and transmembrane potential difference
ΔV in a spherical cell is described in Schwan’s equation
3
ΔV ¼ Er cos ðφÞ (1)
2
where r is the radius of the cell and φ is the polar angle measured from the center of
the cell with respect to the direction of the field (Grosse and Schwan 1992). Relation
between these variables in Eq. 1 shows that induced transmembrane voltage is
proportional to the electric field and the cell radius. For irregularly shaped cells, as
well as for cells close to each other, the induced transmembrane voltage cannot be
derived analytically; thus, numerical approach has to be used.
In dense cell suspension and in tissues, cells are positioned much closer together,
and therefore each of the cells is not exposed to the same electric field as it is in the
case of the low density cell suspension (Fig. 1). Structure of tissues is usually not
homogeneous but rather heterogeneous as tissues consist of cells of different types,
sizes, shapes, and orientations. In addition, cells in tissues are distributed in different
densities and are connected to each other through gap junctions and extracellular
matrix that can additionally influence the process of electroporation (Fear and Stuchly
1998). Still, for all types of cells, it is in common that induced transmembrane voltage
and electroporation process are strongly correlated with the applied electric field.
Exposure of cells to external electric field Eext can lead to four different outcomes
of electroporation process defined by three different thresholds (see Fig. 2):
• Eext < Erev: No electroporation process occurs since the electric field below
reversible electroporation threshold Erev.
• Erev < Eext < Eirrev: Electric field strength exceeds Erev and temporary membrane
permeabilization is established. Electric field is still below irreversible electroporation
threshold Eirrev, and cells remain viable after the end of electric field exposure.
• Eirrev < Eext < Etherm: Permanent membrane permeabilization leads to extensive
leakage of intracellular content and cell death. Still, no thermal damage is present
since electric field is below critical value of Etherm. Here, it is important to note
that combining the cell permeabilization effect of electroporation with the effects
of electrolysis (see ▶ Chap. 91, “Combining Electrolysis and Electroporation for
Tissue Ablation”) can produce effective ablation even at electric fields below
Eirrev (Stehling et al. 2016).
Fig. 1 Fraction of
electroporated cell, as a
function of electric field
strength cell densities of
20 vol.%, 28 vol.%, and
33 vol.%. The results are the
averages of five random
distributions of ternary
core–shell tissue models for a
given cell density. The curves
are the fits of the hyperbolic
tangent law to the calculated
data. Duration of the electric
field was 100 μs (The figure is
adapted from Essone Mezeme
et al. 2012)
Fig. 2 Reversible
electroporation, irreversible
electroporation, and thermal
damage as functions of
electric field strength and
pulse duration. Two points
with different combinations of
pulse parameters but with the
same outcome, i.e., reversible
electroporation, are depicted
• Eext > Etherm: Electric field establishes high electric currents causing temperature
increase and thermal damage to the cell.
The outcomes of electroporation process are not determined solely by electric

field strength but also by the duration of exposure to the field (texp):
texp ¼ tp N p ; (2)
where tp is a duration of a single electric pulse and Np is the number of applied

pulses. Each specific application of electroporation requires to some extent different
settings of pulse parameters, among which the pulse amplitude, tp, and Np have the
largest impact on the outcome of the experiment. In addition, these parameters need
to be adjusted for the targeted cell type, density of cells, and electrode geometry.
Still, the same outcome of experiment can be obtained by using equivalent pulse
parameters which is a compensation of the change in the value of specific pulse
parameter by carefully selecting value of the other parameter, e.g., instead of using a
number of short, high-voltage pulses, one can use longer pulses with lower voltage.
Relation between pulse parameters can be described by simple mathematical expres-
sions, such as power, logarithmic, or exponential functions (Pucihar et al. 2011).
Values of Thresholds
The optimal values of electric pulse parameters depend on the sensitivity of the cells
or tissue to the electric pulses; thus, different threshold values of the electric field for
reversible and irreversible electroporation are being reported in literature. In vitro
electroporation thresholds are different in regard to cell lines. For example, revers-
ible threshold was found to be between 400 and 600 V/cm for different cell lines
exposed to electric pulses with a duration of exposure to the electric field (texp) of
8 100 μs (Čemažar et al. 1998), whereas the majority of electric field strength used
in irreversible electroporation studies fall between 1000 and 2000 V/cm depending
on texp (Jiang et al. 2015). Still, electroporation thresholds cannot be regarded as
discrete values where all cells are either permeabilized or not affected. In reality,
these transitions from non-electroporated to electroporated state (see Fig. 1) and
from reversibly to irreversibly electroporated cells are continuous. These so-called
permeabilization and survival curves (Canatella et al. 2001) can be described using
mathematical models (see ▶ Chap. 63, “Mathematical Models Describing Cell
Death Due to Electroporation”) which allow prediction of electroporation outcomes
not just for electric parameters that were used for determination of the curve but also
for other parameters using interpolation. Some of the best models of cell survival as a
function of treatment time were the adapted Gompertz and the Geeraerd models and,
as a function of the electric field, the logistic, adapted Gompertz and Peleg–Fermi
models.
Permeabilization and survival in vivo curves are similarly shaped as in vitro, but
they are scaled to the thresholds according to the type of treated tissue. Reversible
and irreversible thresholds were mainly determined by overlapping electric field
distribution obtained by mathematical models with experimental observations such
as histological analysis. There are not many reports on in vivo reversible threshold
for different tissues. One of the well-known estimation of reversible threshold
(460 V/cm) was done on a rabbit liver exposed to electric pulses with (texp) of
8 100 μs (Miklavcic et al. 2000; Sel et al. 2005). The estimation was obtained by
overlapping histological results with the electric field distribution obtained by the
numerical model of tissue that incorporated electric field-dependent electrical con-
ductivity. There are more reported observations available for irreversible thresholds
with threshold ranging from 500 to 1300 V/cm (Jiang et al. 2015; Qin et al. 2013).
Similarly as in in vitro studies, it is important to acknowledge that these values
depend on texp, i.e., on the number and duration of applied pulses.
Pulsed electric field (PEF) also determines the outcome and the efficiency of food
processing applications such as improving mass transfer processes. The phenome-
non of mass transfer occurs in many operations of the food industry in which the aim
is extracting a given substance of interest (fruit juices, sugar, colorants, antioxidants,
etc.), removing water from foods (drying), or introducing a given substance into the
food matrix (osmotic dehydration, salting). The electric field required for improving
mass transfer can be achieved at field strength in the range of 50–500 V/cm and
1–10 kV/cm for texp in the range of 100–10,000 μs and texp < 100 μs, respectively.
One of the well-known applications in food industry that employs irreversible
electroporation is microbial inactivation. Since the size of microorganism is around
10–100 times smaller than the size of eukaryotic cells, higher electric field strength
(10–35 kV/cm) needs to be applied for irreversible disruption of cell membrane
integrity of microorganisms (see Eq. 1).
Electric Field Distribution in Biological Tissue
Electric field distribution is established in the biological tissue when electric current
passes through the tissue. In conventional electroporation, electric current is
established by applying electric pulses via electrodes that are in contact with the
tissue. This can be performed either via noninvasive plate electrodes, which embrace
the tissue, or with invasive needle electrodes, which are inserted into the tissue. In
both cases, when electric current passes through the tissue, it is distributed through
the tissue by taking the shortest and easiest path between points of highest potential
differences. In general, distribution of electric current is represented by the amount
of electric current per unit area, also known as current density. In terms of tissue
electrical properties, areas with higher current densities are also areas with highest
electrical conductivity. Highly perfused tissues, such as muscles, have higher con-
ductivity, whereas tissues such as adipose tissues or bone have lower conductivity. In
addition to current density distribution, electrical conductivity also defines electric
field distribution but in opposite manner; electric field will be higher in areas with
lower conductivity and vice versa (see Fig. 3c, d). Another feature that also defines
electric field distribution is the inlet and the outlet of electric current, i.e., electrode
geometry as demonstrated in Fig. 3a, b.
Electric field strength established between plate electrodes is often estimated
using U/d ratio, i.e., voltage applied to the electrodes (U ) divided by the distance
between the electrodes (d ). This estimation is valid only approximately in the area
between and away from the edges of the electrodes. Discrepancy between estimation
and real value of electric field becomes more evident with larger d and smaller
Fig. 3 Four examples of electric field distribution established in homogeneous and heterogeneous
tissue in a section plane perpendicular to plate and needle electrodes. Potential difference between
pair of electrodes (colored in white) in all of four examples is 1000 V. Width of plate electrodes (a, c)
and diameter of needle electrodes (b, d) are 0.5 mm, while the distance between both types of
electrodes is 10 mm. Homogeneous tissue (a, b) consists of single medium with homogeneous
electrical conductivity, whereas heterogeneous tissue (c, d) consists of additional circular-shaped
structure with 10 times higher electrical conductivity. All four examples of electric field distribution
were obtained by numerical modeling
electrode surfaces or if embraced tissue is not homogeneous (see Fig. 3c). Therefore,
for valid prediction of electric field strength and distribution, it is necessary to
introduce numerical modeling, especially in the case of any other electrode geometry
than plate electrodes or if treatment is performed on tissue with heterogeneous
electrical conductivity.
Mathematical Model
By definition, mathematical model is a description of a system composed of vari-

ables and equations that describe relationships between the variables. Mathematical
models are frequently used in studies of the effect of the electromagnetic field
accompanied by coupled phenomena on biological systems such as cells, tissues,
and organs. These systems are usually based on rather complex geometries with
inhomogeneous tissue properties and advanced boundary conditions which prevent
solving mathematical models analytically. Therefore, instead of analytical approach,

approximate solution of equations describing complex mathematical models is
obtained by numerical modeling. A good mathematical model that is validated
with experimental results is a strong tool that offers significant insight into the
understanding of observed processes. This is especially true for electroporation
where mathematical modeling is crucial for determining electric field distribution
established in tissues which electrical conductivity increases due to application of
electric pulses.
Numerical Modeling of Electric Field Distribution
Mathematical model describing electric field distribution in biological systems can

be modeled as steady state by neglecting transient responses that occur during
electric pulse rise time since duration of electric pulses is considerably longer than
cell membrane charging time. This simplifies set of Maxwell equations to Laplace
steady-state equation:
∇ðσ∇V Þ ¼ 0; (3)
where σ is an electrical conductivity of a biological system and V is established

electric potential. The electric field distribution E is then a gradient of electric
potential:
E ¼ ∇V: (4)
The numerical method that has proven to be very efficient in solving mathematical
models of electromagnetic studies is finite element method, a numerical technique
for finding approximate solutions of mathematical model by dividing the geometry
of a model into small finite elements. The solution in these elements can be
considered to be a simple piecewise polynomial function which can then be solved
using standard mathematical techniques.
Numerical models have already demonstrated the importance of contact between
tissue and plate electrodes and the importance of insertion depth of needle elec-
trodes. It has also been shown that for a certain number and duration of applied
electric pulses, the electric field has to be higher than a threshold value for electro-
poration process to occur. Therefore, modeling of electric field distribution can be
used for prediction of the electroporation outcome. Electric field distribution in the
tissue can be determined by the position of electrodes and applied pulse parameters,
both obtained by so-called treatment planning method (see ▶ Chap. 54, “Treatment
Planning for Electrochemotherapy and Irreversible Electroporation of Deep-Seated
Tumors”), which has already proved to have a great potential in clinical use of
electrochemotherapy and tissue ablation with irreversible electroporation of deep-
seated tumors. The outcome of this method is a treatment plan which consists of
instructions for physicians on where to position the electrodes and which voltages
have to be applied between electrode pairs in order to ensure a successful treatment.
The treatment plan is based on a numerical model prepared using CT- or MRI-based
tumor images, which are used for segmentation of the tumor and important healthy
structures in its surrounding. However, deviations in implementation of the treat-
ment plan can occur during the treatment in the clinic, as it is relatively difficult to
insert electrodes precisely according to the anatomy-based numerical treatment plan.
With respect to the inherent nonlinear behavior of biological tissues, it is impor-
tant to include those nonlinearities in the numerical model which contribute to the
outcome of calculations. In the case of electroporation, the most important
nonlinearity to include is the electrical conductivity of the tissue. Namely, the local
electric field in tissues is affected by applied electroporation pulses, which depend on
local electrical conductivity, and vice versa, electroporation increases the conduc-
tivity and consequently alters the electric field distribution. Also, the applied electric
field has to reach reversible electroporation threshold Erev in order to cause conduc-
tivity increase. For the duration of the pulse, this increase is permanent, and electrical
conductivity cannot decrease to its lower value even in the case in which electric
field drops below Erev. This makes it difficult to properly characterize the numerical
model of the treated tissue, and predicting the coverage of the treated tissue with
accurate electric field is a daunting task, since it relies mostly on the accuracy of the
electrical conductivity of the treated tissue used in the numerical model. As there is a
lack of tissue-specific experimental data on tissue properties for numerical models
that would be able to provide accurate and relevant electric field distribution,
electroporation monitoring method that would allow direct measurement of the
electric field strength within the sample would be of great importance for electro-
poration applications.
Monitoring of Electric Field Distribution
A method capable of monitoring electric field distribution during electric pulse

delivery would enable the detection of insufficient electric field coverage before
the end of either reversible or irreversible electroporation treatment. Since electro-
poration depends on local electric field, monitoring of electric field distribution
would increase and assure treatment effectiveness. As there are no available
approaches of measuring electric field distribution in situ, an indirect approach
using magnetic resonance techniques was suggested.
Magnetic Resonance Electrical Impedance Tomography
Magnetic resonance electrical impedance tomography (MREIT) (see ▶ Chap. 32,

“Principles and Use of Magnetic Resonance Electrical Impedance Tomography in
Tissue Electroporation”) enables reconstruction of electric field distribution by
measurement of electric current density distribution and electrical conductivity of
the treated subject during application of electric pulses using MRI and numerical
algorithms. This enables determination of electric field distribution in situ while

taking into account changes that occur in the tissue due to electroporation.
MREIT is a relatively new medical imaging modality based on numerical recon-
struction of electrical conductivity inside a tissue by means of current density
obtained by current density imaging (CDI) (see ▶ Chap. 33, “Current Density
Imaging as Means to Follow Tissue Electroporation”) (Seo and Woo 2011; Joy
et al. 1999). The MREIT algorithm applied for reconstruction of electrical conduc-
tivity of the tissue is based on solving Laplace’s equation (Eq. 3) through iterative
calculation. Electrical conductivity is updated after each iteration (k + 1):
jJCDI j
σ kþ1 ¼ : (5)
j∇uk j
where JCDI is current density obtained by CDI and ∇uk is a solution of Eq. 3. When
the difference between two successive conductivities falls below certain value,
electric field distribution can be calculated using Ohm’s law:
JCDI
E¼ (6)
σ
Still, the main difficulty of using MREIT to determine electric field distribution
during electroporation is associated with the limited capability of MRI scanners for
their use during application of electroporation such as electrochemotherapy and
irreversible electroporation tissue ablation. Hopefully, this could be surpassed in
the near future using open MRI scanner (Wang et al. 2010). Another limitation is
also that MREIT requires an application of at least two electric pulses with a delay of
approximately a second between them in order to deliver complete current density
information. This currently puts a frequency limitation on MREIT, although hope-
fully future improvements of MRI scanners will enable to reduce the required delay
between applied pulses. Conductivity changes that occur during the pulse are at the
moment also too demanding to asses with MREIT as a function of time. Even
though, it is important to be aware that the cumulative effect of electric current on
the MRI signal phase is measured. Therefore, this technique yields an electric field
distribution, which is a time average of its altering time course so that all the
consequences of conductivity alteration, which affect electric current, are not
neglected within obtained electric field distribution.
In Vivo Tissues
Investigation of the feasibility of MREIT for in situ monitoring of the electric field
distribution during in vivo reversible electroporation was performed on mouse
tumors (Kranjc et al. 2015). Electroporation was performed by applying two
sequences of four high-voltage electric pulses with an amplitude of 500 V, duration
of 100 μs, i.e., texp = 800 μs, at a pulse repetition rate of 5 kHz via two needle
platinum–iridium electrodes inserted into the tumor. Electric field distribution in the
tumor during pulse delivery was obtained by means of MREIT. For posttreatment
assessment of reversibly electroporated areas in the tumor, the contrast agent
gadolinium-tetraazacyclododecanetetraacetic acid (Gd-DOTA) was injected before
the application of pulses. After 24 h, T1-weighted images were acquired for the
observation of tumor areas where Gd-DOTA molecules were entrapped inside the
reversibly electroporated tumor cells (Leroy-Willig et al. 2005).
An example of electric field distribution obtained by means of MREIT is
shown in Fig. 4a where it is overlaid to T1-weighted image acquired just before
the application of electric pulses. As expected, the electric field was the highest
around the electrodes where it exceeded irreversible electroporation threshold
(900 V/cm), while it remained under the reversible electroporation threshold
(400 V/cm) in the areas away from electrodes. The average coverage of tumors
with electric field leading to reversible electroporation of tumor cells CMREIT was
calculated by dividing the predicted surface area of the reversibly electroporated
tumor cells with the surface area of the entire tumor, whereas tumor fraction
FGdDOTA was calculated by dividing the surface area of reversibly electroporated
tumor cells as obtained by entrapped MR contrast agent with the surface area of
the entire tumor. CMREIT SD and FGdDOTA SD were 38 9 % and
41 13 %, respectively.
Apparent differences can be observed when comparing tumor coverage with
electric field leading to reversible electroporation of tumor cells (CMREIT) in
Fig. 4b. These differences can be attributed to a varying distance between the
electrodes which lead to different electric field distributions in different tumors.
Another source of varying electric field distribution is believed to be the heteroge-
neous nature of tumor electrical conductivity (Muftuler et al. 2006). However, the
advantage of MREIT is exactly its ability to compensate the effects of different
electrode placements and tumor conductivity heterogeneities which occur in treated
tissues, thus yielding accurate electric field distribution.
Fig. 4 (a) The electric field distribution in the tumor obtained by MREIT superimposed to the
T-weighted image acquired before the application of electric pulses. (b) Scatterplot of the coverage
of five tumors with the electric field of reversible electroporation CMREIT and Gd-DOTA cell
entrapment FGdDOTA
Fig. 5 Illustration of potato tuber sample sliced to cylindrical-shaped samples. Two needle
electrodes with a diameter of 1 mm were inserted in the potato sample and placed in a MR
microscopy probe
Plant Tissues
Electric field distribution during electroporation was successfully reconstructed in

animal tissues and in vegetable tissues. Considering practical value, monitoring of
electric field distribution in vegetable tissues seems interesting since it can poten-
tially enable monitoring of pulsed electric field (PEF) applications in which applied
electric field mostly determines the outcome and the efficiency of PEF applications.
The study was performed on potato tubers (Kranjc et al. 2016) since PEF treatments
are already well established in potato industry for reducing cutting forces oil uptake
and browning during frying. In addition, electroporated areas in potato tubers
become distinctively darker few hours after the treatment and are therefore conve-
nient for observing electroporation effects.
Monitoring was performed on yellow-fleshed potato tubers’ (Solanum
tuberosum) cultivar “Agata” that were sliced to cylindrical-shaped samples
(Fig. 5). Two needle electrodes with a diameter of 1 mm were inserted in the potato
sample and placed in a MR microscopy probe. Potato samples exposed to electric
pulses were treated inside the MRI scanner equipped with micro-imaging system for
MR microscopy. Electroporation treatment of potatoes was performed by applying
two sequences of four high-voltage electric pulses with a duration of 100 μs, i.e.,
texp = 800 μs, a pulse repetition frequency of 5 kHz, and an amplitude of 500 V,
750 V, and 1000 V. Electric field mapping was enabled by two-shot rapid acquisition
with relaxation enhancement (RARE) CDI sequence which enabled imaging of
current density distribution inside conductive samples. Electric field in potato tubers
during application of electric pulses was reconstructed from CDI data by a mathe-
matical algorithm based on solving Laplace’s equation. Potato tubers were also
dynamically monitored by multiparametric MRI protocol. The protocol consisted
of diffusion-weighted imaging (DWI) based on a pulsed gradient spin-echo (PGSE)
sequence for the apparent diffusion coefficient (ADC) mapping and a multi-spin-
echo (MSE) imaging sequence based on the Carr–Purcell–Meiboom–Gill multi-
echo train for the T2 mapping. DWI and MSE images were taken every 45 min
until 12 h after the PEF treatment.
A comparison of digital photographs of treated potatoes taken 18 h after
application of electric pulses and the corresponding measured and simulated
Fig. 6 Digital photographs of three potato tubers from group 1 and one potato from the control
group taken 18 h after the PEF treatment ( first row), measured electric field distributions during the
PEF treatment (second row), and simulations of the electric field distributions obtained by the finite
element method (third row). Potato tubers were subjected to electric pulses of amplitudes 0, 500,
750, and 1000 V
electric field maps is shown in Fig. 6. The darkened region in the treated potatoes
is a result of oxidation that began immediately after the treatment. The extent of
regions with high electric field in the measured electric field maps corresponds to
the results of the simulations, while the electric field distribution deviates from
the simulated. Interestingly, electric field was not distributed evenly on the left
and right side of the treated potatoes. The origin of the effect is most likely
associated with a heterogeneous potato structure as well as its heterogeneous
electrical conductivity, which resulted in an uneven distribution of the electric
field. According to previous studies related to PEF treatment of potatoes, elec-
trical conductivity of untreated potato tubers is considered homogeneous, and,
with the application of electric pulses, conductivity starts to increase following
the sigmoid function (Ivorra et al. 2009). However, even with an electric field-
dependent conductivity, the electric field should be distributed symmetrically,
similar to electric field distributions obtained by simulations in Fig. 6. In results
obtained by MREIT, electric field distributions in potatoes were not symmetrical,
suggesting that electrical conductivity of potatoes was heterogeneous even before
the application of electric pulses (Faridnia et al. 2015). In addition, these differ-
ences between the simulated electric field distribution and the electric field
distribution obtained by MREIT are becoming more distinct with a higher
amplitude of applied electric pulses, presumably due to more pronounced effect
of nonlinear relation between electric field and electrical conductivity of the

potato tuber.
Furthermore, multiparametric MRI allowed additional analysis of induced tissue
changes due to application of electric pulses. These changes are associated with an
increase of extracellular water content on the account of a decrease of intracellular
water content. Differences between extracellular and intracellular water are reflected
in T2 relaxation time of water compartments as shown in Fig. 7. Intracellular water is
more bound and has therefore shorter T2 than the extracellular water. Gradual
decrease of T2 values with time after the application of electric pulses is associated
with a decrease of extracellular water content.
As shown in Fig. 7, T2 values in potatoes are scattered in three groups. First,
lowest values of T2 were measured in untreated potato tubers (E = 0 V/cm) and
were consistent with results from other studies (Nott et al. 2003). Second, scatter of
T2 was obtained in areas of potatoes exposed to an electric field ranging from
200 V/cm to 400 V/cm. Values of T2 were significantly different compared to
untreated potatoes, suggesting that electric field resulted in permeabilization of
cells’ membrane and in the release of water content from potato cells to extracellular
space. Values of T2 and consequently the amount of extracellular water, however,
have not changed significantly within 12 h. Third, scatter of T2 values was measured
in areas of potato exposed to an electric field higher than 400 V/cm. Here, however,
T2 values were linearly increasing with the electric field; hence the amount of
released water increased with the intensity of electric field. In contrast to results
below 400 V/cm, the amount of extracellular water decreased with time. Since the
electric field value of 400 V/cm distinguishes two trends of T2 values, one can
speculate that two electroporation processes were induced by an electric field of
lower and higher value of 400 V/cm, i.e., reversible below 400 V/cm and irreversible
electroporation above 400 V/cm. Obtained range of field strength for reversible
Fig. 7 Scatter of T2 values

(200 V/cm < E < 400 V/cm)
of potatoes exposed to 750 V
was significantly different
compared to untreated
potatoes, suggesting that
electric field resulted in
permeabilization of cells’
membrane and in the release
of water content from potato
cells. Scatter of T2 values
(E > 400 V/cm) was linearly
increasing with the electric
field; hence the amount of
released water increased with
the intensity of electric field
electroporation (between 200 and 400 V/cm) is in accordance with the study by
Galindo et al. in which reversible electroporation was demonstrated by propidium
iodide staining of cell nucleus using comparable electric field exposure time of
texp = 1 ms (Galindo et al. 2009). Cell viability study also showed that field strength
lower than 400 V/cm mostly does not influence the tuber cells, whereas increased
cell death was observed when higher field strengths were applied. Also, field
strengths higher than 400 V/cm are reported to have a considerable impact on potato
tuber microstructure, leakage of ion, and breakdown of the membrane. It seems that
reported significant changes also correspond to changes of T2 values.
Conclusions
Cell electroporation occurs when the threshold transmembrane potential is reached,

that is, when the externally applied electric field is above the electroporation
threshold value. This state is reversible until applied voltage remains under critical
value, whereas when this value is exceeded, an irreversible breakdown of the
membrane occurs. The electroporation process as well as cell viability is governed
by the parameters of applied electric pulses and characteristics of targeted tissue. It is
important to acknowledge that each specific application of electroporation, e.g.,
electrochemotherapy, gene transfer, and irreversible electroporation tissue ablation,
requires to some extent different settings of pulse parameters in order to obtain
desired electroporation process outcome.
Electric field distribution, which is established in biological tissue when elec-
tric current passes through the tissue, is difficult to predict, especially when
targeted tissue has heterogeneous structure. Therefore, for valid prediction of
electric field strength and distribution, it is necessary to introduce numerical
modeling. Still, as there is a lack of tissue-specific experimental data on tissue
properties for numerical models that would be able to provide accurate and
relevant electric field distribution, indirect approach of monitoring electric field
distribution during electroporation can be accomplished using magnetic resonance
techniques.
Cross-References

Deep-Seated Tumors
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Modeling of Electrochemical Reactions
During Pulsed Electric Field Treatment 57
Gianpiero Pataro, Giorgio Donsì, and Giovanna Ferrari
Abstract
In pulsed electric field (PEF) treatment, a biological material (cell suspension,
plant, animal, or human tissue) is placed between two electrodes of a treatment
chamber and here exposed to short (from several nanoseconds to several milli-
seconds) high intense electric field (0.5–80 kV/cm) pulses, with the final aim to
induce the physical electropermeabilization of the cell membranes. However,
when typical conditions for PEF processing are applied, undesired electrochem-
ical reactions, especially those involving metal release from the electrodes,
unavoidably occur at the electrode–electrolyte interface of a PEF treatment
chamber. The occurrence of these electrode reactions is a very complex phenom-
enon, which is affected by several factors, such as PEF chamber design and
electrode material, PEF electrical parameters, as well as composition and
chemical–physical properties of the treated biological material. In this chapter,
the basic concepts of electrochemistry are reported, and a detailed description of
the electrochemical phenomena occurring at the electrode–electrolyte interface of
a PEF chamber is given with a special emphasis to the case of PEF application for
food pasteurization. Finally, particular attention is devoted to metal release from
electrodes in a PEF treatment chamber, and a possible methodological approach
to develop a mathematical model describing the phenomena of the migration of
the electrode material into the treated biomaterial is also presented.
G. Pataro (*)
Department of Industrial Engineering, University of Salerno, Fisciano, SA, Italy
e-mail: gpataro@unisa.it
G. Donsì • G. Ferrari
Department of Industrial Engineering, University of Salerno, Fisciano, SA, Italy
e-mail: donsi@unisa.it; gferrari@unisa.it

DOI 10.1007/978-3-319-32886-7_5
1060 G. Pataro et al.
Keywords
Pulsed electric field (PEF) • Electrochemical reactions • Ionic double layer •
Metal release • Modeling
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1060
Principles of Electrochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1068
Electrochemical Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1069
Electrode Potentials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1070
Electrochemical Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1072
Electrochemical Behavior of a Pulsed Electric Field Treatment Chamber . . . . . . . . . . . . . . . . 1072
Ionic Double Layer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1073
Equivalent Electrical Circuit of an Electrochemical Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1075
Metal Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1076
Metal Release During Pulsed Electric Field Treatment of Food Products . . . . . . . . . . . . . . . . 1076
Modeling of Metal Release Phenomena . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1077
Case Study: Metal Release in a Pulsed Electric Field Treatment Chamber . . . . . . . . . . . . . . . 1077
Methodological Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1078
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1085
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1087
Introduction
Pulsed electric field (PEF) is a well-established nonthermal technology utilized in

several treatments in the area of medicine, biology, biotechnology, and food
processing, which are based on the reversible or irreversible permeabilization of
the membranes of biological cells (microbial, algae, plant, animal, or human)
(Miklavčič et al. 2014; Golberg et al. 2016; Pakhomov et al. 2010).
During a PEF treatment, the biological material (cell suspension, plant, animal, or
human tissue) is placed in direct contact with the metal electrodes of a treatment
chamber, operated either in batchwise or in continuous, and exposed to repetitive
(Hz–kHz) very short (from several nanoseconds to several milliseconds) and intense
(0.5–80 kV/cm) electric field (E) pulses provided by a pulse generator (Fig. 1a)
(Mahnič-Kalamiza et al. 2014). The pulses commonly used in PEF treatments are
unipolar or bipolar, with either exponential or square-wave shape (Figs. 1b). The
application of electric pulses causes a drastic increase in the permeability of the cell
membranes, referred to as electroporation or electropermeabilization, leading to a
loss of their semipermeability, either temporarily (reversible electroporation) or
permanently (irreversible electroporation) (Kotnik et al. 2012) (▶ Chap. 59, “Trans-
membrane Voltage Induced by Applied Electric Fields”).
The extent of cell membrane permeabilization depends, besides on electric field
strength, also on other electrical parameters such as total specific energy input (WT),
or number of pulses (n), or treatment time (tPEF), pulse width (or pulse duration) (τp),
pulse repetition frequency ( f ), and pulse shape. Treatment time refers to the number
57 Modeling of Electrochemical Reactions During Pulsed Electric Field. . . 1061
a Pulse generator
b Pulse shapes
V A
Unipolar Unipolar
V square wave Exponential
I
0.00 kV STOP
U (V) decay wave
STATE 0.00 A
0.00 kW
0.00 Hz
UNIPOLAR BIPOLAR
τp U/e
OFF-ON TRIC STOP RUN τp
Bipolar Bipolar
square wave Exponential
decay wave
E U (V)
Time (ms or ms)
Cell subjected to electric field pulses
Fig. 1 (a) Simplified scheme of a PEF system; (b) typical pulse shapes used in PEF treatments
of pulses applied multiplied by the pulse width, which depends on the pulse shape
(Fig. 1b) (Raso et al. 2016).
In general, at high electric fields (>15 kV/cm), PEF treatments represent a
suitable alternative to traditional thermal processing for the inactivation of alterative
and pathogenic microorganisms and quality-related enzymes in different liquid food
products, which retain, with only slight modification, their organoleptical, nutri-
tional, and health-promoting attributes (▶ Chaps. 127, “Impact of Pulsed Electric
Fields on Enzymes,” and ▶ 113, “Effect of Pulsed Electric Fields on Food Constit-
uents”). The application of low or moderate (0.1–10 kV/cm) electric fields has been
also advantageously proposed to facilitate the mass transfer phenomena of target
molecules, namely, drugs, solutes, water, juice, and bioactive compounds, through
the cell membranes of different biomaterials, such as cell suspension, plant, animal,
and human tissues. Thus, new methods for cancer treatments and gene therapy, as
well as highly efficient food processing operations, such as extraction, juice expres-
sion, drying, and freezing, assisted by PEF technology, have been set up, and some
of them are already in use in specific applications (▶ Chaps. 96, “Electroche-
motherapy of Cutaneous Metastases,” ▶ 23, “Gene Delivery by Electroporation In
Vitro: Mechanisms,” and ▶ 131, “Pulsed Electric Fields as Pretreatment for
Subsequent Food Process Operations”) (Barba et al. 2015; Donsi et al. 2010;
Golberg et al. 2016; Miklavčič et al. 2014; Pakhomov et al. 2010).
However, typical PEF treatment operating conditions cause some unavoidable
electrochemical reactions due to the flow of high current through the electrode–solution
interface of the treatment chamber. The extent of these reactions mostly depends on the
PEF treatment intensity, which, in turn, is of key importance in applications such as the
pasteurization of liquid foods.
Indeed, these undesired electrode reactions should be minimized to guarantee
safety and quality of food products and avoid electrode fouling and corrosion.
For example, as far as food safety and quality aspects are concerned, the electrode
reactions can determine changes of chemical structure and physical properties (e.g.,
pH) of the liquid in the vicinity of electrode surfaces; cause the production of toxic
chemicals, namely, H2O2, HCl, and HClO; and induce the detachment of small
particles of electrode material in the product, with a significant loss in flavor
acceptance (Evrendilek et al. 2004; Meneses et al. 2011; Morren et al. 2003; Pataro
et al. 2014). In addition, the reaction products formed are likely to react with other
compounds in the liquid food, leading to the formation of toxic compounds even
after the pulse treatment has been completed.
Due to the occurrence of electrochemical reactions, a film of food particles at the
electrode surfaces may form. This phenomenon is referred as electrode fouling. The
deposit of material on the electrode surface during extended processing period is also
responsible for several problems, such as local electric field distortion, arcing,
contamination of the system, and, in some case, interruption of the flow of the
product (Bushnell et al. 1995; Morren et al. 2003).
Finally, due to corrosion, the electrodes can be severely damaged, and their
surface roughness can increase due to metal release. This, in turn, can again cause
local electric field distortion and arcing, drastically shortening the lifetime of the
electrodes, thus reducing the technical feasibility of PEF technology (Gad and
Jayaram 2012a; Pataro et al. 2014; Roodenburg et al. 2005a, b).
Nevertheless, although electrochemical reactions and corrosion are well known in
other fields, their importance in PEF treatment systems was studied in detail only
recently (Table 1).
In 1995, Bushnell et al. (1995), based only on a theoretical analysis, suggested a
method to avoid or limit electrochemical reactions and fouling of electrodes in a PEF
treatment system by removing all the residual charge at the electrode interface during
the time elapsing between two consecutive pulses (zero net charge concept). Few
years later, Morren et al. (2003) derived an equivalent circuit to account for electro-
chemical reactions in a PEF chamber and presented the first experimental results on
the release of electrode material into liquid product undergoing a PEF treatment.
Since then, several researchers have addressed the problem of electrochemical
reactions in PEF treatment systems highlighting that electrode reactions are a very
complex phenomena, whose extent depends on many factors, namely, processing
parameters, design parameters, and treatment medium characteristics (Table 2).
An adequate knowledge of the role played by these factors on the occurrence and
extent of the electrochemical reactions at the electrode–solution interface of a PEF
Table 1 Summary of the published data on electrochemical phenomena in PEF treatment systems
Type of Treatment
study chamber Treatment conditions Main results References
Theoretical – – A method to reduce Bushnell
electrochemical et al. (1995)
reactions and the
fouling of electrodes
by removing the
residual charge from
one electrode during
the discharge period
was suggested
Experimental Batch parallel Treatment medium: Bipolar pulses Kotnik et al.
plate Spinner minimum reduced electrolytic (2001)
configuration essential medium contamination (Al3+
with either Treatment and Fe2+/Fe3+) with
aluminum or conditions: respect to unipolar
stainless steel Unipolar/bipolar pulses
electrodes square pulses, Concentrations of
E = 0–2 kV/cm, 1–8 Fe2+/Fe3+ above
pulses, τp = 100 μs 1.5 mM caused
to 1 ms significant loss of
viability of DC-3F
cells; no effect on
cell survival for Al3+
(0–2.5 mM) was
detected
Theoretical/ Tubular Treatment medium: Development of an Morren et al.
experimental configuration Aqueous NaCl equivalent circuit to (2003)
with stainless solution (σ = 4 model the behavior
steel mS/cm) of the double-layer
electrodes Treatment capacitor
conditions: Fe, Cr, Ni, and Mn
Sinusoidal were dissolved in
waveform, 0.15 kV/ the treated liquid
cm, 480 mA, below a certain
f = 1 MHz to 0.001 critical frequency
Hz, 1.5 mL/s (and above a certain
pulse width)
Experimental Colinear Treatment medium: Fe, Cr, Zn, and Mn Evrendilek
configuration Beer were released in the et al. (2004)
with stainless Treatment PEF-treated beer
steel (AISI conditions: samples with
316) Bipolar square significant loss in
electrodes pulses, E = 41–44 flavor acceptance
kV/cm, f = 600 Hz, and mouth feeling of
τp = 4 μs, the products
tPEF = 175 s, flow
rate = 1 mL/s
Theoretical/ Colinear Treatment medium: The amount of metal Roodenburg
experimental configuration Aqueous NaCl release was related et al. (2005a)
with stainless solution (σ = 2.2 to the transferred
(continued)
Table 1 (continued)
Type of Treatment
steel (AISI mS/cm) charge during the
316) Treatment pulse treatment
electrodes conditions: Lower transferred
Unipolar and charge and the use
nonsymmetrical of nonsymmetrical
bipolar pulses, pulse shape reduced
30 kV/cm, 6.7 the release of Fe, Cr,
pulses, τp =1–5 μs, Ni, and Mn
100 mL/s
Colinear Treatment medium: The metal Roodenburg
configuration Orange juice concentrations in the et al.
with stainless (pH = 3.8; σ = 3 PEF-treated juice (2005b)
steel (AISI mS/cm) that were found do
316) Treatment not exceed the
electrodes conditions: legislation values
Nonsymmetrical for fruit juices and
bipolar pulse, 30 kV/ the EU Drinking
cm, 6.7 pulses, τp =1 Water Directive
μs, 100 mL/s Because of the metal
release, the lifetime
of the electrodes
was estimated to be
approximately 760 h
Experimental Batch parallel Treatment medium: The roughness of Saulis et al.
plate 154 mM NaCl the anode surface (2007)
configuration solution increased in a
with polished Treatment manner depending
stainless steel conditions: to the total amount
electrodes Unipolar exponential of electric charge
decay pulses, passed through the
E = 4 kV/cm, unit area of the
60–120 pulses, τp electrode
=20 μs No changes of the
cathode surface
were detected
Continuous Treatment medium: The concentration of Gad and
flow chamber Aqueous NaCl/HCl dissolved Fe and Ni Jayaram
made with solution increased with the (2011)
two stainless (pH = 3.5–7.0, number of pulses
steel (type σ = 2 mS/cm) and peak voltage
316) Treatment applied
electrodes conditions: Low-pH solutions
having Unipolar exponential resulted in higher
opposing decay pulses, concentration of
convex 10–30 kV/cm, metals
surface WT = 0–1000 J/mL,
(biconcave f = 10 Hz,
processing τp = 1.1–1.6 μs,
zone)
(continued)
Table 1 (continued)
Type of Treatment
flow rate = 1500
mL/min
Theoretical/ Batch parallel Treatment medium: Electrode reactions Meneses
experimental electrode Aqueous NaCl induced pH shifts of et al. (2011)
configuration solution (pH = 7.0, up to 4.04 units
with σ = 1 mS/cm) close the electrodes
cylindrical PBS buffer The local change of
stainless steel (pH = 7.1, σ = 1.0 pH at the anode
electrodes mS/cm) (pH = 3.3) resulted
Treatment in considerable local
conditions: PPO inactivation.
Unipolar exponential For the cathode
decay pulses, (pH = 10.9) and the
E = 10–25 kV/cm, center (pH = 7) of
WT = 3–208 kJ/kg, the treatment
τp = 3.4 μs, chamber, no
f = 1–30 Hz inactivation of the
PPO was detected
Experimental Continuous Treatment medium: The amount of Fe Gad and
flow chamber Skimmed milk and Ni released in Jayaram
made with (pH = 7.0, σ = 5.23 the liquid medium (2012a)
two stainless mS/cm) increased with
steel (type Treatment increasing the
316) conditions: energy density, the
electrodes Exponential decay field strength, and
having pulses, 30–40 kV/ the pulse width
opposing cm, WT = 0–1000 No appreciable
convex J/mL, f = 21 Hz, differences were
surface τp = 1.1–1.6 μs detected for
(biconcave chromium
processing
zone)
Experimental Continuous Treatment medium: Highly conductive Gad and
flow chamber Apple cider foods, such as milk Jayaram
made with (pH = 3.8,σ = 2.78 and orange juice, 2012b
two stainless mS/cm), beer experienced a higher
steel (type (pH = 4.3, rate of electrode
316) σ = 0.994 mS/cm), material migration,
electrodes milk (pH = 6.9, which reduced the
having σ = 5.52 mS/cm), lifetime of the
opposing orange juice electrodes
convex (pH = 4.1, σ = 4.54 The effect of pH
surface mS/cm) appeared to be
(biconcave Treatment insignificant
processing conditions:
zone) Exponential decay
pulses, 40 kV/cm,
WT = 0–1000 J/mL,
f = 21 Hz
(continued)
Table 1 (continued)
Type of Treatment
Experimental Continuous Treatment medium: The amount of Pataro et al.
flow parallel Citrate–phosphate dissolved Fe, Cr, (2014)
plate (McIlvaine) and and Ni increased
configuration Trizma-HCl buffer upon increasing WT
with stainless (pH 7, σ = 2 and decreasing E, as
steel (AISI mS/cm) due to the
316 L) Treatment corresponding
electrodes conditions: change in the value
Unipolar exponential of pulse repetition
decay pulses, frequency
E = 12–31 kV/cm, The presence of
WT = 20–100 J/mL, halides in the
τp = 3.1 μs, flow treatment of
rates = 2–4 L/h medium (Trizma-
HCl) accelerated the
corrosion process at
the
electrode–solution
interface
Theoretical/ Continuous Treatment medium: A mathematical Pataro et al.
experimental flow parallel Citrate–phosphate model describing (2015a)
plate (McIlvaine) and the phenomenon of
configuration Trizma-HCl buffer the metal release
with stainless (pH 7, σ = 2 was developed and
steel (AISI mS/cm) validated
316L) Treatment The spatial
electrodes conditions: distribution of the
Monopolar metal concentration
exponential decay in the cross section
pulses, of the treatment
E = 12–31 kV/cm, zone was
WT = 20–100 J/mL, nonhomogeneous
τp = 3.1 μs, flow due to the
rates = 2–4 L/h combination of
hydrodynamic and
kinetic effects
(continued)
Table 1 (continued)
Type of Treatment
Theoretical/ Continuous Treatment medium: Pulse frequency, Pataro et al.
experimental flow parallel Citrate–phosphate rather than the (2015b)
plate (McIlvaine) and applied voltage, is
configuration Trizma-HCl buffer the main parameter
with stainless (pH 7, σ = 2 affecting the
steel (316 L) mS/cm) occurrence of the
electrodes Treatment metal release
conditions: The presence of
Unipolar exponential halides in the
decay pulses, composition of the
E = 12–31 kV/cm, treatment medium
WT = 20–100 J/mL, (Trizma-HCl)
τp = 3.1 μs, flow markedly increased
rates = 2–4 L/h the faradaic current
density, leading to
an intense localized
metal release from
the electrode’s
surface (corrosion)
Table 2 Main parameters affecting electrochemical reactions during PEF treatments

Process parameters Design parameters Treatment medium properties
Electric field strength Chamber configuration Composition
Total specific energy Electrode material Electrical conductivity
Pulsed width Electrode area pH
Polarity Electrode rugosity Halides
Frequency ... ...
Pulse shape ...
Flow rate ...
Temperature ...
chamber is necessary to identify the proper treatment conditions allowing minimiz-

ing the undesired side effects of these reactions. Additionally, another important
factor to be taken into account is that in PEF systems, the current densities occurring
at the electrodes are of orders of magnitude higher than in traditional electrochemical
systems. This raises the question whether the models commonly used to describe the
electrochemical phenomena at the electrodes are applicable to PEF systems.
To this purpose, numerical simulations specifically set up for PEF systems might
significantly help to improve the understanding of fundamentals of the electrochem-
ical phenomena occurring at the electrode–liquid interface of a PEF chamber,
clarifying the effects of the main electrical parameters, chamber design characteris-
tics, and treatment medium composition on electrode corrosion or release of elec-
trode’s materials (Pataro et al. 2015a, b).
Principles of Electrochemistry
Basic principles of electrochemistry and related equations can be traced back before
to the discovery of the electron by J. J. Thompson in 1893. In 1799, Alessandro Volta
invented the first electrical battery, known as voltaic pile, built by alternating stacks
of copper and zinc disks separated by paper soaked in acidic solutions (Ciobanu et al.
2007). Volta’s work stimulated other scientists to further investigations in this field.
It is worth mentioning that, in 1800, William Nicholson and Anthony Carlisle used
the current generated with a battery to decompose water into hydrogen and oxygen
(electrolysis). In 1835, Michael Faraday coined the basic terminology of electro-
chemistry, namely, anode, cathode, electrode, electrolyte, and ions. More impor-
tantly, Faraday discovered the two laws of electrochemistry. The first law states that
the amount of chemical changes or decomposition is proportional exactly to the
quantity of electric charge passing through the electrolyte solution. The second law
affirms that the amount of different substances deposited or dissolved by the same
amount of electric charge is proportional to their chemical equivalent weights.
Specifically, Faraday’s laws can be summarized by
Qi Mi
mi ¼ (1)
F ni
where Qi is the charge in Coulombs [C] involved in the deposition or dissolution of
the chemical species i, mi is the total mass of the chemical species i deposited or
dissolved at an electrode [g], F = 96485 C mol1 is the Faraday constant, Mi is the
molar mass of the substance [g mol1], and ni is the valency number of ions of the
substance (electrons transferred per ion). The ratio Mi/ni is the equivalent weight of
the substance altered.
These findings led ultimately to the development of the area of science nowadays
known as electrochemistry, which is defined as the study of chemical reactions
occurring at the interface between an electrode, usually a solid metal or a semicon-
ductor, and an ionic conductor or electrolyte. These reactions, generally referred to
as electrode processes or electrode reactions or electrochemical reactions, involve
the transport of ions within the electrolyte, accounting for the electrical conductivity,
or ions or electrons across the electrode–electrolyte interface resulting in the inter-
facial potential differences, which may trigger electrotransfer reactions at the
electrodes.
Chemical reactions where electrons are transferred directly between chemical
species (e.g., molecules, atoms, or ions) changing their oxidation state (i.e., the
fictitious charge that an atom would have if all bonds to atoms of different elements
were 100% ionic) are called oxidation–reduction or (redox) reactions.
A redox reaction can be seen as the simultaneous unwinding of two separate

“half-reactions”:
• Reduction half-reactions, which correspond to the gain of electrons from a

chemical species resulting in a decrease of its oxidation state. The chemical
species that gains electrons is called oxidizing agent or oxidant.
• Oxidation half-reactions, which correspond to the loss of electrons from a
chemical species resulting in an increase of its oxidation state. The chemical
species that loses electrons is called reducing agent or reductant.
Given two chemical species, namely, 1 and 2, the reduction and oxidation half-
reactions can be expressed as follows:
Reduction half-reaction
Ox 1 þ ne ! Red 1 (2)
Oxidation half-reaction
Red 2 ! Ox 2 þ ne (3)
where Ox1 and Red1 are, respectively, the oxidized and reduced form of the species 1;
Ox2 and Red2 are, respectively, the oxidized and reduced form of the species 2; and
n is the number of electrons (e) exchanged during the reaction (the same in both
half-reactions).
The overall redox reaction is then represented as
Ox 1 þ Red 2 ! Red 1 þ Ox 2 (4)
Electrochemical Cells
An electrochemical cell is a device capable of either generating electrical energy

from chemical reactions or facilitating chemical reactions introducing electrical
energy.
It generally consists of two half-cells, each containing an electrode in contact with
an electrolyte. The two half-cells may use the same electrolyte, or they may use
different electrolytes, which are typically placed in ionic contact by a salt bridge.
Electrodes can be made from any sufficiently conductive material, such as metals,
semiconductors, graphite, and even conductive polymers. Current flows through the
electrodes, which are connected by a conductor, via the movement of electrons,
while through the electrolyte is carried by ions.
In an electrochemical cell, species from one half-cell lose electrons (oxidation) to
their electrode, while species from the other half-cell gain electrons (reduction) from
their electrode. Michael Faraday defined the anode of a cell as the electrode where
oxidation occurs and the cathode as the electrode where the reduction takes place.
a Current flow b Current flow
Power
V Supply
Voltmeter
e- e-
e- e- flow e- flow
e-
Salt bridge
Anode Cathode Anode Cathode
X+ Y-(aq) Ma-(aq)
X Y X Y
Aa+(aq)
X-(aq) Y
Oxidation Reduction Oxidation Reduction
X Xa+(aq) + ne- Ya+(aq) + ne- Y Aa-(aq) A+ ne- Ma+(aq) + ne- M
Fig. 2 Electrochemical cells. (a) Galvanic cell; (b) electrolytic cell
Electrochemical cells are usually classified as either galvanic or electrolytic

(Fig. 2). In galvanic cells, reactions occur spontaneously at the electrode–electrolyte
interfaces. These cells convert chemical energy in electric energy and are the
components of batteries. In electrolytic cells, reactions are forced to occur at the
electrode–electrolyte interfaces utilizing an external electrical power source
connected to both electrodes. Electric energy is converted in chemical energy of
the products of the electrode reactions. Electrolytic cells are used in most cases to
decompose chemical compounds, and the process is referred to as electrolysis (e.g.,
electrolysis of water).
Finally, a third type of electrochemical cells, such as rechargeable battery, is also
available which acts as galvanic cells when discharged (converting chemical energy
into electrical energy) and as electrolytic cells when charged (converting electrical
energy into chemical energy).
Electrode Potentials
Current and potential are the two electrical variables of major interest in electro-
chemical cells.
Current i), expressed in amperes [A], is related to the electrode reaction rate and is
equal to the variation of the charge with time:
dQ
i¼ (5)
dt
where t, expressed in seconds [s], is the time and Q, expressed in Coulombs [C], is
the total charge passed through the cell.
Reduction half-reaction E∞red(V)

C12(g) + 2e− ↔ 2C1−(aq) 1.36
O2(g) + 4H+(aq)+2e− ↔ 2H2O(1) 1.23
Fe3+(aq) + e− ↔ Fe2+(aq) 0.77
O2(g) + 2H+(aq)+2e− ↔ H2O2(aq) 0.70
2H+(aq) + 2e− ↔ H2(g) 0
Fe3+(aq) + 3e− ↔ Fe(s) −0.04
Oxidizing Power
Reducing Power
Ni2+(aq) + 2e− ↔ Ni(s) −0.26

Co2+(aq) + 2e− ↔ Co(s) −0.28
Fe2+(aq) + 2e− ↔ Fe(s) −0.45
Cr3+(aq) + 3e− ↔ Cr(s) −0.74
Zn2+(aq) + 2e− ↔ Zn(s) −0.76
2H2O(1)+ 2e− ↔ H2(g) + 2OH−(aq) −0.83
Cr2+(aq) + 2e− ↔ Cr(s) −0.91
A13+(aq)+ 3e− ↔ A1(s) −1.66
Mn2+(aq)+ 2e− ↔ Mn(s) −2.37
Na+(aq)+ e− ↔ Na(s) −2.71
Fig. 3 Reduction half-reactions with their standard potentials with respect to SHE (Data from
Arning and Minteer 2007)
Cell potential (Ecell), expressed in volts [V], is a measure of the energy of the cell
reaction [1 V = 1 J C1].
The cell potential is the difference in potential of the two half-cells. Each half-
cell has a characteristic potential, which depends on the couple electrode–e-
lectrolyte, as well as on temperature, pressure, pH, and concentration of the
electrolyte.
The standard potential (E ) is the maximum potential or equilibrium potential
(Eeq), which applies to a half-cell when all the reactants are present at unit activity,
that is, when the concentration of the species in the solution is almost 1 M, at a
temperature of 298.15 K and at a partial pressure of 100 kPa. The standard potential
data are usually listed as reduction potentials (E red) with respect to the standard
hydrogen electrode (SHE), to which is arbitrarily assigned a half-cell potential equal
to zero (Fig. 3).
The reduction potential, E red, is a measure of the tendency of a chemical
species to gain electrons and, thereby, to be reduced. On the contrary, the oxidation
potential, E ox, which is just the negative of the reduction potential, is a measure of
the tendency of a chemical species to lose electrons and, thereby, to be oxidized.
However, the reactions are reversible, and the role of a certain electrode in a cell
depends on the relative oxidation–reduction potential of both electrodes. Gener-
ally, the reaction with the more negative half-cell reduction potential occurs at the
anode as oxidation. The reaction with the more positive half-cell reduction poten-
tial occurs at the cathode as reduction. The cell potential is then calculated as the
difference of the reduction potentials of cathode and anode or, alternatively, as the
sum of the reduction potential for the cathode and the oxidation potential for the
anode.
Eocell ¼ Eored ðcathodeÞ Eored ðanodeÞ ¼ Eored ðcathodeÞ þ Eoox ðanodeÞ (6)
For nonstandard conditions (e.g., difference in temperature, pH, concentration,

and pressure), the cell potential will deviate from the standard potential. The
actual half-cell reduction potential Ered is a function of the solution concentra-
tions and is related to these latter and to the standard potential E red by the Nernst
equation:
RT ½Ox
Ered ¼ Eored þ ln (7)
nF ½Red
where R is the universal gas constant (8.3145 J mol1 K1); T is the absolute
temperature, expressed in Kelvin; and the terms [Ox] and [Red] represent, respec-
tively, the concentrations of the oxidized and reduced species for the generalized
reduction half-reaction:
Ox þ ne Ð Red (8)
Electrochemical Reactions
Electrochemical Behavior of a Pulsed Electric Field Treatment

Chamber
A PEF chamber, which consists of two metal electrodes placed in direct contact with
an electrolytic solution and electrically connected to a pulse generator (Fig. 1a), acts
as an electrochemical cell.
Specifically, during its operation, a PEF chamber initially made with equal
electrodes in contact with an electrolyte with homogeneous concentration can
behave in three different ways.
Before starting the pulse treatment (i.e., without externally applied voltage), no
chemical reactions take place.
During pulse delivery, the PEF chamber acts as an electrolytic cell where an
external source of electrical energy is responsible for driving electrical current through
the cell. This electrical current has an electronic nature (electrons) in the conductive
solid electrodes and an ionic nature (ions) in the conductive liquid electrolyte. As a
result, electrochemical reactions take place at the electrode–electrolyte interfaces to
satisfy the condition of electric current continuity.
After pulsing, due to concentration gradients around the electrodes or to surface
material changes, the behavior of cell may shift from electrolytic to galvanic. Thus, if
the treatment chamber is not dried up, chemical reactions are likely to spontaneously
proceed even with no external voltage applied.
Ionic Double Layer
As in any electrochemical cell, at each electrode–solution interface of a PEF

treatment chamber, an ionic double layer immediately develops in order to compen-
sate any charge imbalance on the electrode surface, which can occur even if no
external voltage is applied (electroneutrality principle). This layer, which consists of
charged particles and/or of orientated dipoles, behaves as an electrical capacitor and
is often called double-layer capacitor (Fig. 4). It is typically described as a form of
different sub-layers. The first layer comprises solvent molecules and (non-hydrated)
ions adsorbed directly onto the electrode surface due to several chemical interac-
tions. The plane formed by the electrical centers of this layer is termed inner
Helmholtz plane (IHP), which lies χ1 than the electrode surface. The second layer
consists of hydrated ions, which are typically located at a distance from the electrode
surface no closer than the radius of the inner solvent coordination sphere (χ2). The
plane formed by these electrical charges is termed outer Helmholtz plane (OHP).
The ionic species in this plane are less compact than the IHP and are distributed in a
region called the diffuse layer, which is extended up to the bulk electrolyte region
where there is no separation of charge (Ciobanu et al. 2007).
The formation of the double layer causes a potential drop (Δϕdl) between the
surface and any point of the mass of the electrolyte solution. This potential drop is of
the order of magnitude of mV and depends on surface charge and on thickness of the
Fig. 4 Schematic diagram of Diffuse layer

OHP
IHP
an ionic double layer at the f

electrode–electrolyte
interface. IHP inner
Helmholtz plane, OHP outer
Helmholtz plane. χ 1 is the
distance between the plane
formed by the electrical
Bulk electrolyte
centers of IHP and the

Electrode
electrode surface; χ 2 is the

minimum distance between
the solvated ions of the second
layer and the electrode
surface. Continuous blue line
represents the potential profile
(ϕ) across the ionic double
layer
Potential (φ)
x1 x2 Distance
Water dipole
Solvated Adsorbed Non-solvated
(arrow points to +
(+) ion (+) ion (-) ion
charge on dipole)
double layer. As shown in Fig. 4, the voltage drop between the electrode and the
OHP is linear and tends asymptotically to zero at the imaginary boundary of the
double layer. To simplify the analysis of electrochemical kinetics, the contribution of
the diffuse layer can be neglected, and it can be assumed that the ionic double layer
extends up to the OHP. Hence, the potential drop at the electrode–electrolyte
interface at equilibrium (Δϕdle), when there is no net current flow, can be assumed
to be equal to the reduction potential of the half-cell reaction (Eored).
When a potential difference between the two electrodes is applied, the charge
buildup across the double layer takes place (Bockris et al. 2002; Gileadi 1993). In
this case, the thickness of the ionic layers at the interface increases leading to an
increase of the potential drop Δϕdl. As far as the voltage across the double layer
remains below the typical threshold voltage (Uth) of the reaction potential of
electrode material (~1–2 V), no electrochemical reactions occur, except some
low-level reactions due to the exchange current, as specified later in this chapter. If
a high enough voltage is applied for a long enough time to let the voltage across the
double-layer capacitor exceed the threshold value, in order to preserve the charge
conservation principle, two independent electrochemical (faradaic) half-reactions
will occur at the two electrodes: oxidation reactions will take place at the electrode
surface behaving as anode (high voltage electrode) and reduction reactions at the
electrode surface behaving as cathode (grounded electrode) (Morren et al. 2003). If
during PEF treatment unipolar pulses are applied, the electrode connected to high
voltage will behave as anode, while the grounded electrode will behave as cathode.
If instead bipolar pulses are applied, the cathode and anode interchange places
according to the pulse repetition frequency. Therefore, in this latter case, the
electrochemical reactions of oxidation and reduction occur alternatively at the
same electrode site.
The current resulting from this change in oxidation state is referred to as faradaic
current (if) because it obeys Faraday’s law. This faradaic current is a direct measure
for the rate of the redox reaction. The current necessary to charge the double layer up
to the threshold voltage, instead, is called charging current (ic), being a non-faradaic
current since it does not obey Faraday’s law, due to the absence of electrochemical
reactions associated (Morren et al. 2003). Quite complicated electrode reactions
occur consisting of a chain of chemical reactions. They involve mass transport of
electro-active species to the electrode, electron transfer across the electrode interface,
and the transport of the reaction products back to the solution. Mass transport and
charge transfer are two consecutive processes whose rate depends, besides the
applied potential, on electrode material, electrolyte, pulse repetition frequency,
pulse polarity, temperature, and pH, among others. The slower of the two processes
is, therefore, the limiting step and determines the overall rate. For example, the rate
of charge transfer is strongly related to the applied potential. When the applied
potential is low, charge transfer is low and it is therefore the limiting mechanism. On
the contrary, when the potential applied is increased, charge transfer is faster and
mass transport is then becoming the limiting mechanism. The mass transfer mech-
anisms occurring are by diffusion (movements of species due to concentration
gradients), migration (movement of charged particles due to an electric field), and
convection (movement of species induced by stirring, flow, or density gradient)

(Wang 2000).
Equivalent Electrical Circuit of an Electrochemical Cell
To obtain a relationship between an external electrical perturbation at the terminals

of a PEF chamber and the response of the double-layer interface, it is necessary to set
up an equivalent electrical circuit (▶ “Chap. 62, Computational Approach for
Electrical Analysis of Biological Tissue Using the Equivalent Circuit Model”). In
Fig. 5, equivalent electrical circuits for two electrode–electrolyte interfaces are
shown for different conditions.
Fig. 5 Equivalent circuit for E

the treatment chamber with
interface. The two ends of the
circuit represent, respectively,
the high voltage (left) and the
ground (right) electrode. (a)
No faradaic reactions; (b)
faradaic reactions and charge
transfer limitation; (c) faradaic
reactions and mass transfer
limitation (Adapted from
Pataro et al. 2015a, with
permission)
Anodic interface Cathodic interface
Electrolyte
Cd1 Cd1
ic i ic
HV
o (a)
Rs
Cd1 Cd1
ic i ic
HV
o (b)
if if
Rs
Rct Rct
Cd1 Cd1
ic i ic
HV
o (c)
if if
Rs
Rct Zw Rct Zw
In these circuits Rs is the bulk resistance of the solution and Cdl is the double-layer
capacitance, while the series of Rct and Zw is the faradaic impedance used to model
the faradaic processes occurring at the electrode–solution interface, with Rct the
charge transfer resistance and Zw the Warburg impedance used to model the mass
transfer limitations (Bockris et al. 2002; Gileadi 1993).
These models do not take into account some peculiar factors such as the hetero-
geneity of electrode surface and the eventual formation of reaction intermediates
absorbed at the electrode surface. Moreover, the circuit is nonlinear being Cdl, Rct,
and Zw depended on potential (Gileadi 1993).
When an external voltage is applied, and the threshold voltage (Uth) at which the
reactions typically start is not yet reached, the model is that of Fig. 5a. In this case,
the interface is said to be ideally polarizable and only a capacitive (non-faradaic)
current is flowing in Cdl.
When the threshold voltage (Uth) is reached, a faradaic current starts passing
through Rct in the branch of the circuit parallel to Cdl, and the circuits appearing in
Fig. 5b or c should be considered, according to the controlling limiting step, namely,
charge transfer and mass transfer.
The time highlighting for how fast the double layer becomes charged is called
threshold time tth and can be evaluated according to Eq. 9:
C0dl U0th
tth ¼ (9)
i
where C0dl = (1/2)∙Cdl is the value of the equivalent capacitance and U0th = 2∙Uth
the voltage at the electrode–liquid interface (Morren et al. 2003).
From Eq. 9, it can be seen that, for a given electrode reaction, the threshold time
depends mainly on the current through the chamber and the value of the double-layer
capacitance. Thus, PEF treatment chamber configurations featuring high intrinsic
electrical resistance (e.g., colinear configuration), which, therefore, operates at a rela-
tively low current, may limit the occurrence of electrochemical reactions. Similarly, the
use of high capacitance electrode materials, such as titanium (Cdl = 50 μF/cm2), glassy
carbon (Cdl = 260 μF/cm2), or dimensionally stable anode (DSA) (Cdl = 2000 μF/
cm2), instead of the most commonly used stainless steel electrodes (Cdl = 35 μF/cm2),
may limit the occurrence of electrode reactions by slowing down the charging process
of the double-layer capacitance (Amatore et al. 1998; Góngora-Nieto et al. 2002).
Metal Release
Metal Release During Pulsed Electric Field Treatment of Food

Products
The migration or release of electrode material into the liquid food under processing
is one of the main consequences of the electrochemical reactions occurring at the
electrode–solution interface of a PEF treatment chamber. The dissolution of the
anode material due to oxidation of the metal of the electrode is responsible of this
phenomenon.
The extent of metal release during pulse treatments must be minimized due to the
undesired effects occurring, which might limit the exploitation of PEF technology in
industrial scale, especially as far as applications for food preservation are concerned.
First of all, type and quantity of materials, which are eventually released from
electrodes and can be detected in PEF-processed foods, must comply with the health
safety rules approved by the food regulatory authorities before being able to intro-
duce the PEF-treated products in the market. Moreover, the organoleptic quality of
PEF-processed foods, especially the flavor of the product, should satisfy the require-
ments of consumers and compete favorably with that of products processed with
conventional preservation methods. The presence of any metallic mouth feeling may
play against consumers’ acceptance of PEF technology (Evrendilek et al. 2004; Gad
and Jayaram 2012a; Roodenburg et al. 2005b; Pataro et al. 2014). Finally, metal
release may cause serious damages to the electrode surface (corrosion), drastically
lowering the performance of PEF treatment systems and reducing the electrode
lifetime (Fig. 6) (Pataro et al. 2014; Roodenburg et al. 2005a, b; Saulis et al. 2007).
Modeling of Metal Release Phenomena
Case Study: Metal Release in a Pulsed Electric Field Treatment

Chamber
The setup of a mathematical model describing the electrochemical phenomena

occurring at the electrode–solution interface of a PEF treatment chamber is very
complex, due to the numerous factors playing an important role in the occurrence of
the electrode reactions during pulse treatment (Table 2). Hereafter, a general simpli-
fied approach, which can be proposed to develop a mathematical model describing
the phenomenon of metal release from the electrodes of a continuous flow PEF
treatment chamber, will be illustrated, according to the methodology described by
Pataro et al. (2015a, b) and schematically illustrated in Fig. 7.
Fig. 6 Corroded electrodes of a PEF treatment chamber after 3 h of operation (21 kV/cm,
WT = 100 J/mL) with Trizma-HCl buffer (pH = 7, σ = 2 mS/cm) (Adapted from Pataro et al.
2014, with permission)
Fig. 7 Schematic of the methodological approach to set up a model describing the phenomenon of
metal release from the electrodes of a PEF chamber
Methodological Approach
Figure 8 shows the 3D view of two typical continuous flow PEF treatment chamber
configurations: parallel plate (Fig. 8a) and colinear configuration (Fig. 8b). In both
model chambers, the high voltage (HV) and grounded (GR) metal electrode are
separated by means of a spacer insulator, which bounds a parallelepiped treatment
zone (in the X–Y–Z space) in the parallel plate geometry and a cylindrical treatment
zone (in the R–Z space) in colinear configuration, through which the treatment
medium flows along the Z axis.
Regardless of the chamber geometry, the phenomenon of metal release from the
electrodes of a PEF treatment chamber into the liquid medium can be regarded as a
mass transport process in an electrochemical system, described with Eq. 9:
@Ci
þ ∇ Ni ¼ r i (10)
@t
Fig. 8 3D view of (a) a HV

continuous flow parallel plate
Anode
treatment chamber (Adapted
from Pataro et al. 2015a with
permission from Elsevier) and
(b) colinear treatment
chamber Cathode
tion GR
Y irec
wd
Flo
Insulator
Z
b HV GR
r
Flow
direction
Z
Anode Cathode
Insulator
where Ci is the concentration of the ionic species i [mol m3] in the treatment
medium, ri is the reaction rate [mo m3s1] of the species i, and Ni is the molar
flux of the charged species i [mol m2s1], which, in turn, is the sum of three
contributions, namely, convection, migration, and diffusion, and is given by the
Nernst–Planck equation:
Ni ¼ Di ∇Ci zi um, i FCi ∇φl þ Ci v (11)
being Di the diffusion coefficient of the species i [m2 s1], zi the charge number of the
ionic species i [unitless], φl the electric potential through the electrolyte [V], v the
velocity vector [m s1], and um,i the ionic mobility of the charged species i [mols
kg1].
To solve Eq. 10, the following initial (I.C.) and boundary (B.C.) conditions are
set:
I.C.: the initial concentration of each metal species i (e.g., i = Fe, Cr, Ni) in the
solution within the PEF treatment zone is zero (or equals to any other initial value
Co)
Ci ðt ¼ 0Þ ¼ 0 (12)
B.C.1: At the electrode–solution interface, the molar flux Ni of the generic charged
species, i, is caused by the faradaic current
vi
n Ni ¼ Ri ¼ ðn ji Þ (13)
ne F
where ji is the faradaic current density associated with the ionic species i and n is the
direction normal to the boundary.
B.C.2: No flux of charged species at the electrode–insulator and
solution–insulator interface occurs:
n Ni ¼ 0 (14)
B.C.3: At the inlet section of the PEF chamber, the concentration of metal ions in the
solution is assumed equal to zero (or equals to any other inlet value Cin).
Moreover, other information and conditions to be included in order to define
completely the model are as follows:
• The electroneutrality of the electrolyte solution

• The continuity equation of the electric current in the electrolyte solution
• The continuity equation of the electric current passing through the electrodes
• The electrical insulation condition at both the electrolyte–insulator and
electrode–insulator
• Physical and chemical–physical properties of all materials (insulator, electrodes,
treatment medium, or electrolyte solution)
• Characteristics of the electric pulses generated by the pulse generator (Fig. 1b)
As shown in Fig. 7, to quantify the metal release from the electrodes, Eq. 10
should be solved by coupling with the equations of conservation of mass and
momentum, primary current, and secondary current distribution.
Fluid Dynamics
The distribution of the velocity field v of the liquid product inside the treatment
chamber can affect the mass transfer of the metals released from the anode surface in
the treatment medium. It can be evaluated by solving the continuity equation
(Eq. 15), which accounts for the conservation of mass, and the Navier–Stokes
equation (Eq. 16), which accounts for the conservation of momentum:
@ρ
þ ∇ ðρvÞ ¼ 0 (15)
@t
h i 2
Dv T ¼
ρ ¼ ∇P þ ∇ μ ð∇vÞ þ ð∇vÞ þ μð∇ vÞ δ þ ρg (16)
Dt 3
where ρ is the density of the fluid, P is the pressure, μ is the dynamic viscosity, and g
is the gravitational acceleration vector. A no-slip condition (v = 0) can be assumed
at the fluid–solid boundary interfaces.
Primary Current Distribution

The primary current defines the electric potential distribution, φl, in the treatment
solution regulating the migration of charged species. Assuming that the distribution
of the primary current is completely controlled by the resistivity of the electrolytic

solution in contact with the electrodes, the equivalent electric circuit representing the
behavior of the electrochemical cell is that sketched in Fig. 5a, where the capacitors
are assumed to be shortcut circuits.
Based on the charge conservation principle, the governing equation for the
electric potential U applied at the terminals of the PEF chamber can be written as
follows:
∇ J ¼ ∇ ½σ ðT Þ∇U ¼ 0 (17)
being J the primary current density vector.

The relation between the electric field E and the electric potential is as follows:
E ¼ ∇U (18)
The time dependence of the potential U(t) of the high voltage electrode is
that imposed by the pulse generator (Fig. 1b), while the other electrode is
grounded, i.e., the potential at the boundary surfaces is zero (U = 0). Finally,
as initial condition, a zero potential throughout the treatment chamber is
considered.
Secondary Current Distribution

The secondary current distribution determines the faradaic current density associated
with each metallic species ji at the electrode–solution interface, which is a direct
measure of the rate of the electrode reactions (ri). It is controlled by both the
resistivity of the treatment medium and the faradaic impedance (Fig. 4b or c).
The overall current density j flowing across the electrode–solution interface is the
sum of the anodic (ja) and the cathodic (jc) current densities:
j ¼ ja þ jc (19)
When no external voltage is applied across the electrodes of the PEF chamber, an
equilibrium potential Eeq is reached, which represents the reduction potential Eored
of the half-cell reaction, Ox + ne$Red. No net current flow is occurring because
the system contains a cathodic current density, given from the half-cell reaction
Ox + ne ! Red, balanced by an equal and opposite anodic current density, given
from the half-cell reaction Red ! Ox + ne:

jo j¼jja Eeq j¼jjc Eeq (20)
being jo the exchange current density, which represents the kinetics of a half-cell
reaction at the equilibrium potential (Eeq Δϕdle), when the so-called overpotential,
η (V), is zero. The overpotential is defined by Eq. 21:
η ¼ Δ∅dl Δ∅dle (21)

Fig. 9 Tafel plot: qualitative

behavior for anodic and
cathodic process. j is the
electrode current density
(A/m2); jo is the exchange
current density (A/m2); and η
is the overpotential (V)
Thus, η indicates the deviation of the potential drop at the electrode–solution

interface, Δϕdl, from the equilibrium potential Eeq and represents the driving force
for a non-spontaneous half-cell reaction to occur. The sign of η is positive for anodic
reactions and negative for cathodic reactions.
For each (anodic or cathodic) electrode reaction, the value jo can be derived
experimentally from Tafel plots (Fig. 9), which describe for an electrolytic process
the dependence of current density from overpotential. This plot shows an anodic
branch for positive overpotentials, where η becomes more positive with increasing
the current density at the anode, and a cathodic branch for negative overpotentials,
where η becomes more negative with increasing the current density at the cathode
(Ciobanu et al. 2007).
When an external voltage is applied to such an extent such that an overpotential is
generated, a net faradaic current density develops because of the occurrence of
non-spontaneous half-cell reactions. In that case, the net faradaic current density
associated with each metallic element i composing the electrode material ( ji,net) can
be calculated by the current density–overpotential equation, known as generalized
Butler–Volmer equation:

COx,s αnF CRed,s ð1 αÞnF

ji,net ¼ jo exp η exp η (22)
COx,b RT CRed,b RT
where A [cm2] is the area of the electrode in contact with the electrolyte solution; COx
and CRed are the concentrations of the oxidized and reduced species, respectively, at
either the surface (subscript s) or at the bulk (subscript b); and α is the charge transfer
coefficient, which is a dimensionless parameter with values between 0 and
1 (Ciobanu et al. 2007).
In Eq. 22, the first and second terms on the right-hand side represent the rates of
the cathodic and anodic parts of the half-cell reaction Ox + ne $ Red.
In a well-stirred cell, it can be assumed that the concentrations of the bulk
electrolyte and the electrolyte at the electrode surface are equal (i.e., COx,s COx,b
and CRed,s CRed,b), and the current–overpotential equation (21) reduces to the
more familiar Butler–Volmer equation:

αnF ð1 αÞnF
j ¼ jo exp η exp η (23)
RT RT
This equation describes the relationship between current density and overpotential
when mass transfer limitation is disregarded and the rate of the electrode reaction is
entirely controlled by charge transfer (Fig. 5b). It is worth noting that even though
the Butler–Volmer equation was derived for one half-cell reaction, it is applicable to
all reactions taking place at the anode and the cathode.
To calculate the values of η to be introduced in Eqs. 22 and 23, the evaluation of
the actual potential drop Δϕdl across the electronic double layer is required, which
can be estimated with Eq. 24 obtained by solving the equivalent circuit of Fig. 5b for
ideally polarizable electrode–solution interface (Fig. 5a).
1 qðtÞ
ΔV Cdl ðtÞ ¼ (24)
2 Ceq
being Ceq = Cdl/2 the equivalent capacitance of the two double-layer capacitors
(Stern 1924) and q(t) the charge accumulated on Ceq at the time t = tk after the
application of the k-th electric pulse, which can be estimated through an iterative
process (Pataro et al. 2015b).
The transient of the double-layer potential drop (Eq. 24) induces a transient in the
net faradaic current density for each metallic element (Eqs. 22 and 23). From these
relationships it is possible to evaluate the average net faradaic current density for
k
each metallic species i ( ji,av) during the k-th pulse as
ð tk
jki dt
tk1
jki,av ¼ (25)
ðtk tk1 Þ
being the time interval (tktk1) the time occurring between two consecutive pulses
applied and represents the reciprocal of the pulse frequency f.
Model Results
The set of partial differential equations introduced in the previous paragraphs,
namely, the coupled equations of mass and momentum conservation, primary
current, secondary current, and mass transfer, with their relative initial and boundary
conditions, can be solved using a finite element method (FEM)-based commercial
software. From the simulation, it is possible to achieve, for different processing
conditions, the dynamics of the spatial distribution of metal concentration in the
treatment medium in each cross section of the treatment chamber. As an example,
Fig. 10 shows a typical predicted spatial distribution of iron concentration in Trizma-
HCl buffer solution (pH = 7, σ = 2 mS/cm) in the cross section at the outlet of a
parallel plate PEF chamber (Fig. 8a) at different processing times (U = 5.6 kV,
E = 21 kV/cm, WT = 60 kJ/kg, τp = 3.1 μs, f = 21 Hz, Reynolds number
Re = 245). The dynamic of the metal release from the anodic surface can be
3.2381x104
4
(ppb) x 10
3
HV HV HV
2.5
1.5
0.5
0 2 4 6 Time (s)
0
0.8367
Fig. 10 Predicted distribution of the iron concentration in the outlet cross section of a parallel plate
PEF treatment chamber as a function of the processing time. HV – high voltage (Adapted from
Pataro et al. 2015a, with permission from Elsevier)
observed, as well as the characteristic nonhomogeneous spatial distribution of metal

concentration in the treatment zone that, for the specific conditions of simulation
adopted, is likely ascribable to the hydrodynamic effect (laminar flow) combined to
the kinetic effect of metal release at the anodic interface (Pataro et al. 2015a).
Moreover, by integrating the spatial distributions of the concentration of metallic
elements i) released from the electrodes (Fig. 10), it is possible to evaluate, for each
processing time, the bulk concentrations of metals Ci,bulk, at each cross section of the
treatment chamber:
ðð
Ci v dS
Ci ,bulk ¼ ðð (26)
v dS
where dS is the differential surface vector normal to the direction of the flow.
For example, in the most common case of electrodes made in stainless steel
AISI316L, the main metallic elements, which are expected to be released from the
electrode into the electrolyte solution, are iron, chromium, and nickel. Figure 11
shows a typical release kinetics of these metallic elements in Trizma-HCl buffer
solution (pH = 7, σ = 2 mS/cm) evaluated at the outlet cross section of a parallel
plate treatment chamber.
From this graph it can be seen that, regardless of the metal species, the amount of
metal released increases with time and a stationary value is reached after almost
3 s. However, although a similar behavior can be observed for all the metallic
Fig. 11 Predicted Ci,bulk profiles of Fe, Cr, and Ni in Trizma-HCl buffer solution at the outlet cross
section of the parallel plate treatment chamber as a function of the processing time. Simulation
conditions: U = 5.6 kV; E = 21 kV/cm, WT = 60 kJ/kg, τp = 3.1 μs, f = 21 Hz, Re = 245
(Adapted from Pataro et al. 2015a, with permission from Elsevier)
elements considered, the final concentrations reached differ, according to the com-
position of the stainless steel electrodes, as well as the specific release kinetic of each
metallic element.
Finally, numerical simulations can be also used to get insights on the effect of the
different processing parameters determining the extent of the electrode reactions. As
an example, Fig. 12 shows a typical release kinetics of iron in Trizma-HCl buffer
solution (pH = 7, σ = 2 mS/cm) as a function of either the applied voltage (electric
field strength) applied when the energy input is kept constant (Fig. 12a) or the total
specific energy input at a fixed applied voltage (electric field strength) (Fig. 12b).
Conclusions
Electrochemical reactions unavoidably occur during PEF treatment especially for

applications (e.g., microbial inactivation) involving the exchange of large amount of
charge at the interface between electrodes and biological material (e.g., cell suspen-
sions, liquid foods). These reactions are undesired and must be minimized, due to
serious problems that they may cause, such as the release of metals from the
electrodes of the PEF chamber. As a consequence of the occurrence of this phenom-
enon, loss of cell viability, contamination of treated products, formation of toxic
compounds, and fouling and corrosion of the electrode surfaces reducing their
lifetime are likely to take place.
Knowledge of basic theory of electrochemistry, including terminology, funda-
mental principles, and equations, as well as of electrochemical cells, are essential for
Fig. 12 Predicted Ci,bulk profiles of Fe in Trizma-HCl buffer solution at the outlet cross section of a
parallel plate treatment chamber as a function of the processing time. Simulation conditions:
(a) WT = 60 kJ/kg, U = 3.0, 5.6, and 7.6 kV, τp = 3.1 μs, f = 59, 21, and 11 Hz, Re = 245;
(b) U = 5.6 kV, E = 21 kV/cm, τp = 3.1 μs, f = 7, 21, and 35 Hz, Re = 245 (Adapted from
Pataro et al. 2015a, with permission from Elsevier)
the comprehension of the complex phenomena occurring at the electrode–electrolyte

interface of an electrochemical cell, such as a PEF treatment chamber. Moreover, the
understanding of the role played by different processing factors, namely, applied
voltage, current magnitude or current density, pulse repetition frequency, pulse
duration, pulse shape, and fluid composition in affecting the value of the double-
layer capacitor and the rate of its charging process, as well as the value of the
overpotential necessary to start the electrochemical reactions, is of utmost impor-
tance. Indeed, on the basis of the fundamental theory, it has been demonstrated that
the cumulative buildup of charge on the double-layer capacitor must be avoided.

This might be done, for example, by selecting electrode material with high capac-
itance, removing the charge after each pulse, applying bipolar pulses, or applying
monopolar pulses at relatively low pulse frequency.
However, due to the large number of parameters affecting the extent of electrode
reactions, the definition of optimal treatment conditions, as well as the selection of
electrode geometry and electrode material able to minimize the occurrence of
undesired electrochemical reactions, requires the development of a mathematical
model specifically set up for PEF treatment systems.
To this purpose, a possible methodological approach to model the phenomena of
metal release from the electrodes of a PEF chamber has been described in the last part
of this chapter. This approach could be used not only to deeply understand the
fundamentals of the electrochemical phenomena taking place at the electrode–solution
interface of a PEF chamber but also to clarify the role played by the main relevant
design and processing parameters, in order to be able to select optimal PEF treatments,
which will allow to prevent or, at least, minimize the occurrence of the undesired
electrode reactions.
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Modeling Transport Across the
Electroporated Membrane 58
Miao Yu and Hao Lin
Abstract
Molecular transport is central to many applications of electroporation. With few
exceptions, delivering target molecules into the cytoplasm and/or the nucleus
defines the very application itself. The pertinent transport phenomena are com-
plex and involve different physical mechanisms which are strongly coupled.
Moreover, there is large variability in molecular properties of the target and the
protocols used in electroporation practice. Such complexity calls for development
of reliable theoretical and modeling tools to assist in understanding, design, and
optimization. In this chapter, a theoretical framework based on a continuum
approach is presented. The model is built by combining and coupling three key
elements: electrodynamics, membrane permeabilization, and species transport. A
detailed description of the governing physics and equations, together with aspects
of numerical implementation methods, is provided. Exemplary results are given
and discussed. The current approach is capable of predicting the permeabilization
status of the membrane and the contributions from various transport mechanisms
with both spatial and temporal resolutions, thus corroborating experimental
results in details. On the other hand, it is limited to simulate the transport of
small- to moderate-sized molecules; further development is required to tackle that
of macromolecules such as DNA.
Keywords
Transport • Electrophoresis • Diffusion • Nernst-Planck equations • Electropora-
tion • Numerical simulation
M. Yu (*) • H. Lin (*)

Department of Mechanical and Aerospace Engineering, Rutgers, The State University of New
Jersey, New Brunswick, NJ, USA
e-mail: miaoyu@rutgers.edu; hlin@jove.rutgers.edu

DOI 10.1007/978-3-319-32886-7_6
1090 M. Yu and H. Lin
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1090
Model Framework: A Continuum Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1091
The Electrical Problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1091
The Permeabilization Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1096
The Coupling of the Electrical and the Permeabilization Model . . . . . . . . . . . . . . . . . . . . . . . . . . 1098
Species Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1100
Numerical Implementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1103
Exemplary Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1105
Other Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1107
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1109
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1109
Introduction
Ionic or molecular transport is a key element in both irreversible and reversible

electroporation. In the former, excessive exchange of the cytosol with the buffer may
contribute significantly to cell death. In the latter, transport plays a central role, as
uptake of target molecules is the very goal of its many applications including cell
transfection, gene therapy, drug delivery, among others. The targets to be delivered
into the tissue or cells range from biologically active agents to fluorescent markers
and are very diverse in molecular properties. Meanwhile, the cell or tissue type and
the electrotransfer configuration also demonstrate large variability. Extensive exper-
imental efforts have been committed to developing optimized delivery protocols. On
the other hand, theoretical and modeling tools are in high demand to offer guidance
and assistance in the understanding and optimization.
Over the past three decades, significant progresses have been made to develop
phenomenological models for membrane permeabilization upon application of a
strong electric field (Weaver and Chizmadzhev 1996; Neu and Krassowska 1999).
These models are able to predict detailed dynamics of each individual pore,
including location on the membrane, creation, destruction, and size evolution.
They serve as the foundation for quantitative prediction of cross-membrane trans-
port. Here the term “cross-membrane transport” refers to ions or molecules
translocating across the electroporated membrane through the aqueous pores –
such is the basic hypothesis throughout this chapter. Evidently, the past research
verified that different models are necessary to account for the very different physics
involved in the transport of small- to moderate-sized molecules as opposed to large
macromolecules. For the former, it is commonly assumed that they can directly
traverse electropores; examples include calcium ions, fluorescent markers such as
propidium iodide, and most anticancer drugs. The latter are typically long polymer
chains that usually have a coil-like effective radius much greater than the typical
pore size; examples are deoxyribonucleic acid (DNA), ribonucleic acid (RNA),
and other macromolecules.
The current state of the art in electroporation research has a relatively strong
consensus in the mechanisms transporting smaller molecules: they are diffusion,
58 Modeling Transport Across the Electroporated Membrane 1091
electrophoresis, or for most cases a combination of the two across the permeabilized
membrane. On the other hand, the mechanism for DNA electrotransfer remains
elusive (Rosazza et al. 2016); possible candidates involve electrophoretic transloca-
tion (Yu et al. 2012) and endocytosis. Endocytosis has been strongly indicated
by recent experimental work (Wu and Yuan 2011), yet direct translocation via
nano-channel-mediated electrophoresis has also been demonstrated in an attempt
to rapidly transport DNA and to bypass the slower endocytosis (Boukany
et al. 2011). In this chapter, the focus is set to the modeling of the transport of
small- to moderate-sized molecules, where both the understanding and model
development are relatively mature.
A continuum approach shall be adopted, which treats variation (in electric poten-
tial, species concentration, pore density, etc.) as continuous and gradual quantitative
transitions in space and time. Discontinuities are only allowed across special bound-
aries (e.g., the cell membrane). Non-continuum approaches, most notably molecular
dynamic models, are presented elsewhere in this handbook.
In what follows, a comprehensive description of the theoretical framework is
given. This framework is comprised of three different physical models tracking
electrodynamics, membrane permeabilization, and molecular transport, respectively.
For electrodynamics, the Ohmic equation is solved for conservation of the electric
current (section “The Electrical Problem”). For membrane permeabilization, the
model is developed based on the Smoluchowski equation in stochastic theory
(section “The Permeabilization Model”). For molecular transport, the Nernst-Planck
equations are solved to account for electrophoresis, diffusion, and chemical reaction
involved in ionic transport (section “Species Transport”). The coupling of these three
models, proper boundary conditions (section “The Coupling of the Electrical and the
Permeabilization Model”), and a brief introduction of numerical implementation
(section “Numerical Implementation”) are also introduced. Exemplary simulation
results are then presented (section “Exemplary Results”). Prior to conclusions
(section “Conclusions”), other alternative transport models are also briefly intro-
duced (section “Other Models”).
Model Framework: A Continuum Approach
The Electrical Problem
To begin with, a simple, one-dimensional model is introduced to familiarize the

readers with the basic physics. A schematic is shown in Fig. 1. A planar lipid bilayer
membrane divides a space filled with an aqueous electrolytic solution of conductiv-
ity λ into two compartments. Planar electrodes parallel to the membrane are placed in
each compartment. When a voltage difference V0 is applied between the electrodes,
an electric field is created in the electrolytic solution. Obviously, the problem can be
approximated to be one dimensional mathematically.
Define Φ(x, t) as the electric potential field (to be solved), and the electric field
strength is E = @ Φ/@ x, where the minus sign results from the fact that the electric
Fig. 1 A schematic of a planar membrane problem. A constant voltage difference V0 is applied on

the electrodes. The reservoir is filled with an electrolytic solution of conductivity λ, creating a
one-dimensional electric potential field Φ(x, t). The membrane has a capacitance of Cm, as well as a
conductance of gm upon electroporation
field directs opposite of the potential gradient. The conservation of Ohmic current λ
E (also called the Ohmic equation) in the electrolytes is the governing equation:

@ @Φ
λ ¼ 0: (1)
@x @x
In most situations, λ can be treated as a constant, so that Eq. 1 is simplified into
@2Φ
¼0 (2)
@x2
This equation holds for both regions left and right to the membrane (namely,
the bulk).
The current balance condition also needs to hold across the membrane. For an
intact lipid membrane, the membrane is purely capacitive, and the cross-membrane
current balance condition is
@Φ dV m @Φ
λ jx¼0þ ¼ Cm ¼ λ jx¼0 ; (3)
@x dt @x
where Cm is the membrane capacitance, V m ðtÞ Φðx ¼ 0þ , tÞ Φðx ¼ 0 , tÞ is the

transmembrane potential (TMP), CmdVm/dt is the capacitive current across the
membrane, and x = 0+ and 0 refers to the right and the left boundary of the
membrane, respectively. The thickness of the membrane ( nm) is ignored. Solving
Eqs. 2 and 3 along with an initial condition for Vm(t = 0) provides a transient
solution of Φ(x,t) and Vm.
Equations 2 and 3 are probably the simplest model for practical electroporation
apparatus. For Vm = 0 at t = 0, the solution is a simple exponential decay,

V m ðtÞ ¼ V 0 1 et=τcharge ; (4)
where τcharge ¼ 2Cm L=λ is the electric charging time of the membrane. It is clear Vm
will eventually approach V0 given sufficient time.
An analytical solution is also available if an alternating current (AC) field of
frequency ω and peak voltage V0 instead of the direct current (DC) field is applied
across the electrodes: V ðtÞ ¼ V 0 cos ðωtÞ. The solution is
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
V0 2
t=τcharge
V m ðt Þ ¼ 2 1 þ ωτchage cos ωt arctan ωτcharge e :
1 þ ωτcharge
(5)
It is straightforward to verify that the solution (Eq. 5) converges to (Eq. 4) in the limit
of ω = 0. More conveniently, the solution can be written in a complex form:
iωt
e et=τcharge
V m ðtÞ ¼ V 0 Re ; (6)
1 þ iωτcharge
pffiffiffiffiffiffiffi
where Re denotes the real part of a complex function, and i ¼ 1 is the imaginary
unit. When ωτcharge 1 or ωτcharge 1, the solution (Eq. 5) for Vm sees an evident
drop in magnitude as well as a notable phase shift from the original AC field. For
both DC and AC electroporation, significant change in behavior of the system is
expected when the electric charging time τcharge becomes comparable to the charac-
teristic timescale of the field, namely, the pulse length tp for both DC and AC pulses,
and in addition 1/ω for AC pulses. This scaling analysis is useful for problems in all
geometries, with τ CmL/λ where L is the characteristic size of the problem.
Electroporation occurs when Vm is greater than a threshold value, typically a
fraction of a volt. This produces a significant increase in membrane conductance, gm,
which is simply ignored for an intact membrane in Eq. 3. The updated cross-
membrane condition becomes
@Φ dV m @Φ
λ j þ ¼ Cm gm V m ¼ λ jx¼0 : (7)
@x x¼0 dt @x
Fig. 2 A schematic of a
spherical-shell model with
axisymmetry. (r, θ) denotes
the spherical coordinate
system. x is the axis of rotation
and is aligned with the
direction of field application.
The field strength is denoted
by E0. The intra- and
extracellular conductivities
are denoted by λi and λe,
respectively
This equation means that upon the occurrence of electropermeabilization, the cross-
membrane electric current becomes a sum of the capacitive current and the conduc-
tive current. Noting that gm(t) is a transient value representing the degree of
permeabilization, it must be derived from the permeabilization model which is
introduced in section “The Permeabilization Model.”
A more frequently encountered geometry is the electroporation of a suspending
cell of approximately spherical shape. This corresponds to a spherical-shell model as
illustrated in Fig. 2. The presumably isolated cell is modeled as a spherical space of
radius R, filled with an electrolyte of conductivity λi, encapsulated by an infinites-
imally thin membrane of capacitance Cm, and surrounded by an extracellular elec-
trolyte of conductivity λe. A pulse with the constant strength of E0 is applied, and
axisymmetry is assumed to be about the direction of the electric field. In principle,
this problem has the same physical governing equations as the planar membrane, but
it needs to be written under the spherical coordinate system, (r, θ). The Ohmic
equation turns out to be

1 @ 2 @Φ 1 1 @ @Φ
r þ sin θ ¼ 0: (8)
r 2 @r @r r 2 sin θ @θ @θ
Again the intra- and extracellular conductivities are taken to be constant. Similar to
Eq. 3, the cross-membrane current balance (in the direction normal to the membrane)
is
@Φ dV @Φ
λi jr¼R ¼ Cm ¼ λe j þ: (9)
@r dt @r r¼R
At r = 1, the field becomes uniform (ambient), so that Φðr ¼ 1Þ ¼ E0 r cos θ.
Note that this is usually a good approximation when the size of cell is much smaller
than the spacing between the electrodes, but may not be valid in, e.g., patch-clamp
experiments.
The solution to Eqs. 8 and 9 can easily be obtained as

V m ðθ, tÞ ¼ 1:5E0 R cos θ 1 et=τcharge ; (10)
where the TMP is defined as V m Φe ðr ¼ Rþ Þ Φi ðr ¼ R Þ, and τcharge ¼ Cm R

1 1
2λe þ λi . Equation 10 is the most widely used formula to estimate the applied
voltage across the cell membrane in electroporation. Typically for Cm 0.01 F/m2,
R 10 μm, and λi,e 0.1 S/m, it can be estimated that τcharge 1 μs. This explains
why in nanosecond electroporation, a much stronger electric field has to be applied.
In such cases, the pulse is too short for the membrane charging process to complete.
In order for the TMP to reach the threshold for electroporation, a much higher E0 is

necessary to compensate for the small factor of 1 et=τcharge .
For conventional electroporation with tens to hundreds of microsecond or longer,
the membrane charging often completes and the TMP reaches a steady state:

V m θ, t τcharge ¼ 1:5E0 R cos θ: (11)
At θ = 0, π, namely, the electrode-facing points, the absolute value of TMP

reaches its maximum 1.5E0R where 1.5 is simply a geometric factor. Generally
speaking, |Vm| E0R serves as a good estimate of the TMP for most conventional
electroporation applications, noting that here E0 is a characteristic field strength, and
R is the characteristic size.
If an AC field E(t) = E0 cos(ωt) is applied, the analytical solution for Vm simply
becomes

eiωt et=τcharge
V m ðθ, tÞ ¼ 1:5E0 Rcosθ Re : (12)
1 þ iωτcharge
It is evident that the solution to the spherical geometry is analogous to the planar
membrane one. Moreover, the occurrence of electroporation also leads to an increase
in membrane conductance, gm, and Eq. 9 needs to be subsequently replaced with
@Φ dV @Φ
λi jr¼R ¼ Cm gm V m ¼ λe j þ: (13)
@r dt @r r¼R
Again a permeabilization model is necessary to complete the problem.
In principle, a continuum model can always be applied to an arbitrary geometry.
The general equations are (in three dimensional)

∇ λi,e ∇Φi,e ¼ 0; (14)
dV m
λi n ∇Φi ¼ Cm gm V m ¼ λe n ∇Φe at the membrane: (15)
dt
Here ∇ denotes the gradient of a scalar field, ∇ denotes the divergence of a vector
field, and n denotes the unit vector normal to the membrane. With appropriate
boundary conditions and numerical approach, the framework is suitable to provide

meaningful predictions for cases with much more complex geometries and/or
experimental conditions.
The Permeabilization Model
The most widely accepted current theory for membrane permeabilization forms from
the perspective of minimizing membrane energy. When the transmembrane potential
exceeds a certain value, hydrophilic membrane pores greater than a particular size
become favorable, because they provide lower energy states than the intact lipid
bilayer which holds otherwise more capacitive energy. The permeabilization model
is subsequently established based on this theoretical understanding, using the
Smoluchowski equation to describe the creation, destruction, and evolution of
pores (Barnett and Weaver 1991):

@nðr, tÞ @ nðr, tÞ @U ðr, V m , tÞ @nðr, tÞ
þD ¼ Sðr, V m Þ: (16)
@t @r kB T @r @r
Equation 16 is a partial differential equation (PDE) governing the distribution of

pore density n(r, t) as a function of pore radius, r, and time, t. Here D is the diffusion
constant for size evolution, U(r, Vm, t) is the pore energy as a function of pore radius
and the TMP, kB is the Boltzmann constant, T is temperature, and S(r, Vm) is the
source term giving the rate of creation and destruction of pores which also depends
on the TMP.
Equation 16 is generally cumbersome to deal with. An asymptotic version (the
ASE model) of this equation is frequency used, which is given by Neu and
Krassowska (1999). Mathematically, the resulting equations are ordinary differential
equations (ODE), hence much easier to analyze and solve:

dN ðtÞ ð V m =V ep Þ
2 N ðtÞ
¼ αe 1 : (17)
dt N eq ðV m Þ
Here N(t) is the pore number density per area (m2), and Neq(Vm) is the equilibrium
pore density for a given Vm. It is straightforward to see that pores are created when
N < Neq and destroyed when N > Neq. α and Vep are model constants, representing
pore creation rate coefficient and characteristic voltage of electroporation, respec-
2
tively. N ¼ N eqðV m =V ep Þ , where N and q are again model constants. The physical
eq 0 0
meaning of these constants and their typical values are listed in Table 1.
In deriving Eq. 17 from Eq. 16, several key features of the dynamic system are
revealed. First, all hydrophilic pores are created at a specific radius r. Second, the
vast majority of pores reach an equilibrium radius rm in typically 5 μs after being
created. In other words, for microsecond electroporation where the pulse length is
much longer than the characteristic timescale of pore evolution, one can simply
Table 1 Typical values of parameters used in the ASE model (Krassowska and Filev 2007)
Symbol Value Definition
α 1 109 m2 s1 Creation rate coefficient
Vep 0.258 V Characteristic voltage of electroporation
q (rm/r)2 Constant for pore creation rate
N0 1.5 109 m2 Equilibrium pore density at Vm = 0
r 0.51 109 m Minimum radius of hydrophilic pores
rm 0.8 109 m Minimum energy radius at Vm = 0
D 5 1014 m2 s1 Diffusion coefficient for pore radius
Fmax 0.70 109 N V2 Maximum electric force for Vm = 1 V
rh 0.97 109 m Constant for advection velocity
rt 0.31 109 m Constant for advection velocity
β 1.4 1019 J Steric repulsion energy
γ 1.8 1011 J m1 Edge energy
σ0 1 106 J m2 Tension of the bilayer without pores
σ0 2 102 J m2 Tension of hydrocarbon-water interface
assume that all pores have reached their equilibrium size at the end of the pulse. The
value of rm can be obtained by finding the local minima of the pore energy U(r, Vm).
If one wishes to capture the pore dynamics in a more accurate manner, another
ODE, also derived in an asymptotic manner from the original Smoluchowski
equation, is needed (Krassowska and Filev 2007):
dr j
¼ U r r j , V m , σ eff (18)
dt
Here rj is the radius of the j th pore and Ur = @U /@r is the driving force of pore
expansion or shrinkage to minimize the pore energy U. Ur is given by
r 4 1
D V m Fmax
ur r, V m , σ eff ¼ þ 4β 2πγ þ 2πσ eff r ; (19)
kB T 1 þ r h =ðr þ r t Þ r r
where D, Fmax, rh, rt, β, r, and γ are model constants. Equation 19 includes four
terms, representing the effect of the electric force, the steric repulsion of lipid heads,
the line tension on the pore perimeter, and the surface tension of the membrane,
respectively. In the last term, σ eff is an effective membrane tension, given by
2σ 0 σ 0
σ eff ¼ 2σ 0 : (20)
ð 1 ρÞ 2
σ 0 and σ 0 are constants, and ρ is the pore area density, namely, the fraction of the local
membrane area occupied by aqueous pores. To solve rm, one may simply solve Ur
(rm,Vm, σ eff) = 0 for given values of Vm and σ eff. Clearly, the equilibrium pore size rm
is a function of the TMP and the membrane tension.
Equations 17, 18, 19, and 20 form a complete permeabilization model providing
description of pore dynamics (radius, number density, location) given Vm. Briefly,
hydrophilic pores will emerge with an initial radius of r, at the number density of N
(t,θ) via Eq. 17, and then each begins to evolve in size per Eq. 18. A numerical
approach is often taken to obtain the specific solution. A number of model constants
are involved, the values of which are either estimated theoretically or measured by
experiments. It is worthwhile to mention that this permeabilization model in practice
usually predicts a resealing timescale much shorter (within microseconds)
(Krassowska and Filev 2007) than what is experimentally observed (tens of milli-
seconds to minutes and even hours) (Sukharev et al. 1992; Khine et al. 2007).
Therefore one should not rely on the model to offer a faithful prediction for the
resealing process upon field cessation.
The Coupling of the Electrical and the Permeabilization Model
The electrical and the permeabilization models above are derived from electro-
dynamics and membrane biophysics, respectively. In electroporation, they are
coupled together at the membrane via interactions between the transmembrane
potential, Vm, and the cross-membrane current density, jm. This is straightforward
to understand: the value of Vm commands creation and evolution of aqueous
pores on the membrane; the creation of such conductive pores in turn increases jm
and “shunts” Vm. Given sufficient time, a new equilibrium between electrody-
namics and pore dynamics can be established. Such equilibrium is usually global
since the Laplace Equation 8 is mathematically an elliptic PDE. This is to say, a
disturbance in the electric current at any membrane locality would influence the
entire domain.
In the previous subsection, details have been given regarding how to update
instantaneous pore information from a local value of Vm(θ, t). The goal of this
subsection is to subsequently show how the transient pore dynamics will in turn
render quantitative changes in Vm. To fulfill this purpose, one needs to solve the
Laplace Equation (8) at every instant with an updated boundary condition at the
membrane (13). In Eq. 13, the only variable that is presumably correlated with pore
dynamics is the membrane conductance gm (in Sm2). Upon electrical breakdown of
the membrane, the value of gm can increase by several orders of magnitude (Neu-
mann et al. 1989). As a result, the conductive current gmVm in Eq. 13 will corre-
spondingly surge and reshape the electric field distribution. Therefore a formula is
needed to calculate gm(θ) from the total number of pores, denoted K(θ), and their
radii, denoted rj=1,2,. . .,K (θ), at a membrane locality, θ.
Intuitively it can be imagined that the electropores are in parallel with each other,
so that gm is the sum of each individual pore conductance Gp divided by the area of a
local membrane element ΔA (see more details about ΔA in section “Numerical
Implementation”):
Fig. 3 Schematic of pore resistance Rp and input resistance Ri. The pore is assumed to be
cylindrical. The solid lines in the extracellular space are equipotential lines, and the dashed are
field lines. Rp is the resistance of the cylindrical pore, and Ri is the resistance of the spreading
electrolytic area outside the pore (on both sides). Rp and Ri are in series to give the total resistance of
an electropore
1 XK
gm ¼ Gp : (21)
ΔΑ j¼1
Several models exist to calculate Gp from the pore radius and conductivity. A simple
and widely accepted approximation is (Powell et al. 1986)
1
Gp ¼ : (22)
Rp þ Ri
Here Rp ¼ h=πλr j 2 is the resistance of a cylindrical aqueous pore with thickness h,

radius rj, and conductivity λ, and Ri = 1/2λrj is the input resistance (also termed the
spreading resistance) resulting from the resistance of the electrolytic solution in
regions next to the pore (Fig. 3). The two resistances add to each other as they are in
series.
In deriving Eq. 22, it is assumed that the conductivities on both sides of a pore are
equal. Such constraint can be removed via an improved formula (Li and Lin 2010):
λeff
Gp ¼ : (23)
h=πr 2j þ 1=2r j
Here λeff is an effective pore conductivity given by λeff ¼ ðλe λi Þ=lnðλe =λi Þ. When
λe = λi, Eq. 23 converges to Eq. 22.
Equations 22 and 23 do not take into account the interaction of ions with the pore
walls or the steric hindrance for ions of comparable size to the pore. These effects
can be introduced into Eqs. 22 and 23 as prefactors in the formula of Rp:
1 h
Rp ¼ : (24)
H r s =r j J r j , V m πλr 2j
In Eq. 24, H is Renkin’s steric hindrance factor, which is a function of the ratio of ion
radius rs (for dominant ions contributing to electrical conductivity) to pore radius rj.
J is a partitioning coefficient accounting for the electrostatic energy barrier for an ion
to enter a pore, which depends on the pore radius and the transmembrane potential.
Both factors converge to unit for large pores, and in most cases they only give rise to
significant modification of Rp for pores smaller than 1 nm. Interested readers are
referred to (Vasilkoski et al. 2006) for details.
More complicated pore conductance models have recently been developed using
molecular dynamics (Casciola et al. 2016) or a continuum Poisson-Nernst-Planck
theory considering the toroidal shape of the pore, pore selectivity for different ionic
species, the electric double layer at the lipid-water interface, and the electroostomic
flow (Rems et al. 2016). These models are expected to provide improved results at
the cost of higher model complexity.
In a brief summary, Eqs. 21, 22, 23, and 24 set up approaches for quantification of
gm(θ). This closes the dynamic system (Eqs. 8, 13, 14, 15, 16, 17, 18, 19, and 20),
and the coupled equations can be solved to obtain temporal and spatial information
of the electric field and pore distribution. In principle, the solution can only be
obtained with a numerical approach, as details will be given in section “Numerical
Implementation.”
Throughout this chapter, the effect of the resting potential, Vrest, is neglected.
Some authors (e.g., (Debruin and Krassowska 1999)) suggest that a constant Vrest
should be added to Vm in Eq. 13 at any moment or place to account for its effects. On
the other hand, there still lacks widely accepted evidence to illustrate how the
occurrence of electroporation modifies Vrest, which is likely to be nontrivial consid-
ering its origin. Readers can add or drop this term – the difference has not been
shown to be substantial.
Species Transport
In this subsection, the governing equations of electroporation-mediated species

transport are presented. Here it is assumed that the transport equation is one-way
coupled from the equation system introduced in sections “The Electrical Problem,”
“The Permeabilization Model,” and “The Coupling of the Electrical and the
Permeabilization Model.” That is, species transport of ions, polymers, and other
molecules does depend on the electric field and the permeabilization state of the
membrane but does not feed back to interfere with the electrodynamic or permeabi-
lization process. This assumption seems to contradict with the fact that the electrical
conductivity derives directly from that of the ionic species. However, in the current
model, only one or multiple species (usually the molecules targeted for delivery)
whose contributions to conductivity are negligible are considered. Taking, for
example, the delivery of propidium iodide, if the extracellular concentration is
100 μM, its contribution to conductivity is 104–103 S m1. This is typically
much lower than the buffer or cytosol conductivity, e.g., 0.1–1 S m1. The
transient dynamics of such species is henceforth unlikely to change the electrical

conductivity, based on which the electrical problem is computed.
The evolution of the major ionic species composing the buffer and the cytoplasm,
on the other hand, does tend to change the bulk electrical conductivities – this is a
very subtle issue. Scaling analysis (Lin et al. 2004) demonstrated that the change of
the bulk electrical conductivity is primarily mediated by a diffusive process, despite
the presence of a driving electric field. The electrophoretic effects between the
cations and anions are largely canceled via electroneutrality which is a strong and
universal constraint. Since the diffusive timescale is typically much longer than
electroporation times, the bulk electrical conductivities being constant are accepted
as a valid approximation in this chapter.
In the bulk of intra- and extracellular medium, the Nernst-Planck equations are
used to describe molecular transport owing to electrophoretic drifting, diffusion, and
chemical reaction:
@ck
¼ ∇ ðμk Fzk ck ∇ΦÞ þ ∇ ðDk ∇ck Þ þ Sk : (25)
@t
Here ck is the molar concentration of the k th species in mol m3; μk, the mechanical
mobility in mol s kg1; zk, the valence number of the species; and Dk, the diffusion
constant of the species in m2 s1. According to Einstein’s relation, Dk = μkRT,
where R is the gas constant, and T is temperature. F is the Faraday constant and Φ
is the electric potential. In Eq. 25, the left-hand side stands for the rate of change of
local concentration; the first and the second term on the right-hand side stand for the
net inward flux into a locally infinitesimal volume owing to eletrophoretic drifting
and molecular diffusion, respectively. Sk is a source term representing local produc-
tion or consumption of the species from any kinetics. Eq. 25 is again written for the
generalized, three-dimensional situation. For a two-dimensional axisymmetric
geometry in a spherical coordinate system, the Nernst-Planck equations become

@ck 1 @ 2 @Φ 1 1 @ @Φ
¼ μk F zk 2 r ck þ 2 sin θck
@t r @r @r r sin θ @θ @θ
(26)
1 @ 2 @ck 1 1 @ @ck
þDk 2 r þ 2 sin θ þ Sk :
r @r @r r sin θ @θ @θ
In Eq. 26, Φ(r, θ, t) is available from the models in sections “The Electrical
Problem,” “The Permeabilization Model,” and “The Coupling of the Electrical and
the Permeabilization Model.” Therefore, the transient species concentration at any
position, ck(r, θ, t), can be obtained by integrating Eq. 26, given appropriate bound-
ary conditions at the membrane. This equation can also be used to solve the transport
of electrically neutral or immobile molecules, by simply setting to zero pertinent
terms.
Another benefit of the simplification made above is that now the species transport
is decoupled from each other, unless via any chemical kinetics there may exist. The
effect of the latter is represented by the source term Sk in each equation. For example,
upon entering the cytoplasm, propidium iodide (PI2+) binds to sites on DNA/RNA
(denoted simply by a single species B) to emit fluorescence. The binding kinetics is
described by
kþ
PI2þ þ B Ð PIB (27)
k
where k+ and k are the association and dissociation rate constants, respectively. This
kinetics leads to the following expressions of Sk, which enters Eq. 25 or Eq. 26:
8
< SPI ¼ kþ cPI cB þ k cPIB
S ¼ kþ cPI cB þ k cPIB : (28)
: B
SPIB ¼ kþ cPI cB k cPIB
In Eq. 28, the right-hand sides are the corresponding production or depletion rates
for each species resulting from the chemical reaction (27).
The above equations, similar to the electrical problem, are solved in both the
intra- and extracellular spaces. These equations are coupled at the permeabilized
membrane to account for cross-membrane transport. An electroporated membrane
only provides limited access to ions and molecules. This access, as hypothesized in
this chapter, depends exclusively on the pore distribution governed by Eqs. (17, 18,
19, and 20) in section “The Permeabilization Model.” In other words, foreign ions or
molecules can only translate across the membrane through electropores. Other
transport mechanisms such as ion channels and endocytosis are not accounted for
by the current model. Another main hypothesis is that ions or molecules can only go
through pores greater than their own sizes, e.g., the hydrated diameter of an ion. A
cross-membrane transport model is derived for fkm, the membrane flux density (per
area) for the k th species, by solving a one-dimensional Nernst-Planck equation
within the pore (Li and Lin 2011):

Dk ðPek ln λr Þ λr 1 cek cik expðPek Þ
fm ¼ ρk : (29)
k
h ln λr λr expðPek Þ
Here Pek ¼ V m =ðRT=Fzk Þ is an effective Péclet number for each species, λr = λe/λi
is the extra- to intracellular conductivity ratio, h is the membrane thickness, and cke, cki
are X extra- and intracellular species molar concentration, respectively.
ρk ¼ πr 2j =ΔΑ is the pore area density summing over pores with radii rj greater
rj >rk
than the effective ionic or molecular radii rk. This term can be conveniently modified
to accommodate other models for ion-selective-passage criteria other than simply by
size comparison.
The flux density is positive when pointing outward and negative when pointing
inward. It has to match the local flux density on both sides of the membrane provided
by the Nernst-Planck equations:
e, i
fmk ¼ fk (30)

where f ek,i ¼ n μk Fzk cek, i ∇Φ þ Dk ∇cek, i in a general coordinate system, or f ek, i

¼ μk Fzk cek, i ∇Φ=@r þ Dk @cek, i =@r in an axisymmetric spherical coordinate
system.
In many cases, Eq. 29 can be greatly simplified. If we ignore the conductivity
difference across the membrane and set λr = 1, Eq. 29 is reduced to
Pek Dk cek cik expðPek Þ

k ¼ ρk
fm : (31)
h 1 expðPek Þ
Considering the situation when Pek = 0 (no electric field or for electrically neutral
molecules), Eq. 31 further reduces to a pure diffusive form:
i
k ¼ ρk Dk ck ck =h:
fm (32)
e
On the other hand, when the electric field is on and |Pek| 1 (this is generally true
because the transmembrane potential required to induce electroporation is much
greater than RT/F 0.025 V), Eq. 31 becomes an upwind scheme as electrophoresis
dominates diffusion:
8
> μ Fz V
< ρk k k m cek for V m zk > 0
h
k ¼
fm (33)
: ρk μk Fzk V m cik
> for V m zk < 0
h
noting that μkFzkVm is the cross-membrane eletrophoretic velocity of the k th species.
Equations 32 and 33 can be used as approximations to Eq. 29 in the specific
simplifying limits.
Numerical Implementation
Equations 14, 15, 25, 29, 30 for a Cartesian coordinate system, or Eqs. 8, 13, 26, 29
and 30 for an axisymmetric spherical geometry, along with the permeabilization
model (17, 18, 19, 20, 21, and 22), provide a complete model description of
electroporation-mediated species transport. This offers a powerful tool that is capa-
ble of capturing the key dynamic behavior of general interest in electroporation
research, such as that of the transmembrane potential, membrane permeability, and
molecular transport. Owing to the continuum approach, details on the variables of
interest have both spatial and temporal resolution, in contrast to the compartment
models which only compute integrated quantities.
In general, the equation system can only be solved numerically. The numerical
implementation of the Ohmic equation and the Nernst-Planck equations follows
standard approaches, typically finite-difference, finite-volume, or finite-element

methods. A transport lattice method is also available to tackle the complex cell
geometry and field interactions in biologically relevant transport problems (Donald
et al. 2005). Noting that both equations to solve are in the form of conservation of
flux, flux-conserving algorithms are preferred. The same rule holds across the
membrane – the flux has to be conserved on both sides of any membrane element
and be equal to the local membrane flux commanded by Eqs. 15, 21 and 22 for the
electric current or Eq. 29 for species transport.
The far-field boundary condition can be approximated by applying it at a distance
far enough from the cell, e.g., 10R from its center. In choosing the time step size, Δt,
one should make it small enough to guarantee stability and convergence, but not
unnecessarily small to reduce computational cost.
The numerical implementation of the permeabilization model needs to be care-
fully and properly executed. After mesh generation, the membrane is discretized into
many membrane elements with an area ΔA (not necessarily uniform). Each area
element is associated with a local pore number density, N(θ, t) (in m2), as governed
by Eq. 17. The value of N(θ,t) is updated by the local TMP, Vm(θ). The value of N
Δ A represents the time-dependent number of pores at this membrane element. When
this value increases to be greater than one or greater integers, creation of new pores
occurs. For example, N ΔA increasing from 5.5 to 8.3 indicates the creation of
three new pores on top of the five existing pores. The radii of newly created pores are
initially at r = 0.51 nm and are subsequently tracked, respectively, as rj (the j th
pore) which is dynamically governed by Eq. 18. Similarly, if the value of N(t) Δ
A decreases and rounds to a lower integer, destruction of existing pores takes place.
For example, when N ΔA drops from 8.3 to 5.5, three pores disappear locally on
ΔA. In practice, one may find three local pores with the smallest size and one may set
rj = 0 permanently for these pores, indicating that they have been destroyed.
This practical approach brings forth an issue for the selection of ΔA. Clearly, each
individual pore is bound within a local membrane element. The value of ΔA should
be moderately large so as to contain electropores. For example, a spherical cell of
R = 20 μm has a total surface area of around 5,000 μm2. The largest electropore
reported in modeling can reach 500 nm in radius or around 1 μm2 in area.
Therefore in practice ΔA should be no less than 1 μm2, and a value around 10 μm2
is recommended. In other words, the mesh size cannot be too small in the simulation
of membrane electropermeabilization. This subtlety is due to the nature of the ASE
model: in fact it combines both continuum and discrete features.
Special treatment is also required if the membrane element is not uniform in the
area upon mesh generation, which is frequently the case for a spherical cell or more
complicated geometries. Spatial variation in ΔA leads to artificial variations in pore
creation. For elements with smaller ΔA, pores will be created earlier than those with
greater ΔA, as the threshold of N to reach N ΔA
1 is lower. Conversely, for
elements with smaller area, pore creation will be delayed. This result is unphysical,
as some part of the membrane is more easily permeabilized than other, with
otherwise no physical reason to indicate so. To eliminate this artificial effect, one
should always prefer to use a uniform ΔA across all membrane elements. However,
when nonuniform elements are unavoidable, such as around certain regions where
high numerical resolution is demanded for accuracy and stability, work-around
approach can also be designed. In this case, one can designate a standard element
area ΔA0, and elements smaller than ΔA0 are designated to be its exact fractions. For
example, if an element has an area of ΔA0/2, it should be combined with a
neighboring element of the same size to evaluate pore dynamics, giving rise to the
same effective area ΔA0. The actual element ΔA0/2 can be used in other parts of the
model system, such as for the electrical problem and species transport. This approach
requires additional care in designing the mesh grid, but is nonetheless
straightforward.
Exemplary Results
Figure 4 provides an example for a whole-cell-level, continuum-based simulation

(Li and Lin 2011). The simulation follows the setup of the experimental work
(Gabriel and Teissié 1999) which investigates the electrotransfer of calcium ions
into a China hamster ovary cell. The simulation uses the same model as introduced in
sections “The Electrical Problem,” “The Permeabilization Model,” “The Coupling of
the Electrical and the Permeabilization Model,” “Species Transport,” and “Numer-
ical Implementation.” A list of model parameters is given in Table 2. The association
and dissociation rate constants are associated with the reaction:
kþ
Ca2þ þ Fluo Ð CaFluo; (34)
k
which describes the binding of calcium ions to Fluo-3 to produce the fluorescent
compound, CaFluo. The simulation is compared with the fluorescence measure-
ments in which a single electric pulse of 6 ms in length and 1 kV/cm in strength was
used. Cells were preloaded with Fluo-3, so that entry of calcium ions into the
cytoplasm led to fluorescence emission owing to the binding reaction (34). The
evolution of the spatial fluorescence distribution inside the cell was recorded during
and after the pulse, as displayed in the bottom row of Fig. 4. The simulation
compares a convolved CaFluo concentration profile with the measurements, and a
reasonable agreement is found.
In Fig. 5, the concentration profiles are plotted along the cell centerline and at the
same moments as in Fig. 4. The cell symmetrically spans from x = 8 to 8 μm.
Figures 4 and 5 display the intricate features of the transport process. Prominently,
the intracellular concentration of Ca2+ is much higher than that in the extracellular
space. This local concentration elevation results from the fact that the extracellular
conductivity in this case is lower than the intracellular conductivity, λe < λi.
Noting that the current balance condition across the membrane (13) commands λe
Ee jr¼Rþ ¼ λi Ei jr¼R, this indicates that the electric field strength is higher on the
lower-conductivity side; hence, Ee jr¼Rþ > Ei jr¼R . Subsequently, according to the
mass transport balance across the membrane (30), Ee ce jr¼Rþ Ei ci jr¼R, which
Fig. 4 Comparison between simulated and experimental results for electroporation-mediated

delivery of calcium ions. The time stamps are the same in all. The top three rows show the
simulated evolution of species concentration at the cell center plane. The fourth row shows a
convolved concentration of the fluorescent compound CaFluo, in an attempt to take into account a
finite optical depth in the measurement system for the observed results. The last row shows
experimental images. In the simulated results, red denotes high, and blue denotes low values.
The single electric pulse is 6 ms in length and 1 kV/cm in strength (Adapted from (Li and Lin 2011))
holds during the pulse when the electrophoretic flux μkFzkckE dominates over the
diffusive flux. This further leads to ce jr¼Rþ < ci jr¼R , explaining the observed high
Ca2+ concentration at the anode-facing (entrance) side of the cell. This effect is due
to a well-known electrokinetic mechanism termed field-amplified sample stacking
(Chien and Burgi 1991). The analysis suggests that using a lower extracellular
conductivity gives higher delivery of small ions or charged molecules in electro-
poration, which has been confirmed by controlled experimental observation (Sadik
et al. 2013).
Similar to the “accumulation zone” of Ca2+ on the anode-facing side, a “depletion
tail” at the cathode-facing side outside the cell is also seen during pulsation. In
addition, calcium ions bind to locally available Fluo-3 almost instantaneously,
causing a receding edge in the Fluo-3 profile (Fig. 5b), accompanied by an
Table 2 List of model Definition Value

parameters for the
Cell radius 8 μm
simulation in Fig. 4
Membrane thickness 5 nm
Intracellular conductivity 0.5 S m1
Extracellular conductivity 0.15 S m1
Association rate constant 80 (μMs)1
Dissociation rate constant 90 s1
Intracellular diffusion coefficient of Ca2+ 250 μm2 s1
Extracellular diffusion coefficient of Ca2+ 790 μm2 s1
Intracellular diffusion coefficient of Fluo-3 20 μm2 s1
Extracellular diffusion coefficient of Fluo-3 90 μm2 s1
Intracellular diffusion coefficient of CaFluo 20 μm2 s1
Extracellular diffusion coefficient of CaFluo 90 μm2 s1
Valence number of Ca2+ +2
Valence number of Fluo-3 5
Valence number of CaFluo 3
Initial intracellular Fluo-3 concentration 2.2 μM
Initial extracellular Ca2+ concentration 1 mM
advancing front in the CaFluo profile (Fig. 5c), and during the pulse. After the pulse
ceases, diffusion begins to smear all profiles within the intracellular space.
This exemplary case demonstrates the complexity in species transport. The
dynamic effects of electric field redistribution, membrane permeabilization, electro-
phoresis, diffusion, and chemical reaction are shown to work in cohort to give rise to
the final profiles observed in measurements. A comprehensive and reliable model is
thus highly valuable in connecting fundamental understanding and experimental
observations.
Other Models
The model framework presented in sections “The Electrical Problem,” “The

Permeabilization Model,” “The Coupling of the Electrical and the Permeabilization
Model,” “Species Transport,” and “Numerical Implementation” has the strength of
resolving detailed spatial evolution. On the other hand, compartment models,
although lacking such details, have the appeal of ease in implementation. Before
concluding the chapter, they are briefly introduced.
A cell-averaged model is proposed by Neumann et al. (1996) to predict the
electrodiffusive transport of DNA across the electroporated membrane:

dnin D0 nin nout zeff je0
c
¼ c
1 Δϕm : (35)
Sdt h Vc v kB T
Fig. 5 Species concentrations along the cell centerline at various times in the simulation. The cell
symmetrically spans from x = 8 to 8 μm. (a) Calcium ion; (b) Fluo-3; (c) CaFluo; (d) convolved
CaFluo. The single electric pulse is 6 ms in length and 1 kV/cm in strength (Adapted from (Li and
Lin 2011))
Here ncin is the amount of delivered DNA into the cell, S is the electroporated surface
area, h is the membrane thickness, D0 is the apparent cross-membrane diffusion
coefficient of DNA, Vc is the cell volume, v is the volume of the bulk solution, nout is
the total amount of DNA in the bulk solution, zeff is the effective charge number of
the DNA-phosphate group, e0 is the elementary charge, kB is the Boltzmann con-
stant, T is the absolute temperature, and Δϕ m is the mean transmembrane potential
across the membrane. Equation 34 is an integrated form of the original Nernst-
Planck equations (25), assuming that the intra- and extracellular DNA concentration,
membrane permeability, and the transmembrane potential are all uniformly distrib-
uted. One may further simplify the model by formulae approximating S and Δϕ m
instead of obtaining them from the solution. Evidently, Eq. 35 can be used for small-
to moderate-sized molecules as well.
Diffusion- and endocytosis-driven transport models, such as a pharmacokinetic
one (Puc et al. 2003) and a phase-transition one (Miklavčič and Towhidi 2010), were
also developed using a volume-averaged manner. These models focus on diffusive
transport and tackle the resealing process with various permeabilization theories.
The readers are referred to further details in the literature.
Conclusions
Transport phenomena within the context of electroporation generally involve mul-

tiple physical, chemical, and biological processes. The intrinsic complexity endows
modeling efforts both necessity and difficulty. Over the past decade, significant
progresses have been made in this area, which lead to whole-field simulations that
are able to directly corroborate experimental measurements with both spatial and
temporal resolution. The framework based on a continuum approach is outlined in
this chapter. The model composes of three main elements, namely, electrodynamics,
membrane permeabilization, and molecular transport. Strong coupling between the
three results in complex patterns in all of field distribution, permeabilization status,
and species concentration evolution. Numerical simulation of the model can capture
much of these features and predict both qualitative and quantitative aspects of the
phenomenon. In particular, one important contribution of such modeling efforts is to
tease out the main transport mechanisms responsible for molecular delivery. In a
broader perspective, the current approach is yet limited to the delivery of small- to
moderate-sized molecules (less than a few tens of thousands of Dalton in molecular
weight). For macromolecules such as DNA, a different approach has to be taken and
is important for the general field of electroporation-mediated molecular transport.
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Biomembr 1818(11):2494–2501
Transmembrane Voltage Induced by
Applied Electric Fields 59
Tadej Kotnik
Abstract
An exposure of a biological cell to an electric field results in the induced
transmembrane voltage (ITV) proportional to the strength of the electric field
and superimposed onto the resting transmembrane voltage for the duration of
the exposure. The ITV can have a range of effects from modification of the
activity of voltage-gated channels to membrane electroporation, and accurate
knowledge of spatial distribution and time course of the ITV is important both
for the studies of these phenomena and for effectiveness of their applications.
Unlike the resting component of the transmembrane voltage, the induced
component varies with position on the membrane, it depends on the shape of
the cell and its orientation with respect to the electric field, and in dense cell
suspensions and tissues also on the volume fraction occupied by the cells.
Inducement of the ITV is a process characterized by a time constant, which
amounts to tenths of a microsecond under physiological conditions. As a
consequence, the time course of the ITV lags the time course of the electric
field that induces it, and for exposures to alternating fields with frequencies
above 1 MHz or to pulses with durations below 1 μs, the amplitude of the ITV
induced by the field of a given amplitude starts to decrease with further increase
of the field frequency or with further decrease of the pulse duration. With field
frequencies approaching the GHz range or with pulse durations in the ns range,
this attenuation of the ITV comes to a halt, and the voltages induced on the
organelle membranes inside the cell can reach the same order of magnitude as
the voltage induced by the same field on the plasma membrane, and under
certain conditions even exceed it. After the description of methods for analytical
derivation and numerical computation of the ITV, the main techniques for
experimental determination of ITV are also outlined.
T. Kotnik (*)
e-mail: tadej.kotnik@fe.uni-lj.si

DOI 10.1007/978-3-319-32886-7_8
1112 T. Kotnik
Keywords
Induced transmembrane voltage • Electric pulses • Plasma membrane • Organelle
membranes • Schwan’s equation • Finite-elements method • Potentiometric dyes •
Electroporation
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1112
Analytical Derivation and Numerical Computation of ITV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1114
Spherical Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1114
Nonspherical Geometrically Regular Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1115
Irregularly Shaped Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1116
Cells in Dense Suspensions and Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1117
High Field Frequencies and Very Short Pulses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1119
Experimental Determination of ITV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1120
Potentiometric Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1121
Image Acquisition and Data Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1123
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1124
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1126
Introduction
From the electrical point of view, the cell can roughly be described as an electrolyte
(the cytoplasm) surrounded by an electrically insulating shell (the plasma mem-
brane), and the natural surroundings of a cell typically also resemble an electrolyte
quite closely. Under such conditions, when a cell is exposed to an electric field, the
field locally concentrates within the membrane, which thus shields the cytoplasm
from the exposure. For this reason, the internal structure of the cell is unimportant,
except with rapidly time-varying electric fields, where, as discussed in section “High
Field Frequencies and Very Short Pulses,” the transient exposure of the intracellular
structures to the field must also be taken into account for proper analysis, and both
the membrane and its surroundings have to be treated as materials with a both
nonzero electric conductivity and a nonzero dielectric permittivity.
The concentration of the electric field inside the membrane results in a consider-
able electric potential difference across the membrane, termed the induced trans-
membrane voltage (ITV), which superimposes onto the resting transmembrane
voltage (RTV) resulting from the imbalances between intracellular and extracellular
ion concentrations and amounting under physiological conditions to tens of milli-
volts. After the electric field ceases, so does the ITV, and only the RTV remains on
the membrane. Modification of transmembrane voltage through ITV can affect the
functioning of voltage-gated membrane channels, thus initiating action potentials
and stimulating excitable cells (Bedlack et al. 1994; Cheng et al. 1999; Burnett
et al. 2003), and when sufficiently large it can also lead to either reversible or
irreversible electroporation of the cells’ plasma membrane (Neumann et al. 1999),
59 Transmembrane Voltage Induced by Applied Electric Fields 1113
with the porated membrane regions closely correlated with the regions of the highest
absolute value of transmembrane voltage (Kotnik et al. 2010). It should be noted that
not only does the ITV affect electroporation, but once the membrane is
electroporated, its significantly increased electric conductivity also affects the ITV,
which decreases from the value induced immediately before the onset of electropo-
ration, and generally the more intense the electroporation, the larger is this decrease.
Using Ψ to denote electric potential, transmembrane voltage is the difference
between the potentials at the inner and the outer membrane surface, Ψint and Ψext,
and in physiology RTV is always defined and measured as
ΔΨm ¼ Ψint Ψext ; (1)
so that a negative value reflects a lower electric potential of the cytosol with respect
to the cell exterior. For superposition of the ITV onto the RTV to be applicable also
formally, one should adhere to Eq. 1 also when characterizing the ITV, and this
chapter does so; thus, the RTV can be superimposed to the ITV by simple mathe-
matical addition to yield the total transmembrane voltage, and this chapter hence-
forth only deals with the induced transmembrane voltage, using the acronym ITV in
the text and the notation ΔΨm in the formulae.
From the geometrical point of view, the cell can be characterized as a geometric
body (the cytoplasm) surrounded by a shell of uniform thickness (the membrane).
For suspended cells, the simplest model of the cell is a homogeneous sphere
surrounded by a spherical shell. For augmented generality, the sphere can be
replaced by a cylinder (e.g., as a model of the axon of a neuron), a spheroid (e.g.,
an oblate spheroid as a model of an erythrocyte or a prolate spheroid as a model of a
bacillus), or an ellipsoid, but with spheroids and ellipsoids the realistic requirement
of uniform membrane thickness complicates the geometrical description of the shell:
if its outer surface is characterized as a spheroid or an ellipsoid, its inner surface lacks
a simple geometrical characterization and vice versa. The steady-state ITV on the
plasma membrane of such cells can, however, still be determined analytically.
Spheres, spheroids, and ellipsoids are reasonable models for most suspended cells
but not for those in tissues. No simple geometrical body can model a typical cell in a
tissue, where furthermore each cell generally differs in its shape from the rest. With
cells of irregular shape and/or close to each other, the ITV cannot be derived
analytically and cannot be formulated as an elementary mathematical function.
This deprives us of some of the insight available from explicit expressions, but
with spatial and temporal discretization on modern computers, the ITV induced on
an irregular cell of interest can still be estimated rather accurately, provided that the
three-dimensional shape of this cell is determined with sufficient accuracy. Numer-
ical computation is generally also required to assess the ITV after the cell is
electroporated, as this increases the electric conductivity of the membrane signifi-
cantly (invalidating its treatment as an insulating shell) and moreover nonuniformly.
Section “Analytical Derivation and Numerical Computation of ITV” provides a
more detailed description of methods for analytical derivation and numerical
1114 T. Kotnik
computation of ITV, while section “Experimental Determination of ITV” outlines

the main techniques for experimental determination of ITV.
Analytical Derivation and Numerical Computation of ITV
Spherical Cells
For an exposure to a homogeneous electric field, the ITV is determined by solving

Laplace’s equation. Although biological cells are not perfect spheres, in the simplest
theoretical treatment they are considered as such, and for an approximation, the
plasma membrane can be treated as nonconductive. Under these assumptions, the
process of solving Laplace’s equation with appropriate boundary conditions (Kotnik
and Pucihar 2010) yields a description of the ITV often referred to as the steady-state
Schwan’s equation (Pauly and Schwan 1959),
3
ΔΨm ¼ E R cos θ ; (2)
2
where E is the strength of the electric field in the region where the cell is situated (i.e.,
the local electric field), R is the cell radius, and θ is the angle measured from the
center of the cell with respect to the direction of the field. The ITV on the membrane
of a spherical cell is thus proportional to the applied electric field and to the cell
radius and has extremal values at the points where the field is perpendicular to the
membrane, i.e., at θ = 0 and θ = 180 (the “poles” of the cell), while in-between
these poles it varies proportionally to the cosine of θ (see Fig. 1, dashed).
The value of the ITV given by Eq. 2 is typically established several μs after the
onset of the electric field. With exposures to a DC field lasting hundreds of
microseconds or more, this formula can safely be applied to yield the maximal,
steady-state value of the ITV. To describe its transient behavior during the initial
microseconds, one uses the first-order Schwan’s equation (Pauly and Schwan 1959),
3
ΔΨm ¼ E R cos θ ð1 expðt=τm ÞÞ ; (3)
2
where τm is the time constant of membrane charging,
R em
τm ¼ σi σe (4)
2d þ Rσ m
σ i þ 2σ e
with σi, σm, and σe the conductivities of the cytoplasm, cell membrane, and extra-
cellular medium, respectively, εm the dielectric permittivity of the membrane, d the
membrane thickness, and R again the cell radius.
In certain experiments in vitro, where artificial extracellular media with conduc-
tivities substantially lower than physiological are used, the factor 3/2 in Eqs. 2 and 3
Fig. 1 Normalized steady-state ITV as a function of the polar angle θ for spheroidal cells with the
axis of rotational symmetry aligned with the direction of the field. Solid: a prolate spheroidal cell
with R2 = 0.2 R1. Dashed: a spherical cell, R2 = R1 = R. Dotted: an oblate spheroidal cell with
R2 = 5 R1
decreases in value, as described in detail in (Kotnik et al. 1997). But generally, Eqs. 3
and 4 are applicable to exposures to sinusoidal (AC) electric fields with frequencies
below 1 MHz and to electric pulses longer than 1 μs.
To determine the ITV induced by even higher field frequencies or even shorter
pulses, the dielectric permittivities of the electrolytes on both sides of the membrane
also have to be accounted for. This leads to a further generalization of Eqs. 3 and 4 to
the second-order Schwan equation (Grosse and Schwan 1992; Kotnik et al. 1998;
Kotnik and Miklavčič 2000a), and the results it yields will be outlined in subsection
“High Field Frequencies and Very Short Pulses.”
Nonspherical Geometrically Regular Cells
Another direction of generalization is to assume a cell shape more general than that
of a sphere. The most straightforward generalization is to a spheroid (a geometrical
body obtained by rotating an ellipse around one of its radii, so that one of its
orthogonal projections is a sphere and the other two are the same ellipse) and further
to an ellipsoid (a geometrical body in which each of its three orthogonal projections
is a different ellipse). To obtain the analogues of Schwan’s equation for such cells,
1116 T. Kotnik
one solves Laplace’s equation in spheroidal and ellipsoidal coordinates, respectively

(Kotnik and Miklavčič 2000b; Gimsa and Wachner 2001; Valič et al. 2003). Besides
the fact that this solution is by itself somewhat more intricate than the one in
spherical coordinates, the generalization of the shape invokes two additional com-
plications outlined in the next two paragraphs.
A description of a cell is geometrically realistic if the thickness of its membrane is
uniform. This is the case if the membrane represents the space between two
concentric spheres but not with two confocal spheroids or ellipsoids. As a result,
the thickness of the membrane modeled in spheroidal or ellipsoidal coordinates is
necessarily nonuniform. By solving Laplace’s equation in these coordinates, one
thus obtains the spatial distribution of the electric potential in a nonrealistic setting.
However, for cells surrounded by a physiological medium and with nonporated
membranes, the electric conductivity of the membrane can be neglected (i.e., the
membrane is treated as an insulator), and the steady-state ITV obtained in this
manner is still realistic, as the electric potential everywhere inside the cytoplasm is
constant, and the geometry of the inner surface of the membrane does not affect the
potential distribution outside the cell, which is the same as if the cell would be a
homogeneous nonconductive body of the same shape; a rigorous discussion of the
validity of this approach is given in (Kotnik and Miklavčič 2000b). Figure 1 shows
the ITV for a spherical cell, as described by the steady-state Schwan’s equation, and
for two spheroidal cells with their axis of rotational symmetry aligned with the
direction of the field, as described by the expressions analogous to the Schwan’s
equation that are valid for such cell shapes (Kotnik and Pucihar 2010).
For nonspherical cells, it is generally more revealing to express the ITV as a
function of the arc length along the membrane rather than as a function of the angle θ
on the membrane (for a sphere, the two quantities are directly proportional). For
uniformity, the normalized version of the arc length is used, denoted by p and
increasing from 0 to 1 equidistantly along the arc of the membrane. This is depicted
in Fig. 2 for the three cells for which the ITV as a function of θ is shown in Fig. 1, and
in this chapter all subsequent plots of the ITV for nonspherical cells will be presented
in this manner.
The second complication of generalizing the cell shape from a sphere to a spheroid
or an ellipsoid is that the ITV now also depends on the orientation of the cell with
respect to the electric field. To deal with this, one decomposes the field vector into the
components parallel to the axes of the spheroid or the ellipsoid and writes the ITVas a
corresponding linear combination of the ITV for each of the three coaxial orientations
(Gimsa and Wachner 2001; Valič et al. 2003). Figures 3 and 4 show the effect of
rotation of two different spheroids with respect to the direction of the field.
Irregularly Shaped Cells
For an irregularly shaped cell, the ITV cannot be expressed as an elementary

mathematical function, since for such a geometry Laplace’s equation is not solvable
analytically, but using modern computers and the finite-elements method
Fig. 2 Normalized steady-state ITV as a function of the normalized arc length p for spheroidal cells
with the axis of rotational symmetry aligned with the direction of the field. Solid: a prolate
spheroidal cell with R2 = 0.2 R1. Dashed: a spherical cell, R2 = R1 = R. Dotted: an oblate
spheroidal cell with R2 = 5 R1
implemented in software packages such as COMSOL Multiphysics, the ITV on a

given irregular cell can be determined numerically (Pucihar et al. 2006; Pucihar
et al. 2009a). With a sufficiently accurately determined three-dimensional shape of
the cell and with sufficiently fine spatial and temporal resolution, the ITVobtained in
this manner is quite accurate, but it should be kept in mind that the set of ITV values
so obtained over the membrane of the cell under consideration is only applicable to
this cell, i.e., for the cell of the particular shape and size for which the three-
dimensional shape has been determined and on which the ITV has been computed.
Figure 5 shows examples of two cells growing in a Petri dish and the ITV computed
on their membranes.
Cells in Dense Suspensions and Tissues
In natural situations the cells are rarely isolated, and when they are sufficiently close
to each other, the mutual distortion of the field caused by their proximity becomes
nonnegligible. Often, the cells are also in direct contact, forming two-dimensional
(monolayers attached to the bottom of a dish) or three-dimensional (tissues) struc-
tures, and they can even be electrically interconnected.
1118 T. Kotnik
Fig. 3 Normalized steady-state ITV as a function of p for a prolate spheroidal cell with
R2 = 0.2 R1. Solid: axis of rotational symmetry (ARS) aligned with the field. Dashed: ARS at
45 with respect to the field. Dotted: ARS perpendicular to the field
In dilute cell suspensions, the distance between the cells is much larger than the
cells themselves, and the local field outside each cell is almost unaffected by the
presence of other cells. Thus, for cells representing less than 1% of the suspension
volume (for spherical cells with radii of 10 μm, this corresponds to up to 2
million cells/ml), the deviation of the actual ITV from the one predicted by Schwan’s
equation is negligible. However, as the volume fraction occupied by the cells gets
larger, the distortion of the local field around each cell by the presence of other cells
in the vicinity becomes more pronounced, and the ITV starts to differ noticeably
from the prediction yielded by Schwan’s equation (Fig. 6), and an accurate estima-
tion of the ITV must be assessed either numerically of by analytical approximations
(Susil et al. 1998; Pavlin et al. 2002). Regardless of the volume fraction the cells
occupy, suspended cells float rather freely, so their arrangement is on average rather
uniform, resembling a face-centered cubic lattice, which is thus the most appropriate
choice for models of dense cell suspensions (Pavlin et al. 2002; Pucihar et al. 2007).
For even larger volume fractions of the cells, the electrical properties of the
suspension start to approach that of a tissue but only to a certain extent; the
arrangement of cells in tissues does not necessarily resemble a face-centered lattice,
since cells differ from each other, form specific structures (e.g., layers), and can also
be directly electrically coupled (e.g., through gap junctions).
Fig. 4 Normalized steady-state ITV as a function of p for an oblate spheroidal cell with
R2 = 5 R1. Solid: axis of rotational symmetry (ARS) aligned with the field. Dashed: ARS at
45 with respect to the field. Dotted: ARS perpendicular to the field
High Field Frequencies and Very Short Pulses
The time constant of membrane charging (τm) given by Eq. 4 implies that there is a
lag between the time courses of the external field and the ITV induced by this field.
As mentioned above, τm (and thus the lag) is in tenths of a microsecond under
physiological conditions but can be longer when cells are suspended in a medium
with electric conductivity substantially below physiological levels. For alternating
(AC) fields with the oscillation period much longer than τm, as well as for rectan-
gular pulses much longer than τm, the effect of this lag on the amplitude of the ITV is
negligible. However, for AC fields with the period shorter than τm, as well as for
pulses shorter than τm, the amplitude of the ITV gets attenuated by the lag, and with
further increase in field frequency or decrease in pulse duration, the amplitude of the
ITV induced by the field of a given amplitude starts to decrease. Figure 7 shows this
attenuation for a spherical cell; the low-frequencies plateau of the amplitude and its
intermediate-frequencies decrease are both described by the first-order Schwan’s
equation, but the high-frequencies plateau is only described by the second-order
version of this equation, for derivation of which all electric conductivities and
dielectric permittivities are taken into account (Kotnik et al. 1998; Kotnik and
Miklavčič 2000a).
1120 T. Kotnik
Fig. 5 Normalized steady-state ITV as a function of p for two irregularly shaped cells growing on
the flat surface of a Petri dish, with the inset showing the top view of the cells
With field frequencies approaching the GHz range or with pulse durations in the
ns range, this attenuation of the ITV comes to a halt, and the voltages induced on the
organelle membranes inside the cell are of the same order of magnitude as the
voltage induced by the same field on the plasma membrane. In certain circum-
stances, particularly if the organelle interior is electrically more conductive than
the cytosol or if the organelle membrane has a lower dielectric permittivity than the
cell membrane, the ITV on the membrane of such an organelle can temporarily even
exceed the ITV on the plasma membrane (Kotnik and Miklavčič 2006). In principle,
this could provide a theoretical explanation for reports that very short and intense
electric pulses (tens of ns, millions of V/m) can also induce electroporation of
organelle membranes (Schoenbach et al. 2001; Beebe et al. 2003; Tekle
et al. 2005; Batista Napotnik et al. 2016).
Experimental Determination of ITV
An alternative to the analytical and numerical methods for determining the ITV are
the experimental techniques – measurements of the ITV with microelectrodes and
with potentiometric fluorescent dyes. Microelectrodes were used in pioneering
measurements of the action potential propagation, first conventionally (Ling and
Gerard 1949) and later as patch-clamp electrodes (Neher and Sakmann 1976),
Fig. 6 Normalized steady-state ITV as a function of p for spherical cells in suspensions of various
densities (intercellular distances). Solid: The analytical result for a single cell as given by Eq. 2.
Dashed: numerical results for cells arranged in a face-centered cubic lattice and occupying (with
decreasing dash size) 10%, 30%, and 50% of the total suspension volume
preferred for their simple use and high temporal resolution. However, the invasive
nature of measurements and low spatial resolution are considerable shortcomings of
this approach. Furthermore, the physical presence of the conductive electrodes
during the measurement affects the distribution of the electric field around them
and thus also the ITV. In contrast, measurements by means of potentiometric dyes
are noninvasive, offer much higher spatial resolution, and do not distort the electric
field and consequently the ITV. In addition, a potentiometric measurement can be
performed simultaneously on a number of cells. For these reasons, the potentiomet-
ric dyes have during the last decades become the predominant method in experi-
mental studies and measurements of the ITV, and the remainder of this chapter
focuses exclusively on this approach.
Potentiometric Dyes
Based on their response mechanism, potentiometric dyes are divided into two
classes: (i) slow potentiometric dyes that are translocated across the membrane by
an electrophoretic mechanism, which is accompanied by a fluorescence change and
(ii) fast potentiometric dyes that incorporate into the membrane, with energy levels
1122 T. Kotnik
Fig. 7 The amplitude of normalized steady-state ITV as a function of the frequency of the AC field.
The dashed curve shows the first-order and the solid curve the second-order Schwan’s equation.
Note that both axes are logarithmic
of their electrons and consequently their fluorescence properties dependent on

transmembrane voltage.
Electric pulses used in electrophysiological and electroporation-based applica-
tions usually have durations in the range of microseconds to milliseconds. To
measure the ITV induced by such pulses, fast potentiometric dyes have to be used.
These respond to changes in the ITV within microseconds or less, which makes them
suitable even for measurements of the transient effects. Slow dyes, on the other hand,
need several seconds to respond to a change in the transmembrane voltage, and while
some of them, particularly rhodamine and several carbocyanines, are efficient in the
measurements of the RTV, their tardiness makes them largely useless for analysis of
the ITV.
One of the most widely used fast potentiometric dyes is di-8-ANEPPS (di-8-
butylamino-naphthyl-ethylene-pyridinium-propyl-sulfonate), developed by Leslie
Loew and colleagues at the University of Connecticut (Gross et al. 1986; Loew
1992). This dye is nonfluorescent in water but becomes strongly fluorescent when
incorporated into the lipid bilayer of the cell membrane, thereby making the mem-
brane highly visible. This enables the construction of spatial numerical models of
cells from a set of cross-section fluorescence images (Pucihar et al. 2006), and thus
provides a possibility to compute the ITV on the same cells on which an experiment
is carried out.
The fluorescence intensity of di-8-ANEPPS varies proportionally to the change of

the ITV; the response is linear for voltages ranging from 280 to +250 mV
(Lojewska et al. 1989; Cheng et al. 1999). Relatively small changes in fluorescence
of the dye, uneven membrane staining, and dye internalization make di-8-ANEPPS
less suitable for measurements of the resting membrane voltage, although such
efforts were also reported (Zhang et al. 1998). It is, however, well suited for
measuring larger changes in membrane voltage, such as the onset of the ITV in
nonexcitable cells exposed to external electric fields (Gross et al. 1986; Montana
et al. 1989) or action potentials in excitable cells (Bedlack et al. 1994; Cheng
et al. 1999). In addition, di-8-ANEPPS allows for determination of the ITV by
ratiometric measurements of fluorescence excitation (Montana et al. 1989) or emis-
sion (Knisley et al. 2000), which increases the sensitivity of the response.
Image Acquisition and Data Processing
As the sensitivity of fast potentiometric dyes to the changes of the ITV is low
(typically, a change by 100 mV results in the change of fluorescence intensity by
2–12%), the fluorescence changes are hardly discernible by the naked eye but
become apparent with appropriate image processing and analysis.
This procedure is performed in several steps (Pucihar et al. 2009b). First, a pair of
images is acquired: the control image (immediately before the exposure to the electric
field) and the exposure image (during the delivery of the electric field pulse); to get a
more reliable measurement, a sequence of pulses can be applied, with both the control
and the exposure image acquired for each pulse. Second, the background fluorescence
is subtracted from both images. Third, the region of interest corresponding to the
membrane is determined for the cell under investigation, and the fluorescence inten-
sities along this region in the control and pulse image are measured. Fourth, for each
pulse, the control data are subtracted from the pulse data, and the result divided by the
control data to obtain the relative fluorescence changes; if a sequence of pulses is
applied, the values of relative fluorescence changes determined for each pulse can be
averaged. Finally, the relative fluorescence changes are translated into ITV using the
di-8-ANEPPS calibration curve; a rough estimation of this curve can be obtained from
the literature, but for higher accuracy, it must be measured for each particular setup, as
shown in Fig. 8. Calibration is performed with either (i) valinomycin, a potassium
ionophore, and a set of different potassium concentrations in external medium
(Montana et al. 1989; Pucihar et al. 2006, or (ii) patch clamp in voltage-clamp
mode (Zhang et al. 1998). For a graphical description of the results, the ITV can be
plotted as a function of the relative arc length. A brief summary of the described
process in given in Fig. 9, showing a cell stained with di-8-ANEPPS, the processed
image reflecting the ITV, and the plot of the ITV along the cell membrane. To remove
some of the noise inherent to potentiometric measurements, the ITV curve can be
smoothed using a suitable filter (e.g., a moving average).
1124 T. Kotnik
Fig. 8 An example of the experimental calibration curve for measurements of the ITV with di-8-
ANEPPS
Conclusions
Knowledge and understanding of transmembrane voltage and the process of its

inducement in cells exposed to electric fields are important in electrophysiology,
and perhaps even more so in research of electroporation and development of
electroporation-based technologies and treatments. As outlined in this chapter,
inducement of the ITV is a process, and although for alternating electric fields
with frequencies up to the kHz range and for electric pulses lasting tens of μs or
longer, it can be approximated as instantaneous, for higher frequencies and shorter
pulses the inducement’s time course must also be taken into account for an accurate
description of the ITV and its effects. In the context of electroporation, the key aspect
is the close correlation between the ITV and the electroporation-mediated transport
across the membrane: the membrane regions with the highest absolute value of the
ITV are commonly also the ones where the transport proceeds the fastest. While the
ITV induced by a given electric field can generally be predicted quite accurately by
means of analytical or numerical calculation, a valuable experimental complement in
determination of the ITV is found in fast potentiometric dyes that allow, with
adequate sample preparation, dye fluorescence calibration, image acquisition, and
data processing to determine the actual ITV and compare it to the calculated
predictions.
Fig. 9 Determination of the steady-state ITV with di-8-ANEPPS. Top left: Raw fluorescence image
of a cell stained with di-8-ANEPPS. Top right: Processed image reflecting the ITV. Bottom: steady-
state ITV as a function of p determined from the processed image using the calibration curve shown
in Fig. 8
Acknowledgment This work was supported by the Slovenian Research Agency (Grant P2-0249)
and conducted in the scope of the European Laboratory of Pulsed Electric Fields Applications (LEA
EBAM) and within networking efforts of the COST Action TD1104 – European Network for
Development of Electroporation-Based Technologies and Treatments (EP4Bio2Med).
1126 T. Kotnik
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S0006-3495(98)77765-3
Mathematical Models of Pulsed Electric
Field Treatment of Plant Tissues and 60
Simulation of Related Phenomena
Abstract
Pulsed electric fields (PEF) are very promising for the treatment of plant
(vegetable, fruits) and animal tissues. PEF-assisted processing which gives
new perspectives for selective extraction and expression, dehydration, drying,
freezing, and osmotic treatment can be marked out. However, the nature of
electroporation in food tissues is very complicated and can include different
scales starting from single membranes and cells to concentrated suspensions
and real plant tissues with complex intrinsic structure and much heterogeneity
in different scales. Effects of electroporation depend on the size of cells, their
orientation and spatial distribution in space, passive and active electro-
physical parameters of cells, pH of media, and the presence in tissue osmotic
agents. Optimization of PEF treatment and PEF-assisted processing of plant
tissues requires precise adaptation of PEF protocols, selection of optimal
electric field strength, pulse duration, repetition time, and temperature of
treatment. Mathematical modeling can provide detailed and valuable informa-
tion for optimization of PEF protocols, power consumption, and preservation
of the food quality. Moreover, numerical simulation can be useful for descrip-
tion of PEF effects in the presence of complex transport processes and changes
N. Lebovka (*)
after F. D. Ovcharenko, NAS of Ukraine, Kyiv, Ukraine
Compiègne, France
E. Vorobiev (*)

DOI 10.1007/978-3-319-32886-7_9
1130 N. Lebovka and E. Vorobiev
in temperature, electrical conductivity, electric field strength, and spatial

distribution of electrolyte inside the tissue during PEF treatment. In recent
decades various models and simulations were applied for optimizing PEF
treatment of liquid foods, geometrical optimization of the treatment chambers,
and prediction of the electric field distribution, flow velocity, and temperature
inside the treatment chamber.
This chapter discusses various models of PEF treatment and computer simu-
lation of related phenomena, which accompanies PEF-assisted processing of
plant tissues.
Keywords
Pulsed electric fields • Plant tissues • Mathematical models • Computer simulation
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1130
Electroporation of Individual Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1131
Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1131
Simulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1133
Electroporation of Individual Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1134
Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1134
Simulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1136
Pulsed Electric Field Treatment of Suspension of Cells and Plant Tissues . . . . . . . . . . . . . . . . . . . 1137
Corrections of Schwan’s Equation for Concentrated Cell Ensembles . . . . . . . . . . . . . . . . . . . . . 1138
Kinetic Models of Inactivation in Suspension of Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1140
Plant Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1142
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1150
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1151
Introduction
Pulsed electric fields (PEF) have been demonstrated in many useful capacities for the
treatment of plant (vegetable, fruits) tissues. Effects of PEF can be explained by
electroporation of cellular membranes and removing the membrane’s barrier functions
(Weaver and Chizmadzhev 1996). PEF-assisted processing has nonthermal nature and
can be achieved by application of moderate electric fields of 500–1000 V/cm and short
treatment times of 104–102 s (Barba et al. 2015). Among recent examples of PEF
applications, selective extraction of valuable components, retention of health-related
compounds, enhancement of juices expression, plant dehydration, drying, freezing,
and osmotic treatment can be marked out. Moreover, PEF treatment of liquid foods
leads to inactivation of microorganisms and quality-degrading enzymes and gives
extension the shelf life of the products (Barba et al. 2015).
However, the nature of electroporation is very complex and can include different
scales starting from single membranes and cells to concentrated suspensions and real
plant tissues with complex intrinsic structure and much heterogeneity in different
scales. The efficiency of electroporation itself as well as different processes assisted by
60 Mathematical Models of Pulsed Electric Field Treatment of Plant. . . 1131
PEF depends critically on many parameters of PEF protocol, variation of these

parameters during PEF treatment, temperature and such properties of the treated
products as the size of cells, their orientation and distribution in space, passive and
active electro-physical parameters of cells, pH of media, the presence in tissue osmotic
agents, and other factors. Optimization of PEF treatment and PEF-assisted processing
of plant tissues requires for accounting of many parameters, which cannot be provided
without mathematical models and numerical simulation. Mathematical models and
numerical simulation can provide detailed and valuable information for optimization
of treatment time, power consumption, and preservation of the food quality. Moreover,
numerical simulation can be useful for description of PEF effects in the presence of
complex transport processes and changes in temperature, electrical conductivity,
electric field strength, and spatial distribution of electrolyte inside the tissue during
PEF treatment. Various models and simulations were already successfully applied for
optimizing PEF treatment of liquid foods, geometrical optimization of the treatment
chambers, and prediction of the electric field distribution, flow velocity, and temper-
ature inside the treatment chamber (Huang et al. 2012; Gerlach et al. 2008).
The current chapter aims to provide a discussion on various models of PEF
treatment and computer simulation of phenomena induced by PEF in plant tissues.
The different scales of electroporation phenomena are also briefly discussed.
Electroporation of Individual Membranes
Models
The electroporation occurs when the potential difference across the membrane, or
the transmembrane potential, um, exceeds a certain threshold, 0.2–1 V (Weaver and
Chizmadzhev 1996). This potential difference corresponds to the electrical field
strength across the membrane of the order of 4.105–2.106 for a typical membrane
thickness of 5 nm. Electroporation reflects the rearrangements of the membrane
lipids leading to increased membrane permeability.
The time of membrane pore creation tc decreases with increasing of the trans-
membrane voltage um (Fig. 1). In general, the time evolution of electroporation
includes:
– Charging and polarization of the membrane (charging time τc > 1 μs)

– Rearrangement of the structure of the membrane and creation of pores (Fig. 1)
– Aggregation of different pores and noticeable increase (by several orders of
magnitude) in ionic, molecular, and nanoparticle transport through the membrane
– Membrane recovery or resealing after the removal of the electric field
Different non-pore models and pore models were proposed for explanation of
the dielectric breakdown of membranes. The non-pore models account for the
electrically induced mechanical, hydrodynamic, wave, osmotic, and viscoelastic
instabilities of membranes. For example, the following relations for critical voltages
Fig. 1 Mean time of

membrane pore creation.
Filled squares are the
experimental data (Melikov
et al. 2001), and the solid line
is obtained by simulations
based on electroporation
theory (Compiled from the
data presented in (Vasilkoski
et al. 2006))
ucm were obtained, respectively, for mechanical compression, hydrodynamic, and

wave instability models:
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ucm ¼ 0:368Y m h2m =eo em (1a)
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ucm ¼ 0:5γhm =eo em (1b)
1=4
ucm ¼ 24Y m γh3m (1c)
Here Ym is a constant of elasticity, hm is the membrane thickness, em is a relative

dielectric constant, and γ is the surface tension of the membrane.
However, these non-pore models fail to describe the stochastic nature of mem-
brane rupture and predict the membrane lifetime dependence from the transmem-
brane voltage τm(um).
Better description of the stochastic nature of membrane rupture can be obtained
using the pore model. This model suggests that the observed increase in membrane
permeability is due to the formation of pores in the lipid bilayer, and it is based on the
analysis of the energy balance in the membrane. This model is supported by recent
molecular dynamics simulations.
For example, the transient aqueous pore model gives the following relation for the
lifetime of a plane membrane τm (Weaver and Chizmadzhev 1996):
h i
2
τm ¼ τ1
m exp ðW m =kT Þ=1 þ ðum =uo Þ ; (2)
where W m =kT ¼ πω2 =kTγ is the relative activation energy of electroporation, ω is

pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
the line tension of a membrane, uo ¼ 2γ=ðCm ðew =em 1ÞÞ is the voltage
Fig. 2 Probability of 1
membrane damage, Z, (see.
Probability of membrane electoporation, Z

Eq. 1) versus the duration of um =1 V
electric field application, t, at
0.8
different values of um. The
membrane lifetime τm was 0.8 V
evaluated using Eq. 2 with
parameters presented in 0.6 0.6 V
(Lebedeva 1987)
0.4 0.5 V
0.2
0
10-6 10-5 10-4 10-3 10-2 10-1
Duration of electric field application, t, s
parameter, Cm is the specific capacitance of a membrane, ew 80 is the a relative

dielectric constant of water, and τ1
m is the limiting value of τm (at um!1 or T!1).
For the typical values of parameters (for general lipid membranes at T = 298 K)
γ 2.103 N/m, ω 1.69.1011 N, em 2, and Cm 3.5.103 F/m2, we have
uo 0.17 V and Wm/kT 109. Experimental estimation for plane membrane gave
also τ1 3.7.107 s (Lebedeva 1987).
The probability of membrane damage, Z, during the electric field application with
duration of t can be estimated from the first-order kinetic equation:
Z ¼ 1 expðt=tm Þ: (3)
Figure 2 presents Z(t) dependences at different values of um. Electroporation can

be depending on pulsed protocol (mainly on the electric field strength and exposure
time). In irreversible electroporation, the pores in the membrane do not reseal. Such
type of electroporation is commonly used for killing of microorganisms and for plant
cell damage in food and biotechnological applications. From the other hand, in
reversible electroporation, the pores can reseal when electric field is switched off.
Such type of electroporation is used in electrochemotherapy, for transdermal drug
delivery and cell fusion (Miklavčič et al. 2014).
Simulation
It is believed that the nature of electroporation is rather similar for the planar lipid
bilayers and biological cell membranes. Up to date the extensive computer simula-
tions of electroporation of lipid bilayers have been performed. The molecular
dynamics models are based on simulation of a trajectory of molecules in time and
allow handling of tens of thousands of particles. The different atomistic and coarse-
grained approaches allowed studying the processes of the electropore formation,
formation of transient water pores induced by ion charge imbalance, influence of
cholesterol content in the bilayer on electroporation time, formation of water chan-
nels, etc. (Rems and Miklavčič 2016). Simulations evidenced that pore formation in
a high intense electric field is driven by local electric field gradients at the water/lipid
interface. The first stage of pore formation includes formation of a water bridge
across the lipid bilayers. Pores up to 10 nm in diameter can be created depending on
the electric field strength and duration of the applied field.
Electroporation of Individual Cells
Models
In the individual biological cell, the thin membrane with low conductivity σ m
separates two high conductive interior and exterior electrolyte media with conduc-
tivities σ d and σ, respectively (Fig. 3). The values of transmembrane potential um
have been analytically evaluated for individual cells of different geometries. For
example, for the spherical cell the Schwan’s equation gives the following expression
for static value of um (Weaver and Chizmadzhev 1996):
um ¼ 1:5f e ER cos φ (4)
where R is the radius of the cell, and φ is the angle between direction of E and
position of the observation point on the surface of a membrane.
The electroporation factor fe(1) depends on the electrical conductivities of all
components σ m, σ, and σ d, as well as on the ratio hm/R, where hm (5 nm) is a
membrane thickness (Kotnik et al. 1998):
f e ¼ ð3hm =RÞσ d σ=½ðσ m þ 2σ Þðσ m þ 0:5σ d Þ ð1 3hm =RÞðσ σ m Þðσ d σ m Þ

(5)
For the spherical cell, the transmembrane potential and lifetime of the membrane
depend upon the local position on the surface of the cell, i.e., on the φ value. The
average lifetime of a spherical cell, τc, can be estimated accounting for the distribu-
tion of um(φ) over the surface of a cell:
ðπ
τ!
c ¼ τ1
m ðφÞd cos φ (6)
0
Figure 4 presents relative lifetime of the cell evaluated by numerical integration, τ*

= τ/τ1, versus relative electric field strength, E* = 1.5ER/uo (Lebovka et al. 2002).
Fig. 3 Induction of
transmembrane potential for
spherical cell in the external
electric field E
External electric field, E
sd
sm hm um(j)
s
Fig. 4 Relative lifetime, τ* =

τ/τ1, versus relative electric
field strength, E*, for spherical
cell (E* = 1.5ER/uo) and
plane membrane (E* = um/uo).
See the text for the detail
description (Compiled from
the data presented in
(Lebovka et al. 2002))
Here τ1 is the limiting value of τ (Eq. 1). For comparison the τm =τ1
m versus E = um/u0
*
dependence for plane membrane is also presented.

For the given E*, the value of τc for the spherical cell exceeds the value of τm for
the plane membrane. The τ(E) dependence for the spherical cell can be fitted by the
formula that is similar to Eq. 1 for the plane membrane:
h i
τc ¼ τ1
c exp ðW c =kT Þ= 1 þ ðE=Eo Þ
d
(7)
with τ1 1
c 2:01τm , d 1.33, and Wc 0.54 Wm (see Eq. 1) (Lebovka et al. 2002).
For the cell with anisotropic shape, the transmembrane potential, um, depends
also on the orientation of the cell in the external fields and shape of the cell (Fig. 5).
For the elongated cell, the value of um has its maximum or minimum when the
longest axis of the cell is parallel or perpendicular to the electric field, respectively.
The more discussion on the problem can be found elsewhere (Kotnik and
Miklavčič 2000).
Simulation
Schwan’s theory predicts the inversely proportional relationship between the cell
radius, R, and the threshold magnitude of the applied electric field, E, i.e., E / 1/R. In
general, this prediction is consistent with existent experimental data. For example,
the experimentally estimated threshold values of electric field strength are of the
order of 100–1000 V/cm for large cells in food tissues (R 30–60 μm) and >10 kV/
cm for small microbial cells (R 1–10 μm) (Barba et al. 2015).
Simulation with a finite element method gave excellent correspondence with the
Schwan’s theory (Zudans et al. 2007). However, the inverse dependence E / 1/R is
not always observed or does not exactly fit Schwan’s equation. To explain this
inconsistency, the membrane permeabilization and conductivity changes during
Fig. 5 Induced transmembrane voltage um in units of ER1 as a function of the polar angle φ for
three spheroidal cells: R2 = 1/5 R1 (solid line), R2 = R1 (spherical cell, dashed line), and R2 = 5R1
(dotted line). Inset: the three cells and the field orientation (From (Kotnik and Miklavčič 2000) with
permission)
the electroporation pulse were accounted (Mercadal et al. 2016). The author
numerically modeled electroporation of a single cell using the finite element
method (FEM) software platform COMSOL Multiphysics 4.4. By modeling the
interplay between the cell membrane conductivity, permeability, and transmem-
brane voltage, the departure from the Schwan’s equation model was observed. It
was particularly significant for high level of cell membrane permeabilization
during the pulse (for the average relative pore area over the whole membrane
higher than 104).
Computer simulation is useful for evaluation of cell electroporation in the
presence of local heterogeneities of membranes related to their passive electrical
properties (local resistance and capacitance) and both passive and active interac-
tion mechanisms (ion pumps, channel gating, electroconformational coupling,
electroporated membrane regions) (Gowrishankar and Weaver 2003). Figure 6
presents the transport two-dimensional lattice model of the individual spherical
cell with two electrolytes separated by a membrane. The model accounted for the
nonlinear dependence of the membrane dynamic conductance versus the trans-
membrane voltage. Transport lattices were solved by Kirchhoff’s laws, using
Berkeley SPICE circuit simulator program for nonlinear DC, nonlinear transient,
and linear AC analyses. The proposed transport model allowed simulation of
cells with irregular geometry and the presence of local regions with different
properties.
Pulsed Electric Field Treatment of Suspension of Cells and Plant

Tissues
Evaluation of um value for the ensembles of cells (suspension of cells and plant
tissues) is more complex and usually requires application of complex mathematical
models and numerical calculations. In general case the calculations should account
a b dM
e1+m+e2
Mele,e1
Re1/2 2Ce1
Rele,e1 Cele,e1
c SW
Mele,e2 Ich(Um) Rlip Rm[ep] Cm Mm
Rele,e2 Cele,e2
Re2/2 2Ce2
Fig. 6 The two-dimensional model of the individual spherical cell with two electrolytes separated
by a membrane (dark curve) (a). Here the charge transport models of exterior electrolyte Me1 (b)
and interior electrolyte Me2 (c) and membrane model, Me1 + m + Me2 (d) are presented (From
(Gowrishankar and Weaver 2003) with permission)
for inhomogeneous distribution in the local electric fields, osmotic flows, resealing
of cells, distribution of cell sizes and orientations, their states and local solute
concentrations, local electrical conductivity, etc. (Pucihar et al. 2007).
Corrections of Schwan’s Equation for Concentrated Cell Ensembles
For concentrated cell ensembles (dense suspensions, plant tissues), the value of um
may be altered owing to the deformation of the applied electric field by the adjacent
cells. For lattice arrays of closely packed cells, the Schwan’s equation was general-
ized as (Pavlin et al. 2002; Qin et al. 2005; Ramos et al. 2006; Susil et al. 1998)
um ¼ 1:5gðϕÞER cos θ; (8)
where g(ϕ) is a correction factor, which is a function of volume fraction of particles,

ϕ.
The finite element method was used to calculate the value of um for different
arrangements of spherical cells including a simple cubic lattice (sc),
two-dimensional planes, and one-dimensional chains of cells both parallel and
perpendicular directions to the applied field (Susil et al. 1998). Numerical simula-
tions using the equivalent circuit method (ECM) were also applied to calculate the
value of um for body-centered cubic (bcc) lattice arrangement of spherical cells
(Ramos et al. 2006). Figure 7 presents a correction factor g versus normalized
volume fraction of particles ϕ/ϕm for spherical cells in sc and bcc packings; here
ϕm = π/6 and ϕm = √3 π/8 are the maximum volume fractions for sc and bcc
packings, respectively. The value of g decreases practically linear with increasing of
ϕm up to g 0.67–0.75 at ϕ = ϕm.
The similar calculations were also extended for cell arrangements in body-
centered cubic (bcc) and face-centered cubic (fcc) lattices (Pavlin et al. 2002).
Later on the computational results for fcc lattice model were used for explanation
of the experimental measurements on the fraction of electroporated cells in dense
suspensions (1 %, 18 %, and 36 % by volume) of CHO cells (Pucihar et al. 2007).
The following expression for g(ϕ) was analytically evaluated using the mean-
field approach (Qin et al. 2005):
3
gð ϕÞ ¼ 1=3 (9)
3=2 3 ϕ=
2 þ ð1 ϕÞ 1þ 4Nπ
Here, ϕ is the volume fraction of cells, and n is the number of particles per unit cell.
For simple cubic lattice (ϕ = π/6 and n = 1) the above equation gives g = 0.859.
Physical modeling of electroporation in close cell-to-cell proximity environments
may be useful for estimation of deviations from Schwan’s equation (Gaynor and
Bodger 2006). The electrically equivalent model, based on aqueous solution filled
thin latex rubber membrane spheroids suspended in a tank filled with water
1
Electric filed, E
0.9
Correction factor, g
bcc
sc
0.8
0.7
0 0.2 0.4 0.6 0.8 1

Normalized volume fraction, f/f m
Fig. 7 Correction factor g versus normalized volume fraction of particles ϕ/ϕm for spherical cells
in sc packing. Here ϕm = π/6 and ϕm = √3 π/8 are the maximum volume fractions for sc and bcc
packings, respectively (Compiled from the data presented in (Ramos et al. 2006; Susil et al. 1998))
(the so-called “balloon” model), was used to investigate membrane permeabi-

lization. The various two-cell, “couplet” arrangements and a 3 3 3 simple
cubic lattice of “cells” were studied. For the case when the external media conduc-
tivity is lower than the cell interior conductivity, the electroporation is initiated at
substantially decreased value of E in regions of close cell-to-cell proximity as
compared to cells in isolation.
The experimental study of electroporation in close cell-to-cell proximity envi-
ronments was performed using optical tweezers for accurate positioning of two cells
in fluidic electroporation device (Henslee et al. 2014). The finite element simulations
of the electrostatic potential distributions were also performed. The situations of two
cells oriented parallel and perpendicular to the electric field were compared. Figure 8
presents the calculated correction factor g versus normalized center-to-center dis-
tance d/2R for two cells aligned parallel and perpendicular to the applied field. For
parallel geometry, the second cell decreases the field on the first where as the
opposite situation is observed for perpendicular geometry. Good theory-experiment
agreement was found for the perpendicular geometry. For the parallel geometry,
experimentally the measured value g was about a factor of two larger than were
obtained in electrostatic calculations. In general, the correction factor is changed
~5–10 % by a nearby, nearly touching second cell.
1.04
Electric filed, E
1.02
Correction factor, g
2R
Electric filed, E
0.98
0.96
1 1.5 2 2.5 3 3.5 4

Normalized center to center distance, d/2R
Fig. 8 Calculated correction factor g versus normalized center-to-center distance d/2R for two cells
aligned parallel and perpendicular to applied field (Compiled from the data presented in (Henslee
et al. 2014))
Kinetic Models of Inactivation in Suspension of Cells
The simplest first-order equation for surviving kinetics was proposed by Bigelow
(Huang et al. 2012):
S ¼ expðt=τc Þ (10)
where S is a fraction of non-electroporated cells and τc is a lifetime of cells. Here, the

lifetime of cells τc is the empirical parameter that can be obtained by fitting of
experimental data on microbial inactivation using Eq. 10.
However, in many cases the Bigelow’s equation fails to describe the microbial
inactivation by PEF treatment. Different more complicated empirical models were
proposed to simulate the survival curves in microbial inactivation, e.g., Fermi,
log-log, log-logistic, and other models.
The very popular is the Hulsheger’s model:
S ¼ ðt=tc ÞðEEc Þ=k (11)
where tc and Ec are the threshold values of treatment time and electric field strength,
and k is the parameter. However, this model is purely empirical and there is no
theoretical justification for the origin of such behavior.
Another model is described by extended exponential (Weibull’s) equation:
S ¼ exp ½ðt=τÞn (12)
where τ and n are also empirical parameters.

The shape parameter n accounts for a concavity of a survival curve. The devia-
tions from first-order kinetics may be explained by different factors such as the
distribution of lifetimes and of cell diameters and/or orientation of cells and many
other factors. For the model of the distribution of lifetimes, the value of
n characterizes the broadness of the distribution. The case n = 1 corresponds to
the single lifetime, and the larger the deviation of n from 1 is, the broader the
distribution of the lifetimes. The mean lifetime <τ> can be estimated from the
following relation:

<τ> ¼ τΓ n1 =n; (13)
where Γ is the Euler gamma function.

The Monte Carlo model was developed for estimation of the kinetics of microbial
inactivation of cells by PEF (Lebovka and Vorobiev 2004). Initial Gaussian distri-
bution of cell radii r was assumed:
pffiffiffiffiffiffiffiffi
f ðRÞ ¼ exp ðR < R >Þ2 =2δ2 = 2πδ; (14)
where <R> and Δ are the mean radius and the standard deviation, respectively.
The surviving kinetics was approximated by Eq. 12. The lifetime of cells
was evaluated using Eq. 2 accounting for the cell radius dependence of trans-
membrane potential um(R), Eq. 4. Figure 9 presents some examples of the
calculated survivor curves S(t) for a suspension of cells with a normal law
distribution of their radiuses. The model predicts the decrease of shape param-
eter with increase of the standard deviation δ and gives upward concavity, i.e.,
n < 1.
The similar Monte Carlo model was also developed for simulation of inactivation
of spheroidal cells by PEF (Lebovka and Vorobiev 2007). The cases of partially and
randomly oriented spheroids were analyzed; it was demonstrated non-exponential
kinetics can reflect the orientational disorder of cells with anisotropic shape.
Figure 10 presents examples of survivor curves S(t) for oblate (a < 1) and prolate
(a > 1) spheroids distributed in the space with random orientations for relative
1=3
electric field strength E* = E/Eo = 10. Here, Eo ¼ uo =1:5R, R ¼ Rz R2 x is an
equivolume radius of a spheroid and a = Rz/Rx is an aspect ratio (major/minor axis).
The model predicts upward concavity, n < 1, for prolate cells, and near-exponential
kinetics, for oblate spheroids. The most pronounced deviations from the exponential
kinetics were observed for disordered suspensions of prolate spheroids at small
electric field strength E and large aspect ratio a. For partially oriented suspensions,
efficiency of inactivation enhances with increasing of order parameter and field
strength.
Fig. 9 Survivor curves S(t) for different values of the relative width of cell radius distribution,
Δ/<R>. Here, <R> is the mean radius of cells, τ1 m is the limiting value of τ (Eq. 1), and the
calculations were made for relative electric field strength E* = 1.5ER/uo = 10 (Lebovka
et al. 2002) (Compiled from the data (Lebovka and Vorobiev 2004))
Plant Tissues
The effects of PEF on electroporation of the plant tissue were studied using the
Monte Carlo computer simulation model (Lebovka et al. 2001). The tissue was
represented by a two-dimensional array of biological cells (Fig. 11). The electrical
potential u was applied between the upper and bottom electrodes, and the potential
distribution of all cells in the lattice uxy was evaluated numerically by solving the
discrete version of Laplace’s equation. For this purpose the successive relaxation
scheme was used. The electroporation probability of all cells during the pulse
application was estimated using the first-order kinetic equation (Eq. 2). The electro-
poration of cells accompanies with replacing the electrical conductivities σ i ! σ d
and updating of the potential uxy in all cells. The effective electrical conductivity of
the tissue was calculated using a highly efficient Frank and Lobb algorithm.
The degree of disintegration Z was defined as the ratio of electroporated cells and
total number of the cell. The application of an external field in the form of an
idealized square pulse sequence with a pulse duration ti and a pulse repetition time
Δt was assumed.
The simulation model accounted also for the possibility of resealing (reversible
electroporation) with time constant of τr and moisture transfer processes (diffusion,
Prolate
100
E*=10 spheroids
10−1
a=10
Surviving fraction, S
10−2
Oblate 5
spheroids
10−3
0
10−4 0.2
0.1
10−5
0 50 100 150 200
Relative time of PEF treatment, t / t m•
Fig. 10 Survivor curves S(t) for oblate (a < 1) and prolate (a > 1) spheroids distributed in the
space with random orientations. The dashed line corresponds to the simple exponential kinetics for
the equivolume with spheroid spherical cell with radius R = (RzR2x)1/3. Here a = Rz/Rx is an aspect
ratio (major/minor axis), τ1
m is the limiting value of τ (Eq. 1), and the calculations were made for
E* = E/Eo = 10, Eo = uo/1.5R (Compiled from the data (Lebovka and Vorobiev 2007))
osmotic flow, and redistribution of moisture inside the sample) with time constant of
τd. Figure 12 shows the example of the simulation of PEF-induced electroporation
tissue in the presence of resealing and moisture transfer processes (Lebovka
et al. 2001). The obtained numerical data were in qualitative correspondences with
experimental data for PEF-treated apples.
It was demonstrated that PEF-induced damage kinetics is very sensitive to the
repetition time Δt. The aggregated groups of the electroporated cell (correlated
patterns) were observed for the case when Δt exceeds the time of moisture transfer
processes τd. On the contrary, when Δt τd, the spatial distribution of the
electroporated cell was observed. In PEF experiments with long repetition time,
Δt = 60 s, the large macroscopic domains of electroporated cells were observed.
Considerable differences between the damage kinetics for long and short interpulse
distances were observed both in experiments and simulations. The experimental data
evidences that PEF protocol with Δt = 60 s was more efficient than PEF protocol
with Δt = 0.01 s.
Electroporation theory predicts higher electroporation for the tissue with larger
radius cells (Eqs. 4 and 8). However, in experiments with selected fruit and vegetable
plant tissues (apple, potato, carrot, courgette, orange, and banana), the direct corre-
lation between PEF-induced electroporation and size of the cell was not always
Electrode Intact cell
si
dm
PEF Electropotated cell
R
sd
Electrode
Fig. 11 The two-dimensional model of the tissue. Each cell is represented by a node with four
conducting bonds. Here, σ d and σ i are the conductivities of intact and electroporated cells,
respectively, R is the size of the cell (“radius”), and dm is the thickness of the membrane (Based
on the model described in (Lebovka et al. 2001))
observed (Ben Ammar et al. 2011). To explain this anomaly, a Monte Carlo model
for simulation of PEF-induced changes in the tissue was developed. Initial Gaussian
distribution of cell radii r was assumed (Eq. 14). Before PEF treatment all cells were
intact. During the PEF treatment (tPEF > 0), some of the cells remained intact, while
others got damaged (electroporated). So, electroporation influenced the distributions
of intact and electroporated cells and they were time dependent. The degree of plant
tissue damage, Z, was defined as the ratio of volume of the damaged cells and the
total volume of cells. In simulation the intact cell was selected randomly. The
damage time evolution under the PEF treatment was approximated by the first-
order equation with accounting for the lifetime dependence upon electric field
strength E, current damage degree Z, and cell radius r (Eqs. 4, 5, and 7):
h i
τc ¼ τ1
c exp ð W c =kT Þ= 1 þ ð 1:5f e ð Z ÞRE=u o Þ d
(15)
with τ1 1
c 2:01τm , d 1.33, and Wc 0.54 Wm (see Eq. 1) (Lebovka et al. 2002).
Note that the electroporation factor fe(Z) in Eq. 15 accounts for the changes of
electrical conductivity of the extracellular medium σ(Z ) during PEF treatment (see
Eq. 5). For estimation purposes, the value of σ d was identified with electrical
conductivity of the totally electroporated tissue with highest level of Z (Z 1).
Such tissue can be obtained using the PEF treatment in the limit of long treatment
time (tPEF 0.1–1 s) and high electric field strength (E > 1000 V/cm).
The value of σ increases with increase of Z from σ (Z = 0) = σ i to σ (Z = 1) σ d
in the limit of high tissue damage. The value of σ m was estimated from the serial
one-dimensional model of cell packing as σ m σ i hm/R, where hm (5 nm) is the
1
tr =10−2 s
Disintegration index, Z
0.8
0.6
0.4
td (s)
Pulse sequence
1
0.2 Dt = 5ti 10−2
ti =1ms
3
Relative conductivity,s/si
Resealing
Electroporation
1
2 4
Pulse number, n
Fig. 12 The example of the simulation of PEF-induced electroporation tissue in the presence of
resealing with time constant of τr and moisture transfer processes with time constant of τd. Degree
of disintegration, Z, and relative conductivity, σ/σ i, versus pulse number, n (Lebovka et al. 2001)
thickness of the membrane. Figure 13 presents examples of calculated dependencies

of electroporation factor fe versus σ/σ d at different values of electrical conductivity
contrast, i.e., the ratio between final σ d and initial σ i conductivities, k = σ d/σ i (>1).
The value of fe is an increasing function of σ and it attains maximum fmax for the
totally electroporated tissue (at σ/σ d = 1 and Z = 1). fmax is an increasing function
of k and fmax 1 at k 1. However, for tissues with small values of electrical
conductivity contrast the transmembrane potential um, the efficiency of electropora-
tion may be noticeably reduced. Finally, for the current damage degree f (Z ),
dependence can be approximated using the general effective medium equation for
electrical conductivity:

1=s 1=s 1=t 1=t
ð1 Z Þ σ i σ 1=s = σ i þ Aσ 1=s þ Z σ d σ 1=t = σ d þ Aσ 1=t
¼ 0; (16)
where s and t are the exponents of the percolation theory, s = 0.73, t = 2.0 for three-
dimensional random materials, and A = (1Zc)/Zc, Zc is the percolation threshold
corresponding to the transition from low (σ = σ i) to high (σ = σ d) conductivity
limits.
Fig. 13 Electroporation factor, fe, versus the electrical conductivity ratio, σ/σ d, at different values
of electrical conductivity contrast, k = σ d/σ i (Compiled from the data (Ben Ammar et al. 2011))
In experimental works to characterize the extent of electroporation, the conduc-

tivity disintegration index is commonly used (Barba et al. 2015):
Zc ¼ ðσ σ i Þ=ðσ d σ i Þ; (17)
where the subscripts i and d refer to the conductivities of the untreated (intact) and
completely damaged (electroporated) tissue, respectively.
Figure 14 shows simulated specific power consumptions W (Zc = 0.8) versus
electric field strength E at two values of electrical conductivity contrast, k = σ d/σ i
(=10 and = 2). The parameters of simulations are specified in Ben Ammar
et al. (2011). The increasing of E accelerates tissue damage in full correspondence
with reported experimental data. Moreover, the model allowed explanation of
experimentally observed correlation between electroporation efficiency and value
of electrical conductivity contrast. The model predicts also the sensitivity of the
evolution of distribution function of intact cells to the values of E and σ d/σ i. The PEF
treatment at relatively small fields allowed of preferential damage of larger cells and
it can be useful for selective extraction of the intercellular medium from larger cells.
The value of W passed through the minimum with increasing of E, and the position
of this minimum at E = Eo was dependent upon the value of k = σ d/σ i. The simulated
behavior is in good correspondence with existing experimental data (Lebovka
et al. 2002). For example, it was estimated Eo 360 V.cm1 (σ d/σ i 14.3) for
Fig. 14 Specific power consumptions, W, required for relatively high disintegration (Z = 0.8)
versus electric field strength, E, at two different values of conductivity ratio σ d/σ i (Compiled from
the data (Ben Ammar et al. 2011))
potato and Eo 976 V.cm1 (σ d/σ i 5.6) for banana, i.e., the value of Eo increases
with decreasing of σ d/σ i (Ben Ammar et al. 2011). The plant tissue with low σ d/σ i
ratio (σ d/σ i 1) requires application of a rather strong electric field (e.g., numerical
estimation gives Eo 3000 V/cm for orange, k = σ d/σ i 1.25).
The Monte Carlo model for simulation of electroporation efficiency induced by
PEF treatment of plant tissues was developed (Lebovka et al. 2014). The PEF
treatments with time-variable protocol are applied in combined PEF pressing
(hydraulic, roll or belt), when the electric field strength E and electrical conductivity
of the sample can change significantly during the operations. The potato was
selected as a model tissue.
The simulations were done using the constant value of electric field strength E =
Ei (model M!) and time-variable protocols with different strategies of E changes:
exponential increase (model M") or decrease (model M#) of E, persistent (model
M""), and anti-persistent (model M"#) evolution of E. In persistent and anti-
persistent strategies, the direction of the E changes was dependent upon efficiency
of tissue electroporation that was characterized by the value of dZ/dW and the sign of
the second derivative d2Z/dW2. Note that application of persistent (M"") or anti-
persistent M"# strategies does not require assignment of the final value of Ef that
makes these strategies more attractive for autoregulation of electric field strength
E during the PEF treatment.
Figure 15 gives examples of simulated specific power consumption, W,

(at disintegration index Z = 0.9) versus initial electric field strength, Ei, for different
exponential evolution of E.
For model M" the electric field strength was increased between Ei and Ef = 2Ei
according to
E ¼ Ei ð2 expðt=tPEF ÞÞ (18a)
For model M# the electric field strength was decreased between Ei and Ef = 0.5Ei
according to
E ¼ 0:5Ei ð1 þ expðt=tPEF ÞÞ (18b)
The data for the constant E model (M!, E = Ei = const) are also presented in
Fig. 16 for comparison (Lebovka et al. 2014).
The computer simulation predicted that application of protocols with exponential
time-variable electric fields would allow optimization of PEF treatment with initial
electric field Ei deviating from the optimal value Eo. In general, the W(Ei) depen-
dencies at nonstationary exponential strategies M" and M# were qualitatively similar
5
M↑: E = (Ef -E i)(1-exp(-tPEF /tE)+Ei
M↓: E = (Ei -E f)(exp(-tPEF /tE )+Ef
Specific power consumption, W, KJ/Kg
M↑
3 Ef = 2Ei
Eo M↓
Ef = Ei /2
M→
Ei = const Z=0.9
2
100 200 300 400 500 600 700 100
Fig. 15 Simulated specific power consumption, W, (at disintegration index Z = 0.9) versus initial
electric field strength, Ei, for exponential models of E evolution (increase of E, M", and Ef = 2Ei
and decrease of E, M#, and Ef = Ei/2), and τE ¼ 3τ1 c , where τ E is the constant characterizing the
time dependence of E, and τ1 c is the limiting lifetime of a cell. The data for the constant E model
(M!, E = Ei = const) are also presented for comparison (Compiled from the data (Lebovka
et al. 2014))
to that of PEF treatment under stationary conditions, E = Ei = const. The value of Eo

was dependent on parameters of the models (i.e., Ef and τE). For PEF treatment at the
constant electric field E (model M!), the optimal value was Eo 335 V/cm and the
value of Eo increased as τE increased for the model M"(Ef = 2Ei), and the opposite
behavior was observed for the model M# (Ef = Ei/2). So, application of
nonstationary protocols with exponential increase or decrease of electric field
strength (models M" and M#) is more attractive in the cases when Ei < Eo or Ei >
Eo, respectively.
Simulations for the persistent M"" and anti-persistent M"# strategies revealed
significant differences in their electroporation efficiency (Lebovka et al. 2014). For
easier adaptation of PEF treatment time-variable protocol, the persistent strategy
M"" was more useful than anti-persistent strategy M"#. At low initial value Ei that
was smaller than the optimal Eo 335 V/cm, the persistent strategy M"" predicted
noticeable benefits in power consumption. For example, the value of W (Z = 0.9) at
Ei = 200 V/cm was 3.2 kJ/kg for M"" model and 5.0 kJ/kg for M! model at
τE = 3–10τ1. From the other side, the persistent strategy M"" resulted in an
increase of power consumption compared to that obtained for M! model at the
initial value of Ei, larger than the optimal Eo value. The obtained data have also
shown that application of anti-persistent M"# strategies did not result in improve-
ment of energy consumption at small values of Ei (<Eo 335 V/cm) and caused
increase of energy consumption at large values of Ei > Eo. Finally, the simulation
results were in reasonable correspondence with experimental data (Lebovka
et al. 2014). The PEF experiments with potato disks at constant values of E gave
W(E) dependence that was in good correspondence with theory predictions for the
model M!, Ei = const. The minimum power consumption was observed at inter-
mediate electric field strength E = Eo 400 V/cm. PEF experiments with expo-
nential increase (M") and decrease (M#) of E values revealed that M" strategy can be
useful at small initial electric field strengths, E < Eo, as far as it allowed significant
improvement of PEF treatment efficiency. From the other side, the exponential
decrease strategy M" with large initial electric field strength Ei (>Eo) was less
efficient than the strategy with constant E at the optimal value of E = Eo 400
V/cm.
The computer simulation and mathematical modeling were used for prediction of
PEF effects on osmotic dehydration apple slices (Yildiz et al. 2016). The data on the
osmotic dehydration times and the final water and solid distributions in apple slices
were in good agreement with experimental data.
It should be mentioned that many attempts were devoted to the studies of
electroporation mechanisms in biological tissues in relation with biomedical
applications of PEF treatment. In several works the potato tissue was used as a
model material (Castellví et al. 2016; Kranjc et al. 2016). Mathematical simula-
tion was used to predict shape and extension of irreversible electroporation (IRE)
(Castellví et al. 2016). The sliced potato tubers were PEF treated and a dye
solution was used to enhance the IRE area. From digitized images the
electroporated area and the resulting treated volume were automatically identi-
fied. A three-dimensional reconstruction of the electroporated tissue was
generated. The model for reconstruction the electric field distribution during PEF
treatment of potato tubers using magnetic resonance electrical impedance tomog-
raphy (MREIT) was also developed (Kranjc et al. 2016). Data shows that MREIT
could be used as an efficient tool for improving the effectiveness of PEF
treatment.
Simulations have been performed in order to determine the distribution of the
electric field and compute the spatial and temporal evolution of electroporation in a
biological tissue during the application of pulse (Langus et al. 2016). An electric
circuit model for the tissue and a phenomenological model for the electroporation
process were used. Data of simulations were in reasonable correspondence with
in vivo experiments made on rabbit livers. Numerical simulations based on a finite
element model were also applied to study the effects of tissue properties, electrode
placement, and electric pulse delivery scenarios in the process of electroporation.
The developed model was able to predict the time evolution of an electric pulse
current for different PEF protocols.
Conclusions
Applications of PEF to processing of plant tissues have many important and

promising perspectives in the food industry. The models of PEF treatment and
simulation of related phenomena can contribute significantly to the development
of efficient PEF protocols and equipments. Nowadays the various approaches on
different spatial and temporal scales have been proposed for such aims. However, the
models and simulations are still rarely explored in studies of the PEF treatment
combined with different food processing techniques such as solid-liquid and press-
ing extraction, drying, freezing, and osmotic dehydration. These studies are required
in the future for facilitation and optimization of the PEF treatment protocol, finding
possible synergy of different mode of treatments, improvement of the power con-
sumptions and preservation of food quality.
Acknowledgments The authors appreciate the support from the COST Action TD1104
treatments; www.electroporation.net).
Cross-References
▶ 3D Tissue Models to Bridge the Gap Between Cell Culture and Tissue in
▶ Application of Numerical Simulation Techniques for Modeling Pulsed Electric
Field Processing
▶ Cell Electroporation Model Using Voronoi Networks
▶ Computational Approach for Electrical Analysis of Biological Tissue Using the

▶ Dual-Porosity Model of Liquid Extraction by Pressing from Plant Tissue Modified
by Electroporation
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Dual-Porosity Model of Liquid Extraction
by Pressing from Plant Tissue Modified 61
by Electroporation
Samo Mahnič-Kalamiza
Abstract
One of the basic challenges in applying electroporation to tissues is the under-
standing of resulting structural property changes and mass transport properties in
the treated material. In applying electroporation to plant tissue, the objective is in
either achieving improved extraction of intracellular compounds or water out of
cells, or facilitating otherwise impractical, impossible, or severely hindered
introduction of molecules into cells. Understanding of and the ability to model
solute and liquid transport phenomena in treated tissues is therefore of great
importance, both in scientific terms as well as for practical purposes that are of
interest to the food processing industry or biorefinery. Electrical and process
parameters that are characteristic of a particular treatment protocol used to
achieve the required mass transport enhancement in tissue should be connected,
theoretically, with the resulting improvement in mass transport and structural
changes in tissue. This theoretical link should be established in order to allow for
optimization of the treatment protocols, thus saving energy, materials (e.g.,
extraction reagents or solvents), reducing time for post-treatment processing
(e.g., diffusion or maceration stage), and improving the quality and safety of
the final product. This chapter presents a modeling approach to understanding
mass transport processes in electroporated tissues, whereby the mass transport
process is mathematically linked to the structural property changes in tissue
resulting from electroporation. The approach has been termed the dual-porosity
model and should be considered as an illustration of the fundamental basis to a
more comprehensive model connecting effects of electroporation on the biolog-
ical membrane with its observable effects on the macroscopic level of tissue. To
allow the reader immediate application and full comprehension of the model, the
S. Mahnič-Kalamiza (*)
Laboratory of Biocybernetics, Faculty of Electrical Engineering, University of Ljubljana, Ljubljana,
Slovenia
e-mail: samo.mahnic-kalamiza@fe.uni-lj.si

DOI 10.1007/978-3-319-32886-7_11
1154 S. Mahnič-Kalamiza
complete mathematical derivation and an analytical solution of the fundamental

model equations are presented, supplemented by an extensive commentary on the
theoretical basis and derivation of individual parameters of the model.
Keywords
Mathematical modeling • Tissue electroporation • Plant tissues • Pressure gradi-
ents • Liquid flow • Consolidation behavior • Bulk properties
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1154
The Dual-Porosity Modeling Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1156
The Dual-Porosity Approach in Theory: Mathematical Problem Formulation . . . . . . . . . . . . 1157
The Hydraulic Permeability of the Electroporated Biological Membrane and
Estimation of Model Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1165
The Analytical Solution of the Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1169
Simulated Liquid Pressure Distributions and Consolidation Kinetics . . . . . . . . . . . . . . . . . . . . . . . . 1170
Model Verification and Application by Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1173
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1174
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1176
Introduction
Models of phenomena in electroporated tissues are scarce in literature. Most

concern the electric field or conductivity distribution and changes in tissue follow-
ing electroporation (Sel et al. 2005; Granot and Rubinsky 2008; Grimi et al. 2010;
Čorović et al. 2012; Dymek et al. 2015; Langus et al. 2016) and do not extend the
modeled properties to consequent mass transport but are mainly concerned with
the objective of ensuring the minimum required coverage of tissue with an electric
field of sufficient strength and the (in)homogeneity of the electric treatment
applied.
Existing mass transport models relating electroporation and tissue are almost
exclusively phenomenological in nature, meaning they are developed by fitting a
suitably chosen mathematical model according to a best-fit criterion to experimen-
tal data, obtained generally by means of a multiparameter experimental study
(Lebovka et al. 2000; Fincan and Dejmek 2003; Grimi et al. 2010). Such models
may be of great practical aid; however, their construction necessitates a multitude
of experiments be performed in numerous repetitions for every varied parameter
value from the chosen parameter range. The downside, apart from the relative
tediousness and time-consuming effort invested in the endeavor, is the limited
applicability of such models in case treatment parameters (electroporation proto-
col) and/or target tissue properties (e.g., humidity, age, structural and textural
61 Dual-Porosity Model of Liquid Extraction by Pressing from Plant. . . 1155
properties, etc.) change for a variety of reasons. Since tissue properties can depend
strongly on the origin of tissue and growth conditions, on what kind of tissue (e.g.,
roots, leafs, fruit, etc.) is being treated, and can vary considerably between species
of plant or indeed between cultivars of the same crop species, this can present a
considerable challenge to the usefulness and applicability of such purely phenom-
enological models.
An alternative approach is to build a model from the ground up; a model can be
constructed by observing the basic physical laws of the processes involved, while
also introducing a minimal sufficient set of parameters that reflect material properties
and model variables of the treatment that is applied, a process known as mechanistic
modeling (Zhu and Melrose 2003). When modeling effects of electric fields on plant
tissues and the resulting mass transport, this rule translates to incorporating, at a
minimum, a mass transport and conservation law or set of laws, a parameter
reflecting the structural changes induced by the treatment in the target tissue, and a
parametrization of the electrical treatment where the link between each of the
treatment parameters and the parameter reflecting structural changes must be
established either theoretically or empirically. The ultimate goal in trying to under-
stand the effects of treatment on tissue is the establishment of a theoretical relation-
ship between any given treatment protocol parameter and the resulting changes in
tissue, a process which remains to date an ongoing effort in the scientific community
concerned with basic principles of electroporation. The modeling paradigm
presented in this chapter is an attempt at formulating such a relationship by
suggesting a theoretical link between one of the most critically important electropo-
ration parameters – the local electric field strength – on the one hand, and the
hydraulic resistance of cell membranes to liquid flow in tissue, on the other. This
formulation is based on previous works in the biomedical field of electroporation
applications and studies done on cell suspensions of animal cells (Smith et al. 2004;
Pucihar et al. 2007; Pavlin and Miklavcic 2008; Kotnik et al. 2010; Li and Lin 2011),
and the mass transport (liquid flow) equations are grounded in the theory of water
relations in soils and fractured rocks, i.e., mechanics of porous materials (Barenblatt
et al. 1990). A similar approach has already been developed and proposed for the
case of mechanical pressing of oleaginous seeds (Lanoiselle et al. 1996) and beds
formed of tissue particles (Petryk and Vorobiev 2013). A completely analogous
formulation, from the mathematical point of view, of a diffusion problem can be
found in Mahnič-Kalamiza et al. (2014a).
The dual-porosity model of liquid expression from tissue that has been
electroporated as presented in this chapter can thus be thought of as a demonstration
of an approach in building a model based on fundamental principles of electropora-
tion and its known effects on cells in tissue and mass transport laws in porous
materials, such as biological (plant) tissues. To keep the account instructive and
comprehensible, the model retains a number of parameters that are fundamentally
phenomenological in nature; however, their initial approximate values can easily be
determined from simple experiments.
The Dual-Porosity Modeling Approach
In order to study mass transport at the tissue level with relation to electroporation or
indeed any treatment, the structural components of tissue that are hindering mass
transport and are affected by the treatment must be identified and introduced into a
model. This can be done by formulating, as a mathematical relation, the dependence
of a property or properties (e.g., permeability, conductivity, etc.) of the identified
structure on the relevant (important) treatment parameters.
As an example, consider the case of the semipermeable biological membrane.
Under normal physiological conditions, the membrane is selectively permeable,
meaning that it is not indiscriminately admitting to transmembrane mass transport.
With electroporation, the selective permeability of the plasma membrane is either
temporarily (case of reversible electroporation) or permanently (in case of irrevers-
ible electroporation) disrupted. Any model attempting to relate electroporation
effects with the resulting transmembrane, mass transport must therefore introduce
the variable permeability of the membrane into the equation(s).
Since tissue comprises a multitude of cells of different shapes and sizes arranged
in a particular structure, where the spaces between the cells are far from empty, one
must account for the specifics of the extracellular matrix as well. This extracellular
network also presents a hindrance of its own to mass transport in tissue, and the
permeability to solute diffusion or liquid flow in this space will be – if not directly, at
least indirectly – a function of the treatment intensity. It is evident that in order to
study mass transport phenomena in tissue, one must, at a minimum, consider the
permeability of the plasma membrane as well as the permeability of the extracellular
space. The dual-porosity approach as described in this chapter follows this logic
closely, with the noted exception of neglecting the variable permeability of the
extracellular matrix that can be ascribed to electroporation. The reduction of extra-
cellular permeability due to tissue compaction, and therefore its temporal variability,
is also not accounted for. The modeled system is schematically illustrated by Fig. 1,
giving a highly simplified representation of tissue with the two compartments or
spaces – the intracellular and the extracellular.
According to the dual-porosity approach, the intracellular and extracellular spaces
are each attributed an intrinsic porosity, i.e., a void-to-total (liquid-to-total) volume
ratio. The void volume is considered to be occupied (filled) with liquid that can be
expressed out of the cells and out of tissue under the driving force of an externally
applied pressure, and the remaining volume is considered to comprise biological
solids. The model does not concern extracellular air, which is supposed to be
expelled out of tissue at the initial stages of the modeled experiment, resulting in
either a slight compaction of the tissue or replacement of air pockets with intracel-
lular liquid at the onset of electroporation.
The transmembrane flow of liquid is regulated by the membrane, of which
hydraulic permeability is a function of electroporation, and the liquid expressed
from cells vacates the tissue via the extracellular space. The cell membrane and the
extracellular space have different hydrodynamic properties. The assumption that
liquid does not reenter cells and does not flow through bulk tissue via the
Fig. 1 A simplified schematic representation of tissue, introducing the minimal structure necessary
for demonstrating the dual-porosity approach. The subfigures correspond to the case of (a) before
electroporation or (b) after electroporation. Coloring is for illustrative purposes only and depicts no
factual property of the biological material
intracellular pathway is based on the assumption that the membrane is the predom-
inant factor in hindering liquid expression from tissue. If so, the intracellular
pressure will always be higher than the extracellular, meaning transmembrane flow
is unidirectional.
What remains in order to arrive at a model formulation is to write mass conser-
vation laws for the two respective spaces.
The Dual-Porosity Approach in Theory: Mathematical Problem

Formulation
Constitutive Equations
In order to introduce the dual-porosity model in the form of its mathematical
formulation, one can consider a particular example of a pressing experiment, set
up thus that the mathematical formulation be kept as simple and as instructive as
possible. For this purpose, consider the model system schematically depicted in
Fig. 2. A sample of plant tissue is supported by a porous support allowing the
passage of liquid only, not that of solids, and pressure is applied to the tissue at the
top surface in the negative z-axis direction according to this particular choice of a
coordinate system.
In this model system, one is interested in determining, theoretically, the pressure
relations for all times t during pressure application and throughout the tissue sample,
i.e., for all z. The problem has been limited to one spatial coordinate along the axis of
pressure application based on the assumption that due to sufficiently large surface
area perpendicular to the z axis of the tissue sample, the effects of liquid displace-
ment in the x and y directions can be neglected. In general, however, and in cases of
strong anisotropy of the material (tissue), it should be noted and considered that the
problem is three-dimensional.
Fig. 2 A simplified schematic representation of plant (sugar beet tap root) tissue exposed to
pressure applied at its top surface while resting on a porous support at its bottom surface; a
model system representing liquid expression from tissue and consequent tissue consolidation.
The magnifying glass is in aid of illustrating the microscopic structure of the material under pressure
Fig. 3 A simplified schematic representation of tissue highlighting the extracellular space in

tissue following electroporation. The dashed line used to represent the membrane of the cell in the
middle is in aid of representing the permeabilized state of this cell membrane after electroporation
Based on the established filtration-consolidation theory of bio-solids (Lanoiselle

et al. 1996; Petryk and Vorobiev 2007, 2013), the first constitutive equation of the
model can be written by observing the law of mass conservation. In the extracellular
space (depicted in blue in Fig. 3), the rate of change in porosity (liquid-to-total-
volume ratio) is proportional to the rate at which the liquid is vacating the extracel-
lular space (the extracellular liquid flow velocity) under pressure (outflow), as well
as to the rate at which intracellular liquid is flowing through the porous cell
membrane into the extracellular space (inflow). The relation just narrated by the
Fig. 4 A simplified schematic representation of tissue highlighting the intracellular space in tissue
following electroporation. As in Fig. 3, the dashed line used to represent the membrane of the cell in
the middle is in aid of representing the permeabilized state of this cell membrane after
electroporation
preceding sentence can be written more succinctly in the form of a mathematical

equation, yielding
@ ðρee Þ @ ðρqe Þ
¼ þ ρυie (1)
@t @z
In Eq. 1, which is a first-order partial differential equation, ρ is the liquid medium
density; ee the extracellular space porosity; qe is the extracellular liquid flow veloc-
ity; and υi-e is the source term that represents the flow of liquid through the cell
membrane from the intracellular into the extracellular space.
Considering pressure relations in the intracellular space (depicted in red by
Fig. 4), one arrives to the second constitutive equation of the dual-porosity model.
Again, by observing the mass conservation law, this yields
@ ðρei Þ @ ðρqi Þ
¼ ρυie (2)
@t @z
In the above Eq. 2, ρ is, as before, the liquid medium density; ei the intracellular
porosity; qi the liquid flow velocity in the intracellular space; and υi-e is the same
source term as in Eq. 1 that represents the flow of liquid through the cell membrane
from the intracellular into the extracellular space. Note, however, the change of sign
from positive to negative in the second term on the right-hand side of Eq. 2 as
compared to Eq. 1. The transmembrane flow is depleting the intracellular space of
liquid, therefore its negative sign (it is an outflow from the intracellular space
perspective).
According to estimates that can be found in literature, the permeability of

extracellular space is several orders of magnitude greater than that of intact cellular
membrane (Tomos 1988; Buttersack and Basler 1991). This should still be a valid
assumption under treatment conditions resulting in tissue damage
(by electroporation) below the threshold that results in, predominantly, irreversible
permeabilization of the cell membranes. If treatment conditions do favor the impor-
tance (in the sense of a limiting factor) of the membrane permeability over that of the
extracellular space, then the bulk liquid pathway will be primarily from within the
cells into the extracellular space and by filtration through the extracellular space out
of the tissue block. Assuming constant juice density ρ, and the supposition that finite
rate of flow inside the cells can be neglected, Eqs. 1 and 2 simplify to
@ee ðz, tÞ @q ðz, tÞ

¼ e þ υie ðz, tÞ (3)
@t @z
@ei ðz, tÞ
¼ υie ðz, tÞ (4)
@t
The above set of equations needs to be reformulated to express the quantities in
terms of variables common to both equations with known initial and boundary
conditions. These two unknowns are the liquid pressures pi and pe, where pressure
pi is the intracellular liquid pressure and pe the extracellular liquid pressure.
On Porosity, Void Ratio, and the Compressibility Modulus

Porosities ee and ei featured in Eqs. 3 and 4 are related to solid pressures pe,S = PE – pe
and pi,S = PE – pi via the compressibility moduli Ge and Gi and the void ratios ee and
ei, where ee = ee/(1 + ee) and ei = ei/(1 + ei). The relationships between void ratios
and solid pressures given by the following equations are taken from literature where
the multiple-porosity modeling principle has been applied to the problem of pressing
of oleaginous seeds (Lanoiselle et al. 1996). These relationships are given as
@ee @ee @pe, S @ee @pe 1 @pe

¼ ¼ ¼ (5)
@t @pe, S @t @pe, S @t Ge @t
@ei @ei @pi, S @ei @pi 1 @pi

¼ ¼ ¼ (6)
@t @pi, S @t @pi, S @t Gi @t
In Eqs. 5 and 6, pe,S and pi,S are the pressures of total insoluble solids in the
extracellular and intracellular space, respectively. In case of constant-pressure
expression, i.e., if @PE/@t = 0, the solid pressures increase in time proportionally
to the decrease in respective liquid pressures, i.e., @pe,S/@t = @pe/@t and @pi,S/
@t = @pi/@t. From Eqs. 5 and 6, it is obvious that Ge and Gi, if they can be assumed
time- and space-invariant, can be estimated by observing that Ge = @pe,S/@ee and
Gi = @pi,S/@ei. This means that compressibility moduli estimates can be based on
the slope of the linear function relating the decrease in void ratio with an increase in
solid pressure in each respective space. In experiments, such as illustrated in Fig. 7,

one thus measures the changes in deformation of the tissue sample due to loss of
liquid, rather than measuring the liquid pressure itself. Consequently, the fundamen-
tal relationship between tissue deformation and loss of liquid pressure should be
established by rewriting Eqs. 5 and 6 not for the liquid-to-solid void ratio, but rather
for porosities ee and ei, thus
@ee @pe, S @ee @p @ee 1 @pe

¼ ¼ e ¼ (7)
@t @t @pe, S @t @pe, S Ge, e @t
@ei @pi, S @ei @p @ei 1 @pi

¼ ¼ i ¼ (8)
@t @t @pi, S @t @pi, S Ge, i @t
The compressibility moduli Ge and Gi as defined in cited literature and Gε,e and
Gε,i as defined by Eqs. 7 and 8 are related, as follows from the following relations

@ee @pe, S @ ee @p @ee 1 1 1 @pe
¼ ¼ e ¼
@t @t @pe, S 1 þ ee @t @pe, S ð1 þ ee Þ2 Ge ð1 þ ee Þ2 @t
(9)
1 @pe
¼
Ge, e @t

@ei @pi, S @ ei @p @ei 1 1 1 @pi
¼ ¼ i ¼
@t @t @pi, S 1 þ ei @t @pi, S ð1 þ ei Þ 2 Gi ð1 þ ei Þ @t
2
(10)
1 @pi
¼
Ge, i @t
According to expressions given by Eqs. 9 and 10, Gε,e = Ge ∙ (1 + ee) 2 and

Gε,i = Gi ∙ (1 + ei) 2. Note, however, that relations ee = ee/(1 + ee) and ei = ei/
(1 + ei) require these quantities to be intrinsic (properties of each respective space)
as opposed to bulk properties that are featured in the dual-porosity model equations,
Eqs. 3 and 4. Further explanation of this issue can be found in subsection “Cell
Volume Fraction and Implications of Its Omission.” By introducing compressibility
moduli defined through porosity, a simplification has been made that comes at a cost,
a trade-off opted for in order to keep the model simple and comprehensive. Conse-
quently, the model results are valid for small piston displacements in cases where
tissue is not severely damaged. The moduli Gε,e and Gε,i that were introduced should
be understood as space- and time-averaged values. The approximate values of
compressibility moduli can be directly estimated from experiments (see subsection
“Estimating Compressibility Moduli Gε,e and Gε,i”), and average values are then
determined based on fitting model results to experimental data.
Another simplification limiting model applicability to small piston displacements
is the assumption of linear elastic deformation of tissue. Compressibility moduli Gε,e
and Gε,i are, more strictly following definitions of thermodynamics and stress
mechanics, in fact non-normalized bulk elastic moduli. A more in-depth and
rigorous treatment in filtration-consolidation theory would also require introduction

of material coordinates (see e.g., Petryk and Vorobiev 2013) to account for time- and
space-variable porosity, i.e.,
dzm ¼ ð1 eÞ dz (11)
Cell Volume Fraction and Implications of its Omission

There is also another important simplification in the model constitutive equations
that requires some attention, and that is the omission of the cell volume fraction.
Plant tissues normally comprise more than 50 % cells, sometimes – as for instance in
the case of fresh sugar beet taproots with high turgidity – much more than 90 %
(close to 97–98 %). Constitutive equations 3 and 4 still hold without accounting for
this large cell volume fraction, but only if all quantities are understood as bulk tissue
properties, i.e., normalized to the entire tissue volume, rather than intrinsic proper-
ties, which would be normalized to the volume fraction of each respective space
(intra- or extracellular). Introduction of the phenomenologically derived compress-
ibility moduli Gε,e and Gε,i has the advantage of avoiding the necessity to explicitly
account for the cell volume fraction. This is advantageous due to the experimental
estimation of parameters Gε,e and Gε,i (refer to subsection “Estimating Compress-
ibility Moduli Gε,e and Gε,i”), which are easier to arrive at, as compared to estimates
on effective cell volume fraction (note that due to irreversible electroporation, cell
volume fraction is a function of treatment intensity).
A noteworthy downside of this omission in the approach using bulk tissue
properties instead of intrinsic quantities is that the literature-based estimate of the
hydraulic permeability coefficient determining extracellular liquid flow rate ke is
likely to require an augmentation in order to arrive at its realistic value. In other
words, if only a few percent of the total tissue volume is available for extracellular
liquid flow while model equations feature bulk-volume normalized parameters, one
can expect the estimated intrinsic value of ke will need to be scaled accordingly.
Another consequence of working with bulk tissue properties is theoretical; the
relations ee = ee/(1 + ee) and ei = ei/(1 + ei) are written for intrinsic properties of
tissue, and by consequence, relations in Eqs. 5, 6, 7, 8, 9, 10, 11, and 12 must also be
understood as written using intrinsic properties. If one would wish to work with
material compressibility moduli Ge and Gi and void ratios ee and ei, one would also
have to account for the cell volume fraction. This point is a current challenge in the
dual-porosity model development that will be addressed by a future, expanded, and
improved formulation of the model.
Constitutive Equations Revisited – Darcy Law and the Final Model

Formulation
Turning the attention to the right-hand side of Eq. 3, the liquid flow velocity in
extracellular space qe is given by Darcy law as
ke @pe
qe ¼ (12)
μ @z
if assuming unidirectional flow in the direction of the principal axis of applied

pressure (z).
The source term υi-e(z,t) representing liquid flow through the porous membrane
can be written in terms of local pressure difference between the intracellular and
extracellular pressure, resulting in
α
υie ¼ ðpi pe Þ (13)
μ
Wherein, the proportionality coefficient α is first introduced, the estimation of which
is discussed in the following subsection.
Combining Eqs. 3, 4, 5, 6, 7, 8, 9, 10, 12, and 13 while dropping the notation of
spatial-temporal dependency of both liquid pressures gives the following final form
of the dual-porosity model equations

1 @pe @ ke @pe α
ð pi pe Þ ¼ 0 (14)
Ge, e @t @z μ @z μ
1 @pi α
þ ð p pe Þ ¼ 0 (15)
Ge, i @t μ i
Note the absence of a spatial derivative in Eq. 15. The above system of equations
is no longer a system of two coupled partial differential equations as was the system
Eqs. 3 and 4, but rather a coupled integrodifferential system with constant coeffi-
cients. Such systems admit readily obtainable analytical solutions. What remain
missing in order to obtain a particular analytical solution are the appropriate initial
conditions and the boundary conditions for Eq. 14.
The initial and boundary conditions for the system of Eqs. 13 and 14 follow from
the particular system setup and choice of coordinate system (refer to Fig. 2) and are
pe0 ¼ pe0 ðz, 0Þ ¼ PE (16)
pi0 ¼ pi0 ðz, 0Þ ¼ PE (17)

@pe
¼0 (18)
@z z¼h
pe jz¼0 ¼ 0 (19)
The boundary conditions in Eqs. 18 and 19 are known as mixed or Neumann-

Dirichlet boundary conditions. They require some attention and care when
assembling the finite difference matrices in the formulation of the numerical solu-
tion, which this chapter, however, does not concern.
A note on initial condition, Eqs. 16 and 17. Externally applied pressure can be
assumed as equally distributed throughout the tissue sample on both the intra- and
extracellular liquid phase in case the sample thickness is relatively small
(in relation to the number of cell layers and piston-tissue contact surface). If the
sample thickness impact to pressure distribution cannot be neglected due to
sample dimensions, a more suitable approximation for pressure distribution
might be linear, for details, see, e.g., Lanoiselle et al. (1996). In experiments
illustrated by Fig. 7, the conditions (use of thin samples) justify the applicability
of this initial condition. One can also suppose that external pressure PE redistrib-
utes itself equally onto the liquid phase of both the extracellular ( pe0) as well as
the intracellular space ( pi0) at the beginning of a pressing experiment, after the
extracellular air is eliminated and replaced by liquid at the beginning of the
consolidation stage.
From Liquid Pressure to Observable Parameters – The Liquid Pressure

Loss Integration and Tissue Deformation/Consolidation
In pressing experiments, as set up as shown schematically in Fig. 7, the observed
quantity is the deformation of a sample block of tissue. Since the dual-porosity
model is describing the dynamics of liquid pressure loss in the extracellular and
intracellular space, an expression giving deformation as a function of the cumulative
change of pressure throughout the sample must be found in order to enable compar-
ison of model results with those of experiments. The relationship between loss of
liquid pressure and deformation is already given in Eqs. 5 and 6.
Since the total deformation is the sum of deformations of the extracellular and of
the intracellular space, the combined total absolute deformation equals
SðtÞ ¼ Se ðtÞ þ Si ðtÞ; (20)
Total deformation can be expressed as a spatial integral of local infinitesimal

differences in void ratio e, therefore
ð h ð ee ðz, 0Þ ð h ð ei ðz, 0Þ ð h ð pe ðz, 0Þ ð h ð pi ðz, 0Þ
1 1
Sð t Þ ¼ dee dzþ dei dz ¼ dpe dzþ dpi dz:
0 ee ðz, tÞ 0 ei ðz, tÞ Ge 0 pe ðz, tÞ Gi 0 pi ðz, tÞ
(21)
However, since compressibility moduli were defined through porosity e instead of
void ratio e, and additionally for reasons of convenience, the relative deformation sε
can be introduced and is defined as
ð 1 ð pe ðz, 0Þ ð 1 ð pi ðz, 0Þ
Se ðtÞ 1 1
se ðtÞ ¼ ¼ dpe dz þ dpi dz (22)
h Ge, e 0 pe ðz, tÞ Ge, i 0 pi ðz, tÞ
where h is the initial tissue sample height. Equation 22 gives relative deformation as
a function of loss of liquid pressure within the tissue. It can be used to obtain model
results from simulated pressure loss profiles, enabling the comparison of the
resulting kinetics with experimental data.
The Hydraulic Permeability of the Electroporated Biological

Membrane and Estimation of Model Parameters
Estimating a – The Hydraulic Permeability of an Electroporated

Biological Membrane
The proportionality coefficient α is a dimensionless parameter relating the intracel-
lular and extracellular deformation due to transmembrane flux with the pressure drop
across the plasma membrane. In accordance with model design and assumptions, it is
a function of membrane permeability ki, multiplied by a corrective geometrical factor
ξ with unit of m2. The corrective factor ξ accounts for the geometrical configuration
of the cell and its porous membrane by relating intracellular space porosity with
volume-averaged (bulk) transmembrane flux (further explanation can be found in the
Appendix of Mahnič-Kalamiza and Vorobiev 2014). For negligible membrane
thickness as compared to the size of the cell, ξ equals the square of specific surface
(surface-to-volume ratio), i.e., ξ = (A/V ) 2. On the level of a biological cell, where
transmembrane fluid transport occurs, the surface A and volume V are those of a
single cell. For an idealized, average, spherical cell of a given radius R, the factor ξ
can be calculated as
9
ξ¼ (23)
R2
Turning now to the effect of electroporation treatment on the hydraulic per-
meability coefficient ki, it should be noted that according to the theory of
electroporation, electric field of sufficient strength increases the permeability of
the plasma membrane. The effect has been attributed to the creation of aqueous
pathways or pores in the membrane. These pores nucleate at some initial radius
and can expand in both number and size during the application of the electric
field. The effect has a transient as well as a long-lasting component, i.e., transient
and long-lasting pores are created in the membrane (Pavlin and Miklavcic 2008).
It has been demonstrated by experiment, see, e.g., Saulis and Saule (2012), that
long-lasting pores permeable to molecules of considerable molecular weight
(tens of kDa) can exist in an electroporated membrane for minutes after the
application of electric pulses, though they are subject to resealing under favorable
physiological conditions. One can, assuming an average stable pore diameter and
pore fraction ratio (i.e., the surface fraction of all pores per one cell), theoretically
estimate how electroporation changes the hydraulic permeability of the cell
membrane.
The derivation of the sought relation starts by first relating membrane permeabil-
ity ki with permeability of a single aqueous pore. The absolute value of membrane
flux is, according to Darcy law,
ki Am Δp
jQ m j ¼ (24)
μ l
However, since the total membrane flux is also the sum of all single-pore fluxes, of
which there are as many as there are pores (i.e., Np), it is possible to write
N p kp Ap Δp
jQm j ¼ N p Qp ¼ (25)
μ l
From equating transmembrane flux in Eqs. 24 and 25 follows the relation
N p k p Ap
ki ¼ ¼ f p kp (26)
Am
where fp = NpAp/Am is the pore surface fraction.
Some estimates on the surface fraction of long-lasting pores can be encountered
in literature (Pavlin and Miklavcic 2008); however, these estimates are based on
experiments on animal cells in vitro and use of a particular treatment protocol
commonly used in biomedical applications of electroporation. As extrapolation of
these results to plant tissues is difficult, one can, as a general guideline, assume that
due to larger plant cells as compared to animal cells and more intense (in the sense of
number, amplitude of pulses, delivered energy) treatment protocols used in plant
tissue electroporation, the corresponding pore surface fraction will be higher than in
experiments reported on in the literature. Since according to the electroporation
theory, pore surface fraction is strongly dependent on induced transmembrane
voltage, more work is needed to help determine the most important parameters
that describe membrane long-term permeability with respect to the treatment
protocol.
The only remaining uncharacterized parameter from Eq. 26 that requires an
estimation is the single pore intrinsic hydraulic permeability kp. To that end, the
Hagen-Poiseuille equation for cylindrical pores of length l and radius rp is employed
in combination with Darcy law. This yields
8μlQp 8μlQp μlQp

Δp ¼ ¼ 2 ¼ (27)
πr p 4 r p Ap k p Ap
Based on Eq. 27, kp can be expressed as rp2/8. The estimate on the radius of an
average stable pore rp can be obtained from literature or some form of special
experiment. Moreover, it allows for coupling of advanced theoretical models of
electroporation (pore formation and evolution – see, e.g., Smith et al. 2004;
Krassowska and Filev 2007) that give estimates on temporal and spatial distribution
of a population of pores with a statistical distribution of radii, to the dual-porosity
model, which is an interesting prospect for future model development.
Estimating Compressibility Moduli G«,e and G«,i

To estimate the compressibility moduli, one can conceptualize two experimental
scenarios. In the first experiment, freshly cut intact tissue is subjected to pressing
under pressure of insufficient strength to cause cell rupture by itself. Under these
conditions, an eventual equilibration between the pressure applied via the piston and
the intracellular liquid pressure can be observed. When this equilibrium is reached,
tissue sample stops deforming, and the sample sustains a certain measurable,
however small, deformation. If the pressure applied is sufficiently large to
completely express extracellular fluid while simultaneously not compromising the
integrity of the cell plasma membrane, this measured deformation is only due to the
compression of extracellular space. The mathematical formulation of this reads
ð 1 ð pe ðz, 0Þ ð 1 ð PE
1 1 PE
s e, 1 ¼ s e ð t ! 1 Þ ¼ dpe dz ¼ dpe dz ¼ (28)
Ge,e 0 pe ðz, t!1Þ Ge,e 0 0 Ge,e
Equation 28 provides means of estimating Gε,e directly from pressing experi-

ments performed using intact, untreated (non-electroporated) tissue. With known
deformation at “infinite” time (complete equilibrium) and known applied pressure
PE, it is estimated using
PE
Ge,e ¼ (29)
se, 1
Since Gε,e is a function of the void ratio, which is not constant in time (and is only
approximately constant throughout the tissue block along z, provided the sample is
thin), the value obtained by Eq. 29 is a rough initial estimate and a good approxi-
mation for untreated tissue. It is expected to decrease with increasing treatment
intensity, since it is not a true material property but depends on void ratio and the
effective cell volume fraction (as established in subsections “On Porosity, Void
Ratio, and the Compressibility Modulus” and “Cell Volume Fraction and Implica-
tions of Its Omission”). Its average value must be determined by optimization
against experimental kinetics.
The second conceptual experiment can be proposed for estimating Gε,i. If the cell
membranes are permeabilized (by e.g., electroporation), liquid will flow from the
intracellular to the extracellular space due to applied pressure and through the
extracellular space out of the tissue block. At complete equilibrium (i.e., after
“infinite” time), all of the liquid will be expressed from the sample, and the
externally applied pressure will be balanced by the sum of solid pressures of the
intracellular and the extracellular space. This is written
ð ð ð ð
1 1 pe ðz, 0Þ 1 1 pi ðz, 0Þ
s1 ¼ se ðt ! 1Þþsi ðt ! 1Þ ¼ dpe dzþ dp dz ¼ ...
Ge,e 0 pe ðz, t!1Þ Ge,i 0 pi ðz, t!1Þ i
ð ð ð ð
1 1 PE 1 1 PE PE PE PE Ge,i þ Ge,e
¼ dpe dzþ dpi dz ¼ þ ¼
Ge,e 0 0 Ge,i 0 0 Ge,e Ge,i Ge,e Ge,i
(30)
If Gε,e is known, e.g., determined previously according to Eq. 29, and deforma-
tion in an experiment is measured when using strongly permeabilized tissue, by
expressing Gε,i from Eq. 30, one obtains
PE Ge, e
Ge, i ¼ (31)
s1 Ge, e PE
which is a function of either previously known (estimated) or measurable parame-

ters. This estimate gives an approximate value for Gε,i in case of damaged tissue. The
average value for untreated or only moderately electropermeabilized tissue is
expected to be much higher, as higher means less compressible. Similarly to Gε,e,
the value corresponding to the particular degree of electropermeabilization can be
determined by obtaining an agreement between model results and experimental data.
Estimating the Intrinsic Hydraulic Permeability Coefficients ke and ki

Hydraulic permeability of tissue is almost always measured rather than calculated
(Buttersack and Basler 1991), due to high complexity of water pathways within
tissue that makes theoretical estimates hard to obtain and the biological diversity,
which renders these estimates unreliable across different plant species and even
across samples of a single species. Measurements on a number of plant tissues and
yeast cells have shown a wide range of permeability, spanning several orders of
magnitude, for both tissue as well as plasma membrane of individual cells (Tomos
1988; Buttersack and Basler 1991). It is nevertheless possible to find several
accounts of measurement of hydraulic conductivity (Lp) of industrially important
plant tissues in literature. The following derivations demonstrate how it is possible to
recalculate these measurements in order to estimate the intrinsic hydraulic perme-
ability coefficients required by the dual-porosity model.
The hydraulic conductivity Lp found in literature is normally calculated based on
an experiment where a tissue sample is subjected to a pressure difference and liquid
volume flux is measured, an experiment which, conceptually, does not significantly
differ from the one represented in Fig. 2. With known flux and pressure, the
hydraulic conductivity is
q
Lp ¼ (32)
Δp
On the other hand, the Darcy law relates the pressure drop across a conduit of
length l with the rate of liquid flow through it, q, as
k Δp
q¼ (33)
μ l
Inserting q from Eq. 32 into Eq. 33 and expressing k gives
k ¼ Lp μ l (34)
Equation 34 can be used to calculate the intrinsic hydraulic permeability of tissue

from measurements obtained via experiments as described above. Assuming negli-
gible symplastic flow (i.e., only apoplastic), this permeability is an estimate of the
hydraulic permeability of extracellular space, ke.
The intracellular hydraulic permeability ki is in fact the hydraulic permeability of
the plasma membrane of thickness l. This coefficient is expected to change when
treatment, be it electrical, mechanical, thermal, chemical, enzymatic or other, is applied
to the tissue. Its initial value (i.e., for untreated tissue) can be estimated from pressure-
probe experiments. Tables of cell membrane hydraulic permeability can be found in
literature on plant physiology for many plants and some yeast species as well.
The Analytical Solution of the Model
The particular solution of the integrodifferential system of Eqs. 14 and 15 for initial
and boundary conditions Eqs. 16, 17, 18, and 19 can easily be obtained, for instance,
by employing the method of separation of variables. A detailed derivation is given
for a mathematically completely analogous case of the dual-porosity model applied
to the problem of solute diffusion in electroporated tissue (found in Mahni-
č-Kalamiza et al. 2014a). The final model equations for the pressing case are almost
identical to the ones obtained for the corresponding solution of the diffusion
problem, after appropriate replacements of parameters have been made. The few
additional exceptions to this analogy are: (i) sine replacing the cosine in the Fourier
series and (ii) a required update in the eigenvalues due to different boundary
conditions.
For extracellular and intracellular liquid pressure, the final equations are,
respectively,

4pi0 X
1
1 γ n, 1 t
γ n, 2 t
ð2n þ 1Þπ
pe ðz,tÞ ¼ γ τ þ 1 C1 e þ γ n, 2 τ þ 1 C 2 e sin z
π n¼0 2n þ 1 n, 1 2h
(35)

4pi0 X
1
1 γ n, 1 t γ n, 2 t τ1 t
ð2n þ 1Þπ
pi ðz, tÞ ¼ C1 e þ C2 e e sin z
π n¼0 2n þ 1 2h
1
þ pi0 eτ t
(36)
where

pe0
1 τ1 γ n, 2
pi0
C1 ¼ (37)
γ n, 1 γ n, 2

pe0 1
1 τ þ γ n, 1
pi0
C2 ¼ (38)
γ n, 1 γ n, 2
and
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2 ffi
τ1 δ þ λn 2 ν τ1 δ þ λn 2 ν 4λn 2 ντ1
γ n1, 2 ¼ (39)
2
For the sake of algebra throughout Eqs. 35, 36, 37, 38, and 39, the following
replacements were made:
ke Ge, e
ν¼ (40)
μ
αGe, i
τ1 ¼ (41)
μ

Ge, e
δ¼ 1þ (42)
Ge, i
and the eigenvalues λn equal
ð2n þ 1Þπ
λn ¼ (43)
2h
Simulated Liquid Pressure Distributions and Consolidation

Kinetics
Employing Eqs. 35, 36, 37, 38, 39, 40, 41, 42, and 43 as given in the preceding
subsection yields pressure distribution profiles throughout the tissue sample for all
times of the simulated experiment. An example of these profiles along the axis of
pressure application is shown in Fig. 5 for six different times taken from the interval
between 0 and 45 min of the simulated experiment.
There is a marked difference in pressure profiles when comparing the calculated
pressure in the extra- with that in the intracellular space. In order to produce these
pressure profiles, the value of the parameter reflecting the degree of permeabilization
Fig. 5 Extracellular (a) and intracellular (b) liquid pressure distributions along the principal axis of
pressure application for different times of the simulated experiment
of the membrane, i.e., the surface fraction of pores, has been chosen from the range
yielding the membrane hydraulic permeability that is not on a comparable order of
magnitude with that of the extracellular space. As a result, there is a noticeable
hindrance effect of the plasma membrane to the liquid flow, and it is evident from the
comparison of pressure profiles that liquid is being retained within the cells for a
longer period while the extracellular space is vacated at a greater rate. The intracel-
lular liquid nevertheless continues replenishing the extracellular space with liquid,
and the resulting final extracellular liquid amount at the end of the simulated
experiment (at t = 2700 s in this case) is still greater as compared to that which
would have been remaining in tissue were it not for the outflow of intracellular juice
from the intra- into the extracellular space.
The pressure profiles shown in Fig. 5 are then integrated according to Eq. 30 to
produce final relative consolidation (i.e., sample deformation) kinetics, an example
of which is shown in the right-hand side of Fig. 6.
The pressure profiles in the intra- and extracellular space that are integrated,
scaled by the compressibility moduli, and then summed to yield the cumulative
tissue deformation therefore determine the dynamics of tissue compaction under
external pressure. The membrane permeability will determine the particular dynam-
ics of this deformation (the shape of the consolidation kinetics), while the com-
pressibility moduli, still partly phenomenological in nature in the presented version
of the dual-porosity model, will determine both the extent of the initial, rapid
deformation (see kinetics in Fig. 6 in range of 0–500 s) as well as of the final or
total attainable extent of compression of the tissue. In this respect, the compress-
ibility moduli are a reflection of the dewaterability of the tissue, and since
dewaterability is dependent on the degree of permeabilization of the membranes
in bulk tissue, these parameters will also depend on the parameters of the specific
electroporation protocol applied.
1172
Fig. 6 Annotated simulated consolidation kinetics, obtained from liquid pressure distributions as shown in Fig. 5 and according to the integration Eq. 30
S. Mahnič-Kalamiza
Model Verification and Application by Example
To demonstrate how the model can be applied, an experiment has been proposed as
schematically shown in Fig. 7, whereby a tissue sample is first exposed to treatment by
pulsed electric fields, then placed into a specially fabricated treatment chamber where
pressure is applied at the top surface and the bottom support is porous, creating a system
as depicted by Fig. 2. This system can be described and analyzed with the dual-porosity
model, and measured kinetics of tissue consolidation (and thus liquid expression) can be
compared against simulated kinetics obtained from the dual-porosity model.
Initial model parameter values are based on literature or experimental estimates as
described in subsection “The Hydraulic Permeability of the Electroporated Biological
Membrane and Estimation of Model Parameters”; however, in order to obtain a good
agreement between experimental and modeled kinetics, these parameters were allowed
to vary within an acceptable range and their exact values were determined in the loop of
an optimization algorithm. The optimization that ensures the model kinetics fit the
experimental consolidation curves is also necessary due to a lack of reliable theoretical
means of estimating such parameters as pore surface fraction, hydraulic permeability of
the plasma membrane (for treated and intact tissue), and extracellular hydraulic per-
meability (note the issues with estimating these parameters noted in subsections “Cell
Volume Fraction and Implications of Its Omission” and “The Hydraulic Permeability of
the Electroporated Biological Membrane and Estimation of Model Parameters”).
It could be argued that due to the high number of parameters that are allowed to
vary and are determined empirically, the model thus applied detracts from its
scientific rigor. However, although the optimal model parameter values are sought
Fig. 7 A schematic diagram of the experiment designed for purposes of verifying the model
for in a multiparametrical sweep with a high number of degrees of freedom, the

parameter values that are determined in this way are then carefully examined and
analyzed in light of the electroporation theory and the assumptions stemming from
the process of model construction. The necessary changes to parameter values
should behave according to the type of tissue that is experimented with, and thus
reflect the characteristics of a particular treatment protocol used. As an example,
during model construction, an implicit supposition was made that compressibility
moduli defined through bulk porosity of each respective space will depend on
treatment intensity, due to the (variable) fraction of cells that are reversibly and
irreversibly electroporated. Moreover, the effective average pore surface fraction ( fp)
should increase non-linearly and experience a sharp sigmoid dependence on treat-
ment intensity (e.g., on applied voltage). These relationships between material and
treatment properties and the parameter values that are obtained from the model after
fitting to experimental data, if well established, are what verifies the dual-porosity
model theory and invites further refinement and improvement of the model with the
prospect of eventually arriving at a truly mechanistic model.
Figure 8 illustrates the results of a model study designed for the purposes of dual-
porosity model verification. Experiments were performed according to the procedure
shown in Fig. 7 using two types of tissue, apple fruit and sugar beet tap root tissue. In
experiments on each type of tissue, only one parameter – the applied voltage to the
electrodes – was varied, and consolidation kinetics was recorded. The second part of
the study involved running dual-porosity model simulations using estimated values
of parameters that were allowed to vary within a predefined range that was deemed
acceptable. Figure 8 demonstrates the good agreement that can be obtained between
model (solid or dashed lines) and experimental (depicted by symbols) consolidation
kinetics. What is more important is the dynamics of parameter value changes that
were required to obtain this agreement if comparing the two different materials and
the six treatment intensities. A detailed commentary on these results and particular
parameter values is outside the scope of this theoretical introductory work and is
given in full in Mahnič-Kalamiza et al. (2014b).
In summary, the model study provides a means of identifying important param-
eters in the dual-porosity model that most strongly depend on material properties and
those that are functions of PEF treatment intensity. These findings can help with
directing future research and model development, and the dual-porosity model of
mass transport in tissue can be considered a promising platform for future study of
electroporation effects on both tissue structure and mass transport properties.
Conclusions
This chapter gives a detailed presentation of the dual-porosity model, which has been
developed with the intent of modeling and studying filtration-consolidation behavior
of electroporated plant tissues. The paradigm it introduces is that of regarding
Fig. 8 Simulated (lines) and experimental (markers) consolidation kinetics for two model tissues;
apple fruit (a) and sugar beet tap root (b) tissue
biological tissue as essentially a two-compartment, two-space, or two-component

material of distinct porosity and hydraulic permeability. The objective is to introduce
the concept of a membrane with variable hydraulic properties into a model of mass
transport in tissue, thus enabling the coupling of membrane-level electroporation
effects to the resulting bulk tissue mass transport processes. More work remains
necessary to help determine the most important parameters that describe membrane
long-term permeability with respect to the treatment protocol. Nevertheless, the
model can at present already be used for evaluation of treatment parameters’ impact
to liquid flow in electroporated plant tissues, but perhaps more importantly, it invites
future refinement and development for purposes of further elucidating basic principles of
pulsed electric field-induced phenomena in biological tissues.
Cross-References
▶ Determination of Pulsed Electric Fields Effects on the Structure of Potato Tubers

▶ Electroporation-Based Applications in Biotechnology
▶ Mathematical Models of Pulsed Electric Field Treatment of Plant Tissues and
Treatment
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Computational Approach for Electrical
Analysis of Biological Tissue Using the 62
Airton Ramos and Daniela O. H. Suzuki
Abstract
The equivalent circuit method is a computational approach for electrical analysis
in which the properties of the medium are modeled using circuit elements such as
conductances and capacitances. Its main application is in the electric field calcu-
lation in biological tissues stimulated by electrical potentials applied with metal
electrodes in contact with the material. This method allows modeling easily
inhomogeneous materials that have dielectric dispersion, anisotropy, and
nonlinear electrical behavior. These characteristics are typical of biological tis-
sues. This chapter presents the mathematical foundations of the equivalent circuit
method and illustrates its main features with a typical example of biological
stimulation for the purpose of cell membrane electroporation.
Keywords
Equivalent circuit method • Field calculation • Dielectric dispersion • Biological
tissues • Electroporation
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1180
Equivalent Circuit for a Nondispersive Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1181
Equivalent Circuit for First-Order Dispersion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1182
Dispersion Mechanism and Equivalent Circuit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1184
Numerical Analysis of the Equivalent Circuit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1188
Modeling Anisotropic, Inhomogeneous, and Nonlinear Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1190
A. Ramos (*)
State University of Santa Catarina (UDESC), DEE/CCT, Joinville, Santa Catarina, Brazil
e-mail: airton.ramos@udesc.br; airton.ramos@hotmail.com
D.O.H. Suzuki (*)
Institute of Biomedical Engineering, Federal University of Santa Catarina (UFSC), IEB/EEL/CTC/
UFSC, Florianopolis, Santa Catarina, Brazil
e-mail: suzuki@eel.ufsc.br

DOI 10.1007/978-3-319-32886-7_12
1180 A. Ramos and D.O.H. Suzuki
Simulation of the Stimulation Effects with Metal Electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1197
Cross‐References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1197
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1198
Introduction
The use of computational approaches to solve electromagnetic problems with

complex geometries and properties of the materials involved has become common
practice in recent decades due to the great development of equipment and software.
In the case of biological materials, electromagnetic analysis presents several diffi-
culties that make the computational analysis practically indispensable. In addition to
the typical inhomogeneity of biological materials, the following important complex
characteristics may be mentioned: biological tissues have strong dielectric dispersion
in a wide range of frequencies extending from zero to hundreds of megahertz;
biological tissues generally exhibit anisotropy in conductivity and permittivity;
biological tissues can present nonlinear electrical behavior as a result of heating or
cell membrane electroporation.
Among the various numerical methods for electromagnetic analysis, the most
commonly used for biological materials are finite difference method (FDM) and
finite element method (FEM). The first one has been widely used in the calculation
of the absorbed energy (specific absorption rate) for biological materials especially
in the range of radio frequencies (Chen and Gandhi 1992; Zhao et al. 2013). In FDM
approach, Maxwell’s equations are solved to obtain the electric and magnetic fields
distributions in the space discretized with a regular grid of cubic elements.
The finite element method is the most used in the field calculation in planning
therapies based on biological electroporation: Electrochemotherapy (Corovic
et al. 2013; Kos et al. 2010; Miklavcic et al. 2010), irreversible electroporation
(Neal et al. 2012; Daniels and Rubinsky 2009), and gene transfer (Lacković
et al. 2009; Županič et al. 2010). In the finite elements approach, the geometry
under the study is discretized by triangular and tetrahedral elements. This provides
greater power to model complex geometries than FDM.
Methods based on the modeling by electric circuit elements have also been
proposed and used in the analysis of biological materials in recent decades. In the
impedance method (IM), the medium is represented by a three-dimensional matrix of
impedances modeling the electrical properties of the medium. The analysis is
performed in the frequency domain using the phasor method (Nadeem et al. 2003;
Ramos 2010).
A similar approach is the equivalent circuit method (ECM) that is based on
modeling the dielectric relaxation through a network of conductances and capaci-
tances associated with a three-dimensional mesh of orthogonal parallelepipeds
(Ramos et al. 2003). In the ECM, field calculation is converted in the analysis of
an electric circuit in the time domain, so that through the Kirchhoff’s first law and
continuity equation, the electric potential and accumulated electric charge in each of
62 Computational Approach for Electrical Analysis of Biological Tissue. . . 1181
the mesh elements are calculated in small time steps. The ECM is particularly useful
in the analysis of electric process in biological materials, since generally they have
high permittivity, low electrical conductivity, and intense dielectric relaxation from
extremely low frequencies (ELF) to high frequencies (HF).
Equivalent Circuit for a Nondispersive Medium
The modeling using equivalent circuit is based on describing the characteristics of a

dielectric material through an electric circuit consisting of conductances and capac-
itances, which depict the properties associated with the electrical conduction and
polarization, respectively, within the material. Consider a small portion of a material
divided in two regular halves as shown in Fig. 1.
If the volumes are small, an approximate calculation of the conduction and
displacement currents can be done by considering uniform electric field in the two
volumes and uniform current density at the contact face between the elements. If the
portion of material involved in this model is homogeneous and linear in the electric
properties, the electric current can be calculated by the simple manner indicated in
Eq. 1:
A A d
inm ¼ σs ðV n V m Þ þ ε1 εo ðV n V m Þ (1)
L L dt
Where inm is the electric current, Vm and Vn are the electric potentials at the center of
elements m and n, respectively, A is the contact area, L is the distance between the
volume elements, σs is the static conductivity, ε1 is the high frequency dielectric
constant and εo is the vacuum permittivity. The first term is the conduction current
due to the movement of ions. The coefficient that multiplies the electric potential
difference is the conductance, whose value depends on the spatial dimensions of the
contact between the elements and the conductivity of the material. The second term
Fig. 1 Geometric model to

obtain the equivalent circuit:
Vn, Vm electric potentials in
the center of the volume
elements; inm electric
current; A, L area and
distance between the volume
elements, respectively
Fig. 2 Equivalent circuit for

a linear, homogeneous, and
nondispersive material: Vn,
Vm node potentials;
inm electric current; A,
L area and distance
between the volume elements;
Gs static conductance;
σs static conductivity;
C1 high-frequency
capacitance; ε1 high-
frequency dielectric constant;
εo vacuum permittivity
is the displacement current due to the rate of change of the electric flux inside the
elements. Therefore, this term is proportional to the derivative of the electric
potential difference in time. The proportionality factor is the capacitance which
depends on the physical dimensions and the dielectric constant of the material.
The symbols “s” and “1” in the conductivity and dielectric constant indicate that
the values are out of the frequency range in which the dielectric dispersion occurs.
The first indicates the static conductivity and the second indicates the high-frequency
dielectric constant.
For this simple system, the equivalent circuit can be constructed with a conduc-
tance and a capacitance connected in parallel, as shown in Fig. 2.
Equivalent Circuit for First-Order Dispersion
Biological tissues generally have strong dielectric dispersion in the frequency range
extending from zero to tens of megahertz mainly related to the space charge
polarization due to the accumulation and diffusion of ions on the surface of the
biological membranes, but also as a result of the dipolar relaxation of the water and
polar macromolecules in the tissue fluids. If the medium has dielectric dispersion, the
transient response of the electric current depends on the intensity of the dispersion
and the relaxation times of the related processes.
The electric polarization of a medium results in a reaction electric field that
opposes the applied electric field. Then, taking into account the effect of polariza-
tion, the change in current density can be described by the following equation:
jd ¼ σd ðE ER Þ (2)
Where jd and σd are increases in the current density and conductivity in relation to
the values considered in the Eq. 1. E is the applied electric field, and ER is the
reaction field. σd describes how the conductivity of the medium is affected by the
electrical polarization. At high frequencies, where the slow polarization processes no
Fig. 3 Equivalent circuit for a first-order dispersion: Vn, Vm node potentials; idnm dispersive
current; Gd, Cd dispersion conductance and capacitance, respectively; σd, εd maximum
increases in conductivity and dielectric constant caused by dispersion, respectively
longer occur, the reaction field becomes zero, and the conductivity σd adds to the
static conductivity of the medium. In a medium whose polarization is proportional to
the applied field, the reaction field should be proportional to the accumulated
electrical charge in the sample volume.
Since Eq. 2 refers to volumetric averaged values, the electric fields E and ER may
be easily associated to the potential difference in sample volume. The dispersive
current may then be described by the following equation:
A
id ¼ σd ðΔV ΔV R Þ (3)
L
Due to the linearity assumption in the polarization process, the reaction
potential can be written as being proportional to the accumulated electric charge
which, in turn, varies with time at a rate equal to the intensity of the dispersive
current:
0 1
ðt
1
id ¼ Gd @ΔV id dt0 A (4)
Cd
0
where Gd and Cd are the conductance and capacitance related to the dispersive
process. This mathematical model is represented by the equivalent circuit shown
in Fig. 3.
In order to obtain the dispersive current in the time domain to an arbitrary voltage
waveform, it can be performed an iterative calculation with small time steps “Δt”
compared to the relaxation time of the circuit τ = Cd/Gd. The potential difference
across the capacitor can be calculated from the following equation:
V c V 0c
Gd ðV n V m V c Þ ¼ Cd (5)
Δt
where Vc0 is the previous value of the potential in the capacitor, i.e., the potential at
time t0 = t Δt.
Vc can be obtained by solving Eq. 5:
ðΔt=τÞðV n V m Þ þ V c 0
Vc ¼ (6)
ð1 þ Δt=τÞ
The dispersive current is then obtained in the following manner:

ðV n V m Þ V c 0
idnm ¼ Gd ðV n V m V c Þ ¼ Gd (7)
ð1 þ Δt=τÞ
Dispersion Mechanism and Equivalent Circuit
In the conductivity and permittivity spectrum of biological tissues, it is generally

possible to distinguish two bands, named alpha and beta, in the frequency range that
extends from ELF to about 1 MHz. The physical mechanisms involved in the
dispersion processes are attributed to diffusion and accumulation of hydrated ions
on the surface of plasma membranes. The diffusion of counter ions in the double
layer around the cells, according to the model proposed by Schwarz (Schwarz 1962),
produces strong dielectric dispersion whose intensity and relaxation time depend on
the radius of the cell (R), ion mobility (μ), temperature (T), and concentration of
counter ions (no), according to the Eqs. 8 and 9 below (Schwarz 1962):
e2 Rno
Δεα (8)
εo K B T
e R2
τα (9)
2μKB T
where “e” is the proton electric charge and “KB” is Boltzmann’s constant. With these
formulas it can be estimated that the alpha dispersion in cell suspensions can produce
variations in the dielectric constant of the order of 105 with relaxation times of the
order of milliseconds.
Similarly, the accumulation of ions on the inner and outer surfaces of the plasma
membrane produces intense dielectric dispersion, which depends on the cell radius,
membrane capacitance (Cm), and internal (σi) and external (σo) conductivity
according to Eqs. 10 and 11 valid for spherical cells in suspension (Foster and
Schwan 1995):
9RCm
Δεβ (10)
4εo

1 1
τβ RCm þ (11)
σi 2σo
With these formulas it can be estimated that the beta dispersion produces varia-
tions in the dielectric constant of the order of 104 with relaxation times less than a
Fig. 4 Equivalent circuit of a

biological material including
first-order dispersions: Vn,
Vm node potentials;
inm total current;
is current in the static
conductance Gs; C1 high-
frequency capacitance; iα, iβ,
iγ dispersive currents; Gα,
Gβ, Gγ dispersion
conductances; Cα, Cβ,
Cγ dispersion capacitances
microsecond. The formulas (Eqs. 8, 9, 10, and 11) are strictly valid for dilute cells in
suspension but were taken as approximations for tissues when considering the
volume fraction very close to unity.
In addition to the alpha and beta relaxations, a third dispersive process with
relaxation time of tens to hundreds of nanoseconds typically shows significant
effects from 1 MHz to higher frequencies in biological tissues. Some different
mechanisms may contribute to this gamma dispersion. However, it is estimated
that the dipolar relaxation of protein or water molecules bound to proteins can be
the most important processes (Foster and Schwan 1995).
The circuit in Fig. 4 incorporates all processes described above. The C1 capac-
itance describes the polarization process with much lower response times than τα, τβ,
and τγ. Conductance Gs describes the conduction at very low frequencies. Dilute
biological cells suspensions often present relaxation process that can be well
modeled by the Debye equation (Foster and Schwan 1995):
_ Δε
ε ð ωÞ ¼ ε 1 þ (12)
1 þ jωτd
where ω is the angular frequency and j = (1)1/2.

Dense biological tissues, however, have different behavior. The irregular shape of
cells and interstitial spaces and the high density of interfaces result in dispersions that
may depart significantly from the Debye formula. In such cases the use of empirical
models involving continuous distributions of relaxation times, although with more
complex formulation, becomes an alternative. For example, one of the most used
models in the interpretation of spectral data in biological materials is the Cole-Cole
function (Cole and Cole 1941):
_ Δε
ε ðωÞ ¼ ε1 þ (13)
1 þ ðjωτo Þ1α
where α is a parameter whose value should be adjusted empirically (α < 1), and τo is
the central relaxation time of the distribution.
A dispersion function based on a continuous distribution of relaxation times can
be described as a linear combination of first order dispersions in which the coeffi-
cients are defined by a distribution function g(lnτ) as follows (Bottcher and
Bordewijk 1978):
ð
1
_ gðlnτÞ dðlnτÞ
ε ðωÞ ε1 ¼ Δεr (14)
1 þ jωτ
0
where g(lnτ) meets the normalization condition:
ð
1
gðlnτÞ dðlnτÞ ¼ 1 (15)

0
In the case of Cole-Cole dispersion, g(lnτ) is given by Eq. 16 (Bottcher and

Bordewijk 1978):
1 senðαπ Þ
gðlnτÞ ¼ (16)
2π cosh½ð1 αÞlnðτo =τÞ cos ðαπ Þ
Figure 5 shows biological tissue dispersion spectra reproduced from parameters

given by Gabriel et al. (Gabriel et al. 1996). The authors performed the adjustment of
a model with four dispersion band described with Cole-Cole function to the exper-
imental values obtained in the frequency range from 10 Hz to 100 GHz for many
human biological tissues. ε1, σs and Δε, τo and α for each band were determined
through a least squares method.
To obtain the spectra of Fig. 5, it was discarded the band with very fast relaxation
of the order of 1011s which not significantly influence the results within the
frequency range of interest. The mathematical expression of the dielectric constant
of the materials listed in this figure can then be described by the following equation:
Fig. 5 Dispersion spectra in human biological tissues according to data published by Gabriel
et al. (Gabriel et al. 1996): (a) and (b) skin; (c) and (d) skeletal muscle (not specified orientation); (e)
and (f) liver. — Model with three bands of Cole-Cole according to Eq. 17; model with three
Debye bands for each relaxation according to the Eqs. 18 and 19
_ Δεα Δεβ Δεγ

ε ð ωÞ ¼ ε 1 þ 1α
þ 1β þ 1γ (17)
1 þ ðjωτα Þ 1 þ jωτβ 1 þ jωτγ
On the other hand, the dashed curves in this figure refers to an approach in
which each band is represented by the sum of three Debye dispersions with time
constants τo, 30τo e τo/30, and amplitudes determined by the distribution func-
tion. For example, in the alpha band, the dielectric constant is calculated as
follows:

_ ao alow ahigh
ε α ð ωÞ Δεα þ þ (18)
1 þ jωτα 1 þ jω30τα 1 þ jωτα =30
gðlnτα Þ
ao ¼
gðlnτα Þ þ gðlnð30τα ÞÞ þ gðlnðτα =30ÞÞ
gðlnð30τα ÞÞ
alow ¼ (19)
gðlnðτα =30ÞÞ
ahigh ¼
The choices of the relaxation times for this model with three Debye bands were
based on minimizing the mean square error calculated in relation to the equation of
the Cole-Cole model.
In an equivalent circuit model containing N Debye bands, the dielectric constant
and conductivity as functions of frequency are calculated by the following equations:
X
N
Δεi
εr ð ωÞ ¼ ε 1 þ (20)
i¼1
1 þ ω2 τ2i
XN
ω2 τ2i Δσi
σðωÞ ¼ σs þ (21)
i¼1
1 þ ω2 τ2i
where Δεi, Δσi, and τi in each band are related by Eq. 22 (Bottcher and Bordewijk
1978).
εo Δεi
Δσi ¼ (22)
τi
The graphs in Fig. 5 show good agreement between models up to 106 Hz.
Naturally, other ways of adjusting the relaxation times and amplitudes can be
implemented to improve the accuracy of the first order model. Anyway, it can be
concluded that it is possible to represent the dielectric dispersion by relaxation of a
biological tissue through a set of Debye bands. This means that the equivalent circuit
of the Fig. 4 can be used in the electrical analysis in a biological tissue if the number
of dispersive branches and the values of Gd and Cd parameters are properly chosen
for correctly representing the dielectric dispersion in the frequency range of interest.
Numerical Analysis of the Equivalent Circuit
Computational analysis of the electric field distribution and electrical current in

biological tissues stimulated by electrical potential applied from electrodes can be
performed using the equivalent circuit of the materials applied to a three-dimensional
array of volume elements that discretize the space under analysis. Figure 6 shows the
Fig. 6 Discretization scheme

using in the modeling with the
equivalent circuit
discretization scheme with cubic blocks. In the center of each block it is defined a
node of the equivalent circuit. There are six neighboring blocks to each block in the
mesh. On the boundaries of the domain some connections are replaced according to
the boundary conditions.
Numerical values of the electric potential in the equivalent circuit are obtained as
the solution of the node equations resulting from applying Kirchhoff’s first law on all
nodes of the mesh. The node equation to the central node in Fig. 6 can be written as
follows:
X6

d
C1nm ðV n V m Þ þ is þ iα þ iβ þ iγ nm ¼ 0 (23)
m¼1
dt
According to the continuity equation, the current summation in Eq. 23 is equal to

the negative of the time derivative of the electric charge in the central node:
X
6 dQn
is þ iα þ iβ þ iγ nm
¼ (24)
m¼1
dt
Substituting Eq. 24 into Eq. 23 and integrating both, the basic equations for
solution of the equivalent circuit are obtained:
" #
1 X
6
Vn ¼ Qn þ C1nm V m (25)
X
6
m¼1
C1nm
m¼1
X
6
Qn ¼ Q0n Δt is þ iα þ iβ þ iγ nm
(26)
m¼1
where Q0 n is the value of the charge at time t0 = t Δt.

The current at the static conductance (is) is calculated by Ohm’s law, while
the other currents in Eq. 26 are calculated according to Eqs. 6 and 7. In order to
obtain the correct convergence in the numerical integration, the time step should be
limited to a value lower than the relaxation times of the circuit. The limit value is
defined by gamma relaxation time or the time constant τs = ε1/σs of the C1||Gs
coupling.
The boundary conditions are of two types: electric potential defined on the
surface of metal electrodes and null electric field perpendicular on the other domain
boundaries. In the internal interfaces between two media with different conductiv-
ities and permittivities, the requirement of continuity of potential and current density
are met naturally.
Modeling Anisotropic, Inhomogeneous, and Nonlinear Media
Anisotropy and inhomogeneity are easily modeled with the equivalent circuit
method. In each volume element of the discretization mesh, the material properties
(σs, ε1, α, Δεα, τα, β, Δεβ, τβ, γ, Δεγ, τγ) should be specified in each direction of the
coordinate system. Thus, the properties can easily vary from one region to another as
well they can be modified according to the direction which they refer. Biological
tissues usually have anisotropy in electrical properties. Skeletal muscle, for example,
has higher conductivity and lower dielectric dispersion in the direction parallel to the
muscle fibers than in the perpendicular direction (Foster and Schwan 1995).
The most important nonlinear effects in biological tissues are caused by electro-
poration and heating. The problem of electrical and thermal coupled calculation
including the heat flow in the medium is beyond the scope of this chapter. But a
simple approach can be used when the electrical stimulation is short. As a result of
heating, electrical conductivity in a biological tissue is increased due to the reducing
viscosity of the fluid in which the ions move both inside and outside the cells. A
simple model is described by Eq. 27 (Neal et al. 2012):
σ ð T Þ ¼ σ o ½ 1 þ χ ð T To Þ (27)
where T is the temperature in each volume element σo is the conductivity at room

temperature To at which the material is considered to be in thermal equilibrium prior
to application of the electric field and χ is the temperature coefficient of conductivity
for the considered medium. Assuming the heat generation by other processes than
Joule effect and the heat flow are negligible during the field application, the
temperature increases with time according to the Eq. 28:
Pdiss Δt
ΔT (28)
cρ
where Pdiss is the power dissipated in each volume element, c is the specific heat
capacity, and ρ is the density of the material.
In the equivalent circuit, the elements that are most affected by heating are those
who depend directly on fluid conductivities. Such is the case of static conductance
Gs, which depends on the external conductivity, and Gβ dispersion conductance,
which depends on both the external and internal conductivity. Thus, Eq. 27 can be
used to correct the values of these conductances:
Gs ¼ Gso þ Gso χ ðT To Þ (29)
Gβ ¼ Gβo þ Gβo χ ðT To Þ (30)
where Gso and Gβo are the values of the conductances at temperature To. The use of
this approach requires that the dissipated power is calculated in every time step Δt
and Eqs. 28, 29, and 30 are used to update the values of the conductances in each
volume element in the mesh.
According to Eqs. 9 and 11, the relaxation times also depend on the temperature,
and in a more detailed approach this can be included in the equivalent circuit where
they influence the calculation of the dispersive currents according to Eqs. 6 and 7.
The other nonlinear effect of most interest is electroporation, which is responsible
for increasing the conductivity of the biological tissue as a result of increasing the
nonselective plasma membrane permeability. The electroporation evaluated in several
biological tissues occurs when the applied electric field is greater than a threshold
value between 8 and 40 kV/m and becomes irreversible when the electric field exceeds
another threshold between 80 and 120 kV/m (Pavselj et al. 2005; Corovic et al. 2013).
When the electroporation occurs, in certain regions of the plasmatic membrane of
the cells the membrane conductance increases rapidly and passes to vary in time
according to the intensity of the transmembrane potential. Part of the electric current
cross the plasmatic membrane through hydrophilic pores created in the electropora-
tion process. Because of this the static conductivity of the biological tissue becomes
a function of time and intensity of the applied electric field. Despite intense exper-
imentation around this phenomenon in recent decades, a general mathematical form
that correctly expresses the time dependence of tissue conductivity during electro-
poration has not yet been obtained. Nevertheless, one way in which a dynamic model
of electroporation may be included in the equivalent circuit is expressed by a first
order equation in time as follows:
A
Gp ðtÞ ¼ Gp ðt ΔtÞ þ gðE, tÞΔt (31)
L
where Gp is the increase in static conductance of the tissue due to the electroporation
process, A and L are explained in Fig. 1, E is the electric field, and the function g(E,t)
describes the dynamics of electroporation from the time rate of change of static
conductivity:
dσs
gðE, tÞ ¼ (32)
dt
Starting with a null value, the electroporation conductance Gp is incremented or
decremented at each time step according to the intensity of the electric field and its
value is added to the static conductance described by Eq. 29:
Gs ¼ Gso þ Gso χ ðT To Þ þ Gp ðtÞ (33)
Simulation of the Stimulation Effects with Metal Electrodes
One of the most common and important analysis refers to biological tissue stimula-
tion with electrodes in the form of needles to produce electroporation of tumors for
treatment purposes. Figure 7 shows the geometry used, and Tables 1 and 2 show
numerical values used in this example (The data in Table 1 were obtained from the
article by Gabriel et al. 1996). The tissue in this case is modeled as a linear and
homogeneous medium. Electrodes in the form of metal needles arranged in two rows
each containing three needles are used for applying an electric potential difference
with pulsed waveform. Three human tissues are used as substrate in these simula-
tions: skin, skeletal muscle, and liver. Its spectra are shown in Fig. 5.
A program in C language was built to perform calculations relating to this
simulation. The equations for the electric potential were solved with the Gauss-
Seidel method. The simulations were running on a computer with Intel i3TM
processor, 4 GB of RAM, and Windows 7TM operational system. Due to the large
amount of volume elements in the discretization mesh (880,000) and the high
number of time steps (50,000), each simulation took about 4 h.
Fig. 7 Geometry for

simulating the electrical
stimulation of biological
tissue using the equivalent
circuit method
Table 1 Tissue properties Parameter Skin Muscle Liver

(Gabriel et al. 1996)
ε1 43 54 43
σs (S/m) 4 104 0.2 2 102
σα (S/m) 1.67 104 9.73 102 1.67 102
τα (s) 1.59 103 2.274 103 1.59 102
α 0.2 0.001 0.05
σβ (S/m) 0.167 3.34 102 1.946 102
τβ (s) 1.59 106 3.18 104 2.274 105
β 0.16 0.1 0.2
σγ (S/m) 0.0311 0.175 0.1
τγ (s) 7.96 108 3.54 107 5.305e-7
γ 0.001 0.1 0.2
Table 2 Simulation Parameter Description Value

parameters
Lx (m) Domain thickness 8 103
Ly (m) Domain width 10 103
Lz (m) Domain length 11 103
Δx (m) Mesh parameter 1 104
Δy (m) Mesh parameter 1 104
Δz (m) Mesh parameter 1 104
d (m) Distance between needle rowsa 5 103
h (m) Needle length 5 103
w(m) Distance between needlesa 2 103
ϕ (m) Needle diameter 4 104
Δt (s) Time step 2 109
Ns Number of steps 5 104
VF (V) Pulse amplitude 500
tr (s) Pulse rise time 20 106
tf (s) Pulse fall time 20 106
thigh (s) Pulse duration in high level 80 106
tlow (s) Pulse duration in low level 80 106
a
Distance center-to-center between needles
In each simulation, the applied voltage between the two rows of needles has the
form of bipolar and symmetrical trapezoidal wave with amplitude suitable to achieve
the electric field required for electroporation in a large region in the space between
the needles. The rise and fall times of the pulse wave were adjusted aiming to
emphasize the effect of dispersion in the transient behavior of the current through the
tissue.
Figure 8a, b, and c show the waveforms of the total electric current through the
tissue in the first period of the applied voltage. The main purpose of this example is
to show the importance of correct modeling of the dispersive properties of the
materials involved in the field calculation in biological tissues. To this end,
Fig. 8 (continued)
simulations were performed with different types of approaches: three Cole bands
(αβγ Cole), three Debye bands (αβγ Debye), only alpha and beta Debye bands
(αβ Debye), only beta Debye band (β Debye), and tissue without dispersion using
either Gs or Gs + Gα + Gβ as the conductance.
It makes sense to discard a dispersion band if the waveform of the applied voltage
contains very different frequencies from the cutoff frequency of that band. Consid-
ering the pulsed waveforms generally used in electroporation experiments, the
gamma band which has relaxation times of tens to hundreds of nanoseconds may
possibly be ignored, and its dispersion capacitance must be added to the capacitance
C1 of the model. Likewise, if the alpha band, with relaxation time of tens of
milliseconds, is ignored, their dispersion conductance Gα should be added to the
static conductance of the model.
The waveforms in Fig. 8 show different aspects that should be highlighted. In the
case of skin (Fig. 8a) which has very small static conductivity, there is not an
adequate conductance value in a model without dispersion which can acceptably
represent the dynamic behavior of the current in the tissue. For liver (Fig. 8c), with
static conductivity fifty times higher, the curves for models with and without
dispersion are comparable but still have significant differences. Only in the case of
skeletal muscle (Fig. 8b), which presents very high static conductivity, is obtained a
suitable modeling in the simulation conditions with a model without dispersion in
which conductance is the sum Gs + Gα + Gβ.
Once the spectrum of the applied voltage concentrates 99% of the intensity in the
frequency range from 5 kHz to 100 kHz while the cutoff frequencies of alpha and
gamma bands in all three analyzed tissues is below 1 kHz and above 1 MHz, respec-
tively, these bands do not influence substantially the dynamic response of the current.
Consequently, in all three cases using only beta dispersion yields a good modeling.
Figure 9 refers to another simulation involving the same previous stimulation
scheme but now including a small spherical volume between the needles. This
volume aims to model a skin tumor with 3 mm diameter located at one millimeter
from the surface. The images 9a and 9c refer to the skin model with alpha and beta
dispersion while the images 9b and 9d relate to the model without dispersion and
using the conductance Gs. It is known that tumors generally have higher conductivity
and permittivity than normal tissues. In this case, it was considered that the spherical
volume presents five times more conductivity and permittivity than normal skin.
The graphs show the distributions of the total electric field in the x and y planes
which pass through the center of the tumor at time t = 105 s which corresponds to
the moment when the voltage pulse reaches its maximum value of 500 V. It is clear
that the field distributions are significantly different between the two models.
Especially inside the tumor where the model with dispersion results in electric
Fig. 8 Total current waveforms obtained in the first cycle of the applied voltage. (a) skin; (b)
skeletal muscle (not specified orientation); (c) liver. Each chart contains six curves corresponding to
the six models used in the simulations. In the graph (a) the curve for Gs + Gα + Gβ is divided by
eight in order to be visually comparable to the other curves
Fig. 9 Electric field distribution in the skin with tumor for the same stimulation scheme of Fig. 7 in
the plane x (a and b) and y (c and d) passing through the center of the tumor: (a) and (c) model with
alpha and beta dispersion; (b) and (d) model without dispersion using the conductance Gs
field higher than 35 kV/m (minimum value around 37 kV/m) and the model without
dispersion results in a large region with lower field (minimum value around
32 kV/m). It was observed that in the end of the positive pulse (t = 9 105 s),
the difference between the results obtained with the two models become almost
unnoticeable, which shows that the dielectric dispersion affects only the transient
electric field distribution.
The images show that a relatively large volume of tissue surrounding the needles
reaches very intense electric field values (E > 95 kV/m) that can result in irrevers-
ible electroporation. It is observed that the tumor is subject to much lower electric
field values, but based on the results obtained from the model with dispersion, there
should be reversible electroporation inside. Moreover, the results obtained with no
dispersion model suggest that would be required to increase the amplitude of the
voltage pulses applied between the needles in order to achieve electroporation across
the tumor volume. Thus, it is emphasized that the use of computer simulation with
suitable electrical models can predict the effectiveness of the electrical stimulation
scheme of a biological tissue for electroporation purposes. These conclusions are
corroborated by a similar but more detailed study by Miklavcic and colleagues
(Miklavcic et al. 2006).
However, one aspect of the problem that was not considered in this analysis due
to the lack of sufficient information is the change in conductivity of the tissue due to
the electroporation. Once the applied electric field exceeds the threshold for revers-
ible electroporation, the conductivity of the tissue increases intensively and rapidly
in a microsecond time scale. Thus, it is expected that the dielectric dispersion has
great influence on the intensity and extent of tissue electroporation and the compu-
tational models used in the electrical analysis in this case should take this into
account. In addition, some studies have shown that due to electroporation, current
and electric field distributions in biological tissues are significantly different from
those obtained in simulations in which the materials are considered linear regardless
of the applied electric field strength (Ramos 2005; Corovic et al. 2013). This makes
the use of numerical methods that describe as realistically as possible the dielectric
dispersion and the dynamics of electroporation in biological tissues, a basic need for
reliable planning therapies based on electroporation.
Conclusions
The computational approach for electrical analysis using the equivalent circuit
model is a relatively simple technique to implement and allows calculating the
spatial distribution of the electric field and electric current in the time domain in
materials in which the dielectric dispersion can be adequately described by one or
more dispersion Debye bands. When applied to the biological tissues, equivalent
circuit method has significant advantages over other numerical techniques, due to the
simplicity in construction of realistic models from dispersion spectra of the materials
involved and applying the boundary conditions. The example presented in this
chapter shows that the dielectric dispersion significantly influences both the temporal
response of the electric current as the electric field distribution in the tissue. The
modeling based on the equivalent electric circuit can be used to study the electro-
poration in biological tissues, since the dynamic conductivity changes resulting from
the electroporation process is correctly modeled and included as variations of the
conductance Gs of the equivalent circuit.
Cross‐References

Field Processing
▶ Assessment of Electroporation by Electrical Impedance Methods
Deep-Seated Tumors
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Mathematical Models Describing Cell
Death Due to Electroporation 63
Janja Dermol and Damijan Miklavčič
Abstract
Various models have been developed to describe microbial inactivation by pulsed
electric field treatment, and they have just recently been used for describing
eukaryotic cell death due to irreversible electroporation. In microbial inactivation,
the mathematical models of cell death enable the adaptation of the pulse param-
eters to achieve sufficient microbial reduction at the lowest energy input while
preserving flavor and sensitive compounds in the food. For precise prediction, the
geometry of the treatment chamber, the fluid flow, the temperature, and the
electric field distribution should also be taken into account. In electroporation-
based medical treatments, currently, a deterministic critical value of electric field
is used to delineate between the destroyed and the unaffected tissue. Conse-
quently, tumor cells which have higher electroporation threshold than the exper-
imentally determined may remain viable and cause incomplete tumor elimination.
On the contrary, the more sensitive surrounding tissue could be damaged. Math-
ematical models of cell death help to achieve sufficient cell death while minimiz-
ing the damage to the surrounding vital structures. In this chapter, different
models are described which were already used for describing microbial inactiva-
tion in liquid foods or eukaryotic cell death (the first order, the Hülsheger, the
Peleg-Fermi, the Weibull, the logistic, the Adapted/Modified Gompertz, the
Geeraerd, the quadratic, the Peleg-Penchina model). The cell death models
have already been used for predicting survival in realistic setups like the treatment
chamber in microbial inactivation and different electrode geometries and tissues
in irreversible electroporation treatments. In conclusion, cell death models are
useful in predicting the treatment outcome. Unfortunately, since the mechanisms
of cell death due to electroporation are not yet fully elucidated, the models are
empiric. There is no direct connection between the parameters of the models and
J. Dermol (*) • D. Miklavčič

e-mail: Janja.dermol@fe.uni-lj.si; damijan.miklavcic@fe.uni-lj.si

DOI 10.1007/978-3-319-32886-7_13
1200 J. Dermol and D. Miklavčič
the biological/electrical parameters. Thus, it is unclear which model is the most

appropriate to use. The models have to be optimized for each specific cell type
and electric pulses separately. The transferability from the in vitro to the in vivo
level is questionable.
Keywords
Microbial inactivation • Predictive models • Numerical modeling • Treatment
planning • Food pasteurization
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1200
Microbial Inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1201
Electroporation-Based Medical Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1201
Models Used to Describe Cell Death in Suspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1204
The First-Order Kinetics Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1205
The Hülsheger Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1206
The Peleg-Fermi Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1207
The Weibull Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1207
The Logistic Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1209
The Adapted/Modified Gompertz Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1210
The Geeraerd Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1210
The Quadratic Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1210
The Peleg-Penchina Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1211
Models Used to Describe Cell Death In Vitro on Attached Cells and Cells in 3D
Tissue-Like Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1211
Models of Cell Death Used in the Treatment Chamber and In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . 1213
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1216
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1216
Introduction
Mathematical modeling is becoming indispensable in the field of life sciences. It

enables description and prediction of a response of a biological system knowing the
excitation and other parameters, and gives insights into the mechanisms of phenom-
ena. The number of biological experiments can be decreased which decreases the
time and costs needed to obtain results. In the field of electroporation, models exist
on different levels – molecules, lipid bilayers, cells, and tissues. Cell death or
inactivation models are used for the description of microbial inactivation and
recently also eukaryotic cell death in irreversible electroporation as nonthermal
soft tissue ablation. In this chapter, the statistical models of cell death are described
on the level of cells and tissues.
Currently, there is no complete explanation of cell death due to electroporation
(▶ Chap. 63, “Mathematical Models Describing Cell Death Due to Electropora-
tion”), neither for prokaryotic nor eukaryotic cells. The cell death is believed to be
caused by the following mechanisms. First, high electric pulses can cause membrane
63 Mathematical Models Describing Cell Death Due to Electroporation 1201
destruction and necrotic cell death. Second, there is the apoptotic cell death, reasons
for which are not yet clear. Third, in vivo, last of the clonogenic cells are destroyed
by the immune system (▶ Chap. 44, “Immunological Response to Electroporation”).
Thus, the currently used cell death models are empirical and were developed on the
basis of the best fit to the experimental results.
Microbial Inactivation
In liquid food pasteurization by pulsed electric fields (PEF), short high-voltage

electric pulses are applied to decrease the number of microbes and quality-degrading
enzymes, and extending the shelf-life while retaining flavor and nutrients (Lelieveld
et al. 2007; Sun 2014). In the literature on cell death models, the expressions
bacterial and microbial cell death are both used. The microbe is a short expression
for microorganisms not visible with a naked eye which includes besides bacteria also
protozoa, fungi, and viruses. Thus, bacteria are a subgroup of microorganisms. Since
cell death models have been used for describing cell death of various microorgan-
isms (i.e., bacteria, yeast), expression microbial cell death models is used through
this chapter. Mathematical modeling of microbial cell death (▶ Chap. 66, “Modeling
Microbial Inactivation by Pulsed Electric Field”) is necessary to adapt the pulse
parameters and achieve sufficient decrease of microorganisms at a minimum energy
input while preserving the sensitive compounds in the food (Huang et al. 2012). In
the food industry, at least five log reductions of bacteria are required for food
pasteurization. Applying too gentle pulses may cause food spoilage and health-
related problems while applying too severe pulses may cause Joule heating and
decrease the quality of food. The cell death models have been used on a variety of
liquid food (e.g., fruit juices, milk, water, liquid egg yolks) or models of such liquids.
When predicting the microbial inactivation by the PEF treatment, the geometry of
the treatment chamber is modeled, and the electric field, the temperature distribution,
and fluid flow are (numerically) calculated (Gerlach et al. 2008). Microbial cell
death prediction can be included using the cell death models as a function of the
electric field, treatment time, the number of the applied pulses, and/or the pulse
repetition frequency (Huang et al. 2013). The inactivation of the quality-degrading
enzymes and the degradation of the health-related compounds can also be modeled
with similar models as the inactivation of microorganisms due to their similar
kinetics.
Electroporation-Based Medical Treatments
When treating eukaryotic cells with electroporation, currently, the three main appli-
cations are the electrochemotherapy, the irreversible electroporation (▶ Chap. 40,
“Tissue Ablation by Irreversible Electroporation”), and the gene electrotransfer for
gene therapy and DNA vaccination (Yarmush et al. 2014). When treating tumors
with electrochemotherapy, pulses of standard parameters and fixed electrode
configurations can be used. However, if the tumor is larger than 2 cm and/or has an
irregular shape, a variable electrode configuration should be used. For the irrevers-
ible electroporation treatments (Jiang et al. 2015), protocols are already set up in the
commercially available electroporators, but the ablated volumes do not always
correlate to the predicted ones (Bhutiani et al. 2016). In the electrochemotherapy,
irreversible electroporation, and also in gene electrotransfer treatments, the need for
patient-specific treatment planning (▶ Chap. 54, “Treatment Planning for
Electrochemotherapy and Irreversible Electroporation of Deep-Seated Tumors”) is
on the rise. The number, the geometry of the electrodes, and the parameters of the
electric pulses must be optimized considering the specifics of the cause and the
treatment (Županič et al. 2012). In electrochemotherapy, all tumor cells must be
reversibly permeabilized, while the surrounding tissue, especially critical structures,
should be undamaged. In irreversible electroporation, the tumor must be irreversibly
electroporated without significant thermal damage, while the surrounding tissue,
especially critical structures like nerves and vessels, should remain largely
undamaged. In gene electrotransfer, cells must be reversibly permeabilized, and
their viability must remain high. Currently, an experimentally determined critical
electric field is used to predict and delineate between the alive and the irreversibly
electroporated tissue – the response is modeled by a step function (Fig. 1). The
assumption of a step function is, however, too simplistic even for cell suspensions of
the same cell line (Canatella et al. 2001; Dermol and Miklavčič 2015) since cells
among other differ in size and position in a cell cycle. In tissues, especially in tumors,
this inhomogeneity is amplified since cells additionally differ in shape and tissues are
comprised of different cell types. In a tumor, in addition to the tumor cells, stromal
cells are present in the microenvironment. Therefore, in reality, the transition from
alive to irreversibly electroporated state is continuous and distributed over a range of
electric field values. The width of this range, as well as the threshold voltage, also
depend on the number and length of the applied pulses (Canatella et al. 2001; Garcia
et al. 2014).
When predicting the survival with the fixed threshold, the cells which have higher
critical threshold than the experimentally determined can survive and cause incom-
plete tumor elimination or its later recurrence. On the other hand, applying more
severe electric pulses than necessary can cause tissue necrosis, excessive Joule
heating, and thermal damage. By using cell death models to predict the treated
volume, the efficiency of the electroporation-based medical treatments and therapies
can be increased. Cell death models allow interpolation and predict survival at
parameters which were not experimentally determined. The last few of the
clonogenic cells are eradicated by the immune system (Yarmush et al. 2014)
which should be considered when determining the tolerated percentage of the
survived cells in electroporation-based treatments. A complete eradication of all
tumor cells may not be needed.
In electrochemotherapy and gene electrotransfer, using cell death models is not
enough to correctly predict the treatment outcome. In the electrochemotherapy
treatments, the application of electric pulses increases the permeability of the cell
Fig. 1 The comparison of the currently used and the proposed way of modeling cell death. The red
line shows the currently used step response where below a critical electric field all cells are regarded
alive and above all dead. The gray and dark lines show the suggested cell death models which
predict a gradual decrease in cell survival. Black circles are the experimentally determined values. It
is clear that the experimental values are better described with different cell death models than with a
step response
membrane to the chemotherapeutics. Two commonly used chemotherapeutics are

cisplatin and bleomycin. Cell death is mostly caused by the cytotoxic effects of
chemotherapeutics, although, in the vicinity of the electrodes, cells can be irrevers-
ibly electroporated. To correctly predict the affected regions, a model of permeability
to cytotoxic drugs and their transport across the membrane as a function of electric
pulses should be included in the treatment planning of electrochemotherapy (Dermol
and Miklavčič 2014). Namely, when the number of the molecules of chemothera-
peutic entering each cell can be calculated, it can be predicted whether the cell will
die or not. In the gene electrotherapy, electric pulses increase the permeability of the
cell membrane to the DNA. The exact mechanisms of gene electrotherapy are
complex and also depend on genes. The presumed steps in the process are electroper-
meabilization of the cell membrane, electrophoretic migration of the DNA towards
membrane, DNA/membrane interaction, DNA translocation across the membrane,
intracellular migration of DNA through the cytoplasm, DNA passage through the
nuclear envelope, and gene expression (Rosazza et al. 2016). To correctly predict
where the transfection will occur, each step should be modeled.
Models Used to Describe Cell Death in Suspension
Ideally, cell death model would include all the parameters, important for cell death.
A problem arises since the exact mechanisms of cell death due to electroporation are
not completely known. Cell death depends on many parameters, among others on the
electric field; the duration and the number of the applied pulses; the pulse repetition
frequency; the properties of the cells; their size, shape, orientation in the electric
field; their concentration in the treated medium; the properties of the treated medium
or food; pH; temperature; configuration of the electrodes or the treatment chamber;
and the concentration of the chemotherapeutics (in electrochemotherapy). The most
important parameters for cell death should be identified and included in the model. It
should also be determined which parameters can be excluded to simplify the models
and the fitting. In summary, the cell death model due to electroporation should
describe the experimental results well, include all the important electrical and
biological parameters, have high predictive power, and a minimum number of
parameters to be optimized.
Various models have been developed and used to describe microbial cell death
due to pulsed electric field (PEF) treatment in liquid food pasteurization (Álvarez
et al. 2003; Peleg 2006; San Martín et al. 2007; Huang et al. 2012). Only recently,
interest to develop and use such models has been expressed in tissue ablation due to
irreversible electroporation, i.e., IRE treatment. Most of the models used, however,
are empirical, and their parameters have no physical meaning and even have no
parameters that could be explicitly linked to treatment parameters such as electric
field, pulse duration, the number of pulses, and pulse shape. There is also no solid
link demonstrated between cell membrane permeabilization and cell death.
In microbial cell death models, the independent variable is usually the treatment
time which is easily determined in the case of thermal, irradiation, high pressure,
ultrasound, or other continuous treatment. The treatment time of the PEF treatment is
more difficult to determine since multiple short pulses are applied at different pulse
repetition frequencies. Nevertheless, the treatment time (t) is usually calculated by
multiplying the number of pulses with the duration of one pulse (t ¼ NT ), where
N denotes the number of the pulses and T the duration of one pulse. This way of
calculating the treatment time assumes that only the product of N and T affects the
survival but not the duration or the number of the pulses by itself. The effect of
the pulse repetition frequency is also neglected. In microbial cell death models, the
electric field, the number of pulses, or their repetition frequency were used as
independent variables. With the exception of the quadratic model, however, no
model so far included more than two independent variables.
The electric field is the most important parameter in electroporation-based treat-
ments (Miklavčič et al. 2006). The survival curves as a function of the electric field are
usually composed of three parts: a shoulder (upper asymptote) which is then followed
by a steep decline in cell number and eventually, a tail (lower asymptote) is reached.
The tail can be a caused by either a resistant subpopulation of cells or the reached limit
of the survival assay. Several different curves can describe the survival data. When
presenting the survival curves and the cell death models, they should be plotted on a
semilogarithmic scale. There, deviations and small percentages of survival are more
easily noticed. The quality of the fit should be evaluated using various statistical
measures: the goodness-of-fit (R2), the root-mean-square error (RMSE), and/or accu-
racy parameter (Af). R2 is a statistical measure of how close the regression line is to the
experimental data; RMSE is a measure of the average deviation between the observed
and predicted data; and Af measures the accuracy of the estimates obtained by the
models. Other criteria for evaluating the suitability of a model are the number of the
models’ parameters (which should be low), the inclusion of more independent vari-
ables, and a high predictive power. In continuation, models which were already used
for describing microbial or eukaryotic cell death are described.
The First-Order Kinetics Model
The first-order kinetics model was developed almost a century ago (Huang et al.
2012). It derives from the assumption that all cells in a population are equally
sensitive to the treatment. When used in PEF treatments, it describes cell death as
a function of the treatment time:
SðtÞ ¼ expðktÞ; (1)
where S denotes the survival, t the treatment time, and k is a first-order parameter,
i.e., the speed of the decrease. The model predicts that the viability decreases
immediately – there is no shoulder at short treatment time. The first-order kinetic
model did not provide a good fit to electroporation treatment of eukaryotic cells
(Dermol and Miklavčič 2015). It has also been observed that it does not describe all
microbial inactivation data well. It was stated that the model describes the data
sufficiently well only when the data is sampled too scarcely (Peleg 2006). The
deviations from the first-order kinetics model could among others be explained by
the statistical distribution of cell radii (Lebovka and Vorobiev 2004).
The first-order parameter k was found to be temperature dependent which could
be modeled in thermal treatments as well as in PEF treatments. In PEF treatments,
the temperature of the sample increases due to the Joule heating and consequently
the electrical conductivity of the sample is also increased. The parameter k could be
modeled by Arrhenius equation as:

EA
k ¼ kt exp (2)
RT
Where kt is the rate constant at the reference temperature, EA the activation energy,
R is the gas constant, and T is the sample temperature in Kelvins.
Traditionally, the sensitivity of microorganisms to static treatments is described
using the decimal reduction time (Dt). It denotes the treatment time needed to obtain
one decimal reduction of the cell population, i.e., 10% of the initial population
survives. It is defined as Dt ¼ 2:303
k since the first-order kinetics model is based on
natural and the decimal reduction time on the decimal logarithm.
If the treated cell population consists of two populations of which each has its
first-order dynamics, the biphasic model can be used:
SðtÞ ¼ f ek1 t þ ð1 f Þek2 t ; (3)
where f denotes the proportion of the first subpopulation in the whole population, and
k1 and k2 are the first order parameters of the first and the second subpopulation,
respectively. In case there are more subpopulations, the decrease of each subpopula-
tion can be modeled with its own exponential factor which is then added to the (Eq. 3).
The Hülsheger Model
Hülsheger studied the effect of electric field on the inactivation of E. coli (Huang
et al. 2012). He derived an empirical formula which has two independent variables –
the treatment time (t) and the electric field (E). As a function of the electric field, the
survival showed a linear decline when the electric field exceeded a certain critical
electric field (Ec) which was modeled as:
SðEÞ ¼ expðbE ðE Ec ÞÞ; (4)
where S is the survival, bE is the regression coefficient, and Ec is the critical value of
electric field below which there will be no inactivation (i.e., the lowest electric field
that causes inactivation). When E < Ec, the model predicts the survival above one
which is incorrect and the survival must be fixed at one.
As a function of the treatment time, the results were modeled as:

t
SðtÞ ¼ exp bt ln ; (5)
tc
where S is the survival, bt is the regression coefficient, and tc is the extrapolated

critical value of treatment time below which there will be no inactivation (i.e., the
shortest treatment time which causes inactivation). For treatment times shorter than
the critical treatment time, the model predicts the survival to be more than 1. Thus,
also here, for t < tc the survival should be fixed at one.
Both models Eqs. 4 and 5 were joined in:
EEk c
t
Sðt, EÞ ¼ (6)
tc
where k is the independent inactivation constant, and other parameters have the same
meaning as in Eqs. 4 and 5. The parameters tc, Ec, and k were proposed to be
microorganism dependent within a certain range of experimental parameters.
Although the Hülsheger model includes two independent variables, it cannot

universally describe the experimentally determined cell death. With the Hülsheger
model, some microbial cell death results were possible to model, and some were not
(Huang et al. 2012), while only one study unsuccessfully fit it to the eukaryotic cell
death data (Dermol and Miklavčič 2015).
The Peleg-Fermi Model
The Peleg-Fermi model has been successfully fit to various microbial inactivation

data (Huang et al. 2012) and was also the first to be used for describing eukaryotic
cell death due to electroporation (Golberg and Rubinsky 2010). It derives from the
Fermi’s equation which is used to describe the behavior of materials at their glass
transition temperature. The Peleg-Fermi model is one of the most promising since it
describes the data well and includes two independent variables – electric field (E)
and the number of the applied pulses (N ):
1
SðE, N Þ ¼ (7)
E Ec ð N Þ
1 þ exp
k ðN Þ
Ec ðN Þ ¼ Ec0 expðk1 N Þ (8)
kðN Þ ¼ k0 expðk2 N Þ (9)
where Ec(N ) is the critical electric field where the survival drops to 50%, k(N) is the
kinetic constant describing the slope of the curve, Ec0 is the intersection of Ec(N) with
the y-axis, k0 (in the same units as the electric field), k1 and k2 are constants which
change depending on the parameters of the pulses and the properties of the cells. The
critical electric field depends on the number and length of the applied pulses (Pucihar
et al. 2011). For values below the critical electric field, the Peleg-Fermi model predicts
survival to be 100%. The Peleg-Fermi model described various experimental data
well, although the k(N) and Ec(N) did not always change exponentially as a function
of the pulse number (Dermol and Miklavčič 2015; Sharabi et al. 2016). Unfortunately,
the pulse length and the pulse repetition frequency are not included in the model, and
the dependency on them is included indirectly via the parameters of the model. An
example of fitting the Peleg-Fermi model to the eukaryotic cell death due to irrevers-
ible electroporation in vitro is shown in Fig. 2.
The Weibull Model
The Weibull model was originally used to describe the time to failure of electronic
devices after stress was imposed on them. A parallel can be made when stress, for
example, thermal, high pressure, or PEF treatment, is applied to biological cells.
Fig. 2 The Peleg-Fermi model, fit to eukaryotic cell death data in a wide range of pulse number and
electric field values. (a) symbols show the experimental values for 100 μs, 1 Hz repetition frequency
at different pulse numbers (8, 30, 50, 70, and 90), lines show the fitted Peleg-Fermi model (Eq. 7).
(b) symbols are the optimized values of Ec and k for different pulse numbers, lines show the fitted
models for Ec(N ) and k(N ) (Eqs. 8 and 9) (Reprinted from Journal of Membrane Biology, Vol
248/Issue 5, Dermol J, Miklavčič D, Mathematical Models Describing Chinese Hamster Ovary Cell
Death Due to Electroporation In Vitro, Pages 865–881, Copyright 2015 Springer Science + Busi-
ness Media New York)
Time to cell death after the stress can be described using the Weibull model (San
Martín et al. 2007). The Weibull model was successfully used to describe microbial
(Huang et al. 2012) and eukaryotic cell death (Dermol and Miklavčič 2015) using
various pulse parameters. Although the Weibull model is very adaptable, no clear
connection between the electrical/biological parameters and the optimized parame-
ters of the model was made. In some cases, the scale parameter b was exponentially
dependent on the electric field. The model is:
x n
SðxÞ ¼ exp ; (10)
b
where x denotes the treatment time or the electric field, b is the scale (in the same
units as x), and n is the shape parameter. The scale parameter determines the
characteristic time or the characteristic electric field at which 63% of the cells die.
With different values of the shape parameter, the shape of the survival curve varies
between the convex (n < 1), linear (n = 1), and concave curve (n > 1) in semi-
logarithmic scale.
The Logistic Model
The logistic model can be used for describing distributions with a sharp peak
and long tails (Cole et al. 1993). When taking into account that survival of
cells before the treatment is 100%, the logistic model can be written as either
Eq. 11 or 12, depending on what is chosen as the independent variable. The
equations are:

ω
SðEÞ ¼ 10^ (11)
1 þ expð4σ ðτ EÞω1 Þ

ω
SðtÞ ¼ 10^ (12)
1 þ expð4σ ðτ log10 tÞω1 Þ
where E denotes the electric field, t the treatment time, ω the common logarithm of
the lower asymptote, σ the maximum slope, and τ the position of the maximum
slope. Survival curves show the cumulative cell death as a function of the indepen-
dent variable, i.e., applied electric field or treatment time. Thus, each data point
includes cells which died due to the corresponding or less severe parameter. The cell
death distribution is obtained as a derivative of the cumulative distribution. Only cell
death due to the corresponding parameter is plotted. In electroporation treatments,
the distribution of cell death with a sharp peak and long tails is obtained when the
independent variable is either the electric field or the common logarithm of the
treatment time. The logistic model was successfully fit to inactivation of microor-
ganisms by PEF treatment (Huang et al. 2012) and to eukaryotic cell death (Dermol
and Miklavčič 2015).
The Adapted/Modified Gompertz Model
The Gompertz model is usually used for describing the growth of a tumor, but in
modified form, it can also describe cell death. Originally, it was used for describing
bacterial (Linton et al. 1995) cell death due to thermal treatment, but later it was
extended to describing bacterial cell death due to pulsed electric field treatment and
eukaryotic (Dermol and Miklavčič 2015) cell death due to electroporation. The
model is written as:

SðxÞ ¼ exp A exp eðB0 þB1 xÞ A exp eB0 (13)
where x denotes either the treatment time or the electric field, A is the natural
logarithm of the survival in the stationary phase (the natural logarithm of the tail),
B0 is the length of the shoulder, and B1 the speed of the increase (when it is positive)
or decrease (when it is negative) in cell number.
The Geeraerd Model
The Geeraerd model (Geeraerd et al. 2000) describes the exponential decrease of
cells including a tail which models the resistant cell subpopulation. Assuming the
initial survival is 100%, the Geeraerd model can be written as:
SðtÞ ¼ ð1 N res ÞexpðktÞ þ N res (14)
Where Nres represents the tail and k is the inactivation rate. Since the model does not
also include a shoulder, it cannot be used universally for all cell death results. The
Geeraerd model was so far successfully fit to experimental data of microbial
inactivation after mild heat treatment and eukaryotic cell death due to
electroporation.
The Quadratic Model
The quadratic model is currently the only model which can model dependency on
two or more independent variables. As the independent variable x, various param-
eters have been used – the electric field, the treatment time, the pulse repetition
frequency, the pH, and the concentration of some compound. The model is written
as:
Sð x Þ ¼ k 1 þ k 2 x þ k 3 x 2 (15)
where the parameter k1 is the central point of the system, k2 is the coefficient of the
linear effect, and k3 of the quadratic effect of the independent variable. It is also
possible to fit more parameters at once by joining several quadratic models into one:
Sðx1 , x2 Þ ¼ k1 þ k2 x1 þ k3 x1 2 þ k4 x2 þ k5 x2 2 þ k6 x1 x2 (16)
where x1 and x2 denote two independent variables which were marked as the x in the
(Eq. 15), k1 is the central point of the system, k2 and k4 represent the linear, k3 and k5
the quadratic, and k6 the interactive effects of the independent variables. If some of
the terms are nonsignificant under certain experimental conditions, they can be
omitted from the model. By quadratic model fitting, the interaction between different
experimental parameters can be determined. The quadratic model was successfully
fit to microbial inactivation results as well as to the inactivation of the enzymes and
health-related compounds like vitamins (Huang et al. 2012).
The Peleg-Penchina Model
The Peleg-Penchina model is also an empirical model but can describe only the
convex curves (Peleg and Penchina 2000; Álvarez et al. 2003) in the semilogarith-
mic scale. It is written as:
SðtÞ ¼ 10 ^ ðm lnð1 þ ktÞÞ (17)
where t is the treatment time of the PEF treatment; m and k are the parameters of the
model, which have to be optimized; and ln denotes the natural logarithm. Together
with the Eq. 17, the authors introduced a way of describing bacterial survival when
the intensity of the lethal agent (e.g., temperature, chemical agent, PEF treatment)
varies either between treatments or during one treatment.
As long as the mechanisms of cell death due to electroporation (PEF treatment)

are not known, it is difficult to decide which model is superior to others, and they can
all be regarded as equivalent. An example of fitting several cell death models to
eukaryotic and bacterial cell death results is shown in Fig. 3.
Models Used to Describe Cell Death In Vitro on Attached Cells

and Cells in 3D Tissue-Like Structure
In liquid food pasteurization, the microorganisms are suspended in the food. How-
ever, they can also grow in layers, for example, on surgical meshes. Surgical mesh is
thin mesh which supports or reinforces damaged tissue. The cell death models were
applied to PEF treatments of medical mesh implants, on which 2D layers of bacteria
can form as a biofilm (▶ Chap. 24, “Electroporation of Biofilms”) (Khan et al. 2016).
The authors successfully described the data using the Weibull model.
The cell death models have not yet been applied to the attached or 3D eukaryotic
cell models. The optimization of the models in vitro would need to be done anew
since the critical electric fields are different for the attached than for the suspended
Fig. 3 (a) Different cell death models as a function of the electric field (the Peleg-Fermi, the
Weibull, the Logistic, the Adapted Gompertz model) were fitted to the experimental data of
eukaryotic cell death when 90, 100 μs pulses were applied to the cell suspension. Symbols show
the experimental values and lines the models. It can be seen that several models describe the
experimental data well Observed values are marked by circles, model 1 by a bold line, model 2 by
a dotted line, model 3 by a thin line, and model 4 by a dashed line. (Reprinted from Journal of
Membrane Biology, Vol 248/Issue 5, Dermol J, Miklavčič D, Mathematical Models Describing
Chinese Hamster Ovary Cell Death Due to Electroporation In Vitro, Pages 865–881, Copyright 2015
Springer Science + Business Media New York 2015). (b) Different cell death models as a function
of the treatment time were fitted to the survival of E. coli after pulsed electric field treatment. Model
1 is the biphasic model, model 2 is the sigmoidal equation, model 3 is the Weibull model, and model
4 is the Peleg-Penchina model. A good fit was obtained with all tested models (Reprinted from
Innovative Food Science & Emerging Technologies, Vol 4/edition number 4, Alvarez I, Virto R,
Raso J, Condon S, Comparing predicting models for the Escherichia coli inactivation by pulsed
electric fields, Pages No 195–202, Copyright (2003), with permission from Elsevier)
cells due to a different shape, density, and connections between cells (Pucihar et al.
2006; Towhidi et al. 2008).
Models of Cell Death Used in the Treatment Chamber and In Vivo
Microorganisms grow in suspensions and 2D layers. Thus, there are no reports on

using the mathematical models to describe PEF inactivation of microorganisms
in vivo. There is, however, an example of modeling the treatment chamber
(▶ Chap. 65, “Application of Numerical Simulation Techniques for Modeling
Pulsed Electric Field Processing”) with included suspension of bacteria. Huang
et al. predicted the inactivation of bacteria in watermelon juice by first building a
2D numerical model of the treatment chamber and using the Hülsheger model to
describe the inactivation (Huang et al. 2013). Authors obtained very good pre-
dictions. Cells respond to the electric field to which they are exposed. In treatment
chambers (as well as in tissue when performing the IRE treatment), the electric field
can be highly inhomogeneous. In future, to obtain the optimal conditions for
microbial inactivation, first optimization of the treatment chambers’ geometry and
the parameters of electric pulses should be done.
There are just a few reports of using cell death models to describe and predict the
extent of eukaryotic cell death due to electroporation in vivo. Golberg and Rubinsky
were first to suggest using statistical cell death models in vivo (Golberg and
Rubinsky 2010). They fit the Peleg-Fermi model to the results of an in vitro study
of electroporation of prostate cell line. Then, the fitted Peleg-Fermi model was
theoretically applied to the 2D case of tissue electroporation using needle electrodes.
The spatial distribution of probability of cell death the authors obtained is shown in
Fig. 4. It was shown that there exists an area where the cell death probability ranges
Fig. 4 The Peleg-Fermi model was fit to the 2D model of prostate tissue treated by irreversible
electroporation using needle electrodes. The legend on the right shows the probability of cell death.
Around the electrodes exists an area where the probability of cell death is between 0% and 100%,
thus the deterministic critical electric field is not sufficient for predicting cell death in vivo. (a–c)
show the predicted cell death when a different number of 100 μs pulses of the same voltage (1.5
arbitrary unit) are applied: (a) 10 pulses, (b) 50 pulses, (c) 100 pulses (Reprinted from BioMedical
Engineering OnLine, Vol 9, Golberg A, Rubinsky B, A statistical model for multidimensional
irreversible electroporation cell death in tissue, Page No 13, Copyright 2010 Golberg and Rubinsky;
licensee BioMed Central Ltd.)
Fig. 5 The probability of cell death due to electroporation around the commercially available
bipolar electrodes at different pulse numbers (a – 30, b – 50, c – 70, and d – 90) when applying
100 μs pulses of 3000 V at 1 Hz. The transition zone where the cell death decreases from 100% to
0% becomes sharper with increasing pulse number (Reprinted from PLoS ONE, Vol 9/Issue
8, Garcia PA, Davalos RV, Miklavčič D, A Numerical Investigation of the Electric and Thermal
Cell Kill Distributions in Electroporation-Based Therapies in Tissue, Pages No e103083, Copyright
2014 Garcia et al.)
from 0% to 100% and the deterministic critical electric field is not an optimal choice
for predicting the electroporation-based medical treatment outcome.
A theoretical study of commercially available bipolar electrodes used in irrevers-
ible electroporation treatments was done by Garcia et al. (2014). The authors
determined the extent of the cell death caused by the heating and by the electropo-
ration when standard IRE pulses were applied. The thermal damage was evaluated
using the Arrhenius integral and the electrical damage using the Peleg-Fermi model.
The electrical damage due to electroporation around the bipolar electrodes is shown
in Fig. 5. Different combinations of pulse number and electric field lead to different
probabilities of cell death as shown in Fig. 6.
Fig. 6 The probability of cell death due to electroporation as a function of pulse number and
electric field. The cell death is predicted by the Peleg-Fermi model optimized for the prostate cancer
cell death. The contours show the predicted cell death (denoted with numbers in bold) using various
combinations of electric field and pulse number. To achieve 99.9% cell death a certain minimal
electric field and a number of pulses should be applied (Reprinted from PLoS ONE, Vol 9/Issue
8, Garcia PA, Davalos RV, Miklavčič D, A Numerical Investigation of the Electric and Thermal Cell
Kill Distributions in Electroporation-Based Therapies in Tissue, Pages No e103083, Copyright
2014 Garcia et al.)
A study in vivo was performed by Sharabi et al., who analyzed the brain
electroporation using the Peleg-Fermi model to describe cell death as well as the
disruption of the blood–brain barrier (Sharabi et al. 2016). The authors concluded
that the Peleg-Fermi model could be successfully used to describe the electropora-
tion of rat brain, although they found that the Ec(N ) was better described using power
than an exponential function, especially in the range of a high number of pulses
(more than 90). As shown in (Eq. 8), Ec(N ) in the Peleg-Fermi model can be
expressed as Ec ðN Þ ¼ Ec0 expðk1 N Þ . Sharabi et al. obtained better fit using the
equation:
Ec ðN Þ ¼ Ec0 N k1 ; (18)
where Ec0 denotes the critical electric field, N the number of the applied pulses, and
k1 is a constant.
In electroporation-based medical treatments, the desired use of the models is in
tissues in vivo. It is questionable to what extent the in vitro optimized models can be
used in vivo. The thresholds for cell death due to electroporation in vivo (Jiang et al.
2015) seem to be different than in vitro (Dermol and Miklavčič 2015). In vivo, the
cells are connected, they are of different size and shape, their density varies, there are
different types of cells in each tissue, and the immune system is present. It seems that
the cell death models optimized in vitro may not be directly transferable in vivo but
would need to be optimized separately.
Conclusions
Using cell death models in treatment planning could increase the efficiency of PEF
microbial inactivation and electroporation-based medical treatments. However, cur-
rently, it is unclear which model is the most appropriate since they are all empirical,
and mostly, there is no clear relation between the electrical/biological parameters and
parameters of the cell death models. Although the cell death models describe data
well, they must be optimized for each cell type/tissue and different electric pulses
separately. A direct translation of in vitro optimized models to an in vivo environ-
ment is questionable. In future, cell death models should be based on mechanisms of
cell death due to electroporation, include all and only the relevant treatment param-
eters, and describe and predict the cell death accurately.
Acknowledgments This study was supported by the Slovenian Research Agency (ARRS) and
conducted within the scope of the Electroporation in Biology and Medicine European Associated
Laboratory (LEA-EBAM).
Cross-References

Field Processing
▶ Electroporation of Biofilms
▶ Immunological Response to Electroporation
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Modeling Transdermal Delivery by
Electroporation: The Thermodynamic 64
Approach
Sid Becker
Abstract
Low voltage long pulse electroporation of the skin results in localized regions
within the stratum corneum of high permeability to mass transfer and electrical
conduction. It is believed that these permeability increases result from resistive
Joule heating that causes lipid phase changes. This chapter considers the physi-
ology of the stratum corneum and the thermodynamic behavior of the lipid
microstructure. A conceptual model of the evolution of the local transport region
is presented in order to clearly illustrate the physics underlying this dynamic
process. The mathematical equations which govern the conservation of charge,
the conservation of thermal energy, and the conservation of drug mass are directly
linked to the thermodynamics state of the stratum corneum lipids. The chapter
concludes with general modeling considerations to capture increased mass trans-
port associated with electroporation of the skin.
Keywords
Lipid, phase transition • Thermodynamic • Transdermal
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1220
The Stratum Corneum and the Lipid Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1221
Stratum Corneum Lipid Thermal Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1222
Conceptual Description of Local Transport Region Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1225
Mathematical Description of Local Transport Region Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1227
S. Becker (*)
Mechanical Engineering Department, University of Canterbury, Christchurch, New Zealand
e-mail: sid.becker@canterbury.ac.nz

DOI 10.1007/978-3-319-32886-7_14
1220 S. Becker
Modeling Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1229

The Skin as a Composite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1230
The Stratum Corneum Defect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1231
Thermophysical Parameter Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1231
Computational Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1232
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1233
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1233
Introduction
Skin electroporation has been proposed to increase the permeability of the skin to
mass transfer (Denet et al. 2004). During skin electroporation the skin is exposed to a
series of intense electric pulses. When the drop in electric potential across the skin
exceeds a critical value, the skin’s permeability can increase by orders of magnitude.
This increase in permeability is attributed to the creation of microscopic aqueous
pores in the skin’s outer microstructure. The nature of the changes to the skin’s
structure depends on electroporation pulse characteristics such as pulse shape, pulse
intensity, and pulse duration (Pavselj et al. 2015).
The most common types of pulse shapes used in skin electroporation are the square
wave pulse and the exponentially decaying pulse. The square wave pulse has a
constant voltage during the entire pulse application. The exponentially decaying
pulse amplitude is initially at its maximum value V0 and then decreases exponentially
in time as: V(t) = V0∙exp(t ∕ τ) where the time constant τ regulates the rate of decay.
Skin electroporation pulses have been classified into two primary regimes: high-
voltage short-duration pulses (HV) and low-voltage long-duration pulses (LV). The
two pulsing regimes result in different physics. While for square wave pulses the
distinction between HV and LV is relatively clear, for exponentially decaying pulses,
this distinction can be less obvious when the tail of the pulse is very long (Pavselj
et al. 2015).
The short HV electroporation pulse durations can reach 1 ms but are typically
100 μs or less. When the drop in electric potential across the skin reaches some
critical value (30–100 V) (Pliquett et al. 1995), skin resistance (both ionic and
molecular) drops several orders of magnitude (Denet et al. 2004). In the short
pulse regime, it is believed that nm-sized pores develop within the barrier of the
skin. The nm-sized pores associated with the short HV electroporation perforate the
skin in a more or less homogenous distribution. This result is very helpful when the
drug is considered to be small (low molecular weight).
This chapter considers the influence of the long duration LV pulses (lasting up to
several hundred ms). Such LV pulses make possible two important secondary
effects: electrophoresis and Joule heating. The longer duration pulse can result in
significant electrophoretic effects that can drive large charged molecules into the
skin. The second important effect of long duration skin electroporation pulses is
Joule heating which results in localized temperature rises that contribute to increased
permeability of the SC by lipid chain melting.
64 Modeling Transdermal Delivery by Electroporation: The Thermodynamic. . . 1221
Early researchers of skin electroporation used the phrase local transport region
(LTR) to describe a phenomenon in which the applied pulses result in “large” μm-sized
regions of increased permeability (Pliquett et al. 1998). The results of in vitro studies
show that within the LTR the electrical and mass permeabilities may be many orders of
magnitude greater than outside the LTR and that development of LTRs is always
associated with thermal effects (Denet et al. 2004). This is believed to be a conse-
quence of the response of the SC lipids to the elevated temperatures that can occur as a
result of resistive heating (also called Joule heating) during the applied LV pulse.
This chapter describes the current understanding of the physics underlying the
relationships between the Joule heating associated with an LV pulse and the perme-
ability increases that are indicative of the growth of the LTR.
The Stratum Corneum and the Lipid Barrier
The outer part of the skin is called the epidermis. Its thickness varies depending on
the location of the body and varies from person to person. The epidermis is thickest
at the palms and soles of the feet reaching about 1.5 mm, while at the eyelids the
epidermis is the thinnest at about 0.05 mm. The epidermis is without vasculature and
acts as a protective barrier preventing infection and molecular transport. The epider-
mis can be considered to be an assembly line of the transformation of cells: the
epidermal base cells are viable and living, while at the outer surface the cells have
transitioned into their dead and flat form. Below the epidermis is the highly vascular
dermis (0.3–3 mm thick). Directly below the dermis, at most sites on the body, lies a
fatty layer that acts to conserve body heat. At most sites on the body, the skin is
perforated by appendages in the form of sweat glands and hair follicles.
The thin outermost layer of the epidermis is the Stratum Corneum (SC). The SC is
composed of 15–20 layers of corneocytes (flat dead cell shells) which are
interconnected by a lipid lamellar bilayer structure in a crystalline-gel phase. The
corneocyte-lipid matrix is depicted in Fig. 1. While the thickness of the SC also
varies depending on body location, consider that at the forearm it is about 20 μm
thick and at the shoulder it is about 10 μm thick.
The SC provides the greatest resistance to transport: the permeability to molecular
transport can be several orders of magnitude lower in the SC than in the adjacent
layers of the skin. Thus the SC receives the most attention in developing transdermal
transport protocols which is the subject of this chapter.
In order to give a conceptual understanding of the architecture and structure of the
SC, a brick and mortar model is sometimes employed. A depiction of a brick and
mortar model is presented in Fig. 1. Here the corneocytes are represented by the
bricks and the lipid sheets are represented by the mortar filled spaces between the
bricks. Note that the lipid structure is interconnected so that, in principle, there is a
continuous lipid-filled pathway through the SC. The pathway that weaves around the
corneocytes through this lipid structure is called the intercellular route. Because the
intercellular route is very indirect and tortuous, the length of the path that a molecule
takes through this route can be 10–50 times longer than the thickness of the SC
1222 S. Becker
Fig. 1 General representation of the stratum corneum, the brick and mortar representation, and the
three primary transport routes
(Lane 2013). By contrast, the much shorter transcellular route passes straight
through the corneocytes and the lipids; this path is possible but not common. A
final potential path through the SC is called the transappedageal route; as its name
indicates, this route follows a pathway along skin appendages such as sweat glands
and hair follicles. However, since the skin appendages occupy only about 0.1% of
the total skin area, this is a relatively restricted route (Lane 2013). In practice it is the
tortuous intercellular route through the lipid-filled spaces of the SC that most
transport follows (Lane 2013).
In order to use this intercellular, i.e., lipid route, researchers in the field of
transdermal drug delivery have gone to great efforts to understand how to manipu-
late the lipid microstructure. This chapter considers the manner in which the
thermodynamic behavior of the lipid structure can be related to LV electroporation.
In order to better understand this relationship, consider next the response of the lipid
structure to thermal state.
Stratum Corneum Lipid Thermal Behavior
Because LV pulses have been shown to result in localized intense temperature rises
at sites on the SC (Denet et al. 2004), this section concerns the behavior of the SC
lipid structure at elevated temperatures.
The composition of the SC lipid structure is responsible for the skin barrier
function. In its undisturbed state, the SC lipid structure is highly organized in a
lamellar phase that is composed of sheets of lipid bilayers (see insert Fig. 1). This
lipid structure is known to become destabilized at elevated temperatures and the
structural rearrangement at elevated temperatures has been shown to positively affect
the SC’s permeability. Different experimental techniques show this restructuring of
the SC lipids at elevated temperatures. For example, x-ray diffraction microscopy
shows that the organized lamellar structure of the SC lipids begins to disappear at
temperatures between 60 C and 70 C (Silva et al. 2006). When SC lipids are
studies using polarized light thermal microscopy, the lipid structures are shown to
dissolve at temperatures around 60 C (Silva et al. 2006). The SC has also been
shown to be much more permeable to water flux at temperatures around 70 C
(Golden et al. 1987).
Perhaps the most useful data that describes the thermal behavior of the SC lipids
is gained from differential scanning calorimetry (DSC). The motivation behind DCS
testing is to identify and define the thermal transitions of the SC. In DSC, a sample of
SC is heated so that its temperature increases at a constant rate ( C/min). The
principle is that during this controlled temperature rise, the rearrangement of the
lipid structures undergoing phase transitions requires additional heating. By care-
fully monitoring the magnitude of the heating, the phase transition temperatures can
be identified.
It is believed that within the temperatures in the range 40–130 C, the SC experi-
ences multiple endothermic transitions. Here the high-temperature transitions of the SC
are considered by adopting the convention: phase changes E, F, and G. Figure 2a
depicts data representative of a DSC study for which the three lipid transition stages are
reflected by the steep portions of the heat versus temperature curve.
Phase change E which occurs from about 65–72 C is associated with the
disordering of the lamellar lipid phase (Tanojo et al. 1999). Phase change E is
believed to be responsible for the noticeable increases in the permeability of trans-
port through the intercellular route. Therefore understanding phase change E is
critically important when attempting to model the permeability increases associated
with thermal effects of skin electroporation.
Secondary lipid melting has been associated with phase change F at temperatures
around 80 C, but this is believed to be related only to the lipids that are bonded to
the corneocytes (Tanojo et al. 1999). Phase transition G is less critical to the barrier
function; it is related to irreversible protein denaturation at temperatures above 90 C
(Van Duzee 1975).
In order to predict the permeability increases, it is necessary to approximate the
degree to which lipid phase transition E has taken place. Consider that DSC data can
provide the total additional amount of heat required for lipid phase transition E. This
is the latent heat ΔHE that is absorbed by the SC as it is heated from TE1 (at which
phase transition E begins) to TE2 (the temperature at which phase transition E
concludes).
One established method that is used to predict the state of the SC represents the
degree of SC lipid alteration as the ratio of heat added to the lipids to the heat
1224 S. Becker
Fig. 2 (a) Representative

differential scanning
calorimetry data indicating the
three phase changes. (b)
Rectangular specific heat
versus temperature
representation
required for lipid phase transition. The function referred to as the lipid melt fraction
is used to describe the degree of lipid disorder. It is mathematically defined as:
ðH cSC ðT T E1 ÞÞ
φ¼ (1)
ΔH E
where cSC is the SC specific heat capacity and ΔHE is the latent heat associated with
phase transition E. The total enthalpy, H, is defined as:
ðT
H¼ cSC, APP dτ ½T E1 < T T E2 (2)
T E1
where TE1 and TE2 are the temperatures over which transition E takes place, and
cSC,APP is the apparent specific heat of the SC.
Equations 1 and 2 can provide an estimate of the lipid state when the temperature
dependence of the specific heat is approximated directly from the DSC data. As an
alternative to the direct representation of the heat versus temperature curves of
Fig. 2a, it is often more convenient to approximate the DSC data using a simple
rectangular specific heat versus temperature curve as depicted in Fig. 2b. In this way,
the apparent SC specific heat, cSC,APP, can be represented as the sum of the specific
heat capacity and the latent specific heat as:
cSC, APP ¼ cSC þ cSC, L (3)
where cSC,L is the latent specific heat:
ΔH E
cSC, L ¼ (4)
T E2 T E1
When the specific heats are treated as constant over the phase transition temperatures
(as in Fig. 2b), Eqs. 3 and 4 can be substituted into Eq. 2 to arrive at:

ΔH E
H ¼ cSC þ ðT T E1 Þ (5)
T E2 T E1
When Eq. 5 is substituted into the melt fraction of Eq. 1 and simplifies to a ratio of
temperature differences, the result is:
T T E1
φ¼ (6)
T E2 T E1
By approximating the DSC data of Fig. 2a as the rectangular shaped specific heat curve
of Fig. 2b, it is possible to predict the state of the lipids by the simple ratio of temperature
differences of Eq. 6. The lipid melt fraction is a thermodynamically based function that
can be used to represent the degree of lipid disorder in the range 0 φ 1.
When φ = 0, the lipids are in their unaltered state. In this case, the intercellular
route is composed of lipid in their orderly lamellar structures which is very resistive
to transport. Additionally this organized structure is associated with a SC that is
electrically resistive. When the SC is heated above lipid transition temperatures, the
melt fraction increases so that φ > 0, and the SC experiences an increase in
electrical conductivity and permeability to mass transport. The limit of these
increases occurs at temperatures above TE2 for which φ = 1. At a melt fraction of
unity, the lipid architecture is in its completely unstructured state so that the SC is
very electrically conductive and is much more permeable to mass transport. A melt
fraction of φ > 0 is associated with the permeability increases that are indicative of
the LTR; in this way, within the LTR φ > 0, while outside of the LTR φ = 0.
Conceptual Description of Local Transport Region Evolution
In order to better understand the model of the LTR development during LV electro-
poration that results as a consequence of thermally induced lipid phase transition, it
is helpful to begin with a simple conceptual representation of the process. This
1226 S. Becker
Fig. 3 Conceptual representation of local transport region evolution by electroporation
process is depicted in Fig. 3. Consider the unaltered SC as in Fig. 3a. Here the
electrically resistive SC surrounds a small region that is representative of an
appendageal pore or some defect in the SC microstructure. This defect region exists
prior to the application of the pulse and it has a higher electrical conductivity than the
surrounding SC. Recall that in the case of skin appendageal macropores, they only
occupy a small fraction of the total skin area, so that the existence alone of a defect
does not preclude the need for increased permeability. When the LV electric field is
applied, the current will prefer the high conductivity pathway through the
preexisting pore to the surrounding electrically resistive SC (Fig. 3b). This high
current density will result in resistive heating (also called Joule heating). The LV
pulse duration is long enough to sustain the temperature rises required for the lipids
in the surrounding SC to experience phase transition. As the lipid structures are
altered, the SC electrical conductivity within this region increases allowing the
electric current to penetrate this part of the SC (Fig. 3c). The current density is
proportional to the area affected by lipid phase transitions; thus as the affected
area grows, the current density within this area decreases. The area affected by
lipid phase changes (the LTR) continues to grow either until the end of the electric
pulse or until the current density within the LTR is so low that further lipid melting
cannot be sustained. This process is representative of the growth of the LTR; within
the regions of the SC that have experienced lipid thermal phase transitions, the
permeability is much higher than in the surrounding unaffected regions of the SC
(Fig. 3d).
Mathematical Description of Local Transport Region Evolution
As the electric field is applied to the skin, the current attempts to cross the electrically
resistive SC. Any SC location with a lower electrical resistance (as would be
associated with a sweat gland or hair follicle) will result in a much higher current
density. This high current density results in the Joule heating that provides the
necessary power for the sudden intense local temperature rises. As the lipids
experience phase transition, the electrical conductivity of the SC in these regions
increases. This section presents the governing equations that capture this coupled
relationship between the electrical field and the thermal field.
The temperature distribution is governed by the conservation of thermal energy:
@T
ρSC cSC, APP ¼ ∇ ðkSC ∇T Þ þ QJ (7)
@t
where ρ is the density, k is the thermal conductivity, T is the temperature, and t is the
time. To fully capture the effect of phase change, the apparent specific heat, cSC,APP,
is represented by Eq. 3.
The latent specific heat of Eq. 3 depends on the lipid temperatures (it is phase
dependent). Recalling the discussion of lipid thermodynamic behavior of section
“SC Lipid Thermal Behavior” and using a rectangular specific heat versus temper-
ature approach (Fig. 2b), the latent specific heat may be represented as:
8
>
> 0 for T < T E1
>
> T T E1
>
> ΔH E for T E1 T T E2
>
>
> T E2 T E1
< T T E2
cSC, L ðT Þ ¼ ΔH F for T E2 T T F2 (8)
>
> T F2 T E2
>
> T T F2
>
> ΔH G for T F2 T T G2
>
>
>
: T G 2 T F21
0 for T F2 < T
Here the subscripts refer to the phase transition E, F, or G. Note that DSC data can
provide the heat of transition ΔH values for each phase change as well as the
associated temperature ranges which are provided in Table 1.
1228 S. Becker
Table 1 Differential scanning calorimetry data of stratum corneum lipid phase change (Adapted
from Cornwell et al. (1996) and Silva et al. (2006))
Phase change [T1,T2] C ΔH (J/kg)
E [65,75] 5500
F [75,90] 4000
G [90,110] 4700
The term, QJ, of Eq. 7 is the Joule heat generated from the electric field. It is the
Joule heating that results in the intense temperature rises that are required for thermal
phase transitions. The magnitude of the Joule Heating may be represented as:
QJ ¼ σ SC j∇ϕj2 (9)
Here σ SC is the SC electrical conductivity and ϕ is the electric potential of the applied
electric field.
Consider that once the local temperature is determined, the extent of lipid melting
can be expressed through the melt fraction of Eq. 6 which is rewritten here for
emphasis:
8
>
< 0T T for T < T E1
E1
φð T Þ ¼ for T E1 T T E2
>
: T E2 T E1
1 for T E2 < T
The electric field’s transient non-Ohmic behavior occurs at very short time scales
(sub ms) (Chizmadzhev et al. 1998) relative to those of thermal (ms to s) and mass
transport (min to hour) and can be neglected for time steps around 1 ms
(Chizmadzhev et al. 1998). To model the macroscale electrical behavior of the SC
during LV electroporation is often represented by the Laplace equation:
∇ ðσ SC ∇ϕÞ ¼ 0 (10)
The electrical conductivity of the SC has been shown to experience increases during
the disruption of the SC architecture. Experimental observations on the thermal
dependence of SC electrical resistance have shown that the magnitude of the electrical
resistance can drop by two orders of magnitude at lipid thermal phase change
temperatures (Craanevanhinsberg et al. 1995). Large changes in the SC electrical
conductivity will strongly influence the electric potential of Eq. 10 as well as the
Joule heating in Eq. 9 (Becker et al. 2012, 2014; Becker 2011, 2012; Becker and
Kuznetsov 2008). To capture this effect, the studies have related the local SC effective
electrical conductivity increase directly to lipid melt fraction by a linear relation:

σ SC ¼ σ SC, U þ φ σ SC, M σ SC, U (11)
where σ SC,U is the SC electrical conductivity associated with the unaltered lipid
structure (φ = 0), and σ SC,M is the electrical conductivity associated with the SC
after phase change E is complete (φ = 1).
Electrically driven transdermal delivery is negotiated by the three modes of
transport: electrophoresis, electroosmosis, and diffusion (Denet et al. 2004). Studies
focusing on electrically driven transport of large charged molecules show that
electroosmotic effects are negligible compared to electrophoresis forces (especially
for the relatively short electroporation pulse). Homogenous tissue in vivo electro-
poration studies show that electrophoretic forces dominate in the transport of large
molecules (Pavselj and Preat 2005). The transport of solute through the SC and into
the underlying domain is formulated from the Nernst–Planck equation:
@C
¼ ∇ ðmSC C∇ϕÞ þ ∇ ðDSC ∇CÞ (12)
@t
Here the term C is the concentration of the drug which is expressed in units of mass per
unit volume. The transport coefficient mSC is the effective electrophoretic mobility of
the solute (drug) in the SC, and DSC is the effective diffusion coefficient in the SC. The
magnitude of the transport coefficients depend on the drug mass, the drug shape, and
the drug charge as well as the microstructure characteristics of the medium (here of the
SC). Their values can be approximated experimentally or theoretically.
In order to show the link between the increase in the SC lipid transport coefficients
and the lipid state, consider once again the melt fraction. The experimentally observed
increases in permeability to mass transport can be satisfied by relating the transport
coefficients directly to the lipid melt fraction of Eq. (6): mL ¼ f ðφÞ and DL ¼ gðφÞ. A
linear dependence of transport coefficients on the lipid melt fraction has previously
been posed, so that

mSC ¼ mSC, U þ φ mSC, M mSC, U (13)
and

DSC ¼ DSC, U þ φ DSC, M DSC, U (14)
The transport coefficients mSC,U and DSC,U correspond to the low magnitudes
of mobility and diffusion that are associated with the drug in the SC whose
lipids have not experienced any thermal transition φ ¼ 0. The transport coefficients
mSC,M and DSC,M represent the highest values of transport coefficient after full lipid
melting φ ¼ 1.
Modeling Considerations
This chapter has so far focused only on the SC during skin electroporation. In order
to model LTR evolution and the subsequent transport of drug, it is necessary to
consider the skin as a whole, the associated parameter values, and the computational
and numerical methods used to model the conservation equations.
1230 S. Becker
The Skin as a Composite
In practice, electroporation is conducted on the entire skin (not just the SC).
Consider the composite representation of the skin presented in Fig. 4 that includes
the different layers of the skin as well as an applicator gel at the surface of the SC. In
such a case the conservation of charge, the conservation of thermal energy, and the
conservation of drug mass must be considered in each of the different composite
layers.
In the gel layer
@T
ρG c G ¼ ∇ ðkG ∇T Þ þ σ G j∇ϕj 2
@t |fflfflffl{zfflfflffl}QJ
∇ ðσ G ∇ϕÞ ¼ 0 (15)
@C
¼ ∇ ðmG C∇ϕÞ þ ∇ ðDG ∇CÞ
@t
Note that in the conservation of thermal energy, the symbol representing Joule
heating, QJ, has been replaced by its definition.
In the epidermis
@T
ρED cED ¼ ∇ ðkED ∇T Þ þ σ ED j∇ϕj2
@t
∇ ðσ ED ∇ϕÞ ¼ 0 (16)
@C
¼ ∇ ðmED C∇ϕÞ þ ∇ ðDED ∇CÞ
@t
In the dermis
@T
ρD c D ¼ ∇ ðkD ∇T Þ þ σ D j∇ϕj2 ωcb ðT T b Þ þ QM
@t
∇ ðσ D ∇ϕÞ ¼ 0 (17)
@C
¼ ∇ ðmD C∇ϕÞ þ ∇ ðDD ∇CÞ
@t
Fig. 4 Composite
representation of the skin
The conservation of thermal energy in the dermis has been structured to include the
effects of blood perfusion and of volumetric energy generation. The perfusion rate ω
describes the mass flow rate of blood per volume of dermis. While dermis perfusion
rates can fluctuate, a good approximation is about ω = 2 kg·m3s1. The term Ta is
the arterial blood temperature (assigned a value of 37 C), and the parameter cb is the
specific heat of blood: cb = 3800 kJ/kgK. In the living tissue layers, the relative
contribution of the volumetric metabolic energy generation term, QM, is small
compared to the heat supplied by Joule heating so that it is often neglected.
Experimentally it has been shown that, depending on pulse intensity, when
intense electric pulses are applied to living tissue, the perfusion of blood may be
locally temporarily inhibited (80% reduction) for time scales ranging from minutes
to hours (Jarm et al. 2010). Even if perfusion is uninhibited it is likely that during the
short time interval of the application of the pulse, the presence of perfusion makes a
negligible contribution to the thermal profile.
The Stratum Corneum Defect
Recall the conservation of electric charge of Eq. 10 that states
∇ ðσ SC ∇ϕÞ ¼ 0
Note that in the conceptual description of Fig. 3, the electrical conductivity of the SC
is higher within the SC defect than in the surrounding SC. This is required in order to
achieve the concentrated current density that is needed for the resistive Joule heating
of Eq. 9. With this in mind, it is very important to note that this thermodynamic
approach requires that the electrical conductivity of the SC prior to the application of
the pulse is not uniform throughout the SC and that there is at least one region in the
pre-electroporated SC whose electrical conductivity is higher than in the surrounding
SC. To account for this, one can simply assign a higher value of electrical conduc-
tivity in a small region of the SC. An upper limit of conductivity value in the defect is
the conductivity of the applicator gel. In this way:
Inside the defect: σ SC ¼ σ G (18)
In previous studies, the defect region has been modeled as a small cylindrical volume
of SC with a diameter of 5–20 μm.
Thermophysical Parameter Values
The thermodynamic properties of the skin layers such as density, heat capacity, and
thermal conductivity are well established. Electrical properties can be a bit more
challenging to find reliable estimates for. This is because the electrical properties of
biological tissue are often sensitive to the condition of the tissue (hydration level and
1232 S. Becker
Table 2 Representative values of the thermophysical parameters of the skin (Adapted from Zorec
et al. (2013))
Gel SC Epidermis Dermis
Thickness, L (μm) – 20 50 300
Thermal conductivity, 0.6 0.2 0.21 0.29
k (W/mK)
Density, ρ (kg/m3) 1000 1500 1110 1115
Heat capacity, c (J/kgK) 4180 3600 3600 3800
Electrical conductivity, σ 1 σSC-U = 1 104 0.1 0.15
(S/m) σ SC-M = 5 102
Electrophoretic mobility, 1.5 108 mSC-U =1 1015 1 109 1 109
m (m2/Vs) mSC-M = 1 1010
Diffusion coefficient, 1.5 1010 mSC-U =1 1017 3 1011 3 1011
D (m2/s) mSC-M = 1 1012
how it is stored). Generally order of magnitude estimates have been used to represent
the conductivity values. The transport coefficients, electrophoretic mobility and the
diffusion coefficient, can be strongly influenced by tissue state. These are also very
dependent on the drug molecular weight and size.
Some representative thermophysical property values are provided in Table 2. The
electrical conductivities listed are order of magnitude estimates. The SC conductivity
values corresponding to the unaltered lipid state, σ SC,U, and the electrical conductivity
associated with total alteration of the SC lipids, σ SC,M, have been chosen to represent
the two order of magnitude increase in electrical conductivity with lipid restructuring
as suggested by the results of reference (Craanevanhinsberg et al. 1995).
The transport coefficient values are also order of magnitude representations and
are representative of calcein. The properties of the gel are representative of a
phosphate buffer solution (pH 6.5, 100 mM).
Computational Considerations
The solution of Eqs. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17 must be evaluated
numerically. In the past, these equations have been recast in discrete finite volume
forms. The computational domain representing the space occupied by the skin is
divided into individual control volumes, and the electrical charge, the thermal
energy, and the mass are conserved in each of these control volume. When the
space is discretized, it is reasonable to refine the mesh in regions that are anticipated
to experience large variations in parameters values. In the case of LV skin electro-
poration, refine the mesh within the SC around the defect region. This will be
particularly helpful when attempting to resolve the lipid melt fraction and its
influence on the transport coefficients of Eqs. 11, 13, and 14 (Becker 2012; Becker
and Kunetsov 2007, 2008).
The conservation of thermal energy is coupled directly to the conservation of
charge through the Joule heating term. Within the SC, the transport coefficients
(conductivity, mobility, and diffusion coefficient) are temperature dependent

through the melt fraction. Because the equations are so strongly coupled, they
must be solved iteratively. These coupled equations have previously been solved
using a combined explicit-implicit approach. The implicit formulation requires an
iterative approach between time steps. To speed convergence it has been previously
proposed that all properties within the SC of the current time step are evaluated
using the melt fraction of the previous time step (Becker 2012; Becker and
Kunetsov 2007, 2008).
Note that the Laplace equation that is used to represent the conservation of
charge of Eqs. 10, 15, 16, and 17 is not explicitly transient (it has no time
derivative). This is generally an accepted approximation because the electric field
develops on a time scale that is so much shorter than the thermal field. Numerically
what can be done is that the steady Laplace equation is reevaluated at each time step
(Sel et al. 2005).
Conclusions
Permeability increases resulting from the thermal effects associated with skin
electroporation-related Joule heating can be explained in terms of the lipid chain
melting. In this way, the transport coefficients can be related directly to the lipid
state. By considering differential scanning calorimetry data of stratum corneum
thermal behavior, the phase transition E has been proposed to represent the thermal
alteration of the lipid microstructure. When the stratum corneum thermal data is
recast in a rectangular specific heat versus temperature representation, the degree of
lipid melting can be approximated by a ratio of temperature differences. This
principle allows the transport coefficients and the electrical conductivity of the
stratum corneum to be predicted from the local temperature distribution.
Cross-References
▶ Mass Transfer of Electrolytic Species During Electric Field-Based Tumor

Treatments
References
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Becker SM (2012) Transport modeling of skin electroporation and the thermal behavior
of the stratum corneum. In: Vafai K (ed) 4th international conference of porous media
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and its applications in science, engineering, and industry, Potsdam. AIP Conf Proc, pp
365–370
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Application of Numerical Simulation
Techniques for Modeling Pulsed Electric 65
Field Processing
Thies Wölken, Johannes Sailer, Francisco Daniel Maldonado-Parra,

Tobias Horneber, and Cornelia Rauh
Abstract
The application of numerical simulation techniques is becoming more and more
popular with the progress of computational capabilities. It finds use in the context
of pulsed electric field (PEF) research in various ways, especially to predict
treatment efficacy and homogeneity. This contribution aims to be an orientation
guideline for numerical investigations. It provides a mathematical framework as
well as an understanding of the interrelations of fluid dynamic, thermal, and
electric fields that are important to investigate PEF processes. Emphasis is put on
the versatility of numerical models: modeling of permeabilization and inactiva-
tion of enzymes and spoiling agents, prediction of treatment homogeneity,
modeling of particulate fluids, modeling of microscopic aspects of the permeabi-
lization process, and geometry and process optimization are described exemplary
for the broad range of possible applications to motivate novel approaches.
The presented theory is used for a case study: the flow fields, temperature
fields, and electric field strengths are simulated for three different coaxial treat-
ment chamber geometries. They vary in the shape of the central insulator. The
geometry with an arc-shaped insulator provides the highest treatment intensity
with regard to the electric field strength and treatment times but also has the
largest coefficient of variation of the electric field strength in the treatment area.
The results suggest that a trade-off between treatment effect and homogeneity is
necessary. However, the fields can be used to compute the residual activity of the
enzyme pectinesterase (PE) with a balance equation in a second simulation. A
sink term in the balance equation is used to couple in the effect the treatment has
on the enzyme to predict its inactivation spatially. The approach offers a more
T. Wölken (*) • J. Sailer • F.D. Maldonado-Parra • T. Horneber • C. Rauh

Food Biotechnology and Food Process Engineering, Technical University of Berlin, Berlin,
Germany
e-mail: thies.woelken@campus.tu-berlin.de; johannessailer@gmail.com;
f.maldonado-parra@tu-berlin.de; tobias.horneber@tu-berlin.de; cornelia.rauh@tu-berlin.de

DOI 10.1007/978-3-319-32886-7_42
1238 T. Wölken et al.
quantitative basis for process evaluation. Comparing the residual activities, the
arc insulator indeed provides the largest treatment effect. It also reveals that the
differences in treatment homogeneity are smaller than the electric field strength
distributions suggest. Balancing the residual activity increases the conclusiveness
of the numerical investigation.
Keywords
Pulsed electric fields • PEF • Numerical simulation application • Computational
fluid dynamics • Residual activity • Geometry optimization • Process
optimization
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1238
Governing Equations and Constitutive Relations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1241
Thermophysical Fluid Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1244
Application Examples for Numerical Simulation Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1247
Modeling of Permeabilization and Inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1247
Treatment Homogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1250
Dense Particulate Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1252
Modeling of Microscopic Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1253
Treatment Chamber Geometry and PEF Process Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1254
Finite Volume Case Study: Influence of Insulator Geometry on Treatment Intensity
and Homogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1256
Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1256
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1260
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1263
Notation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1264
Latin Characters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1264
Greek Characters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265
Introduction
The food industry aims to provide high quality and safe food at minimal resource
demand. It is the task of food technology to find appropriate methods to achieve
these goals. The importance of minimal food processing is strongly related to the
food quality. Common high-temperature treatment runs the risk of reducing valuable
nutrients, but sufficient treatment is inevitable in order to guarantee a safe and shelf
stable food. Thus, nonthermal and nonchemical treatment alternatives are sought
after to preserve taste, functionality, and nutrition of the food while assuring its
safety.
Pulsed electric field (PEF) processing is known as a nonthermal food processing
method. This designation is not to be mistaken with an isothermal process. Temper-
ature increase caused by internal energy production due to the electric field is a side
effect of the treatment which can be considered for process design. Recent PEF
65 Application of Numerical Simulation Techniques for Modeling Pulsed. . . 1239
Fig. 1 Schematic representation of a collinear PEF treatment chamber
treatment chambers consist of special arrangements of electrodes and insulators that

are both in contact with the treated matter. Therefore, they represent an electric
circuit with resistive properties. The concept of PEF treatment can be explained with
the example of a collinear treatment chamber schematically given in Fig. 1. The
dotted line represents the rotational axis of the axisymmetric geometry. The insulator
in the middle separates the cathode (+) from the anode ground (GND) which causes
an electric field to occur in the region of the central insulator during a pulse of high
voltage on the cathode. This region is where the food is treated. The liquid food
enters the chamber from the left, resulting in an overlap of the electric field, thermal
field, and fluid dynamic field of the fluid transport. The electric field strengths
(voltage-to-distance ratio) used for microbial inactivation by PEF treatment are
typically in the range of 10–50 kV/cm and for the extraction of plant materials and
pretreatment of meat for processing only about 0.7–3 kV/cm (Toepfl et al. 2006).
In high-temperature treatment, the heat is transported by convection and heat
conduction through liquid or solid food. Local overheating and under-heating can
occur especially if the process is dominated by heat conduction. Nonhomogeneous
food treatment is an important problem of the process and its design. Volumetric
food treatment methods like Joule heating, microwave processing, high-pressure
thermal sterilization, and pulsed electric field processing support the reduction of
such local effects. The electric field as a driving force of PEF processing acts without
the need of material transport processes on macroscopic level. However, as the use of
volumetric methods does not naturally ensure homogeneous treatment, particular
attention is required for homogeneous process conditions as is discussed in section
“Treatment Homogeneity.”
Besides a change in temperature, PEF causes different effects depending on the
treated material. By inducing an electric field in the treated matter structural changes,
chemical reactions, reversible and irreversible permeabilization of cells, and inacti-
vation of enzymes and microorganisms can occur. These effects can be used for
various applications of PEF treatment. Applications reach from medicine, where
reversible permeabilization of cell membranes is used to introduce active com-
pounds, to large installations in food technology, where PEF is mainly used to
permeabilize cell membranes to make their content more easily available or inacti-
vate enzymes and microorganisms. Plant cell permeabilization aims to extract
valuable cell contents, whereas inactivation of enzymes and microorganisms is
used to prevent spoilage (Krauss et al. 2011). Numerical simulation aspects of
inactivation are discussed in section “Modeling of Permeabilization and
Inactivation.”
The main parameters of membrane permeabilization are identified as the electric

field strength, the treatment time (the time the media is exposed to the electric field,
see (28)), and the treatment temperature. The local treatment time is a result of the
velocity distribution throughout the chamber. Therefore, a simultaneously gathered
data of the electric, thermal, and fluid dynamic field, and preferably the spatiotem-
poral treatment history is desirable for complete analysis. Experiments capable of a
volumetric data acquisition of all these fields have not been conducted due to a lack
of available techniques. In contrast, modeling and simulation with numerical
approaches provide locally resolved knowledge of the three physical fields. There-
fore, modeling is a promising approach to further enhance the understanding and
optimization of PEF processes. Analysis conducted and verified on laboratory-scale
chambers can be scaled up to industrial processes by varying the geometry and
boundary conditions with respect to representative dimensionless parameters.
From a biological, physical, and chemical perspective, PEF is a very complex
process which includes microscopic and macroscopic phenomena. Microscopic
phenomena like chemical reactions or permeabilization of cell membranes take
place on the length scale of nanometers to micrometers and on the time scale of
nanoseconds to microseconds. Macroscopic phenomena like the velocity profile of
the fluid or the distribution of the electric field are observed on a length scale from
millimeters to meters and on a time scale from milliseconds to seconds.
Food can be represented as a multicomponent mixture of solid and fluid matter,
with each component having its own mechanic, thermodynamic, chemical, and
electrical properties. Most food systems contain hundreds up to thousands of
components. Assuming it is possible to write down all equations describing the
relevant phenomena from microscopic up to the macroscopic scale, a numeric
simulation of PEF treatment fails because of a lack of computational resources.
This does not imply an impossibility of modeling microscopic aspects. In fact,
microscopic studies of treatment effects exist and expand the understanding of the
underlying mechanisms, as will be recalled in section “Modeling of Microscopic
Aspects.”
From an engineering point of view, the macroscopic fields are of interest, and a
complexity reduction of the modeled system to macroscopic scales is reasonable.
Using appropriate simplification assumptions and models is an essential issue to
generate data in good agreement with reality. The reduction enables a simulation of a
full-scale geometry of the process. Microscopic aspects like the membrane
permeabilization can still be considered by the implementation of representative
empirical models. Such a procedure makes numerical simulations a versatile tool for
PEF application analysis. Research topics which can benefit from the flexibility of
numerical simulations will be given in section “Application Examples for Numerical
Simulation Techniques.” Before discussing the applications, the theoretical back-
ground for modeling is given in section “Governing Equations and Constitutive
Relations.” It provides an overview of the mathematical equations such as mass
conservation, momentum conservation, balance of internal energy, and the numer-
ically modeled transport of matter. They are reduced to a system of equations that
must be found to be in good agreement with experimental results and are sufficient
for chamber design and description of treatment effects.
It is followed by a discussion of thermophysical material properties in section
“Thermophysical Fluid Properties” to provide an understanding of the inter-physical
relations of the different fields.
Finally, a model case of simulating continuous PEF treatment in two collinear
chambers with different insulator geometries is explained and presented as an
application example in section “Finite Volume Case Study: Influence of Insulator
Geometry on Treatment Intensity and Homogeneity.”
Governing Equations and Constitutive Relations
The basic equations to describe PEF processes from a macroscopic point of view are
provided by continuum thermodynamics and electrostatics. Together with constitu-
tive models that relate the constitutive quantities to the field variables, they form a
closed system of partial differential equations that can be solved numerically. In the
present context, the fields of temperature, pressure, and the three velocity compo-
nents, as well as the electric potential from which the electric field can be calculated,
are of interest.
The basis for the derivation of equations are the principles of conservation of
mass, momentum, energy, and charge. For a brief overview, the thermodynamic
balance equations in their general forms are listed, followed by a description of the
modeling assumptions.
The balance equations for mass, momentum, and internal energy are:
@ρ @ρuj
Mass: þ ¼0 (1)
@t @xj
@ρui @ρui uj τij

Momentum in ith direction: þ ¼ f i ði ½1, 2, 3Þ (2)
@t @xj
@ρe @ρeuj þ qj @ui

Internal energy: þ ¼ ρτij þ ρπ e (3)
@t @xj @xj
Here, ρ is the density, t is the time, and u and x are the velocity and coordinate
vectors, respectively. The indices i and j represent the considered directions. The τ is
called the stress tensor, e is the internal energy, and q is the heat flux vector. These
three so-called constitutive quantities must be related to the field variables. The
internal energy production term π e on the right-hand side accounts for internal
working and a production of internal energy by electrical effects. Together with
the force density vector f, they must be prescribed or related to the field variables. In
contrast to the total energy, the internal energy is not a conserving quantity.
The system of Eqs. 1, 2, and 3 provides five equations for the five unknown field
variables and internal energy, mass density, and velocity components in each of the
three dimensions.
The additionally occurring a priori unknown nine components of τ, three com-
ponents of q, and the value of π e must be modeled with constitutive equations to
achieve a closed system of equations.
The investigated liquid food is a multicomponent system with varying component
densities. However, when the fluid is assumed as a single continuum, it can be
represented by one density field and its temporal change. When particles within the
liquid food, for example, fruit pieces, are considered as a particulate phase because
their spatial distribution is of interest, further sets of balancing equations become
necessary.
In case the media is considered to be incompressible, which is a common
assumption for water-like liquids, the mass balance reduces to:
@uj
¼0 (4)
@xj
For a Newtonian, incompressible fluid, the stress tensor has the form:

@ui @uj
τij ¼ pδij þ μ þ (5)
@xj @xi
Here p is the static pressure, δ is the Kronecker delta, and μ is the dynamic
viscosity. There is no general need to consider only Newtonian liquids. In food
technology, there are fluids that must be described by non-Newtonian stress tensors,
like a pseudo plastic strawberry juice. Such a case is discussed in section “Dense
Particulate Fluids.”
The magnetic and electric fields cause energy production and changes in momen-
tum by the forces acting on charged moving particles. There are general coupling
terms for momentum and total energy balance that affect the forces f in the momen-
tum balance and also coupling terms for the internal energy balance. For the internal
energy balance, only effects due to Joule heating are considered (Meneses et al.
2011a). The remaining force term is the momentum supplied by gravity g, which
here is assumed to act in the negative direction of the third coordinate component.
With Eqs. 4 and 5, the momentum balance can then be written in the form:
@ui @ui @p @ 2 ui
ρ þ ρuj ¼ þ μ 2 þ ρf i , f i ¼ ð0, 0, gÞ (6)
@t @xj @xj @xj
The heat flux is modeled by Fourier’s law which postulates the temperature
gradient to be linearly related to the heat flux by the thermal conductivity k. For an
incompressible fluid, the internal energy can be modeled as a function of tempera-
ture. The internal energy balance of Eq. 3 can be rewritten using the temperature
T and the specific heat capacity cp:

@T @T @ @T
ρcp þ ui ¼ k þ πe (7)
@t @xi @xi @xi
The dissipation, transformation of kinetic energy into thermal energy, which is a

part of the source term π e, is assumed to have a negligible contribution compared to
the internal energy production due to the heating caused by the current induced by
the electric field and is therefore not considered. This assumption has been shown to
produce reliable temperature predictions compared with experimental results
(Meneses et al. 2011b). The current induced heat production is described by the
formula for Joule heating:
j2i ðσEÞ2
πe ¼ ¼ ¼ σE2 (8)
σ σ
The formula uses the electric current density vector j, the electric field strength E,
and the electrical conductivity σ.
The inactivation process is found to depend on the strength, the frequency, and
the shape of the electric field pulse (Barbosa-Cánovas et al. 2011). Since in this
modeling approach the food system is handled as mono-component, there is no
relative velocity between the cells and the water assumed. This implies the absence
of diffusive transport of cells in the fluid.
From the partial mass balance without diffusion, the following scalar transport
equation can be derived as:
@c @c
þ uj ¼ πm (9)
@t @xj
π m is the production rate of the molar concentration of the transported quantity
concentration c. The form of the production term is discussed in section “Modeling
of Permeabilization and Inactivation.”
At last, an equation for the electric field is needed. Following (Meneses et al.
2011a) the electric field can be derived from charge conservation of free charges. In
an electrostatic case, there is no temporal change of electric charges, and if the fluid
is treated to be electrically neutral, there is no free charge density, and the conser-
vation law reduces to:
@jfree
i
¼0 (10)
@xi
In combination with Ohm’s law for the electric current jfree follows:
@σEi
¼0 (11)
@xi
A divergence-free vector field can be represented as a gradient of a scalar field. In
case of electrostatics, this field ϕ is called electric potential:
@jfree @σEi @ @ϕ
i
¼ ¼ σ ¼0 (12)
@xi @xi @xi @xi
For cases where the electrical conductivity can be assumed as constant, it reduces
to the well-known Laplace equation:
@ @ϕ
¼ Δϕ ¼ 0 (13)
@xi @xi
The Laplace equation has a unique solution if fixed values of ϕ are prescribed at
the boundary of the domain or if section-wise ϕ and gradients of ϕ are prescribed.
The electrodes can be modeled by assuming a fixed potential on their boundary.
At the insulator, the idea of continuity of the normal component of the electric
free current is used (Meneses et al. 2011b):
nF ¼ σ F EnF ¼ σ F ∇n ϕ ¼ jnI 0
jfree free
(14)
The indices F and I indicate the domains fluid and insulator. If there is no free
current on the insulator domain, a boundary condition for the electric field in normal
direction to the wall is obtained:
EnF ¼ ∇n ϕ 0 (15)
This corresponds to a zero-gradient boundary condition.
Thermophysical Fluid Properties
The presented equations and their algebraic formulation form the numerical basis for
a coupled solution of the fluid dynamic, thermal, and electric fields in PEF chambers.
These fields have strong interrelated effects on each other. An overview shall show
the benefits of solving the coupled equations simultaneously.
Material properties like viscosity and density, thermal and electrical conductivity,
and heat capacity are temperature dependent. The degree of dependence helps to
decide whether modeling of the behavior is necessary to respect significant interre-
lations. Due to the high complexity of the constitution, often empirical models for
the temperature behavior of a liquid food’s properties are used by fitting experimen-
tal data. Exemplary models are shortly listed here but cannot be generalized. They
have to be representative for the actually investigated liquids and conditions.
Resulting interrelations are discussed afterwards.
For some liquids, estimations using the values for water or aqueous solutions can
be adequate. A formulation for the density of water at a temperature TC in degree
Celsius is given in (Kessler 2002):
Table 1 Coefficients for Liquid Slope a [mS/cm C] Offset b [mS/cm]

linear conductivity
Apple juice 0.037761 1.38071
equations of liquid foods
(Toepfl 2006) Red grape juice 0.060964 1.11286
Orange juice 0.0695 1.53
Tomato juice 0.3169 3.31
Carrot juice 0.14125 2.775
Milk (3.5% fat) 0.080119 2.37714
ρWater ¼ 1000:22 þ 1:0205 102 T C 5:8149 103 T 2C þ 1:496 105

T 3C (16)
Al-Shemmeri (2012) gives a formulation for the dynamic viscosity μ of water

with a discrepancy of less than 2.5% with the temperature T in Kelvin:
μWater ¼ 2:414 105 10T140K Pa s

247:8K
(17)
For the electrical conductivity σ and temperature TC, a linear relation with a slope
a and offset b is given in Toepfl (2006) for various liquids:
σ ¼ aT C þ b (18)
Coefficients for selected liquid foods are given in Table 1 from Toepfl (2006). The
positive slope means an increase in conductivity with temperature and a decrease in
resistance. The table reveals a significant difference between different liquids.
The behavior of thermal conductivity k and specific heat capacity cp of food at
different temperatures is described in depth in Sahin and Sumnu (2006). As an
alternative to an experimental evaluation of a specific liquids property, a generali-
zation approach based on the property of the pure substances making up the liquid is
presented. In a range of 0–90 C, formulations of the thermal conductivity for pure
water, carbohydrate, protein, fat, and ash are given by Choi and Okos (1986):
kwat ¼ 0:57109 þ 1:7625 103 T C 6:7036 106 T 2C

kcar ¼ 0:20141 þ 1:3874 103 T C 4:3312 106 T 2C
kpro ¼ 0:17881 þ 1:1958 103 T C 2:7178 106 T 2C (19)
kfat ¼ 0:18071 2:7604 103 T C 1:7749 107 T 2C
kash ¼ 0:32961 þ 1:4011 103 T C 2:9069 106 T 2C
To calculate the property behavior of the whole liquid, different formulations

combining the properties of the pure substances exist. The simple parallel model
sums up the values of every ingredient weighted by its volume fraction. Additional
models are a series model, the Krischer model, the Maxwell-Eucken model, the
Kopelman model, and advanced improved prediction models. A discussion of these
models is beyond the focus of this section but is given in Sahin and Sumnu (2006).
Fig. 2 Thermophysical property models for liquids, normalized by value at TC = 10 C
For the same range of 0–90 C, Choi and Okos (1986) give a volume fraction-
weighted parallel model appropriate to predict the heat capacity cp for liquids when
their ingredients’ heat capacity is determined by:
cp, wat ¼ 4176:2 0:0909 T C þ 5:4731 103 T 2C

cp, car ¼ 1548:8 þ 1:9625 T C 5:9399 103 T 2C
cp, pro ¼ 2008:2 þ 1:2089 T C 1:3129 103 T 2C (20)
cp, fat ¼ 1984:2 þ 1:4373 T C 4:8008 103 T 2C
cp, ash ¼ 1092:6 þ 1:8896 T C 3:6817 103 T 2C
The given formulations are plotted in Fig. 2 for a temperature range of 10–90 C
normalized by their value at 10 C. The electrical conductivity is modeled for apple
juice. All other properties are modeled for water. With a difference of the maximal
and minimal values of less than 5% for the density and less than 1% for the specific
heat capacity, these dependencies can be neglected to reduce modeling effort. All
other properties are significantly temperature dependent, and representative models
are recommended.
An effect known as thermal runaway results from these thermophysical relations:
the temperature field is influenced by the electric field through Joule heating. The
food is heated by a transfer of energy from the electric field when it is subjected to
electric current. Areas with local temperature peaks also have an increased local
electrical conductivity. This results in increased current and energy input which
further promotes a temperature increase. Local overheating of the food may result
which is against the nonthermal nature of PEF treatment, causing off-flavors and
reducing the overall product quality.
The flow field as the driving force of convective transport of heat has a significant
influence on the temperature field distribution. Especially areas with long fluid
residence times should be prevented as they can enable overheating due to a lack
of heat exchange with the main flow. This is discussed further in section “Treatment
Homogeneity.”
An increased process temperature is beneficial for the electroporation effect

because membrane fluidities are increased. Models for PEF efficiency can respect
that by including temperature into the inactivation or membrane permeabilization
prediction formulae.
Application Examples for Numerical Simulation Techniques
The equations given in section “Governing Equations and Constitutive Relations”

are the framework for numerical investigations of PEF applications. They are of
general nature and have a dense data output as their solution can provide spatially
highly resolved fields for flow, electric field strength, temperature, and concentra-
tion. Therefore, numerical simulation techniques can be used for manifold applica-
tions in modeling PEF processes. This section serves to give an overview of recent
applications and to point out the high potential of numerical modeling for novel
investigations. Due to the amount of possibilities for numerical simulations in PEF
research, this section cannot be comprehensive. Instead, application examples with
various focuses are given to emphasize the diversity of feasible applications.
Modeling of Permeabilization and Inactivation
For evaluation of treatment efficacy, a prediction of the effect of the process is

fundamental. Whether inactivation of spoiling agents, enzymes, or whether cell
membrane permeabilization to improve content extraction is of interest, predictive
models are necessary to enable numerical approaches on a macroscopic scale. Using
a numero-experimental approach, empirical models based on experimental data can
be combined with the simulation results of the physical fields to predict the treatment
effect. For steady physical fields, this can be achieved in two steps. First, a static
solution for the electric, thermal, and flow field is simulated. This result can be used
in a second, time-resolved simulation, which solves a transport equation for residual
activity. The treatment effect depending on the local field values is coupled into this
transport equation as a negative source term for residual activity, using predictive
models for cell disintegration. A separation into a stationary field simulation and a
transient permeabilization or inactivation simulation is not inevitable as the equation
system for both can be solved coupled, but it is a suitable way to reduce computa-
tional effort.
As an alternative to thermal pasteurization, PEF treatment can be used to inacti-
vate microbes in food that induce spoilage due to electroporation of their cell
membranes. The inactivation can be modeled either by the means of a transport
model or by purely kinetic approaches. No generally valid model is known by now.
Specific models are based on empirical data for specific fluids and conditions.
A residual activity RA after a treatment time tt with an electric field strength E of
the considered quantity can be calculated as the ratio from final activity A to initial
activity A0:
AðE, tt Þ
RA ¼ (21)
A0
Here, the residual activity depends on the treatment time and the electric field
strength but additional influences have been documented. Sensoy et al. (1997) report
a lower survival rate for food with lower conductivity. A decrease in food conduc-
tivity enlarges the conductivity difference between food and microorganism cyto-
plasm, which makes the organisms more vulnerable to PEF processing. Kotnik et al.
(2003) identify the treatment time above a maximum voltage-to-distance ratio as a
crucial parameter. Toepfl et al. (2007) find a clear increase in inactivation for process
temperatures above 40 C.
A common kinetic formulation for the residual activity is given in Hülsheger et al.
(1981):
ðEEc Þ=λ
tt
RA ¼ (22)
ttc
It uses the electric field strength and treatment time as dominant parameters but
additionally introduces a spoiling agent-depending constant λ and critical values for
the treatment time ttc and the electric field strength EC as thresholds above which the
inactivation is initiated. With these additional parameters, the model can be adapted
to different organisms. They need secondary models depending on further input
information like temperature and medium properties.
The residual activity can also be modeled with a Weibull function of the form:
tt β
RA ¼ 10ð2:303Þð α Þ
1
(23)
This model uses the treatment time along with two parameters, α for scaling and β
for shaping of the function. The parameters make the model adaptable. It can be used
for different treatment methods because it does not depend on the electric field
strength.
The models directly depending on the electric field strength can be evaluated with
the information provided by the numerical simulation. For the Weibull approach, the
parameters must be adapted to the local electric field strength. The residual activity
can be interpreted as the local concentration of active microorganisms or enzymes
being transported through the simulation domain using a transport model approach
as presented in Krauss et al. (2011):

@RA @RA @ @RA
þ ui ¼ D þ SðT, E, tt Þ (24)
@t @xi @xi @xi
The temporal change of the residual activity is balanced by the convective and
diffusive transport depending on the diffusion coefficient D and source term S, both
depending on temperature, electric field strength, and treatment time. When diffu-
sion is neglected, this equation reduces to the scalar transport (Eq. 9). The source
term can be used to couple the empirical inactivation model into the balance equation
by a negative value. The local finite volume residual activity represents the treatment
history the spoiling agent has experienced while being transported through the
domain.
The source term S is based on empirical evidence. The experimental data base
must be broad enough to allow interpolation from discrete input like the electric field
strength or the treatment time to the occurring values in a numerical analysis. An
example from literature for enzymatic residual activity of pectinesterase PE sub-
jected to PEF treatment with varying intensity is documented in Giner et al. (2005).
Six field strengths (voltage-to-distance ratio) in the range of 19 kV/cm to 38 kV/cm
are tested, and models are fitted to the resulting residual activity. In addition to a
Weibull and the Hülsheger approach, a first-order decay kinetic is tested. It has the
general form:
RA ¼ RA0 eλ1 tt (25)
The initial residual activity RA0 is reduced depending on the value of the
inactivation rate constant λ1. The constant is used to couple the electric field strength
with the model:
cm 1
λ1 ¼ ð4:2 0:5Þ 103 þ ð266 15Þ 106 E (26)
kV μs
The electric field strength must be given in kV/cm and the resulting inactivation rate
value in 1/μs. Since the initial residual activity should either be constant or reduced by
the treatment, λ1 must be negative or zero. This gives a lower limit for the electric field
strength of about 16 kV/cm. An upper limit cannot be determined from the given data.
Due to the homogeneous nature of the fields investigated by Giner et al. (2005),
the results are useful to model local inactivation considered in the source term of
Eq. 24. The source term as the time derivative of the model is:
dRA cm 1
¼ SðEÞ ¼ RA0 4:2 103 266 106 E τf p RA (27)
dt kV μs
The derivative uses the following relation between the real-time t, the pulse width
τ, the pulse frequency fp, and the treatment time tt:
tt ¼ tτf p (28)
The source term in Eq. 27 will be evaluated with the results of the static electric
field simulation and used as input for Eq. 24 to calculate the local residual activity.
Additionally to inactivation, the PEF-induced increased electroporation of cells
can be used to extract intracellular content. Enhancements in the yield of juice or oil
expression from fruits and vegetables are addressed in section “Dense Particulate
Fluids.” Predictive models for the effect of PEF treatment on valuable extraction
have to be found in the same way as described for inactivation by empirical models.
Treatment Homogeneity
A common goal of PEF process analysis is the optimization of treatment homoge-

neity. A homogeneous treatment is desirable to ensure product quality and safety
with respect to inactivation. Over-processed parts of the food may differ in appear-
ance, taste and integrity of the food, and its ingredients. Under-processed parts
impose a risk of spoilage on the processed product and can reduce the energy
efficiency of the process, when the electric field strength is too weak. Energy is
still transferred from the electric field to the product by heating without contributing
to the desired treatment.
To increase extraction yield, a homogeneous treatment is similarly desirable to
ensure a high-yield expression from the whole treated food.
The treatment intensity depends on the food’s local specific energy input and the
treatment time. For batch chambers, the treatment time can be defined by the number
of pulses np with a constant pulse width τ for which the food is subjected to the
electric field pulses:
t t ¼ np τ (29)
For continuous processing, the number of pulses can be estimated by the pulse
frequency fp, the treatment area length L, and the average flow velocity ū or residence
time tr:
fp L
np ¼ ¼ f p tr (30)
u
This estimation is only accurate in the ideal case of a homogeneous velocity field.
In general, significant velocity differences can occur throughout the treatment area,
resulting in inhomogeneous treatment times. Areas with flow separation and
recirculation can occur, depending on the flow condition and the chamber geometry.
A shear layer separates these areas from the main flow. The fluid exchange across the
shear layer is limited and fluid can be trapped in recirculation areas. Even without
recirculation areas, the flow velocity close to the chamber walls is slower and the
residence time of food in these areas correspondingly longer. This effect is pro-
nounced for laminar flow conditions, where the momentum exchange perpendicular
to the main flow direction is weak. The part of the fluid continuum in the low
velocity region close to the wall is less likely transported to a more centered position
due to the lack of perpendicular mixing.
Under turbulent conditions, the differences in the velocity profile normal to the
wall are reduced and the mixing is increased. Alkhafaji and Farid (2007) tested
microbial inactivation for the same treatment times but different frequencies and
flow rates. The results reveal higher inactivation for high-frequency- and high-flow-
rate treatments compared to low-frequency-low-flow-rate treatments for the same
treatment times. The increased mixing of the fully developed flow and therefore a
more homogeneous residence time distribution is given as an explanation.
Consequently, over-processing in recirculation or low velocity areas or under-
processing in the center can result even from an ideal homogeneous electric field.
Without knowledge about the fluid dynamics and therefore the mixing intensity and
local treatment times, the information given by the electric field distribution is less
conclusive and can even be misleading.
The two-way relation between the electric and the temperature field is explained
in section “Thermophysical Fluid Properties”: The electric field influences the
temperature field through Joule heating and the electrical conductivity is temperature
dependent. Moreover, the temperature field is strongly influenced by the convective
transport and mixing due to the flow field. An increased process temperature is
beneficial for permeabilization because membrane fluidity is increased.
Optimization of treatment homogeneity is most expedient when these interrela-
tions of temperature, flow, electric field, and treatment effect are considered concur-
rently to predict treatment intensity.
Recent work has used numerical simulations for geometric improvements to
achieve a more homogeneous treatment. Jaeger et al. (2009) compared simulations
of a baseline continuous PEF chamber with simulations where additional rectangular
grids were inserted in front and behind the insulator as a static turbulence generating
device. An increase in turbulent kinetic energy as a result of the grid insertion is
documented as well as a more homogeneous and intense electric field for the case of
metal grids. Both effects contribute to a higher inactivation of E. coli bacteria. The
generated turbulence also smoothed out the temperature distribution and reduced
local peaks, resulting in a gentler treatment of the used milk as quantified by a
reduced temperature-induced inactivation of alkaline phosphatase.
Schroeder et al. (2009) compared different insulator shapes of collinear treatment
chambers, also with respect to the electric field strength, turbulent kinetic energy, and
temperature. The simulations are validated with a comparison of simulated and
measured temperatures at the outlet of the chamber and at the insulators. The most
homogeneous electric field strength distributions are simulated for rounded insula-
tors, while sharp edges lead to local strength peaks. A rounded elliptical geometry is
found to provide the most uniform turbulent kinetic energy throughout the treatment
area. Sharp edges however result in areas with larger turbulence and therefore a better
distribution of the temperature. Due to the more homogeneous electric field strength
distribution, the elliptic geometry is recommended. Meneses et al. (2011c) conducted
CFD simulations with varying inlet velocities. For a constant energy input through
the electric pulses, a higher inactivation rate was found when the inlet velocity was
increased from 3 to 10 l/h. Further increase was not beneficial. The authors explained
the higher inactivation with a more homogeneous treatment because of a less laminar
velocity profile. This research used a residual activity balance as given in Eq. 24. It is
possible that the mixing effect of a configuration with higher turbulence can com-
pensate for a less homogeneous electric field strength distribution.
Dense Particulate Fluids
Fluid expression from fruit and vegetable cells can be enhanced by additional
treatment before or while pressing. The food is treated to increase the juice or oil
yield, conventionally with thermal methods or enzymatic or chemical treatment.
Usage of PEF offers an advantageous alternative. The permeability of the membrane
increases and the cell content can be expressed with less effort. No additives are
necessary and thermal degeneration of flavor, color, and nutrient contents is avoided.
The reduction of necessary mechanical cell destruction also reduces the transfer of
cellulose or pectins, resulting in a clearer product (Vorobiev and Lebovka 2006).
A beneficial way to combine PEF treatment and juice expression in a continuous
process is successfully reported in Jaeger et al. (2012): Carrot mash and apple mash
were pumped through a colinear PEF chamber where they were subjected to critical
electric field strengths of 2.1–7 kV/cm throughout the treatment area. Depending on
mash coarseness and press type, juice yield was improved by 0–11% for apple mash
and 8–31% for carrot mash compared to a control run without PEF treatment.
A comparison of juice yield and juice composition between juice expressed from
apple mash treated with PEF or pectolytic enzymes and an untreated control group is
presented in Schilling et al. (2007). Compared to the untreated control group, the
PEF treatment increased the juice yield between 4% and 9% for a field intensity of at
least 3 kV/cm. The yield increase due to enzyme maceration compared to the control
group was slightly less than for PEF treatment with at least 3 kV/cm but higher than
for PEF treatment with 1 kV/cm. A comparison of the component composition of the
juice did not reveal significant differences between the PEF-treated group and the
control group.
As these research studies prove, mash treatment with PEF is a promising
approach and a worthwhile aim for further studies. To complement experimental
investigations with the rich data of numerical simulations, challenges of handling the
constitution of the mash must be met. A Lagrangian approach for the particulate
phase fails simply due to the high particle density. A Eulerian approach is not
feasible due to the very high modeling effort necessary to account for heterogeneity
in particle sizes and the numerous particle-particle interactions.
A change of concept is helpful to open the problem up for numerical analysis. The
limitations of a multiphase approach can be avoided by modeling the mash as a
single-phase fluid. Particles are not explicitly modeled, and local interactions of the
phases are disregarded. Instead, the heterogenic concept of substances solved in a
liquid is replaced by a numerically homogeneous mash fluid model. The influences
the secondary phases have on the rheological behavior of the mash are considered by
giving the mash fluid averaged physical properties. Additionally to determining the
thermophysical models for the mash as presented in section “Thermophysical Fluid
Properties,” an implementation of a shear-dependent viscosity model may be
necessary.
Experimental data for mashes is barely accessible in literature. Empirical models
must be determined for a specific investigated liquid. A behavior comparable to
purees is expected, which is well studied for different types of fruit and vegetables. A
comprehensive review is given in Diamante and Umemoto (2015). A

non-Newtonian behavior is reported with experimental results, which can be char-
acterized by the Herschel-Bulkley model:
τ ¼ τO þ dγ n (31)
The yield shear stress τ0 is added to the Oswald de Waele power law with a
consistency coefficient d and a flow index coefficient n to describe the shear stress τ
with the shear rate γ. For n < 1 the fluid behaves shear thinning, which is the reported
case for all reviewed pulps, purees, and jams. The three parameters, i.e., yield shear
stress, consistency coefficient, and flow index coefficient, all change when the
process temperature varies. Secondary models for the influence of the temperature
changes on these parameters are necessary for an implementation in a numerical
simulation solving the temperature field. Maceiras et al. (2007) find a linear increase
of the flow index coefficient, while the other two parameters decrease linearly when
the temperature of the fluid is increased.
With a fitting model of the rheological behavior, the single-phase approach of a
homogenous mash fluid can be implemented. In the same way, heterogeneities due
to phase changes can be neglected for the conductivity models. These simplifications
make the model inappropriate for a detailed local study of the treatment. Neverthe-
less, investigations of the fluid model with the spatially averaged properties can be
used for computation of global fields, which are of great importance in process
optimization. The described approach expands the field of numerical simulation
applications to dense particulate flows like mash and puree.
Modeling of Microscopic Aspects
For investigations aiming to optimize quality of the treated food and the PEF
process, a macroscopic numerical approach with empirical models for the treatment
effect is state of the art as it is a trade-off between feasibility and significance of the
provided data. A clear majority of numerical investigations therefore is conducted on
a macroscopic scale. This does not mean that microscopic investigations are cate-
gorically impossible. Computational resource limitations do not yet allow a simula-
tion that resolves the effects on cell scale level while conducting a full-scale
treatment chamber or process time analysis. Nevertheless, an investigation of
selected microscopic aspects is possible by reducing the domain size to cell scale
level. Even though influences of changes in chamber geometry, treatment intensity,
process temperature, or pulse shape and duration on the treatment effect are explored
and known, the data foundation for and knowledge about underlying PEF effects on
the treated cells is still inconclusive. From a scientific point of view, future in-detail
simulations of microscopic behavior can contribute to close this gap of information.
A microscopic aspect that became the subject of numerical investigations is the
pore formation in cell membranes due to electroporation. The mathematical
framework for these studies is given by molecular dynamic (MD) models.
Many-body systems of molecules and atoms forming the lipid bilayer of a cell
membrane are exposed to electric fields. The membrane is modeled as two layers
of lipid molecules with polar heads and nonpolar tails. The hydrophobic tails are
facing toward each other so that the hydrophilic heads make up the boundaries of the
membrane which is in contact with the surrounding aqueous solution (Kotnik et al.
2012).
Dynamics of the system are computed by solving Newton equations of motion.
Therefore, the relevant forces acting on an atom or molecule must be chosen and
calculated. These include inter- and intramolecular potentials as well as external
forces, in particular the Lorentz force, as in equation without a magnetic field.
Molecular dynamic investigations including the works of Delemotte and Tarek
(2012), and many others, report a transient structural change of the membrane
constitution resulting in pore formation. The process is initiated by the formation
of what the authors name water wires, water fingers, or defects: water molecules
reach into the membrane through the lipid headgroups. The process is followed by a
rearrangement of the hydrophilic headgroups: they move inside the membrane
following the water wires and thereby stabilize the emerging structures. The pore
formation is the result of this process. The water wires grow until they span the
membrane, and the lipid heads establish these channels. The hydrophilic pores
increase the membrane permeability and favor content injection into or extraction
from the cell.
The time duration of the pore formation is found to be on the scale of nanosec-
onds, the pores’ diameter spans over nanometers. At this spatial and temporal scales,
the numerically gathered information of the membrane perforation is hard to validate
by experiments but helps understand the electroporation processes. MD simulations
allow insights into aspects that need further investigations and prove to be valuable
for fundamental research on microscopic levels.
Treatment Chamber Geometry and PEF Process Optimization
Gerlach et al. (2008) show the importance of the optimization of the treatment
chamber geometry in order to improve the uniformity of the electric field and
consequently avoiding intensity peaks and temperature heterogeneities. Barbosa-
Cánovas and Altunakar (2006) demonstrate that the level of electric field homoge-
neity depends on the different geometry arrangements of the treatment chamber. The
arrangement can be either parallel plate, coaxial, or collinear. Therefore, Gerlach
et al. (2008) confirm the significance of an optimum design of the chamber geometry
to enhance the homogeneity of the electric field in the chamber.
The geometry optimization can be carried out by applying a systematic optimi-
zation or by parametric studies (Gerlach et al. 2008). Misaki et al. (1982) optimize
the chamber geometry using a numerical algorithm, which automatically searches
the minimum of the local peaks of the electric field strength. Lindgren et al. (2002)
perform parametric studies using numerical simulations of different chamber geom-
etries by varying two geometrical parameters: the insulator length and the inner
insulator radius. Both approaches are performed by only taking into consideration
the simulation of the electric field and not solving the momentum and energy
balances in the chamber. Optimizing the treatment chamber geometry and simulta-
neously considering and finding a solution for the momentum and energy balances
are challenging tasks because both the mathematical formulation complexity and the
necessary computer resources to solve the optimization problem increase.
Numerical investigations are well suited for optimization tasks. Once a simulation
is set up, a variation of parameters can often be done effortlessly and can be
automatized.
As an example of numerical PEF process optimization, the work of Krauss et al.
(2011) is presented here. The authors optimize the electrode and insulator geometry
and the PEF processing conditions by solving the three-dimensional momentum,
energy, and electric field balances in the treatment chamber, as well as considering
the distribution of the spoiling agent activity in the chamber. With an evolutionary
algorithm, the chamber geometry and process optimization is described using the
simulation software OpenFOAM ® coupled with the optimization software
modeFRONTIER. OpenFOAM ® is used to simulate the electric, fluid dynamic,
and thermal fields as well as the spoiling agent activity field for a given chamber
geometry and process condition. The software modeFRONTIER finds the optimum
chamber geometry and process conditions, considering process restrictions for
excessive local temperatures in the chamber and minimal and maximal values for
the internal radius Ri of the chamber geometry.
An optimization problem is formulated with the objective function f1 to minimize
the residual activity of the spoiling agent in the treatment chamber. Additional
restrictions for the average temperature TAvg, the maximum temperature Tmax, and
the internal radius Ri are necessary to bound the optimization problem:
minf1 ðRI Þ ¼ RAavg

Restrictions:
T avg 313:15 K (32)
T max 353:15 K
1:2 mm RI 2:5 mm
The average residual activity RAavg is evaluated at the outlet. Krauss et al. (2011)
find that increasing the internal radius of the insulator reduces the average residual
activity RAavg but both the average temperature in the volume of the insulator and the
maximum temperature in the whole treatment chamber increase.
Furthermore, Krauss et al. (2011) show that there is a relationship between the
relative standard deviation of the electric field strength and the average residual
activity RAavg. This result suggests that the optimization problem could be
reformulated by directly minimizing the relative standard deviation of the electric
field strength.
For a given geometry of the treatment chamber, Krauss et al. (2011) minimize the
residual activity and maximize the mass flow rate ṁ using the treatment time tt, the
mass flow rate ̇ m, and the electric potential ϕ as variables. Since the treatment time tt
is proportional to the pulse frequency fp, and the pulse width τ, the treatment time tt
can be written as a function of these process parameters and the number nc of
consecutive treatment chambers. For these variables limiting values are given to
restrict the optimization process. The multi-objective process optimization problem
can be described as:
minf1 ðτÞ ¼ RAavg

maxf2 ðm_ Þ ¼ m_
Restrictions:
T avg 313:15 K
(33)
T max 353:15 K
3 kg=h m_ 7 kg=h
1 nf p τ 500 Hz μs
10 kV ϕ 15 kV
Krauss et al. (2011) find that fulfilling this multi-objective optimization leads to
contradictory requirements. A compromise between maximum throughput and min-
imum residual activity is necessary. Thus, two possible optima were proposed.
The presented example applications for numerical approaches in PEF simulations
are not comprehensive. The numerous models offer an extensive toolbox for inno-
vative approaches and give an insight into the possibilities of numerical applications.
This versatility is encouraging for future research and novel investigations.
Finite Volume Case Study: Influence of Insulator Geometry

on Treatment Intensity and Homogeneity
Solving the governing equations for mass, momentum, internal energy, and residual
activity can produce valuable numerical data for the understanding and improvement
of PEF processes. An example of a numerical investigation is given here in order to
present the steps necessary for a practical application. Flow fields, thermal fields, and
electric fields are computed for three different chamber geometries. Their influence
on enzyme inactivation is compared by solving a balance equation for the residual
activity afterwards.
Method
The general design of the chambers is shown in Fig. 3. The laboratory-scale collinear
chambers are axisymmetric and differ only in the shape of the central insulator in the
treatment zone. All chambers have a main diameter of 6 mm, a smaller treatment
diameter of 4 mm in the treatment area, and an insulator length of 4 mm. For the
baseline geometry A, the insulator has an edge radius of 1 mm, reducing the chamber
diameter more rapidly. The insulator used in geometry B has the shape of an arc with
Fig. 3 Rotational symmetric chamber geometries A, B, and C. Dimensioning in millimeter
a radius of 2.5 mm, and geometry C has rectangular edges with no smoothing radius.
The treatment area of geometry C is the smallest, and the fluid experiences the
strongest displacement and acceleration, followed by geometry A.
The modeling equations are partial differential equations. The differential oper-
ators are defined as limits between neighboring points of infinitesimal distance in
space or time and must be approximated by discrete forms of the concerning
operators for numerical solutions. These discrete operators contain values at distinct
points in time or space and form the computational grid of the domain. The result is
an algebraic expression that can be solved with different strategies like the finite
volume, finite difference, finite element, or boundary element method. Each method
has distinct advantages and limits that cannot be discussed here. The presented
simulations use the finite volume method. The algebraic system is solved to get a
good numerical approximation of the partial differential equation evaluated at
discrete points.
A computational grid is necessary, which represents the investigated geometry at
discrete points in space. The investigated geometry is split up into a finite number of
small volumes on which the transport equations are solved locally. For each field,
each volume element contains at least one value at the center of the volume that
represents the field value in form of a mean value. For higher-order methods, more
than one value is possible for each cell.
The computational effort of a simulation increases with the number of cells, but a
coarse grid can have a significant influence on the results. A trade-off between effort
and accuracy has to be made. Taking advantage of symmetries to split up the
geometry and negligence of unnecessary small-scale phenomena is a common way
to reduce the computational effort while ensuring appropriate grid quality.
Regions with large gradients of simulated quantities are typically sensitive to
inappropriate grid cell sizes and deserve particular refinement. In practice, grid
independence of the results is investigated by a stepwise refinement of an initially
coarse grid until a further refinement does not have an impact on the simulation
results.
Fig. 4 Mesh refinement at chamber wall
The grids of the three geometries are generated with the mesh generation utility
snappyHexMesh which is part of the open-source simulation software package
OpenFOAM ®. The generation procedure is the same for all three grids. The surfaces
shown in Fig. 3 along the symmetry line of the chamber are meshed with rectangular
cells. Afterwards, the five layers of squares closest to the wall are bisected in both
directions resulting in ten layers of finer resolution at the chamber walls. Exemplary,
the grid at the wall at the beginning of the treatment area is shown for geometry A in
Fig. 4.
This surface mesh is then rotated around the rotational axis by 5 to create a
second, equal surface. The resulting wedge between the two surfaces is resolved
with a single cell. The wedge domain can be used to solve the physical fields of the
whole treatment chamber by setting periodic boundary conditions at these two
surfaces, reducing the computational domain size significantly. In OpenFOAM ®
this boundary type is called wedge.
The remaining boundary conditions necessary to calculate the three physical
fields are given in Table 2. The fluid enters the domain at the inlet with a constant
velocity of 0.05 m/s and a temperature of 283.15 K. The electric potential between
the two electrodes is set to 15 kV, which results in a voltage-to-distance ratio of
37.5 kV/cm.
To solve the balance equations for mass, momentum, and internal energy on these
grids, the noncommercial finite volume solver software OpenFOAM ® is used and
customized. A stationary and laminar flow is assumed because the Reynolds number
of around 200 is well within the laminar domain for pipe flows.
Models for the temperature-dependent material properties are necessary. For
liquid food, properties of water are assumed. As described in section “Thermo-
physical Fluid Properties,” the influence of temperature on density and specific heat
capacity can be neglected. A fixed value of ρ = 999.756 kg/m3 and cp = 4,176.2 J/
(kg K) corresponding to the values for a temperature of 10 C is given. Furthermore,
in this example case, the temperature dependency of the electrical conductivity of the
treated matter is unknown and therefore neglected. A constant electrical conductivity
of σ = 1.32 mS/cm is assumed. Note that this simplification is not valid for most
Table 2 OpenFOAM ® boundary conditions used for the example cases

Insulator Electrode
Inlet Outlet walls Electrode + GND
Velocity [m/s] Fixed value Zero Fixed value Fixed value Fixed value
(0.05 0 0) gradient (0 0 0) (0 0 0) (0 0 0)
Pressure [Pa] Fixed flux P Fixed value Fixed flux P Fixed flux P Fixed flux P
100,000 100,000 100,000 100,000 100,000
Temperature [K] Fixed value Zero Zero Zero Zero
283.15 gradient gradient gradient gradient
Electric potential Zero Zero Zero Fixed value Fixed value
[V] gradient gradient gradient 15,000 0
Residual activity Fixed value Zero Zero Zero Zero
[] 1 gradient gradient gradient gradient
liquid foods as shown in Table 1 because the effect of thermal runaway heating is not
considered. Experimental data for water is fitted with polynomial functions to model
the temperature depending properties dynamic viscosity μ(T) and thermal conduc-
tivity κ(T):
μðT Þ ¼ 3:62971422206 1011 T 4 4:9477498369759 108

(34)
T 3 þ 2:5327 105 T 2 5:7765342 103 T þ 0:4962579846
κðT Þ ¼ 8:34216392161674 106 T 2 þ 6:5319021 103 T

0:5989611328 (35)
The residual activity at the outlet of the domain is used as a direct indication of the
treatment efficiency. Therefore the transient distribution of the residual activity of PE
is calculated in a second run by solving Eq. 24 using the inactivation model given by
Eq. 27. The residual activity is initialized with a value of 1 throughout the domain.
The fluid flows into the channel with a residual activity of 1 at the inlet, is transported
through the domain by the flow field, and altered by the source term accounting for
the inactivation driven by the electric field strength in the treatment area. The
boundary conditions are listed in Table 2. The evaluation of the treatment homoge-
neity is made after a simulation time of 10 s. The frequency fp and pulse width τ of
the electric field pulses needed to determine the source term in Eq. 27 are set to 20 Hz
and 10 μs, respectively. Their product is additionally multiplied by a factor 10 to
increase the treatment intensity for the evaluation in the context of the presented
cases. Krauss et al. (2011) interpret this factor as consecutive treatment with a
number nc of chambers. For this assumption, a good mixing of temperature and
residual activity after a treatment chamber is necessary to guarantee constant profiles
at the inlet of each chamber. Another way to interpret the factor is to increase the
treatment frequency or pulse width.
Results
A more rapid reduction of the channel diameter and a smaller volume of the
treatment area result in a stronger acceleration of the fluid and higher velocities in
the treatment area. As visualized in Fig. 5, this leads to the highest flow velocities in
geometry C followed by geometry A. Consequently, the treatment time is highest
when insulator geometry B is used. At the end of the central insulator of all
geometries, an adverse pressure gradient occurs caused by the sudden area increase,
and the main flow separates from the wall. Recirculation areas with low velocity
magnitudes form. The streamlines show that geometry B has the smallest
recirculation area because the flow can shortly follow the less rapid diameter
expansion at the end of the insulator. In these recirculation areas, the liquid can be
trapped and treated for a long time because the fluid exchange with the main flow is
weak.
Such flow conditions are known to cause problems of overtreatment. Figure 6
shows the temperature distributions and reveals a clear increase in the recirculation
area for the geometries A and B of up to 65 K. Even though the strength of the
electric field decreases behind the insulator, the fluid throughout the whole
recirculation area is heated up. It is transported in and out of the treatment area
repeatedly and has long residence times due to the low velocities in the recirculation
areas. Especially for geometry B, where the temperature increases up to 348 K, this
can result in a temperature-induced alteration of the treated matter, contradicting the
supposed nonthermal nature of PEF treatment. The food in the recirculation area
behind geometry C is also subject to a temperature increase but with values of up to
305 K an overtreatment is not expected.
The electric field strength in the treatment area is presented in Fig. 7. The spatial
distribution is shown on the left side. The thin black line in the middle of the central
insulator marks the line along which the electric field strength is plotted on the right
side of the figure. For all geometries, the field strength reaches maximum values of
Fig. 5 Velocity magnitude distribution and streamlines

Fig. 6 Temperature distribution
Fig. 7 Electric field strength distribution (left) and profile along insulator center line (right)
up to 40 kV/cm. The area size where such high values are reached is the largest for
geometry B. It provides the strongest treatment with respect to high field strengths
and long treatment times.
However, this does not give any conclusive information about treatment homo-
geneity. The plot of the field strength along the center line on the right side of Fig. 7
shows that the intensity reduces for all geometries with increasing distance to the
insulator. The coefficient of variation Cv, defined as the ratio of the field strength’s
standard deviation by its mean value, is a normalized rate of deviation in the field
strength data sets. The coefficient for geometry A and geometry B is three and ten
times higher, respectively, compared to geometry C.
When high treatment intensity is preferred, the results of treatment time and
electric field strengths suggest preferring geometry B followed by A and C. When
treatment homogeneity is desired, the presented results suggest precisely the reverse
ordering. However, basing a prediction of treatment homogeneity only on the field
strength distribution along the center line is not sufficient, as it does not take the
whole treatment area into account, and the treatment time is neglected. Furthermore,
additional parameters like a threshold critical treatment time, critical electric field
strength, or a temperature influence on the treatment effect are not considered. A
Fig. 8 Temporal development of residual activity distribution for geometry A
more quantitative basis for the evaluation is given by the residual activity as
described in section “Modeling of Permeabilization and Inactivation.”
The residual activity RA at the outlet of the PEF chamber can be used to compare
the treatment results of the two geometries. The temporal development of the
residual activity distribution in Fig. 8 is shown for geometry A. It is a representative
for all three geometries as the behavior is similar in all cases. The figure shows the
spatial distribution of residual activity after three different treatment times. After
0.1 s the inactivation has started in the treatment area and is especially strong close to
the insulator where the flow velocity is low and the electric field strength is high. By
convection of the treated matter, the decreased RA is transported downstream of the
treatment area.
The distribution after 1.5 s reveals that the exchange perpendicular to the main
flow direction is so weak that the matter with the lowest RA values is mostly
transported along the shear layer between main flow and recirculation area.
After 4 s the treated matter is mixed into the recirculation area and transported
back upstream resulting in residual activities below 0.5 throughout the recirculation
area. A further transient simulation results in a quasi-static solution of the residual
activity distribution.
The distribution of RA after the simulation reached a steady state (about 30 s) is
shown on the left side of Fig. 9. The results confirm that geometry B is the most
efficient in reducing the residual activity, but qualitatively the results are similar. On
the right side of the figure, the values of the residual activities are plotted across the
chamber radius. The thin black lines close to the outlet mark the location from which
data is taken. The treatment effect is weakest in the center of the channel. From the
maximum values at the center, the residual activity decreases in the direction of the
walls until a minimum is reached at the wall.
At the outlet, the mean residual activity of geometry C across the channel radius is
0.67. Compared to this value, the mean residual activity is 5.3% less when geometry
A is used and 10.8% less when geometry B is used. This corresponds to the
assumptions made from the electric field strength and velocity magnitude distribu-
tions but offers a more direct comparison basis. As expected, the coefficient of
variation for the residual activity is lowest for geometry C. However, the difference
Fig. 9 Spatial residual activity distribution after 10 s of treatment (left) and plot of residual activity
across the chamber radius close to the outlet (right)
is small compared to the differences in the coefficients of variation for the electric
field strength shown in Fig. 7.
Conclusions
Without the simulation of the residual activity, the decision between the geometry
resulting in the most efficient treatment and the most homogeneous treatment is
inconclusive. With the knowledge about the residual activity at the outlet, geometry
B is preferable because it offers the highest treatment intensity while the differences
in homogeneity are small. However, the residual activity distribution has to be based
on a reliable empirical model. The additional information comes at the cost of
extensive experimental data.
When a temperature increase of up to 60 K is not acceptable for the treated matter,
geometry A and B cannot be used despite their more efficient treatment. Note that for
food with a temperature-dependent electrical conductivity, the temperature peaks can
further increase due to thermal runaway.
The presented example case points out the advantages of solving an additional
balance equation for residual activity. The model can be used for further detailed
optimization studies as described in section “Treatment Chamber Geometry and PEF
Process Optimization.”
The presented work contributes to highlighting the purpose and benefits of
numerical techniques for investigations of PEF processes. The numerical equation
framework for studies of fluid dynamic, thermal and electric fields, and transported
quantities like the residual activity is given and explained. The interrelations of these
different physical fields are described. The importance of these interrelations is
pointed out, and it was shown that a coupled computation of these fields enhances
the reliability and conclusiveness of investigation results. This is done in order to
provide an orientation guideline. The introduced various application fields of numer-
ical approaches can be used as examples for further numerical research of PEF
treatment processes. They show the versatility of modeling approaches and therefore
encourage finding novel research topics that can be solved by means of numerical
simulation techniques.
Cross-References

▶ Impact of Pulsed Electric Fields on Enzymes
▶ Modeling Transdermal Delivery by Electroporation: The Thermodynamic
Approach
▶ Pulsed Electric Fields as Pretreatment for Subsequent Food Process Operations
Notation
Latin Characters
Character Description Unit

a Linear equation slope [mS/(cm C)]
A, A0 Activity, initial activity []
b Linear equation offset [mS/cm]
B Magnetic flux density [N/(mA)]
c Quantity concentration []
cp Specific heat capacity [J/(kgK)]
Cv Coefficient of variation []
D Diffusion coefficient [m2/s]
e Internal energy [J]
E Electric field [V/m]
E, Ec Electric field strength, critical [V/m]
f Force density vector [N/m3]
fp Pulse frequency [Hz]
g Standard gravity [m/s2]
j Electric current density vector [A/m2]
k Thermal conductivity [W/(mK)]
L Treatment area length [m]
ṁ Mass flow rate [kg/s]
n flow index []
nc Number of consecutive chambers []
(continued)

np Number of pulses []
p Pressure [Pa]
q Electric charge [C]
RI Internal treatment chamber radius [m]
RA, RA0, RAavg Residual activity, initial, average []
t Time [s]
tt, ttc, tr Treatment time, critical, residence [s]
T, Tavg, Tmax Temperature in Kelvin, average, maximum [K]
TC Temperature in degree Celsius [C]
u Velocity vector [m/s]
ū Average velocity magnitude [m/s]
x Coordinate vector [m]
Greek Characters

α Weibull function scaling parameter [s]
β Weibull function shaping parameter []
γ Shear rate vector [1/s]
δ Kronecker delta tensor []
λ Specific spoiling agent constant [V/m]
μ Dynamic viscosity [Pas]
π Production rate [W/m3]
ρ Density [kg/m3]
σ Electrical conductivity [S/m]
τ Pulse width [s]
τ Stress tensor [Pa]
τΟ Yield shear stress [Pa]
ϕ Electric potential [V]
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Modeling Microbial Inactivation by
Pulsed Electric Field 66
Micha Peleg
Abstract
Modeling microbial inactivation by pulsed electric fields (PEF) can be
approached at different scrutiny levels. The starting point can be the biophysical
effects on an individual cell’s membranes and organelles, their manifestation at
the cellular level, and in turn on the population’s survival curve. This chapter
focuses on different kinds of population models. It presents empirical phenome-
nological dose–response models and a method to translate PEF survival data into
equivalent thermal inactivation curve at a chosen lethal reference temperature. It
includes a proposal to combine deterministic continuous survival and injury
models and demonstrations of how stochastic events at the cellular level are
manifested in observed inactivation patterns at the population level. The potential
utility of the stochastic and deterministic kinetic models in describing PEF
inactivation, especially of individual or a very small number of a pathogen’s
cells, is evaluated and their theoretical and practical limitations highlighted.
Briefly assessed are the potential benefits and limitations of models that attempt
to account for various degrees of damage that a PEF treatment might cause to a
microbial pathogen’s or spoilage organism’s cells.
Keywords
Food preservation • Microbial survival • Cell injury and recovery • Kinetics •
Stochastic models
M. Peleg (*)
Department of Food Science, University of Massachusetts, Amherst, MA, USA
e-mail: micha.peleg@foodsci.umass.edu

DOI 10.1007/978-3-319-32886-7_43
1270 M. Peleg
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1270
Modeling Microbial Inactivation by a Pulsed Electric Field Treatment . . . . . . . . . . . . . . . . . . . . . . 1271
Comparison of the Logistic and Weibullian Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1272
Equivalency of a Pulsed Electric Field to Thermal Inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1274
Continuous Models of Microbial Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1274
Stochastic Models of Mortality and Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1276
A Stochastic Model of Microbial Cells Mortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1278
A Stochastic Model of Injury and Mortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1279
Recovery from Injury and Delayed Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1281
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1284
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1285
Introduction
Foods exposure to high voltage pulsed electric fields (PEF) is a promising nonther-
mal food preservation technology. The treatment inactivates pathogens and spoilage
organisms by electroporation of their cell walls with little or no adverse effect on the
food’s flavor and nutritional value and hence its appeal. The various aspects of the
technology, including the inactivation kinetics, have been thoroughly investigated
(e.g., Zhang et al. 1995; Vega-Mercado et al. 1977; Wouters et al. 2001; Heinz
et al. 2002; Toepfl et al. 2007; Zhao et al. 2013; Pataro et al. 2015; Kotnik
et al. 2015). In this chapter the focus is on three issues: the mathematical description
of the dose–response curve in PEF preservation of foods, the possibility of
expressing a PEF process’s lethality in terms of an equivalent isothermal heat
treatment at a chosen reference temperature, and a proposed probabilistic approach
to modeling cells injury caused the treatment.
Most if not all the theories and mathematical models of microbial inactivation in
foods, notably by heat but also by nonthermal preservation method such as the
application of ultra high hydrostatic pressure or exposure to a chemical disinfectant
or preservative, were developed for systems where the microbe’s exposure to the
lethal agent is continuous. In contrast, and by definition, a cell’s exposure to a pulsed
electric field is not a continuous treatment at least on a certain pertinent time scale.
Moreover, since the number of electric pulses as well as the field’s intensity, the
pulses’ duration and their “shape” can and do vary, what constitutes a “dose” is not
always easy to define. Yet, for quantitative comparison of different PEF treatments
intensity, one can treat the pulse’s voltage as loosely analogous to a lethal temper-
ature level or chemical agent concentration. Similarly, where all other things are
equal, one can treat the number of pulses as loosely analogous to a thermal or
chemical treatment’s duration and vice versa. Either way, since time is not the
independent variable, the term “dose–response curve” seems to be more appropriate
than the more commonly used term “survival curve.” However, since the term “dose
response curve” in food microbiology usually refers to the relationship between the
66 Modeling Microbial Inactivation by Pulsed Electric Field 1271
number of ingested pathogen cells and the probability of acute sickness or death, the
term “survival curve” can still be used in our context. Admittedly, from a mechanistic
viewpoint, the mentioned above analogies are far from being satisfactory. Yet, they
can be helpful in adapting concepts and kinetic models originally developed for
traditional methods of microbial inactivation to food preservation by the PEF method.
Modeling Microbial Inactivation by a Pulsed Electric Field

Treatment
A “survival curve,” by definition, is a plot of the number or fraction of surviving

individuals as a function of time, which in microbial inactivation by PEF translates
to the number of fraction of microbial cells as a function of the number of pulses
(of given shape and intensity) or the field intensity for a given number of pulses.
Either way, the survival curve is a graphical description of the cumulative form of the
death events’ distribution with respect to the number of pulses or field intensity. In a
traditional survival curve of a microbial population exposed to a lethal agent such as
heat, the curve’s local slope has time reciprocal units. Therefore, the local slope can
be viewed as the momentary mortality rate and hence the relation to inactivation
kinetics. Regardless of the death cause or causes, the temporal distribution of the
mortality events, and hence the mortality kinetics, is primarily determined by the
targeted organism, its growth phase, the medium in which it is treated, and of course
the lethal agent’s intensity. The most commonly encountered distribution in micro-
bial thermal inactivation is the Weibull distribution (Peleg and Cole 1998; van
Boekel 2002), a kin to the stretched exponential model. When applied to microbial
inactivation by PEF it can assume the form (Rodrigo et al. 2003):
mt
t
SðtÞ ¼ Exp (1)
tc
where S(t) is the survival ratio after time t (in μs), S(t) = N(t)/N0 where N(t) and N0
are the momentary and initial number of recoverable cells, respectively, tc a charac-
teristic time (“scale factor”) and mt a constant (“shape factor”).
The model can also be written in the form of a dose–response relationship where
the dose w is expressed in terms of released energy per unit mass (Alvarez
et al. 2003), for example, i.e.,
mw
w
SðwÞ ¼ Exp (2)
wc
where wc a characteristic energy and mw a constant. The Weibullian model can also
be applied to survival as a function of the number of pulses of equal voltage and
shape, n, i.e.,
1272 M. Peleg
mn
n
SðnÞ ¼ Exp (3)
nc
where nc a characteristic number of pulses and mn a constant, or as a function of the

field intensity, E, for a given number of pulses
mE
E
SðEÞ ¼ Exp (4)
Ec
where Ec is a characteristic field intensity and mE a constant. Examples of the fit of

Eq. 4 to published inactivation data on cells of Candida (yeast) and Listeria
(bacterium) are given in Fig. 1a, b.
Notice that for the same organism, at the same growth stage, and treated in the
same medium, the model parameters’ magnitudes also depend on the field intensity
and uniformity, waveform’s shape, the intensity and/or number of pulses, etc. Also
notice that Eqs. 1, 2, 3, and 4 all imply that S(0) = 1, or Log[S(0)] = 0, and that
S(1)!0, or Log[S(1)]!1.
Comparison of the Logistic and Weibullian Models
Sigmoid survival data of the kind shown in Fig. 1 can be also fitted by the simplified
logistic model (Peleg 1995)
1
Sð nÞ ¼ (5)
1 þ Exp½kn ðn nc Þ
where nc marks the inflection point’s location and kn the curve’s steepness around it,
or
1
S ð EÞ ¼ (6)
1 þ Exp½ kE ðE Ec Þ
where Ec marks the inflection point’s location and kE the curve’s steepness around
it. The fit of Eq. 6 to the same data is also shown in Fig. 1c, d. As can be seen by
visual comparison of the corresponding plots, the fitted curves produced by the
Weibullian and logistic models are practically indistinguishable. They also had
similar fit as judged by statistical criteria. The regression coefficients (r2’s) of the
Candida inactivation data are in the range of 0.9939–0.9999 versus 0.9930–0.9998,
respectively, and of those of the Listeria’s 0.9808–0.9997 versus 0.9809–0.9997.
Thus, at least for the two sets of analyzed inactivation data, the two models can
indeed be used interchangeably. Nevertheless, the Weibullian and simplified logistic
models as described above differ in an important way. While nc or Ec in Eqs. 5 and 6
marks the inflection point location independently of kn or kE, the inflection point’s
location according to the corresponding Weibullian models is determined by both
a b
c d
Fig. 1 The fit of the “stretched exponential” and logistic (Fermi’s) models to published PEF
inactivation data on Candida and Listeria after 2, 4, 10, and 30 pulses. The original data are from
Hulsheger et al. (1983)
nc and mn or Ec and mE. Therefore, the inflection point’s location cannot be always
deduced intuitively from the Weibullian parameters’ magnitudes. Both the
Weibullian and simplified logistic models (as described by Eqs. 5 or 6) imply that
S(1)!0. However, the logistic model, unless appended by a correction term, is
mathematically inconsistent with the condition of S(0) = 1. The discrepancy’s
absolute magnitude, however, as with the data shown in Fig. 1 can be too small to
be of any practical significance in which case it is up to the user to choose between
the models. The logistic model, sometimes known as Fremi’s distribution (Peleg
1996; Peleg et al. 1997), has clear advantage over the Weibullian model when used
to describe dose–response or survival curves that resemble a step function, i.e.,
where the drop in the number of survivors appears instantaneous at time tc or if there
is such a case in PEF treatments at field intensity Ec. Where the magnitude of k in the
logistic equation is sufficiently large, then for all t < tc or E < Ec, including t = 0,
the magnitude of S(t) or S(E) would be indistinguishable from one. Similarly, for all
t > tc or E > Ec, including, the magnitude of S(t) or S(E) would be indistinguishable
from zero. [This continuous logistic model was used by Fermi to describe the very
sharp density distribution in an atom. It is very high and constant within the nucleus
radius (rc) and practically zero beyond it.]
1274 M. Peleg
Equivalency of a Pulsed Electric Field to Thermal Inactivation
The widely used concept of F0 in thermal processing notwithstanding the correct

criterion of microbial inactivation efficacy of a preservation process is the accomplished
number of decades reduction in the targeted microbial population’s size. This reduction,
mathematically expressed as –Log10[Sfinal], is an observed quantity and more impor-
tantly model independent. When the survival ratio, or its base ten logarithm, is used as a
process’s lethality measure, one can compare the efficacy of treatments of different
kinds, thermal or nonthermal. To do the comparison all one needs is to construct an
equivalency curve (Corradini and Peleg 2010). A schematic example of which is given
in Fig. 2. The figure shows the translation of survival data recorded during a PEF
treatment into corresponding times at a reference lethal temperature. Thus if it is known
from experience that a certain isothermal (or nonisothermal) heat treatment is safe, one
can design a PEF process that will match or exceed the thermal safety level with respect
to the particular organism in the particular food or medium. Obviously, this can also be
achieved by simply setting the targeted value of –Log10[Sfinal] and check whether it has
been surpassed. The advantage of the equivalency plot is that it helps visualize what
happens during the examined PEF process. It also provides a lethality measure
expressed in term of equivalent time at the chosen temperature, which is familiar and
intuitively meaningful to practitioners, i.e., food microbiologists and technologists. This
“equivalent time” might be especially useful to highlight the benefits of the PEF
technology, which is aimed at reducing the adverse effects of conventional heat treat-
ments on the nutritional value and other quality attributes of thermally processed food.
Continuous Models of Microbial Injury
PEF “survival curves” of the kind shown in Fig. 1 depict the three ways relationship
between the number of recovered viable cells (colony-forming units or CFU’s),
expressed as a survival ratio, the electric field’s intensity, and number of pulses.
Fig. 2 Construction of an equivalency curve between a PEF treatment and thermal inactivation at a
constant reference temperature (Adapted from Corradini and Peleg (2010))
Recovery of the survivors is done in a favorable growth medium and under optimal
growth conditions. Consequently, the count might include injured cells, which in the
actual food under realistic storage conditions might not be able to repair the damage
and would eventually perish. If so, an experimental survival curve so determined
represents a worst-case scenario, and its use to establish the process’s safety provides
an extra margin of safety. Nevertheless, it is not inconceivable that since the formed
colonies are counted after 24–48 h of incubation, injured cells that would have been
able to repair the damage if given longer time are not represented in the count. At
least theoretically, therefore, such uncounted cells might still pose a health or
spoilage hazard and it would be prudent to take this possibility into account.
Apart from direct microscopic observation, the number of injured cells after a lethal
treatment has been traditionally determined by counting the cells recovered under
stressful conditions, which presumably only intact cells can withstand. This number is
subtracted from that of the cells recovered under optimal conditions and the result is
sought number of the injured cells. This method is based on the assumption that the
injured cells are unable to form colonies under less than optimal conditions. The principle
of the method is demonstrated in Fig. 3. The figure shows the screen display of an
interactive Wolfram Demonstration posted on the Internet where the user can generate the
two Weibullian survival curves: “total” – under optimal conditions, and “intact” – under
stressful conditions, and the resulting (dashed) “injury curve.” To access the demonstra-
tion open: http://demonstrations.wolfram.com/InjuryInMicrobialInactivation/.
[The program CDF Player which runs the demonstration (and over 10,850 other
demonstrations to date) is freely downloadable following instructions on the screen.]
The posted demonstration is based on the assumption that both the inclusive survival
curve, i.e., that of the injured as well as intact cells and that of the intact cells only can
be described by the Weibullian (“stretched exponential”) model. The validity of this
assumption in thermal inactivation has been demonstrated by the analysis of published
microbial mortality and injury data (Corradini and Peleg 2007). The analysis also
indicated that most of the survivors were injured to at least some extent. Notice that the
“subtraction method” on which this demonstration is based lumps together all cell
injuries regardless of the damage severity. Theoretically at least, this weakness of the
method can be remedied by recovering the surviving cells under conditions that create
different stress levels. If and when done, the results would enable the researcher to
quantify the numbers or fraction of surviving cells that sustain different degrees of
injury. An example of a program that simulates cell injuries of various levels is given
in Fig. 4. It shows the screen display of a different Wolfram Demonstration, especially
written for the purpose and can be found at: http://demonstrations.wolfram.com/
DegreesOfMicrobialInjuryAndSurvival/. Here too the assumption is that the inclusive
survival curves, i.e., those of the injured (at the different levels) as well as the intact
cells, and that of the “intact cells only” all follow the Weibullian model. Whether
application of such a method, especially routinely, would be feasible in future PEF
inactivation research is uncertain. However, the existence of such an option should be
kept in mind, especially if unexpected survival is discovered long after the treatment.
Apart from obvious logistic considerations, the main issue concerning the multiple
recovery conditions incorporation in PEF studies would be the proper choice of
1276 M. Peleg
Fig. 3 Screen display of the Wolfram Demonstration where the “subtraction method” is used to
determine the fraction of injured cells after an antimicrobial treatment
recovery media and time-temperature conditions for the incubation. Since there is little
known about the relationship between an organism’s degree of injury and its ability to
recover in different media and incubation conditions, one would have to establish it
experimentally, which might require a considerable time and effort. Again however,
the concept should not be dismissed outright, not the least because it could be used to
examining hypothetical situations using computer simulations based on assumed
recovery efficiencies.
Stochastic Models of Mortality and Injury
Almost universally, reported microbial survival curves have been determined by

exposing a large population of the targeted vegetative cells (or spores) to the lethal
agent under investigation and counting the survivors after different times. The same
Fig. 4 Screen display of the Wolfram Demonstration that calculates the fractions of cells having
three levels of injury
is true for PEF except that the independent variable has been the numbers of pulses at
constant electric field intensity, electric field intensity at constant number of pulses,
or energy at a constant numbers of pulses. The rationale for starting with a large
inoculum is that it enables the detection and counting of survivors after the targeted
population size has been reduced by several orders of magnitude. Obviously, this
makes perfect sense and there is no reason to do otherwise. Yet, the number of a
virulent pathogen’s cells actually encountered in foods, unless intentionally contam-
inated, rarely if ever matches the huge numbers used in microbial inactivation
studies. It would therefore be interesting to investigate at least some of the potential
implications of the relation between the inactivation (and growth) patterns of
individual or very small groups of cells and those of large cell populations of the
same organism in the same food or a surrogate medium.
1278 M. Peleg
A Stochastic Model of Microbial Cells Mortality
Consider a scenario where a microbial cell exposed to PEF can be in one of only two
states, alive (countable) and dead (uncountable). After the first pulse it can be dead
with a mortality probability rate Pm1 and alive at a probability 1- Pm1 as shown in the
Markov tree presented in Fig. 5. If the cell has survived the first pulse, then after the
second pulse it can be dead at a probability Pm2 and alive (1- Pm2), and so on after
pulses i = 3, 4, . . ., n. The probabilities, Pm1, Pm2, Pm3, Pm4, . . ., Pmn, need not be
the same and one would expect that they will rise as the cell is weakened by the
repeated assault. This scenario can be simulated by drawing a random number
between zero and one, which will determine whether after a given pulse i the cell
dies and leaves the game, or survive for the next pulse, depending on the mortality
probability Pmi. The process can be repeated for a number of cells, each tested with a
new set of drawn random numbers, and the total number of survivors after each pulse
recorded. A plot of these survivors’ numbers divided by the initial number vs. the
pulse’s number will be the corresponding survival curve.
Figure 6 shows that, at least qualitatively, almost all the common microbial
inactivation patterns encountered in food preservation can be expressed in terms of
an underlying Pm(t), a function that describes how the mortality probability rate
changes with time (Horowitz et al. 2010; Corradini et al. 2010). The procedure to
generate such survival curves with the stochastic model, which allows the user to
choose the initial number of cells, has been automated and posted on the Internet as a
Wolfram Demonstration, open: http://demonstrations.wolfram.com/ProbabilisticMo
delForMicrobialMortality/. Its screen displays for a large initial population is shown
in Fig. 7 and for a small population in Fig. 8. Although the demonstration was
written for arbitrary time units instead of the number of pulses, it can still be used to
highlight the difference between the survival patterns of large and small microbial
populations. As expected, the simulated survival curve of a large population shown
Fig. 5 The Markov tree of

microbial cells mortality by
Markov tree of cell mortality
PEF. Notice that the mortality
probability Pmi and hence that No. of
Pulses (i) Dead [Pm(i)] Intact [1- Pm(i)]
of the survival 1- Pmi can vary
with the number of pulses
I =0
i=1
i=2
i=3
i=4
Log-linear survival curves without and Tailing with residual “Absolute death
with a flat “shoulder” survival time”
“Tailing” with finite Complete inactivation
“First order kinetics” “First order kinetics” with “shoulder” number of survivors (no survivors after td)
1 1 1 1
0.8 0.8 0.8 0.8
0.6 0.6 0.6 0.6

Pm(t)
Pm(t)
0.4 0.4 0.4 0.4
0.2 0.2 0.2 0.2
0 0 0 0
0 5 10 15 20 0 5 10 15 20 0 10 20 30 0 10 20 30
1 1 1 1
Survival Ratio S(t)
Survival Ratio S(t)
0 0 0 0
0 5 10 15 20 0 5 10 15 20 0 10 20 30 0 10 20 30
0 0 0 0
–2 –2 –2
–1
LogS(t)
LogS(t)
–4 –4 –4
–2
–6 –6 –6
–8 –8 –3 –8
0 5 10 15 20 0 5 10 15 20 0 10 20 30 0 10 td1 td2 td3 30
Time (arbitrary scale) Time (arbitrary scale) Time (arbitrary scale) Time (arbitrary scale)
Fig. 6 Simulated microbial inactivation patterns produced with different mortality rate functions
in Fig. 7 (n = 1000) is smooth and deterministic until there is only a score of

survivors left. In contrast, the curves of small groups of cells shown in Fig. 8
(n = 10) – compare plots a and b – are visibly jugged and more importantly
irreproducible – compare the left and right displays generated with exactly the
same model parameters. Notice that if Pm(t) or Pmi) = constant, the result is
log-linear inactivation (Fig. 7). If Pm(t) or Pmi) varies with time as shown in
Fig. 8, the survival curve has a “shoulder” regardless of whether the population is
large or small. What is important is that one can retrieve the probability rate function
Pm(t) or Pmi) from experimental survival data obtained with a large cell population
and use it to generate realistic scenarios involving individual or small groups of cells
[ibid].
A Stochastic Model of Injury and Mortality
The described approach can be extended to situations where the inactivation is

manifested in both injury and mortality. In such cases, the Markov tree would be
based on two underlying probability rate functions, Pm(t) and Pinjury(t), or for PEF
Pmi) and Pinjuryi). These probabilities are allowed to vary independently with time or
1280 M. Peleg
Fig. 7 Screen display of the Wolfram Demonstration showing the mortality curve of a large cell
population (n = 1000) that follows first order inactivation kinetics and no injury, generated with the
stochastic model. Notice the log-linear curve’s smoothness until only a few survivors are left
the number of pulses – see Fig. 9, which also describes the resulting deterministic
survival curve of a relative large population (n = 1000), except when it reaches only
a score of survivors.
Examples of the concept’s use to simulate microbial injury and mortality of a
small microbial population (n = 10) are given in Fig. 10.
This figure provides another demonstration of the irregularity and irreproducibil-
ity of the survival pattern of very small microbial populations when exposed to a
lethal agent – compare plots a and b. Figures 9 and 10 describe a grossly
oversimplified and perhaps even unrealistic scenario where there is only a single
level of injury, and where the mortality and injury probability rate Pm(t) and
Pinjury(t), respectively, or in the case of PEF the actual probabilities Pmi) and Pinjuryi)
are only functions of time, or number of pulses, regardless of position on the Markov
tree’s branch. In reality, one would expect both the existence of different degrees of
injury, and that cells which have already sustained damaged by a previous pulse or
pulses could become more vulnerable to the treatment.
Fig. 8 Two screen displays of the Wolfram Demonstration showing the mortality curves of a very
small cell population (n = 10) generated with the stochastic model. Notice that the curves are both
irregular and irreproducible
In principle, one can introduce several injury levels into the PEF mortality/injury
model as shown schematically in Fig. 11, with corresponding new probabilities Pmi)
and Pinjuryi) at the beginning of each branch of the Markov tree. Either way, the
resulting stochastic model would be so cumbersome mathematically, and the assign-
ment of the different probabilities so difficult, that the model might be rendered
impractical even for computer simulations. It also remains to be seen whether the
simplified version of the stochastic mortality/injury model that was used to generate
the survival patterns shown in Figs. 9 and 10 can capture the essence of microbial
inactivation by PEF at least qualitatively. But even without such experimental
demonstration, simulations of the kind that the Wolfram Demonstration offers can
still provide some insight into how the treatment affects small and large microbial
cell populations and how different survival patterns emerge.
Recovery from Injury and Delayed Growth
In principle, the stochastic approach can be extended to cells’ recovery after being
damaged by a PEF treatment. It can also be extended to cells division in the
1282 M. Peleg
Fig. 9 Screen display of the Wolfram Demonstration showing survival curve of a large cell
population (n = 120) that suffers mortality and injury, generated with the stochastic model. Notice
the logarithmic curve’s smoothness until only a few countable survivors are left
survivors, be they those who succeeded to remain intact or those who managed to
repair the damage. Schematic view of the Markov trees of such scenarios is given in
Fig. 12. If the approach is adopted in future research, how to assign the
corresponding probabilities and time dependencies will be a serious challenge. But
regardless of the model chosen, i.e., with or without the distinction between damage
severity levels and whether cell division commences or not, time would have to
replace the number of pulses as the independent variable. In other words, the steps in
the stochastic process, the Δt’s, would have to be expressed in pertinent time units
such as hours or days which are orders of magnitudes larger than the treatment
duration let alone that of an individual pulse. The probabilities of mortality, recovery,
and cell division would then become probability rates, i.e., the probability that a
cell’s state would change during the chosen unit time. Again, the practicality of the
probabilistic approach to such scenarios is yet to be demonstrated. But, again, even if
it would never serve as a starting point for any quantitative model development, it
could still highlight the theoretical possibilities that certain unexpected patterns of
Fig. 10 Two screen displays of the Wolfram Demonstration showing the survival curves of a very
small cell population (n = 10) generated with the stochastic model. Notice that the curves are both
irregular and irreproducible
Pulse Markov tree of cell mortality and two injury levels

number
i=1
i=2
i=3
i=4
intact cell slightly injured cell gravely injured cell dead cell
Fig. 11 The Markov tree of an intact cell that can have two levels of injury. Notice that the
mortality and injury probabilities are likely to be functions of the number of pulses
1284 M. Peleg
Markov trees of cell mortality, injury, recovery and division

a Recovery of a slightly b Recovery of a severely c Growth of an intact or
injured cell injured cell recovered cell
Fig. 12 The Markov trees of three scenarios: the recovery of a slightly injured cell (left), the
recovery of a severely injured cell (middle), and the division of an intact surviving cells (right).
Notice that the dividing cell can be a recovered injured cell
recovery and growth can emerge. This is probably true not only for assessing the
microbial safety of foods but also for at least certain medical applications of the PEF
technology where the possibility of damage repair and renewed cell division cannot
be totally ruled out (Garcia et al. 2014; Dermol and Miklavčič 2015).
Conclusions
Traditional survival models used in quantitative microbiology can be adapted for the
dose–response curves encountered in food preservation by PEF. Two prominent
models, the Weibullian and logistic (or Fermi’s distribution), are particularly suitable
for the purpose, and in many situations can be used interchangeably.
For reference or quantitative comparison, a dose–response (or survival) curve
observed in a PEF treatment can be converted into an equivalent isothermal survival
curve at a chosen lethal reference temperature by a simple graphical procedure.
Unlike the traditional F0 which is used as measure of a thermal process’s lethality, the
procedure is based on the number of decades reduction in the targeted organism and
it is model independent, i.e., neither the thermal inactivation nor the lethality caused
by the PEF treatment has to follow first order or any other particular kind of kinetics.
The kinetics of microbial inactivation by PEF treatments, as by other lethal
processes including heat, can be described by stochastic (probabilistic) models
where the underlying probability of a living cell dying (or become uncountable) is
a function of the pulse number. For large populations, such models predict smooth
and deterministic survival patterns, except when the targeted population is reduced
to the last few individual cells. Simulations of small groups of cells, such as of
pathogenic bacteria when present in foods, show that the survival curve is inherently
irregular and irreproducible which is consistent with many observed patterns of food
spoilage and poisoning episodes.
Both the deterministic and stochastic models can be extended to include injury,
recovery from injury, and even delayed cell division. The practicality of such models
is in doubt, but they can be used for simulations with assumed parameters. Although
such simulations are unlikely to provide quantitative predictions, they might provide
insight into how events at the cellular level translate into inactivation and survival
patterns at the population level.
Cross-References

Field Processing
▶ Application of Pulsed Electric Field for Treatment of Fish and Seafood
▶ Detection of Electroporation in Microbial Cells: Techniques and Procedures
▶ Different Cell Viability Assays Following Electroporation in Vitro
▶ Effect of Pulsed Electric Fields on Food Constituents
▶ Electroporation of Biofilms
▶ Pulsed Electric Fields for Food Industry: Historical Overview
▶ Pulsed Electric Field Treatment for Beverage Production and Preservation
Cells
▶ Pulsed Electric Fields Effects on Health-Related Compounds and Antioxidant
Capacity of Tomato Juice
▶ Pulsed Electric Fields for Inactivation of Endogenous Enzymes in Foods
▶ Pulsed Electric Fields for Pasteurization: Defining Processing Conditions
▶ Stress Induction and Response, Inactivation, and Recovery of Vegetative Micro-
organisms by Pulsed Electric Fields
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Cell Electroporation Model Using
Voronoi Networks 67
Qin Hu and Ravindra P. Joshi
Abstract
A simulation study to probe cell membrane electroporation in clusters by a high-
intensity, nanosecond voltage pulse is presented. The focus is on assessing effects
associated with the variability in shape and randomness of the cells within
clusters, the effects on heterogeneous tissues, and the electric strength versus
pulse duration behavior. A two-dimensional Voronoi network approach is used to
obtain quantitative, time-dependent results for the cluster collection. The method
takes account of the random structure in terms of cell shape and heterogeneous
tissue placement. Results demonstrate that in an inhomogeneous system
containing a connected mass of tumor cells, electroporation is likely to start at
the periphery of the unhealthy cells within the tissue. This arises from the
difference in electrical properties between normal/healthy and tumor cells. Sim-
ulations also show that the time for poration is the largest for the configurations
having the lowest disorder.
Keywords
Electroporation • Biological membranes • Voronoi network • Pulse • Electric
fields
Q. Hu
School of Engineering and Technology, Central Michigan University, Mt. Pleasant, MI, USA
e-mail: hu1q@cmich.edu
R.P. Joshi (*)
Department of Electrical and Computer Engineering, Texas Tech University, Lubbock, TX, USA
e-mail: ravi.joshi@ttu.edu

DOI 10.1007/978-3-319-32886-7_141
1288 Q. Hu and R.P. Joshi
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1288
Modeling Details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1290
Simulation Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1294
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1300
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1301
Introduction
The effects of pulsed electric fields on biological cells and tissues have been the
topic of research since the late 1950s. It is now well known that externally
applied electric fields of sufficient magnitude can cause nonlinear changes in
cells. The first such report was the electric-field-assisted reversible breakdown of
cell membranes by Stampfli (1958). The orders-of-magnitude increase in plasma
membrane permeability of a biological cell was termed electroporation (Neu-
mann and Rosenheck 1972). The electric fields that are required to achieve such
effects depend upon the applied pulse duration. Values range from tens of
milliseconds to a few microseconds with an electric field of 100 V/cm to several
kilovolts per centimeter. Electroporation involves (Stampfli 1958; Tsong 1991;
Weaver and Chizmadzhev 1996) rapid structural rearrangement of the membrane,
which leads to the rapid increase of electrical conductivity (Abidor et al. 1979).
In most applications (Meglic and Kotnick 2016), the electric pulses have been
used as tools to modify various properties and responses of cells such as attaining
increases in membrane permeability for introducing various molecules and drugs
into cells, fusion of cells, achieving physical separation of different cell types, the
killing of non-healthy cells, and neuromuscular manipulation for therapy. The
interest in this area continues (Joshi and Schoenbach 2010; Rols and Teissie
1998) and has expanded to numerous subareas in biomedical engineering, drug/
gene delivery, and membrane poration (both reversible and irreversible). More
recently, high-intensity (~100 kV/cm), nanosecond duration pulsed electric fields
have been shown to be useful tools for cellular electroporation, electrically
triggered intracellular calcium release, shrinkage of tumors, temporary blockage
of action potential in nerves, and activation of platelets for accelerated wound
healing. Conceptually, short pulse durations offer the possibility of triggering
purely electrically driven responses without much thermal heating. In principle
then, fast processes such as electron transfers between molecules, electrophoretic
separation and self-organization, or field-induced changes in reaction kinetics
could be fashioned (Kranich et al. 2008), though detailed analyses and a com-
plete understanding remain. An even newer field of research opens up when the
pulse duration is decreased even further, into the subnanosecond range. This push
toward further pulse shortening is driven in part by the possibility of using
wideband antennas, rather than direct contact electrodes, to deliver electrical
energy and create fields (albeit at much lower power levels) to tissues (Seminov
et al. 2015).
67 Cell Electroporation Model Using Voronoi Networks 1289
Of the various effects and cellular responses mentioned above, irreversible

electroporation (IRE) is perhaps one of the most important from a practical stand-
point. IRE occurs if a cell subject to an external electric field cannot recover from the
membrane disruption and undergoes apoptotic cell death (Ben-David et al. 2012).
This technique is emerging as a relatively safe, effective, and minimally invasive
ablation modality for treating tumors (Jourabchi et al. 2014). The nonthermal nature
of this excitation (unlike the heating caused by microwave or radiofrequency
ablation) allows for successful treatment even in close proximity to critical structures
and/or large vessels (Ahmed et al. 2011).
Most of the bioelectric studies reported in the literature have focused on single,
isolated cells (Joshi and Schoenbach 2010; Pucihar et al. 2008). This is a relatively
simple system and an important first step that provides for direct observations,
fundamental understanding, and finer resolutions. However, from the standpoint of
realistic and practical applications of such a technology, it becomes necessary and
germane to assess such high-field effects in tissues and cell clusters. For example, the
application of nanosecond-pulsed electric fields for tumor treatment requires bio-
electric analyses in the context of a multitude of cells and different types of tissues.
Only a few simulation studies have begun to probe bioelectric effects at a higher
level of discretization, including tissues and cell clusters (Pavlin and Miklavcic
2003; Smith et al. 2006). These calculations have evoked time-domain methods
with distributed circuit element representations (Smith et al. 2006; Joshi et al. 2007;
Ramos and Suzuki 2016) and frequency-domain approaches (Kotnik and Miklavcic
2006) and included finite element (FE) analyses (Garcia et al. 2014). Even though
these studies have targeted various aspects, such as electroporation, effects of
suspension conductivity, dye uptake following electroporation, etc., an important
gap exists in assessing the bioresponse with regard to: (a) the variability in cell
density within a given volume, (b) the shape and randomness of the cellular clusters,
(c) the influence of heterogeneous tissue types, (d) the role of pulse width on
fractional electroporation within a given bio-volume, and (e) the density and cell
shape effects on electric strength versus pulse duration characteristics.
A critical analysis of electroporation-based treatments (e.g., electrochemotherapy,
gene therapy and DNA vaccination, tissue ablation with IRE, and transdermal drug
delivery) will provide a good understanding and precise prediction of the local
electric field distributions within tissues. Such information would clearly be an
important tool in planning the treatment and ensuring optimum results. The requisite
electric fields need to develop within the tumor mass embedded in a heterogeneous
cluster with varying electrical properties. Furthermore, it is not immediately clear
where electroporation might start within a heterogeneous mass and, further, whether
electroporation would progress within a desired subregion or subsequently be
initiated within a different tissue type. The aim of the study is to probe some of
the above aspects through numerical simulations. Electric field development in a
heterogeneous tissue with different cell types is considered. The random structure in
terms of cell shape and tissue placement is taken into account here based on a
Voronoi network approach. Details of this method, its implementation, results
obtained, and related discussion are presented next.
Modeling Details
The heterogeneous tissue was modeled using a Voronoi network model which was
previously discussed and applied to charge transport in semiconductor varistors
having heterogeneous microstructure (Zhao et al. 2008). The Voronoi structure is
set up by generating N random seeds that correspond to N locations (xi, yi) within the
entire simulation region. Polygons are then formed by the intersection of perpendic-
ular bisectors from lines that connect neighboring seeds. The above includes gran-
ularity, and variations in the internal disorder can be controlled through the positions
of the N random seeds. Two parameters – the disorder length d (d > 0) and an angle
θ (0 θ 2π) – vary the heterogeneity. The random-shaped, granular Voronoi
cluster models of the form shown in Fig. 1 with varying disorder parameters were
used for the simulations. For concreteness, four different values of the disorder
parameter (d = 0, 0.3, 0.5, and 0.7) have been shown. The mesh generation algo-
rithm was based on an open-source MATLAB mesh generator (Smith et al. 2006).
Fixed nodes were placed on the cell membranes with a uniform spacing. Nodes were
then distributed throughout the domain and connected by Delaunay triangulation.
a b
200 200
180 180
160 160
140 140
Width (µm)
Width (µm)
120 120
100 100
80 80
60 60
40 40
20 20
0 0
0 50 100 150 200 0 50 100 150 200
Length (µm) Length (µm)
c 200 d 200
180 180
160 160
140 140
Width (µm)
Width (µm)
120 120
100 100
80 80
60 60
40 40
20 20
0 0
0 50 100 150 200 0 50 100 150 200
Fig. 1 Voronoi diagrams with different values of the disorder parameters d: (a) d = 0, (b) d = 0.3,
(c) d = 0.5, and (d) d = 0.7
Fig. 2 Distributed representation for the electrical analysis. (a) Circuit schematic for nodal analysis
and (b) various R-C electrical elements across membranes
Between each set of connected nodes, a force was imposed that was a function of the
position between the node and the membrane. The nodes were displaced in propor-
tion to this force, and the system was then re-triangulated. This process was repeated
until the system reached equilibrium.
The random-shaped, granular model sketched in Fig. 2a forms the basis for
electrical calculations for assessing possible membrane electroporation in tissues and
cell clusters. The external electrical voltage was applied between the top and bottom
edges of the Voronoi network. The boundary of each polygon represents a cell wall
and, hence, is electrically very resistive under normal conditions. If the electric field
across any segment exceeds a critical threshold (typically amounting to a transmem-

brane potential difference of 1 V), then physically, the local conductivity would rise
dramatically. It is, thus, important to compute the transmembrane voltages at each time
step and then to make dynamic modifications to the local membrane conductivity.
Electrically, current flow through the entire volume (including both the cells and the
extracellular fluid) is best represented in terms of a distributed circuit model that
consists of capacitive and conductive elements. This approach has been used previ-
ously in bioelectrics (Hu et al. 2005) and accounts for both displacement and conduc-
tion currents. In this scheme, voltages at the center of each polygon are calculated. This
is done by applying Kirchhoff’s current law (KCL) and treating each polygon center as
the ith node of an N-node circuit. The region between each pair of nearest neighbor
nodes is electrically represented in terms of parallel resistance-capacitance (R-C)
elements, as indicated schematically by the rectangular blocks in Fig. 2b.
For completeness, it may be mentioned that the above scheme, though appropri-
ate and physically representative of tissues, overlooks more complicated and realistic
geometries that may have irregular or jagged boundaries and edges. However, by
using finer resolutions and an ever-increasing number of grid points, the present
method could be extended to treat such situations. In addition, a fixed (and hence
“hard”) threshold of 1 V for electroporation has been implemented here for simplic-
ity and computational speed. However, treatments that include pore formation
dynamics based on a time-dependent transmembrane potential do exist and have
been used for several researchers, including our group. Hence, it would be possible,
as the next step up toward a more rigorous analysis, to incorporate such a “soft”
voltage-dependent poration behavior.
The relevant equations connecting any node (say #1) to its neighbor (say #2) can
easily be obtained based on circuit analysis. Thus, for example, continuity of the
total (conduction and displacement) currents between nodes #1 and A, those
between nodes A and B, and, finally, between B and #2 [as shown in Fig. 2b] yields:
d ðV 1 V A Þ V 1 V A d ðV A V B Þ V A V B
C1A þ ¼ CAB þ (1a)
dt R1A dt RAB
d ðV A V B Þ V A V B d ðV B V 2 Þ V B V 2
CAB þ ¼ CB2 þ (1b)
dt RAB dt RB2
where Cij and Rij represent the capacitance and resistance, respectively, between
points “i” and “j” while Vi is the voltage at point “i.” The two above equations
involving the four voltages V1, VA, VB, and V2 yield expressions for both VA and VB in
terms of V1 and V2. Simple algebraic manipulation of Eq. 1a leads to:

Δt 1 1 Δt 1 n CB2 n CB2 n
V nþ1 ¼V n1þ
V nA þ Vn þ V þ V V
A
C1A R1A RnAB A C1A R1A 1 C1A B C1A 2
Δt 1 n Δt 1 n Δt 1 n CB2 nþ1 CB2 nþ1
V þ V þ V þ V V þ V nþ1
C1A RB2 B C1A RnAB A C1A RB2 2 C1A 2 C1A B 1
(2a)
where VAn+1 and VAn represent the voltages at node A at the current and previous
times, respectively. Eq. 2a can symbolically be expressed as:
CB2 nþ1 CB2 nþ1

V nþ1 ¼Kþ V V þ V nþ1 (2b)
A
C1A 2 C1A B 1
where

Δt 1 1 Δt 1 n CB2 n CB2 n
K¼ V n1 þ
V nA þ n V nA þ V þ V V
C1A R1A RAB C1A R1A 1 C1A B C1A 2
(2c)
Δt 1 n Δt 1 n Δt 1 n
VB þ n VA þ V
C1A RB2 C1A RAB C1A RB2 2
Similarly from Eq. 1b, the following expression results:

CAB CB2 nþ1 CB2 nþ1 CAB þ CB2 nþ1
Kþ V2 V B þ V nþ1
1 VB
Δt C1A C1A Δt

CAB n CAB þ CB2 n CB2 n 1 1 Vn
¼ VA VB þ V2 þ þ V nB nA
Δt Δt Δt n
RAB RB2 RAB
V n2 CB2 nþ1
V (3a)
RB2 Δt 2
which has the form:
CAB CAB n
T 1 V nþ1 ¼ T 2 V nþ1
1 T 3 V nþ1
2 Kþ V
B
Δt Δt A
CAB þ CB2 n CB2 n 1 1
VB þ V2 þ n þ Vn (3b)
Δt Δt RAB RB2 B
n n
V V
nA 2 T 2 V nþ1 1 T 3 V nþ1
2 þ K
RAB RB2

þCB2 n Vn Vn
where K ¼ CΔtAB K þ CΔtAB V nA CABΔt V B þ CΔtB2 V n2 þ R1n þ R1B2 V nB RnA RB22 .
AB AB
Using Eqs. 2b and 3b, the current (I12) flowing from node #1 to node #2 in terms
of the voltages V1 and V2 then is given as:

C1A CB2 nþ1 CB2 T 2 V nþ1
1 T 3 V nþ1
2 þ K
I 12 ¼ V1 þ K þ
nþ1
V þ V1
nþ1
Δt C1A 2 C1A T 1
V n1 V nA
V n1 þ V nA g þ (4)
R1A
Thus, Eq. 4 above connects the current I12 flowing between the two node voltages
V1n+1 and V2n+1. If node #1 had multiple neighbors, a simple extension involving an
Table 1 Parameters used Voronoi cell number 90

in the present simulations
Simulation length Lx 200 μm
Simulation width Ly 200 μm
Normal cells
Cell inclusion permittivity 55 Ɛ0
Membrane permittivity 7 Ɛ0
Cell inclusion conductivity 1.2 S/m
Membrane conductivity 5.6 105 S/m
Cancer cells
Cell inclusion permittivity 50 Ɛ0
Membrane permittivity 4 Ɛ0
Cell inclusion conductivity 0.38 S/m
Membrane conductivity 9.1 106 S/m
appropriate sum over all the appropriate paths from node “1” would need to be
implemented.
In the present simulations, whenever the electric field at any polygon segment
reached a magnitude in excess of the electroporative threshold, the membrane
conductivity was switched over to a low level dictated by the cellular fluid. For
healthy cells, the conductivities of the membrane σ m and intracellular fluid σ fi were
taken to be 5.6 105 and 1.2 S/m, respectively. Dielectric permittivities of the
membrane and the fluid were assigned values of 7 e0 and 55 e0. Cell membranes were
deemed to have been porated if the corresponding transmembrane potential reached
1 V. Upon such poration, the membrane conductivity was taken to increase by a
factor of 106. A summary of the various values used in the present simulations is
given in Table 1, which includes parameters for both normal/healthy and cancer
cells. It is understood that there are different types of cancers and the values used
represent averages reported for some common cancer cells.
Simulation Results and Discussion
Simulations results for the response of a homogeneous Voronoi cell cluster to an

electric pulse of amplitude 320 kV/cm are discussed first. Homogeneity implies that
all the cells were taken to have the same electrical parameters and correspond to
normal, healthy cells in this case. The lengths along and x- and y-axes were taken to
be 200 μm. Time-dependent variations on the various transmembrane potentials
across the various nodes were numerically computed. Snapshots of Fig. 3a, b, and c
show snapshots of the Voronoi network with the various edges at three different
times of 40 ns, 60 ns, and 100 ns. In Fig. 3, unporated edges are shown in blue, while
porated membrane segments are in red, pink, or green. A red edge represents a
heavily porated membrane, pink is for moderately porated segments, and green
a 200 b 200
180 180
160 160
140 140
Length (µm)
Length (µm)
120 120
100 100
80 80
60 60
40 40
20 20
0 0
0 50 100 150 200 0 50 100 150 200
c d
200 1.4
ton = 40 ns
180
Transmembrane Potential(V) 1.2 ton = 60 ns
160 ton = 100 ns
140 1
Length (µm)
120
0.8
100
0.6
80
60 0.4
40
0.2
20
0 0
0 50 100 150 200 0 20 40 60 80 100 120 140
Length (µm) Time (ns)
Fig. 3 Snapshots of unporated (blue) and porated (red, pink, or green) for membrane edges. Red
edge represents a heavily porated membrane, pink is for moderately porated segments, and green
edges denote lightly porated ones. (a) t = 40 ns snapshot, (b) t = 60 ns snapshot, (c) t = 100 ns
snapshot. (d) Time-dependent transmembrane potential for the segment indicated by the red arrow in
(a) for three different pulse durations of 40 ns, 60 ns, and 100 ns. The applied E field was 320 kV/cm
edges denote lightly porated membranes. Figure 3d shows the time-dependent

transmembrane potential for the segment indicated by the red arrow in Fig. 3a for
three different pulse durations of 40 ns, 60 ns, and 100 ns. Figure 3a shows only two
membranes to be porated within the entire Voronoi network at 40 ns. The result
shown in Fig. 3b represents a 40 ns snapshot. Four more membranes are predicted to
be lightly porated. Two features emerge out of this analysis. First, a relatively high
electric field value is necessary, and a delay of about 40 ns can be expected for the
onset of poration at this high 320 kV/cm field. Second, once poration is initiated, an
ever-increasing number of membrane gradually get porated over time, as long as the
external pulse remains applied. Figure 3c predicts two neighboring cells to be
porated after 100 ns. This is roughly in line with expectations, since the poration
of any cell segment dramatically reduces its voltage drop, thereby increasing the
electric fields at the neighboring cells.
a b
200 200
180 180
160 160
140 140
Length (µm)
Length (µm)
120 120
100 100
80 80
60 60
40 40
20 20
0 0
0 50 100 150 200 0 50 100 150 200
c d
200 200
180 180
160 160
140 140
Length (µm)
Length (µm)
120 120
100 100
80 80
60 60
40 40
20 20
0 0
0 50 100 150 200 0 50 100 150 200
Fig. 4 Electroporation of one tumor cluster (shown in green) at different time instants. (a)
t = 50 ns snapshot, (b) t = 51 ns snapshot, (c) prediction at t = 52.5 ns, and (d) a snapshot at
t = 53 ns. The applied electric field amplitude was 60 kV/cm
Next, simulations were carried out to study poration in a heterogeneous cluster.

For simplicity, a two-region Voronoi model was used, with a contiguous region of
cancer cells (all having the same electrical parameters), embedded within a simula-
tion region containing normal, healthy cells. The healthy cells were all assigned
separate but equal values for their parameter set according to the recipe given in
Table 1. For convenient comparisons, snapshots of the Voronoi cluster at different
time instant have been shown in Fig. 4 with regards to electroporation of the single
tumor cluster. The applied electric field amplitude was 60 kV/cm. The green region
in the simulation areas shown in Fig. 4 denotes cancer cells with electrical param-
eters that differ from those assigned to the unshaded healthy cells. Figure 4a is a
snapshot at t = 50 ns. It shows about eight membrane segments on the outer
periphery on the right side of the tumor cluster to be porated. The simulation results
predict these membranes to be connected. The snapshot at t = 51 ns shows slightly
more poration, again at the boundary of the tumor cluster. Furthermore, the time
difference between Fig. 4a, b is only about 1 ns and, hence, relatively very short.
Thus, once poration is initiated, it is predicted to develop and progress quite quickly.
The prediction of membrane poration at the tumor boundary is not unexpected

considering the differences in the electrical parameters between healthy and cancer
cells. Cancer cells typically tend to have lower cytoplasm conductivity and also
lower membrane capacitance values. As a result, the voltage drop across the plasma
membranes of tumor cells is likely to be larger than in normal/healthy cells. Hence,
initiation of poration at a plasma membrane of a cancer cell is very likely.
The rapid development of the poration process becomes more evident in Fig. 4c, d.
The t = 52.5 ns snapshot shows membrane poration to continue at the tumor cells,
with some inroads into the tumor mass. In addition, one segment of a healthy cell
adjoining the tumor mass toward the right is also predicted to be porated. Finally,
there is near-complete poration of all membranes as seen from the t = 53 ns
snapshot. The poration at the later time is essentially a domino effect since the
breakdown on any membrane segment causes the transmembrane potential to
collapse, thereby increasing the fields across the neighboring cells. The results,
though, do collectively indicate two important aspects. First, cancer cells can be
expected to be target first by the electric field application over healthy cells. This
would be the desired outcome. Second, much lower values of the external electric
fields would be necessary to porate a heterogeneous cluster containing tumor cells.
The previous results of Fig. 3 for a homogeneous population required much higher
field values. Finally, since a fairly rapid deterioration and killing of the cells are
predicted (e.g., after about 51 ns in this case of Fig. 4), it would be prudent to tailor
the external pulse width. In particular, if the external pulse could have been termi-
nated around 51 ns, most of the healthy cells would not have been porated, thereby
suppressing any collateral damage. Thus developing a recipe with quantitative pre-
dictions of the requisite pulse duration for a given applied field depending on the cell
properties becomes a very meaningful and practical exercise.
After simulating a single cluster embedded in a matrix, a heterogeneous system
consisting of two clusters of cancer cells separated from each other and lying in a
matrix of healthy cells was analyzed. Thus three regions within a Voronoi model
were probed as shown in Fig. 5, with the green-colored space denoting the tumor
cells. The same applied electric field amplitude of 60 kV/cm was used in these
simulations. Electroporation of the various membranes at different time instants is
shown in Fig. 5a–d. At the earliest time of 25 ns, shown in Fig. 5a, poration is just
initiated at one membrane lying toward the right bottom part of the left cluster. Fairly
soon thereafter at t = 26 ns, two edges on the left cluster and one edge on the right
cluster are predicted to be porated. As with the results of Fig. 4, a fairly rapid
progression is then predicted to occur. Figure 4c, representing a snapshot at
t = 27.5 ns, shows more poration, including three segments of healthy cells in the
neighborhood of the left cluster toward the right side. In Fig. 4d at t = 28 ns, almost
all the cells, both normal and tumor, are predicted to be completely porated.
Finally, the strength-duration (S-D) curves were obtained for the two cancer cell
cluster scenarios (as shown in Fig. 5), separated from each other in a matrix of
healthy tissue. Electroporation, being a dynamic phenomenon, depends on applied
electric field and time. It, therefore, becomes germane and important to consider the
pulse-width aspect and to evaluate the underlying strength-duration curves for
a 200
b 200
180 180
160 160
140 140
Length (µm)
Length (µm)
120 120
100 100
80 80
60 60
40 40
20 20
0 0
0 50 100 150 200 0 50 100 150 200
c d 200
200
180 180
160 160
140 140
Length (µm)
Length (µm)
120 120
100 100
80 80
60 60
40 40
20 20
0 0
0 50 100 150 200 0 50 100 150 200
Fig. 5 Electroporation of two tumor clusters at different time instants of: (a) t = 25 ns, (b)
t = 26 ns, (c) t = 27.5 ns, and (d) t = 28 ns. The applied electric field amplitude was 60 kV/cm
various tissue parameters and shapes. Traditionally, the S-D curve plots the threshold
voltage necessary for the onset of an electrically activated bioresponse, as a function
of the pulse duration (Blair 1932). The interest, in this context, is to obtain the
threshold voltage (or average electric field) required to electroporate at a given pulse
duration for a specified distortion factor d and intracellular parameters. Results
obtained are shown in Fig. 6 for four different situations. Two different cases, one
for a homogeneous volume of healthy cells and the other for a single tumor cluster
embedded in a matrix of healthy cells, were simulated. For each of the cases, two
different values of the disorder parameter (d = 0.5 and d = 0.7) were used. The
salient points from the set of curves are the following: (a) For a homogeneous case,
the disorder parameter does not play a significant role. (b) With one tumor cluster,
when the applied electric field is reduced, the time for poration increases as may be
expected. (c) Furthermore, the time for poration is the largest for the single-cluster
configurations having the lowest disorder. With disorder, one is more likely to get
greater variability in the spatial distribution of the electric fields. Hence, the prob-
ability of encountering large electric fields at some locations is higher, leading to
faster electroporation. The cluster starts to fail at such weak points, thus requiring a
Fig. 6 Strength-duration curves obtained for two scenarios involving two different values of the
disorder parameter
lower duration for a given electric field strength. Such poration can then cascade
toward a much greater fraction of affected cells in the tissue cluster.
Finally before closing, a comparative discussion between electroporation in
individual cells and a cluster within a tissue is briefly presented. Basically, as the
density of cells in a suspension increases, the induced transmembrane potential
(TMP) across each cell begins to be more influenced by the proximity of other
cells. If spherical cells are packed together such that they are in contact, the
maximum induced TMP will become equal to the product of the cell radius and
the electric field strength. This corresponds to a 1.5 reduction with respect to the
predicted value in isolated cells. As a result of this reduction, cells in dense
suspensions or tissues need higher electric field strengths to be electroporated than
cells in dilute suspensions. This has been corroborated by experiments (Pucihar et al.
2007; Chopinet et al. 2012). The results of transfection efficiency have demonstrated
striking differences between tissues and individual cells. While about 24% of cells
could be transfected when in suspension, only about 1% were seen to be express the
transfected gene affected in spheroids. Part of the issue is that cells on the periphery
(and those next to the suspension which has higher conductivity and/or permittivity)
are subject to much higher electric fields. Such cells porate first, and as the potential
drop at these locations collapses, those in the interior subsequently get subjected to
high electric fields. The electroporation thus follows a domino process, and this was
shown in the present simulations.
The use of nanosecond electric pulses with pulses shorter than the charging time
of the plasma membrane (<< 1 μs) seems beneficial in this regard for tissue
electroporation This has to do with the fact that the electric current associated with
pulses longer than the charging time of the plasma membrane (1 μs) is confined to
narrow, high-resistance extracellular spaces and does not lead to quick electropora-
tion. The shorter pulses, on the other hand, can penetrate the various cells and layers,
powered by the displacement currents, and the total current flows through both
extracellular and intracellular spaces. The lower membrane permittivity of cancer
cells (as shown in Table 1) would also be advantageous in this regard for building
higher fields toward local electroporation. Also, with shorter duration pulses, more
cells present in an organ or tissue, regardless of their packing and morphology,
would experience a higher and more homogeneous electric field distribution, though
microscopic nonuniformities in the electric field would still be present. However, a
greater number of pulses may be necessary for better results. A solution for enhanc-
ing the cellular breakdown through application of the external electric pulses in
tissues may be to initiate additional electrochemical processes. For example, the
possibility of a synergistic combination of electrolysis and electroporation could
produce more effective poration, ablation, or damage. Furthermore, this combination
would lend itself to the design of clinical protocols that employ lower voltages.
Conclusions
Results were obtained based on two-dimensional, time-dependent, random Voronoi

network analyses. The following salient features were observed from the simula-
tions. First, in an inhomogeneous system containing a connected mass of tumor
cells, electroporation is likely to start at the periphery of the unhealthy cells within
the tissue. This arises from the difference in electrical properties between normal/
healthy and tumor cells (Qiao et al. 2010). Cancer cells typically have lower
cytoplasm conductivity and also lower membrane capacitance. As a result, the
voltage drop across the plasma membranes of tumor cells is likely to be larger
than that in normal/healthy cells. The largest differential can be expected to occur at
the boundary between healthy and tumor cells, leading to the preferential poration of
tumor cell membranes at the boundaries of a connected tumor cluster. This result has
been demonstrated here through numerical Voronoi network calculations for both a
single and a double cluster within a healthy cell matrix. These results also show that
high-intensity, ultrashort pulse application is likely be to a useful tool for electroche-
motherapy and that the cell killing will likely be initiated from the periphery. This is
precisely the region where not only is poration predicted to be stronger, but the
availability of the external drugs (such as bleomycin) will be the strongest. From the
technological standpoint of drug delivery, this is an extremely beneficial outcome. It
ensures the preferential entry of anticancer drugs starting from the outer boundary of
the unhealthy tissue mass. Furthermore, the results suggest that moderately high
fields would be most beneficial as cancer cells would be porated, while leaving the
healthy cells intact. High fields or even moderate values applied over longtime
durations would likely cause collateral damage to the healthy tissue.
The simulation results were also used to obtain the strength-duration curves.
Results showed that the time for poration is the largest for the configurations having
the lowest disorder. With disorder, one is more likely to get larger variability in the
spatial distribution of the electric fields. Hence, the outcome of encountering large
enough electric fields at some locations is more probable, leading to faster electro-
poration. Once electroporation of any cell occurs, the voltage drop across all other
neighboring cells along the field axis increases, thereby accelerating the poration
process. Since most cells in tissues are not symmetric or regular, the implication of
the above bodes well from a practical standpoint for electroporation therapies.
Finally, though not explicitly stated, intracellular conductivity turns out to be a
more important parameter influencing cell membrane poration, with membrane
permittivity having a secondary effect. As a result, though not explicitly probed
here, the injection of nanoparticles to modulate the local conductivity could be used
to accentuate and engineer stronger cell-killing strategies. Some aspects of this
concept have already been demonstrated experimentally (Huang et al. 2014).
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(ed) Handbook of electroporation. Springer International Publishing, Cham
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91:1188–1193
Part VI
Laboratory Techniques Used in Electroporation
Research
Marie-Pierre Rols, Boris Rubinsky, and Javier Raso

Fluorescent Indicators of Membrane
Permeabilization Due to Electroporation 68
Tina Batista Napotnik
Abstract
The plasma membrane represents a barrier between a cell and its surroundings. The
membrane’s selective transport enables the cell to maintain solute concentrations
different from the outside medium. However, in the process of electroporation, the
function of plasma membrane barrier and transport selectivity is at least partially
lost and the molecules that are impermeant to intact membrane can enter into the
cell’s cytoplasm. The uptake of such molecules can be indicative for plasma
membrane permeabilization. Most commonly used indicators for this purpose are
fluorescent molecules: their cellular uptake can be detected by fluorescence micros-
copy as well as by methods suitable to analyze bulk cells, such as spectrofluorom-
eters, plate readers, and flow cytometers. The use of fluorescent indicators is a
simple, noninvasive, highly sensitive, and safe method to monitor the extent of
membrane permeabilization, allowing real-time dynamic imaging or evaluating
response of an average response to electric pulse exposure, depending on the
mode of detection used. There are many fluorescent indicators that are used in
electroporation experiments. Among them, the most widely used fluorophore is
propidium iodide: because it significantly increases its fluorescence by binding to
nucleic acids, it enables microscopic observation of rapid transport across
permeabilized membrane without any washing. In addition to nucleic acid binding
dyes (e.g., PI, YO-PRO®-1 Iodide), there are also other fluorescent indicators of
membrane permeabilization: nonbinding, low molecular weight (such as lucifer
yellow), and large indicators (such as dextrans and quantum dots) available on the
market. With the use of different fluorescent indicators and detection methods,
scientists can gain insight on different perspectives of the electroporation
T. Batista Napotnik (*)

Faculty of Electrical Engineering, Department of Biomedical Engineering, Laboratory of
Biocybernetics, University of Ljubljana, Ljubljana, Slovenia
e-mail: Tina.BatistaNapotnik@fe.uni-lj.si

DOI 10.1007/978-3-319-32886-7_133
1306 T. Batista Napotnik
phenomenon (e.g., asymmetric uptake of molecules, the effects of pulse parameters

on membrane permeabilization, the size of pores, and response variability in cells).
Keywords
Electroporation • Cell membrane • Membrane permeabilization • Fluorescent
indicators
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1306
Fluorescent Indicators of Membrane Permeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1308
Nucleic Acid Binding Fluorescent Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1309
Nonbinding, Low Molecular Weight Fluorescent Indicators (Lucifer Yellow,
Calcein) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1313
Large Fluorescent Indicators (Dextrans, Quantum Dots, Nanoparticles) . . . . . . . . . . . . . . . . . . 1314
Fluorescent Ion Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1315
Fluorescent Conjugates of Annexin V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1316
Fluorescently Labeled DNA and Green Fluorescent Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1316
Detection Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1318
Microscope Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1319
Bulk Cell Detection Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1319
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1321
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1322
Introduction
The plasma membrane represents a barrier between the cell interior and surrounding
environment which enables the cell to maintain different concentrations of solutes,
compared to those in extracellular media. It is a selective barrier: lipid bilayer is not
permeable for most of large uncharged polar molecules and charged ions and
molecules. For the transport of some of these substances, the cell has evolved various
transport mechanisms such as transport proteins (carriers and channels) and a
number of endocytotic pathways; however, most exogenous molecules lack specific
transport mechanisms and are therefore confined to extracellular environment.
Simple diffusion of molecules across a membrane can be defined as a flux from a
region of high concentration toward a region of lower concentration along a con-
centration gradient. It is described by the first Fick's law of diffusion where diffusion
flux (the amount of substance per unit area per unit of time) is proportional to the
diffusion coefficient and concentration gradient of the molecule over thickness of the
membrane. In other words, flux is proportional to the permeability coefficient of the
membrane for the molecule under consideration and its concentration gradient. The
permeability coefficients are determined experimentally.
In the process of electroporation, the barrier function and selectivity of plasma
membrane are at least partially compromised and membrane permeability for mol-
ecules that are deprived of transport mechanisms is transiently increased (the
permeability coefficient increases) (Puc et al. 2003). Therefore such molecules can
68 Fluorescent Indicators of Membrane Permeabilization Due to Electroporation 1307
be indicators of an electroporation extent (▶ Chap. 12, “Measurement of Molecular

Transport After Electropermeabilization”). Charged non-permeant exogenous mol-
ecules are transported through the electroporated plasma membrane by molecular
diffusion due to the concentration gradient and for the duration of the electric pulses
also by electrophoretic forces on charged molecules. The amount of these molecules
that are transported across the permeabilized plasma membrane is proportional to
electroporation extent; however, the transport (▶ Chap. 27, “Mechanistic Descrip-
tion of Membrane Electropermeabilization”) is not just a simple electrochemical
gradient based diffusion across the membrane (more thoroughly described in Part I).
Interactions of indicator molecules with pore walls and molecules in a surrounding
medium and the pore shapes and dynamics also influence molecular transport across
permeabilized plasma membrane. The transport of molecules across electroporated
membrane can be described with different numerical models (▶ Chap. 58, “Model-
ing Transport Across the Electroporated Membrane”). Nevertheless, with the use of
indicator molecules, the scientists can get an important insight into the electropora-
tion phenomenon: observe spatial and temporal dynamics of membrane permeabi-
lization, the effects of pulse parameters and other experimental conditions on
electroporation extent, estimate pore size and thresholds of reversible and irrevers-
ible electroporation, and explore variability of electroporation in different cell types
and biological species.
The entry of these indicator molecules can be demonstrated with different
detection methods that depend on intrinsic properties of molecules, e.g., they are
radioactive, magnetic, or possessing a certain biological function and can be detected
by radioactivity detectors, MRI, or biological tests, respectively. However, our
understanding of biological systems depends on visualizing cellular events; there-
fore the use of color and fluorescent indicators enables us to observe electroporation
under a microscope in real time. Although some of the color stains (trypan blue,
phenosafranine, and erythrosine B) were used as optical imaging probes for electro-
poration detection, a vast majority of experiments are done by using fluorescent
indicators. Their advantages over color stains are higher contrast providing higher
image resolution, higher sensitivity, and higher selectivity toward the target –
fluorescence microscopy is thus a preferential optical-based microscopy technique.
The use of fluorescent probes and fluorescence microscopy possesses several
advantages over other imaging methods. The major advantage is that it can be
compatible with living cells and therefore requires minimal invasiveness and allows
real-time dynamic imaging, i.e., there is no need for cell fixation. Fluorescence
imaging is highly sensitive, with safe detection systems, and allows site-specific
targeting: with the use of fluorescent probes with intrinsic binding specificity or
hybrid systems – a probe is attached to a target specific molecule (peptide/protein
labeling, enzyme-mediated protein labeling). Two or more fluorescent probes can be
used on the same specimen revealing interconnected cellular events. Moreover,
fluorophores with intrinsic ability to change spectral properties upon irradiation
with light of a specific wavelength (photoactivatable, photoshiftable fluorophores)
have been developed over the last two decades. Exploring properties of fluorescent
probes and detection systems is leading to a vast variety of fluorescence imaging
methods (immunolabeling, fluorescence in situ hybridization or FISH, fluorescence

resonance energy transfer or FRET, multiphoton fluorescence, super-resolution
imaging techniques), improving site specificity, sensitivity, and spatial resolution
(which is the main weakness of fluorescence microscopy) (Fernández-Suárez and
Ting 2008; Thomas 2015).
Fluorescent indicators of electroporation can be used with different imaging
techniques, using widefield epifluorescence or confocal microscopes and more
complex, super-resolution imaging techniques. Electroporation in single, individual
cells can be observed in real time with high spatial and temporal resolution.
However, even cells in a cell culture are not completely homogeneous, the effects
of the electric field may vary from cell to cell. Therefore, the use of other means of
detection that enables analysis of a large number of treated cells leads to gaining
information on the average response in a cell population. Spectrofluorometry and
microplate readers with fluorescence detection can be used for bulk cell investiga-
tions; however, background effect plays a significant role in fluorescence detection.
Flow cytometry can be used to determine individual fluorescence in a large number
of cells (more than 104). This way, we can get the distribution of responses in a
population; however, rapid events cannot be observed in real time since they are,
compared to microscopy, time-consuming.
Fluorescent indicators that are used to detect membrane permeabilization during
electroporation are presented in section “Fluorescent Indicators of Membrane
Permeabilization.” In the first part, the basic characteristics of fluorescent probes
are described, and in the second part, the main groups of fluorescent indicators for
electroporation detection are outlined together with their modes of use in laboratory
experimenting. In section “Detection Methods,” the detection methods, their advan-
tages, and drawbacks are described. The detection methods range from microscope
imaging for observing single cell electroporation to bulk cells detection methods:
spectrofluorometry, microplate readers, and flow cytometry.
Fluorescent Indicators of Membrane Permeabilization
A fluorescent probe or a fluorophore is a molecule that absorbs light of a certain

wavelength and re-emits light at a longer wavelength (and therefore, lower energy).
Each fluorescent probe has an absorption (=excitation) and emission maximum
(EX/EM) wavelength of spectra (expressed in nm) (Fig. 1), and the difference
between them is called Stokes shift.
The efficiency for light absorption and emission at a certain wavelength is
represented by extinction coefficient ε (determined by measuring the absorbance
of 1 M concentration at 1 cm cuvette path, measured in mol 1cm 1) and quantum
yield QY (number of photons emitted per photons absorbed), respectively. Fluores-
cence intensity per fluorophore is proportional to the product of ε and
QY. Fluorescence can be temporarily, reversibly decreased due to fluorophore
interactions with nearby molecules (quenching), including the same fluorescent
molecules (self-quenching). It can also be irreversibly decreased in high-illumination
100
75
Relative Intensity (%)
50
25
0
300 400 500 600 700 800 900
Wavelength (nm)
Fig. 1 Excitation and emission spectra of propidium iodide. Excitation spectrum is shown with a
dashed line and emission spectrum is shown using a solid line (Source: Fluorescence
SpectraViewer, Molecular Probes, Thermo Fisher Scientific Inc.)
conditions due to irreversible destruction of fluorophore (photobleaching, also

termed as fading) (Drummen 2012). Fluorescent indicators that are used in electro-
poration studies are listed in Table 1.
Nucleic Acid Binding Fluorescent Indicators
Propidium iodide (PI) is by far the most often used fluorescent indicator for
membrane permeabilization in electroporation studies. There are two main reasons
for this wide use of PI and other nucleic acid binding (intercalating) fluorophores
from phenanthridinium and cyanine dye groups in electroporation experiments:
(1) they are nonpermeant for intact cells and can only enter cells with increased
membrane permeability (caused by electroporation), and when they enter cells they
have a high affinity for binding to nucleic acids (DNA, RNA) and (2) when bound to
nucleic acids, they exhibit a large fluorescence enhancement. This way, the detection
of electroporation is very sensitive and the removal of extracellular medium
containing fluorescent indicator is not necessary. This allows us to observe electro-
poration phenomenon in real time under a fluorescence microscope. Moreover, they
can be used with spectrofluorometers, microplate readers, and flow cytometers,
bearing small background effect.
Propidium iodide is a small dye with a molecular mass of 668 Da and fluorescence
excitation and emission spectral maxima at 535 and 617 nm, respectively (EX535/
EM617, Fig. 1). It dissociates in water to a 2+ charged propidium ion that is subjected
to detection (molecular mass: 542 Da) and iodide anions. Despite the dissociation of
PI, the term “PI uptake” is still used traditionally in publications (Nesin et al. 2011).
In cell biology, PI is widely used as indicator for determining viability: it is
membrane impermeant for intact viable cells and only cells with compromised
membranes (confined to cell death) are labeled. Since electroporation is a reversible
1310
Table 1 Fluorescent molecules of membrane permeabilization, with their excitation and emission maxima, molecular mass, estimated minimal electropore size
for uptake, and their main characteristics. Abbreviations: FITC Fluorescein isothiocyanate, AM Acetoxymethyl, nsEP nanosecond electric pulses, GFP green
fluorescent protein
Excitation Emission Molecular mass,
Indicator group Indicator max (nm) max (nm) minimal pore size Notes
Nucleic acid binding fluorescent Propidium iodide 535 617 668 Da (dissociated Enhance fluorescence upon binding
indicators propidium: 542 Da), to DNA, washing before detection
~1–1.5 nm not needed
Ethidium bromide 300/520 605 394 Da,
~1–1.5 nm
Ethidium 528 617 857 Da,
homodimer 1 ~1–1.5 nm
YO-PRO ®-1 491 509 629 Da,
Iodide ~1–1.5 nm
SYTOX ® Green 504 523 600 Da,
~1–1.5 nm
Nonbinding, low molecular weight Lucifer yellow 428 536 522 Da, Washing before detection needed
fluorescent indicators ~1–1.5 nm
Calcein 495 515 623 Da, Washing before detection needed,
~1–1.5 nm calcein AM ester: leakage out of cells
T. Batista Napotnik
68
Large fluorescent indicators Dextran 495, 519, other 1000 Da to To estimate pore size
conjugates (FITC, other 2 MDa,
other) up to 30 nm
Quantum dots Various Various 2–6 nm High quantum yield and large Stokes
shift, possible aggregates in cells
Fluorescent ion indicators Ca2+ ions + 340/380 520 Ca2+: 0.5 nm Ratiometric measurements,
Fura-2 recommended for nsEP
Ca2+ ions + 506 531 Ca2+: 0.5 nm Recommended for nsEP
Calcium green
Ca2+ ions + 350 405/485 Ca2+: 0.5 nm Ratiometric measurements
Indo-1
Tl+ 490 525 Tl+: 0.4 nm Sensitive, recommended for nsEP
ions + FluxORTM
Annexin V FITC-annexin V 495 519 PS externalization, recommended for
nsEP
Fluorescently labeled DNA and DNA + 514 533 Various DNA (~650 Da Tracking DNA uptake and gene
green fluorescent protein (GFP) TOTO ®-1 Iodide for each Bp) electrotransfer
GFP 395/475 509 GFP plasmid: ~4–6 Indirect determination of DNA
kBp uptake and gene electrotransfer
(~2–4 MDa)
Fluorescent Indicators of Membrane Permeabilization Due to Electroporation
1311
Fig. 2 Transport of PI to B16 F1 mouse melanoma cells after application of 20 electric pulses
(60 ns, 45 kV/cm, 100 Hz) (a) before pulse application, (b) 1 s after pulse application, (c) 1 min after
pulse application (Image by the author, not published previously)
event, PI cannot be used for determining viability in time range until complete
resealing has occurred (at least an hour after electric field application).
In electroporation studies, PI can be used for observation under the fluorescence
microscope as well as for spectrofluorometry and flow cytometry investigations. PI
is added to electroporation medium prior to electric field exposure in concentrations
ranging mainly between 30 and 100 μM (Golzio et al. 2002), according to a
threshold of detection system. In real-time monitoring, fluorescence labeling of
different parts of the cell can be observed: this way, we can get information of
spatial distribution of plasma membrane permeabilization related to electric field as
well as its temporal kinetics (Fig. 2). The dynamics of plasma membrane resealing
(▶ Chap. 4, “Membrane Permeabilization Lifetime in Experiments”) can be studied
by adding PI at different periods after electric field application. When using flow
cytometry for detecting membrane permeabilization, the analysis has to be
performed within a certain time frame after electroporation (usually 5–15 min after
electric field application). In this time, PI enters the cells and binding to DNA fully
enhances PI fluorescence; however, longer periods of incubation time are not
recommended since PI is toxic and can damage cells which leads to false positive
PI staining.
When using short, nanosecond electric pulses (nsEP, or nanosecond pulsed
electric field – nsPEF), the scientists have to be aware that single or a small number
of pulses lead to the emergence of smaller pores (termed as “nanopores”) that are too
small to allow PI to pass through and therefore special care has to be taken at
interpreting the results of PI detection (Pakhomov et al. 2009). In this case, the use of
smaller molecules or ions as indicators of plasma membrane permeability is
recommended.
Apart from in vitro studies, PI was also used in vivo: to gain information of
plasma membrane permeabilization in vivo, PI solution was injected to mouse
skeletal muscle prior the delivery of electric pulses, and the extent of membrane
permeabilization in the whole muscle was evaluated using a stereomicroscope
(Čorović et al. 2010).
In a similar way as PI, other phenanthridinium (such as 394 Da, EX300 or
EX520/EM605 ethidium bromide, and 857 Da, EX528/EM617 ethidium
homodimer 1) and cyanine (such as 629 Da, EX491/EM509 YO-PRO ®-1 Iodide,
and 600 Da, EX504/EM523 SYTOX ® Green, both Molecular Probes™, Thermo
Fisher Scientific Inc.) nucleic acid binding dyes were used for the detection of
membrane permeabilization in electroporation studies. All of these exhibit brighter
fluorescence when bound to nucleic acid (especially to DNA). YO-PRO ®-1 Iodide
was used in experiments in nsEP studies: compared to PI, YO-PRO ®-1 Iodide is a
smaller molecule and it enters the cell at lower nsEP pulse count than PI (Vernier
et al. 2006), therefore its use represents higher sensitivity for the detection of
membrane permeability than PI in the case of nanosecond electric pulse application.
Nonbinding, Low Molecular Weight Fluorescent Indicators (Lucifer

Yellow, Calcein)
Some other small fluorescent indicators of membrane permeabilization such as

lucifer yellow (522 Da, EX428/EM536) and calcein (623 Da, EX495/EM515) are
also used in electroporation studies. They are both nonpermeant for intact plasma
membrane so they can be used as markers for fluid-phase endocytosis, but they can
also enter cells during electroporation. However, in contrast to nucleic acid binding
fluorophores (from section “Nucleic Acid Binding Fluorescent Indicators”), they
remain confined to cell cytoplasm, they do not bind to any cell structures, and they
do not enhance their fluorescence intensity when they are transported into cells.
Therefore, the fluorescent molecules in the surrounding medium pose a significant
background problem at detecting membrane permeabilization and cells have to be
washed before detection is made. On one hand, if cells in vitro are attached, the
medium is simply replaced by a fresh medium, on the other hand, cells in suspension
have to be centrifuged several times in order to minimize background fluorescence
which takes a certain amount of valuable time. Moreover, these dyes can also leak
out of the cells during washing if the cell membrane is not resealed yet. Electropo-
ration has to be done quickly after adding dye to the electroporation medium since
these dyes tend to accumulate in endocytotic vesicles over time. These dyes cannot
be used for real-time imaging of membrane permeabilization and for precise spatial
and temporal studies of electroporation phenomenon. However, they can be used for
evaluating the effect of pulse parameters on electroporation and membrane perme-
ability and detecting variability between cell types and biological species (Canatella
et al. 2001; Puc et al. 2003).
Another possible use of calcein as an indicator of membrane permeabilization is
in the form of acetoxymethyl (AM) ester: this is a cell-permeant derivative of calcein
that is cleaved by esterases in live cells into a nonpermeant form. When cells
are porated, the efflux of calcein out of the cells can be monitored as a sign of
membrane permeabilization (Khine et al. 2005). The leak of lucifer yellow out of
endocytotic vesicles (where it was trapped during fluid-phase endocytosis) also
demonstrated permeabilization of vesicles with nsEP (Napotnik et al. 2010). Calcein
is also used in studying transdermal delivery by electroporation of the skin (Zorec
et al. 2015).
Large Fluorescent Indicators (Dextrans, Quantum Dots,

Nanoparticles)
The size of the pores (▶ Chap. 11, “Experimental Determination of Lipid

Electropore Size”) that emerge during electroporation range from a few tens of nm
to less than a nanometer (after nsEP application). To determine the size of these
openings that cause an increase in membrane permeability, larger fluorescent indi-
cator molecules than standard PI and other nucleic acid binding dyes have been used.
Among larger fluorescent indicators of membrane permeabilization, quantum dots
and other nanoparticles are found; however, dextrans were mainly used for the
purpose of determining pore size.
Dextrans are water-soluble polysaccharides, polymers of glucose with high
molecular weight (usually more than 1000 Da, up to 2000 kDa). A linear backbone
consists of α-1,6 linked glucopyranosyl repeating units and the branches with α-1,2,
α-1,3, and α-1,4 linkage. They have varying chain length and side branching and
are therefore of different molecular weight. They are biologically inert, with
low toxicity and immunogenicity. Commercially available dextrans are conjugated
to a wide variety of fluorophores such as fluorescein isothiocyanate (FITC)
which can be detected with fluorescence microscopy, flow cytometry, or
spectrofluorometry.
Fluorescent dextran conjugates of different molecular weight (and size) are often
used for determining pore size: smaller dextrans pass plasma membrane more
efficiently than larger ones; if their size is bigger than pore size, they cannot enter
cells (Yumura et al. 1995). This way, one can estimate the approximate size, spatial
distribution, and dynamics of membrane openings for certain pulsing conditions.
Dextrans can also be used together with other indicators. Moreover, dextrans can be
used ex vivo and in vivo to study transdermal delivery and blood vessel permeability
in electroporation experiments.
Quantum dots (QD) can also be delivered to cell interior via electroporation;
hence, they can serve as indicators of membrane permeabilization. Without electro-
poration or other delivery methods, QD are transported to cells by endocytosis and
remain confined to endocytotic vesicles. QD are semiconductor crystals that fluo-
resce at narrow discrete wavelengths with high quantum yield and large Stokes shift.
They are photostable and resistant to quenching and therefore their use in fluores-
cence microscopy is increasing. Moreover, their surface can be modified in order to
gain water solubility, biocompatibility, and conjugation with functional biomole-
cules which makes QD interesting for biomedical applications, although their
cytotoxicity due to core elements (cadmium, zinc, tellurium, selenium) remains
questionable (Breger et al. 2015). Because of larger size (typically between two
and six nm), the delivery of QD to cells requires higher electric field intensity and
duration compared to smaller dyes such as PI (Sun et al. 2014; Santra et al. 2014).
Grouping of QD into aggregates in cells often occurs in electroporation delivery,
however can be overcome by proper QD surface coating.
Fig. 3 Monitoring electroporation with fura-2. (a) CHO cells – bright field. (b) Ratio of fluores-
cence at two excitation wavelengths (EX345/EX385) for cells in control (non-porated cells). (c)
Cells 1 min after electroporation with a 250 V/cm, 10 ms pulse. Brighter cells were electroporated.
Arrow denotes the field direction. Bar represents 20 μm (Source: (Pucihar et al. 2011), # 2011
IEEE. Reprinted with permission of a copyright holder (IEEE))
Fluorescent Ion Indicators
Permeabilization of plasma membrane in electroporation can also be detected by an

influx of ions that are present in cytoplasm at very low concentrations (such as
calcium, a concentration of free calcium in cytoplasm is kept at below 100 nM due to
its signaling role) or not present at all (thallium). For this purpose, the use of
fluorescent indicators is indirect: they are introduced to cells and change their
fluorescence upon ion binding. Fluorescent ion indicators such as fura-2 (calcium
indicator) are introduced to cells in the forms of AM esters which are cell permeant.
They diffuse passively into cells where they are cleaved by intracellular esterases to
cell impermeant dyes. Compared to fluorescent dyes such as PI, ions are much
smaller and can therefore be used for detecting smaller pores such as those that
emerge during nsEP application. Hence these ion indicators (i.e., fura-2, calcium
green and indo-1 for calcium, FluxOR™ from Molecular Probes™, Thermo Fisher
Scientific Inc., for thallium, for EX, and EM please see Table 1) in combination with
ion influx can be considered a very sensitive detection of membrane
permeabilization.
Moreover, some indicators have a shift in their excitation (fura-2) or emission
(indo-1) spectra upon ion binding and can therefore be used for ratiometric quanti-
fication. With ratiometric measurements, the variations and artifacts due to uneven
loading to cells, variable specimen thickness, dye leakage, and photobleaching can
be minimized. In ratiometric measurements, the ratio between fluorescence intensi-
ties at maximum wavelengths for bound and unbound indicator is calculated. For
fura-2 which is the most often used ion indicator in electroporation experiments,
emission at EM520 is measured for two excitation wavelengths (EX340 and EX380)
and the ratio between the two fluorescence intensities is determined (Fig. 3). With the
use of solutions of known ion concentrations for calibration, the concentrations of
free cytosolic calcium can be determined.
The influx of ions due to the increased membrane permeability can be detected by
the use of fluorescence microscope: ion uptake can be monitored both spatially and
temporally to gain an insight on electroporation phenomenon (Pucihar et al. 2011).
When using calcium uptake for plasma membrane permeabilization detection, we
have to bear in mind that calcium ions (Ca+) can also be released from internal stores
(endoplasmic reticulum, mitochondria), or more complex pathways involving cal-
cium, such as calcium-induced calcium release or store-operated (capacitive) cal-
cium entry, can be activated. They all can contribute to the elevation of intracellular
calcium concentrations and can therefore lead to false detection of plasma membrane
permeabilization. To avoid that, thallium ions (Tl+) are used instead of Ca+. Thallium
is not present in cells in any detectable concentrations and Tl+ influx is always
related to plasma membrane permeabilization. Since thallium ions are even smaller
than calcium ions (0.392 nm and 0.462 nm diameter, respectively), they are even
more suitable for nanopore detection when using nsEP (Bowman et al. 2010).
Fluorescent Conjugates of Annexin V
One of the interesting and unconventional uses of fluorescent indicators of mem-

brane permeabilization is the use of fluorescent conjugates of annexin V. Annexin V
is a vascular protein that binds to phosphatidylserine (PS), a phospholipid membrane
component that is confined to the inner leaflet of plasma membrane. During apo-
ptosis, PS is no longer restricted to the inner leaflet and is translocated to the outer
leaflet; hence, annexin V is widely used as a tool to detect PS externalization and
serve as an assay for apoptosis (Vermes et al. 1995). However, it can serve also for
plasma membrane permeabilization detection since it was hypothesized that PS can
be translocated from the inner to the outer leaflet of plasma membrane along the
walls of electropores (Vernier et al. 2004). PS externalization detection with FITC-
annexin V was used for detection of plasma membrane permeabilization. PS exter-
nalization occurs at much lower electric field intensities, compared to dyes such as PI
and YO-PRO ®-1 Iodide, and is therefore considered as a very sensitive fluorescent
indicator of membrane permeabilization, especially in the case of nsEP (Pakhomov
et al. 2009). However, for the purpose of membrane permeabilization detection due
to electroporation, annexin V has to be used within a short time window after
electroporation. If it is used more than 20 min after applying electric pulses, PS
externalization can be rather a sign of apoptosis than of electroporation.
Fluorescently Labeled DNA and Green Fluorescent Protein
DNA and RNA can also be introduced to cells by using electroporation, transferring
foreign genes to cells in the process of gene electrotransfer. Fluorescence labeling related
to nucleic acids can be used for detection of membrane permeabilization in two ways.
One way is to tag DNA with a fluorophore such as TOTO ®-1 Iodide nucleic acid
stain (Molecular Probes™, Thermo Fisher Scientific Inc.). This way, the
Fig. 4 Detecting cells with expressed green fluorescent protein (GFP) with fluorescence micro-
scope and determining the rate of transfection. (a): Light microscopy. (b): Fluorescence micros-
copy. Electric pulses used in gene electrotransfer: 8x 5 ms, 700 V/cm, 1 Hz (Image by the author,
not published previously)
interactions of DNA with plasma membrane and its transport into cell interior can
be directly observed and monitored. However, DNA is a large molecule and its
cellular uptake with electroporation is a complex process. Although not yet
completely understood, the basic steps of gene electrotransfer (▶ Chap. 23,
“Gene Delivery by Electroporation in Vitro: Mechanisms”) are already known:
when applying an electric field, DNA interacts with plasma membrane, and then
DNA is transported across the membrane and through the cytoplasm to enter the
nucleus. Only after DNA reaches the nucleus, gene electrotransfer is successful and
leads to the expression of transferred genes (Rosazza et al. 2016b). The mechanism
by which DNA is transported across the plasma membrane is not yet elucidated.
Possible mechanisms include transport of DNA through large electropores, drag-
ging into cells by electrophoresis or different modes of endocytosis (Rosazza
et al. 2016a). Because of the complexity of DNA uptake, gene electrotransfer is
not always easy to achieve.
The second way to detect plasma membrane permeabilization with fluorescence
labeling of nucleic acids is indirect. An electric field is applied to cells in electropo-
ration medium containing the GFP plasmids (small circular DNA that encode a gene
for green fluorescent protein – GFP, EX395 and EX475, EM509) and the plasmids
are transferred to cell interior. In the case of successful DNA transfer, the fluorescent
GFP protein is expressed. This occurs about 24 h after electroporation; therefore the
researchers have to be aware that gene electrotransfer requires sterile work. Cells
with expressed GFP can be detected simply under the fluorescence microscope: they
appear green (Fig. 4). The rate of transfection can be determined by counting green
cells versus all cells in the cell culture. The analysis can also be done by using flow
cytometry. However, we have to bear in mind that not only plasma membrane
permeability but numerous other conditions influence gene expression (Rosazza
et al. 2016b).
Table 2 Methods for fluorescence detection used in electroporation studies, their advantages and
drawbacks
Mode of
detection Detection method Advantages Drawbacks
Microscope Fluorescence For spatial and temporal Only single or a few cells
imaging microscopy position and dynamics of analyzed, not for an
fluorescent indicators in average response of a
single cells, real-time population
monitoring, rapid,
individual response
Bulk cell Spectrofluorometry For an average response of Not for spatial and
detection a population temporal position and
methods dynamics of fluorescent
indicators in cells, not for
real-time monitoring,
background signal (can be
decreased by washing or
disintegration), not rapid,
time-consuming,
measuring one sample at a
time
Microplate readers For an average response of Not for spatial and
a population, multiple temporal position and
sample analysis, for dynamics of fluorescent
attached cells or in indicators in cells, not for
suspension real-time monitoring
Flow cytometry Response of the average of Not for spatial and
the whole population as temporal position and
well as single cells, dynamics of fluorescent
analysis of a high number indicators in cells, not for
of cells (104 or higher) real-time monitoring
Detection Methods
There are different methods and systems for detecting photons that are emitted from
fluorophore molecules. The transport of fluorescent indicators in single cells can be
observed with various fluorescence microscopy imaging techniques or plasma
membrane permeabilization in bulk cell suspensions can be determined with spec-
trofluorometers, microplate readers, or flow cytometers. All the methods have
advantages and weaknesses which define the choice of protocol for each of our
experiments (see Table 2).
Each detection method has a detection threshold above which a fluorescence signal
is detected as positive. With the proper choice of fluorescent indicators and their
concentration, plasma membrane permeabilization can be detected, although the scien-
tists have to be aware that the use of different detection methods can lead to slightly
different results. For example, flow cytometry leads to higher measurements of gene
electrotransfer yield (with GFP) than fluorescence microscopy (Marjanovič et al. 2014).
Microscope Imaging
The most common fluorescence microscopes are epifluorescence ones: the light for
excitation and the emitted light pass through the same optical path (through the
objective). The light source is usually xenon arc or mercury vapor lamp; however,
nowadays it can be replaced by high-power light-emitting diodes (LEDs). Light
from the light source passes through an excitation filter or a monochromator which
allows the passage of light of certain wavelength appropriate for each fluorophore.
Emitted light passes back through the objective and through an emission filter which
transmits only emitted light and (with the help of a dichroic mirror) separates it from
much stronger excitation light.
Each microscope has a few objectives leading to different magnifications and
optical resolutions where events can be observed on the cellular and subcellular level
in real time. Higher magnifications require the use of immersion oils and thin bottom
cell chambers. The highest lateral resolution obtainable with current epifluorescence
microscopes is around 200 nm.
Axial resolution is much lower than lateral. The use of confocal microscopy
provides optical sectioning – with the use of a pinhole, noninvasive optical sections
of thick specimen can be obtained and hence 3D images can be created. Lasers that
are mostly used as a light source in confocal microscopes have great spatial
coherence and high irradiance which, together with the pinhole and optics in
confocal laser-scanning microscopes (CLSM), greatly improve the resolution. How-
ever, only with super-resolution imaging techniques (such as stimulated emission
depletion, STED; reversible saturable optical linear fluorescence transitions,
RESOLFT; photo-activated localization microscopy, PALM; and stochastic optical
reconstruction microscopy, STORM) the resolution is increased beyond the diffrac-
tion of light by which the conventional light microscopy is limited.
Fluorescence microscopy techniques enable to monitor a rapid transport of fluo-
rescent indicators across the permeabilized plasma membrane providing information
on precise spatial and temporal dynamics in single or a small number of cells. The
individual response of cells can be evaluated, either in attached cells or cells in
suspension.
Bulk Cell Detection Methods
For the average response of population, bulk cell detection methods which analyze
the whole population of cells (a large number) have to be used. However, rapid
events cannot be monitored since they are mostly time-consuming and require
translocation of cells from electroporation to detection chambers. The intracellular

spatial dynamics of plasma membrane permeabilization cannot be observed and we
have to take into an account that background signal can also significantly contribute
to the results.
In spectrofluorometry, an excitation light beam passes through a sample in cuvette
and emission light from the sample is collected by a photomultiplier tube, photodi-
ode, or charge-coupled device detector. This way, not only cells but the surrounding
medium too contributes to the emission signal. Therefore, a strong background
signal can be expected. Cells that take up fluorescent indicators after electroporation
can be washed (after resealing of plasma membrane) and disintegrated, and the
amount of indicator taken up in cells can be measured in a sample solution by a
spectrofluorometer (Puc et al. 2003). Calibration can be done by using known
concentrations of the indicator.
With microplate readers, more than one sample can be analyzed at a time
(in contrast to spectrofluorometry where only one sample can be analyzed at a
time): multiwell plates of up to 96 wells can be used, or even higher density
microplates (having 384- or 1536-well plates) are available on the market. They
can be used for cells in suspension and for attached cells or even for measuring the
fluorescent indicator from disintegrated cells (the same as with spectrofluorometer).
Emission light is collected from various points in a microplate well and average
intensity is determined for each well.
In flow cytometry, cells in suspension are aligned in a flow-through system (using
laminar flow of sheath fluid to carry the cells) where they are analyzed for fluores-
cence one by one. The response of single cells as well as the average of the whole
population can be obtained. Cells reach the interrogation point where they are
illuminated with excitation light derived from lamps (xenon, mercury) or lasers.
Intensity of emitted light is determined for each cell. Contemporary flow cytometers
can analyze thousands of cells in each second, gaining measurements of a large
population of cells (104 to 106) in a very short period of time. Besides the possibility
of using more fluorescent indicators at the same time (multicolor flow cytometry),
the cells are also analyzed for their size (by forward light scattering, FSC) and cell
granularity or internal complexity (by side light scattering, SSC). Some of flow
cytometers can even sort cells based on their characteristics (fluorescence-activated
cell sorters –, FACS).
The individual data obtained are subjected to computerized analysis. Usually, the
results are presented in the form of a single parameter histogram or a two-dimensional
dot plot (Fig. 5). With manual gating, different subsets of cells in the sample can be
analyzed separately; however, gating can be complex and requires an involvement of
a skilled cytometrist. Recently, a variety of novel automated computational analyses
were introduced to bypass manual gating (Mair et al. 2016).
The flow cytometry is a fast, multiparametric method that has many applications
in basic research and clinical practice. With flow cytometry, the scientists can get
important information on plasma membrane permeabilization. However, rapid intra-
cellular events can only be observed by microscopic imaging.
Control
Electroporation
106
500
105
Propidium fluorescence
400
104
Number of cells
300
103
200
102
100 101
0 100
100 101 102 103 104 105 106 0 200K 400K 600K 800K 1.0M
Propidium fluorescence Forward scatter
Fig. 5 Detection of electroporation with flow cytometry, presented by histogram (left) and dot plot
(right), using propidium iodide as a fluorescent indicator. Electroporated B16F1 mouse melanoma
cells (pulse parameters: 8 100 μs, 1 kV/cm, 1 Hz, shown in red) have much higher emission
fluorescence than control cells (blue). Courtesy of Janja Dermol, University of Ljubljana, Faculty of
Electrical Engineering
Conclusions
There are numerous fluorescent indicators that can be used for membrane permeabi-
lization detection to study the effects of pulsed electric field on plasma membrane
and cells, membrane, or cell structure and function. The indicators used in these
experiments fit to the group of molecules that cannot be transported to intact cells by
any transport mechanisms and are therefore introduced to cells when plasma mem-
brane is electroporated. In some cases, fluorescent indicators are used indirectly,
connected through the transport of other ions and molecules (fluorescent ion indi-
cators, e.g., fura-2, annexin V, GFP). Different fluorescent indicators for membrane
permeabilization can be used in various ways to gain an insight on the electropora-
tion phenomenon (e.g., asymmetric uptake of molecules, the effects of pulse param-
eters on membrane permeabilization, the size of pores, response variability of cells
of different type and species). Moreover, different detection methods for evaluating
changes in spatial and temporal positions of fluorescent indicators are available for
different purposes: to observe transport of molecules into single cells in real time
(fluorescence microscopy) or to gain overall response of the population (bulk cells).
Nevertheless, the use of fluorescent indicators for membrane permeabilization rep-
resent fast, precise, relatively inexpensive, and safe detection of plasma membrane
permeabilization in electroporation experimental work.
Acknowledgment The work was supported by the Slovenian Research Agency (ARRS), grant
P2-0249. It was conducted in the scope of LEA EBAM: the European Laboratory of Pulsed Electric
Fields Applications (2011–2018) and COST Action TD1104: European network for development of
electroporation-based technologies and treatments (EP4Bio2Med) (2011–2016).
Cross-References

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Atomic Force Microscopy to Explore
Electroporation Effects on Cells 69
Etienne Dague
Abstract
Atomic force microscopy (AFM) is more and more used in life science. Its ability
to provide images of living cells as well as mechanical or adhesion maps makes it
a technology that cannot be ignored. In the context of electroporation (EP) which
undoubtedly affects the cell membrane or wall, a technology able to probe the cell
surface is more than interesting. This chapter describes the principle of the AFM
technology and especially the latest multiparametric imaging modes developed
recently. It then demonstrates that AFM can be used to probe cell’s morphology
modifications induced by electric pulses. We then show that EP modifies cell’s
nanomechanical properties and that the actin cytoskeleton plays a major role in
this process. Finally we shed light on the effects of EP on bacteria as probed by
AFM. In this latest example, it must be noticed that no mechanical modifications
are induced, but the adhesion properties of the bacteria are dramatically reduced
by pulsed electric fields (PEFs). Altogether the chapter shows the interest of
applying AFM on cells exposed to EP, in order to get a better fundamental
understanding of EP effect on cells or bacteria.
Keywords
Atomic force microscopy • Electroporation • Elasticity • Cell topography •
Cytoskeleton • Bacteria • Adhesion
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1326
Atomic Force Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1326
Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1327
E. Dague (*)
LAAS-CNRS, Université de Toulouse, CNRS, Toulouse, France
e-mail: edague@laas.fr

DOI 10.1007/978-3-319-32886-7_134
1326 E. Dague
Force Spectroscopy: Nanomechanics and Adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1328

Multiparametric Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1329
Imaging Effects of EP on Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1329
Probing Biophysical Modifications Induced by EP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1332
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1336
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1336
Introduction
Electroporation (EP) consists in applying electric fields of various intensity and/or

duration on cells in order to modify their interface and let non-permeant molecules go
through the plasmic membrane (Wong and Neumann 1982). EP has proven to be
efficient to potentiate cytotoxic molecules (electrochemotherapy in cancer treatment)
and has the potential to be used in gene transfer (Testori et al. 2012; Bodles-Brakhop
et al. 2009). However the mechanisms of cell membrane modifications are poorly
understood and limit the applications of this technology (Teissie et al. 2005). Explor-
ing and understanding the modifications induced by EP on cell’s membrane is indeed a
challenge that has been addressed by measuring the entry of fluorescent or radioactive
molecules in pulsed cells or by transmembrane potential measurements. However,
using these techniques, the direct physical effect on the membrane is not obvious, and
the word electroporation itself is questionable as no pores were ever reported.
Electropermeabilization is therefore sometimes preferred. The cell membrane perme-
ability is, indeed, modified, and understanding electroporation effects is therefore a
matter of cell membrane exploration. In this context, the atomic force microscope is a
pertinent technology as it makes it possible to analyze cell surface in their native
environment, in this case before and after electric pulse application. Atomic force
microscopy (AFM) is an imaging technology developed in the 1980s by physicists
(Binnig and Quate 1986; Hansma et al. 1988; Gerber and Lang 2006). It has been
more and more applied on biological samples (virus, bacteria, mammalian cells, DNA,
proteins, etc.) since the 1990s (Pillet et al. 2014) and recently to explore the effects of
EP on cell morphology and nanomechanical properties (Chopinet et al. 2013a, b;
Louise et al. 2014; Pillet et al. 2016; Thompson et al. 2014).
But more than an imaging technology, AFM is also a force machine able to
measure forces in the pN range (Neuman and Nagy 2008). This capability can be
used to measure the nanomechanical properties of cells and especially after EP.
In this chapter, I will describe the AFM technology and present its application to
better understand EP.
Atomic Force Microscopy
The AFM principle (Binnig and Quate 1986) relies on the measure of interaction
forces between a sharp tip and the analyzed sample. The tip is scanned over the
sample, or conversely, the sample is scanned over the tip, thanks to a piezoelectric
69 Atomic Force Microscopy to Explore Electroporation Effects on Cells 1327
ceramic that can be moved in the three spatial directions: x, y, and z. In order to allow
a displacement of the tip in the z direction, it is mounted on a cantilever, whose
deflection is monitored via an optical device based on a laser and a four-quadrant
photodiode. The resolution is therefore directly linked to the tip size. The sharpest
tip, for example, carbon nanotubes, will achieve the highest resolution. The draw-
back is that such tips are both stiff and delicate. As a consequence, they are not
adapted for living cells, and tips presenting pyramidal geometry (curvature radius
around 20 nm) are preferred for living cell exploration. Another parameter that can
limit AFM resolution is the scanning velocity. Images or force maps are recorded
line by line or point by point, and this is a time-consuming process. A 512-line image
recorded at a scan rate of 1 Hz requires 8 min and 30 s (each line is scanned two
times). A solution to increase the resolution can be to decrease the scanned area.
Classical piezo can scan areas up to 100 100 μm2. By decreasing the analyzed
surface, the resolution is mechanically increased. However, this has a limit linked to
the tip size. In a 10 10 μm2 picture made of 512 lines, the thickness of each line is
of 19 nm, which is close to the typical curvature radius of tips used for living cells. In
this case, decreasing the scan size will not result in increasing the resolution so
much, each line being smaller than the tip size.
The main advantage of AFM in life science is its ability to work in liquids, like
growing media at a controlled temperature, i.e., 37 C, under gas flow, typically CO2
5%. In these conditions, living cells can be observed during several hours, in their
native condition, and/or after a treatment (drug, EP, temperature modification, etc.).
Imaging
AFM images represent the topography of the sample, which is deduced from the
cantilever deflection. In contact mode (Fig. 1), the tip is applying a constant and
arbitrary force on the sample during scanning. The force is kept constant by
modulating the tip or sample height trough a feedback loop. This mode is easy to
use and provides high-quality images. However, the tip, while scanning, induces
lateral forces that can damage the sample, especially in the case of a soft sample like
a cell. Conversely the lateral forces induce a friction, which is recorded as a lateral
deflection of the tip. This lateral deflection is material dependent and can therefore be
used to decipher between materials under investigation. The drawback of these
lateral forces is that the biological sample has to be firmly immobilized on the
surface to overcome them (Fig. 1). Thus, strategies were developed to perform
non-denaturing immobilization (El Kirat et al. 2005; Touhami et al. 2003; Dague
et al. 2011; Formosa et al. 2015). It must be noticed that mammalian cells are usually
spreading on their support and that no special immobilization procedure is needed
(Fig. 1). However, these cells are very soft (Young modulus (YM) are ranging from
1 to 100 kPa) and are usually difficult to image in contact mode.
To overcome this limitation, the historical AFM manufacturer developed a
non-contact mode named Tapping ® mode. Here the tip is oscillated near to its
resonance frequency, and the oscillation amplitude is kept constant during scanning.
1328 E. Dague
Fig. 1 Schematic representation of three AFM modes: contact, force volume (FV), and quantitative
imaging. These modes can be used to explore loosely immobilized sample, trap samples in holes of
a polydimethylsiloxane (PDMS) stamp, or soft samples such as mammalian cells (Adapted from
Chopinet et al. (2013b))
The phase shift can also be interpreted as material dependent and the phase signal
used to decipher between different materials.
Force Spectroscopy: Nanomechanics and Adhesion
As already stated, the AFM is a force machine. In the force spectroscopy mode, the
tip is no longer scanned in the x and y direction but in the z direction. While
approaching the sample, the tip is first encountering no forces, then it contacts the
surface and finally the cantilever, and the sample (if it is soft) is deflected. The
approach force curve can be converted into an indentation force curve. From the
indentation of the tip in the sample, the mechanical properties can be deduced, using
models like the Hertz model. In this model, as in many others, the applied force is a
function of the sample elasticity or Young modulus. A higher value of E means that a
higher force is required to achieve the same indentation in the sample. Thus the link
between elasticity and the Young modulus is biologically unintuitive: high Young
modulus do not mean easily deformable. The Young modulus is a pressure measured
in Pa (N.m 2). The exact absolute value of a sample Young modulus is a complex
question. The sample anisotropy, the shape of the tip sample contact, the dispersed
energy, the sample viscosity, etc., should be taken into account to achieve an exact
measure. For this reason, Young modulus absolute values should be used with
caution. Nevertheless, the comparison of Young modulus values measured on the
same sample but in different conditions is valuable and provides interesting insight
into the nanomechanical modifications induced by a treatment.
During the retraction process, the cantilever comes back to zero deflection, but if
an interaction has occurred between the tip and the sample, a higher force will be
needed to detach the tip from the sample. The adhesion forces can be due to
electrostatic or hydrophobic interactions (Ahimou et al. 2002; Dague et al. 2007)
or to specific biological interactions between a ligand and a receptor, for example
(Hinterdorfer et al. 1996; Hinterdorfer and Dufrêne 2006). Measuring specific forces
requires to functionalize the tip, which can be performed through the use of linkers
grafted on the AFM tip.
In the force volume (FV) mode (Fig. 1), force curves are recorded according to a
matrix of point. Each force curve is recorded and their analysis leads to adhesion
and/or elasticity maps.
Multiparametric Modes
Multiparametric modes (Dufrêne et al. 2013) (Fig. 1) were developed recently by

two AFM manufacturers. They are optimizing the FV by increasing the number of
force curves recorded in the same time. The resolution is therefore increased and/or
the acquisition time is reduced. In this new mode, and thanks to an increased
resolution, both valuable topography and adhesion and elasticity maps are recorded
at the same time. Moreover, multiparametric modes are convenient to image soft and
loosely immobilized biological samples. Indeed, as the tip is retracted between each
force curve, thus no or low lateral forces are exerted on the sample. The main
limitation remaining in these modes is the z piezo course. It must be large enough
to detach the tip from the cell; however, this inconvenience can be avoided by
selecting the appropriate tip (shape) and cantilever (spring constant).
Imaging Effects of EP on Cells
In order to explore EP effects on mammalian cells shape, Chopinet et al. (2013a), in

2013, first imaged, by AFM, cells immediately fixed after EP. No specific morpho-
logical alterations were detected neither in front of the electrode nor on regions
perpendicular to the electrodes. Globally the measured elasticity is decreased after
EP (from 30.0 +/ 2.0 kPa to 20.5 +/ 1.0 kPa), but no link with the electrodes
position appears. Then living cells were imaged across time. Figure 2a presents the
same living cell imaged during 50 min and Fig. 2c cross sections of the cell measured
after 9, 24, and 50 min of imaging. These topographical images were recorded in the
multiparametric quantitative imaging mode. It demonstrates that the cells are not
changing their shape during a 50 min period of time. At higher resolution (see insets
in the downright corners and supplementary material in Chopinet et al. (2013a)),
images show that the membrane is smooth and homogeneous and cytoskeleton fibers
are detected. The membrane and cytoskeleton remain stable during all the
experiment.
1330 E. Dague
Fig. 2 AFM QI images of living cells before and after EP. (a) 1–3: three AFM height images of the
same untreated cell across time. B1: AFM height image of an untreated cell. (b) 2–3: AFM height
images of the cell presented in B1, respectively, 18 and 50 min after EP. The scale bar in the insets
represents 1 μm. The insets present details of the membrane and are numerical zoom. (c, d) Cross
sections of the cells taken along the lines indicated for the control cell (c) and the pulsed cell (d); the
black line corresponds to non-pulsed cells, light gray 15 min later and dark gray 30 min later. The
double arrow in D marks the cell swelling. The color scale is ranging from 0 to 7 μm (Adapted from
Chopinet et al. (2013a))
In a second step, Fig. 2b, EP (8 square-wave electric pulses of 5 ms duration at

400 V/cm) was applied at a frequency of 1 Hz through stainless steel parallel
electrodes directly on the Petri dish in buffer after the first image of the cell. The
next two images and the cross sections presented in Fig. 2d (black arrow) demon-
strate a swelling of the cell induced by EP. At higher resolution, cell membrane
Fig. 3 AFM QI images of Bacillus pumilus vegetative cells (a, b, c, and d; scale bars, 200 nm) and
spores (e, f, g, and h; scale bars, 100 nm) before (a, b, e, and f) and after (c, d, g, and h) EP. The
dotted scales indicate the position of images b, d, f, and h, respectively, on images a, c, e, and g. The
color scale, representing the highness, is given on the right of each image (Adapted from Pillet et al.
(2016))
modifications appear to be reversible. After 18 min, (i) many extensions rippling

membrane can be seen, and (ii) no cytoskeleton fibers are observed any more. At the
end of the experiment (50 min), the cell membrane seems to recover, looking like
before EP.
EP is also used in food industry to eradicate undesirable bacteria. Pillet et al.
(2016), in 2016, explored the effect of EP (5 μ s-pulses at frequency of 1 kHz in a
4.1 mM NaCl solution, with a conductivity of 500 μ S/cm and a pH of 7), on Bacillus
pumilus cell surface and shape. They both analyzed vegetative bacteria and spores.
Figure 3a, b, c, and d presents AFM height images of vegetative cells before (Fig. 3a,
c) and after EP (Fig. 3b, d).
The surface roughness has been determined to be 1.4 +/ 0.5 nm before EP and
6.1 +/ 5.2 after EP. Similarly to mammalian cells, EP induces a swelling of
vegetative bacteria: the mean cell volume is 1.9 +/ 0.7 μm3 before EP and increases
to 3.0 +/ 1.6 μm3 after pulse application. Vegetative bacteria have thus a smooth
surface that is dramatically impaired by EP. The effect is emphasized on higher-
resolution images (Fig. 3b, d).
As far as spores are concerned (Fig. 3e, f, g, h), their surface structure is also
disturbed by EP. Bacillus spores are covered by coat proteins forming parallel
nanostructured named ridges (see doted lines in Fig. 3f). These structures are clearly
affected by EP (Fig. 3h), but no impact on roughness or cell volume was quantified
(roughness was 5.5 +/ 2.9 nm for untreated spores, 4.2 +/ 1.7 nm after EP
exposure, and volume was 0.9 +/ 0.2 μm3 for untreated cells and 0.9 +/
0.3 μm3 for exposed spores).
These two examples demonstrate the interest of AFM imaging for the fundamen-
tal elucidation of EP effect on cell morphology, shape, volume, and membrane
ultrastructures.
1332 E. Dague
Probing Biophysical Modifications Induced by EP
More than an imaging tool, AFM is a force machine, and when indenting the tip in
the sample, nanomechanical parameters are measured. As already stated and
observed on the AFM images, the mammalian cell cytoskeleton seems to be
transiently destabilized by EP. The nanomechanical properties of the cells should
therefore be altered too. To make this point sure, Chopinet et al. (2013a), in 2013,
measured the Young modulus of Chinese hamster ovary cells before EP and mon-
itored the cell recovery across time. Figure 4a presents five elasticity maps of the
same CHO cell across time. EP is applied at 0 time and the same area on the cell is
monitored during 35 min after EP. Figure 4b shows indentation force curves where
the applied force is plotted as a function of the tip indentation in the cell. It clearly
demonstrates the softening of the cell after EP and the recovery 35 min after
EP. Indeed, as seen on Fig. 4c (histogram in the center), the mean YM drops from
18.8 +/ 2.0 kPa before EP to 11.2 +/ 0.5 kPa just after EP. On fixed cells, the YM
of native cells is 30.0 +/ 2.0 kPa and decreases to 20.5 +/ 1.0 kPa after EP. The
same relative difference is thus found, emphasizing the pitfall that absolute values of
Fig. 4 Probing nanomechanical properties of electroporated cells across time. EP is applied at time
0, except on C where EP is not applied. (a) YM maps of the same region acquired during 50 min
(the color scale given on the right ranging from 0 to 36 kPa). (b) Typical force curves extracted from
the maps presented in (a). Black lines, before EP; light-gray line, 8 min after EP; dark-gray lines,
35 min after EP. (c) Evolution of the YM of a non-pulsed cell across time. (d) Evolution of the YM
of a pulsed cell (EP applied at time 0). Unpaired t-test, *P value = 0.05; (e) % of permeabilized
cells across time after EP (Adapted from Chopinet et al. (2013a))
YM can constitute and the high attention that should be paid to the conditions of YM
comparisons.
During the 35 min of the experiment on living cells, the authors show that the
mean YM increases slowly to roughly reach the initial value again. The authors
explored different areas of the cells according to their position in regard to the
electrodes. The hypothesis was that areas facing the positive or the negative elec-
trodes would be differently affected. However, no differences in the YM were found
on these different areas of the cell membrane, suggesting a mechanism involving
more than the cell membrane. It must be noticed that the mean YM is remarkably
constant if no stress is applied to the cell (left histogram on Fig. 4c). Conversely the
cell permeability induced by EP is shorter than the modification of the cell YM. The
histogram on the right of Fig. 4c demonstrates that 5 min after EP, the cell membrane
is no more permeabilized and that small molecules like propidium iodide cannot
anymore go through.
Therefore, EP modifies the cell membrane, but there is another mechanism
responsible for the long-term modification of the cell YM. To explore the role of
the actin cytoskeleton in this mechanism, the YM of latrunculin-treated cells has
been measured (Louise et al. 2014). Indeed, latrunculin is a drug inhibiting the actin
polymerization. The YM of latrunculin-treated cells was found to be close to the
value of cells submitted to EP: 10.6 +/ 0.7 kPa. Interestingly, removing the drug
(latrunculin) from the medium results in an increase of the YM (Fig. 5d) toward
values similar to cells having recovered from EP: 17.3 +/ 1.7 kPa. The cytoskel-
eton fibers are reconstructed during this process as observed on both the AFM height
images and the elasticity maps, respectively, on Fig. 5a, b, e and f. Combining the
two treatments (latrunculin and EP) provided surprising results. When applied first,
latrunculin induces a dramatic decrease in the mean YM and EP, applied in a second
step, has no more impact on the cell’s elasticity. On the contrary, when applied first,
EP induces the usual YM decrease and latrunculin and applied in a second step has
still an impact, reducing the mean YM of the cells toward 8.3 +/ 0.3 kPa. This
result means that EP and latrunculin both have an effect on the cytoskeleton, but not
the same one! The author’s interpretation is that EP destabilizes the cortical actin
linked with the membrane, whereas latrunculin inhibits the actin polymerization in
the whole cell. This hypothesis was then reinforced by an experiment consisting in
applying EP after removing latrunculin from the medium (Fig. 5h). In this case, cells
were able to reconstruct their actin network but were unable to establish the link
between the cortical actin network and the membrane, resulting in a low mean YM of
9.2 +/ 0.6 kPa even 32 min after EP application. The AFM height image (Fig. 5c)
and elasticity map (Fig. 5g) confirm that, even if the actin polymerization occurs, no
cytoskeleton fibers can be observed.
In 2014, Thompson et al. (2014) used a similar strategy to explore the effect of
nanosecond pulsed electric fields (nsPEFs) (50–100 pulses of 10 ns, 150 kV/cm) on
the actin cortex of cells. In this work, CHO-K1 cell elasticity is measured using a
colloidal probe before and after EP, on native- or latrunculin-treated cells. Colloidal
probes are AFM cantilevers terminated by a spherical bead (here made of borosil-
icate, and of 5 μm in diameter). The contact area between the probe and the sample is
1334
Fig. 5 Topography and elasticity of cells exposed to latrunculin and EP. (a, b, and c) are AFM QI height images of a latrunculin-treated cell (a), the same cell
35 min after latrunculin removal (b) and a cell 35 min after latrunculin removal but electroporated at time 0. (e, f, and g) are the respective elasticity maps of the
precedent conditions. The color scale given on the right of (e) ranges from 0 to 60 kPa. (d) Evolution of the YM after latrunculin removal. Unpaired t-test relative
to the point before t = 0; *P value < 0.05. (h) Evolution of the YM after latrunculin removal if EP is applied at time 0 (Adapted from Louise et al. (2014))
E. Dague
Fig. 6 AFM stiffness (a, d) and adhesion (b, c, e, and f) maps recorded on Bacillus pumilus
vegetative cells (a, b, d, and e) and spores (e, f). The white bar represents 200 nm in (a, b, d, and e)
and 100 nm in (c and f); the color scale next to each image corresponds to the stiffness (a, d) or the
adhesion force (b, c, e, and f) (Adapted from Pillet et al. (2016))
thus much higher than with pyramidal probes which is an advantage to measure
global modifications but is a drawback when it comes to measure fine structures like
cytoskeleton fibers. Despite the different applied pulses and probe, they found quite
similar results: nsPEFs induce a decrease in the cell elastic modulus of 50%. And
latrunculin A has the same qualitative effect but is quantitatively higher. The cell
elasticity of latrunculin-treated cells is, indeed, 75% lower than for healthy cells.
Interestingly, the application of nsPEFs on latrunculin-treated cells had no effect on
the cell elasticity. These results are in line with other reports already described here
and confirm that EP has an action on the cell mechanical properties that is most
probably mediated by modification at the cytoskeleton level.
In the case of microorganisms, the biophysical properties of the interface are
driven by the cell wall (Pillet et al. 2014). To continue with Bacillus pumilus, which
is a Gram-positive bacterium, the cell wall of the vegetative form is made of a thick
(50 nm) layer of peptidoglycan. The peptidoglycan has been described to be coiled
and super coiled around the cells (Hayhurst et al. 2008). The stiffness map presented
in Fig. 6a adapted from Pillet et al. (2016) confirms this nanoscale organization of
the peptidoglycan (dotted lines correspond to peptidoglycan cables). After EP
(Fig. 6d), the stiffness map does not show any peptidoglycan organization anymore.
The mean stiffness did not really change (increasing from 0.08 N/m before EP to
0.09 N/m after EP), but the surface became heterogeneous as evidenced by the
standard deviation increasing from 0.01 N/m before EP to 0.03 after EP. In this work,
the authors also studied the cell surface hydrophobicity as probed by chemical force
microscopy (CFM) (Dague et al. 2007) (Fig. 6b and e). They measured a mean
adhesion force of 2.1 +/ 1.2 nN (Fig. 6b) on untreated vegetative bacteria, 0.2 +/
1336 E. Dague
0.3 nN on untreated spores (Fig. 6f), 0.2 +/ 0.2 nN on pulsed vegetative cells
(Fig. 6e), and 0.1 +/ 0.1 nN on pulsed spores (Fig. 6f). The CFM technology
clearly demonstrates in this study the impact of EP on both vegetative forms and
spores of Bacillus pumilus hydrophobicity, which is a key parameter in adhesion and
biofilm formation.
Conclusions
Very few studies devoted to EP are using AFM. Works presented here nevertheless
demonstrate the interest of this quite new technology in the field. Indeed, AFM has
proven its ability to image the modifications induced by EP both on mammalian and
bacteria cells. Moreover it gives access to nanomechanical information, with a
spatial resolution, that is crucial to better understand the fundamental mechanisms
of EP. On bacteria, the measure and mapping of adhesions, in this case of hydro-
phobicity, could have important applied consequences, e.g., in the food industry.
In the future, it would be useful to develop a device to apply electric pulses on the
AFM setup. Then combining AFM and high-resolution optical imaging, it would be
interesting to analyze the permeabilization of cell membranes toward anticancerous
drugs or nucleic acids.
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Electroporation in Microfluidic Devices
70
Séverine Le Gac and Iris van Uitert
Abstract
Crossing the plasma cellular membrane for loading of exogenous substances or
accessing the intracellular medium is essential for cell engineering and transfec-
tion, cell analysis, or controlled extraction of the cellular content. Various chem-
ical and physical techniques have been developed to open up the cell membrane
and allow molecular exchange between the extra- and intracellular environments.
Electroporation, which relies on the use of a high external electric field to
permeabilize the cell membrane, is the most popular physical technique: not
only it avoids the use of viral material, but the cell transfection yield is also
enhanced compared to chemical approaches. However, while electroporation is
currently used on a daily basis for the transformation of a great variety of cells, it
still suffers from a low success rate when it is performed in bulk in a cuvette, at
the level of an mL-sized cell population. Furthermore, the use of high voltages in
the kV range as required in such cuvettes gives rise to various issues, such as
Joule heating, creation of bubbles through electrolysis of water, and generation of
reactive species, which all compromise the success of the electroporation treat-
ment. Using miniaturized and/or microfluidic devices helps solving these issues
while enhancing the overall electroporation success rate, by bringing enhanced
control on the process and requiring voltages as low as a few volts. In this chapter,
after a short introduction to microfluidics, the unique features this technology can
offer for cellular electroporation are discussed. Next, different classes of
S. Le Gac (*)
Applied Microfluidics for BioEngineering Research, MESA+ Institute for Nanotechnnology,
MIRA Institute for Biomedical Engineering and Technical Medicine, University of Twente,
Enschede, The Netherlands
e-mail: s.legac@utwente.nl
I. van Uitert
Demcon bv, Enschede, The Netherlands
e-mail: iris.van.uitert@demcon.nl

DOI 10.1007/978-3-319-32886-7_136
1340 S. Le Gac and I. van Uitert
microfluidic devices for cell electroporation are presented, which are suitable for
the treatment of individual cells or small cell populations. Finally, promising
applications of microscale cellular electroporation are discussed.
Keywords
Microfluidics • Miniaturization • Single-cell electroporation • Flow-through
devices • Integrated electrodes
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1340
Microfluidic Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1342
Microfluidics for Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1343
Classification of Microfluidic Devices for Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1345
Single-Cell Electroporation Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1345
Flow-Through Electroporation Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1348
Promising Applications of Microfluidics for Cell Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1352
Fundamental Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1353
Intracellular Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1353
Food and Biotechnology Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1354
Single-Cell Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1355
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1355
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1356
Introduction
Electroporation is a widely employed physical technique to transiently

permeabilize cell membranes, and it finds multiple applications, e.g., for the intro-
duction of foreign material into cells, for partial or entire extraction of the intracel-
lular content, or for inactivation of microorganisms. Upon exposure to an external
electric field, the cell transmembrane potential increases. Provided a threshold value
(▶ Chap. 2, “Critical Electric Field and Transmembrane Voltage for Lipid Pore
Formation in Experiments”) of 0.2–1 V is reached, this increase is accompanied
by the formation of conducting pores within a few microseconds, after
rearrangement of the molecular structure of the membrane (Fig. 1). Depending on
the strength of the electrical treatment, this process is reversible and aqueous pores
reseal. Molecular transport can take place through these pores, this being governed
mainly by diffusion for small species or involving more complex transportation
mechanisms (▶ Chap. 23, “Gene Delivery by Electroporation In Vitro: Mecha-
nisms”) for plasmids or large entities.
Conventionally, electroporation is performed in bulk at the level of a cell popula-
tion. In bulk electroporation, a cell suspension (> 106 cells, up to a few mL) is
introduced in a cuvette equipped with two facing electrodes, on which a high voltage
is applied in the form of short pulses (exponentially decaying or square pulses, in the
70 Electroporation in Microfluidic Devices 1341
Fig. 1 Principle of cell electroporation. When a cell is exposed to short pulses of a high electric
field, the cell transmembrane potential increases. If the latter reaches a critical value of 0.2–1 V,
pores are created in the cell membrane. These pores allow molecular exchange between the extra-
and the intracellular media and, for instance, loading into the cell of foreign substances for which
the cell membrane is normally impermeable. In case of reversible electroporation, the pores in the
cell membrane reseal (Single Cell Analysis, Single Cell Electroporation using Microfluidic
Devices, 2012, page 66, S. Le Gac and A. van den Berg, “with permission of Springer”)
low ms range). In such a cuvette, voltages in the kV range are applied to reach the
required threshold for poration. Furthermore, in bulk electroporation, the viable
poration rate, corresponding to cells being porated but remaining viable after the
treatment, is low (40–70%) (Chen et al. 2008). In general, three scenarios concom-
itantly take place, which explains this overall low success rate. In a first scenario, there
is no membrane poration. In a second scenario, too many pores or too large pores are
created so that the cell is not able to recover, which is known as irreversible poration.
Finally, in the most favorable case, the membrane is permeabilized and reseals.
Bulk electroporation suffers from a number of issues, which explains this hetero-
geneity in the response of the cells to the electric field and the overall low success
rate. First, the electric field is not homogeneous in the cuvette, and cells are randomly
oriented with respect to the electric field direction, so that they experience different
treatments. Next, the use of high voltages brings about a number of problems. Upon
application of high voltages, reactive chemicals, which are toxic to the cells, are
produced. Furthermore, Joule heating can induce a significant change of temperature
in the cell suspension. Similarly, water electrolysis can happen, with the creation of
bubbles, which can also result in a loss of cell viability. Finally, toxic metal ions
released from the large plate electrodes also endanger cell viability, depending on the
nature of the electrode material. Moreover, this cuvette-based approach is not
optimal for the treatment of all kinds of cells. Adherent cells must undergo a (bio)
chemical treatment to be placed in solution, which may damage the cell membrane
and sensitize the cells to the electric field. Furthermore, rare cells, such as stem cells
or induced pluripotent stem cells (iPSCs), are available in too limited amounts to be
handled in an mL-sized cuvette. A promising approach to solve some of the

aforementioned issues while bringing better control on the treatment to eventually
enhance its success rate is to use microfluidic technology and miniaturized devices.
In this chapter, microfluidics is shortly introduced, and the unique features this
technology has to offer for cell electroporation in comparison with a bulk process are
discussed. Thereafter, different microfluidic-based strategies for cell electroporation
are presented as two distinct categories of devices, which are especially suitable for
single-cell electroporation and for flow-through treatments, respectively, and the
specific advantages and limitations of these two types of devices are discussed.
Finally, different fields of applications of cellular electroporation that can especially
benefit from the use of microfluidic technology are briefly reviewed.
Microfluidic Technology
Microfluidics, as pointed out by Georges Whitesides, can be defined as “the science

and technology of systems that process or manipulate small (10 9 to 10 18 l)
amounts of fluids using channels with dimensions of tens to hundreds of microme-
ters” (Whitesides 2006). Microfluidic devices – also known as lab-on-a-chip devices
– are miniaturized devices, which have a footprint of a few square centimeters. These
devices include structures with dimensions in the 1–100 μm range such as channels
or chambers, in which such small volumes of liquids can accurately be transported
and processed. The field of microfluidics initially focused on the miniaturization of
analytical devices. The motivations to use miniaturized devices for biomolecule and
medical analysis were manifold, e.g., the possibility to work with small-sized
samples (in the sub-microliter range), the rapidity and sensitivity of the analysis,
and the portability of the systems to eventually yield autonomous point-of-care
devices. Since their introduction in the 1990s, microfluidic devices have become
more and more popular, and their applications have greatly diversified toward, for
instance, the field of chemistry and synthesis, and also, more and more, for cell
biology-related and biological research.
Since the field of microfluidics directly originated from those of microelectronics
and microelectromechanical systems (MEMS), microfluidic devices have first been
fabricated from silicon and silicon-based materials using processes from the micro-
electronics industry (i.e., photolithography, wet and dry etching, etc.) in a clean room
environment. Glass quickly came in the picture and was preferred to silicon for its
transparency and electrical insulating properties. As the field has been further
evolving and expanding, materials and associated fabrication processes have been
changing. Traditional materials now have to compete with polymers, which are
much cheaper and already ubiquitous in laboratories. Polymer processing relies on
alternative approaches such as replication or ablation techniques (Becker and
Gartner 2008), which do not require having access to dedicate and expensive clean
room facilities, but which can readily be implemented in a standard wet lab.
The popularity of microfluidic devices is explained not only by the numerous

advantages they present compared to their lab-scale counterparts but also by the fact
that they offer new experimentation opportunities, especially in the fields of chem-
istry and cell biology. Among commonly cited advantages linked to their small
dimensions are reduced consumptions of samples and reagents and associated lower
operational costs. Similarly, all processes that scale with the dimensions of the
device become more efficient and faster like mass transport, heat transfer, gas
exchange, molecular diffusion, etc. For experimentation involving cells, the
micrometer-sized structures make these devices ideal tools for the manipulation
and isolation of individual cells and to conduct experimentation at the single-cell
level (Le Gac and van den Berg 2010). Next to this, microdevices enable to emulate
the confined environment cells experience in vivo. At the micrometer scale, flows
also behave in a very different way than at the macrometer scale, which can be
evaluated by examining the dimensionless Reynolds number (Re). The Reynolds
number compares inertial and viscous forces and indicates the transition from a
turbulent to a laminar regime. In miniaturized devices, as a result of the increased
surface-to-volume ratio, surface forces dominate bulk phenomena, and the flow is
mostly in the laminar regime. Consequently, flows are predictable and highly
controlled in microfluidic devices. A downside of a laminar flow regime however
is that mixing only proceeds via molecular diffusion and is therefore not as efficient
as when turbulences are involved. Altogether, microfluidic devices offer the possi-
bility to accurately control any physical and chemical parameter both spatially and
temporally. This ability is essential in biology to fine-tune the cellular microenvi-
ronment, as well as in chemistry to yield more homogeneous reaction mixtures.
Finally, microfluidic devices present a high level of integration. One device can, for
instance, comprise a series of identical systems (horizontal integration) for assay
parallelization. Alternatively, a series of distinct operations can be implemented in
one single device (vertical integration) for the accomplishment of a complete
(analytical) process. Furthermore, microfluidic structures can be combined with
add-on capabilities such as electrodes or sensors (smart integration), e.g., for electrical
detection of cells or biomarkers, for controlling the temperature, or for fluid actuation.
Microfluidics for Electroporation
For cellular electroporation, microfluidic technology brings a number of attractive

features. First, distances in microfluidic devices are in the micrometer range, and
since the required electroporation voltage scales with the device dimensions, a few
volts are typically sufficient to reach the kV/cm electroporation threshold. Further-
more, microstructures can be designed to locally enhance and/or shape the electric
field so that even lower voltages are required. For instance, channels can include
constrictions in which the electric field is enhanced, since the E-field strength scales
with the cross section of the channels. Alternatively, micrometer-sized structures can
be added to isolate cells, which allows locally creating hot spots in electric field
across the trapped cell. Similarly, electrodes can be directly integrated in the device
and their shape and geometry optimized toward the same goal. For instance,
saw-tooth electrodes in a microchannel have been reported to locally enhance the
field strength (Lu et al. 2005). Furthermore, 3D electrode structures provide a more
homogeneous electric field distribution across a channel compared with planar
electrodes. Altogether, using microfluidics, the electroporation treatment becomes
much milder, and all adverse effects originating from the use of high voltage, as
normally encountered in bulk electroporation, are drastically reduced or entirely
alleviated using such miniaturized devices.
As mentioned earlier, microfluidics lends itself well to single-cell experimenta-
tion, so that the electroporation treatment can be downscaled to the level of a single
cell – or even to the subcellular level – by isolating individual cells at specific
positions between two electrodes. This configuration brings about a high control on
the electroporation treatment, and all parameters (electric field strength, duration of
the treatment, etc.) can be adjusted for every single cell. Arguably, this single-cell
approach opens the doors for a 100% theoretical success rate of the treatment.
Furthermore, single-cell electroporation is particularly attractive for rare cells,
which can be treated individually in a highly controlled and customized fashion,
with a high success rate and no loss in viability. Finally, this single-cell approach is
interesting to control molecular delivery and/or extraction following cell membrane
poration, as discussed in more detail later in this chapter.
The high level of integration offered by microfluidics allows combining the
electroporation treatment with another process. Cells can be tracked in situ to monitor
the process of pore formation in real time and the outcome of the treatment, on a
longer time scale. To that end, fluorescent probes are typically employed to instan-
taneously follow molecular transport across the membrane resulting from its success-
ful permeabilization. Alternatively, the process of pore formation can be detected
electrically, by monitoring the membrane conductance or the impedance (▶ Chap. 38,
“Assessment of Electroporation by Electrical Impedance Methods”) of individual
cells, using either the same electrodes as for the electroporation or a second pair of
electrodes. For longer-term examination, cells can be cultured in the same device and
their state evaluated in situ after one or more days using bright-field and/or fluores-
cence microscopy. A common approach to validate a cell electroporation protocol
consists of transfecting a plasmid coding for the green fluorescent protein (GFP); this
strategy however implies keeping the cells in culture for about 1 day to verify not only
that they have survived the treatment but also that they are functioning properly and
able to produce proteins from the exogenous and transfected plasmids. This whole
sequence can be implemented in a microfluidic device, with cells being tracked to
examine the success of the electroporation/transfection treatment at the single-cell
level. The electroporation treatment can finally be coupled to a cell-sorting step, e.g.,
using dielectrophoresis, either prior to electroporation to select cells to be exposed to
the electric field or after, to isolate successfully porated cells.
Classification of Microfluidic Devices for Electroporation
In this section, different microfluidic-based cell electroporation strategies are

discussed to illustrate the potential of this technology for cell electroporation.
Microfluidic devices for cell electroporation are classified in two distinct categories
depending on how the treatment is performed: either at the single-cell level or using a
flow-through configuration on cell populations. For each of the two approaches, the
concept underlying the strategy is explained, and possible variations around the
strategies are presented, together with a short discussion on the specific advantages
of these respective devices and their possible limitations.
Single-Cell Electroporation Devices
Principle
Single-cell electroporation devices all rely on the isolation of individual cells, which
are typically secured in trapping microstructures. These mechanical trapping sites
consist of either microholes, which are located underneath the cells (Huang and
Rubinsky 2001, 2003), or lateral trapping slits connected to another microchannel
(Khine et al. 2005; Valero et al. 2008; van den Brink et al. 2011). In both cases, the
trapping structures present a characteristic dimension of 2–3 μm, which is several
times smaller than the cell diameter. Importantly, these low micrometer-sized struc-
tures also allow shaping the electric field and creating hot spots of electric field
across the isolated cells. Moreover, a microsystem can easily include arrays of these
structures for the electroporation of a series of individual cells.
Trapping of the cells is a key step in this single-cell electroporation protocol.
Typically, a negative pressure is applied across the trap after injection of the cell
suspension in the device to attract and immobilize individual cells in the trapping
sites. A too high negative pressure may damage the cells and increase their vulner-
ability and associated risks for cell death. On the contrary, if this force is too low, the
cell may not be tightly immobilized in the trapping site and can easily escape.
Furthermore, if a cell is loosely trapped, a leakage pathway is created so that a
higher potential must be applied for cell poration. This leakage pathway also
obviously prevents the electrical detection of pore formation.
Once the trap(s) is (are) filled, the cell(s) can be electroporated. To apply the
electroporation voltage, electrodes are either integrated in the device or inserted in
the access reservoirs. In the former case, electrodes can be placed in the close
vicinity of each trapping site, so that a voltage of a few volts is typically needed to
achieve cell electroporation. Furthermore, in this configuration, the electric field
parameters can be adjusted for each individual cell, would the device include an
array of trapping sites and individually addressed electrodes for each trapping site.
Therefore, this strategy is particularly attractive to optimize the treatment for every
single cell to eventually give a 100% (viable) poration yield.
Bottom Trapping of Cells

A first type of single-cell electroporation devices includes a horizontal substrate with
an array of micrometer-sized apertures in which cells are trapped. Two channels or
chambers are placed above and underneath this perforated substrate to, respectively,
introduce the cell suspension and apply a negative pressure for cell trapping.
The first microfluidic cell electroporation device worked along this principle of
single-cell trapping in a horizontal microhole (Huang and Rubinsky 2001, 1999).
This device, reported by Huang and Rubinsky, was composed of two silicon
chambers separated by a horizontal 1-μm-thick silicon nitride membrane containing
a 2–10-μm diameter orifice (Fig. 2a). The electrodes, which were integrated in the
device, consisted of n+-polysilicon membranes placed in the two silicon chambers,
and they were separated by 900 μm. The semiconductive and translucent properties
of this material allowed both electrical and optical monitoring of the trapping and
poration processes. Trapping of a cell and sealing of the cell in the orifice resulted in
a great reduction of the current through the trap – so-called leakage current – while
cell electroporation was accompanied by an increase in the conductance across the
orifice. Using this device, the electroporation voltage was successfully lowered to a
few 10s V (single square pulse of 60 ms).
Adaptations have been brought to this first device, which proved the concept of
single-cell microfluidic electroporation while being limited to one single cell.
Addition of another pair of electrodes allowed detecting cells arriving on the
micro-orifice to trap them. After electroporation, reversing the pressure released
the cell, so that another cell could be attracted in the orifice and subsequently
treated (Huang and Rubinsky 2003). This planar configuration of orifices is easily
amenable to parallelization to increase the poration throughput through the use of
2D arrays of orifices, in which populations of cells are immobilized and submitted
to an electric field. However, in most of these planar devices, common electrodes
are used for all orifices in the array so that the electric field treatment cannot be
customized per cell, and similarly, the poration process cannot be monitored
electrically.
Lateral Trapping of Cells

In a second configuration, trapping is achieved using lateral structures separating the
channel, in which the cell suspension is injected, and another channel (Valero et al.
2008) or a series of individual channels (Khine et al. 2005; van den Brink et al.
2011), from which the negative pressure is applied (Fig. 2b, c). This lateral design
lends itself better to electrical decoupling of the traps and to multiplexed single-cell
electroporation. Furthermore, provided the different trapping sites are connected to
individual channels, the lateral approach is also compatible with parallel single-cell
analysis (van den Brink et al. 2011). A second main advantage of this lateral
approach, when individual channels and electrodes are used, is that the electropora-
tion process can be monitored electrically; thereby, as soon as pore formation is
detected through a change in the measured membrane conductance, the electropo-
ration voltage can be stopped. Similarly, individual channels and individually
addressed electrodes can be employed to assist and enhance the introduction of
a Bottom Micro
Top
electrode
electrode hole
Top layer Al wires
Middle layer
Circuit board
Bottom layer Acrylic substrate
Fluids inlets/outlets
b c
50
200µm
Ag/AgCl electrode
Conductive
PDMS media
Trapping channel
Cell
PDMS 4µm
Fig. 2 Cell trapping devices. (a) Three-layer single-cell electroporation device consisting of two
microfluidic chambers in silicon, separated by a 1-μm-thick silicon nitride membrane containing an
aperture (2–10 μm in diameter) on which the cell is trapped before electroporation. The top and
bottom electrodes are formed by a transparent n+ polysilicon membrane (Courtesy Prof. Rubinsky).
(b) Top: Top view of a device for lateral cell trapping. Two wide inlets on either side of the large
chamber in the middle are employed to fill it with the solution containing the cells. By applying a
suction force via the small radiating side channels, cells are trapped at the channel entrances. The
electrodes employed for electroporating the cells and detecting the increase in membrane conduc-
tance are connected via the large chamber and to the small cell trapping channels, enabling single-
cell electroporation. Bottom left: Schematic cross section of the chip displaying a trapped cell and
the two electrodes employed to generate the required field. The trapped cell is pulled into the small
opening due to the applied suction force. Bottom right: Photograph of a trapped cell (Reproduced
from (Khine et al. 2005) with permission of The Royal Society of Chemistry). (c) Top: Top view of a
foreign substances into the cells, after pore formation, using electrophoresis
(Ionescu-Zanetti et al. 2008).
Advantages and Limitations

The main advantages of these single-cell miniaturized electroporation devices are the
use of milder electrical parameters and the higher control on the poration process,
which altogether enhances the success yield in cellular treatment. Since the elec-
trodes are very close to each other and the cells can be tightly trapped, the required
potentials become very low compared to conventional systems, ranging from 20 V
(Huang and Rubinsky 2001) down to ca. 1 V (Khine et al. 2005), while the success
rate can go up to 100% (Ionescu-Zanetti et al. 2008). Moreover, plasmids can be
actively brought into the cells by electrophoresis (Ionescu-Zanetti et al. 2008), which
is not possible in bulk electroporation. Furthermore, the electric field can be tuned to
match the cell properties, which further increases the poration yield. Finally, the
electric field distribution across the trapped cell can easily be modeled; the cells are
considered as spherical objects (Khine et al. 2005; Valero et al. 2008) (Fig. 2b, c),
and their position in the electric field is well defined. These modeling aspects also
ultimately contribute to the improvement of the poration treatment. All these advan-
tages make these (single)-cell trapping devices the best suited for the treatment of
rare cells.
An important drawback of these devices is their level of complexity: they include
multiple channels or chambers with low micrometer-sized trapping structures and,
sometimes, integrated electrodes. Therefore, their realization involves sophisticated
fabrication processes. However, the main limitation of (single)-cell electroporation
devices is their treatment capacity; they mostly include a small number of traps, and
this, in turn, gives a low cell treatment throughput. It is still worth noticing that a few
recent devices including 2D arrays of micro- and nanopores have scaled up the
process to tens of thousands of cells in parallel (Chang et al. 2016 ).
Flow-Through Electroporation Devices
Principle
A second category of microfluidic devices for cellular electroporation, which
addresses this issue of throughput, consists of flow-through systems (Bao et al.
Fig. 2 (continued) silicon-glass microfluidic single-cell electroporation device. Two microfluidic

channels are connected by nine cell traps where cells are captured with the help of a suction force.
The electroporation signal is individually applied to each cell with nine stimulation electrodes
located above the traps and the common ground electrode placed below the traps. The inset shows a
picture of trapped single cells between the electrodes. Bottom: Bright-field and fluorescent micros-
copy images of four trapped cells (C2C12) before and 24 h after transfection with eGFP (enhanced
green fluorescent protein), respectively. The transfection resulted in a 100% success rate
(Reproduced from (Valero et al. 2008) with permission of The Royal Society of Chemistry)
2008; Kim et al. 2007; Wang and Lu 2008). In these devices, a cell suspension is
continuously perfused in a microchannel, and the cells are exposed one by one to the
electrical treatment, the voltage being applied again using integrated or external
electrodes. With this approach, cells are still treated individually, but larger cell
populations can be processed compared to the single-cell electroporation devices.
Cells can also be collected at the outlet of the device for further and off-line studies.
The channels or structures in which cells are flowing comprise most of the time
geometric constrictions, in which the electric field is locally enhanced, so that
electroporation takes place in those areas of higher electric field. Therefore, the
flow rate at which the cell suspension is perfused in the system is essential, since it
directly relates to the time the cells are exposed to the electric field and the
electroporation treatment strength and thus, in turn, to the success yield and cell
viability rate (Kim et al. 2007). Specifically, increasing the flow rate translates into
shorter exposure of the cells to the electric field, which is equivalent to the use of
shorter pulses.
Flow-through devices can be further divided into two subcategories, depending
on how the cell suspension is flown: (i) using a continuous column of liquid or
(ii) using a discrete flow based on droplet microfluidics, where (single) cells are
encapsulated in sub-nanoliter aqueous droplets in a continuous flow of an immisci-
ble liquid (i.e., an oil phase).
Continuous Flow Approach

A first option relies on the use of a simple microchannel with electrodes placed at a
short distance from each other, to locally create – between the electrodes – a high
electric field.
Most of the flow-through devices include in their design constricted areas, where
the electric field is concentrated (Bao et al. 2008; Wang and Lu 2006) (Fig. 3a). The
electric signal in this configuration is mostly applied using external electrodes, which
are inserted in the reservoirs of the device. The width ratio between the channel and
the constricted areas determines the electric field enhancement at the poration area
(s). The length of the constricted area and the flow rate define the pulse length for the
electrical treatment cells are exposed to. Furthermore, multiple pulses can be applied
by implementing a series of constrictions in a single channel.
In case electrodes are integrated, a design with both electrodes on the same
substrate is preferred from a fabrication point of view, even if it gives an inhomo-
geneous distribution in the electric field. If electrodes are placed on the bottom and
top substrates, a more homogeneous electric field is created, but their fabrication is
more delicate, especially for the alignment of the electrodes. Furthermore, in this
configuration, electrodes are obstructing the view so that the electroporation process
cannot be monitored in real time. Another configuration of integrated electrodes has
been proposed to solve both issues, where electrodes were placed at the half height
of the channel (Sukas et al. 2014) (Fig. 3b). A last alternative, which yields better
results in terms of uniformity and strength of the electric field while providing
optical access, relies on the use of 3D electrode structures. Liquid-based coupling
strategies have also been reported to apply a homogeneous electric field through the
a b Isometric view Top view A
1350
Receiving Sample
50 µm cell
Reservoir Reservoir
Cell movement electrode fused silica
W1 A9
Cross section:AA9
W2 GND
cell
L1 L2 L1 electrode
fused silica
e- cell
c Ag/AgCl d Power supply
Cell electrode
injection Buffer with Cells and DNA Gold Electrode
Ionic
flux 100 µm 60 µm
Hydrogel
Hypertonic plug
solution
Electroporated cell
cell
Fig. 3 Flow-through devices. (a) Right: Schematic representation of a channel comprising a constricted area to create a region of high electric field to
electroporate the cells. This device employs the principle that the electric field scales inversely with the channel cross section, i.e., when a region in the
microfluidic channel is smaller, the electric field across it is higher. Inset: Picture of the microfluidic channel at the constricted area (Reprinted with permission
from (Wang and Lu 2006). Copyright 2006 American Chemical Society). (b) Schematic representation of a flow-through microfluidic device consisting of a
S. Le Gac and I. van Uitert
cell suspension while using external electrodes and avoiding any direct cell-
electrode contact. In those cases, the electric field is enhanced at the intersection
between the main channel, in which the cell suspension is perfused, and a side
channel in which the electrodes are introduced. In a first liquid-based strategy, the
cell suspension is hydrodynamically focused as a single-cell line in the main channel
using lateral co-flows of a high ionic strength solution (Zhu et al. 2010); cells are
exposed to the electric field when passing the channel intersection, and the voltage
drop across the cells is determined by the width of the focused flow. Alternatively, a
fluidic junction using a polyelectric salt bridge was created between the main
channel where cells flow and the side channels which were filled with a high ionic
strength buffer (Kim et al. 2007); here the main channel was made narrower at the
channel intersection to further enhance the electric field at the place of cell electro-
poration (Fig. 3c).
Droplet Microfluidic Approach

Another class of flow-through microfluidic devices uses a discrete flow, which is also
known as droplet microfluidics (Baroud et al. 2010). In droplet microfluidics, an
emulsion is created between two immiscible liquids – an aqueous phase and an oil
phase – to yield water-in-oil or oil-in-water emulsions, with both an exquisite control
on the droplet size and a very high production frequency of more than 1000 droplets/s
(Baroud et al. 2010). Thereby, an extremely large number of identical picoliter-to-
nanoliter-sized droplets reactors are created in a very short period of time. Water-in-
oil emulsions have found a variety of applications in biology (Baroud et al. 2010).
They particularly present a number of advantages for single-cell experimentation
such as (i) the ability to confine one cell and its environment in a volume 100–1000
times larger than the volume of a cell and (ii) the massive parallelization of the
assays. As such, droplet microfluidics allows high-throughput electroporation of
individual cells, which are encapsulated in a small aqueous volume in a continuous
nonconductive oil phase (Fig. 3d). In droplet microfluidic devices, electrodes are
Fig. 3 (continued) fused silica microchannel equipped with integrated electrodes placed at half
height of the channel to provide an homogeneous electric field across the channel while allowing
optical inspection of the cells during their exposure to the electric field (Reproduced from (Sukas
et al. 2014) with permission of The Royal Society of Chemistry). (c) Schematic drawing of the
electroporation chip including hydrogel plugs that function as salt bridges to form an integrated
electrode junction in the channel. Cells are exposed to the electric field and porated in the region
between the salt bridges (Reprinted with permission from (Kim et al. 2007). Copyright 2007
American Chemical Society). (d) Droplet microfluidics platform for cell electroporation. Using a
T configuration, a water-in-oil emulsion is created with high control on the aqueous droplet size.
Each droplet contains one single cell – or no cell – as well as a well-defined amount of DNA to be
loaded into the cells. The device includes two electrodes for cell electroporation in the droplets and
cell transfection (Reprinted with permission from (Zhan et al. 2009). Copyright 2009 American
Chemical Society)
mostly integrated. As before, the electrical treatment depends on the number of

electrode pairs (which correlates with the number of applied electric field pulses on
the cells), the distance between the electrodes, the strength of the applied signal and
the flow velocity, as well as on the droplet volume (Zhan et al. 2009).
The confinement offered by droplet microfluidics brings specific advantages
compared to other flow-through approaches: a high control on the number of cells
and substances to be delivered in the cell, which are encapsulated per droplet, which
gives additional control on the molecular delivery process; the possibility to incubate
the cells and, for instance, DNA to be transfected in the cell prior to electroporation,
which can enhance the transfection efficiency (Qu et al. 2012); the possibility to
monitor the cell fate and conduct post-electroporation functional assays at the single-
cell level in the droplets; and, for molecular analysis, limited dilution of the mole-
cules extracted from the cell, which is essential for the detection of
low-concentration species.

The main interest of flow-through devices is the throughput in cell treatment, which
can reach values of up to 104–108 cells/min. In flow-through devices, cells are treated
individually, and the electroporation efficiency rate is as high as for (single)-cell
devices. Similarly, voltages as low as a few volts are required, depending on the
exact device geometry and dimensions and on how the electric field is applied.
However, parameters of the electrical treatment cannot really be optimized for
each individual cell so that the whole population is exposed to the same treatment.
Still, this configuration can be further developed to couple cell characterization to
cell electroporation, prior or after application of the electrical treatment. For
instance, the cell size and morphology can first be evaluated, e.g., using impedance
spectroscopy and another pair of integrated electrodes, to tune the electroporation
parameters. Most of the flow-through devices do not provide any active control on
molecular delivery, and molecular exchange after cell poration only relies on
diffusion phenomena. Another limitation associated with these devices is the diffi-
culty to precisely model the electric field distribution across the cells, as the latter
keep a certain degree of freedom in their position and orientation with respect to the
electrodes, and most of the time the electric field is not homogeneous through the
entire channel cross section. Lastly, this flow-through approach is still not suitable
for the treatment of both adherent and rare cells, but it is more appropriate for large-
scale cellular poration.
Promising Applications of Microfluidics for Cell Electroporation
Implementing the process of electroporation in microfluidic or miniaturized devices

is particularly attractive for certain fields of application, as discussed in the follow-
ing. Furthermore, depending on the application and the required throughput, one
specific category of devices, single cell or flow through, may be preferred.
Fundamental Research
Since microfluidic devices bring excellent control on the poration parameters and
allow varying them in a precise way, they are ideally suited to conduct fundamental
studies on the process of electroporation. Furthermore, cells can be monitored in situ
to evaluate the effect of the electrical treatment, both in real time or in a longer term,
using either electrical or optical means. This set of assets is instrumental, for
instance, in gaining new insight into the processes of pore formation and membrane
recovery. Similarly, parameters for the electroporation-based treatment can be
screened in one device on multiple cells exposed to different conditions and subse-
quently optimized for various cell types and applications. For such screening,
geometrical variations can be implemented in simple channels (e.g., a tapered
shape, different constriction dimensions) to locally or gradually alter the electrical
treatment and to ultimately study in situ the influence of specific parameters on the
poration outcome.
The combination of cell electroporation and microfluidics can also benefit
fundamental research in the field of cell biology, for instance, to elucidate targeted
signaling pathways, and the activation of specific signaling pathways has notably
been studied using reversible electroporation. For instance, the translocation of
specific kinases in a cell has been followed using “electroporative flow cytometry,”
which involves reversible cell poration and analysis of molecular species that
diffuse out of the cells through the created pores (Wang et al. 2008). In another
approach, cells were transfected with plasmids coding for a protein of interest
extracellular signal-regulated kinase 1 (ERK1) coupled to enhanced green fluores-
cent protein (eGFP), and the accumulation of the resulting fusion protein in the
nucleus was tracked in situ in the device using time-lapse imaging after stimulation
of the cells with specific growth factors (Valero et al. 2008). The possibility to
porate and transfect cells locally in multicellular constructs such as an embryo is of
particular interest in the field of developmental biology to unravel processes
involved in the development of embryos and to follow cell migration (Mazari
et al. 2014).
Intracellular Delivery
The combination of microfluidics and electroporation has been explored for loading
cells with very different substances, ranging from small molecules such as drugs and
siRNA to larger substrates such as DNA, proteins, or even nanoparticles and
quantum dots (Sun et al. 2014). A clear benefit brought by miniaturized strategies
for the introduction of foreign substances in cells is the possibility to better control
the delivery process and to dose the amount of foreign material loaded in the cells.
Enhanced DNA delivery can be achieved, for instance, using electrophoresis-
assisted molecular loading after the cell membrane electroporation (Ionescu-Zanetti
et al. 2008). Specifically, a single cell can be trapped in a micro- or even nanometer-
sized constriction connected to a microchannel, in which the substances to be loaded

into the cell are introduced. Upon application of a second low-voltage electrical
signal across this channel and constriction and after poration of the cell membrane,
the substances are pushed into the cell in a highly controlled manner. Furthermore,
using electrophoresis-assisted delivery, the transfected DNA is in a free state in the
cytoplasm, so that it is more available for its translocation to the nucleus. Similarly,
preincubation of individual cells with DNA in confined volumes created using
droplet microfluidics proved not only to enhance the transfer efficiency but also to
yield more homogeneous transfection in a cell population (Qu et al. 2012). Further-
more, the need in DNA or other substances to load in the cell is drastically reduced
since the volumes involved in microfluidic devices are more than three orders of
magnitude smaller than in the traditional electroporation cuvettes. For such cellular
delivery applications, both types of devices, single-cell electroporation and flow-
through devices, can be used depending on the throughput required and number of
cells to be treated.
The possibility to use milder conditions for cell electroporation, together with the
higher success rate, and the ability to manipulate small populations of cells or even
single cells make a microfluidic format particularly attractive for the treatment of rare
and fragile cells such as stem cells, primary cells, or induced pluripotent stem cells, with
plethora of applications in various fields such as regenerative medicine (▶ Chap. 82,
“Tissue Engineering with Electroporation”) and gene therapy (▶ Chap. 81, “Principles
of Electroporation for Gene Therapy”). Similarly, the excellent control on the electro-
poration parameters brought by the micrometer scale has proven to benefit the electro-
poration of hard-to-transfect cells (Qu et al. 2012).
Food and Biotechnology Applications
Flow-through microfluidic devices find multiple applications in the food industry for
pulsed electric field (PEF) treatment for the pasteurization of liquid food samples
(▶ Chap. 133, “Pulsed Electric Field Treatment for Beverage Production and Pres-
ervation”) like dairy products and fruit juices. There, all bacteria and microorgan-
isms must be inactivated in the sample, which is achieved through irreversible
electroporation. PEF also consists of an attractive low energy-consuming treatment
for the permeabilization of microalgae for the extraction (▶ Chap. 37, “Selective
Extraction of Molecules from Biomaterials by Pulsed Electric Field Treatment”) of
various cellular components such as the lipid biomass. As microorganisms and algae
are much smaller than mammalian cells, higher voltages must be applied than for
other applications, which are accompanied by a significant production of heat
through Joule effects. In this context, the advantage of using microfluidic devices,
which are characterized by higher surface-to-volume ratios, is linked to their ability
to dissipate heat in a more efficient way. A shortcoming however of using microscale
systems is their relatively low throughput since these applications require processing
of large volumes.
Single-Cell Analysis
Microfluidic technology shows great promises for the molecular analysis of single
cells due to its ability to accurately manipulate individual cells and small-sized
samples down to the low picoliter range (Le Gac and van den Berg 2010). To access
a cell molecular content, the plasma membrane must be ruptured. To that end,
electroporation, whether it is reversible or irreversible, offers specific advantages
compared to conventional chemical approaches: it suppresses issues associated with
sample dilution and contamination, which can hamper molecular analysis; it allows
on-demand and targeted lysis of certain cells if electrodes are individually addressed;
it is ideally suited to look at transient species, since electric pulses can rupture the
plasma membrane within milliseconds against seconds to minutes for chemical
processes; and the membrane poration process can be tuned to be either reversible
and noninvasive or irreversible. When electroporation is combined with micro-
fluidics, the retrieved cellular content can be confined in small volumes for further
processing or analysis, in situ or off-line, without any extensive dilution and
sample loss.
For instance, capillary electrophoresis has been employed, in a proof-of-concept
experiment, to separate two dyes previously loaded into the cells and that were
detected by fluorescence (Han et al. 2003). Using droplet microfluidics, the entire
content of individual cells can be retrieved in individual micrometer-sized reactors
with limited dilution of the cell content. Using this approach, de Lange et al. success-
fully performed enzymatic assays on the content of individual E. coli cells to assess
the activity of β-galactosidase at the single-cell level (de Lange et al. 2016). Finally, of
particular interest for single-cell analysis is the so-called technique of “electroporative
flow cytometry” (Bao et al. 2008), which relies on the selective extraction of
molecules located at the periphery of the cytoplasm after reversible cell poration.
Using this technique, controlled extraction of small molecules as well as proteins was
reported without compromising the viability of the cells (Zhan et al. 2012).
Conclusions
Cell electroporation can benefit considerably from its implementation in miniatur-

ized and/or microfluidic devices. The whole process is better controlled, down to the
single-cell level or even subcellular level, and the success yield is greatly enhanced
to virtually reach 100%. Moreover, the risks associated with the treatment are
diminished. Indeed, when the dimensions of the devices are reduced to the microm-
eter range, much lower voltages are required, and signals as low as < 1 V have led to
successful permeabilization of the cell membrane. These low potential values make
the devices not only safer to work with but also less energy-consuming while
avoiding significant temperature changes induced by Joule heating. Furthermore,
miniaturized devices bring enhanced protocols for molecular exchange, i.e., by
assisting intracellular delivery using electrophoresis, for instance, and dosing the
amount of substances loaded in the cell or by confining the extracted cellular content
into sub-nanoliter volumes for their analysis or further processing.
Microfluidic electroporation devices can be classified into different categories
depending on the way the cells are manipulated: whether they are individually
trapped in dedicated microstructures, flown in a channel either as a single-cell file
between two electrodes or as individual cells encapsulated in sub-nanoliter droplets.
Interestingly, these different types of devices are complementary to each other.
Devices from the first category are the best suited for treating single or rare cells
and for following the response of each individual cell to the electric field, which is
particularly interesting for fundamental studies on the process of electroporation.
These devices are also particularly attractive for single-cell study using either
imaging of intact and living cells or molecular analysis approaches after cell lysis
and extraction of the cellular content. Flow-through devices better apply for middle-
to-high-throughput treatment of populations of cells. In these devices, cells can be
characterized online before and after exposure to the electric field, and they can
easily be retrieved from the device for off-line analysis or utilization. Promising
applications of flow-through devices are the inactivation of bacteria and microor-
ganisms for the food industry, the transformation of plant cells, or the treatment of
algae.
Cross-References

▶ Critical electric field and transmembrane voltage for lipid pore formation in
experiments
▶ Principles of electroporation for gene therapy
▶ Pulsed electric field treatment for beverage production and preservation
Treatment
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Detection of Electroporation in Microbial
Cells: Techniques and Procedures 71
Diego García-Gonzalo and Rafael Pagán
Abstract
Application of an electric field to microbial cells can cause electroporation of their
membranes, leading to permeabilization and formation of pores in these struc-
tures. Assessment of electroporation in microbial cells, such as fungi and bacteria,
could be hindered by the presence of additional structures located externally to
the cytoplasmic membrane, such as the cell wall and outer membrane.
The most common methods to assess the electroporation of these membranes
can be divided between those based in the entrance of external compounds into
the bacterial cytoplasm (e.g., a fluorescent probe) and those which detect the
leakage of intracellular compounds to the environment (e.g., nucleic acids, pro-
teins or ATP). Moreover, other techniques could be used to understand the
mechanism of electroporation in microbial membranes, such as direct visualiza-
tion of damages in cell membranes by electron and atomic force microscopy,
evaluation of membrane damages by a selective medium plating technique,
measurement of osmotic response, Fourier transform infrared spectroscopy, and
molecular tools (including transcriptomics and site-directed mutagenesis). In
addition, several methods are proposed in order to avoid interference of the cell
wall and/or outer membrane in the evaluation of cytoplasmic membrane
permeabilization: (pre)treatment of cells with cationic agents or chelators and
preparation of protoplasts or spheroplasts.
The described methods to detect electroporation of bacterial cells have their
own advantages and disadvantages. Therefore, analysis of results obtained with
different methods is recommended to provide a more accurate knowledge in the
mechanism of microbial permeabilization by electroporation.
D. García-Gonzalo (*) • R. Pagán

Tecnología de los Alimentos, Departamento de Producción Animal y Ciencia de los Alimentos,
Facultad de Veterinaria, Instituto Agroalimentario de Aragón– IA2 (Universidad de
Zaragoza-CITA), Zaragoza, Spain
e-mail: diego.garcia@unizar.es; pagan@unizar.es

DOI 10.1007/978-3-319-32886-7_137
1360 D. García-Gonzalo and R. Pagán
Keywords
Outer membrane • Peptidoglycan • Irreversible and reversible permeabilization •
Fluorescent probes • Leakage of intracellular material • Sublethal injuries • DNA
microarrays • Site-directed mutagenesis
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1360
Entrance of External Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1362
Fluorescent Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1363
Antimicrobial Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1365
Leakage of Intracellular Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1366
Electron and Atomic Force Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1367
Selective Medium Plating Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1367
Measurement of Osmotic Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1369
Fourier Transform Infrared Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1369
Molecular Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1370
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1371
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1372
Introduction
The effects of electric fields in cell membranes have been extensively documented,
due to their extended use in cell biology, biotechnology, or medicine (Prasanna and
Panda 1997). Although the exact molecular mechanisms are not fully understood, it
is known that application of an electric field to cell membranes can cause electro-
poration, involving the formation of pores on the membranes of cells and organelles.
Depending on the intensity of the applied electric field, electroporation can induce
the formation of transitory and/or permanent pores leading to reversible or irrevers-
ible electroporation, respectively (Teissie et al. 2005). Reversible pores are resealed
when the electrical field ceases, and the electrically stimulated cells survive. How-
ever, at high intensities, permeabilization of cell membranes might be irreversible
and even cell membranes and other structures might break down. Under these
circumstances, electric fields cause permanent membrane damage and cell death
which is the basis for the success of pulsed electric fields (PEF) as a novel processing
method for food preservation. In addition, application of electric fields has also been
proposed as an excellent method to extract compounds of interest from the cyto-
plasm of bacteria and unicellular organisms (Luengo et al. 2015).
The promising use of PEF as a nonthermal process for food preservation resides
in its potential to inactivate microbial cells without altering sensorial and nutritional
food properties. The design of effective PEF treatments which provide safe food
with a prolonged shelf life requires, among others, knowledge of how PEF kill
microorganisms (i.e., the mechanism of microbial inactivation by PEF). A better
understanding of this mechanism would help to define PEF treatments as alternatives
to traditional heat preservation.
71 Detection of Electroporation in Microbial Cells: Techniques and Procedures 1361
Fig. 1 Structure of cell membranes of Gram-positive and Gram-negative bacteria: (1) the outer
membrane with lipopolysaccharides, (2) cell wall with peptidoglycan, (3) cytoplasmic membrane
Knowledge of the mechanisms of electroporation has been mostly obtained using

artificial membranes, theoretical models, and eukaryotic or mammalian cells. How-
ever, its occurrence and relation to microbial inactivation by PEF is less understood.
In contrast to ideal systems, such as artificial membranes or mathematical models,
biological membranes are complex and dynamic systems: their composition and
structure could be modified as a response to stressing conditions, such as temperature
variations or exposure to chemical compounds. In addition, microorganisms, such as
fungi and bacteria, have additional structures located externally to the cytoplasmic
membrane (Neidhardt et al. 1990). For the sake of simplicity, this chapter will focus
on the techniques used to detect electroporation in bacteria, although most of the
techniques could be easily adapted to fungal cells.
Composition of bacterial membranes differs between species and strains, being
highly noticeable between Gram groups. Gram staining technique is based on
different chemical and physical properties of peptidoglycan present in cell walls:
after a washing step, Gram-positive cells retain crystal violet dye, but the dye is
washed out of Gram-negative cells. In the next counterstaining step, safranin only
stains the washed Gram-negative cells. This differential staining is due to the
presence of the outer membrane in Gram-negative cells. In Gram positives, outside
the cytoplasmic membrane, there is a cell wall formed by several peptidoglycan
layers and teichoic acids (Fig. 1), which confers rigidity and physical resistance to
the bacterial cell. In Gram negatives, the cell wall is thinner, but surrounded by a
protective outer membrane (Fig. 1).
The outer membrane differs from the cytoplasmic membrane since its external
leaflet is made up of lipopolysaccharides (LPS) instead of phospholipids, and it
confers special resistance against the entrance of some molecules such as antimicro-
bial compounds, bile salts, and some fluorescent probes. In both Gram groups, but
more especially in Gram negatives, these external structures create a zone outside the
cytoplasmic membrane with a different chemical composition from the environment,
known as periplasmic space. This particular disposition of bacterial membranes has
to be taken into account for the evaluation of electroporation. For example, PEF
could cause electroporation of the cytoplasmic membrane, but not of the outer
membrane, and consequently access of some fluorescent probes to the cytoplasm
could be hindered, as described for Gram-negative cells at acid pH (Garcia
et al. 2007).
Maintenance of the integrity and functionality of cell membranes is of key
importance for bacterial cells. First of all, these structures protect microorganisms
from the external conditions acting as a semipermeable selective filter. In addition
the cytoplasmic membrane is involved in other cellular processes such as the control
of the passage in both directions of small molecules, nutrients, and end products of
metabolic activities; synthesis of the RNA, protein, and cell wall; control of DNA
synthesis; and electron transport and oxidative phosphorylation (Neidhardt
et al. 1990). Thus, the cytoplasmic membrane controls the cellular metabolic
activities by maintaining an effective osmotic boundary between the cell interior
and the surrounding environment. Any damage in the integrity or functionality
of this membrane could impair one or more of these processes, resulting in
bacterial death.
This chapter presents an overview of the techniques and procedures used to
provide direct and indirect evidences of electroporation in microbial cells. The
presented methods could detect irreversible and/or reversible permeabilization as
well as modifications in cell envelopes as a consequence of PEF treatments.
Considering that cell membranes separate the cytoplasm from the outside envi-
ronment, the methods used to assess the electroporation of these membranes can be
divided into (1) those based in the entrance of external compounds into the bacterial
cytoplasm (i.e., a fluorescent probe) and (2) those which detect the leakage of
intracellular compounds to the environment (i.e., nucleic acids). In addition, there
are other excellent methods, highlighting (3) direct visualization of damages in cell
membranes by electron microscopy, (4) evaluation of membrane damages by a
selective medium plating technique, and (5) measurement of osmotic response of
PEF-treated cells or (6) Fourier transform infrared spectroscopy. These techniques
could be complemented by (7) molecular tools, such as directed mutagenesis or
transcriptomics, to understand the mechanism of electroporation in microbial
membranes.
Entrance of External Compounds
As compared to evaluation of leakage of intracellular compounds, the determination

of the access of external compounds to the cytoplasm is easier because of the
possibility to modify the concentration of the latter compounds. Consequently,
techniques based on the detection of foreign substances in the bacterial cytoplasm
have been widely used to measure electroporation efficiency and quantify
electroporated cells. Two main classes of substances have been used with this
objective: fluorescent probes and antimicrobial agents.
Fluorescent Probes
The measurement of the increased uptake of a fluorescent dye, which normally does
not penetrate the membrane of healthy cells, is a direct and sensitive technique to
evaluate membrane integrity and/or functionality. In addition, this methodology
could allow for single-cell analysis and identification of permeabilized cell fraction
by the use of fluorescence microscopy or flow cytometry (FCM). FCM assists in the
study of microbial behavior and responses to PEF with single-cell resolution, since it
quantifies small differences between cell populations analyzing thousands of cells in
seconds. A great advantage of fluorescent probes is the delivery of rapid and
sensitive results. Even more, in comparison to radioactive probes, these probes
present a lower toxicity for lab operators. The most used fluorescence-based
methods to evaluate electroporation are based on the addition to the treatment
medium of cell-impermeant nucleic acid stains, such as propidium iodide (PI),
cyanine monomers, or SYTOX ® dyes.
PI is a small (660 Da), strongly hydrophilic molecule, showing a high sensi-
tivity and potential to provide quantitative results by using a spectrofluorometer, to
identify stained cells by using an fluorescence microscope and to analyze individ-
ual cells in combination with FCM or image analysis. PI has been commonly used
to assess membrane electroporation of PEF-treated microorganisms (Wouters
et al. 2001; Aronsson et al. 2005; Garcia et al. 2007; Jaeger et al. 2009; Sagarzazu
et al. 2013). Moreover, PI could be added to the treatment medium before and after
PEF treatments (Fig. 2) in parallel experiments to detect reversible or irreversible
permeabilization (Garcia et al. 2007). PI has also been used in combination with
other probes to successfully identify sublethally injured populations in
Escherichia coli and Lactobacillus plantarum PEF-treated cells (Jaeger
et al. 2009; Zhao et al. 2011). For future research, FCM might also be used to
assess heterogeneity in stress survival, including damage repair capacity and PEF
targets.
Ethidium bromide (EtBr) can also be used to evaluate electroporation, although it
is less reliable than PI, because PI binds to naked DNA with a higher affinity than
EtBr. This is because PI is doubly charged as compared to EtBr. However, this
disadvantage could be overcome by the use of ethidium homodimer-1, with high
affinity for DNA (it binds to nucleic acids 1,000 times more tightly than does EtBr,
and its fluorescence increases 40-fold upon binding) and low membrane permeabil-
ity in intact cells (Johnson and Spence 2010). Due to its high affinity, low concen-
trations needed for fluorescence experiments lead to the use of small quantities of
potentially hazardous compounds.
Cyanine monomers, such as TO-PRO ® dyes, are among the best fluorescent
probes available for nucleic acid staining because of their high affinity for nucleic
acids and high fluorescence enhancement and quantum yield upon binding (Johnson
and Spence 2010). In addition, cyanine monomers have narrow emission band-
widths, making them a good choice for multicolor experiments. For example, in
FCM applications, the complex formed by nucleic acids with TO-PRO ®-3 can be
Fig. 2 Protocol for propidium iodide (PI, ) staining of microorganisms to detect reversible and
irreversible electroporation. Addition of PI after electroporation detects only cells with irreversible
permeabilization of their membranes. However, the presence of PI during the treatment allows for
detecting not only irreversible cell permeabilization but also the presence of reversible pores that are
repaired immediately after the treatment. Addition of PI at different times after electroporation (e.g.,
immediately, 1 min, 5 min, etc.) would also allow for the determination of cells that are repairing
their pores after the treatment, i.e., the dynamics of reversibility of pores. It should be noted that this
protocol could also be used with other membrane-impermeant probes
directly excited by the He-Ne red laser. The binding affinity to dsDNA, RNA, and
ssDNA of TO-PRO ® dyes is lower than that of other dyes, with lower fluorescence
quantum yields.
SYTOX® nucleic acid stains are also good cell-impermeant dyes to assess elec-
troporation because they show high affinity to nucleic acids and little base selectiv-
ity. They are especially useful for staining both Gram-negative and Gram-positive
bacteria, providing a bright green, blue, or orange signal (Johnson and Spence 2010).
These dyes could be used simultaneously with cell-permeant SYTO® stains (blue,
green, orange, or red), which passively diffuse through membranes and show a lower
affinity to nucleic acids than SYTOX ® dyes.
The aforementioned probes have been widely used to assess membrane permeabi-
lization caused by antimicrobial compounds, such as antibiotics. However, it should
be taken into account that the action of most antimicrobials, such as disinfectants, is
inward to the cytoplasm, i.e., they should pass through the outer membrane and/or
cell wall to access the cytoplasmic membrane. However, PEF might cause the
electroporation of the cytoplasmic membrane without altering the outer membrane,
or the size of pores caused by PEF in the outer membrane might be smaller than the
size of the probes and thus their pass through the outer membrane would be
prevented. Under these circumstances, it is difficult to evaluate electroporation of
the cytoplasmic membrane by these fluorescent techniques: it can be concluded that
all stained cells are permeabilized, but not that all permeabilized cells are stained and
can be detected. A possible solution could be to permeabilize the outer membrane by
cationic agents (e.g., polymyxin derivatives, lysine polymers, or benzalkonium

chloride) or chelators (e.g., ethylenediaminetetraacetic acid (EDTA), sodium
hexametaphosphate, or acetylsalicylate), although these compounds could also
alter the cytoplasmic membrane. Alternatively, bacteria with mutations disrupting
the outer membrane permeability barrier, such as E. coli lptD4213 (Ruiz et al. 2006),
could be used to prevent interferences of the outer membrane in the assessment of
cytoplasmic membrane permeabilization by fluorescent techniques.
Moreover, prevention from protective role of the cell wall in the assessment of
cytoplasmic membrane permeabilization of Gram-positive cells could be achieved
by preparation of protoplasts. Treatment of cells with lysozyme degrades the
peptidoglycan component of cell walls, resulting in the formation of protoplasts.
Since protoplasts are osmotically sensitive, cell wall digestion, storage, and further
experiments must be done in an isotonic solution. The method to obtain protoplasts
from Gram-negative cells is complicated by the presence of the outer membrane
which prevents access of lysozyme to the cell wall. As a consequence, EDTA should
be added to the isolation medium as a destabilizer of the outer membrane. This
technique is not capable to totally remove the outer membrane and the cell wall is
only partially degraded, and so the usual products obtained from Gram-negative
cells are spheroplasts (with spherical shape) rather than protoplasts.
On the other hand, permeabilization of the outer membrane could be eval-
uated using probes that are usually excluded by this membrane, but once the
integrity of the outer membrane is compromised, they can access the cytoplas-
mic membrane or the bacterial cytoplasm. Most used probes for this purpose
are 1-N-phenyl-naphtalene (NPN) and hexidium iodide (HI). NPN (currently
found as N-phenyl-1-naphthylamine) is a hydrophobic fluorescent probe which
binds to hydrophobic regions of the bacterial cytoplasmic membrane: it fluo-
resces weakly in aqueous environments, but becomes very strongly fluorescent
in nonpolar or hydrophobic environments. HI is a cell-permeant nucleic acid
dye which selectively stains Gram-positive bacteria because it is blocked by the
LPS layer of Gram-negative bacteria and thus only permeable to Gram-positive
bacteria and Gram-negative bacteria with an altered LPS layer. Therefore, the
increase of fluorescence upon addition of NPN or HI to PEF-treated Gram-
negative cells would indicate the electroporation of the outer membrane.
Antimicrobial Agents
This method is based on the uptake of antimicrobials into the cytoplasm or the
cytoplasmic membrane which normally would not access to these structures. The
increase of sensitivity of the target cell to lysozyme or to hydrophobic antibiotics,
such as actinomycin, is an indirect assay to determine electroporation of the outer
membrane (Sánchez-Gómez et al. 2008). In this context bile salts could be included,
but the use of these compounds will be commented in section “Selective Medium
Plating Technique.” Lysozyme damages peptidoglycan layers of the cell wall, and
actinomycin inhibits transcription by binding the DNA at the transcription initiation
complex and preventing elongation of RNA chain by RNA polymerase in Gram-

positive bacteria. However, their access to cell targets is hindered by the outer
membrane in Gram-negative cells. Consequently, sensitization of PEF-treated
Gram-negative cells to these antimicrobial agents would demonstrate the permeabi-
lization of the outer membrane.
Leakage of Intracellular Compounds
The most commonly used techniques to evaluate the presence of intracellular

material outside the cell are the measurement of UV-absorbing material such as
nucleic acids and proteins at 260 and 280 nm, respectively, and the presence of
adenosine triphosphate (ATP) by luciferin–luciferase assay. The sensitivity of these
methods could be increased by the use of fluorescent probes (such as nucleic acid
stains mentioned above) which bind to leaked compounds. However, the occurrence
of various degrees of cell permeabilization complicates to find a relationship
between the amount of intracellular components outside the cell and the number of
inactivated cells (Wouters et al. 2001; Aronsson et al. 2005). Furthermore, this
technique would not allow for quantification of electroporated cells.
An alternative method with a higher sensitivity than detecting cell components
in the environment is the evaluation of leaked fluorescent probes that have
been previously introduced in the bacterial cytoplasm. For this purpose, esterase
substrates could be used, such as calcein acetoxymethyl ester (calcein AM),
20 ,70 -bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester
(BCECF AM), and carboxyfluorescein diacetate (CFDA). These nonfluorescent
substrates freely diffuse into the cytoplasm (cell permeants) where they are
converted by intracellular esterases into fluorescent compounds which are retained
by cells with intact cytoplasmic membranes (Johnson and Spence 2010). There-
fore, these probes assess enzymatic activity (for hydrolysis of incorporated sub-
strates) and integrity of the cytoplasmic membrane (for intracellular retention of
fluorescent products). As explained in the previous section, these probes may also
be combined with other probes (e.g., with PI) for multicolor experiments.
Calcein AM is usually the first choice because of its notable cell retention and its
stability at physiological pH. Calcein is well retained in viable cells due to its six
negative charges and two positive charges at pH 7. Other chemical variations with
different emission wavelengths can be found in the market: calcein blue AM, calcein
violet AM, and calcein red-orange AM.
Hydrolysis of BCECFAM leads to formation of BCECF with four to five negative
charges. These charges improve its cell retention, but the emission intensity is
reduced at acid pH, making it less optimal for some applications.
CFDA, originally conceived as a probe to measure intracellular pH but promptly
adapted as an indicator of cell viability and membrane integrity, forms
carboxyfluorescein upon hydrolysis. Extra negative charges as compared with
fluorescein improve cell retention of carboxyfluorescein, making it preferred for
these assays than fluorescein diacetate.
Electron and Atomic Force Microscopy
Because of its higher resolving power, electron microscopy has been used to
examine morphological changes in cells, either at cytoplasmic organelles or cell
envelopes, after PEF treatments. With regard to membrane damage, the technique
allows detecting pores from 1 nm in diameter. However, it should be noted that it
would not always allow observing lysed cells since their debris might be removed
during the washing and cell concentration steps. Furthermore, PEF-induced pores
smaller than 1 nm would be undetected. The use of this technique has not confirmed
any relationship between membrane damage and microbial inactivation by PEF
because frequency of morphologically damaged cells did not correspond to the log
reductions of viability. Nevertheless, electron microscopy could be useful as a
complementary technique to gain insight into PEF action, by revealing effects not
only in cell membranes but also in intracellular modifications.
Atomic force microscopy could also be used to visualize cell changes. This
technique provides a three-dimensional image in real time of living cells with a
resolution of fractions of a nanometer, reflecting modifications in the ultrastructure
of the electroporated cells (Pillet et al. 2016). Apart from surface topography, atomic
force microscopy provides information related to electrical and mechanical proper-
ties, such as surface potential or adhesion strength.
Selective Medium Plating Technique
Membrane damage measured using a selective medium plating technique includes

the loss of both membrane integrity and functionality. The method consists of plating
survivors after treatments into two culture media: a nonselective one, which allows
cells to repair sublethal damages and recover, and a selective one, in which survivors
are not capable of repairing their damages and finally die (Mackey 2000). Those
membrane damages that are repairable under suitable conditions and detected
following this methodology are commonly called sublethal injuries. As illustrated
in Fig. 3, sublethally injured cells in their membrane(s) are estimated by the
difference in the number of survivors obtained after plating treated cells in both
nonselective and selective media. The most common selective media used to detect
damage to the cytoplasmic membrane is agar medium with sodium chloride added
(Mackey 2000). In addition, bile salts can be used in order to detect sublethal
damages in the outer membrane of Gram-negative bacteria. As noted for hydropho-
bic antibiotics, the outer membrane is also an excellent barrier to membrane active
agents such as bile salts, with LPS playing a major role in resistance to bile salts. The
loss of resistance to sodium chloride and bile salts is likely to be multifactorial.
However, sensitivity to sodium chloride implies a loss of osmotic functionality of the
cytoplasmic membrane, whereas sensitivity to bile salts reflects the loss of outer
membrane integrity and/or impairment of multidrug efflux systems by the loss of
proton motive force (Thanassi et al. 1997; Mackey 2000; Begley et al. 2005).
Fig. 3 Fundamentals of selective medium plating technique for detecting sublethal damages in
microbial membranes. After an electroporating treatment, there could be alive (intact), dead
(inactivated), and cells with sublethal injures in their membranes (damaged). Plating these treated
cells in a nonselective medium allows the recovery of both alive and sublethally injured cells. However,
if these cells are plated in a selective recovery medium, only alive cells will produce visible colonies.
For example, if 1 mL of a microbial suspension at 109 CFU/mL is PEF treated and survival counts in
nonselective medium are 108 CFU/mL, the treatment caused 1 log10 of inactivation. If survival counts
of the same treated sample in a selective medium with sodium chloride are 106 CFU/mL would indicate
that 2 log10 cycles of the survivors to PEF are sublethally damaged in their cytoplasmic membranes.
This methodology could be performed with higher or lower cell concentrations
Sublethally injured bacteria in their cytoplasmic membrane after PEF treatments

were firstly detected by the use of this technique. Moreover, the growth of suble-
thally injured cells in nonselective media indicated that, in some cases, damages
inflicted in cell membranes by PEF are repairable (Garcia et al. 2006). The addition
of inhibitors such as chloramphenicol, cerulenin, penicillin G, rifampicin, and

sodium azide to the repair medium would indicate the biosynthetic requirements to
repair these damages in the cell envelopes.
In comparison to the previous mentioned techniques, it is more time consuming
and requires at least 48 h to get the final results. Nevertheless, this technique shows
an extraordinary sensitivity because it can be used to evaluate the damages in treated
suspensions with a high number of bacterial cells (e.g., 109 CFU/mL), allowing the
detection of several log cycles of cells with sublethal damages in their membranes.
Moreover, the recovery of cells in media with different proportions of selective
agents would allow the identification of several degrees of membrane damages.
Measurement of Osmotic Response
This rapid and inexpensive technique evaluates alterations in membrane integrity

through the loss of the ability of the cell to plasmolize. When a cell with an undamaged
membrane is suspended in a hypertonic medium, such as a strong salt or sucrose
solution, water diffuses from the cytoplasm to the external medium. This initial passive
response causes gross morphological changes with the cytoplasm shrinking and the
plasma membrane contracting from the cell wall, leading to a strong condensation of
the cell material, which can be measured through the increase in optical density
[usually at 600 nm (OD600)] of cell suspensions (Korber et al. 1996). On the contrary,
in cells whose membrane is altered, salt and water diffuse freely through the mem-
brane, and no response is detected (i.e., no changes in OD). Additionally, FCM and
microscopic methods, such as low-magnification dark-field microscopy, high-
magnification phase-contrast microscopy, and scanning confocal laser microscopy,
used with or without image analysis are suitable to determine membrane integrity
through direct visualization and/or the measurement of the cell area.
Fourier Transform Infrared Spectroscopy
Fourier transform infrared (FTIR) spectrum in mid-infrared range provides bands

from all the cellular components of microorganism (e.g., cell membrane and wall
components, proteins, and nucleic acids). Therefore, the obtained spectral signatures
or “fingerprints” can be used for detection, classification, and identification of
microorganisms (Alvarez-Ordóñez et al. 2011). Thus, FTIR is a suitable tool to
detect the changes occurring in bacterial cells and indicate the physiological state in
response to electroporation. Actually, most of the studies assessing the effects of
stressing conditions on the bacterial biochemical composition of bacterial cells by
FTIR have been focused on the examination of the membrane properties, such as
changes in the membrane phase behavior, the presence of longer and/or more
saturated acyl chains, and structural changes of membrane phospholipids. Similarly,
this method indicated that PEF treatments would mainly target LPS cell fraction or
outer membrane proteins of E. coli, as a function of the treatment pH (Gelaw

et al. 2014).
Molecular Tools
Molecular microbiology tools are at the vanguard of most cutting-edge scientific

research because they allow investigating and manipulating microbial cells at a
molecular level. Among the high-throughput techniques that are available, both
DNA microarrays and site-directed mutagenesis methods could be most useful to
study permeabilization of microbial cells.
DNA microarrays provide information at a molecular level: transcriptome analysis
of cells (i.e., all RNA molecules in the bacterial population) under different conditions
may reveal differences in factors contributing to PEF resistance and damage repair
systems. A DNA microarray contains thousands of microspots on a glass slide. Each
spot contains identical single-stranded DNA oligonucleotide-capturing probes, whose
locations are fixed during the process of hybridization and detection. This high-
throughput technique can be used to measure changes in gene expression after
exposure to PEF. RNA will be isolated from the samples at preset times and convert
it into cDNA. Each DNA oligonucleotide spot represents a specific gene sequence to
which cDNA will bind by complementarity. After labeling, a microarray reader will
provide the intensity of signal per gene that can be compared between untreated and
electroporated cells. Thus, the transcriptome at the moment of the RNA isolation will
be inferred. By comparing the gene expression profiling before and after electropora-
tion would reveal sets of genes upregulated or downregulated and subsequently the
proteins activated and repressed by PEF (Chueca et al. 2015). This knowledge will
allow to better understand the mechanism of inactivation and the mechanisms of
resistance that the cell uses to cope with PEF treatments by the identification and
selection of specific targets for further characterization, e.g., assessment of their role
(s) in stress adaptation and damage repair. While DNA microarrays will yield a vast
amount of data that will undoubtedly give insight into the electroporation of bacterial
membranes, one major limitation is its inability to detect the cells within the bacterial
population that are differentially expressing a gene. Therefore, the combination of
FCM with transcriptional fusions using fluorescent proteins (e.g., green fluorescent
protein controlled by a promoter which activity is modulated by electroporation)
would show those cells affected by electroporation.
Construction of deletion mutants in selected genes or gene clusters (site-directed
mutagenesis) allows the creation of microbial cells with a modified composition and
structure of membranes. As explained above, a mutation in lptD gene of E. coli (Ruiz
et al. 2006) altered the outer membrane, avoiding the potential interferences of the outer
membrane in electroporation. Moreover, candidate genes which could play a role in
membrane permeabilization could be modified or deleted to assess the influence of
certain proteins and/or structures in cell electroporation. Deletion mutants in E. coli can
be constructed using P1 phage transduction derived from an E. coli single-gene
knockout library (Baba et al. 2006). Positive P1 transductants would be confirmed by
acquisition of kanamycin resistance and polymerase chain reaction (PCR). Subse-

quently, the kanamycin-resistance cassette would be removed using the previously
described pCP20 plasmid and confirmed by PCR. Effect of mutations could be initially
studied by comparing survival counts in differential plating media between wild type
and mutant strains after exposure to PEF. Next, their transcriptomes could be compared
by DNA microarrays after PEF treatments. These important tools will help to identify
not only cellular targets but also to discover (novel) stress-resistance mechanisms.
Conclusions
In summary, there is a wide variety of available techniques and methods to detect and
evaluate the electroporation of microbial cells (Table 1).
Table 1 Summary chart with described procedures for detection of electroporation in microbial
cells with their main advantages and disadvantages
Procedure Advantages Disadvantages
Entrance Fluorescent Rapid and sensitive results Some probes are potentially
of external probes Possibility of single-cell hazardous
compounds analysis Permeabilization of all cell
envelopes is required
Antimicrobial Easy and fast technique Normally incubation of plates
compounds (24 h) is required
Indirect evidence of
permeabilization
Leakage of intracellular Rapid results Difficult correlation between
compounds Sensitivity could be increased leaked components and
with fluorescent probes permeabilized cells
Electron and atomic force Visualization of Difficult identification of all
microscopy morphological changes permeabilized cells
High resolution (1 nm) Extensive training and
special equipment are
required
Selective medium plating Easy and fast technique Normally, it is more time
technique Extraordinary sensitivity consuming, because a long
(allows for discrimination incubation of plates (48 h)
between 99 and 99.99% of is required
damaged cells)
Measurement of osmotic Rapid and inexpensive Low sensitivity
response technique
Fourier transform infrared Detection of biochemical Extensive training and
spectroscopy changes in PEF-treated cells special equipment are
required
Molecular tools Identification of metabolic Extensive training and
pathways for cell survival and special equipment are
repair required
Discovery of resistance Indirect evidence of
mechanisms to PEF permeabilization
Since they evaluate different membrane modifications, all of them have their
limitations in assessing cell electroporation. Consequently, analysis of results
obtained with different methods could provide a more accurate picture of membrane
permeabilization. Moreover, it should be noted that the techniques and methods
described in this chapter have already been used to confirm the occurrence and
relation of the electroporation to microbial inactivation by PEF. Some techniques
such as electron microscopy examinations or the measurement of leakage of intra-
cellular material have allowed obtaining visual or direct evidence of the phenome-
non; others, such as the measurement of dye exclusion/uptake and selective medium
plating technique, have allowed finding out correlation between electroporation and
microbial inactivation. Available molecular tools will be extremely valuable to
increase our knowledge on the mechanism of electroporation and consequently
complement our knowledge in the mechanism of bacterial inactivation by PEF.
Acknowledgments Authors thank to CICYT (Spanish Interministerial Commission of Science

and Technology, Projects AGL2012-32165 and AGL2015-69565-P), European Social Fund, and
Aragonese Departamento de Ciencia, Tecnología y Universidad for their financial support.
Cross-References

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The Use of Electroporation in
Developmental Biology 72
Charlie Gosse, Xuan Zhao, I. Migeotte, Diana Suárez-Boomgaard,
Isabelle Hue, Séverine Degrelle, Aitana Perea-Gomez, and E. Mazari
Abstract
During the formation of a complex organism, cells divide, die, migrate, and
differentiate. Biologists have established tools to observe those phenomena but
also to change their course, which subsequently enables to infer causal relation-
ships between various events occurring in different cell groups. More precisely,
present approaches mostly rely on modifications of gene expression. For instance,
cells are labeled with fluorescent proteins and tracked within the embryo,
C. Gosse (*) • X. Zhao

Laboratoire de Photonique et de Nanostructures, LPN-CNRS, Marcoussis, France
e-mail: charlie.gosse@lpn.cnrs.fr; xuan.zhao@lpn.cnrs.fr
I. Migeotte • D. Suárez-Boomgaard
Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université Libre de
Bruxelles, Bruxelles, Belgium
e-mail: imigeott@ulb.ac.be; boomgaard83@gmail.com
I. Hue
Biologie du Développement et Reproduction, UMR 1198 INRA, ENVA, Université Paris-Saclay,
Jouy-en-Josas, France
e-mail: isabelle.hue@inra.fr
S. Degrelle
Physiopathologie et Pharmacotoxicologie Placentaire Humaine, INSERM UMR-S1139, Université
Paris Descartes, Sorbonne Paris Cité, Paris, France
e-mail: severine.degrelle@inserm.fr
A. Perea-Gomez
Institut de Biologie Valrose, CNRS UMR7277, Inserm U1091, Université Nice Sophia Antipolis,
Nice, France
e-mail: apereagomez@unice.fr
E. Mazari
Center for International Research on MicroMechatronics, Institute of Industrial Science, The
University of Tokyo, Tokyo, Japan
e-mail: mazari@iis.u-tokyo.ac.jp

DOI 10.1007/978-3-319-32886-7_139
1376 C. Gosse et al.
molecular signals are switched on and off to perturb regulatory pathways. Impor-
tantly, in all those experiments, the exogenous genetic material must be delivered
at the right place and with the appropriate timing: requirements that can both be
fulfilled by electroporation. After 15 years of constant refinement, this technique
has now superseded methods like viral infection, microinjection, and lipofection.
Applications encompass a large number of model organisms, targeted anatomical
structures, and molecular biology techniques.
Keywords
Embryogenesis • Organogenesis • Cell labeling • Genetic engineering • Numerical
simulations • Microfabrication
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1376
Specificities Related to the Scientific Context . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1377
Typical Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1377
Organisms and Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1381
Molecular Biology Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1382
Competing Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1384
Strategies to Spatially Restrict Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1387
Orientation of the Embryo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1387
Containment of the Nucleic Acids Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1389
Generation of an Electric Field Gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1390
Combination of both Molecular Localization and Electric Field Focusing . . . . . . . . . . . . . . . 1391
Engineering Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1393
Setup Conception . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1393
Material Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1395
Reagents Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1397
Performances Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1398
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1401
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1403
Introduction
The use of electroporation to study embryogenesis and organogenesis was initiated

around 1995, and most of the associated technical developments took place in the
following decade (Nakamura and Funahashi 2013). Therefore, the book Electropora-
tion and Sonoporation in Developmental Biology edited by H. Nakamura in 2009
displays a quite complete overview of the possibilities offered by this technique, with
detailed accounts on manipulations performed on model organisms such as chick and
mouse (Nakamura 2009). Since then electroporation has become a widespread tool
enabling embryologists to answer questions specific to their discipline.
More than a hundred of original articles have been published on the sole meth-
odological advances. Additionally, many reviews and focus papers have already
extensively summarized and discussed this large corpus of studies (Itasaki et al.
72 The Use of Electroporation in Developmental Biology 1377
1999; Swartz et al. 2001; Ogura 2002; Krull 2004; Odani et al. 2008; Sauka-
Spengler and Barembaum 2008; Takahashi et al. 2008a; Tanaka et al. 2014). Thus,
the present chapter has been conceived along somewhat different lines: it aims to be a
primer that presents the utilization of electroporation in developmental biology from
an engineering point of view. To start with, the fundamental objectives pursued by
embryologists will be described, as well as the typical experiments they carry out.
How electroporation can fulfill some of the corresponding technical needs will also be
explained. Next, a comprehensive summary of the realizations so far published will be
provided, using the strategy devised to localize transfection as an organizing principle
for the redaction. Finally, some of the practical issues related to the implementation of
electroporation in this particular field of life sciences will be discussed.
The bibliography will be more illustrative than exhaustive. It will mainly focus on
seminal publications and on well-detailed technical papers, leaving aside reports on
biological questions in which electroporation is marginally employed. Furthermore,
references for many reviews will be included, to be used as introductions to more
specific parts of the literature. We will also try to discuss as much as possible the
advances posterior to the 2009 publication of Nakamura’s book. Noteworthily, in the
last 10 years both Nature Protocols and the Journal of Visualized Experiments have
released a few dozens of detailed protocols, old and new. In the case of the latter
journal, each article is associated with videos that really bring a plus, because skilled
manual experimentation is often a prerequisite in embryology. Finally, for comple-
mentary technical and bibliographical information, one can profitably check the
websites of some of the main providers of electroporation apparatus (e.g., BTX
Harvard Apparatus, NepaGene).
Specificities Related to the Scientific Context
Typical Experiments
The formation of a multicellular organism from a single cell, the fertilized egg
(or zygote), involves fundamental processes like differentiation, which produces
diverse cell types such as neurons or insulin producing cells, and morphogenesis,
which allows their spatial and functional organization into organs such as the brain
or the pancreas. During embryonic development, a tight regulation of cell division,
cell death, and cell migration is also at play to control the global growth (Slack 2006;
Gilbert 2013). Importantly, developmental processes are not restricted to the time of
the embryonic life. Indeed, in human for instance, some organs like the skin or the
surface of the intestine are constantly renewed. Moreover, in animals with high
regenerative capacities like amphibians, an entire adult limb or a tail can be replaced
after injury.
Understanding the development of a complex organism requires acquiring a
precise knowledge of the origins and of the dynamics of the cell populations that
form the different tissues of the embryo. So-called fate maps are then drawn by
tracing the descendants of a particular cell population (cell lineage analysis) or even
Fig. 1 Examples, in developmental biology, of seminal experiments relying on tissue transplan-

tation. (A1–2) Lineage tracing performed on avian embryos to decipher the origin of the skull
components (Adapted from Couly et al. 1993). (A1) A piece of the anterior part of the neural fold is
dissected from a quail embryo at the three-somite stage and grafted in a chick one. (A2) The fate of
the tissues issued from this isotopic and isochronic substitution is analyzed by inspection of head
sections at later stages. Quail cells can indeed be distinguished from chick ones thanks to differ-
ences in the structure of their interphase nuclei. The xenograft is here at the origin of the upper part
of the skull. (B1–2) Induction experiment evidencing the presence of the organizer in newt embryos
(Adapted from Kretzschmar and Watt 2012) and inspired from the work presented in (Spemann and
Mangold 1924). (B1) A piece of tissue from above the dorsal blastopore lip is collected on a Triton
cristatus gastrula and ectopically transplanted in the ventral marginal zone of a Triton taeniatus one
(the two species differ by their pigmentation). (B2) The signals produced by the graft subsequently
lead to the apparition of a secondary body axis
of a single cell (clonal analysis) (Stern and Fraser 2001; Kretzschmar and Watt
2012). Classically, marking selected cell populations relied on the injection of
organic dyes (Bhattacharyya et al. 2008) or the grafting of cells possessing specific
characters such as a pigmentation or a specific nuclear organization (Le Douarin
et al. 2008 – Fig. 1a). In modern developmental biology, cells are labeled by
modifying their gene expression so that they produce specific proteins like the
enzyme β-galactosidase, which product can be detected in fixed tissues or fluores-
cent proteins, that allow live imaging experiments (Fig. 2a).
Another important experimental paradigm in developmental biology is to modify
the behavior of a given cell population in order to understand its role in the formation
of a given tissue. In classic embryology it was achieved by the grafting or the
ablation of specific cell groups. This approach is best illustrated by the Spemann
and Mangold experiment, demonstrating that grafting some specific dorsal cell
population of an amphibian embryo, the organizer, is sufficient to modify the
72
The Use of Electroporation in Developmental Biology
Fig. 2 Examples, in developmental biology, of contemporary experiments relying on tissue electroporation. (A1–4) Lineage tracing performed on a chick
embryo to evidence somitogenesis (Adapted and reproduced from Iimura and Pourquié 2008). (A1) Schematic dorsal view of the injection of the DNA solution
(in green) between the epiblast and the vitelline membrane at stage HH4. Using a fine glass needle, the anterior first third of the primitive streak groove is
targeted. (A2) Transverse representation of the electroporation. (A3) Overlay of fluorescence and bright-field images evidencing the area over which the dsRed is
protein expressed 4 h after electroporation with the corresponding plasmid. (A4) Same result after 24 h. (B1–4) Induction experiment demonstrating that the
ectopic expression of the Hoxb-8 transcription factor can yield “mirror image” digit duplication in chick (Adapted and reproduced from Oberg et al. 2002).
(B1) Schematic dorsal view of the injection of the DNA solution (in green) into the limb bud mesoderm at stage HH19. (B2) Transverse representation of the
1379
behavior of the adjacent tissues in the receiving host, so that a secondary axis is
induced (Spemann and Mangold 1924 – Fig. 1b). Later on, the molecular nature of
the communication between cell groups was further evidenced by grafting polymeric
beads releasing so-called morphogens, i.e., small molecules or proteins that induce
the formation of anatomical structures from a distance (Eichele et al. 1985;
Stern et al. 1990). Nowadays, local modification of gene expression is commonly
used to change the characteristics of targeted cells (Fig. 2b). Gain- or loss-of-
function strategies result in the respective up- or downregulation of the activity of
the protein of interest, by modulating its synthesis or by interfering with its normal
molecular behavior (section “Molecular Biology Tools”). The consequences of these
changes on the modified cell population or on the adjacent tissues can then be
addressed in the context of the whole developing organism (Sauka-Spengler
and Barembaum 2008; Tanaka et al. 2014). The ultimate goal of such perturbative
approach is to explain morphogenesis thanks to wiring diagrams that emphasize the
spatiotemporal coupling, either chemical or mechanical, between intracellular
genetic regulatory networks and intercellular communication pathways (Streit
et al. 2013).
Local modification of gene expression to trace cell populations or to change their
behavior can be achieved in several ways. In some animal models like the Drosoph-
ila, powerful genetic tools allow the production of lines where clones of cells harbor
stable transgenes or mutations. In contrast, some other models like the chick or the
Xenopus do not offer such possibilities. Finally, in some cases like the mouse,
genetically modified embryos can be obtained but it requires much time and labor
(Itasaki et al. 1999; Slack 2006; Sharpe and Mason 2008). In addition, when a stable
line can be produced, one also has to cope with the lethality associated with
mutations affecting most developmental genes – a protein might have several roles
at different stages and in different tissues but the death of the embryo during early
regulatory processes prevents the study of the following ones. Therefore, conditional
expression systems have been devised, relying on the response to an external
chemical trigger (Danielian et al. 1998; Shin et al. 1999) and/or on the activation
of tissue-specific cis-regulatory elements (Jaenisch 1988). However, these tech-
niques are quite heavy, and alternative methods have been developed to modify
gene expression with high spatial and temporal resolutions (section “Competing
Techniques”). Electroporation is one of them, which enables embryologists to
perform experiments that are basically the same than a century ago but in the context
of functional genomics and with the incredible precision associated with modern
molecular biology – compare Figs. 1 and 2.
Fig. 2 (continued) electroporation. (B3) Fluorescence image displaying the area over which the
GFP is expressed 24 h after electroporation of a mixture of plasmids encoding for the latter
fluorescent protein and for Hoxb-8. The dark spot indicates the injection site where, in this
technique, some oil remains. (B4) Anatomical results after trichloroacetic acid fixation and alcian
green staining at stage HH35-36
Organisms and Tissues
As stated above, the scale of embryology is the one of cell populations and tissues.
Therefore, we will not consider here transfection studies that are performed on
zygotes in order to produce stable lines of transgenic animals. In a similar way,
researches on isolated embryonic cells will be ignored. Conversely, we will take into
account manipulations realized on explants to understand organogenesis. We will
also include experiments related to regeneration (of tail and limb in fishes and
amphibians) and neonatal development (of brain in mouse and rat).
The early implementations of electroporation in developmental biology were
directly inspired by the protocols devised for cell suspensions. Transfections were
mainly performed in cuvette equipped with parallel plate electrodes, relying on
apparatuses providing exponentially decaying voltage pulses. This type of experi-
ments was well adapted to very early stage embryos, containing from one to a few
cells. Among the first scrutinized animals were fishes (Inoue 1992; Buono and
Linser 1992; Powers et al. 1992; Müller et al. 1993; Murakami et al. 1994), insects
(Kamdar et al. 1995; Leopold et al. 1996), and ascidians (Corbo et al. 1997).
Soon after, electroporation was demonstrated on chick embryos in ovo
(Muramatsu et al. 1996, 1997). This advance was made possible thanks to the use
of a generator delivering square voltage pulses of low amplitude and long duration
(section “Material Selection”), which enabled to significantly reduce cell death
(Itasaki et al. 1999; Ogura 2002; Krull 2004; Nakamura and Funahashi 2013).
Naturally, the fact that chick was characterized by a long tradition in microsurgery
as well as in in ovo culture was also important for those initial trials (Momose et al.
1999). Now that this animal model had become amendable to gene expression
manipulation, developmental studies including gain- and loss-of-function could be
performed, especially on neural tissues (Itasaki et al. 1999; Swartz et al. 2001; Odani
et al. 2008). Transfection protocols adapted to other tissues (Oberg et al. 2002; Krull
2004) or to in vitro culture (Iimura and Pourquié 2008; Voiculescu et al. 2008) were
further published.
Two years after the pioneering work of Muramatsu and his colleagues, mouse
embryo electroporation was successfully undertaken (Akamatsu et al. 1999; Itasaki
et al. 1999), once more focusing on nervous system formation. In these in vitro
protocols transfection was followed by whole embryo culture (Takahashi et al.
2008a). However, not all developmental stages could be studied that way – in
particular, it was impossible to obtained living pups. Consequently ex and in utero
electroporation techniques were conceived (Tabata and Nakajima 2001; Saito and
Nakatsuji 2001; Fukuchi-Shimogori and Grove 2001). Finally, the preimplantation
(Grabarek et al. 2002; Soares et al. 2005) and the early post-implantation (Mellitzer
et al. 2002; Davidson et al. 2003; Soares et al. 2008) stages were also transfected
in vitro.
Until now, chick and mouse embryos are the ones that have been the most often
electroporated. These two species, respectively, Gallus domesticus and Mus
musculus, belong to the group of the six most popular animal models in develop-
mental biology, with the zebrafish Danio rerio, the frog Xenopus laevis, the
nematode worm Caenorhabditis elegans, and the fruit fly Drosophila melanogaster

(Slack 2006; Sharpe and Mason 2008). Reports on electroporation of the two latter
organisms can hardly be found in the literature because genetic tools have been
available for long, and establishing a new technique was unnecessary (section
“Typical Experiments”). Conversely, methods to control gene expression were also
eagerly required for the two remaining species, and therefore electroporation was
readily transferred to zebrafish (Tawk et al. 2002; Teh et al. 2003; Cerda et al. 2006;
Hendricks and Jesuthasan 2007; Tawk et al. 2009; Kera et al. 2010) and Xenopus (Eide
et al. 2000; Haas et al. 2001, 2002; Sasagawa et al. 2002; Falk et al. 2007; Lin et al.
2007; Chernet and Levin 2012) after it had been established in chick and mouse.
Subsequently, more confidential animal models could also be transfected by
electroporation. It includes the silkworm Bombyx mori (Moto et al. 1999; Thomas
2003; Ando and Fujiwara 2013), the sea squirt Ciona intestinalis (Corbo et al. 1997),
the freshwater polyps Hydra vulgaris and Hydra magnipapillata (Smith et al. 2000;
Bosch et al. 2002), the axolotl Ambystoma mexicanum (Echeverri and Tanaka 2003,
2005), and the newt Notophthalmus viridescens (Kumar et al. 2007).
As far as in vitro organ electroporation is concerned, the proof of concept was
realized on chick retinal explants (Pu and Young 1990) – in fact it was the first use of
the technique in a developing tissue. Then, the transfection of a whole heart
(Harrison et al. 1998) as well as pieces of gut (Fukuda et al. 2000) and slices of
brain (Hashimoto-Torii et al. 2003) followed. To study organogenesis in mouse,
protocols are now available for the intestine (Abud et al. 2004), retina (Donovan and
Dyer 2006), kidney (Alie et al. 2007), palate (Lee et al. 2008), cochlear (Driver and
Kelley 2010), genital ridge (Tanaka et al. 2014), mandibular arch, tail bud, neural
plate, and lung endodermal bud (Mazari et al. 2014).
Finally, in regenerative biology, electroporation has enabled to better understand
the regrowth of tentacle in Hydra (Smith et al. 2000), spinal cord in Xenopus tadpole
(Lin et al. 2007) and axolotl (Echeverri and Tanaka 2003), limb in newt (Kumar et al.
2007) and axolotl (Echeverri and Tanaka 2005), as well as fin in zebrafish (Tawk
et al. 2002).
Molecular Biology Tools
From the physicochemical point of view, there are only four kinds of molecules that
are electroporated in embryos to perform gain- and loss-of-function experiments: the
plasmids (pDNA) which are double-stranded circular DNA polymers (up to 14,000
base pairs long), the messenger RNA (mRNA) which are single-stranded RNA
polymers (up to 1,500 nucleotides long), the small interfering RNA (siRNA)
which are double-stranded RNA molecules (20–24 base pairs long), and the
morpholinos (MO) which are oligomeric single-stranded analogues of DNA (around
25 morpholino units long). For the sake of simplicity, all these chemical species will
be denoted as nucleic acids in the following, although morpholinos do not have a
phosphate backbone. In fact, the latter oligomers are neutral, and to favor their
delivery to the targeted cells by electrophoresis, they are often conjugated to a
charged dye (section “Performances Validation”). The difference in size character-

izing those four reagents is also noteworthy when one considers the transfection
mechanism: siRNA are small enough to first enter through the pores opened by the
electric pulses and then diffuse freely in the cytoplasm before reaching their biolog-
ical target (Paganin-Gioanni et al. 2011); in contrast, large plasmids form aggregates
at the cell surface (Golzio et al. 2002; Mazari et al. 2014) before being actively
transported to the nucleus (▶ Chaps. 23, “Gene Delivery by Electroporation In Vitro:
Mechanisms,” and ▶ 19, “Nucleic Acid Electrotransfer in Mammalian Cells: Mech-
anistic Description”). Information on mRNA and MO are scarce (Chabot et al.
2013), although processes should be similar to the ones occurring in the siRNA case.
From the biological point of view, the nature of the obtained biological pertur-
bation, i.e., either an up- or a downregulation, is not unambiguously related to the
nature of transfected molecules, as reviewed for chick (Krull 2004; Sauka-Spengler
and Barembaum 2008; Streit et al. 2013) and for mouse (Takahashi et al. 2008a;
Tanaka et al. 2014).
Gain-of-function experiments consist in the overexpression of the gene of inter-
est, either in its wild-type form or in a constitutively activated one. This is commonly
achieved through the electroporation of a plasmid encoding for the corresponding
protein; relying on mRNA is more expensive and thus rarer (Sasagawa et al. 2002;
Cerda et al. 2006; Bansal et al. 2009; Chernet and Levin 2012).
On the other hand, the ways to realize loss-of-function experiments are more
diverse. First, one can use a plasmid that encodes for a dominant-negative form of
the targeted protein, e.g., a transcription factor with an inactive transactivation
domain or a receptor without transmembrane domain. Consequently, the latter
molecule will act as a competitive inhibitor of the wild-type form and perturb the
associated regulatory pathway (Akamatsu et al. 1999; Bartkowska et al. 2007,
Barembaum and Bronner-Fraser 2007). Another common strategy consists in
blocking the transcription of the gene of interest, which is classically achieved
through morpholino electroporation (Mellitzer et al. 2002; Kos et al. 2003). These
nucleic acids are in fact antisense oligonucleotides which are either directed against
the proximal region of the translation initiation site, to sterically inhibit the initiation
complex, or designed to encompass the intron/exon boundaries and thus interfere
with RNA splicing. The last well-established loss-of-function approach involves
siRNA. These nucleic acids operate within the RNA interference pathway, a suc-
cession of several enzymatic reactions catalyzed by various protein complexes, the
final result being that the sequence complementarity between one of the RNA strand
and a given mRNA drives the degradation of the latter molecule and thereby
prevents protein synthesis. siRNA molecules can be introduced as such in the
cytoplasm by electroporation (Mellitzer et al. 2002; Calegari et al. 2002; Pekarik
et al. 2003) or one may have them produced by the cell from a transfected plasmid
(Katahira and Nakamura 2003; Peng et al. 2012). In this case, a promoter for RNA
polymerase III is used, and the sequences are chosen to yield small hairpin RNA
(shRNA) that are then processed as siRNA.
To conclude, we would like to mention that up to now, the CRISPR/Cas9 genome
editing technology has only been scarcely used in embryos in conjunction with
electroporation, except for transgenic line production (Sato et al. 2016). Concerning
developmental biology, knockout experiments have so far been realized in ascidia
(Stolfi et al. 2014; Sasaki et al. 2014), chick (Véron et al. 2015), and mouse
(Tsunekawa et al. 2016).
The differences in mechanism of action outlined above come with differences in
timing of perturbation. In experiments using plasmids encoding fluorescent proteins,
expression is detectable 2–4 h after electroporation, it then reaches a maximum
4–7 days later, and finally it fades within 1–2 weeks (Momose et al. 1999; Teh et al.
2003; Echeverri and Tanaka 2003; Oberg et al. 2002; Davidson et al. 2003; Falk
et al. 2007; Takahashi et al. 2008a; Kera et al. 2010; Sauka-Spengler and
Barembaum 2008; Simkin et al. 2014). However, these numbers may strongly
vary with the cell type: for instance, a signal still measured after 4 months in
postmitotic neurons (Saito 2006). Such an observation thereby indicates that
pDNA displays a fair chemical stability and that the loss of expression is likely
due to the dilution of the vector in the dividing cells. To tackle the latter issue, Tol2
transposon-mediated gene transfer has been devised, which provides a stable inte-
gration into the host genome (Takahashi et al. 2008b; Simkin et al. 2014).
With mRNA, expression can be observed as early as 1 h after electroporation
(Sasagawa et al. 2002). Yet, this technique is more costly and more challenging due
to the poor stability of mRNA and its degradation by cell nucleases. As far as siRNA
are concerned, things are even worst since the dicer enzyme cut them into pieces.
Therefore, to sustain the knockout activity, plasmids permanently enabling shRNA
production are utilized (Katahira and Nakamura 2003; Peng et al. 2012). Finally,
MO are stable but their antisense effect tends to disappear due to the dilution
associated with cell division.
Competing Techniques
Different methods have been developed to introduce nucleic acids into cells, so as to
produce genetically modified animals or to cure hereditary diseases (Kaestner et al.
2015; Sato et al. 2016). Among the ones listed in the first column of Table 1, four
are today routinely used in embryology laboratories: viral infection, lipofection,
microinjection, and electroporation. Incidentally, a few articles report on
sonoporation (Ohta et al. 2003; Skachkov et al. 2014) and on gene biolistics
(Muramatsu et al. 1997; Thomas et al. 2001; Lee et al. 2005). On the other hand,
the Table 1 header gives some of the parameters that have to be taken into account
when selecting a technique. It is here worth noting that protocols are not exclusive.
For instance, lipofection has been coupled to sonoporation (Lee et al. 2005) or to
magnetofection (Svingen et al. 2009); grafting experiments, as described in Fig. 1,
have been performed after transfection of the donor tissues (Iba 2000; Iimura and
Pourquié 2008).
Gene transfer by viral infection has been important in chick embryology, as a way
to cope with the lack of genetic tools characterizing this model (Iba 2000; Sauka-
Spengler and Barembaum 2008). With retrovirus, the transgene is integrated in the
72
Table 1 Comparison of the various methods utilized to introduce nucleic acids in embryos or in embryonic tissues, in order to achieve gene expression
modulation. This synthesis is partly based on the results and discussions presented in the following comparatives studies and reviews: for chick (Muramatsu
et al. 1997; Fukuda et al. 2000; Oberg et al. 2002; Ohta et al. 2003; Scaal et al. 2004; Sauka-Spengler and Barembaum 2008), for mouse (Giroux et al. 2007;
Sakai and Trainor 2014; Tanaka et al. 2014), and for both (Ogura 2002; deCastro et al. 2006; Geetha-Loganathan et al. 2011)
Drawback Timing for
Possible risk for the Transfected Localization overexpression Problematic
Technique Efficacy for the tissue researcher molecules Spatial resolution strategy experiments cell type
Biological transfection
Retroviral High Biological Labor; cost; Insert Initially Injection Start after 12–24 h; Postmitotic
infection toxicity; biohazard <5 kbp 100 umm; Stable cells
mutagenic Ulterior spreading
Lentiviral High Biological Labor; cost; Insert Stable
infection toxicity; biohazard <8 kbp
mutagenic
Adenoviral Variable Labor; cost Insert Transient
infection <8 kbp
Chemical transfection
Lipofection Chemical pDNA; 100 μm Injection Start after 2–3 h;

toxicity siRNA Stop within days
Physical transfection
Gene biolistics Mechanical pDNA 500 μm Focusing Start after 2–3 h; Buried cells
damage Stop within days
Microinjection Good Mechanical Labor All Single cell Injection Small cells
damage
Electroporation High Electrical All Single cell Injection; Mesenchyme
damage focusing
Sonoporation Good Mechanical pDNA; dye 500 μm Injection;
damage focusing
1385
host genome, which leads to a stable expression. However, such initial process is
only possible when cells are dividing. It also significantly delays the onset of
expression. Furthermore, the infectious nature of the method results in spreading
outside the initially targeted area and limits the possibility of subsequent gene
transfer (through a phenomenon called interference). The latter issue becomes
problematic when associated with the reduced space available for the inserts. The
use of other types of viruses has enabled to overcome numbers of the preceding
shortcomings: lentivirus can carry two genes (Semple-Rowland and Berry 2014) and
replication-defective vectors cannot infect neighboring cells (Iba 2000). Despite all
these progresses, the viral infection technique is getting superseded. Indeed, virus
production remains too demanding in time, money, and expertise. Additionally,
stable gene integration can now also be achieved with the other transfection tech-
niques, relying for instance on the Tol2 transposon-mediated gene transfer
(Takahashi et al. 2008b; Simkin et al. 2014).
Chemical and physical transfection protocols are in fact much easier to imple-
ment, from both an economical and a practical point of view: only basic embryology
laboratory equipment is necessary, plus a machine costing less than 15,000 € for
electroporation and sonoporation. These methods are also more polyvalent since
they are not restricted to any organism, or even cell type, for biological reasons.
Similarly, the variety of molecules they can deal with is larger (Table 1). For
lipofection all negatively charged nucleic acids can be used since they all assemble
with cationic lipids to form transfection complexes that will cross the cell membrane
(deCastro et al. 2006). In comparison, electroporation suffers even less limitations
since many molecular species are actually able to diffuse through the electropores
(▶ Chaps. 68, “Fluorescent Indicators of Membrane Permeabilization Due to Elec-
troporation,” and ▶ 19, “Nucleic Acid Electrotransfer in Mammalian Cells: Mech-
anistic Description”). Finally, microinjection is adapted to any kind of marker since
the apertures created in plasma membranes by micropipettes are larger than a
micron. Accordingly, fluorescently labeled polymers like dextrans, proteins like
the horseradish peroxidase, and small cyanine dyes like DiI have all been micro-
injected into embryonic tissues to establish fate maps (Bhattacharyya et al. 2008).
Nevertheless, this technique is quite time consuming and is better suited to target
large cells, as in the classical manipulations on early Xenopus embryos, that involved
mRNA injection (Slack 2006).
To end, the different methods can be compared with respect to their temporal and
spatial targeting abilities (Table 1). In fact, the former property mostly depends on
molecular biology as modern constructs allow the sustained or the conditional
production of both proteins and interfering RNA, independently of the utilized
transfection technique (sections “Molecular Biology Tools” and “Conclusion”).
Concerning the spatial resolution, because some intimate contact between the nucleic
acids and the cells is necessary, locally injecting the reagent always offers an
opportunity to restrict the transfected area to a given tissue. However, molecular
diffusion prevents the precision to be better than 100 μm, as observed for
viral infection and for lipofection. Electroporation further permits to focus the electric
field and to approach the single cell resolution. Interestingly, this limit is more
surely reached when the DNA solution fills a patch-clamp pipette and when
the tip of the latter is in close contact with the plasma membrane: microinjection
driven by iontophoresis and focal electroporation then become surprisingly similar.
Strategies to Spatially Restrict Transfection
Orientation of the Embryo
Whereas electroporation provides a straightforward control of the timing of gene

expression (section “Molecular Biology Tools”), managing the spatial extent over
which transfection is achieved is more complicated. In the following, the case of a
6.5-day-old (E6.5) mouse embryo placed between parallel plate electrodes will be
considered as an illustration (Fig. 3a, b). The latter organism roughly consists of a
closed double-layered epithelium and can be modeled by two concentric prolate
spheroids (section “Setup Conception”): the first one corresponds to the interface of
the embryonic tissues with the buffer and the second one with the proamniotic cavity
content (Mazari et al. 2014; Zhao et al. 2014). Pores will open at the surface of cells
only if the induced transmembrane voltage (ITV) generated upon electric field
application exceeds a given threshold, ITVpore (▶ 59, “Transmembrane Voltage
Induced by Applied Electric Fields,” and ▶ 2, “Critical Electric Field and Trans-
membrane Voltage for Lipid Pore Formation in Experiments”). Since the ITV is
maximal for plasma membrane regions facing the electrodes, it is possible to
selectively transfect a given area by adjusting the orientation of the biological object
(Fig. 3c). However, controlling the extent of the porated zone additionally requires to
tune the amplitude of the voltage pulses, Vanode (Fig. 3d). Unfortunately, this
adjustment may not be easy to achieve because, in comparison with what occurs
on clearly bumped tissues, ITV variations are gentle on flat surfaces. As a conse-
quence, to significantly restrict poration, the ITVpore threshold should not be
exceeded by much, which in turn may result in suboptimal transfection efficiency.
The former example shows how problematic localized transfection may become
when the embryo, or the explant, is simply soaked in the solution of nucleic acids
and when a homogeneous electric field is applied. Therefore, cuvettes with parallel
plate electrodes is mainly used to electroporate early embryos, from zygotes to
cleaving stages and blastocysts (Powers et al. 1992; Buono and Linser 1992;
Corbo et al. 1997; Grabarek et al. 2002; Soares et al. 2005; Peng et al. 2012). Data
analysis is here often performed while taking into account the mosaic nature of the
transfection, and a precise localization of the expression at the surface of those
spherical objects is not a prerequisite. Moreover, cuvette protocols can deal with tens
to hundreds of organisms in parallel and thus are well suited to the better embryo
availability at those stages – it compares favorably with microinjection in terms of
throughput. Despite this general trend of using parallel plate electrodes only for
simple biological objects, studies on larger and more complex organisms or tissues
Fig. 3 Computation by FEM simulations of the ITV at the surface of an E6.5 mouse embryo
electroporated between parallel plate electrodes. (A) Bright-field image of the conceptus and
schematic representation of the corresponding numerical model. The anatomical annotations refer
to the three main tissues, i.e., the epiblast (Epi), the extraembryonic ectoderm (ExE), and the
visceral endoderm (VE), as well as to the proamniotic cavity (PAC) and to the ectoplacental cone
(EPC). The embryo is approximated by two concentric prolate spheroids of respective length and
width 400 and 200 μm for the buffer/VE interface and 300 and 100 μm for the PAC/Epi+ExE one –
see Mazari et al. (2014) for technical details on the used electrical parameters. (B) Schematic top
view of the 4 mm cubic electroporation cuvette with the conceptus positioned at its centered (not to
scale). The embryo revolution axis forms an angle α with the two planes of the electrodes and the
curvilinear coordinate along its largest horizontal cross-section is denoted p. (C) View of the
simulated ITV on the anterior surface of the embryo, for orientations corresponding to α = 15
and 45 . The applied voltage difference, Vanode, was adjusted in order to induce pore formation over
exactly 6% of the total surface of the outer spheroid. The black line delimitates the area where the
ITV is superior to the permeation threshold ITVpore = 0.65 V. (D) ITV as a function of p for various
α, Vanode being tuned as indicated above
have to be mentioned. It concerns whole Xenopus tadpole (Eide et al. 2000),

silkworm brain (Moto et al. 1999), and mouse cochlea explant (Driver and Kelley
2010), as well as chick limb bud, gut, and heart explants (Harrison et al. 1998;
Fukuda et al. 2000; Bobick et al. 2014). In some of these experiments, a simple
orientation of the tissue could yield a fair enough targeting.
Containment of the Nucleic Acids Solution
A first strategy to restrict the size of the transfected area is to inject the nucleic acid
solution in a natural body cavity with a glass micropipette mounted on a microma-
nipulator. The electric pulses are next applied in between parallel plate or wire
electrodes, either set at fixed positions in a chamber or attached to the movable
arms of tweezers. Whatever the configuration, the electric field is roughly homoge-
neous and poration is non-localized. However, gene expression is only modified in
the lining of the cavity containing the molecules to transfect. Incidentally, with such
a technique molecular biology reagents can be used in very small amount, and their
concentration can be quite well controlled.
Brain vesicles are the most commonly harnessed cavities for site-specific deliv-
ery, both in vitro (Calegari et al. 2002; Takahashi et al. 2008a) and in utero (Saito
2006; De la Rossa and Jabaudon 2015) in mouse but also in chick (Momose et al.
1999), zebrafish (Teh et al. 2003; Hendricks and Jesuthasan 2007; Kera et al. 2010),
and Xenopus (Haas et al. 2002). Again in the nervous system, one has relied on the
central canal of the spinal cord in chick (Pekarik et al. 2003) and Xenopus (Lin et al.
2007). Other examples include, at earlier stages, the proamniotic (Soares et al. 2008),
the amniotic (Mellitzer et al. 2002), and the yolk sac (Giroux et al. 2007) cavities in
post-implantation mouse embryos as well as the space between the vitelline mem-
brane and the epiblast (Cui et al. 2006, Iimura and Pourquié 2008, Hatakeyama and
Shimamura 2008, Muramatsu et al. 1996, 1997 – Fig. 2a), the lumen of the neural
tube (Kos et al. 2003; Krull 2004), and the somitocoels (Scaal et al. 2004) in avian
embryos. Likewise, utilization has been made of the subretinal space in mouse pup
(Donovan and Dyer 2006), of the rays of the caudal fin in zebrafish (Tawk et al.
2002), and of the hemocoel (Thomas 2003) and of the body cavity (Ando and
Fujiwara 2013) in silkworm.
Conversely, reports can be found where the injection is performed in the
bulk of a tissue, an approach often necessary when targeting the mesenchyme.
Structures that have been treated this way are for instance the limb bud (Oberg
et al. 2002 – Fig. 2b) and the cranial mesenchyme (Momose et al. 1999) in
chick, the blastema in the regenerating limb of axolotl (Echeverri and Tanaka
2005) and newt (Kumar et al. 2007), as well as palatal (Lee et al. 2008), genital
ridge (Tanaka et al. 2014), and kidney (Alie et al. 2007) explants from mouse
embryos. Mesenchymal cells are reputed hard to transfect by electroporation,
for reasons that may be from both biological and biophysical origins (Bobick
et al. 2014). Still, the low success rate has equally been explained by the
diffusion of pDNA in between the loosely attached cells of this conjunctive
tissue (Krull 2004). Therefore, it has been advised to end the injection with an
oil bolus that should act as a plug, preventing nucleic acids to escape (Oberg
et al. 2002).
Another ingenious method for situations where no cavity is available makes
use of agarose beads that have been soaked in the transgene solution and that are
applied on the targeted site during the electroporation (Hatakeyama and Shimamura
2008; Simkin et al. 2009).
Generation of an Electric Field Gradient
Instead of controlling the place where the nucleic acids are applied, restraining the
area over which cells are porated constitutes an alternative strategy to spatially limit
gene overexpression or knockdown. More explicitly, the molecules to transfect can
now be present everywhere since they will not cross the plasma membrane if the ITV
is lower than the ITVpore threshold.
Such localization effect can only be obtained if the electric potential varies a lot
in close proximity to the targeted tissue. Laying the embryo in between much
smaller electrodes that have been microfabricated on a flat substrate is an option
(Murakami et al. 1994). Nevertheless, the most popular implementation of electric
field focusing relies on tungsten microneedles (Fig. 4a): strong electric potential
gradients are here generated around the highly curved conductors upon charging
(Olofsson et al. 2003). Applications to chick (Momose et al. 1999; Brown et al.
2012) and mouse (Davidson et al. 2003; Quinlan et al. 2008) embryos have for
instance been reported.
To achieve an optimum reduction of the porated area size, the metal tip must be
very close to the tissue. However, chemical species are produced by electrolysis at
the surface of both the anode and the cathode during pulses, which is problematic
(▶ Chap. 91, “Combining Electrolysis and Electroporation for Tissue Ablation”;
Wang and Lee 2013). Indeed, metal ions are directly toxic for cells, whereas protons
and hydroxide ions yield dangerous pH changes. Furthermore, gas like H2, O2, and
Cl2 are also released. Thus, bubbles may form (Cerda et al. 2006 – Fig. 4b), which
can be detrimental to the plasma membrane integrity and which alter the geometry of
the electric field lines. A way to reduce harmful species production is to limit the
current, by lowering the buffer conductivity for instance (▶ Chap. 75, “Parameters
Affecting Cell Viability Following Electroporation In Vitro”). Incidentally, it will
also prevent excessive temperature increase due to Joule heating.
In order to bypass the lethality issues associated with metal microneedles, one can
employ “liquid electrodes,” as it has been done for single cell electroporation
(Olofsson et al. 2003, 2007). More precisely, the electric field is channeled by a
dielectric hollow structure which opening has a size close to the one of the targeted
area. Therefore, the ITVpore threshold is exceeded only locally, before electric
potential gradients weaken in the container welcoming the embryo or the explant
(Fig. 5a). Such dielectric guides result in a focusing effect similar to the one
associated with microelectrodes (Mazari et al. 2014), with the advantage that
harmful species are not generated in proximity to the studied tissue but around
metal electrodes located afar. Applications involving microsystems have been
reported for mouse (Mazari et al. 2014; Zhao et al. 2014) and zebrafish (Bansal
et al. 2009) embryos. Interestingly, the latter article demonstrates that it is possible to
electroporate the surface of a blastula with various arbitrary patterns.
Fig. 4 Examples of strategies used to restrict the number of electroporated cells and to control their
location. (A) Electroporation of the distal tip of an E7 mouse embryo with a tungsten needle. A
platinum plate is used as a counter-electrode, and the conceptus is fully immerged in the DNA
solution (Reproduced from Quinlan et al. 2008). (B) Electroporation of the midbrain of a zebrafish
embryo with tungsten needle electrodes, after microinjection of the molecular biology reagent in the
targeted brain vesicle (Reproduced from Cerda et al. 2006). The white arrow head indicates a
bubble that has formed by electrolysis. (C1–2) Electroporation of a few cells in the distal epiblast of
an E7.5 mouse embryo (Adapted and reproduced from Huang et al. 2015). (C1) The amniotic cavity
is microinjected with the DNA solution (in green), and the electric field is focused at the tip of a
glass micropipette, which is 20 μm in diameter. (C2) Overlay of confocal and bright-field images
displaying the nuclear distribution of the GFP 2 h after electroporation with the corresponding
plasmid. (D1–2) Single neuron electroporation in the Xenopus tadpole brain (Reproduced from
Haas et al. 2001, Haas et al. 2002). (D1) A micropipette filled with the DNA solution (in green) is
introduced into the brain of an anesthetized animal, and electrical pulses are delivered between the
metal wire inserted in the pipette and the external counter-electrode. (D2) Confocal image of an
optic tectal neuron expressing the EGFP 48 h after electroporation with the corresponding plasmid
Combination of both Molecular Localization and Electric Field

Focusing
In order to more robustly restrict the spatial extent over which transfection is
achieved, researchers have simultaneously enforced the two previous approaches
1392
Fig. 5 Prediction of the extent of the electroporated area by FEM simulations. (A1–4) Localized transfection of the anterior visceral endoderm of an E6.5
mouse embryo thanks to a dielectric guide device (Partially adapted from Mazari et al. 2014). (A1) Schematic representation of the embryo in position, with the
targeted cells in front of the 110 μm high and 60 μm large cathodic aperture. The electroporation chamber is filled with the DNA solution (in green). (A2) View
of the norm of the simulated electric field (in log scale) in a cross-sectional plane 70 μm above the chamber surface. Arrows indicate the field direction. (A3)
Confocal and bright-field images displaying the nuclear distribution of Venus 24 h after electroporation with the corresponding plasmid (anterior is to the left).
(A4) Confocal images of the same embryo observed from the anterior side after counterstaining with Hoechst (in blue) and Alexa555-conjugated phalloidin
C. Gosse et al.
(sections “Containment of the Nucleic Acids Solution” and “Generation of an

Electric Field Gradient”). For instance, the nucleic acid solution can be micro-
injected into a cavity (Momose et al. 1999, Cerda et al. 2006, Falk et al. 2007,
Quinlan et al. 2008 – Fig. 4b), or in the bulk of a tissue (Momose et al. 1999; Chernet
and Levin 2012), and strong electric potential gradients generated by a microneedle
electrode.
Likewise, the use of a pipette acting as a dielectric guide can be coupled with the
injection in a body cavity to further improve localization (Huang et al. 2015 – Fig. 4c). Yet,
the most common strategy consists in backfilling the hollow glass needle itself with the
solution containing the molecule to transfect (Fig. 4d). The equipment is here similar to the
one used in patch-clamp and applications mainly concern neural tissues, in Xenopus (Haas
et al. 2001, 2002), axolotl (Echeverri and Tanaka 2003), zebrafish (Tawk et al. 2009), and
chick (Hashimoto-Torii et al. 2003), for instance. Although the formation of a seal does
not seem to be required, the single cell resolution is often reached. Of course with larger
pipette openings more cells are transfected, as demonstrated in Hydra (Smith et al. 2000),
chick (Atkins et al. 2000), and rat (Barker et al. 2009).
Finally, an original approach based on three electrodes has been devised to allow
more flexibility in electric field lines engineering during in utero rat embryo elec-
troporation (dal Maschio et al. 2012). After plasmid injection it then became possible
to target, at the surface of brain vesicles, areas that were poorly addressed in previous
studies (Fig. 5b).
Engineering Practices
Setup Conception
Computation of the ITV is valuable to interpret electroporation experiments when

dealing with isolated cells. Although analytical calculations provide satisfactory
results for simple geometries, finite element method (FEM) simulations are required
for more complicated situations, like the one involving plated cells with irregular
shapes (▶ Chap. 59, “Transmembrane Voltage Induced by Applied Electric Fields”).
Similarly, numerical tools soon become necessary when departing from the case of
the homogenous electric field generated in between parallel plate electrodes, for
instance, when a metallic needle or a dielectric guide is used to yield a strong voltage
Fig. 5 (continued) (in red). (B1–3) Localized transfection of the motor cortex of an E17 rat embryo
thanks to a three-electrode probe (Reproduced from dal Maschio et al. 2012). (B1) Schematic
representation of the in utero electroporation procedure after injection of the DNA solution in the
lateral ventricle (in green in the insert). (B2) Coronal view of the norm of the simulated electric field
(in log scale). Arrows indicate the field direction. (B3) Confocal image of a brain slice displaying
the cytoplasmic distribution of the EGFP 6 days after electroporation with the corresponding
plasmid (the sample was counterstained with Hoechst (in magenta)
gradient (Olofsson et al. 2003, 2007). For more than 15 years now, programs such as
Comsol Multiphysics are thus harnessed to assist engineers in the design of instru-
ments and microdevices dedicated to cell electroporation – see the large majority of
the references reviewed in (Fox et al. 2006, Wang and Lee 2013, Geng and Lu 2013;
▶ Chap. 70, “Electroporation in Microfluidic Devices”). Surprisingly, the use of
FEM simulations have little pervaded in developmental biology, even if the struc-
tural complexity of embryos would make this practice pertinent (Murakami et al.
1994; Huang et al. 2007; dal Maschio et al. 2012; Mazari et al. 2014; Zhao et al.
2014). Two examples detailed in the following will illustrate such an opportunity.
Figures 3 and 5a display studies performed on an E6.5 mouse embryo. This
developing organism has roughly the shape of a hollow spheroid and is made of
three tissues organized in a double-layered epithelium (Fig. 3a). Since GAP junc-
tions electrically connect adjacent cells (Iimura and Pourquié 2008; Mazari et al.
2014), it was possible to only model the interface of the tissues with the electropo-
ration solution on the outside and with the liquid of the proamniotic cavity on the
inside (Fig. 3b). In this approach an epithelium thus behaves as a kind of macro-
scopic cell: without surprise, cuvette electroporation of the mouse embryo yields
results similar to the ones found for spheroidal cells – compare (Fig. 3d) and the data
in (▶ Chap. 59, “Transmembrane Voltage Induced by Applied Electric Fields”).
Despite its simplicity, such a numerical model has also permitted to better under-
stand the working principle of a microsystem far more complicated than a basic
cuvette with two plate electrodes (Mazari et al. 2014 – Fig. 5a). In addition, it helped
to devise an upgrade of this device (Zhao et al. 2014).
Simulations have also been helpful in the conception of a triple-electrode system
dedicated to site-directed in utero transfection of the rat brain (dal Maschio et al.
2012) (Fig. 5b). More precisely, electric field computation has allowed the authors to
orient each of their three plates and to define the applied voltage values necessary to
achieve a well-controlled electrophoretic motion of the plasmid toward a precisely
defined area at the surface of given brain vesicles. The neural tissue was here
modeled with macroscopic electrical parameters, without taking into account any
details present at the cell scale.
Incidentally, calculations are always run in stationary mode since it provides a
very good approximation for pulses that are usually tens of milliseconds long
(▶ Chaps. 2, “Critical Electric Field and Transmembrane Voltage for Lipid Pore
Formation in Experiments,” and ▶ 59, “Transmembrane Voltage Induced by
Applied Electric Fields”).
Although numerical estimates relying on coarse representations of embryonic
tissues agree with experimental outcomes, FEM simulations still need to be backed
up by systematically acquired data to gain in popularity. For instance, measurements
could be performed on simpler systems to validate the electrical properties of the
basic tissular units found in developing organisms, e.g., mesenchymal tissues,
single-layered and stratified epithelia. In this way, one may be able to understand
why particular anatomical tissues, like the mesenchyme, are more difficult to
electroporate (Itasaki et al. 1999; Oberg et al. 2002; Scaal et al. 2004; Krull 2004;
Iimura and Pourquié 2008) and why complex organs, like kidney explants, have
been reported to respond to electrical pulses differently (Alie et al. 2007). Thus, a
deeper knowledge of the biophysics at work would certainly favor the rational
design of more efficient transfection protocols (▶ Chaps. 26, “3D Culture Models
to Assess Tissue Responses to Electroporation,” ▶ 15, “3D Tissue Models to Bridge
the Gap Between Cell Culture and Tissue in Assessing Electroporation,” and ▶ 18,
“Effects of Electroporation of Mammalian Cells on Cytoskeleton and Intercellular
Connections”).
Material Selection
Electroporation of embryos and embryonic tissues is mainly performed relying on

trains of square voltage pulses (section “Organisms and Tissues”). The most com-
monly used machines are the CUY21 and the NEPA21 from NepaGene (Ishikawa,
Japan), the TSS20 Ovodyne from Intracel (St Ives, UK), and the ECM830 and the
ECM2001 from BTX Harvard Apparatus (Holliston, USA) – see the provider’s
webpage for detailed specifications; Echeverri and Tanaka (2003), Krull (2004), and
Cerda et al. (2006) for comparative analyses; and Ando and Fujiwara (2013) and
Bullmann et al. (2015) for instructions on the homemade assembly of cheaper
apparatuses (costing less than 200 €).
Relatively low pulse amplitudes are selected to limit cell death, whereas long
durations allow nucleic acids to reach the targeted area upon electrophoresis
(▶ Chap. 23, “Gene Delivery by Electroporation In Vitro: Mechanisms”). More
precisely, a series of three to six pulses is generally applied. Their amplitude of
course depends on the electrodes geometry and spacing as well as on the tissue shape
and nature (section “Setup Conception”) (Krull 2004; Chernet and Levin 2012);
however, it usually falls within the 10–100 V range. Concerning the length of the
pulses and the dwell time between them, the respective 50 ms and 1 s values
established during early studies seem today consensual (Itasaki et al. 1999); they
consequently represent a good starting point for any optimization in a new biological
system. Larger amplitudes and longer durations will be associated with higher
poration efficiency but also with lower cell, or even embryo, viability (Muramatsu
et al. 1997; Momose et al. 1999; Eide et al. 2000; Echeverri and Tanaka 2003; Teh et
al. 2003; Abud et al. 2004; Hendricks and Jesuthasan 2007; Kera et al. 2010; Mazari
et al. 2014; Simkin et al. 2014; ▶ Chaps. 23, “Gene Delivery by Electroporation In
Vitro: Mechanisms,” and ▶ 75, “Parameters Affecting Cell Viability Following
Electroporation In Vitro”). Note also that more specific adjustments may be required
in the case of organs with a more complicated structure (Alie et al. 2007).
Despite the success of square-wave electroporation, some experiments are based
on other electrical sequences: exponentially decaying voltage pulses are extensively
used on zygotes and on embryos including only a small number of cells (Buono and
Linser 1992; Kamdar et al. 1995; Leopold et al. 1996; Corbo et al. 1997; Bosch et al.
2002); rapid trains of pulses produced with neurophysiological simulators look more
appropriate when utilizing a patch-clamp pipette (Atkins et al. 2000; Haas et al.
2001; Echeverri and Tanaka 2003; Tawk et al. 2009).
Concerning the electroporation setup per se, platinum is the material of choice for
electrode fabrication. Although expensive this noble metal is inert and thus resistant
to corrosion. Furthermore, it is quite soft, which enables one to easily shape foils and
wires in house (Haas et al. 2002; Krull 2004; Scaal et al. 2004; Quinlan et al. 2008).
This latter property obviously becomes a drawback when penetration in a tissue is
necessary. In this case one will favor tungsten, which is harder and which can be
electrochemically sharpened to yield microneedles (Momose et al. 1999; Quinlan
et al. 2008). Options cheaper than platinum include stainless steel and aluminum
(Leopold et al. 1996; Thomas 2003) but these two materials are prone to fast
electrochemical degradation (Wang and Lee 2013). Other noble metals can equally
be considered: silver is utilized to build bulk electrode, whereas gold is only plated.
Finally, standard procedures exist in microelectronics to deposit thin film of platinum
or gold; therefore, those two materials are often used to apply voltage pulses in
miniaturized electroporation devices (Murakami et al. 1994; Huang et al. 2007;
Bansal et al. 2009; Mazari et al. 2010).
Dielectric guides usually consist in a glass micropipette in which a tungsten,
silver, or platinum wire has been inserted (Haas et al. 2002; Echeverri and Tanaka
2003; Tawk et al. 2009; Huang et al. 2015). The former part of the setup can simply
be pulled out from a glass capillary thanks to a microforge (Huang et al. 2015) – for
more complex injection scheme, one can even rely on double-barreled microtubes
(Atkins et al. 2000; Barker et al. 2009). When the structure that focused the electric
field is integrated in a microsystem, as reviewed in Fox et al. (2006), Geng and Lu
(2013), Wang and Lee (2013) (▶ Chap. 70, “Electroporation in Microfluidic
Devices”), options in terms of material are more diverse. Glass is often used, alone
(Zhao et al. 2014) or in association with polymers like photoresists (Mazari et al.
2014), photocurable glues (Mazari et al. 2011), plastics (Huang et al. 2007), elasto-
mers (Falk et al. 2007; Bansal et al. 2009), and/or paraffins (Mazari et al. 2014). In
fact, the choice of a given material is first related to the microfabrication procedure,
itself selected in terms of laboratory know-how as well as of desired spatial resolu-
tion and planed functionality. Eventually, it is compulsory to consider the compat-
ibility between the foreseen experiment and the properties of the material, such as its
biological innocuity (Mazari et al. 2010, 2014) and its wettability (Mazari et al.
2014; Zhao et al. 2014).
To conclude this instrumentation section, it is interesting to discuss the electro-
poration protocol itself. Targeting a given area can be achieved by simple orientation
of the tissue with respect to plate electrodes or thanks to a setup enabling electric
field focusing, e.g., a microelectrode or a dielectric guide (section “Strategies to
Spatially Restrict Transfection”). Whatever the selected option, two main strategies
are available: in the first one the electrodes and their appendages are set in place
around the embryonic tissue; in the second one the position of the biological object is
adjusted within a fixed device. Naturally, some situations enforce a procedure:
tweezers terminated by paddle or needle electrodes are mandatory when surgery is
involved, e.g., when performing in ovo (Momose et al. 1999; Krull 2004; Scaal et al.
2004) or in utero electroporation (Saito 2006; dal Maschio et al. 2012; De la Rossa
and Jabaudon 2015); on the opposite, non-localized electroporation of isolated cells
or of small cellular clusters is simply realized in batch in cuvette (Corbo et al. 1997;
Bosch et al. 2002; Bobick et al. 2014). However, in many cases the choice remains
open. On one hand, manipulating the electrodes allows the experimenter to easily
adapt the protocol to individuals, which can be important when considering the
non-negligible size and shape variability encountered in embryology – especially for
higher organisms or when a lack of accessibility makes it difficult to synchronize the
experimental timing with the developmental one. On the other hand, adjusting the
position of the electrodes can be tricky and thus time consuming (Kos et al. 2003;
Krull 2004; Scaal et al. 2004). Furthermore, relying on a well-defined experimental
device may be preferable since it enables some standardization, synonym of a better
reproducibility – this is especially the case when using microsystems that had been
fabricated with a precision close to the micrometer (Fox et al. 2006; Geng and Lu
2013; Wang and Lee 2013; ▶ Chap. 70,“Electroporation in Microfluidic Devices”).
Reagents Preparation
After plasmids have been produced in bacteria, they must be purified while taking
into account two important recommendations: endotoxin-free kits must be employed
(Scaal et al. 2004; Saito 2006; Mazari et al. 2014) and nucleic acids recovery must be
performed in water (Haas et al. 2002; Krull 2004; Falk et al. 2007; Mazari et al.
2014). In particular, the use of EDTA in buffers is not recommended, albeit contra-
dictory information may be found (Krull 2004; Huang et al. 2007; Odani et al. 2008).
As far as the other nucleic acids are concerned, mRNA are produced by in vitro
transcription, whereas siRNA and morpholinos are chemically synthesized. Hence,
they come clean of harmful contaminants.
The titer in pDNA of the electroporation solution must be at least around 1 μg/μL
(Krull 2004). It can be increased to improve the transfection yield but the benefit will
plateau around 5 μg/μL, i.e. ~1 μM, while the reagent preparation step will become
more laborious (Momose et al. 1999; Haas et al. 2002; Sasagawa et al. 2002;
Davidson et al. 2003, Scaal et al. 2004; Cerda et al. 2006; Voiculescu et al. 2008;
Takahashi et al. 2008a; Kera et al. 2010; Tanaka et al. 2014; Simkin et al. 2014).
Interestingly, coelectroporation is a very efficient strategy to introduce two different
plasmids in the same cells, which can be useful when bi-cistronic constructs are not
available or when some toxicity due to the pDNA size is observed (▶ Chap. 75,
“Parameters Affecting Cell Viability Following Electroporation In Vitro”). Relying
on such protocol, a gene encoding for a fluorescent protein can be transfected with
the nucleic acids of interest in order to precisely locate the modified cells (Calegari
et al. 2002; Oberg et al. 2002; Mellitzer et al. 2002; Takahashi et al. 2008b; Odani
et al. 2008; Streit et al. 2013). This approach is also necessary when working with the
CRISPR/Cas9 system, the many components of which are scattered over several
plasmids (Sasaki et al. 2014; Véron et al. 2015; Tsunekawa et al. 2016). As far as
mRNA is concerned, its typical concentration in the electroporation buffer is
0.5–2 μg/μL, i.e., ~1 μM (Sasagawa et al. 2002; Cerda et al. 2006; Bansal et al.
2009; Chernet and Levin 2012). For siRNA the recommended titer varies between
0.05 and 5 μg/μL, i.e., 3 and 300 μM (Mellitzer et al. 2002; Grabarek et al. 2002;
Pekarik et al. 2003; Soares et al. 2005, 2008; Tanaka et al. 2014), whereas for
morpholinos a consensus exists with values around 0.5–1 mM (Kos et al. 2003;
Cerda et al. 2006; Voiculescu et al. 2008; Tawk et al. 2009; Peng et al. 2012).
Whereas in developmental biology most of the ex vivo cultures are carried out in
media rich in salts, growth factors, and nutriments, electroporation is usually
performed in dilute buffers (e.g., phosphate buffer saline, HEPES buffer saline)
(▶ Chap. 75, “Parameters Affecting Cell Viability Following Electroporation In
Vitro”). Whatever the selected components, relying on low salinity solutions enables
to reduce Joule heating and thus tissue degradation. In the particular case of sea
water organism, such as ascidians, mannitol is used to replace some of the dissolved
ions while keeping the osmolarity constant (Corbo et al. 1997).
The nucleic acid solution may be supplemented with various adjuvants. For
instance, a dye is often employed to facilitate delivery by microinjection close to the
cells to be transfected. Fast green is the far most popular molecule (Haas et al. 2002;
Krull 2004; Saito 2006; Huang et al. 2015; De la Rossa and Jabaudon 2015) but the
use of methyl green (Momose et al. 1999), trypan blue (Pekarik et al. 2003), and
phenol red (Krull 2004; Cui et al. 2006) has also sporadically been reported –
interestingly, some authors have pointed out that fast green may inhibit genetic control
by morpholinos (Kos et al. 2003) or be toxic to embryos (Krull 2004). Other additives
may include carboxymethyl cellulose which, by making the injected mixture more
viscous, prevents its spreading outside of the targeted tissue or cavity (Scaal et al.
2004; Falk et al. 2007). Trans-cyclohexane-1,2-diol has also been mentioned, its role
being to disrupt the selectivity of the nuclear pores and thus to favor the passive
diffusion of plasmids into the nucleus (De la Rossa and Jabaudon 2015).
Performances Validation
In test experiments, transfection success, both in terms of efficiency and localization,

is assessed thanks to plasmids that encode readily detectable proteins. Early on,
enzymes such as the β-galactosidase (Muramatsu et al. 1997, Momose et al. 1999,
Itasaki et al. 1999, Swartz et al. 2001, Oberg et al. 2002 – Fig. 6a) or the luciferase
(Murakami et al. 1994; Muramatsu et al. 1996; Harrison et al. 1998; Sasagawa et al.
2002) were used, the first one producing a colored product and the second one
releasing photons upon reaction with its substrate. Nowadays, most technical
developments are undertaken relying on constructs driving the expression of a
fluorescent protein, like the GFP or the mCherry (Fig. 6b). Those markers are not
only more sensitive and more practical; they also enable live imaging (Fig. 6c). The
onset of expression can even be determined quite precisely provided one utilizes a
fast maturating protein (Shaner et al. 2004; Chudakov et al. 2010). Fluorescence
72
Fig. 6 Utilization of molecular markers to investigate electroporation in embryos. (A) Detection of the β-galactosidase 18–24 h after in ovo electroporation of a
chick embryo at the 8–10 somites stage. The plasmid carrying the LacZ gene had been microinjected between the neural tube and the somites, and parallel wire
electrodes were used (Reproduced from Itasaki et al. 1999). (B) Confocal image of the nuclear distribution of Venus 24 h after cuvette electroporation with the
corresponding plasmid of a D10 bovine embryo. Counterstaining was performed with DAPI (in blue) and Alexa555-conjugated phalloidin (in red). (C1)
Overlay of bright-field and single confocal slice of visceral endoderm cells from a transgenic Hex-GFP-positive E6.5 mouse embryo electroporated at E5.75 in a
dielectric microdevice with a plasmid encoding FusionRed-LifeAct. Hex-GFP (green) marks anterior visceral endoderm cells and FusionRed-LifeAct (red) is an
actin reporter. (C2) Zoom on a dividing cell, extracted from the previous picture. Red fluorescence enables to image cortical actin structures, and cytoplasm is in
green. (D) Incorporation of FITC-dextran in the cells of a D9 bovine embryo after cuvette electroporation. (E) Repartition of a lissamine-tagged morpholino at
1399
starts to be detected 2–4 h after electroporation, as reported in zebrafish (Teh et al.

2003; Kera et al. 2010), Xenopus (Haas et al. 2002; Falk et al. 2007), chick (Oberg
et al. 2002; Sauka-Spengler and Barembaum 2008), quail (Cui et al. 2006), axolotl
(Echeverri and Tanaka 2003), and mouse (Abud et al. 2004).
Nevertheless, one may fail to observe any modification in gene expression upon
electroporation. Several reasons may then be invoked, among which pores that do
not open in the plasma membrane, nucleic acids that do not enter through them, or
transfected cells that die. Each of these issues can in fact be systematically addressed
with the help of appropriate fluorescent probes.
The technique to detect pore formation is the same than the one working for
isolated cells (▶ Chap. 68, “Fluorescent Indicators of Membrane Permeabilization
Due to Electroporation”). It consists in applying the voltage pulses in presence of a
molecule that normally does not enter into the cytoplasm. Dextrans labeled with dyes
such as fluorescein, tetra-methyl-rhodamine, or Texas Red are the most popular
markers (Haas et al. 2002, Bosch et al. 2002, Grabarek et al. 2002, Barker et al. 2009,
Hendricks and Jesuthasan 2007, Bansal et al. 2009, Tawk et al. 2009, Mazari et al.
2014, Zhao et al. 2014 – Fig. 6d). Their molecular weight is usually ~ 4 kDa.
Interestingly, such size also prevents the molecule to diffuse from cell to cell through
GAP junction, thereby allowing one to trace lineage (Stern and Fraser 2001;
Bhattacharyya et al. 2008; Kretzschmar and Watt 2012). Other reporters of poration
include propidium iodide (Harrison et al. 1998; Eide et al. 2000; Barker et al. 2009),
trypan blue (Huang et al. 2007; Bansal et al. 2009), and even quantum dot (Huang
et al. 2007).
Evaluating if nucleic acids enter the targeted cells is the next step in developing an
efficient transfection protocol. The neutral morpholinos are most often tagged with a
fluorescent dye that provides them a charge and consequently enables their transport
by electrophoresis to the tissue to be transfected. As a result they can be tracked
thanks to labels like lissamine (Cerda et al. 2006; Falk et al. 2007; Sauka-Spengler
and Barembaum 2008 – Fig. 6e), fluorescein (Kos et al. 2003; Falk et al. 2007;
Voiculescu et al. 2008; Tawk et al. 2009), or carboxyfluorescein (Kos et al. 2003).
Although it is not necessary to favor their electroporation, siRNA have also been
conjugated to fluorophores, and their presence in embryos could then be monitored
(Soares et al. 2005). Concerning plasmid labeling, the strategy involving
non-covalent ligands of the double helix, such as TOTO-1, is very popular to
investigate electroporation at the cell level (Golzio et al. 2002; ▶ Chap. 23, “Gene
Delivery by Electroporation In Vitro: Mechanisms”). However, it has never been
Fig. 6 (continued) staged HH10 after unilateral electroporation of neural crest and neural pro-
genitors of a chick embryo at stage HH4 (Reproduced from Sauka-Spengler and Barembaum 2008).
(F) Formation of nucleic acids aggregates at the surface of an E5.5 mouse embryo after electropo-
ration in a dielectric guide device with a plasmid that had been previously chemically conjugated to
FITC. (G) Detection of dead cells using propidium iodine uptake and morphological analysis after
electroporation of an E6.5 mouse embryo in a dielectric guide device
experimented in embryos. Instead, utilization has been made of reacting dyes able to
covalently bind to pDNA (Mazari et al. 2014 – Fig. 6f).
The third issue to be studied is whether the cells that have been exposed to the
voltage pulses are still healthy or not. Immediate death by plasma membrane
disruption is suspected when darker areas appear (Huang et al. 2015) or when
cellular morphology is abnormal (Mazari et al. 2014 – Fig. 6g). However, to secure
the diagnosis, a post-electroporation staining procedure can be performed (▶ Chap.
73, “Different Cell Viability Assays Following Electroporation In Vitro”), which
involves dyes that do not cross intact phospholipidic bilayers and that also stain
genomic DNA inside the nucleus, e.g., propidium iodide (Haas et al. 2002, Barker
et al. 2009, Mazari et al. 2014 – Fig. 6g), Sytox Green (Barker et al. 2009), and
RedDot1 (Huang et al. 2015). More complex assays can further be realized to detect
cells entering apoptosis: TUNEL reports on DNA fragmentation (Krull 2004; Falk
et al. 2007; Peng et al. 2012), propidium iodide staining informs on nuclei structure
(Eide et al. 2000), and immunohistochemistry is used to evidence the activation of
the caspase3 signaling pathway (Brown et al. 2012). Yet, the ultimate test is
functional: one has to check if the electroporated cells behave as expected, with
respect to their division rate, their migration route, and their differentiation pathway
for instance (Echeverri and Tanaka 2003; Mazari et al. 2014). Indeed, electropora-
tion is known to alter both the cytoskeleton and the intercellular connections, which
may result in subtle and/or delayed effects on the tissue structure and/or on its
function (▶ Chap. 18, “Effects of Electroporation of Mammalian Cells on Cytoskel-
eton and Intercellular Connections”).
Conclusions
The successful implementation of electroporation in developmental biology may be

attributed to the following characters: the protocols are quite straightforward to set up,
time-saving, low cost, and relatively harmless for the manipulated organisms
(Table 1). It took roughly 15 years of methodological research to be able to introduce
nucleic acids in embryonic tissues in a save and efficient way (section “Organisms
and Tissues”). Meanwhile, molecular biology tools were conceived in order to
perform loss-of-function experiments more easily, consequently adding to the already
existing constructs allowing gain-of-function experiments (section “Molecular Biol-
ogy Tools”). As a result, electroporation is now a mature technique routinely
employed to decipher the workings of the genetic networks involved in the transfor-
mation of a unicellular fertilized egg in an autonomous multicellular organism.
Although the previous assertion holds for the most popular animal models (e.g.,
mouse, chick, frog, zebrafish), the situation is rather different for numerous species
which embryology and functional genomics are less advanced, despite their eco-
nomic importance (e.g., silkworm, cow, sheep, pig, horse). This situation is para-
doxical since electroporation represents an effective methodology to tackle the lack
of genetic tools. However, things should change soon thanks to the large corpus of
studies now available: numerous instruments have been invented that can be
inspiring to design more specific devices (sections “Strategies to Spatially Restrict

Transfection” and “Material Selection”), and clear procedures have been published
that describe how to validate an electroporation protocol or at least how to under-
stand what is going wrong with it (section “Performances Validation”).
Besides those applied issues, a few challenges remain in the field of biophysics.
For example, through the literature it has been reported that mesenchymal tissues are
difficult to transfect (Itasaki et al. 1999; Oberg et al. 2002; Scaal et al. 2004; Krull
2004; Sakai and Trainor 2014). However, the invoked reasons have not been so far
strongly backed up by experimental facts. Although some have raised concerns
about the leaking of the nucleic acids solution out of the injection site, deeper causes
related to the structure of the mesenchyme have been proposed. Indeed, dealing with
cells dispersed into a conjunctive matrix (Itasaki et al. 1999; Krull 2004), or with a
tissue far less electrically coupled than the surrounding epithelia, may be problem-
atic (Oberg et al. 2002). Therefore, more fundamental studies are needed that would
analyze results acquired on massive tissues and on small cellular systems like
spheroids, cysts, and organoids (▶ Chaps. 26, “3D Culture Models to Assess Tissue
Responses to Electroporation,” and ▶ 15, “3D Tissue Models to Bridge the Gap
Between Cell Culture and Tissue in Assessing Electroporation”). These data would
also benefit the conception of more accurate numerical models (section “Setup
Conception”).
Finally, one of the last frontiers that may present opportunities for technological
development is the one of resolution, both in space and in time. As a matter of fact, the
physical tools that have been devised so far have reached their intrinsic limitations. The
patch-clamp pipette technique is already able to transfect a single cell. Similarly, each
molecular biology tool has its own response time (Table 1), which can hardly be reduced
because it depends on how long it takes for the nucleic acids to reach the concerned
cellular machinery and interact with it. Thus, a paradigm shift appears necessary. For
example, controlling the protein synthesis or functionality can be achieved well after
electroporation relying on some external trigger. The latter signal can be molecular, like
in the activation by the 4-hydroxytamoxifen of a Cre/lox-mediated inducible expression
system (Matsuda and Cepko 2007) and by the doxycycline of Tet-On or Tet-Off ones
(Takahashi et al. 2008b). Alternatively, light proves useful when it drives photo-
activation of fluorescent proteins (Stark and Kulesa 2007; Bhattacharyya et al. 2008).
Noteworthily, using photons also allows one to act deep inside organisms, in places that
are normally reluctant to genetic modulation by methods requiring some kind of
physical intimacy between the targeted cells and the nucleic acids (e.g., microinjection,
viral infection, and electroporation). A last approach to manage both the timing and the
location of expression involves the use of tissue-specific cis-regulatory elements; in this
case the protein is only produced when cells engage into a given developmental
pathway (Itasaki et al. 1999; Matsuda and Cepko 2007).
Acknowledgments This book chapter was written in the context of collaborative research projects
funded by the grants “ElectroMice” (CNRS PI: Physique Chimie Biologie 2008), “Electro-
TagMam” (Cnano IdF 2011), “ElectroDev” (PHC Tournesol 2014), and “RhoGTPases and collec-
tive migration” (FNRS MIS 2013).
Cross-References
▶ 3D Culture Models to Assess Tissue Responses to Electroporation

▶ 3D Tissue Models to Bridge the Gap Between Cell Culture and Tissue in
Experiments
▶ Effects of Electroporation of Mammalian Cells on Cytoskeleton and Intercellular
Connections
▶ Electroporation in Microfluidic Devices
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Different Cell Viability Assays Following
Electroporation In Vitro 73
Saulius Šatkauskas, Baltramiejus Jakštys, Paulius Ruzgys, and
Milda Jakutavičiūtė
Abstract
Exposure of cells to high intensity electric fields can lead to death of some cells
due to irreversible electroporation or due to electroporation-induced secondary
effects. If cells are exposed to these fields in the presence of various exogenous
molecules, cell death can also be induced due to their activity in the cells. In all
these cases, mechanisms of cell death as well as the time periods over which death
of these cells occur are highly variable. Therefore, application of different cell
viability assays can lead to inconsistent results, either depending on the assay
itself or the application time. The aim of this chapter is to provide a brief
presentation of viability assays that have been employed in electroporation-
based applications and provide a short discussion on choosing the specific cell
viability assay to properly interpret the obtained results. Since most of cell
viability assays (all tetrazolium salt-based assays, resazurin assay, calcein
acetoxymethyl ester (calcein AM) assay, crystal violet (CV) assay, evaluation
of necrosis and/or apoptosis, etc.) are applied at time points when death of some
cells in the treated population is still in progress, they cannot provide precise
viability results. Therefore, those who seek to evaluate cell viability as precisely
as possible should rely on end-point clonogenic assay. Other cell viability assays
can be applied in parallel with clonogenic assay or at least should be tested in pilot
experiments before they are employed routinely in a specific set of experiments.
Keywords
Electroporation • Cell death • Irreversible electroporation • Cell viability assay
S. Šatkauskas (*) • B. Jakštys • P. Ruzgys • M. Jakutavičiūtė

Biophysical Research Group, Faculty of Natural Sciences, Vytautas Magnus University, Kaunas,
Lithuania
e-mail: s.satkauskas@gmf.vdu.lt; b.jakstys@gmf.vdu.lt; p.ruzgys@gmf.vdu.lt;
m.jakutaviciute@gmf.vdu.lt

DOI 10.1007/978-3-319-32886-7_140
1412 S. Šatkauskas et al.
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1412
Different Routes of Cell Death Exposed to an Electric Field in the Absence or Presence
of Exogenous Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1413
Types of Cell Viability Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1416
Metabolic Activity Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1416
Membrane Permeability Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1418
Colony Formation and Direct Cell Counting Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1418
Assays to Detect Specific Death Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1419
Functional Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1420
Methods of Detection for Cell Viability Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1422
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1422
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1423
Introduction
Electroporation as a physical method that reversibly increase the permeability of

plasma membrane was described in early 1970s (Neumann and Rosenheck 1972;
Kinosita and Tsong 1977) (for review see also (Venslauskas and Satkauskas 2015)).
The method uses short, high voltage electric pulses, which, according to prevailing
hypothesis, lead to formation of transient aqueous pores (see ▶ Chap. 2, “Critical
Electric Field and Transmembrane Voltage for Lipid Pore Formation in Experi-
ments”). These pores allow the delivery of exogenous molecules into the cells or
molecule extraction from the cells. Due to its versatility, electroporation has gained
wide array of application in various biotechnological and biomedical fields; some of
these are electrochemotherapy, DNA electrotransfer (see ▶ Chap. 19, “Nucleic Acid
Electrotransfer in Mammalian Cells: Mechanistic Description”), electrogenotherapy
(see ▶ Chap. 81, “Principles of Electroporation for Gene Therapy”), food pasteur-
ization (see ▶ Chap. 122, “Pulsed Electric Fields for Pasteurization: Defining
Processing Conditions”), pulsed electric field (PEF) treatment for enhanced extrac-
tion of a product from plant raw material or micro algae (see ▶ Chaps. 37, “Selective
Extraction of Molecules from Biomaterials by Pulsed Electric Field Treatment,” and
▶ 121, “Pulsed Electric Fields-Assisted Extraction of Molecules from Bacterial and
Yeast Cells”).
In many electroporation applications, especially in antitumor therapy or gene
delivery, it is crucial to precisely evaluate cell viability. Currently, many different
cell viability assays, varying in their principles and methodologies, are available.
Researchers working in the field of electroporation apply different cell viability
assays, basing their choice on the familiarity with the assay, availability of the
equipment, or time consumption. Some of these assays have been designed over
half of the century ago but are still routinely used in laboratories, including those
working with electroporation; among these assays are the clonogenic assay
(Franken et al. 2006), crystal violet assay (Gillies et al. 1986), trypan blue assay
73 Different Cell Viability Assays Following Electroporation In Vitro 1413
(Altman et al. 1993), and MTT (methylthiazoletetrazolium) assay (Berridge et al.

2005). All of these assays are well known and time tested in laboratory practice.
However, none of these tests are without drawbacks. Clonogenic assay is time
consuming: final results are obtained only 5–7 days after the treatment. Crystal
violet and trypan blue dyes enter both alive and dead cells, given sufficient time, so
swiftness of the procedure must be ensured. In addition, crystal violet assay is
complex to perform and it is difficult to get repeatable, robust results. MTT assay
has gained popularity among researchers due to its easy application and high-
throughput capabilities; however, MTT test results only represent the metabolic
state of the cells but not viability as number of viable cells. Use of the assays based
on metabolic activity of the cells (tetrazolium salt-based assays (MTT, WST –
water soluble tetrazolium, XTT – xanilide tetrazolium), calcein AM assay, and
others) introduce problems in data analysis, since data reflecting the metabolic
activity might be affected by a specific treatment (Li et al. 2012; Jakstys et al.
2015). Therefore, this may lead to inaccuracies when the metabolic activity data is
extrapolated to reflect cell number or viability in general.
Despite methodological differences, many researchers working in the field of
electroporation apply these assays without paying much attention to the fact that
poor choice of the assay can lead to inaccuracy of cell viability estimation. Indeed,
various studies have demonstrated that cell death occurring after exposure of cells to
the electric field in the absence or presence of exogenous molecules is not necessar-
ily immediate and can take place within wide range of time periods after the
treatment (Gabriel and Teissie 1995; Jakstys et al. 2015). Therefore, to get robust
viability results it is important to consider both the assay to be used and the time of
application of the assay.
The aim of this chapter is to provide a brief presentation of viability assays that
have been employed in electroporation-based applications and provide a short
discussion on choosing the right cell viability assay in order to properly interpret
the obtained results.
Different Routes of Cell Death Exposed to an Electric Field

in the Absence or Presence of Exogenous Molecules
Cells exposed to an electric field can follow one of the three routes: (1) remain
unaffected; (2) be affected by reversible electroporation, when pores formed in the
plasma membrane reseal in time, (3) be affected by irreversible electroporation,
characterized by cell inability to restore membrane barrier function (reseal the
pores), leading to short-term cell death. If cells are treated by electric field in the
presence of exogenous molecules (e.g., anticancer drug), cell death can also occur
due to the activity of these molecules (Fig. 1).
In the case of irreversible electroporation, cells do not manage to restore the
membrane barrier function and start dying shortly after the induction of electropo-
ration (Fig. 1, middle pathway). During reversible electroporation (Fig. 1, lower
Fig. 1 Schematic representation of the dynamics of cell death processes taking place after cell
exposure to high-intensity electric field pulses in the presence of anticancer drug bleomycin. Cells
that are unaffected by electric field do not uptake bleomycin and remain viable (upper pathway).
After the induction of electroporation, some cells do not restore their membrane barrier function
(middle pathway, irreversible electroporation) and die shortly (within tens of minutes) thereafter.
Those cells that undergo electroporation but manage to restore membrane barrier function (lower
pathway) die either due to secondary effects induced by electroporation or due to bleomycin activity
inside the cells. Note, that in both cases cells start dying at least 1 h after electroporation. This
progressive cell loss continues at least up to 72 h
pathway) electric field induced pores start to shrink after the field is switched off and
reseal in tens of minutes thereafter (see ▶ Chap. 7, “Lipid Electropore Lifetime in
Molecular Models”). Some of the cells that are able to restore the barrier function of
the membrane start dying at later time periods. Indeed, as reported in Jakštys et al.
(2015) study, after exposure to four 1200 V/cm, 100 μs pulses, some cells that
became impermeable to propidium iodide (PI) 10–15 min after electroporation were
lost as early as 6 h after the treatment. Decrease in cell number continued for a period
lasting up to 72 h (Jakstys et al. 2015). The causes of delayed cell death presumably
are different and dependent on the electrical parameters used to induce electropora-
tion. Cell death after electroporation may be caused by the loss of metabolites,
generation of reactive oxygen species, damage or modification of cytoskeleton
function, or initiation of secondary processes leading to cellular demise (see
▶ Chaps. 17, “Cell Stress Responses to Pulsed Electric Fields,” and ▶ 18, “Effects
of Electroporation of Mammalian Cells on Cytoskeleton and Intercellular Connec-
tions”). Because the causes and timing of cell death due to irreversible electropora-
tion or electroporation-induced secondary effects are different, the observed viability
of the same cell samples can differ depending on the assay itself and the assay
application time. Moreover, membrane permeabilizing nature of electroporation

means that certain viability assays that rely on evaluation of membrane integrity
as a cell viability marker should be applied with caution, taking into account
that complete resealing of transient pores can take tens of minutes. Therefore, if
the assay is applied before the complete resealing of the pores occurs, part of the
cells that are in the process of resealing can be misevaluated as being on late
apoptosis or necrosis. This is just one example to show that the selection of the
assay and especially time of application of the assay is crucial to obtain reliable
results.
Additional level of complexity on the proper evaluation of cell viability is added
when electroporation is performed in the presence of exogenous molecules, like
anticancer drugs or other agents that can alter cell survival (Gehl et al. 1998;
Hansen et al. 2015). If cells take up low dose of the agents, they can remain viable
after membrane resealing (Fig. 1, lowest pathway). However, if cells take up high
enough dose of the agent, they are killed due to its effect. Nevertheless, cell death
induced by these agents is not necessarily immediate. For example, in the case of
the delivery of bleomycin, an anticancer drug most often used in antitumor
electrochemotherapy in clinical trials, reversibly electroporated cells that uptake
the drug are not killed immediately (Jakstys et al. 2015) (Fig. 1, lower pathway).
Indeed, since bleomycin acts as DNA and RNA degradation agent, cell death
occurs progressively after some time of bleomycin activity within the cell. Same
happens after electroporation with another anticancer drug, cisplatin, that is also
used in antitumor electrochemotherapy (see ▶ Chap. 93, “Electrochemotherapy
and Its Clinical Applications”). Since cisplatin or its analogues also induce DNA
damage, cell death is not immediate and therefore the efficiency of the treatment
(the drug alone or in combination with electroporation) in terms of cell viability
depends on selection of the assay application time. Therefore, all assays that rely
on measurement of membrane integrity (like PI assay) are not advisable, since
application of the assay too early can lead to overestimation of cell death (due to PI
diffusion into cytoplasm through the induced pores), while application at later
stages can lead to underevaluation of cell death because some cells may be already
degraded. On the other hand, application of tetrazolium-based assays (e.g., MTT,
XTT, WST-1) that measure metabolic activity of the cells can also lead to mis-
leading results. Since the inhibition of DNA expression or damage to the DNA is
not directly and immediately related to efficiency of MTT conversion to formazan
(Berridge et al. 2005), cell viability estimated via metabolic activity-based assay
can significantly differ from the actual percentage of viable cells. Moreover since
membrane electroporation together with induction of oxidative stress (Gabriel and
Teissie 1995) initiates various processes to restore membrane and cell integrity,
metabolic activity – as well as formazan production – after electrotransfer of
bleomycin, cisplatin, or other cytotoxic compounds can be altered by these pro-
cesses. One more reason why metabolic activity assays can also lack accuracy in
electroporation setting is the fact that these assays evaluate the energetic level of
the cell, which may be affected by the loss of energy-storing molecules such as
ATP (adenosine triphosphate), which has been demonstrated after cell electropo-
ration (Rols and Teissie 1990).
In addition to these widely used methods, other approaches to measure cell
viability can be used. One of these approaches is based on cell enumeration using
manual, semi-automated, and automated methods. Another approach is based on
detection of cell signaling markers related to self-destruction (apoptosis, necroptosis,
autophagy). These assays can provide quantitative as well as qualitative results
related to specific cell death pathway.
Types of Cell Viability Assays
Metabolic Activity Assays
Metabolic activity assays are based on the enzymatic activity of the cells, which is
indicated by conversion of dyes or other markers during metabolic reactions in cells
that causes changes in colorimetric or fluorimetric features of these markers, such as
shift in color, fluorescence, or their intensities.
Tetrazolium salt assays utilizes the conversion of tetrazolium salts into purple-
colored formazan through a series of specific bioreactions involving bioreduction of
tetrazolium salts by NAD(P)H cofactors (Berridge et al. 2005). The color changes
are detected via absorption measurements (colorimetric method). The data is nor-
malized to the control, whose absorption is equalized to 100%, and either normalized
to cell number to measure cell proliferation or expressed as a percentage variation to
evaluate cell metabolic activation or cytotoxicity of the treatment. The method is
praised as being rapid, accurate, and allowing high-throughput.
The best-known of tetrazolium salts, MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide), is a water soluble, cell permeant yellow tetrazo-
lium salt. MTT is reduced by piridine nucleotides (NADH – reduced form of nicotin-
amide adenine dinucleotide, NAD(P)H- reduced form of nicotinamide adenine
dinucleotide phosphate) linked to dehydrogenases in the mitochondria, and in endo-
plasmic reticulum (Berridge et al. 2005) forming purple-colored, cell impermeant
formazan. MTT-derived formazan absorbance can be measured in 550–610 nm wave-
lengths after formazan solubilisation with organic solvents such as alcohol.
WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene
disulfonate) sulfonic acid hydrate is another tetrazolium salt, which, like MTT, can
also be reduced to formazan. However, WST-1 derived formazan has different
spectrum and its absorption maximum is at 450 nm. Unlike MTT, WST-1 is reduced
mainly at the cell surface by dehydrogenases with participation of intermediate
electron acceptors that are reduced by cellular NADH and NAD(P)H and, in turn,
are used by dehydrogenases to reduce WST-1 (Yin et al. 2013). Therefore, WST-1-
derived formazan accumulates in extracellular volume, rendering formazan solubi-
lization (which is required in MTT assay) unnecessary.
MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium) is a compound of tetrazolium salt family that is reduced by electron
coupling reagent, such as phenazine methosulfate, to MTS formazan. MTS-derived
formazan has absorption maximum of 490 nm in PBS (phosphate-buffered saline)
solution. Like WST-1, MTS does not require any additional steps for solubilization
and is convenient for using with adherent or suspension cell cultures.
XTT (2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide
salt) assay is similar to WST-1 and MTS as it also yields water-soluble formazan,
abolishing the step of formazan solubilization (Wang et al. 2011). XTT assay exhibits
higher sensitivity in comparison to MTT. Unlike MTT, inhibition of reduction of
WST-1 and XTT tetazolium salts was observed if mitochondria were present in the
extracts of cells. XTT and WST-1 can be reduced by succinate if mitochondrial
succinate dehydrogenase is present in the medium. This proved that different meta-
bolic pathways are responsible for reduction of different tetrazolium salts (Berridge
et al. 2005). It should be noted that the use of tetrazolium salt assays can introduce
artifacts as the signal is influenced by the pH of the medium as well as glucose content
and other medium characteristics (Marshall et al. 1995).
Resazurin assay. 7-hydroxy-10-oxidophenoxazin-10-ium-3-one, also known
as diazo-resorcinol, azoresorcin, resazoin, alamar blue is water-soluble, non-toxic
compound that passes through plasma membrane by diffusion gradient and is
stable in culture medium. The resazurin assay is based on the action of resazurin
as intermediate electron acceptor. After reduction by NAD(P)H or FADH, blue
nonfluorescent resazurin is converted to pink fluorescent resorfurin (Page et al.
1993). This allows the measurement of the data, which reflects cell health or
viability, either spectrometrically by resazurin absorbance or fluorimetrically by
resorfurin fluorescence. Assay has to be performed in uniform 37 C temperature,
neutral pH (7.0–7.4), on cells in exponential growth phase, shielded from light to
prevent photobleaching of fluorescent resorfurin, which limit the usage of the
assay. However, resazurin assay allows the monitoring of cells repeatedly in time
course.
Calcein AM assay. Calcein acetoxymethyl ester (calcein AM) is plasma mem-
brane permeant nonfluorescent hydrophobic dye. In viable cells, calcein AM is
hydrolyzed by esterases to form hydrophilic, membrane nonpermeable green-
fluorescent calcein. Produced calcein is trapped inside the cells and accumulates,
allowing the estimation of cell viability or compound cytotoxicity. Calcein fluores-
cence can be measured by flow cytometry, multiwell plate reader, or fluorescent
microscopy. Calcein AM assay has several advantages: the procedure takes about
two hours to perform (Fonseca et al. 2006), compound used is not radioactive, assay
can be used for both proliferating and nonproliferating cells, different platforms can
be used to measure the results and high-throughput can be achieved, which made
calcein-AM assay a suitable method for cell viability assessment for different
purposes, such as evaluating multi-drug resistance, electroporation, cytotoxicity,
cell adhesion.
Membrane Permeability Assays
This assay type is employed to sort cells according to plasma membrane permeabil-
ity to specific molecules that are cell impermeant under normal conditions. The best
representative of these assays is propidium iodide (PI) assay.
Propidium iodide assay. Propidium iodide is a substitute for highly volatile
ethidium bromide. Propidium iodide has 1.8 fold higher fluorescence compared to
ethidium bromide; it has excitation maximum at 535 nm and emission maximum at
617 nm. PI stoichiometrically binds to both DNA and RNA (one molecule of PI per
4–5 base pairs), resulting in 20–30 fold increase in PI fluorescence. If only DNA
content is of interest, for example, when evaluating cell cycle, RNA has to be
degraded before measurement. PI cannot cross the plasma membrane of intact
cells but can freely diffuse inside through compromised membrane of necrotic
cells (Jakstys et al. 2015). Therefore, it is used to reflect ratio or quantity of necrotic
cells in a sample.
Propidium iodide is used in electroporation to measure cell membrane perme-
ability after electric pulse exposure. However, the permeabilizing nature of electro-
poration does not allow the use of propidium iodide as dead cell indicator since the
signal from dead cells (PI positive cells) can overlap with propidium iodide influx to
still living cells permeabilized by electric field, leading to possible misinterpretation
of results.
Colony Formation and Direct Cell Counting Assays
Clonogenic assay. The premise of clonogenic (cell colony formation) assay is that
each viable cell remaining after exposure will form a colony (cluster of daughter
cells of the primary cell seeded after the treatment) after sufficient time. This assay
cannot be used with all cell lines as some cells do not form colonies; however, for
cell lines that do, clonogenic assay is a best option for assessment of exact number of
viable, proliferating cells after treatment. Clonogenic assay is attractive due to its
accuracy, which comes from the simplicity and low cost (Franken et al. 2006;
Cepurniene et al. 2010). It only requires considerable amount of growing medium,
appropriate and meticulous dilution of cells to be seeded, and additional space in
incubator. However, this test has three major disadvantages: (1) it is not applicable
for cell lines that do not form colonies; (2) it is impossible to assess the dynamics of
cell loss over time; (3) it takes a long time to get results sufficient for cell viability
assessment: clonogenic assay usually takes 5–7 days, but on slowly dividing cell
types it may take 14 days or more. It is a major goal to find assay suitable for
substituting clonogenic assay that would get accurate results after shorter time, but
up to this day, clonogenic assay is essential tool to evaluate final percentage of dead
cells.
Trypan blue (TB) assay. Trypan blue assay is cell viability assay based on cell
ability to exclude (actively transport outside) the dye if cells are viable and retain the
dye if cells are dying. Trypan blue dye was originally used for tissue staining and
visual evaluation of viable cells by using haemocytometer under microscope. Nev-

ertheless, TB assay has a significant disadvantage: the accuracy of the method
decreases if the measurement of the assay takes more than 3–5 min after trypan
blue addition to cell suspension, as even viable cells start to retain the dye in time
(Altman et al. 1993). Trypan blue assay is used to estimate excessive cell viability
changes due to factors such as prolonged trypsinization, abruption, or treatment with
chemical compounds.
The interest in trypan blue has increased again because of fluorescence properties
of trypan blue. TB turns from nonfluorescent form in aqueous solution to a fluores-
cent compound when trypan blue is bound to biomolecules such as bovine serum
albumine (BSA), proteins, etc. Trypan blue is excited under blue (490 nm) or green
(560 nm) light and has an emission maximum at 670 nm (bright red fluorescence).
Trypan blue is suitable for cell viability determination by fluorimetry, if suitable
concentration of TB for each of cell line is determined in preliminary experiments.
After calibration, TB assay might be exploited for flow cytometry, fluorescent
microscopy, or cell counting using haemocytometer.
Trypan blue assay can be used in tandem with calcein AM and PI to determine
cell viability after electroporation. The premise of this combined assay relies on the
assumption that reversibly electroporated cells are calcein and PI positive and TB
negative, while irreversibly porated (dead) cells are calcein negative but PI and TB
positive.
Crystal violet (CV) assay. This assay was originally designed for histological
staining and detection of Gram-positive bacteria at the end of the nineteenth century.
The molecules of CV pass plasma membrane by simple diffusion and bind to DNA
in the cells. For adherent cell lines, CV is generally used at 0.1% final concentration
and various times of incubation. For this assay, cells have to be attached to the
bottom of the plates when the solution of CV is added. After incubation, CV solution
has to be removed carefully. Crystal violet assay allows high throughput as the
absorption of CV can be read by plate readers in multiwell plate format. The assay
also allows determining cell number at desired time period after exposure. Absorp-
tion spectrum of CV has maximum absorption at 540–610 nm, dependent on the
medium CV is dissolved in, which can be read by most of high-throughput plate
readers. The absorption of the well after subtracting the background (control well
with no cells) is correlated to the number of cells. One of the advantages of CV assay
is that it does not require large number of cells to be seeded in order to statistically
significantly evaluate cell number after treatment, which also increases the high-
throughput capability of the assay (Gillies et al. 1986).
Assays to Detect Specific Death Pathways
These assays employ specific markers to detect different cell death pathways,
namely, apoptosis, necrosis, necroptosis, or autophagy.
Acridine orange/ethidium bromide (AO/EB) staining. For determination of
live, apoptotic, and necrotic cell content in the sample using fluorescent microscopy,
AO/EB double staining can be used. This assay is based on different plasma
membrane permeability of the different dyes. AO stains both live and dead cells.
EB, similarly to PI, can enter only dead cells. Therefore, alive cells have green-
fluorescent nucleus, early apoptotic cells have green-fluorescent nucleus with dense
green spots where chromatin is condensing, late apoptotic cells have orange-
fluorescent nucleus with compact chromatin structures, and necrotic cells have intact
orange nucleus with red EB fluorescence when viewed under a fuorescent micro-
scope (Atale et al. 2014).
Annexin V/PI assay: Annexin V is a 35–36 kDa protein that selectively binds to
phosphatidylserine, a phospholipid that is located on the inner layer of plasma
membrane. During apoptosis, phosphatidylserine is translocated from inner layer
of plasma membrane to the outer layer facing extracellular medium (Wu et al. 2006).
Annexin V protein can be labeled using different fluorescent probes; one of the most
popular is fluorescein isotiocyanate (FITC) probe which emits green fluorescence.
This makes FITC probe convenient for simultaneous usage with PI as the emission
spectra of these two dyes do not overlap. Annexin V/PI assay is based on annexin V
labeling early apoptotic cells and PI labeling necrotic cells, thus detecting both dyes
enables the calculation of ratio between live, apoptotic, and necrotic cells. Apoptotic
cells exhibit green FITC fluorescence, necrotic cells show red PI fluorescence, and
viable cells are both PI and annexin-V negative.
Functional Assays
It is possible to assess whether the cells are suffering or dying by observation under a
light microscope, carried out by trained personnel. There are specific processes that
can be easily noticed by simple observation of the cells. Some of these are blebbing,
swelling, changes in cell shape, inner compartmentation, loss of the plasma mem-
brane integrity, adhesion of cells, etc. (Pehlivanova et al. 2012). However, observa-
tion under a light microscope is only a qualitative indicator of cellular behavior after
the treatment and cannot be used to quantify the ratio of live and dead cells. In
conjunction with these observations, it is advisable to perform ATP assay that
measures ATP level changes in the treated cells that can reflect the observable
changes in cell shape or functions.
ATP assay (Luciferin-luciferase) can be used in tandem with fluorimetric
detection but generally is based on luminescence of luciferin-luciferase reaction.
The assay is conducted lysing the cells, which causes the release of ATP that is used
by luciferase enzyme to cleave luciferin. The luminescence produced by this reaction
is then measured with microplate reader (Cali et al. 2008). Modern ATP assays tend
to use genetically modified forms of luciferase that exhibit lesser loss of signal in
time when compared to wild-type form of luciferase, in which luciferase activity
sharply dropped in a short period of time after initiation of the reaction. However,
ATP measurements to enumerate the cells can be inaccurate as ATP is vital cell
compound that stores cellular energy supplies. The content of ATP in cell can be
Table 1 Comparison of different cell viability assays. For more details see description of the
specific assay
Usual time
between the Approximate time
experiment and consumption for
Name of Basis of the cell viability performing the assay (for
the assay assay results 30 experimental points) Reliability
MTT Metabolic 24–48 h 2–4 h Variable
activity
WST-1 Metabolic 24–48 h 2–4 h Variable
activity
MTS Metabolic 24–48 h 2–4 h Variable
activity
XTT Metabolic 24–48 h 2–4 h Variable
activity
Resazurin Metabolic 24–48 h 2–4 h Variable
activity
Calcein Metabolic 24–48 h 2–4 h Variable
AM activity
ATP ATP 30 min – 24 h <1 h Low
(Luciferin- concentration
luciferase) measurement
PI Membrane 30 min – 24 h ~30 min Low
(propidium permeability
iodide)
Clonogenic Cell 5–7 days ~30 min High
proliferation
Trypan Counting of Within 1 h ~1 h Low
blue cells
Crystal Counting of 24–48 h ~1 h Low
violet cells
Acridine Specific 12–72 h <3 h Variable
orange/ markers/
ethidium membrane
bromide permeability
Annexin Specific 12–72 h <3 h Variable
V/PI markers/
membrane
permeability
affected by various conditions that require the cell to use more energy. In the context
of electroporation, it has been shown that ATP leaks from the cell after electropo-
ration (Rols and Teissie 1990) and can be depleted even further after calcium
electroporation (Hansen et al. 2015; see ▶ Chap. 85, “New Drugs for Electroche-
motherapy with Emphasis on Calcium Electroporation”).
Detailed description of some other cell viability assays can be found in a review
presented by Stoddart (2011). The comparison of different cell viability assay
indicating their advantages and disadvantages is given in Table 1.
Methods of Detection for Cell Viability Tests
Multiwell plate-based systems. Many tests described above, such as tetrazolium

salt assays (Berridge et al. 2005), ATP assays (Cali et al. 2008), and resazurin assays
(Page et al. 1993) can be used in high-throughput manner, utilizing multiwell (96 or
384 well plates) and modern plate readers (Marjanovic et al. 2014).
Flow cytometry. Flow cytometry enables simultaneous distinguishing, enumer-
ation, and sorting of cells based on forward scattering (FSC), which is related to cell
size or side scattering (SSC) that depends on cell granularity and fluorescence in one
or more channels. Thus, an actual number of cells, together with a number of
fluorescent markers, e.g., acridine orange and propidium iodide, calcein AM and
propidium iodide, etc., can be evaluated simultaneously, allowing to assess both
markers of cell death and the number of cells in a defined volume of the sample.
The method of flow cytometry is being used more frequently as time goes by;
however, there are still some hardships. First of all, there is still the need of
development for more affinity-based markers that would allow the evaluation for
more specific processes in cells and discrimination of single cells versus doublets
and triplets. Also, the amount and complexity of data generated by flow cytometry
requires trained personnel to interpret properly. While flow cytometry allows for
simultaneous usage of more than one fluorescent marker, the markers have to be
evaluated closely to make sure there are no interactions or overlaps of fluorescence
spectra between the markers to insure the accuracy of the results.
Fluorescent microscopy. Fluorescent microscopy-based approaches offer the
ability to visualize the fluorescent markers as well as cell morphology, which can
be used to aid the classification of dying cells as apoptotic. However, the use of
fluorescent microscopy techniques to quantify the amount of viable cells requires
tedious work (or sophisticated automated counting techniques). Cells with low level
of fluorescence can be misinterpreted as not being fluorescent due to somewhat low
sensitivity of technique (in comparison to flow cytometry), thus underestimating the
percentage of fluorescent cells (Marjanovic et al. 2014).
Conclusions
Cell treatment with high intensity electric fields alone or in combination with various
exogenous molecules can lead to cell death. Depending on the intensity of the
electric field and/or the type and concentration of exogenous molecules (anticancer
drugs, calcium, nucleic acids, etc.), the mechanisms of cell death as well as length of
the periods of cell population death may vary greatly. This shows the importance of
selecting both the cell viability assay as well as the timing of application of the assay.
For example, since cell death due to electroporation induced secondary effects or due
to activity of the bleomycin is progressive (see Fig. 1), application of MTT assay at
24, 48, and 36 h after cell treatment leads to estimation of different results (Jakstys
et al. 2015). Therefore, it can be concluded that in order to get most reliable cell
viability results, clonogenic assay that measures cell proliferation capability should
be considered in the first order. Other viability assays can be applied in parallel with
clonogenic assay or at least should be tested in pilot experiments before starting them
to use routinely to asses efficiency of special amendment of the treatment.
Acknowledgements This work was funded by Research Council of Lithuania.
Cross-References

Experiments
▶ Effects of Electroporation of Mammalian Cells on Cytoskeleton and Intercellular
Connections
▶ New Drugs for Electrochemotherapy with Emphasis on Calcium Electroporation
Cells
▶ Pulsed Electric Fields for Pasteurization: Defining Processing Conditions
Treatment
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Patch Clamp in Use of Electroporation
Mechanisms Studies 74
Lars H. Wegner
Abstract
Exposure of cells to strong pulsed electric fields shifts the membrane potential to
values far outside the physiological voltage range and elicits membrane
permeabilization, purportedly due to aqueous pore formation (“electroporation”).
These pores allow passage of usually membrane-impermeable solutes, offering
various biomedical applications. In this tutorial essay, the reader is introduced to
patch clamp techniques (both “classical” manual patch clamp and more advanced
automated versions). Benefits as well as potential drawbacks of these techniques
are explained with respect to studying membrane properties at poorly studied
“supraphysiological voltages” that are hardly accessible by any other method.
Using a glass microelectrode, the cellular membrane is homogenously charged to
defined voltages (known as “voltage clamp”). The current response elicited by a
voltage pulse provides information on dielectric properties of the membrane and
on membrane permeabilization by putative membrane pores. Recent scientific
progress in this field is briefly summarized. Analysis of current voltage relations
covering a broad voltage range allows to exactly determine the threshold poten-
tials of pore formation (separately at both positive and negative voltages). In a
more refined biophysical approach, data are interpreted in terms of a transition
between a low-conductance, non-porated and a high -conductance, porated state
of the membrane described by Boltzmann distributions. Ion selectivity of electro-
pores can be probed by polarizing the membrane to induce pore formation and
subsequently shifting the membrane voltage to opposite polarity at a fast, approx-
imately constant rate (voltage ramp protocols). These experiments indicated
cation selectivity of electropores. By using cations of various molecular diameter,
a pore size of about 1.8 nm was extrapolated. Moreover, post-pulse recordings
L.H. Wegner (*)

Karlsruhe Institute of Technology, Institute for Pulse Power and Microwave Technology (IHM),
e-mail: lars.wegner@gmx.net

DOI 10.1007/978-3-319-32886-7_147
1426 L.H. Wegner
with the patch clamp technique revealed that the membrane remains
permeabilized for a long time (in the range of minutes) after field exposure.
Among other things, this effect is amplified by repetitive pulsing, suggesting that
a memory effect is involved.
Keywords
Manual patch clamp • Automated patch clamp • Whole-cell configuration •
Voltage divider effect • Transient electroporation • Persistent electro-
permeabilization • Cation selectivity
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1426
The Patch Clamp Technique: The Original Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1427
The Patch Clamp Technique: More Recent Advances of the Technique . . . . . . . . . . . . . . . . . . . . . 1429
Benefits and Limitations of Using the Patch Clamp Technique in Electroporation
Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1430
Electrical Properties of Cellular Membranes at Supraphysiological Voltages . . . . . . . . . . . . . . . . 1436
Ion Selectivity of Field-Induced Membrane Pores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1439
Electroporation or Electropermeabilization: Acute Field Effects Versus Long-Term
Conductance Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1441
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1445
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1446
Introduction
When aqueous pores are transiently or permanently formed in a lipid bilayer, usually
non-permeant solutes can pass the membrane barrier separating two aqueous com-
partments. In a biomedical context, this refers most likely to the cellular (plasma)
membrane separating the cytosol from the extracellular solution. This effect is
exploited to channel large non-permeant solutes into cells such as the antitumor
drug bleomycin (preferentially) into cancer cells. Pore formation “on demand” is
most conveniently achieved by exposing the membrane to strong pulsed electric
fields (known as electroporation). According to the generally accepted theory (Saulis
2010; Yarmush et al. 2014), field exposure leads to the generation of a large,
nonphysiological voltage difference across the membrane that forces water mole-
cules into the bilayer until membrane-spanning threads of water are formed. Subse-
quently, lipid head groups reorient until lipid-lined membrane pores are formed;
these processes that occur within a nanosecond have been investigated in detail by
in silico studies using molecular dynamics simulations (e.g., Delemotte and Tarek
2012). Experimental verification of models derived from theoretical and simulation
studies of electric field-induced membrane processes turns out to be very difficult,
though. Techniques are required that allow monitoring of membrane properties at a
very high time resolution, ideally at a cellular or even subcellular level. Optical
techniques have frequently been used to visualize membrane permeabilization;
passage of membrane-impermeant fluorescent dyes such as propidium iodide
74 Patch Clamp in Use of Electroporation Mechanisms Studies 1427
(PI) usually provide very high spatial resolution, but time resolution is critical
because a sufficient number of dye molecules has to permeate through the pores to
allow optical detection, and time resolution is restricted by the speed of the camera
(Tekle et al. 1994; but see also Gabriel and Teissie 1999). A good time resolution can
be obtained by monitoring membrane conductance during and/or after the exposure
of cells to pulsed electric fields. Lipid bilayers, including biological membranes,
usually have a low electrical conductance (usually a few mS cm2, depending on the
presence and activity of ion channel proteins). However, the conductance increases
dramatically upon formation of aqueous pores, when ions can pass through them
following the gradient of the electrical field. Therefore, electrophysiological tech-
niques are most suitable for studying changes in membrane properties. Impedance
spectroscopy has been used to monitor membrane conductance changes in cell
suspensions and tissues treated with pulsed electric fields (Ivorra and Rubinsky
2007; Pliquett and Schoenbach 2009). For measuring electric field effects on mem-
brane conductance at the single cell level, the patch clamp technique is the method of
choice.
In the following, both manual and automated patch clamping are introduced to
the reader and technical aspects of using them in electroporation research (which
includes membrane polarization to extreme voltages) are critically discussed. Sub-
sequently, the most important recent results with respect to the effects of pulsed
electric fields on cellular membranes that have been obtained with this technique are
briefly summarized. Finally, an outlook is given on potential future use of the patch
clamp technique in electroporation research.
The Patch Clamp Technique: The Original Design
The advent of the patch clamp technique in the seventies of the last century
revolutionized electrophysiology, since many (small) cells became accessible to
electrical measurements for the first time. Originally, the technique was designed
to record tiny current fluctuations of a few pA generated by single ion channel
proteins in a very small membrane area (“patch”). The challenge was to obtain a
sufficient signal to noise ratio – i.e., to reduce background noise accordingly – and to
eliminate “leak” currents by-passing the membrane. E. Neher and B. Sackmann, the
inventors of this technique, conceived the idea to position a glass capillary on the
membrane surface and to aspirate a tiny membrane bleb (still in contact with the cell)
into the very tip of the capillary by applying slight suction (subatmospheric pres-
sure). It turned out that the membrane surface tends to attach tightly to the inner wall
of the glass capillary forming a high electrical resistance in the GΩ range (termed
“gigaseal”). This process is favored by the presence of divalent cations, especially
Ca2+. Usually, capillaries prepared from borosilicate glass are used for the fabrica-
tion of patch clamp electrodes; for more details, see Hamill et al. (1981) and Walz
et al. (2002). The successful formation of a gigaseal (usually abbreviated to “seal”)
ends up with the so-called “cell-attached configuration” (Fig. 1a). Currents passing
through the small area of the membrane bleb facing the electrolyte filling solution of
1428 L.H. Wegner
a Glass pipette -
‚cell-attached‘ ‚whole-cell‘
‚inside-out‘ ‚outside-out‘
ΔU
Fig. 1 Patch clamp procedures and configurations. (a) Manual patch clamping. A glass pipette is
positioned firmly on the membrane surface of a cell. Subsequently, suction (subatmospheric pressure)
is established in the pipette, and a bleb is sucked into the very tip. The membrane surface becomes
tightly attached to the inner wall of the capillary, forming a gigaohm resistance (“gigaseal”). This
status is called “cell-attached configuration.” From this configuration, the membrane area separating
the cell interior (cytosol) from the pipette lumen can be destroyed by a brief suction pulse to establish a
low-resistance access to the cell interior (“whole-cell configuration”), or the pipette can be removed
mechanically from the cell, leading to a cell-free patch with the physiological inside of the membrane
being exposed to the bath (“inside-out configuration”). A cell-free patch can also be formed starting
from the whole-cell configuration. In this case, the original orientation of the membrane is retained
(“outside-out configuration”). Bottom left bright yellow-2 (BY-2) tobacco culture protoplast attached
to a patch pipette. (b) Automated patch clamping. A cell is positioned on a flat support (“chip”) with a
conical borehole. After establishment of the whole-cell configuration, voltage pulses are applied to the
cell by a rectangular deflection of the voltage difference across the support
the microcapillary can now be recorded. Ideally, a single channel protein is “trapped”
in this membrane area, and spontaneous fluctuations of the currents can be recorded
that reflect the stochastic transitions between (at least) two configurations, “closed”
and “open.”
The first single-channel recordings were a milestone in physiology that earned the
inventors the Nobel Prize in 1991. However, it became soon obvious that applica-
tions of the technique reached far beyond this particular use it was originally
designed for. By breaking the membrane area separating pipette solution and cytosol
(e.g., by applying a suction pulse), the whole-cell configuration can be established
that provides a low-resistance access to the cell interior (Fig. 1a). This configuration
offers the possibility to record ionic currents passing the entire surface of the plasma
membrane, e.g., when a rectangular voltage pulse is applied via the pipette. In the
physiological voltage range (i.e., at voltages close to the intrinsic membrane poten-
tial of the cell), these currents reflect the ensemble of all ion channels and other
electrogenic transporters in the membrane. A subset of ion channels can be studied
separately by arranging the composition of the electrolyte solutions used for filling
the pipette and the bath in a way that deprives “unwanted” channels from their
substrate. Alternatively, selective ion channel inhibitors can be added to the bath; this
aspect is beyond the scope of this review, though. The whole-cell configuration soon
became the most widespread and popular application of the patch clamp technique in
physiology. Single ion channels are nowadays mostly studied on so-called excised
patches, i.e., a membrane patch is removed from the cell. This can be done simply by
dragging the pipette away from the cell (which implies that the cell is immobilized to
its support). It has been shown empirically (and confirmed numerous times since it
was first reported) that the patch is oriented either “inside-out” or “outside-out”
depending on whether patch excision was initiated from the “cell-attached” or the
“whole-cell” configuration, respectively (Fig. 1a). “Inside-out” means that the inner
leaflet of the membrane in the intact cell is exposed to the bath, whereas in the
outside-out configuration the “natural” orientation of the membrane is retained.
The Patch Clamp Technique: More Recent Advances

of the Technique
The “traditional” patch clamping is a relatively cumbersome and time-consuming

procedure – the electrode tip has to be placed on an individual cell under microscopic
control, with the help of a micromanipulator. After the technique became popular at
the end of the eighties of the last century, attempts started to facilitate the procedure
and to speed up the process of seal formation in order to make it suitable for high-
throughput screening. Technical advancement focused on the idea to firmly place a
cell onto a support, called “chip,” with a central small drilling (Fig. 1b). Silicon chips
were tested successfully, but eventually glass chips turned out to be superior. The cell
is attracted onto the support by applying suction via this drilling, and a suction pulse
can be used to produce the whole-cell configuration. A prerequisite is that a gigaseal
between the membrane and the chip has to be established, much as between pipette
tip and membrane in the original design. Indeed that turned out to be the most serious
obstacle to the so-called “automated” or “planar” patch clamping, particularly since
for low-cost screening of cells it was required to sequentially test as many cells as
possible on a single chip to minimize efforts and costs. Among several concepts
1430 L.H. Wegner
presented around the turn of the millennium, the one introduced by N. Fertig and
coworkers (Fertig et al. 2002; Behrends and Fertig 2007) and commercialized by
Nanion Technologies turned out to be among the most successful.
Automated patch clamping aimed at a most convenient and rapid investigation of
cellular electrical properties, e.g., for drug screening. Doubtlessly, this approach
turned out to be very successful, and several versions of automated patch clamp
devices are on the market now. But so far the new technology has not (yet) made the
original patch clamp design with a microglass pipette obsolete, at least not in the
academic world. For scientific purposes that do not work with a high-throughput
approach but rely on an exact control of voltage, manual patch clamping is still
superior to the more recent automated concepts. In this review, the focus will be on
work performed with the original patch clamp approach, using a micropipette
instead of a planar chip.
Benefits and Limitations of Using the Patch Clamp Technique

in Electroporation Research
One way of applying the patch clamp technique in electroporation research is to take
advantage of the whole-cell configuration in order to establish a relatively
low-resistance electrical access to the cell interior and to administer voltage pulses
via this pathway. In fact, the whole-cell configuration is most suitable for studying
the effect of pulsed electric fields on cellular membranes in a controlled way (even
though the other configurations have also been successfully applied, as explained in
more detail below). The counter-electrode is positioned in the bath filled with a
conductive medium in such a way that a shallow, homogenous field gradient is built
up around the cell. The micropipette is likewise filled with a medium of high electric
conductivity. It will equilibrate with the cell interior by diffusion within little more
than a minute (Pusch and Neher 1988). Hence, when a field pulse is imposed via a
chlorinated silver wire inside the microcapillary, the low-conductive cellular mem-
brane is charged homogenously (at least to a first approximation). Electric currents
elicited by a transient shift of the membrane potential reflect dielectric membrane
properties and allow to infer the features of electrogenic transport proteins such as
ion channels and of electric field-induced membrane pores. Since all parts of the
cellular membrane “see” the same voltage (within limits of accuracy), the current
response of a cell directly reflects the properties of the membrane, and a current
density can be calculated by normalizing the current to the cell surface area (gener-
ally well represented by the cell capacitance). In that respect, the technique offers
unique options to drive the membrane potential to values far outside the physiolog-
ical range and to establish extreme, so-called supraphysiological voltages
(as opposed to the range of native membrane potentials generated by electrogenic
membrane transport). However, some limitations and pitfalls of the technique when
being employed in electroporation research have to be taken into account to arrive at
a meaningful interpretation of the data.
First of all it is stating the obvious that (manual) patch clamping is a research tool
only (in a broader sense) that can, however, help in optimizing pulse parameters for
routine treatment. Being a single cell technique, though, it is not directly suitable for
medical treatment.
Secondly, it has to be pointed out that the voltage established in the micropipette
(with the help of sophisticated amplifier technology (Sigworth 1995)) is not identical
with the actual potential drop at the plasma membrane of a cell. Rather, both values
can deviate considerably. Whole-cell patch clamping provides a “low resistance”
access to the cell interior (compared with the resistance of a membrane patch of the
same size), but that does not mean that this resistance (the so-called access resis-
tance, Raccess) is negligible. Raccess is associated with the pipette tip or rather, with the
junction of pipette lumen and cell interior, shaping the effective field pulse that the
membrane is actually sensing in two respects: It defines the time course of membrane
charging (and in turn, the “flank” of the field pulse), and it decides on the amplitude
of the voltage drop at the membrane that can deviate considerably from the one
imposed by the patch clamp amplifier (termed “clamped voltage,” Vclamp; Fig. 2a, b).
When the voltage difference between the microelectrode and the reference elec-
trode in the bath, as imposed by the amplifier, is changed stepwise (implying that a
new steady voltage is established within about 20–50 μs), the change in membrane
voltage will lag behind considerably. To understand this delayed response, it has to
be recalled that the membrane can be represented, from an electrical point of view,
by a capacitor and a membrane resistance acting in parallel (see also analog circuit in
Fig. 2b). The capacitive current response to a voltage step, Icap(t), starting at the
initial current amplitude I0, is best described by single-exponential time course
(Fig. 2a)
I cap ðtÞ ¼ I 0 expðt=τÞ (1)
with the time constant τ being:
τ ¼ Raccess CM (2)
CM is the membrane capacitance. For obvious reasons, Raccess cannot be much

lower than the resistance of the cell-free pipette tip once it is lowered into the bath,
but it can be considerably higher (up to about twofold) due to membrane material or
cellular organelles accumulating in the very tip during and after seal formation.
Assuming Raccess to be about 10 MΩ and with a typical cellular capacitance of 12 pF,
a time constant of 0.12 ms is calculated, and full membrane charging is achieved at
five times the time constant, that is roughly at 0.6 ms. Taking some “safety margin”
into account, it can be concluded that steady state conditions are established after
about 1 ms. This implies that whole-cell patch clamping using standard amplifier
technology is not suitable for studying effects of sub-ms pulses on the cellular
membrane. This is a serious limitation in view of the fact that ultrafast pulses of ns
field exposure (“nanosecond pulsed electric fields,” nsPEFs) have become more and
more popular in recent years, especially for medical applications, since they avoid
a Vclamp versus VM Current response b
1432
capacitive spike Membrane attached

τ = Raccess*CM to glass surface
CM
100 mV 5 nA glass
micropipette
Raccess
RM
Vclamp
5 ms
c d 120 e Electroporated Low-conductance Electroporated

25 KCl state (physiological) state
state
20 120 30 KCl 50
K-gluc
15 −0.4 −400 120
30 K-gluc 40
10 53 nS KCl
5 −236 mV −0.2 −200 30 30 KCl
I (nA)
0 −0.0 0
1.2 nS 20
F-F0/F0
−5 55 nS 172 mV
Gchord (nS)
VM (mV)
−0.2 200
−10 10
−15 −0.4 400 0
−20
−600 −400 −200 0 200 400 600 −600 −400 −200 0 200 400 600 −600 −400 −200 0 200 400
VM (mV) Vclamp (mV) VM (mV)
Fig. 2 Exploration of membrane properties during pulsed electric fields by patch clamp experiments. (a) Effect of short voltage pulses (10 ms) of different
amplitudes on the current response of a BY-2 protoplast. The voltage imposed by the amplifier (Vclamp, dotted lines) is contrasted with the actual voltage drop at
the membrane (VM, continuous lines) for a strongly and a moderately depolarizing pulse (560 and 100 mV clamped voltage, respectively). The latter voltage
L.H. Wegner
heating of biological material. Patch clamp amplifiers usually allow for compensa-
tion of voltage loss at the access resistance. The shape of the applied voltage pulse is
adjusted in such a way that the requested pulse form drops at the membrane, but this
is based on an estimation of the underlying analog circuit and, hence, is not exact.
Moreover, at unfavorable time constants, oscillations are produced. Krassen et al.
(2007) tailored hardware that could cope with drastic changes in membrane con-
ductance around threshold voltages of pore formation (see below). This technical
modification allowed to apply voltage pulses in the sub-ms range (tens to hundreds
of microseconds), but the ns range was still out of reach. Fortunately, evidence is
accumulating (including molecular dynamics simulations) that at the molecular level
there is not too much difference between the effects of field pulses ranging from the
ns to the ms range (as long as the electrical energy remains comparable), so it is
reasonable to expect that the results of patch clamp experiments remain relevant for
field pulse application irrespective of pulse length (Delemotte and Tarek 2012).
When no hardware compensation is used to correct for a voltage drop at the
access resistance, the actual voltage difference across the cellular membrane, VM,
can be calculated using Kirchhoff’s law based on the respective analog circuit. Since
Raccess is in series with the membrane resistance, both act as a voltage divider
(Fig. 2b): The fraction of the clamped voltage (Vclamp) that is actually dropping at
the membrane depends on the ratio of Raccess and RM, the membrane resistance,
according to
Fig. 2 (continued) pulse just induces a transient, capacitive response of the current (lower trace),
whereas the larger voltage amplitude elicits a rapidly decaying capacitive spike (orange box) which
is superimposed on a slower current increase (green box) representing the formation of membrane
pores until a new steady state is reached (arrow). (b) The analogue circuit of a cell clamped in the
whole-cell configuration is depicted. Raccess Access resistance associated with the pipette tip, RM
membrane resistance, CM membrane capacitance (After Wegner et al. (2013), reproduced with
permission by the publisher). (c) Current-voltage relations of a BY-2 tobacco protoplast exposed to
a train of 10-ms pulses in the whole-cell configuration of the patch clamp technique. Pipette and
bath solution were mainly composed of K-Gluconate (K-Gluc) as indicated in the figure (concen-
trations in mM). Note that the current level at the end of the pulse was plotted against the actual
voltage drop across the membrane as calculated by Eq. 4 (After Wegner (2015), reproduced with
permission by the publisher). (d) Membrane voltage calculation based on the voltage divider effect
(b) can be verified by staining protoplasts with the voltage-sensitive dye ANNINE-6. Data for one
representative, stained BY-2 tobacco protoplast investigated in the whole-cell configuration, are
shown. Main electrolyte in pipette and bath solutions was KCl (schematic drawing). Relative
fluorescence measured at 475 nm (solid symbol) and calculated membrane potential (Eq. 4; open
symbol) are plotted against the clamped voltage. Note perfect match of both curves, confirming the
procedure of voltage correction. Insets show the fluorescence signal recorded at the extreme
clamped voltages for this protoplast (Figure obtained from Wegner et al. (2013), with permission
by the publisher. See also there for more experimental details). (e) Plot of the chord conductance as a
function of membrane voltage for a BY-2 tobacco culture cell. Gchord was calculated according to
Eq. 5. Data in the negative and the positive voltage range were separately fitted with Boltzmann
distributions (Eq. 6). Continuous lines represent best fits (After Wegner (2015), reproduced with
permission by the publisher; see there for more details)
1434 L.H. Wegner
RM
VM ¼ V clamp (3)
Raccess þ RM
While Raccess can be considered to be nearly constant across the whole voltage
range, this is not true for RM. In fact, in the non-porated membrane in the physio-
logical voltage range around zero mV, RM is quite high, at least ten times the access
resistance, and, hence, VM deviates only slightly from Vclamp (compare Fig. 2a, b).
However, when the membrane is polarized beyond a certain threshold potential, the
membrane conductance increases strongly (by a factor of up to 100, see more details
below) and RM decreases accordingly. Under these conditions, just about 50% of the
applied voltage actually drops at the plasma membrane. The voltage drop at Raccess
has to be subtracted from Vclamp to arrive at a meaningful value. Raccess is directly
accessible from the current amplitude I0 at an infinitesimal time dt after imposing a
voltage step ΔVclamp. At this time point, the clamped voltage is exclusively dropping
at Raccess. I0 is accessible by fitting an exponential function to the recorded current
relaxation (Eq. 1). With Raccess = ΔV/I0 one arrives at the following expression for
the transmembrane voltage difference:
I ðt Þ
V M ðtÞ ¼ V clamp Raccess I ðtÞ ¼ V clamp ΔV clamp (4)
I0
A typical experiment on a tobacco culture cell protoplast (a plant cell after

enzymatic removal of the cell wall) is shown in Fig. 2a, c. After establishing the
whole-cell configuration, the cell was challenged with a train of 10-ms pulses that
covered a broad voltage range. Positive and negative voltage pulses were applied
alternately. Starting at +/1000 mV, the amplitude decreased by 40 mV in succes-
sive pulses of the same polarity. Between pulses, the cell was clamped to 80 mV
for 20 s. Two current responses are depicted in Fig. 2a; when the membrane was
stepped to a voltage that did not induce pore formation, a capacitive current spike
was elicited that decayed with an exponential time course. Current decreased to a
low level deviating only slightly from the prepulse level. However, when the
membrane was porated, the current response was biphasic: Following the capacitive
spike, the current did not decay to the background level but passed through a
minimum and increased again – this second phase was due to the activation of
membrane pores. By plotting the current level at the end of the pulse against voltage,
a current-voltage relation of the cell could be extracted from these data. Figure 2c
shows a plot of the current level at the end of the voltage pulse against the effective
voltage drop at the membrane as calculated according to Eq. 4. The current-voltage
(IV) curve shows the characteristic varistor-like shape with a drastic increase in slope
conductance upon charging the membrane beyond a threshold potential. Very similar
results had previously been obtained on vacuoles isolated from plant cells (Meissner
1998). Plotting the current against the clamped voltage (instead of VM) would render
a similar result (not shown), but would represent the properties of the cell less
accurately. However, another potential source of error could potentially lead to a
fatal misinterpretation of the data: A sudden increase in conductance at extreme
voltages could also be induced by a (reversible) breakdown of the seal, i.e., the
adherence of the membrane to the pipette could be affected – in this case, one would
be looking at an experimental artefact rather than at authentic membrane properties.
Reversibility of the conductance increase once the voltage is stepped back into the
physiological range provides evidence that the seal was not damaged (although the
possibility remains that the seal restored spontaneously). More direct evidence that
Eq. 4 provides an accurate description of membrane voltage (which would not be the
case if currents induced by strong field pulses were by-passing the membrane due to
a bad seal) is provided by optical membrane potential measurements using the
voltage-sensitive dye ANNINE-6. This dye intercalates into the membrane, and
the spectrum of fluorescence emittance is affected by the membrane potential;
hence, the intensity of the fluorescent light emitted by these dyes when excited by
a certain wavelength is affected by the voltage gradient across the membrane (for
more details, see Silve et al. 2015). In order to test the method, whole-cell patch
clamp experiments like those depicted in Fig. 2a, c were repeated on tobacco
protoplasts that had been stained with ANNINE-6. Figure 2d shows a plot of the
voltage-dependent fluorescence intensity and the calculated membrane potential
(Eq. 4) as a function of Vclamp (amplitude of the applied voltage pulse) for a single
protoplast derived from bright yellow-2 (BY-2) tobacco culture cells. Note that both
curves match perfectly, including the characteristic saturation at voltages beyond the
threshold potential of pore formation and redistribution of voltage among RM and
Raccess (for more details, see Wegner et al. 2013). The voltage-sensitive dye exper-
iment provides an independent test of the interpretation of whole-cell patch clamp
data presented so far. Further support comes from diffusion monitoring of PI into
cells with a high spatial resolution (see further below, Fig. 5).
Another way of using the patch clamp technique in electroporation research has
been employed by Pakhomov and coworkers (Pakhomov et al. 2007, 2009; André
et al. 2010). These authors used external electrodes to impose pulsed electric fields
on single cells; subsequently the whole-cell configuration of the patch clamp tech-
nique was applied to monitor changes in the electrical membrane properties. With
this protocol, the patch clamp technique could be employed to study the effect of
ultrashort (ns) PEFs on cells, but unfortunately not during the pulse – a delay of
70–100 s between nsPEF application and the onset of patch clamp recordings was
unavoidable. Another unfavorable corollary of pulse application by external elec-
trodes is highly inhomogeneous membrane charging (for a review, see, e.g., Saulis
2010). Therefore, whole-cell recordings on field-exposed cells represent an average
of membrane areas that have been exposed to very different field strengths, ranging
from full impact at the cellular poles facing the electrodes to only minor deflections
of membrane potentials at membrane areas oriented perpendicular to the direction of
the external electrical field.
The above considerations refer to the use of the whole-cell configuration, but in
principle the other configurations can also be employed to study electroporation of
biological membranes (Tovar and Tung 1992). However, the cell-attached configu-
ration should be used with care. When pore formation is only elicited in the patch,
the outcome may be affected by the rest of the cellular membrane, which is in series
1436 L.H. Wegner
with the patch and may still have retained its low conductive state. Hence, the
experimental situation is not well defined. Cell-free patches (inside-out or outside-
out) avoid these problems. When “whole-cell experiments” were repeated on these
excised patches, qualitatively similar results to those reported above were generally
obtained (Meissner 1998; Wegner et al. 2011; Wegner 2015). Quantitatively results
could deviate somewhat, possibly because of “unnatural” bending of the membrane
in the patch and a contribution of the membrane attached to the glass surface that
would have a relatively stronger impact on total currents than in whole-cell record-
ings. Therefore, it was assumed that whole-cell experiments would represent cellular
membrane properties in a better way than isolated patches, even though the “voltage
divider effect” would be absent in the cell-free patches (Wegner et al. 2011; Wegner
2015).
Another technical alternative to the approach presented here should also be men-
tioned: In addition to “voltage clamp,” patch clamp amplifiers also allow to control the
current instead (“current clamp”). Controlling (and thus limiting) the current across an
(electroporated) membrane may be a more gentle approach under some conditions, but
here the focus will be on results obtained in the “voltage clamp” mode.
Electrical Properties of Cellular Membranes at Supraphysiological

Voltages
When the cellular membrane is polarized to extreme voltages, its electrical properties
change dramatically, as revealed by current-voltage relations that cover a broad
voltage range (Fig. 2c). At “moderate” membrane potentials, the membrane serves
as an efficient and highly selective diffusion barrier to electrolytes (in accordance
with its biological function), which is associated with a low membrane conductance.
Polarization beyond this intermediate voltage range leads to a dramatic increase in
membrane conductance, presumably due to the formation of aqueous membrane
pores that facilitate electro-diffusion of ions (and other solutes) into and out of the
cell. Wegner et al. (2011, 2015) provided evidence against a prominent role of ion
channel proteins in this process. In BY-2 cells, activity of K+ channels that dominate
the membrane conductance in the physiological voltage range was reduced, rather
than enhanced, by pulsed electric fields, and the K+ channel blocker tetra-
ethylammonium did not affect the outcome. Moreover, mammalian Chinese hamster
lung cell line (DC-3F) cells showed extremely low rates of electrogenic transport in
non-porated membranes, but the response to extreme voltages was nearly identical to
that of the BY-2 cells on which a high channel activity was recorded.
Transition from a low-conductance to a high-conductance “electroporated” state
occurs within a narrow voltage range, most conveniently reflected by defining a
“threshold voltage” at which pore formation is initiated. The low-conductance state
is adequately described by a linear fit of the current-voltage relation at moderate
membrane potentials, and any deviation from linearity (with currents being larger
than calculated by linear extrapolation from the low-conductance range) indicates
that the membrane is polarized beyond the threshold potential and pore formation
Table 1 Comparison of DC-3F cells and BY-2 protoplasts with respect to the conductance increase
induced by supraphysiological voltages (Depol, depolarization; Hyperpol, hyperpolarization)
Tobacco culture cells
DC-3F cells (BY-2)a
Threshold potential (mV) Depol +201 7 (n = 18) +205 14 (n = 9)
Hyperpol 231 8 (n = 18) 273 16 (n = 9)
Conductance increase Depol 8–100 ~56
Hyperpol 6–100 ~52
a
Table from Wegner et al. (2015), reproduced with permission by the publisher
has started. Hence, the threshold potential can be interpolated by successively

stepping the voltage to more extreme values. If the IV curve can be extended into
the electroporated states at positive and/or negative voltages (which is not always
possible, see below), these segments can also be fitted by linear approximation. In
this case, the threshold potential corresponds to the intersection points of linear slope
lines for high- and low-conductance states of the membrane (Fig. 2c and Wegner
et al. 2011). Table 1 summarizes threshold potential values obtained on two types of
culture cells, the BY-2 and DC-3F from plant and mammalian origin, respectively
(taken from Wegner et al. 2015). With BY-2 cells the voltage step required to induce
electroporation was significantly lower with positive- than with negative-going
voltage pulses, and a slight asymmetry was also observed with DC-3F cells in the
presence of Ca2+ in the bath (facilitating seal formation), but not in the absence of
Ca2+ (not shown). One reason for a slight asymmetry of threshold potentials may be
the presence of negative surface charges on the external face of the membrane that is
affected by the Ca2+ concentration in the bath.
The definition of a threshold potential provides a convenient approach to interpret
patch clamp data dealing with electroporation, and it is of practical relevance in an
applied context, e.g., for tailoring PEFs for medical purposes. Arguably though, this
approach is purely empirical and has no molecular biophysical justification. There-
fore, Wegner (2015) reevaluated current voltage data obtained on BY-2 protoplasts
part of which can be polarized to extreme voltages in a fully reversible way. From the
current-voltage curve, the conductance was calculated using an “ohmic approxima-
tion”: For each data point on the current-voltage curve, the current level was divided
by the respective voltage. Zero voltage was set to the potential at which no current
passes through the pores (reversal potential, Erev) that has to be determined sepa-
rately using ramp protocols (see below). This is denoted as the “chord conductance,”
Gchord (in contrast to the “slope conductance” which represents the first derivative of
the IV-curve).
I
Gchord ¼ (5)
V M Erev
In order to facilitate a quantitative evaluation, the background membrane and
seal conductance were eliminated, i.e., Gchord exclusively reflects the activity of
electropores. An example for a plot of the chord conductance of a single
1438 L.H. Wegner
protoplast determined in this way against VM is shown in Fig. 2e. Gchord is zero at
low membrane potentials and increases strongly with both de- and hyperpolari-
zation of the membrane. This behavior can be described quantitatively by
adapting a model that interprets electroporation as a transition between a
non-porated and a porated state adopted from Neumann et al. (1999). Data
were fitted by the following equation (for more details, see the derivation by
Wegner 2015):
Gchord,max
Gchord ðV Þ ¼ (6)
1 þ expððV 0:5 V M ÞSÞ
With the slope factor S being
S ¼ RTd
M
In mV1
Gchord,max is the maximum chord conductance at which the membrane is

completely porated, and V0.5 is the voltage at which 50% of the membrane area is
in the porated state; d is the membrane thickness, R is the gas constant, and T is the
absolute temperature. The parameter M denotes the increase in the dipole moment
during administration of an electric field, due to the intrusion of water into the membrane
(termed “reaction moment” by Neumann et al. 1999). Note that this approach bears
some similarity with the formalism used to analyze the voltage-dependent open prob-
ability of ion channels (e.g., Wegner and De Boer 1999). Qualitatively similar results
were observed for the negative and the positive “branch” of the curve, but fit parameters
differed quantitatively; mean values SEM for n = 6 independent experiments are
given in the following; with depolarization and hyperpolarization, Gchord,max was
40 5 nS and 33 4 nS, and U0.5 was 294 21 mV and 332 14 mV, respec-
tively. The slope was steeper with negative than with positive voltages (0.05
vs. +0.027 mV1). Consequently, a higher value for the reaction moment M was
obtained with hyperpolarization than with depolarization (6.19 0.5 104 C m
mol1 and +3.35 0.37 104 C m mol1, respectively), suggesting that more water
was dragged into the membrane by negative than by positive membrane potentials.
The opposite polarity of the dipole moment change can be explained by an
alignment of water molecules inside the membrane by the electric field. When
the direction of the field is reversed, water molecules (and, hence, the direction of
the dipole) also reorient, following the direction of the field. Interestingly, for
Chinese hamster ovary (CHO) cells, Krassen et al. (2007) and Pliquett et al. (2005)
observed a saturation of conductance-voltage curves only at membrane hyperpo-
larization, but not with depolarization. Instead, positive membrane potentials
induced a progressive increase in membrane conductance. These authors con-
cluded that the membrane responded differently to supraphysiological membrane
potentials, depending on the polarity of the pulse. An alternative explanation could
be that saturation did also occur with membrane depolarization in CHO cells, but
not in the voltage range tested in that study. Limitations, e.g., of seal stability may
have prevented screening of a broader voltage range.
Ion Selectivity of Field-Induced Membrane Pores
Biological membranes are characterized, among other things, by a high selectivity

for solutes, which can be variable and is tightly regulated by physiological require-
ments. While the lipid bilayer is a most effective diffusion barrier for ions and most
uncharged molecules, highly specialized membrane transport proteins mediate the
passage of solutes in a controlled way. It is generally believed that this selectivity is
transiently overcome by the formation of field-induced, aqueous membrane pores.
Consistently, penetration of large organic molecules like the antitumor drug
bleomycin is much facilitated during the exposure of tissues to pulsed electric fields.
However, systematic investigations into the permeability of cellular membranes in
the electroporated state remain rather scarce. Electrophysiological techniques have
been identified as the method of choice to measure the selectivity of membranes for
ions rapidly and with a high precision. The analysis is based on the voltage at which
the direction of currents turns from inward to outward (or vice versa). If just one
permeating ion of positive or negative charge (i) is considered, the reversal potential
depends on the concentration gradient across the membrane as described by the
Nernst equation:
RT ai,out
Erev ¼ ln (7)
zF ai,in
With R, T, and F being the gas constant, the absolute temperature, and the Faraday
constant, respectively; z is the valence of the ion (negative sign in case of anions),
and ain and aout are ion activities inside and outside the cell, respectively. When
several ions are involved (which is usually the case in complex media), the reversal
potential depends on both ion activities and permeabilities p as described by the
Goldman equation (only monovalent anions (an) and cations (cat) are considered):
P P
RT i¼1:::n pcati acati ,out þ p aan ,in
Erev ¼ ln P P i¼1:::n ani i (8)
F i¼1:::n pcati acati ,in þ i¼1:::n pani aani ,out
For prerequisites and limitations of this approach see, e.g., Hille (2001). A more
complicated version has to be used when divalent Ca2+ is included in the analysis
(Wegner 2013). The Goldman equation has successfully been employed to quantify
the selectivity of ion channels. In principle it should be possible to measure the ion
selectivity of field-induced membrane pores in the same way. However, the reversal
potential cannot be obtained from current-voltage curves as those shown in Fig. 2c,
since the electropores are only manifest at extreme voltages and do not contribute to
currents around zero mV. This problem can be overcome by first driving the
membrane to extreme voltages that induce pore formation and then shifting it rapidly
to zero voltage and beyond at a constant rate (so-called “ramp protocol,” slope about
40 mV/ms). A fraction of pores will remain open during the voltage ramp, and ions
can pass through them. The reversal potential is obtained by generating a current-
voltage curve from these data. To this end the current response of the cell is plotted
1440 L.H. Wegner
600
400
200
Vclamp (mV)
1. 2.
0
−200
−400
−600
−800
0 10 20 30 40
Time (ms)
2 120
KCl
30 KCl 2.
Current (nA)
1 −26 mV
1.
0
EK+
ECl−, ECa2+
−1
−60 −40 −20 0 20
VM (mV)
Fig. 3 Probing ion selectivity of electropores with voltage ramp protocols. (a) Symmetrical ramp
protocols (top) and current responses of a cell plotted against the concomitant change in membrane
potential (bottom). Prior to changing the voltage continuously at a rate of 40 mV/ms, the clamp
voltage was adjusted to 100 mV (first ramp, no pore formation) and 600 mV (second ramp,
electroporation induced). Current response to the first ramp (After subtracting capacitive currents as
described in the text) reflects seal resistance and ion transporters in the membrane only, and any
difference between the two curves is ascribed to the contribution of the electropores. Hence, the
intersection potential (arrow) is taken as the potential at which no current is passing through the
pores, corresponding to the reversal potential (Erev.). The reversal potential can be used to calculate
relative ion permeabilities as described in detail by Wegner (2013). Main electrolyte concentrations
in mM indicated in the figure
against the concomitant change in membrane potential (that can be estimated from
the clamped voltage as described above). It should be noted that the current response
to this ramp protocol includes a capacitive component reflecting the dielectric
properties of the membrane that has to be subtracted from the raw data to retain
the “resistive component” of the current response that renders the aspired value for
Erev. In order to exactly determine Erev, two ramp protocols are successively applied
(Fig. 3): the first one reflects the seal resistance and ion transporters in the probed
membrane, whereas the second one includes the activity of electropores; Erev is
obtained from the intersection point of the two IV curves (Fig. 3). Details are beyond
the scope of this essay but can be taken from previous publications (Wegner et al.
2011; Wegner and Schönwälder 2012; Wegner 2013). A reversal potential of zero
mV (irrespective of the ion gradients across the membrane) would indicate utterly
non-specific pores. Unexpectedly, though, the reversal potential of electric field-
induced membrane pores obtained from ramp protocols deviated significantly from
zero; data analysis suggested an about six times higher selectivity for K+ compared
to chloride or gluconate. This was explained by negatively charged head groups of
lipids lining the pores, and at the mouth of the pore, facilitating passage of cations
in comparison to anions. For a transfer of ions from the bulk phase to the
membrane surface, work against the Born energy has to be performed that is
lowered by fixed negative charges for cations, but increased for anions. Entrance
of cations is also preferred over that of anions by negative charges at the head
groups lining the pore. Observations on short-lived membrane electropores are
also in close agreement with results obtained on permanent nanopores etched, e.g.,
into polyethylene terephthalate (PET) foils (Siwy et al. 2002). Selectivity is a
matter of charge, but also of the size of the cation. This was revealed by a detailed
study using various inorganic and organic cations (Wegner 2013). Permeability of
the field-induced pores to small cations like Li+ and Ca2+ was significantly higher
compared to large organic cations such as tetraethylammonium (TEA+) and tetra-
butylammonium (TBA+). This finding could be used to estimate the effective pore
diameter: The relative permeability (normalized to that of Rb+) is negatively
correlated with the ionic diameter. An apparent pore diameter of +1.8 nm was
extrapolated from a cumulative plot of relative permeability data (not shown here;
for more details, see Wegner 2013). It is interesting to note that the permeability of
the membrane to Li+ was roughly two times that of K+. Consistently, the ionic
diameter of Li+ is much smaller than that of K+ (0.12 nm vs. 0.27 nm). However,
the hydrodynamic diameter of Li+ is considerably larger than that of K+ (0.48
vs. 0.25 nm). This observation provides evidence that the hydration shell of the
ions is stripped off while passing through the pore.
Electroporation or Electropermeabilization: Acute Field Effects

Versus Long-Term Conductance Changes
So far, this review focused on changes of electrical membrane properties that

occurred during the (extremely short) exposure to an electric field, and no attention
was paid to effects that lasted after the end of the pulse. But from an applied point of
view, these persistent effects may be more relevant. This is, e.g., indicated by the
finding that cell mortality was much increased compared to SHAM controls when
the membrane-impermeable drug bleomycin was added to cell cultures shortly after
cells had been exposed to PEFs, suggesting that the membrane remained
permeabilized for some time (Silve 2011). The fraction of cells that survived the
treatment increased with the time increment between the end of the pulse and the
administration of the drug. First direct evidence for changes of the membrane
conductance that prevailed after the end of nsPEF application was provided by
1442 L.H. Wegner
a 0.6
0.3
4 0.0
−0.3
2 −0.6
Current (nA)
−200 −100 0 100 200
0
120
KCl −2 1
2
3
PBS medium 4
−4
+ 0.1 CaCl2
−300 −200 −100 0 100 200 300
Trans-membrane voltage difference (mV)
320 mV
b
80 mV
0 mV
Specific membrane conductance
2.5
2.0
(nS/pF)
1.5
1.0
0.5
0.0
2 4 6 8 10 12 14 16 18 20
Pulse number
Specific membrane conductance
3.0
2.5
2.0
(nS/pF)
1.5
1.0
0.5
0.0
2 4 6 8 10 12 14 16 18 20
Pulse number
Fig. 4 (continued)
Pakhomov’s patch clamp studies on growth hormone 3 (GH3) and CHO cells
(Pakhomov et al. 2009). Post-pulse whole-cell IV curves were characterized by a
pronounced increase in membrane conductance compared to control cells. Fre-
quently, current-voltage curves recorded in the physiological voltage range after
exposure to pulsed electric fields displayed a characteristic inward rectification (i.e.,
the membrane conductance decreased when the membrane potential was shifted
from negative to positive voltages). Miniature current fluctuations reminiscent of ion
channel activity were observed. Trivalent cations (e.g., Gd3+) tended to block these
current fluctuations and stabilized the membrane after field exposure. Unfortunately,
this approach did not allow to link post-pulse changes in membrane conductance to
processes induced during the application of the pulse. This aspect was investigated
more recently by Wegner et al. (2015) in a patch clamp study performed on DC-3F
cells, another mammalian cell type that is frequently used as a model system in
electroporation research. Cells were clamped in the whole-cell configuration, and the
current response of the membrane before, during, and after the administration of
voltage pulses that induced the formation of electropores was recorded. Before
treatment, gigaohm resistances were frequently recorded even after the whole-cell
configuration was established, indicating very low ion permeability of the membrane
due to the apparent absence of any ion channel activity. Apparently, the response of
the cell to extreme voltages largely reflected the response of the lipid bilayer,
potentially facilitating the interpretation of the data (compare Wegner et al. 2011).
A strong increase in membrane conductance was observed when the membrane was
hyper- or depolarized for 5 or 10 ms beyond threshold potentials of about 230 and
+200 mV, respectively (Table 1). In most of the clamped cells, field-induced pore
formation (termed “transient electroporation”) was fully reversible (membrane
resealing within ~100 ms) when one pulse or a few pulses slightly exceeding the
threshold potential were applied, and identical results were obtained when the same
pulse protocol (consisting of pulses trains with alternating polarity and increasing
Fig. 4 Repetitive exposure of DC-3F cells clamped in the whole-cell configuration to pulsed
electric fields applied via the pipette: Evidence for a memory effect. (a) Current-voltage relations
obtained in four successive runs (different symbols, see legend in figure) of the same pulse protocol
that includes membrane polarization to voltages slightly exceeding the threshold potential of pore
formation. Basic information on pipette/bath medium given in the figure, numbers denote concen-
trations in mM. Inset: detail of the curve showing the shift in the slope of the current-voltage relation
in the physiological voltage range with third and fourth repeat of the same pulse sequence. The
conductance increased at least twofold with respect to the first two sweeps (Reproduced from
Wegner et al. (2015), with permission by the publisher). (b) Effect of repetitive administration of the
same, depolarizing voltage pulse (see schematic drawing of the pulse sequence; not to scale) on the
membrane conductance. A depolarizing voltage pulse of 320 mV clamp voltage was imposed for
10 ms, followed by a clamp to 80 mV for 25 or 100 ms. Subsequently, the membrane was stepped
back to 0 mV for 5 s before an identical pulse was applied. The membrane conductance at the
physiological voltage range was calculated from the difference in steady-state current level induced
by stepping the voltage from 80 back to 0 mV. Two examples were selected to reflect the variability
among individual cells. The conductance increased stepwise (dotted lines), either in several steps
(upper plot; see pulse 3, 4, and 10) or in one, single step (lower plot; pulse 15)
1444 L.H. Wegner
a −1:28 + 0:57 +1:41 + 4:56 + 7:29 + 8:53
6000 7000 8000

t=0
120
KCl 100 mV
PBS medium b
+ 0.1 CaCl2
Specific conductance (nS/pF)

1.0
20 nA 0.5
Prepulse specific
conductance: 0.07 nS/pF
0.0
00:00 00:07 00:14 00:21 00:28 00:36 00:43
Time after poration pulse (hh:mm)
2 ms
Fig. 5 Persistent membrane permeabilization of DC-3F cells by a single, strong depolarizing

voltage pulse. (a) Sequence of images showing propidium iodide (PI) fluorescence before and at
several intervals after pulse application. Time is given in minutes relative to pulse application.
Fluorescence intensity is visualized by pseudocolors (see scale bar, arbitrary units). The voltage
pulse (dotted line clamped voltage, continuous line actual transmembrane voltage drop) and the
current response of the membrane are also depicted below. Basic information on pipette/bath
medium given in the figure, numbers denote concentrations in mM. Initial fluorescence increase
(arrow) did not coincide with the position of the pipette, indicating that PI did not enter the cell
because of poor contact between membrane and glass (i.e., a bad seal) or membrane damage at the
contact site of the pipette. Spots of high fluorescence intensity are associated with the position of the
nucleus and started to appear with a delay of about 5 min after pulsing (For more details, see Wegner
et al. (2015)). In (b), long-term changes of membrane conductance (normalized to cell capacitance)
of a different cell challenged with the same type of pulse is shown. Membrane conductance
remained elevated for more than 40 min after the pulse, but decreased slightly with time (After
Wegner et al. (2015), reproduced with permission by the publisher)
amplitude; increment about 40 mV) designed for probing the threshold potentials
was imposed a second time (Fig. 4a). However, when this protocol was repeated a
third or fourth time, or single pulses of higher amplitude were imposed, membrane
properties tended to undergo a pronounced change: The membrane conductance
started to increase over the whole voltage range, including physiological voltages
that tended to exhibit a high membrane resistance. This was apparently due to the
formation of persistent membrane pores that pertained after the electric field
disappeared, with properties similar to those described by Pakhomov et al. (2009).
Monitoring uptake of the fluorescent dye PI into those persistently porated cells at a
high spatial resolution revealed that diffusion into the cell was not initiated at the site
where the pipette contacted the membrane (Fig. 5a), providing evidence for a “true”
membrane phenomenon, rather than a mere patch clamp artefact. In order to study
this feature in a more systematic way, pulse trains of identical amplitude
(VClamp = 280–360 mV) were applied (Fig. 4b; porating pulse length 10 ms,
between successive pulses the membrane was clamped to 0 mV for 5 s). Results
differed depending on membrane polarity: While hyperpolarizing pulses induced a
gradual increase in membrane conductance with pulse number until a maximum was
obtained (not shown), depolarizing pulses were associated with a stepwise increase
in membrane conductance. The number of pulses of positive polarity preceding a
particular conductance step differed considerably and was non-predictable. Persis-
tent electropermeabilization could also be achieved by a single, strong voltage pulse
(Fig. 5). The permeabilized state prevailed for 40 min and longer. Apparently, the
patch clamp technique is well suited for separating transient and persistent effects of
pulsed electric fields; the latter is probably more relevant for practical applications,
e.g., for channeling usually membrane-impermeable substances into cells.
The dual response of cellular membranes to pulsed electric fields was also
recently investigated in a theoretical study by Leguebe et al. (2014). These authors
distinguished between (transient) electroporation which is ascribed to the direct
physical impact of the field on the membrane, and the (persistent) electro-
permeabilization, that is explained by a more enduring chemical modification of
the lipids, possibly an oxidation of fatty acid chains. Further experimentation is
required to test this hypothesis in more detail.
Conclusions
The patch clamp technique has been firmly established as a most sensitive method to
monitor effects of PEFs on (biological) membranes on a single-cell basis and to
study membrane properties at supraphysiological voltages. So far, it has been used as
a tool in basic research, but the technique may also prove useful in designing cell-
specific pulse protocols in medical applications, e.g., in electrochemotherapy. For
this purpose, new protocols are required to optimize the efficiency of the technique
and to reduce the effort that is needed to obtain the desired information. Automated
patch clamping may prove useful in that respect, but the range of cell types that can
conveniently be studied with this technology is still limited.
A further severe limitation of the technique is still associated with the minimum
pulse length required to charge the membrane until a more or less stable voltage is
established. With standard patch clamp techniques, pulses of ms length are required;
the time constant τ that governs the charging process depends on membrane
capacitance and access resistance (the resistance of the pipette tip contacting the
membrane). Reducing one of them (or both) by orders of magnitude seems to be a
challenging (if not impossible) task. To circumvent this problem, the field pulse can
in principle also be applied via the external electrode, either by using 3-D arrays of
microelectrodes that were originally designed to establish field cages, or by using
fluid electrodes that ensheath the cell, separated from the membrane by a thin
fluid layer.
1446 L.H. Wegner
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annurev-bioeng-071813-104622
Parameters Affecting Cell Viability
Following Electroporation In Vitro 75
Marie-Pierre Rols
Abstract
Cell membrane permeabilization to nonpermeant molecules can be obtained by
applying electric field pulses, which parameters have to be adapted to each cell
type according to the purpose of the experiments. Electropermeabilization is
associated with an uptake or release of molecules between external medium and
cell interior and may cause cell death. Roughly speaking, any parameters that will
increase either the size of membrane defects, leading to the permeable state of the
cell membrane, or their lifetime, regulating the amount of exchanged molecules,
will have severe effect on cell viability. However, other parameters, not directly
correlated to the electric pulses conditions, can also have dramatic effect and
jeopardize cell viability. Indeed, the ways experiments are conducted (tempera-
ture, pulsing buffer, electrode material) and the characteristics of the cells (size,
shape, orientation, ATP content, normal primary cell or cancer cells, sensitivity)
have direct consequences on membrane permeabilization and cell viability. Short-
term direct effects, usually associated with strong electric pulses, and long-term
effects, linked to the ability of the cells to repair or not the damages, can be
present, which all have consequences not only on cell survival but to cell
responses to electric pulses. Optimization of the protocols of electroporation,
according to the objectives of the experiments, is therefore a necessity.
Keywords
Electroporation • Cell death • Cell viability • Electric pulses • Cell sensitivity
M.-P. Rols (*)


DOI 10.1007/978-3-319-32886-7_149
1450 M.-P. Rols
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1450
Basics Aspects of Electropermeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1452
Cell Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1453
Cell Permeability Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1455
Cell Viability Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1455
Effects of Physical Parameters on Cell Viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1456
Orientation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1456
Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1456
Electrochemical Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1457
Electroporation Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1458
Presence of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1458
Effects of Cell Parameters on Cell Viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1459
Size and Shape Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1459
Normal Versus Tumor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1459
Reactive Oxygen Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1460
Bystander Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1460
Some Tips to Preserve Cell Viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1461
Addition of Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1461
Addition of Poloxamer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1461
Cell Centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1461
Cryopreservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1462
Mathematical Models to Predict Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1462
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1462
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1464
Introduction
Electroporation-based treatments are powerful medical and biotechnological tools.

They are based on the use of well-defined electric field pulses that can cause the
reversible or irreversible permeabilization of the cells. Reversible electropermeabi-
lization of the cell membrane is used for electrochemotherapy and gene therapy,
allowing to deliver cytotoxic drugs or plasmid DNA into tissues. The aim is to
permeabilize the cell membrane while preserving the cell viability. Irreversible
electropermeabilization resulting in cell death can be useful and is under clinical
evaluation for treating tumors. Electroporation is also of high importance in food
industry and bioeconomy. Pulsed electric fields (PEF) and high-voltage electric
discharges can efficiently assist extraction of bio-compounds from fruits, grapes,
and crops (▶ Chap. 134, “Pulsed Electric Field Treatment for Fruit and Vegetable
Processing”). They can also be used to inactivate microorganisms.
Electropermeabilization, also named electroporation, does not just consist on
“pushing a button on a generator” but can be controlled by a number of parameters
such as the electric field strength, the pulse duration and number, the interval
between pulses, and the shape of the pulse and its polarity. For reversible electro-
poration, the electroporation parameters must be carefully selected to preserve the
viability of the cells. Cells may lose their viability due to direct or indirect effects.
75 Parameters Affecting Cell Viability Following Electroporation In Vitro 1451
Fig. 1 Effects of electric pulses on cell permeabilization and viability. A cell is placed between two
parallel electrodes. Electric pulses induce the permeabilization of the cell. Different scenarios can be
observed: reversible permeabilization leading to viable cell; irreversible permeabilization leading to
cell apoptosis or necrosis
Permanent damages to the plasma membrane can occur during pulse delivery.
Indirect metabolic long-term effects, from which cells are not able to recover, can
also occur up to hours following pulse delivery. After resealing, the recovery of cells
depends on their ability to achieve biochemical balance after the induced intense
influx and efflux of molecules. If that balance is not attained, cell viability may be
lost. Other factors may contribute to a reduced survival rate such as the increase in
temperature, the production of reactive oxygen species, and/or the release of elec-
trolytes from electrodes. The survival of the cells may be influenced by the properties
of the cells as their size and shape and their sensitivity to the electric pulses.
Cells can die by two main physiological different processes, necrosis and apo-
ptosis, that have different characteristics. While apoptosis is associated with cell
shrinkage, chromatin condensation, nuclear fragmentation, and the formation of
apoptotic bodies, necrosis is associated with cell swelling and plasma membrane
rupture. Depending on the reversibility or irreversibility of electropermeabilization,
different outcomes can be induced as shown in Fig. 1.
A high number of cell viability assays can be used to assay membrane perme-
ability, metabolic activity, cell function (motility, deformability, ATP (adenosine
triphosphate) level), or colony formation. Simple observation of cells under wide
field microscope (cell shape, ability to adhere and divide) may be also indicative of
inflicted damage. Morphological changes in electrically treated cells have indeed
been investigated many years ago by light and scanning electron microscopy. The
application of 100-μs rectangular pulses leads to the formation and growth of
1452 M.-P. Rols
spherical and hemispherical protuberances of the cell membrane. The formation of

such electro-induced blebs was however not associated with the cells’ death and was
reversible. Increasing the tonicity of the medium prevented the bleb formation,
indicating the osmotically dependent nature of the processes involved (Gass and
Chernomordik 1990). Questions about cell organelle alterations remain unanswered
for years. A recent publication gives evidence for a number of ultrastructural
alterations in cells exposed to electric pulses. Specifically, cells subjected to trains
of 5 ms, i.e., under conditions leading to gene transfer and expression, were observed
to undergo morphological alterations of the mitochondria and nucleus. These mod-
ifications, detected in the minutes following pulse delivery, were transient and have
direct long-term consequences on molecule delivery and therefore may explain
various aspects of the mechanisms of DNA electrotransfer (Phez et al. 2016).
The aim of this chapter is to discuss how experimental conditions can affect cell
viability. Among electric pulse parameters, pulse amplitude, duration, number, and
repetition frequency can significantly affect electroporation. When these parameters
exceed their optimal values, cell viability is affected and irreversible electroporation
takes place. For introduction of small and large molecules, different electric pulse
parameters need to be used. Small molecules are efficiently introduced into the cell
by application of short electric pulses in range of tens to hundreds of microseconds.
The transport of small molecules takes place predominately after the pulse by
diffusion. On the other hand, for macromolecules, long 5–10 ms pulses with
relatively low pulse amplitude are used. Besides, for successful gene electrotransfer,
DNA has to be present in the medium before electric pulses are applied, while small
molecules can enter the cell even if added after the pulse. For irreversible electro-
poration that is used for inactivation of microorganisms, the electric pulse parameters
should exceed critical value, as cell death is the desired result of such application.
Basics Aspects of Electropermeabilization
Square-wave electric pulse generators are the most popular and commonly used in
electropermeabilization protocols. Compared to exponential decay electric pulse
generators, they permit to adjust the different pulse parameters (E, pulse
strength; T, pulse duration; N, number of pulses; and f, repetition frequency)
independently one to each other and whatever the ionic strength conditions. Electric
pulses are delivered through a set of electrodes connected to the generator. As will be
described in this chapter, in addition to the electric pulse parameters, the choice and
the placement of electrodes have to be carefully selected with respect to the charac-
teristics of the cells.
The distribution of the electric field is dependent on the electrode geometry. The
most applicators used for cells in culture are plate electrodes. These electrodes can be
used both for cells grown in cultures on Petri dishes and in suspension. If the cells to
be treated are grown on a surface that is larger than the interelectrode distance, the
entire surface will obviously have to be treated by successive placements of the
electrodes to cover the entire surface by repetitive application of electric pulses.
Fig. 2 Effects of electric pulse parameters on permeabilization extent and efficiency. A cell is
placed between two parallel electrodes. Electric pulses induce the permeabilization of the mem-
brane area facing the anode (gray area). Increasing the field intensity E further increases this area,
while, at constant E, increasing the pulses number N or duration T has no effect in the size of this
area but increases its permeability (darker gray)
Permeabilization of cells can therefore be conducted on different ways. For cells

grown on Petri dish, culture medium can be replaced by a so-called pulsation
medium that, as will be detailed later, has to preserve cell viability. The bottom of
the Petri dish serves as an electropulsation chamber. For cells in suspension,
centrifugation allows to replace culture medium by pulsation one. Special cuvettes
can be purchased, but “home-made” chambers can be easily designed by the plate
electrodes placed directly on the bottom of the Petri dish (▶ Chap. 23, “Gene
Delivery by Electroporation In Vitro: Mechanisms”).
Cell Electroporation
As more detailed in other chapters of this handbook, electropermeabilization occurs

only on the part of the plasma membrane where the potential difference has been
brought to a critical value close to 200–300 mV (▶ Chap. 2, “Critical Electric Field
and Transmembrane Voltage for Lipid Pore Formation in Experiments”). Membrane
permeabilization is controlled by the electric strength. This means that field intensity
E larger than a critical value, Ep, must be applied. Ep is dependent on the size of the
target cells. Large cells are permeabilized at lower electric field values than smaller
cells and appear more “sensitive” because the induced transmembrane voltage is
higher for large cells than for small ones. Therefore, electric field values have to be
carefully adapted to each cell line in order not to affect their viability. In addition, for
a given cell line, all the cells don’t have the same size due to size variation along the
cell cycle.
As tentatively cartooned in Fig. 2, the field strength triggers membrane permeabi-
lization: when E > Ep, it controls the area of the cell surface, which is affected.
Therefore, only the localized parts of the membrane surface facing the electrodes are
affected and will, eventually, be permeabilized. Within these regions, the extent
1454 M.-P. Rols
efficacy of permeabilization (i.e., the flow rate of molecules across the membrane) is
controlled by the number and duration of electric pulses. So, membrane permeabi-
lization only occurs for electric field values E higher than the threshold value Ep,
whatever the number and duration of electric pulses. Increasing E, above Ep, leads to
increase in the extent of membrane area where permeabilization can take place, and
in that specific area, the extent efficacy of permeabilization is determined by the
number and duration of electric pulses (Rols and Teissie 1990).
Electropermeabilization of the cell membrane can therefore be quantified in terms
of the flow of molecules S (Fs), which cross the plasma membrane. For small
molecules, Fs obeys Fick’s law but occurs only on the permeabilizable cell surface:

Ec
FsðtÞ ¼ Ps yðN, T ÞAt=2 1 ΔS exp kðN, T Þt (1)
E
Where Ps is the permeation coefficient of the solute S, y is a function that depends

on the duration and number of pulses, N is the number of pulses, T is the pulse
duration, At is the cell surface, E is the electric field intensity, ΔS is the concentration
difference of S between cytoplasm and external medium, k is the resealing process
constant (in the case of small molecules), and t is the time after the pulse. At=2
ð1 Ec=EÞ defines the size of “macro-domains” where electrotransfer can take
place; y (N, T ) defines the size of “micro-domains” where electrotransfer takes place.
The electro-induced permeabilized state of the cell membrane is transient and
disappears progressively after the application of the electric field pulses. Its lifetime
is under the control of the electric field parameters. The rate constant of the annealing
phase is shown to be dependent on both pulse duration and number but is indepen-
dent of electric field intensity which creates the permeabilization. The phenomenon
can be described in terms of membrane organization transition between the natural
impermeable state and the electro-induced permeable state of the membrane.
Membrane electropermeabilization to small molecules depends on the different
parameters of the electric field. The electric field intensity E is the deciding param-
eter inducing membrane permeabilization which controls the extent of the cell
surface where the transfer can take place. An increase in the number of pulses
enhances the rate of permeabilization. The pulse duration parameter is crucial for
the penetration of macromolecules into cells under conditions where cell viability is
preserved (Rols and Teissie 1998). The main difference between the electrotransfer
of small size molecules and macromolecules is the duration of the pulses. Micro-
second pulses are sufficient to obtain cytotoxic drug transfer. Millisecond pulses are
required to obtain efficient gene expression with a good cell viability. Long pulse
durations limit electric field intensities required when short pulses are used. DNA is a
large molecule; therefore the size of membrane areas allowing its transfer must be
sufficient enough. This is obtained by applying pulses of low intensity but long pulse
duration. Under that optimized conditions, cell viability is generally preserved.
So, the optimization of electroporation protocols highly depends on the objective
of the experiments: the size of the molecules to be electrotransferred and the effect on
cell viability. If the objective is to affect a large area of cell, then high electric field
values have to be used. If cell viability has also to be preserved, this can be obtained
by using short pulse duration.
Cell Permeability Measurements
It is obvious that, under “classical” conditions of electroporation, cell membrane

permeabilization precedes cell viability loss. Therefore, detection and quantification
of permeabilization have to be first evaluated to define the electrical conditions that
will be further used to determine cell viability. The use of fluorescent dyes allows
detecting membrane permeabilization (▶ Chap. 68, “Fluorescent Indicators of Mem-
brane Permeabilization Due to Electroporation”). Visualization can be performed at
the single-cell level under fluorescent microscopes, while flow cytometry permits to
quantify the uptake of molecules on a large number of cells. Quantification can be
subject to artifacts as the sensitivity of the detection method highly depends on the
fluorescent dyes (size, fluorescent quantum yield). Consequently, the statement that a
cell has been successfully permeabilized or not requires additional information
including the type of molecule used in the assay.
Cell Viability Measurements
Cell viability can be assayed by different methods (▶ Chaps. 63, “Mathematical

Models Describing Cell Death Due to Electroporation,” and ▶ 73, “Different Cell
Viability Assays Following Electroporation In Vitro”). However, results can be
inconsistent if assays are performed without any idea of the process of electroper-
meabilization, i.e., the fact that membrane can stay permeable after a while following
pulses application and that both short-term and long-term effects can be present. In
their study, Satkauskas and collaborators compared different and commonly used
cell viability assays after mammalian cell electroporation in the presence of
bleomycin. Cell viability was measured in vitro using clonogenic assay, propidium
iodide, and MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide), employing flow cytometry modality. Results showed that although
clonogenic cell viability drastically decreased, PI assay performed a few minutes
after the treatments indicated nearly 100% cell viability. Assays performed at 6–72 h
time points revealed that cell viability is highly dependent on evaluation time point
and decreased with later evaluation time points. In comparison to clonogenic cell
viability, MTT cell viability was significantly higher, except if used at later times,
2–3 days after the treatment, allowing reliable evaluation of cell viability. In overall,
their results showed that in order to estimate cell viability after electroporation, the
most reliable method is clonogenic assay. Improper use of PI and MTT assays can
lead to misinterpretation of the experimental results (Jakstys et al. 2015).
1456 M.-P. Rols
Effects of Physical Parameters on Cell Viability
Different effects of pulsed electric field are present during cell electropermeabi-
lization. In addition to the transmembrane potential increase, that is mandatory to
induce membrane permeabilization, other effects can be induced that may have
direct or indirect consequences on cell viability.
Orientation
The transmembrane potential of the cell is critical for successful permeabilization. In

that context, cell shape and orientation are two parameters that therefore may impact
both permeabilization efficiency and cell viability. The transmembrane potential of
prolate and oblate spheroidal cells has been calculated for various orientations with
respect to the electric field direction, both numerically and analytically. The induced
transmembrane potential is maximum when the longest axis of the cell is parallel to
the electric field and is minimum when the longest axis of the cell is perpendicular to
the electric field (▶ Chap. 2, “Critical Electric Field and Transmembrane Voltage for
Lipid Pore Formation in Experiments”). The dependency on orientation is highly
pronounced for elongated cells. It is negligible for spherical cells. The biological
relevance of these theoretical results was confirmed with experimental results of the
electropermeabilization of plated Chinese hamster ovary cells, which are elongated.
Both theoretical and experimental results showed that permeabilization is not only a
function of electric field intensity and cell size but also of cell shape and orientation
(Valic et al. 2003). Since in practical use of electropermeabilization, especially for
gene transfection, we need to use the electric field high enough to achieve permeabi-
lization but at the same time low enough to keep the viability of the cells, the
orientation of the cells in the applied electric field is an important parameter that has
to be taken into account.
Temperature
Another key parameter that can be easily adjusted is the temperature. Experiments
are usually performed at room temperature, but incubating the cells on ice before
exposing them to electric pulses or during the minutes following it can improve the
uptake of molecules. This is due to the fact that membrane can stay permeable for a
certain delay following pulse delivery and that this delay is increased by decreasing
the temperature. However, for preserving cell viability and depending on the goal of
the experiment, one of the tips to preserve cell viability can be to place the cells
immediately after the pulses at 37 C.
This is specially recommended for gene electrotransfer experiments. Former
protocols in the literature prescribed to perform electropulsation at low temperature.
This was before knowing the process of DNA delivery into cells, where only DNA
present during pulses will eventually enter the cells. There is no rational need to let
the cells at low temperature following pulse delivery that instead of enhancing DNA
delivery, affect cell survival and as a consequence jeopardize gene expression. The
effect of the preincubation temperature has indeed been shown not to significantly
affect the survival rate of cells while strongly affecting the transfection efficiency.
Lowering the temperature from 37 C to 4 C can induce up to a tenfold increase in
the percentage of cells expressing the electrotransferred activity. When cells are
preincubated at 4 C before pulsation, transfection is shown to highly increase with
post-pulse incubation temperature, while viability is affected at a lower extent.
Optimum conditions thus are a preincubation on ice, followed by a post incubation
at 37 C, which is in conflict with what is currently performed, but in line with
reports where post-pulse incubation at room temperature has been reported to
increase transfection yield. These observations can be explained as follows.
Preincubation of cells with plasmid DNA before pulsation increases the interfacial
concentration of plasmid at the cell membrane. Performing this step at low temper-
ature limits DNA degradation by extracellular nucleases. As it has been shown that
transfection only occurs when DNA is present during pulsation, stability of the
transient permeable structures with time by lowering the temperature is not required
to increase transfection of cells. It appears that, associated with the increase in cell
viability, a 37 C post-pulse temperature highly increases transfection efficiency. The
data indicate that the post-pulse incubation at low temperature which is routinely
performed is detrimental to transfection (Rols et al. 1994).
In a more recent publication, Heller and collaborators evaluated high and low
electric field conditions on cells preheated up to 45 C. Gene expression and viability
were measured. They clearly demonstrated that heat can in fact assist in increasing
gene expression levels. Their results indicated a two- to fourfold increase in gene
expression that was temperature and field dependent. By doing that, electric field can
be reduced 20–30% while maintaining or slightly increasing gene expression and
increasing viability (Donate et al. 2015).
Electrochemical Reactions
Electrochemical reactions take place at the electrodes. Electropermeabilization by

symmetrical bipolar rectangular pulses may therefore cause some electrolytic con-
tamination due to the electrodes. This can have direct consequences on cell viability.
A comparative study of the contamination of a cell suspension by ions released from
aluminum cuvettes (Al3+) and stainless steel electrodes (Fe2+/Fe3+) during cell
membrane electropermeabilization by unipolar and by symmetrical bipolar rectan-
gular electric pulses revealed that the released concentrations of Al3+ and Fe2+/Fe3+
were always more than one order of magnitude lower with bipolar pulses than with
unipolar pulses of the same amplitude and duration. Electrolytic contamination and
its detrimental effects can therefore be largely reduced with no loss in efficiency of
electropermeabilization, if bipolar rectangular pulses of the same amplitude and
duration are used instead of the commonly applied unipolar pulses (Kotnik et al.
2001).
1458 M.-P. Rols
Electroporation Medium
In addition to the modulation of the transmembrane potential, electric pulse delivery

to cells may induce significant increase of the temperature due to Joule effect. As an
electrical current is flowing, Joule heating is taking place and the temperature of the
sample is going to increase. When the temperature reaches values above 42 C, cell
viability may be affected. Temperatures between 46 C and 60 C are associated with
irreversible cellular damage, proportional to the exposure time. Between 60 C and
100 C, protein coagulation occurs instantly with irreversible damage of key cyto-
solic and mitochondrial enzymes and nucleic acid-histone complexes. Temperatures
above 105 C result in tissue boiling, vaporization, and carbonization. Therefore,
one of the key aims of any experimental protocols for electropermeabilization should
be to control temperature. This is particularly the case when long duration pulses are
applied. This deleterious side effect can be controlled by using a low ionic content
pulsing buffer and by delivering a limited amount of energy. A commonly used
so-called pulsation medium is a low ionic, isoosmotic buffer that allows to limit the
Joule effect. The composition of this medium is: 10 mM phosphate buffer, 250 mM
sucrose, and 1 mM MgCl2, pH 7.4.
As one of the steps involved in gene electrotransfer is the interaction of DNA with
the cell membrane, divalent cations may influence the anchoring of DNA to the cell
membrane and therefore control gene electrotransfer efficiency. The effect of differ-
ent concentrations of Mg2+ on electropermeabilization, gene electrotransfer, and
viability of cells has been studied. For that, different pulsation media with increasing
Mg concentrations have been used and revealed that high Mg concentrations lead to
both high electropermeabilization and viability while causing low gene
electrotransfer. This was due to the fact that Mg2+ ions can bind DNA at cell surface
at such strength that cannot pass into the cell during application of electric pulses,
which can lead to lower gene transfection (Haberl et al. 2010).
Presence of DNA
The permeability of cell membranes submitted to electric pulses was shown to

increase noticeably if the cells are pulsed in the presence of DNA (Klenchin et al.
1991). Therefore, plasmids can be toxic in combination with electric pulses. This can
lead to a decrease in survival and transfection efficiency directly linked to the
physical size and concentration of each individual molecule of plasmid: a single
copy of a large plasmid is more toxic and harder to introduce into the cell than a
single copy of a small plasmid or than the number of copies of a small plasmid
equivalent in mass to the single copy of the large plasmid (Lesueur et al. 2016). This
toxicity of large plasmids electrotransfer is not caused by plasmid expression. This is
of remarkable practical importance to reach high levels of transfection and survival
for both small and large plasmids. In another study, electrotransfer of pDNA
(plasmid devoid of therapeutic genes) was shown to lead B16F10 melanoma
tumor cells to secrete IFNβ (interferon) and to die by necrosis (Znidar et al. 2016).
B16F10 cells in culture indeed express cytosolic DNA sensors, and the expression of
IFNβ mRNA (messenger ribonucleic acid) and protein is increased after pDNA
electrotransfer. These changes in mRNA and protein levels are accompanied by
DNA concentration-dependent cell death. These effects may be due to the activation
of signaling pathways mediated by the upregulated cytosolic DNA sensors.
Electrotransfer of pDNA can cause necrotic and/or apoptosis cell death. Not only
pulse parameters but also plasmid DNA can be responsible for triggering cell death.
Effects of Cell Parameters on Cell Viability
Cells have all the equipment and expertise necessary to carry out the functions of
life. But, despite these similarities, cells are not equal. Cells by essence have
different morphological and metabolic characteristics. They can in particular differ
in size and in shape and can be obtained from cancer cell lines or from primary cell
cultures; all these parameters may have direct or indirect consequences on their
response and sensitivity to electric pulses.
Size and Shape Effect
Computer simulations predicted that large cells would be specifically permeabilized

in a mixture with smaller cells. This was examined on a mixture of Chinese hamster
ovary (CHO) cells and erythrocytes. CHO cells were permeabilized to Trypan blue
without any occurrence of hemolysis. A similar “size” specificity was observed on
blood samples. This agreement between prediction and experimental observation
indicates that induction of electropermeabilization is mainly under the control of the
size of the target cell. Its physiology plays only a minor role, if any. Treating blood
induced the permeabilization of 70% of the leucocytes (polymorphs and monocytes)
but did not affect erythrocytes (Sixou and Teissie 1990). Therefore, in agreement
with theory, the larger the cells, the more sensitive to electric pulses both in terms of
permeability and, as a direct consequence, loss in viability.
The differences in gene electrotransfer and membrane electropermeabilization
between cells grown plated on Petri dishes or in suspension have been investigated
in two different cell lines (CHO and B16F1) in addition to viability. Results showed
that there is a marked difference in the efficiency of gene electrotransfer between
suspended and plated cells (Marjanovic et al. 2010). CHO cells had a slightly better
survival rate at higher electric fields than B16F1 cells, both in suspension and plated,
that was due to differences in cell size and shape.
Normal Versus Tumor Cells
Using a 3D spheroid cell culture mode, the effect of calcium electroporation has been
tested on three different human cancer cell lines as well as on primary normal human
1460 M.-P. Rols
dermal fibroblasts. The results showed a clear reduction in spheroid size in all three
cancer cell spheroids. Strikingly, the size of normal fibroblast spheroids was not
affected (Frandsen et al. 2015). One very important issue is the relation between
tumor cell kill efficacy and normal cell sensitivity, calcium electroporation being a
promising anticancer treatment method (▶ Chap. 93, “Electrochemotherapy and Its
Clinical Applications”).
Another difference between normal primary and cancer cell lines comes from
their repair capacity that could explain their various sensitivities. Membrane repair
was investigated by monitoring dye entry over time after electroporation. The
normal primary cell line exhibited the slowest rate. A viability assay performed
1 day after plasma membrane electropermeabilization revealed that viability in the
primary normal cell line was higher than in the three tested cancer cell lines. These
data suggest more effective membrane repair in normal, primary cells and supple-
ment previous explanations why electroporation-based therapies and other therapies
permeabilizing the plasma membrane are more effective on malignant cells com-
pared to normal cells in cancer treatment (Frandsen et al. 2016).
Reactive Oxygen Species
Exposure of cells to the electric pulses induces generation of reactive oxygen species
at the electropermeabilized cell membrane level (▶ Chap. 28, “Involvement of
Reactive Oxygen Species in Membrane Electropermeabilization”). This generation
has been shown to be directly associated with the part of the membrane surface
which is electrically restructured, as shown by its dependence on electric parameters.
The electro-induced cell process is activated by Ca2+ and Mg2+ ions and by
exogenous adenosine 50 -triphosphate. A metal-ion-catalyzed Haber-Weiss reaction
occurs. The use of antioxidant products such as dimethylsulfoxide and sodium
L-ascorbate showed that cell survival after electric treatment was directly correlated
to the oxidative jump intensity. This observation had to be associated with the cell-
damaging action of oxygen-reactive species that have a dramatic effect on cell
viability (Gabriel and Teissie 1994).
Bystander Effect
Bystander effect, a known phenomenon in anticancer therapies, where cells release

signals which cause damage to nearby cells, has been very recently explored in
electroporated melanoma cells in vitro. Viability of non-electroporated cells exposed
to medium from electroporated cells and the release of microvesicles have been
determined as potential indicators of the bystander effect. Results showed that cells
exposed to electric pulses mediated their damage to the non-electroporated cells,
thus decreasing cell viability. The murine melanoma B16F1 cell line was found to be
more electrosensitive and thus more prone to bystander effect than the canine
melanoma CMeC-1 cell line. Shedding microvesicles may be one of the ways
used by the cells to mediate the death signals to the neighboring cells. The effect
depends on pulse amplitude, repetition frequency, and cell type (Prevc et al. 2016).
Some Tips to Preserve Cell Viability
In addition to the electric pulse conditions that have to be carefully defined,

according to the cell characteristics, some tips can be used to enhance cell viability
for cells which are easily permeabilized but too much affected by electroporation.
Addition of Serum
Serum present during electric pulse delivery can have a positive effect on viability.
Increase in cell viability is due to a protection of cells by serum that limits the flow of
exchange of molecules across the permeabilized membrane. In particular, the ATP
leakage is less pronounced when serum is present. ATP leakage can be reduced from
40% to 10%, thanks to the presence of 20% serum. Serum addition can therefore
have a positive effect on cell transfection when added after the pulses. This depends
on the physiology of the cells; the more their viability will be preserved, the more
efficient transfection will be obtained. By acting as a protector of cell viability, serum
indeed can help to increase gene transfer and expression (Delteil et al. 2000).
Addition of Poloxamer
Triblock copolymers as pluronic L64 are used to improve gene transfer mediated by
several agents into muscle tissue. Their mechanism of action is still under investi-
gation. The combination of electrotransfer and triblock copolymers, in allowing
softening electric field conditions leading to efficient DNA transfection, could
potentially represent a milder and more secure transfection method. A pretreatment
of cells with L64 has been showed to significantly improve transfection efficiency.
This pretreatment was indeed shown to increase cell viability, and this was partly
responsible for the improvement of transfection efficiency (Wasungu et al. 2011).
Cell Centrifugation
Viability of electrotransfected adherent and suspended cells can be improved if

pelleting by centrifugation is performed immediately after pulsing. The protection
effect on cell viability is cell line and pellet thickness dependent. For forming CHO
cell pellets, centrifugation force (300–13,000 g) and duration are not crucial; about
five to ten cell layers in the pellet provided the optimal protection effect. NK-L,
K-562, L1210, and MC2 cell pellets are optimally formed by centrifugation at
13,000 g. Pelleting improves the cell viability. When this pelleting method is applied
1462 M.-P. Rols
to load CHO cells with FITC-dextran (41,000 MW), not only is the success rate close
to 100%, but the growth rate is similar to the control, which is far better than the
conventional electroporation method. Furthermore, the transfection efficiency of the
cell lines in pellet could be significantly higher than that in suspension (Li et al. 1999).
Cryopreservation
New cryopreservation approaches for medically applicable cells are of great impor-
tance in clinical medicine. Current protocols employ the use of dimethyl sulfoxide
(DMSO), which is toxic to cells. Trehalose is a nontoxic disaccharide that can be
used as a cryoprotectant. The efficiency of combining reversible electroporation and
trehalose for cryopreservation of human adipose-derived stem cells was assayed.
After thawing, 80% cell recovery was obtained at 250 mM trehalose and cells’
functionality was preserved. Successful cell growth and efficient adipogenic and
osteogenic differentiation were achieved. Electroporation seems to be an efficient
method for loading nonpermeable trehalose into stem cells, allowing long-term
cryopreservation in DMSO-free and xeno-free conditions (Dovgan et al. 2016).
This approach is also developed for plant. The impregnation of trehalose by the
combined actions of vacuum impregnation and pulsed electric field is a technology
with strong potential for improving the freezing tolerance of leaves and, therefore,
keeping their fresh-like characteristics after freezing and thawing (▶ Chap. 114,
“Pulsed Electric Fields in Combination with Vacuum Impregnation for Improving
Freezing Tolerance of Vegetables”).
Mathematical Models to Predict Cell Death
Mathematical models of survival in which the probability of cell death is predicted

are of interest (▶ Chap. 63, “Mathematical Models Describing Cell Death Due to
Electroporation”). Various mathematical models (first-order kinetics, Hülsheger,
Peleg-Fermi, Weibull, logistic, adapted Gompertz, Geeraerd) were shown to fit to
experimental data using a nonlinear least-square method. The most appropriate
models of cell survival as a function of treatment time were the adapted Gompertz
and the Geeraerd models and, as a function of the electric field, the logistic, adapted
Gompertz and Peleg-Fermi models (Dermol and Miklavcic 2015). This approach is
of interest for in vivo situations where viability is of high importance to preserve
healthy tissues integrity.
Conclusions
Electroporation can be detrimental to cell viability, not only by increasing the

permeability of the cell membrane but also by inducing side effects as the increase
in temperature, the release of ATP, and the formation of electrolytes near the
Table 1 Examples of parameters that can preserve cell viability following electroporation
Parameters Effect on cell viability References
Temperature of Placing the cells at 37 C after PEF decreases the release of Rols et al.
experiment ATP and preserves cell viability (1994)
Preheated cells before PEF can reduce electric field values Donate et al.
needed for membrane permeabilization and preserve cell (2015)
viability
Electrodes Electrochemical reactions can induce the release of Kotnik et al.
cytotoxic compounds (2001)
Reversing the polarity may decrease this deleterious
phenomenon
Pulsation Joule heating can damage the cells; it can be reduced by Haberl et al.
medium using a low ionic, isoosmotic medium (2010)
External DNA can be toxic in combination with electroporation Lesueur et al.
molecules (2016)
Triblock polymers can increase cell viability Wasungu
et al. (2011)
Serum has a positive effect on cell viability Delteil et al.
(2000)
Cell size and Large cells are generally more easily permeabilized and Sixou and
shape therefore affected by PEF than small cells Teissie 1990
Marjanovic
et al. (2010)
Normal and Viability in primary normal cell lines is higher than in Frandsen et al.
cancer cells cancer cell lines (2015)
Membrane repair is more effective in normal, primary cells Frandsen et al.
than in cancer cells (2016)
electrodes or reactive oxygen species near cell membranes. Some of these side
effects can be partly reduced in vitro by optimizing the electric pulse parameters
and the experimental conditions according to the cell characteristics and sensitivity
to electric pulses (Table 1).
Therefore, long-term viability that is required for gene transfer and expression can
be obtained. In in vivo environments, one must also consider cells and tissues
viability, which in turn can also strongly affect gene electrotransfer efficiency due
to DNA damage/denaturation or decreased cell viability. Gene electrotransfer rep-
resents a promising delivery system for introducing foreign genes into cells for a
range of medical applications. Though numerous strides forward have been made in
defining the mechanisms of DNA electrotransfer, several challenges remain for
researchers in this field of study (Rosazza et al. 2016). It is therefore necessary to
define the relationship between findings in vitro and in vivo, as cell responses to
electric pulses can differ between in vitro and in vivo cell cultures, where the
microenvironment itself may influence the cell response.
Acknowledgments This research was performed in the scope of the EBAM European Associated
Laboratory (LEA) and is a result of networking efforts within COST TD1104 (www.electropora
tion.net). It was supported by the Centre National de la Recherche Scientifique (CNRS), the Agence
1464 M.-P. Rols
Nationale de la Recherche (ANR), the Projet PIERGEN ANR-12-ASTR-0039, and the Direction
Générale de l’Armement (DGA).
Cross-References
Experiments
▶ Involvement of Reactive Oxygen Species in Membrane Electropermeabilization
▶ Pulsed Electric Fields in Combination with Vacuum Impregnation for Improving
Freezing Tolerance of Vegetables
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Techniques to Detect Electroporation in
Food Tissues 76
Abstract
Recent academic experiments provided many evidences for disintegration of
membrane in biological tissues with pulsed electric fields (PEF) having the
strength and duration of order of E = 100–1000 V/cm and tPEF = 10 μs–0.1 s,
respectively. Commonly, this disintegration is selectively applied to the mem-
branes without significant impact on the cell walls and other constituents of the
tissues. The phenomenon of membrane electroporation is intensively studied
during the last decades. Many practical efforts were initiated in the field of PEF
application to the processing of food tissues (fruits, vegetables, animal tissues).
PEF-assistance for selective extraction, pressing, osmotic treatment, freezing, and
drying was already tested. The advantages of PEF for the retention of quality,
color, texture, aroma, and other positive qualities of fresh foods were demon-
strated. Up to date the different techniques to detect electroporation in food
tissues were proposed. They evolve analysis of the microscopic structure and
changes in electrical, diffusion, textural, and acoustic properties. In this chapter,
the examples of estimation of the membrane disintegration index by different
techniques are illustrated. The chapter is concise and covers various type of
N. Lebovka (*)
after F. D. Ovcharenko, NAS of Ukraine, Kyiv, Ukraine
Compiègne, France
E. Vorobiev

DOI 10.1007/978-3-319-32886-7_150
tissues and as well as the benefits and disadvantages of techniques. Practically all
techniques to detect electroporation are destructive (invasive), and they can
violate independently the structure of the tissues and misrepresent extend of
electroporation. Moreover, the applied procedures of measurements are not
universal and require careful adaptation for the selected type of food tissue. In
each part of the chapter, emphasis is placed on the particular details of application
of these techniques, and their advantages and disadvantages are compared in the
conclusion part.
Keywords
Electroporation • Disintegration index • Detection of degree of electroporation •
Pulsed electric fields
Nomenclature
C Membrane capacity F
D Diffusion coefficient m2/s
E Electric field strength V/cm
fe Electroporation factor
f Pulse frequency
Gm Elasticity modulus Pa
F Force N
n Number of pulses
PF Fracture stress Pa
R Radius of the cell μm
S Acoustical index of firmness g2/3/s2
ti Pulse duration μs
tPEF Time of PEF treatment s
T Temperature K
um Transmembrane potential V
W Specific energy consumption Wh/kg
Z Membrane disintegration (damage) index
Za Acoustic disintegration index
Zc Electrical conductivity disintegration index
ZD Diffusion coefficient disintegration index
Zt Texture disintegration index
Greek Symbols
φ Phase shift rad
e* Complex relative permittivity
er Relative dielectric constant
e0 Permittivity of the free space = 8.854 187 817. . . 1012 F m1
σ Electrical conductivity S/cm
θ Angle between direction of the external field E and position of the obser-
vation point on the surface of a membrane
76 Techniques to Detect Electroporation in Food Tissues 1469
τc Time constant reflecting of charging the membrane capacity C μs

ω Angular frequency rad/s
Subscripts
d Damaged completely electroporated
i Initial intact
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1469
Different Techniques to Detect Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1470
Optical Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1470
Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1476
Measurements of Electrical Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1476
Diffusion Coefficient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1482
Texture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1483
Acoustic Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1484
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1486
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1487
Introduction
Phenomenon of electroporation was discovered in 1960th, and starting from the

early 1990th, the electroporation in food products by pulsed electric fields (PEF) was
tested for microbial killing, food preservation, and improvement of process opera-
tions. Nowadays PEF-assisted processing aimed for better selectivity of solute
extraction, pressing, osmotic treatment, freezing, and drying attracts great attention.
Electroporation requires some threshold value of the potential difference across a
membrane, or the transmembrane potential, um, typically, 0.5–1.5 V (Barba
et al. 2015). Depending on conditions of treatment, the value of um and PEF
exposure time tPEF, a temporary (reversible) or irreversible loss of barrier function
can occur. The electroporation is rather complex phenomena, and it includes mem-
brane charging and polarization (charging time tc > 1 μs), expansion of pore radii
and aggregation of different pores (during 100 μs). The existing theories of
electroporation account for the electrically induced mechanical, hydrodynamic,
osmotic, and viscoelastic instabilities. The value of um can be analytically evaluated
for individual cells of different geometries. For example, for spherical cell
um ¼ 1:5REf e cos θð1 expðt=τC ÞÞ; (1)
where R is the radius of the cell, θ is the angle between the external field with
strength of E and position of the observation point on the surface of a membrane, τC
(1–10 μs) is the time constant reflecting the process of charging the membrane
capacity C, and fe(1) is the electroporation factor that depends on the electrical
conductivities of the membrane, external medium, and cytoplasm. For anisotropic or
irregularly shaped cells, the value of um can depend on the cell shape, its orientation,
and other details.
Evaluation of value of um in foods (suspensions or tissues) is more complex and
usually requires application of complex mathematical models and numerical calcu-
lations. The calculations account for inhomogeneous distribution in the local electric
fields, osmotic flows, resealing of cells, distribution of cell sizes, their states and
local solute concentrations, local electrical conductivity, etc. In general, the effi-
ciency of electroporation increases with increasing of the cell size. For example, the
experimentally estimated threshold values of Et are of the order of 100–1000 V/cm
for food tissues (R 30–60 μm) and >10 kV/cm for microbial cells (R 1–10 μm)
(Barba et al. 2015). Both theory and experiments evidenced that efficiency of
electroporation strongly depends on electrical conductivity of food materials before
and after treatment (Ben Ammar 2011).
This chapter will concentrate on the existing techniques for detection and evalua-
tion the degree of electroporation in different food tissues. An overlook on the methods
based on analysis microscopic structure, changes in transport, textural, and acoustic
characteristics is presented. In general case, the task is very complicated accounting for
the complexity of food structure, and the evaluated membrane disintegration index is
dependent on the applied procedures of measurements. In each part of the chapter,
emphasis is placed on the particular details of application of these techniques, and their
advantages and disadvantages are compared in the conclusion part.
Different Techniques to Detect Electroporation
The membrane disintegration (damage) index Z can be defined as the fraction of

completely electroporated cells in the food tissue. The level of electroporation
induced by application of PEF can be estimated by using the different experimental
techniques.
Optical Microscopy
The optical microscopy is the most direct technique for visualization of PEF impact
on the structure of individual electroporated cells, suspensions of cells, and food
tissues.
The electroporation of individual NG108-15 cell was studied using fluorescence
microscopy (Ryttsen et al. 2000). The carbon-fiber microelectrodes (with 5-μm-
outer-diameter) were used to apply monopolar 1-ms rectangular pulses with field
strength E = 1.1–8.1 kV/cm. The cells were incubated in a buffer, supplemented by
5 mM fluorescein, and then were washed several times in order to remove extracel-
lular dye. The experiments revealed that the PEF-treated cells were more swollen
and granulated when exposed to higher fields. The estimated threshold transmem-
brane potential for breakdown was 0.250 V. Transmembrane potential distribu-
tion um(θ) on the surface of individual cell (sea urchin egg) stained with the voltage-
sensitive dye RH292 was obtained using a pulsed-laser fluorescence microscope

(Kinosita et al. 1988). It was demonstrated that the membrane recovers quickly after
the external field is removed.
PEF effects on aqueous suspensions of Chinese hamster ovary cells were studied
(Valic et al. 2003). The direct observation evidenced that electroporation of elon-
gated cells was more intensive when the longest axis of the cell was parallel to the
electric field. The light and fluorescence microscopy were used to visualize electro-
poration transformations in culture cells from Schistosoma japonicum (Yuan
et al. 2005). Fluorescence microscopy was used in order to study the intracellular
pH inside yeast cells in suspensions of Saccharomyces cerevisiae (Herman
et al. 2005). The pyranine was used as pH-sensitive fluorescent probe. It was
demonstrated that electroporation can be used for accurate in vivo pH calibration
in living cells.
PEF efficiency at high concentration of yeast cells in aqueous suspensions of
Saccharomyces cerevisiae was studied using optical microscopy from (El Zakhem
et al. 2006). The data had shown the presence of strong aggregation in PEF treated
suspensions (Fig. 1). The phenomenon was explained by attraction between killed
and alive cells that have surface charges (ζ-potentials) of different sigh.
PEF-induced changes in color of onion epidermis stained with neutral red dye
(NRD) were observed using optical microscopy (Fincan and Dejmek 2002). The
directly counted number of electroporated cells was proportional to the increase in
electrical conductivity. The studies evidenced that PEF does not affect noticeably the
structure of cell walls, while the damage of membranes was confirmed by free
colorant diffusion inside electroporated cells. The similar studies of onion tissues
stained with NRD were done (Asavasanti et al. 2011; Ersus and Barrett 2010).
About 92% of onion cells were electroporated after PEF treatment
(E = 333 V/cm, ti = 100 μs, n = 100 pulses) (Ersus and Barrett 2010). At
E = 167 V/cm the rupture of cell walls was not observed. However, air spaces
Fig. 1 Images of untreated (a) and PEF treated (b) aqueous suspension of Saccharomyces
cerevisiae. PEF treatment was done at T = 328 K, E = 7.5 kV/cm, and tPEF = 0.04 s. In order
to visualize the damaged cells, the probes of S. cerevisiae yeast suspensions were immersed in the
methylene blue stain solution (With permission from (El Zakhem et al. 2006))
Fig. 2 Images of red beet tissue after aqueous extraction at 30 C: untreated (a) and PEF pretreated
(b, Z 0.9, electric field strength E of 1000 V/cm, pulse duration ti of 1000 μs and total time of
PEF treatment of 0.1 s) (Loginova 2011)
around cell walls disappeared. Small plant cells that are located near the vascular
bundles and upper epidermis showed higher resistance to PEF treatment. It was
observed that intracellular convective motions (cytoplasmic streaming) after PEF
application were strongly correlated with PEF frequency (Asavasanti et al. 2011).
Direct visualization of the effects demonstrated that lower frequencies ( f < 1 Hz)
cause more damage to tissue integrity than higher frequencies ( f = 1–5000 Hz).
Effects of PEF were visualized in red beet tissues (Loginova 2011). Figure 2
shows images of cambium rings after 3 h of the aqueous extraction for nontreated
(Fig. 2a) and PEF pretreated (Fig. 2b) tissues. After some time of extraction, the
patterns for nontreated tissue were a mixture of intensively colored and almost
colorless pale cells. The intensively colored cells corresponded to undamaged cells
and colorless pale cells corresponded to the damaged cells with lost vacuolar sap and
diluted pigment.
The simultaneous studies of apple parenchyma using the optical microscope and
measurements of electrical conductivity revealed correlations between the extent of
electroporation, cell shape, and orientation of the applied electric field (Chalermchat
et al. 2010). Different tissue regions were sampled and the results showed that fields
of lower intensity were required for electroporation of cells when the field was
applied parallel to the longest axis of the cells. The spherical cells in the outer region
of apple became permeabilized regardless of field orientation.
The similar observation was reported for onion epidermis stained with NRD (Ben
Ammar 2011). Figure 3 presents evolution of cell colors after PEF treatment for
electric field applied parallel to the longest axis of the cells (left column) and
perpendicular to it (left column). The stronger PEF effects were observed for parallel
orientation.
Optical microscopy was used to compare effects of PEF on Nicotiana tabacum
cells with and without cell walls. Isolated protoplasts were obtained by enzymatic
cell wall degradation (Janositz and Knorr 2010). The cells were stained with the vital
dye phenosafranine. The data demonstrated higher sensibility of protoplasts to the
electric field in contrast to cells with the cell walls. The protoplast cell size was
Fig. 3 Evolution of images of onion epidermis after PEF treatment (electric field strength E of
250 V/cm, pulse duration ti of 100 μs and number of pulses n of 8) for different orientations of
electric field (shown by arrows) (Ben Ammar 2011)
reduced after irreversible cell disruption and slightly increased after reversible
permeabilization.
The effects of PEF treatment on grape-skin (Cabernet Sauvignon) histo-
cytological structures and on the organization of skin cell wall polysaccharides and
tannins using two protocols PEF1 (E = 4 kV/cm; tPEF = 1 ms, W = 3.99 Wh/kg) and
PEF2 (E = 0.7 kV/cm; tPEF = 200 ms; W = 31 Wh/kg) were studied (Cholet
et al. 2014). Samples for light microscopy were stained via the Periodic Acid Schiff
Fig. 4 Evolution of the skin cross sections during 14 days for untreated, PEF1-treated and PEF2-
treated grape skins. Here eE is external epidermis, oH is outer hypodermis, iH is inner hypodermis,
S is skin; and VPC is vacuolar phenolic compounds (With permission from (Cholet et al. 2014))
(PAS) method for the detection of polysaccharides. Figure 4 shows evolution of the
skin cross sections for different PEF protocols.
The data evidenced that depending on the PEF parameters, cell-wall structure was
differently impacted. For example, PEF1 had less pronounced effect on the content
of phenolic compounds and skin cell wall pectic fraction skeletons, whereas,
whereas PEF2 profoundly modified the organization of skin cell walls, providing
variable performance in terms of polyphenol extraction and wine quality.
Optical microscopy was also utilized to study PEF effects on the freezing of onion
tissue (Ben Ammar 2011). Figure 5 compares the typical images, captured during
freezing of untreated (Z = 0) and PEF-treated (Z 1) onion upper epidermis
Fig. 5 Images of onion upper epidermis tissue captured during freezing at T 261 K. The cell size
is about 400 μm 45 μm. The tissue was untreated (Z = 0) and PEF-treated (Z = 1, electric field
strength of 400 V/cm, pulse duration of 1000 μs and total time of PEF treatment of 0.5 s) (Ben
Ammar 2011)
tissues. Cooling of untreated onion tissue in the temperature range between -10 C
and -15 C provoked the turbidity of the cytoplasm and appearance of opacity, which
reflects commencement of freezing.
The cells of untreated tissue (Z = 0) were frozen progressively, i.e., one after
other. The process of freezing was lasting approximately 60 s. In contrast, the cells of
PEF-treated tissue got frozen practically instantly, in 2–3 s.
Electron Microscopy
Scanning (SEM) and transmission electron microscopy (TEM) are very powerful
techniques for quantification the morphological properties of membranes and cell
walls, and they were widely applied in research for the study the PEF effects in
materials of different biological origin (Condello et al. 2013). Applications of SEM
and TEM techniques to reveal the PEF-induced effects in food tissues were recently
reviewed (Fazaeli et al. 2012). SEM was used to study effects of irreversible
electroporation (IRE) in rabbit and swine liver (Lee et al. 2012). IRE induced
nanopores in membranes, as shown by SEM. The pore diameters for IRE were
larger than nanopores created by RE. SEM microscopy was also used to study the
effects of electroporation on erythrocytes (Spugnini et al. 2007). Untreated red cells
shown their typical discocytic shape where as most treated undergo evident mor-
phological changes and some of them shown a star-like shape. The effects were
explained by alterations of the molecular organization of the whole plasma mem-
brane and connections between the membrane proteins and the underlying cytoskel-
etal proteins.
SEM data evidenced the presence of similarity in structure of the cell walls, area
of cells, and morphology of starch granules for untreated and PEF-treated potato
samples (Fig. 6a; Ben Ammar 2011). However, the sequential PEF + osmotic
treatment can provoke cell wall damage and changes in the surface morphology of
starch granules (Fig. 6b). The starch granules became rougher, perforated, and
crushed that was also typical for granules exposed to multiple freezing and thawing
cycles or deep freezing in liquid nitrogen. The most significant changes were
observed for air-blast frozen and freeze-dried potato samples (Fig. 6c, d). For
PEF-treated samples, starch granules were still observed (Fig. 6c). However, PEF +
osmotically treated starch granules were completely damaged (Fig. 6d).
Measurements of Electrical Characteristics
A number of useful methods for characterization of electroporation in food materials

are based on measurements of their electrical characteristics. The main electrical
characteristics are electrical conductivity σ and dielectric constant or permittivity e
(Reilly 2012). The simplest electrical model of individual cell implies that it is
surrounded by an insulating membrane with capacitance, while the extracellular
medium and cytoplasm are conductive electrolytes. The complex relative permittiv-
ity, e*, is defined as
e ¼ er jσ=ðωeo Þ (2)
where er is relative dielectric constant that reflects the capacitance of the material
between electrodes, ω is the angular frequency (ω = 2πf), and e0 is the permittivity
of the free space.
The ratio σ/(ωeo) is called the loss factor and the useful quantity is also the phase
shift
Fig. 6 The SEM images of potato tissues: PEF treated (a, c) and PEF + osmotically treated (b, d).
The samples (c) and (d) air-blast frozen and then freeze-dried (Ben Ammar 2011)
φ ¼ arctanðωer eo =σ Þ (3)
Typically, the values of er, σ, and φ are strongly dependent on f. Different types of
dispersions (α-, β-, and γ- dispersions) related to different polarization mechanisms
were identified in biological materials Fig. 7.
The α-dispersion ( f = 1–104 Hz) is related with relaxation of counterions in double
electric layers at the electrodes and relaxation of membranes. The contribution from
electrode polarization at small frequencies may be essential, and estimation of dielec-
tric parameters of the cellular tissue requires introduction of special corrections. The
β-dispersion ( f = 104–108 Hz) reflects polarization resulting from the charging mem-
branes through intra- and extracellular electrolytes. The γ -dispersion ( f > 109 Hz)
reflects rotation of molecules having a permanent dipole moment (water, proteins).
Up to date the three different methods based on measurements of electrical
parameters were applied to determine extend of electroporation in tissues. The
main idea is that the extent of electroporation is in a linear proportion with the
measured electrical parameter.
It should be noted that the electrical parameters of biological materials can reflect
different characteristics of the tissue, details of spatial distributions of air, and
moisture contents. Changes in these characteristics during or after PEF treatment
107 101
Relaxation of near-electrode
counterions
106
Polarization from the
charging membranes
a
Relative dielectric constant (er)
Electrical conductivity (σ, S/m)

105 through intra- and
extracellular fluids
104
100
b
103
102
g
101 Rotation of
permanent dipoles
(water, proteins)
100 10−1
103 105 107 109 1011
Frequency (f, Hz)
Fig. 7 Frequency dependencies of the values of er and σ of biological tissue (Compiled from
(Reilly 2012))
can affect the electrical parameters indirectly related to electroporation. So, the
electrically based methods can be applied only with some precaution.
Low-Frequency Method
The very popular method of estimation of membrane disintegration in cells is based
on the measurements of electrical conductivity σ at the low frequency at the
boundary between α- and β-dispersion range, f 1–10 kHz. Figure 8 presents
typical behavior of electrical conductivity σ in the course of electroporation. Elec-
trical conductivity disintegration index Zc was defined as (Barba et al. 2015):
Z c ¼ ðσ σ i Þ=ðσ d σ i Þ (4)
where σ is the electrical conductivity measured at low frequency (1–10 kHz) and
indexes “i” and “d” refer to the conductivities of intact and completely damaged
tissues, respectively. This definition gives Zc = 0 and Zc = 1 for intact and
completely damaged tissue, respectively.
The low frequency method requires the knowledge of σ d. It can be estimated from
the data obtained in the limit of long treatment time (tPEF 0.1–1 s) and high
electric field strength (E > 1000 V/cm) or by the measurement of electrical con-
ductivity of the freeze-thawed material. However, the strong PEF treatment or
freeze-thawing procedure can also differently affect the structure of cell walls. The
0.7
sd
0.6
Electrical conductivity (s, S/m)
Completely
0.5
electroporated
0.4
Intact sd - si
0.3
s - si
0.2
0.1
si
0
10−4 10−3 10−2 10−1 100
Time of PEF treatment, (tPEF,S)
Fig. 8 Illustration for the application of low-frequency electrical conductivity method for deter-
mination of the electrical conductivity disintegration index. Here, the typical dependence of
electrical conductivity σ (at f 1–10 kHz) versus PEF treatment time tPEF is presented
existing textural data evidenced the different softening of PEF-treated and freeze-
thawed potato, apple, and carrot tissues. So, theσ d value in the low-frequency
method can not be uniquely determined, and the value of electrical conductivity
disintegration index Zc can be also dependent on the selected way for determination
of σ d.
Figure 9 presents typical Zc(tPEF) dependencies for potato and orange (Ben
Ammar 2011). For potato tissue more efficient electroporation was observed as
compared with orange. It is surprising because the radius of the orange cells
(65 μm) is larger than of the potato cells (35 μm) and one expected the opposite
behavior (see, Eq. (1)). However, the potato and orange have significantly different
electrical conductivity contrasts, k = σ d/σ i 14.3 and σ d/σ i = 1.3, respectively. It
was demonstrated that differences in electroporation efficiency for these tissues can
be explained by the differences in the electrical conductivity contrast.
Low-High Frequency Method

This method is based on the measurements of electrical conductivity σ of intact and
electroporated materials at low (1–10 kHz) and high (50 MHz) frequencies; it is
based on the model of bio-impedance. At low frequency, cell membranes have very
1
sd /si ª14
Potato
Conductivity disintegration index (ZC)
0.8
t
0.6
0.4 Orange
sd /si =1.3
E, V.cm–1
0.2 400
1000
0
10–3 10–2 10–1 100
Time of PEF treatment (tPEF, S)
Fig. 9 Electrical conductivity disintegration index Zc versus the time of PEF treatment tPEF for
potato and orange. PEF treatment was done with bipolar pulses duration ti =1 ms and pulse
repetition time Δt =10 ms. The data were obtained using low frequency method (Ben Ammar 2011)
high resistance and reactance, and their electroporation can result in noticeable
changes in the electrical conductivity spectrum in the ranges of α-and β-dispersions.
However, at high frequencies, the effect of intact membranes on the conductivity
spectrum becomes negligible. That is why electroporation of material results in
weakening of frequency dependence of σ.
Figure 10 presents typical frequency dependencies of electrical conductivity σ for
intact and partially electroporated materials. For the totally electroporated material,
the electrical conductivities measured in low-frequency range σ and high-frequency
range σ 1 should coincide and to be equal to that for intact material σ 1 i . The
measured frequency dependence σ m( f ) commonly required corrections on changes
in temperature, tissue porosity, and electrolyte concentration. Such correction can be
easily done using correction coefficient α ¼ σ 1 1
i =σ m that accounts for the difference
1 1
between σ i and measured value of σ m . Electrical conductivity disintegration index
ZC for low-high frequency method was defined as (Angersbach et al. 2002)

Z c ¼ ðασ m σ i Þ= σ 1
i σi (5)
Partially electroporated
0.8 sm∞
• •
Measured a = si / sm
0.7
Corrected si ∞
0.6
Electrical conductivity (s ,S/m)
sm
sc ( f ) = asm ( f )
0.5
sc
0.4
si •- si
Intact
0.3
asm - si
0.2
0.1
si
0
103 104 105 106 107 108
Frequency ( f, Hz)
Fig. 10 Application of low-high frequency method for determination of the electrical conductivity
disintegration index. Here, the typical dependencies of electrical conductivity σ versus the fre-
quency f for intact and partially electroporated materials are presented
Thus determination of ZC requires measurements of electrical conductivity of

intact and electroporated materials at low (σ i, σ m) and high (σ 1 1
i , σ m ) frequencies.
This equation gives Zc = 0 for intact and Zc =1 for completely electroporated
material.
Method of Phase Shift

The electric impedance method and measurements, based on the frequency depen-
dency of the phase shift φ in the range of 500 Hz–10 MHz, were also used for
estimation of the electroporation effects in PEF-treated mash from the wine grapes.
Figure 11 shows example of frequency dependencies of specific conductivity and
phase shift for mash from Muscatel grapes without (1) and after (2) electroporation
(Sack et al. 2009). The maximum phase shift was approximately located at approx-
imately 300 kHz, and the remaining phase shift for electroporated sample indicates
that not all cells have been opened. Good correlations were observed between the
measurements of the complex impedance and color intensity of the must. However,
the qualitative parameter for characterization of disintegration index on the base of
phase shift was not proposed.
Electrical conductivity
( σ, S/m) 0.3
0.2
1
2
0.1 3
10 104 105 106 107
Frequency (f, Hz)
15
Phase shift (j °)
10
0 3
10 104 105 106 107
Frequency (f, Hz)
Fig. 11 Frequency dependency of specific conductivity σ and phase shift φ for mash from
Muscatel grapes without (1) and after (2) electroporation (specific energy:42 kJ/kg) (Compiled
from (Sack et al. 2009))
Diffusion Coefficient
The level of electroporation induced by PEF treatment can be also estimated from
studies of mass transport characteristics in solid–liquid extraction and convective
drying processes. The diffusion coefficient disintegration index ZD was defined as
(Barba et al. 2015):
Z D ¼ ðD Di Þ=ðDd Di Þ; (6)
where D is the diffusion coefficient and the subscripts “i” and “d” refer to the values
for intact and completely electroporated tissue, respectively.
As for electrical conductivity low-frequency method this method also requires the
knowledge of Dd. It can be estimated from the data obtained in the limit of long
treatment time (tPEF 0.1–1 s) and high electric field strength (E > 1000 V/cm) or
the data for the freeze-thawed material. So, the value of Dd cannot be uniquely
determined. For example, drying experiments with potato tissue have shown that Dd
value of a freeze-thawed tissue is noticeably higher than Dd of a PEF-treated tissue
with Zc 1.
Texture
The textural experiments can be rather useful for qualitative characterization of

PEF-induced changes, and they were frequently used for characterization of PEF-treated
tissues (Barba et al. 2015; Fincan and Dejmek 2003). The stress–deformation and
relaxation tests can be applied to analyze the texture of intact and PEF-treated tissues.
The differences in the pressure-displacement curves P-e of PEF-treated and intact tissues
were observed in many experimental works.
For example, after PEF treatment (E = 1.1 kV/cm, tPEF = 0.1 s), the tissues
(carrot, potato, and apples) lose a part of their textural strength, and their elasticity
modulus and fracture stress decrease with increase of the conductivity disintegration
index. PEF-induced changes in elasticity modulus Gm and fracture stress PF were
significantly smaller than those observed in the freeze-thawed and thermally
(T = 45 C, 2 h) pretreated tissues. The tissue structures were less affected by the
PEF treatment as compared to the freeze-thawing or heating. This conclusion was
confirmed by the textural studies of sugar beet tissue treated by PEF (Shynkaryk
2006). Linear dependency between the fracture pressure and the value of ZC was
observed for PEF-treated apple samples. The PEF impacts on compression and
solid–liquid expression of different vegetable tissues were also extensively studied
(Barba et al. 2015). PEF-treatment accelerated the stress relaxation of tissues. The
relaxation behavior reflected membranes damage, and it was sensible to the state of
the cell walls and turgor pressure. In order to quantify electroporation, the texture
disintegration index was introduced as
Zt ¼ ðF Fd Þ=ðFi Fd Þ; (7)
where F is the force measured after sufficient time of textural relation (e.g., at
t = 50 s), and subscripts i and d refer to the values of intact and completely damaged
tissue, respectively. The force relaxation tests for PEF-treated and freeze-thawed
tissues had shown that at t 50 s the changes in values of force are unessential.
The studies evidenced that PEF-treated tissue with high level of disintegration
(Zc 1) has stronger texture compared with the texture of maximally disintegrated
freeze-thawed tissue. Texture disintegration index Zt is convenient for understanding
of the viscoelastic properties of tissue. Unfortunately, the textural parameters reflect
the PEF-induced changes in an indirect manner and the definitive relations between
these parameters and fraction of damaged cells Z are still absent. Moreover,
the results of textural tests can be dependent on the mode of textural experiment.
For example, they can different for uniaxial (1d) and three dimensional (3d) tests,
and each tissue requires careful tests and comparison with other characterization
techniques.
Note that atomic force microscopy (AFM) technique can be also used for
characterization the texture and local mechanical properties of food tissues
(Cybulska et al. 2013). Particularly, application of AFM technique demonstrated a
decrease in membrane elasticity (living CHO cells) by 40% due to electroporation
(Chopinet et al. 2013).
Fig. 12 Acoustical disintegration index Za versus the time of PEF treatment tPEF for different fruit
and vegetable PEF-treated tissues. PEF treatment was applied to whole unpeeled samples at electric
field strength E = 200 V/cm and pulse duration ti =100 μs in an aqueous medium (0.06 S/m, tap
water) (Grimi et al. 2010)
Acoustic Tests
The acoustic technique is traditionally used for characterization of the quality of

different agricultural products. The acoustical index of firmness S (or stiffness
coefficient) shows good correlations with the quality and maturity of fruits
and vegetables. Acoustic techniques have been applied for characterization of
the different fruit and vegetable PEF-treated tissues (Grimi et al. 2010). The
acoustic disintegration index Za was defined in a similar manner to electrical
conductivity disintegration index Zc (Eq. 4) and diffusion disintegration index, Zd
(Eq. 6):
Z a ¼ ðS Si Þ=ðSd Si Þ, ; (8)
where subscripts i and d refer to the indices of firmness of the intact (untreated) and
completely damaged tissues, respectively.
Examples of acoustic disintegration index Za versus time of PEF treatment tPEF
for different fruit and vegetable tissues are presented in Fig. 12. Acoustic technique
Table 1 Advantages and disadvantages of different techniques for estimation of PEF-induced

electroporation in food tissues
Technique Advantages Disadvantages
Optical The direct visualization is very It is destructive technique;
microscopy attractive, demonstrable and decisive; The computerized visual
The precise mechanism and spatial fractionalization for counting of the
distributions of different components cells is not easy task;
can be revealed; The preparation of the samples,
It can be applied to the visual study of mounting of the epidermis and
different processes: diffusion, osmotic staining with dyes requires special
impregnation, drying, and freezing strategies, individual adaptation of pH
and conductivity of used solutions for
each selected type of the material
Electron SEM/TEM technique is very It is destructive technique;
microscopy illustrative and powerful for It requires application of complex
quantification the morphological individual methods of specimen
properties of membranes and cell preparation (staining, fixation,
walls dehydration, ultrathin sectioning
etc. . .)
Electrical This electrical impedance technique is It is destructive technique;
characteristics straightforward, relatively simple and The changes in distributions of the air
does not require expensive and moisture content inside the tissue
equipment; can affect the results;
It is express and can be easily applied The direct proportionality between
for continuous monitoring of mechanical disintegration and
electroporation during PEF treatment measured electrical characteristic is
questionable;
The estimated values of disintegration
index depend on the procedure used
for the determination of electrical
conductivity of completely damaged
tissue, σ d, (freezing, heating or
complete electroporation), and from
the temperature of the medium
Diffusion This mass transfer technique is It is destructive technique;
coefficient straightforward, relatively simple and The direct proportionality between
does not require expensive disintegration and measured
equipment; diffusivity characteristic is
It is useful for the estimation of PEF questionable;
impact on solute and moisture The estimated values of disintegration
diffusivities in solid–liquid extraction index depend on the procedure used
and drying experiments for the determination of solute
(moisture) diffusivity in completely
damaged tissue, Dd, (freezing, heating
or complete electroporation) and
temperature of the medium
Texture It is straightforward and relatively It is destructive or partially technique;
simple; The textural parameters reflect the
It is useful for qualitative PEF-induced changes in an indirect
determination of PEF-induced manner and there is no
changes in mechanical characteristics: direct proportionality between
disintegration and measured
(continued)
Table 1 (continued)
Technique Advantages Disadvantages
cutting strength, stress–deformation characteristic;
and relaxation The results of the textural tests depend
on the mode of textural experiment
(e.g., they are different for uniaxial
(1d) and three dimensional (3d) tests)
Acoustic It is straightforward and relatively Justification of direct proportionality
test simple; between electroporation
It is nondestructive and applied to disintegration index and measured
whole roots or fruits, e.g., sugar beet, acoustic characteristics is still
potato, tomato, apple required
allows estimations of the whole unpeeled samples. It can be important when fruits
and vegetables are processed as whole unpeeled samples in existing industrial
examples of PEF application to whole samples, e.g., sugar beet, potato, tomato.
Conclusions
The different experimental techniques for estimation of PEF-induced electroporation

in food tissues were proposed in recent years. Their advantages and disadvantages
are summarized in the Table 1. Practically all techniques are destructive (invasive),
and they can violate the structure of the tissues and can affect the evaluated index of
disintegration by applied procedure of measurements.
The details of the sample preparation and individual adaptation for each selected
type of the material can be also very important. Unfortunately, there is no linear
dependence between fraction of damaged (electroporated) cells Z and any of pro-
posed indexes Zc, Zd, Zt, or Za. The correlations between different indexes, for
example Zc and Zd, and Zc and Za, were discussed for some foods (Barba
et al. 2015), but further studies for different types of food materials are still required.
In general, for characterization of electroporation, one can advice using the combi-
nation of different techniques and compare them using the same PEF-protocols and
the same food materials.
Some other methods to detect electroporation phenomena can be used, including
different tomography techniques (NMR, impedance, microwave) (see,
section Cross-References). Such techniques require more qualified service, and
they were only applied in particular studies aimed for elucidation of electroporation
mechanisms.
Acknowledgments The authors appreciate the support from the COST Action TD1104
treatments).
Cross-References

▶ Detection of Electroporation in Microbial Cells: Techniques and Procedures
▶ Diffusion Weighted Magnetic Resonance Imaging for Detection of Tissue Elec-
troporation In Vivo
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Determination of Pulsed Electric Fields
Effects on the Structure of Potato Tubers 77
Indrawati Oey, Farnaz Faridnia, Sze Ying Leong, David J. Burritt,
and Tingting Liu
Abstract
Potato tubers are often considered by food processors as being “homogenous”
plant structures. However, tubers are complex organs made up of many tissue
types, including a complex vascular system, each with different functions and
properties that can affect the influence of pulsed electric fields (PEF) on cell and
tissue structures. Many research investigations evaluating the effects of PEF on the
microstructure of potato tuber have used samples that have been mechanically
fragmented prior to PEF treatment and unfortunately the internal structure of
vascular system inside the tuber has been ignored during sample preparation,
PEF treatment, and microstructure analysis. This chapter discusses different meth-
odologies that have been used to assess the effect of PEF treatment on potato
tubers, including electrical impedance and conductivity measurements, cell
I. Oey (*)
Department of Food Science, University of Otago, Dunedin, New Zealand
e-mail: indrawati.oey@otago.ac.nz
F. Faridnia (*) • S.Y. Leong (*)
e-mail: farnaz.faridnia@otago.ac.nz; sze.leong@otago.ac.nz
D.J. Burritt (*)
e-mail: david.burritt@otago.ac.nz
T. Liu (*)
e-mail: liuti506@student.otago.ac.nz

DOI 10.1007/978-3-319-32886-7_151
1490 I. Oey et al.
viability staining, enzymatic browning, and microscopic techniques such as light

microscopy, fluorescence microscopy, scanning electron microscopy, energy dis-
persive spectroscopy, and also magnetic resonance imaging. The limitations of
each technique and how these techniques have been used in published studies are
discussed. In addition, this chapter demonstrates how sample pretreatments such
as peeling or cutting influence the assessment of PEF effect on the microstructure
of the tuber and how several techniques should be used and combined in order to
understand how the PEF effects are distributed throughout the whole potato tubers.
Keywords
Potato • Tubers • Microstructure • Staining • Browning • Microscopy • Magnetic
resonance imaging • Electroporation
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1490
An Overview of the Use of Electrical Impedance and Electrical Conductivity
Measurements to Determine Cell Membrane Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1492
Determination of the Distribution of the Pulsed Electric Field Effect Across Potato
Tubers Using Cell Viability Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1496
Determination of the Distribution of Pulsed Electric Field Effect Across the Tuber
Using Enzymatic Browning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1498
Microscopic Techniques Used to Assess the Microstructure of Potato Tubers After
Pulsed Electric Field Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1501
Light Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1501
Fluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1502
Scanning Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1502
Combining Scanning Electron Microscopy and Ion Migration Using Energy
Dispersive Spectroscopy Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1503
Determination of the Electric Field Distribution During electroporation Using
Magnetic Resonance Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1504
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1506
Introduction
Pulsed electric fields (PEF) technology offers promising opportunities to improve

product quality in the potato industry. PEF technology induces electroporation,
leading to increased cell membrane permeability which also affects tissue structure
in a way to soften potato tissues resulting in better cutting quality and accuracy
(Ignat et al. 2015). In addition, electroporated potato cells are more prone to release
of intracellular compounds such as reducing sugars and low-molecular substrates
involved in the Maillard reaction and hence reduce the tendency of fried potatoes to
brown (Janositz et al. 2011). How PEF processing affects the potato tuber, which is a
complex heterogeneous structure, is not completely understood. Important questions
still to be addressed include whether the effects of PEF treatment are distributed
77 Determination of Pulsed Electric Fields Effects on the Structure of. . . 1491
Potato tuber
Stem end
Outer medulla
Medullary ray
Pith
(Inner medulla)
Skin
Vascular ring
Bud end
Fig. 1 Cross section of a potato tuber showing the internal structure
uniformly across the whole tuber and which of the cell types that make up tubers are
most affected by PEF.
Being a living intact plant organ, a potato tuber (Solanum tuberosum) is made up
of several tissue types with different cellular properties (Fig. 1) and functionalities.
The stem tubers of S. tuberosum have a vascular system, in which the vascular
bundles form a ring beneath the skin and the pith (or inner medulla) forms branched
medullary rays that terminate in vegetative buds (Reeve et al. 1969). The pith is
comparatively low in starch, but is high in free amino acids, resulting in the pith
having a relatively high electrical conductivity. Sweet potato (Ipomoea batatas) is a
distant relative of S. tuberosum that produces tubers from the root system. The root
tubers of I. batatas plants are structurally different than the stem tubers of
S. tuberosum and initially are structurally similar to the roots of carrots. However,
during the transitional process to be a storage structure, additional cambia within the
secondary xylem produce mostly storage parenchyma cells in both directions,
resulting in xylem and phloem composed mostly of storage parenchyma cells,
which make up most of the ground tissues (Fig. 2).
From the above description of potato and sweet potato tubers, it is clear that tuber
should not be considered to be “homogenous” plant structures, and this fact needs to
be taken into consideration when investigating PEF-induced structural changes in
plant cells, tissues, and organs. To date, previous work evaluating the effects of PEF
on potato tuber microstructure has used tuber samples that have been mechanically
fragmented prior to PEF treatment, e.g., slices (Mhemdi et al. 2013), cylinders
(Boussetta et al. 2013), strips (Shayanfar et al. 2013), or cubes (Ignat et al. 2015).
These forms of sample preparation result in potato tuber tissues that have already
1492 I. Oey et al.
Fig. 2 Cross section of a sweet potato tuber showing the internal structure
experienced considerable tissue damage prior to PEF treatment, which could mask
the nature of any PEF-induced changes. More importantly, these earlier works did
not consider the complex nature of plant organs and neglected the fact that potato
tubers are heterogeneous structures.
Evaluation of cell electroporation due to PEF treatment in multilayer biological
tissues such as potato tubers is challenging. Most research has been based on theories
derived from experiments carried out on model systems, such as liposomes, planar
bilayers, and phospholipid vesicles. These models are undoubtedly useful when
explaining the exact mechanisms associated with irreversible and reversible electro-
poration and/or electrically induced membrane breakdown (Zimmermann 1986).
However, there is a need for simple high-throughput experimental approaches to
assess cell membrane permeability and integrity in multicellular plant tissues and to
study how electric fields and their effects are distributed throughout complex plant
tissues and organs. This chapter will discuss different experimental approaches to
assess the effect of PEF treatment on the structure of potato tubers. The limitations of
each technique will be discussed and examples about how these techniques have
been used in the published literature are given.
An Overview of the Use of Electrical Impedance and Electrical

Conductivity Measurements to Determine Cell Membrane
Electroporation
The measurement of electrical impedance and electrical conductivity considers plant

tissue to be an electrical system (circuit) with both the resistive and capacitive
properties (Zhang and Willison 1993). When plant samples are treated with PEF,
the sample is considered to be an “electrical object,” the electrical properties of

which can be changed by PEF treatment (Angersbach et al. 2002). An increase in the
cell membrane permeability due to PEF would lower membrane resistance allowing
the cell contents to pass through the membrane into the intercellular spaces, thus
changing the impedance of the plant cell (Angersbach et al. 2002). Therefore, the
impedance of a plant sample is a useful parameter to immediately assess the extent of
cell membrane electroporation or cell damage due to PEF. Determining the extent of
cell damage by measuring the impedance has been reported for potato tubers
(Gachovska et al. 2015) and other plant samples (Donsì et al. 2010), and is a well-
established technique used to gain an overview of the condition status of a plant
sample (Zhang and Willison 1993).
Cell membranes exhibit frequency-dependent electrical properties (Angersbach
et al. 2002). At low frequencies (1 kHz), the cell membrane acts as a capacitor to
prevent the flow of electric current in the intracellular medium, which is a classical
example of ohmic-capacitive behavior. At high frequencies (10–100 MHz), the cell
membrane shows no further resistance to electric current flow, resulting in a pure
ohmic behavior in which the absolute value of impedance does not vary greatly
between intact cells and cells with ruptured membranes. Taking the advantage of the
changes in the electrical properties of plant samples at low and high frequencies,
impedance measurements can be performed. At the laboratory scale, a plant sample
is placed in a test vessel consisting of two parallel plate electrodes or cylindrical
electrodes. The electrodes are connected to a generator that produces a sinusoidal
waveform at a specified effective voltage (usually 1–5 V, peak-to-peak) with a
frequency ranging between 1 kHz and 50 MHz. A frequency-impedance spectrum
is then obtained, revealing the transition of the untreated and PEF-treated samples
from an intact to ruptured state within the frequency range investigated (Donsì
et al. 2010).
The electrical impedance (unit in Ohm, Ω) is the ratio of the voltage drop across
the sample and the current crossing it, when the measurement is taken (Eq. 1).

Vs
Cell impedance, Z ðΩÞ ¼ Rf (1)
Vf 1
Rf (Ω) is the reference resistor that is connected in series to the impedance instrument
generating the sinusoidal voltage, while Vs and Vf represent the peak-to-peak voltage
of the sinusoidal waveform, i.e., voltage applied and the voltage measured on the
reference resistor, respectively. As an alternative, measuring the electrical conduc-
tivity response of the sample at the low-to-high frequency range is also a good
method to evaluate a change in cell impedance due to PEF (Angersbach et al. 2002).
To allow a better interpretation of the impedance result and to quantify cellular
damage caused by independent PEF treatments, a polarization coefficient termed the
cell disintegration/permeabilization index (Zp) has been introduced (Lebovka
et al. 2002). This coefficient is calculated based on the ratio of the measured
impedance or electrical conductivity measured at low (Z1 1 kHz) and high
frequencies (Z2 10 MHz) (Eq. 2).
1494 I. Oey et al.
Zp ¼ ðZ1untreated Z 1PEF-treated Þ=ðZ1untreated Z2PEF-treated Þ (2)
Based on this equation, Zp = 0 and Zp = 1 represent the intact and maximally

damaged cells, respectively. There is strong evidence that a higher PEF treatment
intensity always leads to a higher degree of cell membrane permeabilization.
Another way to interpret the impedance result is to determine the degree of damage
(Sd), which is the ratio of impedance before and after PEF treatment. In the case of
potato tubers, the use of Sd to compare the effectiveness of increasing pulse number
on potato tuber cell damage was first implemented by Gachovska et al. (2015). In
general, the degree of cell damage due to PEF can be more clearly observed at low
frequencies (1 kHz). This is because where PEF-induced cell electroporation
occurred, the ion concentration in the extracellular spaces would be already high
enough to contribute to a change in the impedance values at low frequencies.
Generally, permeabilization of cell membranes causes diffusion of intracellular
cell components, containing various ionic species, from the intracellular to the
extracellular space. The measurement of electrical conductivity (σ, unit in S/m)
considers the total ion leakage from plant cells. This is also another simple method
to estimate the degree of cell damage and to generate a cell disintegration/permeabi-
lization index (Zp) for plant tissues after PEF treatment (Eq. 3).
ðσPEF σintact Þ
Zp ¼ (3)
σdamaged σintact
σ is the measured electrical conductivity at low frequencies (usually 0.5–1 kHz) of

PEF-treated samples (σPEF), intact cells (untreated samples, σintact), and maximally
damaged cells (σdamaged). Similar to Eq. 2, Zp = 0 and Zp = 1 represent the intact
and totally damaged cells, respectively, on the basis of electrical conductivity
disintegration. In all cases, the electrical conductivity increases with PEF treatment
intensity. When the applied PEF treatment is sufficient to cause maximal cell
membrane permeabilization (Zp 1), no further increase in cell damage with
more intense PEF treatments can be obtained (Lebovka et al. 2002). This is helpful
when defining optimal PEF treatment conditions to achieve the desired level of cell
damage in the final product, without excessive energy consumption.
In an attempt to improve PEF processing efficiency, previous works have
employed the measurement of electrical conductivity to (i) predict the characteristic
damage time of potato tubers, with the PEF treatment condition required for
attaining one half of the maximum Zp value (i.e., Zp = 0.5) (Lebovka et al. 2002),
(ii) recommend the combined use of mild preheating (<50 C) and low electric field
strengths (<0.1 kV/cm) that guarantee high cell permeabilization degree for potato
tubers (Lebovka et al. 2005), and (iii) predict the viscoelastic properties of potato
tuber tissues (Fincan and Dejmek 2003). Moreover, through the use of electrical
conductivity measurement, it is clear that the efficiency of PEF-induced cell damage
of potato tuber tissues can depend on temperature (Boussetta et al. 2013) and tissue
structure, cell size, the electrical properties of cell fluids and membranes, and the
apparent density of the potato tissue during PEF (Mhemdi et al. 2013).
In most research aimed at using PEF for structural modifications of potato
tissues, measurement of electrical conductivity is typically performed during the
pause period, Δtt, between two consecutive series of pulses at an optimal fre-
quency (i.e., 0.1–1 kHz) that would minimize polarization effects on the elec-
trodes and the sample (Lebovka et al. 2002). More recently, Faridnia et al. (2015)
suspended whole potato tubers (either untreated or PEF treated) in an osmotically
neutral (isotonic) solution of mannitol (0.2 M) that allowed real-time monitoring
of the leakage of ions from tubers, with changes in electrical conductivity deter-
mined in the solution surrounding the potato tubers as a function of time, up to 8 h
post-PEF.
However, the measurement of electrical conductivity and electrical impedance
has limitations including:
• The value of σdamaged (from Eq. 3) is established by measuring the conductivity of

freeze-thawed potato tissue or PEF-treated potato tissue at high electric field
strengths (E 1 kV/cm) and long treatment durations (tPEF 0.1–1 s). As a
result, the value of σdamaged is not well defined and the real degree of damage to
these tissues can be ambiguous in some cases, since there is no consensus
achieved on a standard protocol of treatment to obtain the totally damaged cell
value.
• A destructive technique where a totally damaged sample needs to be obtained for
Zp determination (based on Eqs. 2 and 3).
• Lack of selectivity with respect to the different ions that contribute towards the
change in conductivity.
• High sensitivity towards the air spatial redistribution (e.g., the porous structure of
a plant tissue and air content inside the tissue) that eventually influences mea-
surement accuracy.
• Ion leakage takes time since PEF induces many long-lasting transition processes
(e.g., mass transfer, moisture, and air redistribution among the microscopic pores
of the matrix surrounding the cells and resealing of cell membranes) that may take
from seconds to hours after PEF to occur and hence measurements cannot be
performed immediately after PEF.
• A lack of suitability for solid plant-based material that are known to have thick
skins (e.g., sweet potato) due to the protective effect of the dermal cells, for
which it can be very difficult to establish totally damaged cells for Zp
determination.
• Plant materials often need to be fragmented before PEF treatment and measure-
ment, which makes interpretation on the degree of cell damage in intact samples
difficult, since the current flow during electrical impedance or conductivity
measurement will also depend on the tissue structure including the degree of
vascularization in the fragmented samples.
1496 I. Oey et al.
Determination of the Distribution of the Pulsed Electric Field

Effect Across Potato Tubers Using Cell Viability Staining
While the electric field generated between parallel plate electrodes is often consid-
ered homogenous, it may not necessarily produce homogenous electric field in the
sample/plant material being treated. Plant-based food samples such as potato tubers
are heterogeneous material that consists of irregular shaped cells and a complex
tissue structure, including variations in vascularization. During electroporation, a
current is forced to flow across different tissue layers and those tissues with lower
resistivity will be exposed to lower electric fields (Davalos et al. 2003). This
implies that some tissues are less prone to electroporation/damage than others
and so could survive PEF-induced electroporation. Therefore, it is critical to use
experimental techniques that reveal the cell viability status of PEF-treated potato
tuber tissues. In this regard, cell viability assays, e.g., cell viability staining can be
used to quickly determine whether the cells that make up complex tissues/organs
remain viable after PEF. Viability staining is also a useful technique to track the
distribution of PEF-induced effects across a whole potato tuber composed of
different tissue types.
Neutral red dye, as a staining agent, has been used in previous studies to estimate
cell viability in plant tissues after various PEF treatments (Ersus and Barrett 2010).
Neutral red is an uncharged and unprotonated dye in alkaline solution that diffuses
across plant membranes due to its lipophilic nature. The dye crosses the tonoplast
membrane of the vacuole, where it ionizes and accumulates in the acidic vacuolar
medium, appearing as dark red-colored vacuoles in intact/viable cells (Ersus and
Barrett 2010). Penetration of this dye into the tissue depends on the integrity of the
cell membranes and the capacity to maintain pH gradients (Repetto et al. 2008).
Recently, a standard method of cell viability staining for plant cells has been
adapted to visualize areas where a potato tuber still contained living cells after a PEF
treatment (Faridnia et al. 2015). Potato tissues were stained by completely immers-
ing samples in a water-soluble tetrazolium salt (2, 3, 5-triphenyltetrazolium chloride)
solution (0.5% (w/v)). Reduction of the tetrazolium salt by oxidoreductase enzymes
to the insoluble red formazan is directly proportional to the number of living or
metabolically active cells (Berridge et al. 2005). The cellular oxidoreductase
enzymes are present in the mitochondria of living cells, but absent in dead cells,
will catalyze the conversion of the yellow tetrazolium salt to the red formazan. In
other words, cells without formazan deposits are considered to be dead cells with no
metabolic activity.
Based on the tetrazolium cell viability staining assay, it was found that the
presence of different cell and tissue types within the potato tubers, having particular
electrical and topological properties, has a pronounced influence on the effectiveness
of a PEF treatment. The electric current appeared to flow through the pith and
towards each vegetative bud (Faridnia et al. 2015). Moreover, the orientation of
the tuber with respect to the electrodes also influenced the response of the potato
tubers to PEF treatments. When the stem end of the tuber (that was attached to the
Fig. 3 Representative photographs of potato slices obtained from untreated and PEF-treated
(at different electric field strengths) potato tubers stained with tetrazolium salt. Slice thickness
(1–2 mm). *FE facing the electrode: the stem end was arranged so as to be perpendicular to the high
voltage electrode (parallel to the electric current); NFE not facing the electrode
stolon) faced to an electrode, the effect is more pronounced compared to an orien-

tation where the stem end is not facing the electrodes. The skin or periderm of a
potato tuber was also shown to have a protective effect against the electric fields
applied. Peeled potato tubers showed a decrease in the proportion of viable cells
compared to unpeeled potato tubers, and this effect was enhanced when the PEF
treatment intensity was increased. Therefore, through the use of cell viability
staining technique, the disruptive pattern of current flow across and within the tissues
that make up a potato tuber was shown not to be uniform and depends on sample
pretreatments (peeled versus unpeeled), the electric field strength applied, and tuber
orientation within the treatment chamber (Fig. 3). This staining technique can be
combined with image analysis to calculate quantitatively the proportion of dead and
viable cells and has the potential to map the global distribution of electric current
flow across a whole potato tuber.
However, the limitations of this staining technique are as follows:
• The cell viability assay using tetrazolium salts is only suitable for plant materials
with white or light yellow flesh. This is due to the fact that oxidoreductase
enzymes in the living cells will react with tetrazolium salt to form red formazan,
and hence the presence of natural red color or similar pigment of the tuber itself
1498 I. Oey et al.
will interfere with the analysis to differentiate areas of viable cells with natural
pigments.
• It is important to assess the cell viability status of the whole potato tuber prior to PEF
treatment to map the metabolically active/viable regions of the tuber and to ensure
factors other than the PEF have not influenced the tubers prior to PEF treatment. A
high level of replication of potato tubers treated with same PEF treatment conditions
is also required to take into consideration biological variation between the tubers.
• This technique necessitates careful handling as the time delay between PEF treat-
ment and staining should be minimized to avoid environmental influences that could
interfere with the chemical reaction involved during staining. Otherwise, staining
artifacts could lead to an underestimation of the influence of PEF on cell viability.
• Dye should be taken up by plant cells through passive diffusion, and the staining
duration will depend on sample size and type, which will differ between plant
species and cultivars.
Determination of the Distribution of Pulsed Electric Field Effect

Across the Tuber Using Enzymatic Browning
Potato tubers contain the enzyme polyphenoloxidase (PPO) and phenols, with both
compounds located in separate cell compartments, i.e., plastids and vacuoles,
respectively. PPO activation occurs instantly when these compartments are disrupted
after tissue wounding. The pores formed due to PEF treatment facilitate the interac-
tion between PPO and its phenol substrate allowing the accumulation of brown
pigments. Thus, observing the extent of the naturally occurring enzymatic browning
on the potato tissues is a simple approach to assess how PEF-induced changes in cell
electroporation may affect the movement of compounds between cellular
compartments.
Castellví et al. (2016) studied the distribution of PEF-induced irreversible elec-
troporation in a potato tuber by analyzing the enzymatic browning area. In their
study, a pair of parallel needle electrodes (diameter 1.27 mm, length 15 mm) was
inserted inside potato tubers, in which the distance between the two electrodes and
the length of the electrodes were smaller than the tubers. As a result, the PEF-treated
tissue was only a portion of the whole potato tuber. Following PEF treatment at
various intensities, the treated potato tubers were sliced into thin layers. An image of
each slice was taken and the individual images were stacked, using an image
processing software, to generate a three-dimensional representation of the tuber. A
three-dimensional model was computed based on the stacked images to illustrate the
position and volume of tissue affected by PEF inside a whole potato tuber. Results
from this novel experimental approach showed an almost linear increase of
PEF-induced darkening volume as the applied voltage between electrodes is raised.
Using this method of visualization, it is difficult to interpret images of fragmented
samples without a clear indication of how the electric current is delivered inside the
potato tubers. An example of PEF-induced enzymatic browning is depicted in Fig. 4.
Figure 4 illustrates nonhomogenous enzymatic browning when the potato tubers are
Enzymatic browning after 24 hours
No PEF treatment PEF: 2.3 kv/cm, 4.7kj/kg

Surface browning
Browning within samples
Medullary ray (extending from the pith) Medullary ray (extending from the pith)
Fig. 4 Representative photographs of enzymatic browning after 24 h for different potato rectan-
gular prisms treated with PEF treatment (at 2.3 kV/cm, pulse width of 20 μs, and 50 Hz). The
authors acknowledge Steffi Supangkat for providing the potato pictures
cut into tissue blocks/rectangles of the same size, immersed in water that act as the
electric current transferring medium, and placed between electrodes inside the PEF
chamber. These pictures illustrate that the tissues present within the tuber samples
varied greatly in the browning reactions, with strong pith-associated enzymatic
browning clearly visible. Hence, the tissue composition should be completely
controlled or carefully considered when interpreting the images of samples showing
enzymatic browning.
Rather than using potato blocks, the distribution pattern of electric fields
visualized using enzymatic browning should be more representative if unpeeled
or intact potato tubers are used and the tubers are positioned inside the PEF
chamber with the same orientation for all the PEF parameters tested. However,
the weakness of this approach is that a large PEF chamber is required to fit large
potato tubers and the use of a large chamber can restrict the range of electric field
strengths that can be used.
In the example shown in Fig. 5, the distribution of PEF-induced browning is
observed in sweet potato tubers that were PEF-treated as a whole, sliced, and then
incubated at room temperature. Images of each potato slice were then taken imme-
diately after slicing and every 30 min. In addition to image analysis of PPO-induced
browning, PPO activity was determined in the sweet potato tubers immediately after
PEF treatment. Results showed that the browning caused by PEF increased in both
area and depth of color with increasing PEF intensity. In addition, it was observed
that browning was most pronounced in the medullary rays and surrounding tissue.
1500 I. Oey et al.
0 min 30 min 60 min 120 min

Control
0.5kV/cm
1.2 kV/cm
Fig. 5 Representative photographs of enzymatic browning as a function of reaction time for sweet
potato slices obtained from untreated and PEF treated (at different electric field strengths) sweet
potato tubers
It is important to note that while this approach is promising as a tool to investigate

PEF promote enzymatic browning of cut potato tubers, the exact distribution pattern
of PEF-induced damage throughout the whole tuber is difficult to determine as:
• The extent of enzymatic browning depends on the distribution of phenolic

compounds and PPO enzymes in tubers. The amount of phenolic compounds
can differ between cultivars, between individual tubers, and between tissues
within the same tuber. For instance, in the sweet potato tubers, the stem end has
significantly more phenolic compounds than root end and middle part of the
tuber, and the flesh near to skin was reported to contain higher amount of
phenolics (Jung et al. 2011).
• PPO enzymatic activity can also be influenced by the presence of enzyme
inhibitors and enhancers (such as metal ions) and by the availability of oxygen.
These factors could mask potential enzymatic browning caused by PEF.
Enzymatic browning combined with cell viability staining could be used to assess
the PEF-induced cell viability within potato tubers. Following PEF treatment of
whole tubers at various intensities, the treated tubers are thinly sliced. One slice is
stained with tetrazolium salt and then the other slice closed to the one that is stained
with tetrazolium salt is incubated to allow enzymatic browning to occur. Figure 6
Fig. 6 Representative photographs of potato slices obtained from PEF treated (at 0.5 kV/cm,
50 Hz, pulse width of 20 μs) potato tubers indicating viable cells (red) after stained with 0.5 %
tetrazolium salt (top row) and enzymatic browning (bottom row)
shows that enzymatic browning does not occur in areas of the tuber where viable
cells (red) are detected, as these areas have no or limited cell damage. In addition,
slices from peeled potato tubers have more area of enzymatic browning and dead
cells than the unpeeled tubers.
Microscopic Techniques Used to Assess the Microstructure of

Potato Tubers After Pulsed Electric Field Treatment
Light Microscopy
This technique is based on the assumption that electroporated cell membranes will
partially lose their ability to function as a barrier and may allow the free move-
ment of cytoplasm contents out of damaged cells. For example, leaching of
red/purple anthocyanin pigments from electroporated red onion cells can be
observed using light microscope. Since potato tubers are made up of white storage
cells containing no colored pigments, this technique would only be of value for
the less commonly grown tubers with pigmented flesh. However, light micros-
copy combined with staining cells for viability could provide additional useful
information concerning the electroporation effects of PEF at the cellular level.
Fincan and Dejmek (2002) have made in situ observation of changes in plant cell/
tissue microstructure caused by PEF, which involved placing tissue samples
between electrodes, staining them with neutral red and directly observing the
cells using light microscopy during or after PEF treatments. Direct observation of
neutral-red-stained tissues allowed the detection and observation of individual
1502 I. Oey et al.
permeabilized cells. The result obtained using the technique supported the esti-
mated degrees of damage and cell permeabilization index (Zp value, Eq. 3)
determined by conductivity measurements.
Fluorescence Microscopy
Fluorescence microscopy has also been used to study the effects of PEF on the cells
of potato tubers by visualizing the diffusion of the fluorescent dye (FM1-43) through
the cells of potato tissues (Galindo et al. 2008). PEF, in the range of 0.1–0.5 kV/cm
(one 1 ms rectangular pulse) slowed down the diffusion of the dye through the cells
of potato tuber tissues, suggesting a significant decrease in cell wall permeability.
This phenomenon was thought to be related to the production of H2O2 by cell wall-
associated peroxidases, which resulted in increased cross-linking within the cell wall
matrix. Janositz et al. (2011) investigated the effect of a PEF treatment (E = 5 kV/cm,
n = 20, τ =100 μs, 2 Hz, d = 0.2 cm) on potato cell wall pectin using a microscope
to evaluate direct cell wall structural changes during PEF treatment. Although it was
observed that the cell wall was affected by PEF treatment, it was not clear whether
cell wall components were changed directly due to the PEF treatment or due to cell
membrane disintegration and the subsequent release of cell contents into the cell wall
from the cytoplasm.
Scanning Electron Microscopy
Cryogenic scanning electron microscopy (cryo-SEM) is a useful tool to observe cell/

tissue microstructure as it does not require pretreatment steps, such as staining and/or
chemical fixation, thereby reducing preparation time and minimizing artifacts. Faridnia
et al. (2015) studied the effect of PEF treatment at different electric field strengths on
potato tuber cells using cryo-SEM. While no significant differences were observed
between untreated and samples treated at 0.2 kV/cm, noticeable changes in tissue
structure were observed when samples subjected to 0.5 or 0.7 kV/cm field strengths.
Tissue shrinkage, loss of plasma membrane functionality, and loss of turgor were found
to be greater at higher electric field strengths, compared to the lower field strengths
applied. Moreover, the observed distribution of permeabilized cells was found to be
nonhomogenous (Fig. 7). This technique can be used to view variation in the shape and
size of the cells and also the location of starch granules after PEF treatment.
One of the advantages of this technique is the preservation of the triphasic structure
of a sample with the distribution of solid, liquid, and gas in a state closer to its natural
state (James 2009). Another advantage is that large areas of an intact specimen can be
studied, both at low and high magnifications. SEM images also have a relatively good
depth of field, which is useful for viewing biological samples. When cutting specimens,
the use of a sharp razor blade is highly recommended to reduce excessive damage to the
surface of the specimen, and it is often necessary to carefully wash off any surface cell
debris remaining after cutting as they can interfere with SEM observations.
Fig. 7 Representative SEM images with magnified view (150) of potato tissue showing the thin,
transparent cell walls of several parenchyma cells and clusters of starch grains, untreated and treated
at different electric field strengths
However, the major drawbacks of this technique include:
• High operation cost

• Expensive equipment
• Choosing an appropriate specimen from each potato tuber for SEM observation
that is representative of PEF-induced effect
Combining Scanning Electron Microscopy and Ion Migration Using

Energy Dispersive Spectroscopy Analysis
Combining scanning electron microscopy and energy dispersive spectroscopy anal-

ysis (Cryo-SEM-EDS analysis) is a useful tool to study ion distributions within plant
tissues/organs and will help to give a better understanding of how the ions migrate
from cells within a tissue. Recently, it has been used to investigate potassium ion
(K+) distributions in potato tubers following PEF treatments at different electric field
strengths (Faridnia et al. 2015). Potassium ion retention has successfully been related
to cell integrity after PEF. By means of SEM-EDS analysis, it was demonstrated that
PEF, at certain electric field strengths, affects cell permeability within the potato
tuber in a nonuniform manner. The heterogeneous structure of potato tubers and the
resultant heterogeneous electrical conductivity result in an asymmetric distribution
of the electric field and can influence ion distribution within tissues. Hence, the
combination of cryo-SEM and EDS analysis is another method that can be used to
1504 I. Oey et al.
give important insights with respect to the within organ/tissue uniformity of the PEF
response. EDS, when combined with cryo-SEM, can provide elemental analysis on
areas as small as nanometer in diameter for a range of elements present in a sample.
Hence, EDS analysis can also be used to determine the elemental composition of
individual region or to map out the lateral distribution of elements across an entire
sample.
However, the main limitations of this combined technique are:
• High operational cost and expensive equipment.

• The size of the sample that can be used is no more than 10–15 mm in diameter;
therefore selecting an appropriate specimen from each potato tuber that is repre-
sentative of PEF-induced effect is very crucial.
• The voltage setting used for EDS analysis should be standardized between the
measurements.
Determination of the Electric Field Distribution During

electroporation Using Magnetic Resonance Imaging
Nuclear magnetic resonance (NMR) spectroscopy technology has been proposed as

a method to assess cell membrane integrity in PEF-treated plant samples (Ersus
et al. 2010). The main benefit of this methodology is that it is a noninvasive and can
be used to obtain cell damage information for in-line optimization of PEF parame-
ters. Recent advancements in this technique have enabled the electric field distribu-
tion in a potato tuber during PEF treatment to be determined (Kranjc et al. 2016). The
approach is monitoring the electroporation process through magnetic resonance
imaging (MRI) complemented with electrical conductivity measurement by electri-
cal impedance (EIT) tomography, which has led to the development of magnetic
resonance electrical impedance tomography (MREIT) technique. In brief, MREIT
can predict electroporated areas in a PEF-treated potato tuber (Kranjc et al. 2016) by
reconstructing the electric field distribution, by means of electric current density and
electrical conductivity measurements, of the treated tissue during the application of
electric pulses, followed by numerical data analysis.
The PEF-treatment of potato tubers was conducted inside the MRI instrument to
enable treatment monitoring by means of electric field mapping. Electric field
mapping was acquired through current density imaging (CDI) and MRI, which
measures magnetic field changes caused by applied currents inside conductive
samples (Sersa et al. 1994). Magnetic field changes lead to a proportional change
in signal phase shift and subsequently, maps of image signal phase shift are acquired
by CDI with vector components of the induced magnetic field change extracted from
the phase shift stored in the acquired images. The electric current density in the
sample is then calculated from the magnetic field change vector maps using
Ampere’s Law. In the next step, the electric field distribution in the PEF-treated
potato tuber is reconstructed from CDI data by a MREIT mathematical algorithm.

For comparison with the experimental results obtained via MREIT, a numerical
model for the same sample/electrode configuration was constructed using the finite
element method (Kranjc et al. 2016).
Overall, MREIT is capable of determining regions of electric field distribution
corresponded to visibly darkened areas of PEF-treated potato tubers (due to enzy-
matic browning, see section “Determination of the Distribution of PEF Effect Across
the Tuber Using Enzymatic Browning”). Results from the work of Kranjc
et al. (2016) showed that the electric field was not distributed symmetrically in a
potato tuber, suggesting that the electrical conductivity was heterogeneous. This
finding also confirms that the complexity of tissue system found in tubers influences
the prediction and interpretation of result obtained using this methodology. MREIT
can be an efficient tool to detect regions within a potato tuber exposed to insufficient
electric field coverage of the applied PEF treatment. This near-real-time information
is of practical use to fine-tune PEF treatment parameters instantly to ensure effective
electric field distribution. Nonetheless, some limitations of using MREIT technique
are as follows:
• MREIT requires a skillful operator with a sound knowledge of the mechanism of

cell electroporation induced by PEF, and expertise in MRI.
• The ability to perform and understand the mathematical algorithm required for the
computational simulation analysis is also needed.
Conclusions
Different methodologies have been developed to understand the complex effect of

PEF on the structure of potato tubers. This chapter shows that sample preparation
prior to PEF treatment affects the result interpretation and sometimes creates arti-
facts. Sample preparation (e.g., cutting, slicing, peeling) results in damage to the
surface of potato tubers, which can be more pronounced than the PEF treatment.
Therefore, whole potato tubers can be used to eliminate or avoid this problem. The
response of potato tissue to the application of PEF was shown not to be uniform and
strongly dependent on the PEF intensity, the pattern of vascular system, potato size,
potato variety, potato age, tissue type, and the viability of cells within the tissue.
Smart combinations of different methodologies and sample preparation techniques
have been found useful to evaluate cell electroporation due to PEF treatment for
biological materials with complex structure such as potato tubers.
Acknowledgment The authors thank the Riddet Institute Center of Research Excellence Program
for providing PhD scholarship to Tingting Liu. This research was carried out as part of the Food
Industry Enabling Technologies Programme funded by the New Zealand Ministry of Business,
Innovation and Employment (contract MAUX1402).
1506 I. Oey et al.
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Part VII
Preclinical Development for Biomedical
Application of Electroporation
Richard Heller and Gregor Serša

Preclinical Studies on Electrochemotherapy
78
Gregor Serša, Masa Bosnjak, Maja Čemažar, and Richard Heller
Abstract
Electrochemotherapy is the combination of chemotherapy and local application
of electric pulses. Application of electric pulses, i.e., electroporation, is used as a
means to increase drug delivery to cells. Electrochemotherapy has the potential to
improve the efficacy of drugs that have impeded transport through the plasma
membrane. While enhancement has been demonstrated for some chemotherapeu-
tic drugs that were tested, only cisplatin and bleomycin have been evaluated
clinically. Recently, calcium electroporation has also entered clinical testing. The
increased cytotoxicity of bleomycin and cisplatin was demonstrated by exposure
of cells to electric pulses in vitro, and potentiation of their antitumor effectiveness
in vivo was also demonstrated. Preclinical studies have explored several different
parameters that could affect effectiveness of electrochemotherapy, such as the
dose and route of drug administration, timing of application of electric pulse, and
coverage of the tumor with electric field. These aspects were all evaluated on
different primary or transplanted tumors models in mice, rats, and hamsters. Most
importantly, the underlying mechanisms of electrochemotherapy were explored,
G. Serša (*) • M. Bosnjak (*)

e-mail: gsersa@onko-i.si; mbosnjak@onko-i.si
M. Čemažar (*)
University of Primorska, Faculty of Health Sciences, Izola, Slovenia
R. Heller (*)
School of Medical Diagnostics and Translational Sciences, Old Dominion University, Norfolk, VA,
USA
e-mail: rheller@odu.edu

DOI 10.1007/978-3-319-32886-7_45
from increased drug uptake to vascular effects and involvement of immune

system. These findings enabled swift translation of electrochemotherapy into
human and veterinary oncology.
Keywords
Electrochemotherapy • Bleomycin • Cisplatin • Cells • Experimental tumors •
Blood flow • Immune response
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1512
Preclinical Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1513
In Vitro Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1513
In Vivo Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1515
Mechanisms of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1519
Electroporation Mediated Drug Uptake in the Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1519
Antivascular Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1520
Induction of Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1520
Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1521
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1523
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1524
Introduction
Treatment of cancer requires a multidisciplinary approach since several different

types of treatment are used in combination, simultaneously or sequentially. In
general, there are three major treatment modalities available: surgery, radiotherapy,
and chemotherapy.
While surgery and radiotherapy represent good local ablative treatments,
chemotherapy is a systemic treatment modality. Drugs used for chemotherapy in
general easily cross the plasma membrane and are cytotoxic once they reach their
intracellular targets. However, there are still a few chemotherapeutic drugs that
have hampered transport through the plasma membrane. Those drugs may be
good candidates for electrochemotherapy. Electrochemotherapy is a local abla-
tive treatment modality combining chemotherapy and application of electric
pulses (i.e., electroporation) to the tumor tissue. Electroporation performed at the
time of the highest extracellular drug concentration facilitates the uptake of
hydrophilic chemotherapeutic drugs into the cells, which leads to good antitumor
effectiveness (Sersa et al. 2008a). Therefore, electrochemotherapy possesses the
qualities of an ablative technique, although it uses chemotherapeutics that can be
given systemically, but are in such low doses that they act only at the site of
electroporation and have no or minimal systemic effectiveness and negligible or
no side effects.
78 Preclinical Studies on Electrochemotherapy 1513
Preclinical Data
In Vitro Studies
Electroporation is used to facilitate transport of different molecules across the plasma

membrane with an aim to study different biochemical and pharmacological phe-
nomena. The application of external electric field changes the transmembrane
potential of plasma membrane, and when the threshold value is exceeded, structural
changes of the plasma membrane occur. Thus, the plasma membrane permeability is
increased (Yarmush et al. 2014). In the case of chemotherapeutic drugs, intracellular
drug accumulation is increased and it results in increased drug cytotoxicity. Since
electroporation is used to facilitate drug transport through the cell membrane only for
poorly permeant or nonpermeant molecules, electrochemotherapy is limited to
hydrophilic drugs and/or drugs that lack a transport system to enter the cell.
Among chemotherapeutic drugs several candidates were tested in vitro for potential
application in electrochemotherapy (Jaroszeski et al. 2000). Electroporation proved
to be an effective delivery method to introduce and increase intracellular concentra-
tion of several chemotherapeutic drugs, such as bleomycin, cisplatin, melphalan,
methotrexate, doxorubicin, etc. (Table 1). Among these, only bleomycin and cis-
platin were identified as potential candidates for electrochemotherapy of cancer
patients (Miklavcic et al. 2014).
Bleomycin is a hydrophilic molecule with very restricted transport through the
cell membrane, limited almost exclusively to endocytosis; its cytotoxicity after
electroporation can be potentiated up to 700 times (Orlowski et al. 1988). Several
other anticancer drugs were also tested, among them also melphalan, netropsin,
methotrexate, and Actinomycin D, but neither of them was as potentiated as
bleomycin after electroporation. Survival of different tumor cell lines was evaluated
in in vitro studies on electroporation combined with bleomycin or cisplatin.
Increased uptake of the molecules after exposure of cells to electric pulses was
also demonstrated. This extraordinary potentiation of bleomycin cytotoxicity was
systematically demonstrated in an in vitro study (Orlowski et al. 1988). In in vivo
studies the effect was documented on electrochemotherapy (Okino and Mohri 1987;
Mir et al. 1991), including clinical application (Sersa et al. 2008a).
Cisplatin similarly possesses hampered transport through the cell membrane,
which is mainly regulated by transporters controlling intracellular copper homeo-
stasis, Ctr1 for influx and ATP7A and ATP7B for efflux, and partially also by passive
diffusion. Electroporation increases cisplatin cytotoxicity by up to 70-fold (Sersa
et al. 1995). Therefore, extensive preclinical and clinical research focused on
electroporation predominately with bleomycin and cisplatin.
Studies of electrochemotherapy with bleomycin, cisplatin, and doxorubicin have
also been conducted on 3D multicellular tumor spheroids, to visually and molecu-
larly assess the effect of electrochemotherapy. Electrochemotherapy of all three
tested drugs led to macroscopic morphological changes of spheroids, tumor spheroid
Table 1 Drugs tested for in vitro and in vivo potentiation in combination with electroporation
(Adapted with permission from Miklavcic et al. (2014))
In vivo
Drug tested In vitro potentiation potentiation
Bleomycin Yes; 100–5000-fold Yes
Cisplatin Yes; 1.8–70-fold Yes
Calcium Yes; more hundred-fold Yes
Netropsin Yes; 200-fold –
Carboplatin Yes; 1.6–13-fold –
2-N-methyl-9-hydroxy-ellipticinium Yes; 4-fold –
(NMHE)
Vincristine Yes; 1.3–3.4-fold –
Actinomycin D Yes; 2–3-fold –
Cytarabine Yes; 2-fold –
Oxaliplatin Yes –
Platinum (II) complex 3P-SK No Yes
Platinum (II) complex PtAMP No –
Mitomycin C Yes but low; 1.3–1.4-fold –
Vinblastine Yes but low; 1.1–1.3-fold –
5-fluorouracil No or low; 1.25-fold –
Paclitaxel No –
Doxorubicin Yes but low; 0.67-fold –
Ruthenium-based anticancer agent KP1339 No Yes
Nimustine hydrochloride (ACNU) No –
Methotrexate No –
9-OH-ellipticine No –
Didemnin B No –
Melphalan No –
Mithramycin No –
Taxotere No –
Daunorubicin No –
Adriamycin No No
Etoposide No or ND –
Ancitabine ND –
Gemcitabine ND –
ND – drug was tested but the potentiation could not be determined due to methodological
limitations
growth arrest, and finally to its complete apoptosis-mediated dissociation. Morphol-

ogy of spheroids was not changed after drug treatment alone. After electroche-
motherapy individual cells or clusters separated from spheroid surface.
Electrochemotherapy first led to a decrease in size of spheroid and then to its total
destruction, regardless of the drug used. The study also showed that underlying
mechanism of cell death and spheroid growth arrest involves apoptosis. The results
on spheroids are very valuable, because the spheroid model mimics the behavior of
tumors very well (Gibot et al. 2013).
In recent years in vitro electroporation studies focused on calcium electropora-
tion. Dose-dependent decrease in cell viability was illustrated for all tested cell lines.
Cell viability after calcium electroporation was decreased to a similar level as cell
viability after 0.1 μmol/L bleomycin electroporation. Further studies with calcium
electroporation on spheroids demonstrated reduced size of three different cancer cell
spheroids: human colon, breast, and bladder cancer cell spheroids. In contrary,
calcium electroporation had no effect on the size of human fibroblast cell spheroids,
thus indicating a different effect on normal and malignant cells (Frandsen
et al. 2015).
The effectiveness of electrochemotherapy with bleomycin was tested also on
BRAF V600E mutated melanoma cells (SK-MEL-28) and nonmutated melanoma
cells (CHL-1). The survival of both cell lines was reduced, with more pronounced
effect of electrochemotherapy on BRAF V600E mutated SK-MEL-28 cells, which
required half lower concentration of BLM at IC90 compared to nonmutated cells
CHL-1. Furthermore, concomitant treatment with electrochemotherapy with
bleomycin and 0.5 μM vemurafenib was also tested in vitro. The potentiation of
combined treatment was more than additive; it was calculated to be synergistic
(Dolinesk et al. 2016).
In Vivo Studies
Based on encouraging antiproliferative results of bleomycin and cisplatin after cell

electroporation in vitro, these two drugs were the first tested on murine tumor models
and later translated to veterinary oncology. The antitumor effectiveness of
electrochemotherapy was demonstrated on transplantable or spontaneous tumors in
mice, rats, rabbits, hamsters, cats, dogs, and horses. Extensive studies on tumors in
different locations, either cutaneous or subcutaneous or located in the muscle, brain,
or liver, were performed. The treatment was tested on tumors of different histology:
melanomas, carcinomas, gliomas, and sarcomas (Gothelf et al. 2003; Sersa
et al. 2008b). The effectiveness of electrochemotherapy relies on the chemothera-
peutic drug administration that by itself does not have any antitumor effectiveness,
and application of electric pulses to the tumor that do not exert any antitumor
effectiveness as well, but when combined together excellent antitumor effectiveness
is obtained (Fig. 1).
Therefore, the effectiveness of electrochemotherapy relies on two factors:
the drug and the electric field. The appropriate drug dose and administration,
timing of the application of electric pulses is needed, as well as the parameters
of electroporation and coverage of the tumor with electric field (Fig. 2). Based
on the extensive preclinical studies, several important factors were identified
that need to be known and taken into consideration when performing
electrochemotherapy.
Fig. 1 Example of effective electrochemotherapy in B16F10 tumors. Cisplatin was given

intratumoraly (4 mg/kg) and 8 electric pulses were applied to the tumor with plate electrodes.
Electric pulses were applied in two directions: 4 pulses in one and the other 4 in the perpendicular
direction. Six days after treatment antitumor effectiveness of electrochemotherapy with cisplatin is
evident, compared to cisplatin only group
• Two different administration routes are used in electrochemotherapy: intrave-

nous or intratumoral injection of chemotherapeutic drug. Each of the routes has its
advantages and disadvantages. The intravenous route provides better drug distri-
bution in the tumors, when they are well vascularized, whereas the intratumoral
route can provide higher drug concentration in the tumors and can be used in
small and poorly vascularized tumors, such as recurrent tumors in
pre-irradiated area.
• At the time of application of electric pulses sufficient amount of the drug at the
tumor site is mandatory. After intravenous drug administration (5 mg/kg of
bleomycin or 4 mg/kg of cisplatin), the maximal drug concentration in the tumors
is reached in small laboratory animals after 3 min. Even shorter interval is needed
Fig. 2 Schematic
presentation of
electrochemotherapy. First the
drug (yellow circles) is
injected intravenously or
intratumorally. At the time of
maximal chemotherapeutic
drug concentration at the
tumor site, electric pulses are
applied either by plate or
needle electrodes. The whole
tumor and appropriate safety
margins should be covered
with a sufficient electric field
for optimal electroporation of
cells in the tumors
after intratumoral administration of cisplatin, so the electric pulses have to follow

the administration of the drug within a minute, while for intratumoral adminis-
tration of bleomycin the optimal time of application of electric pulses after
intratumoral injection was 10–15 min (Heller et al. 1997; Miklavcic et al. 2014;
Sersa et al. 2008a).
• Electrode selection is crucial for achieving good tissue electroporation and thus
good antitumor effectiveness. Electrode geometry and tissue composition control
electric field distribution in the tissue, which is responsible for plasma mem-
brane permeabilization. The electric field distribution in the tissue can be
improved by rotating the electrodes so that higher fraction of cells is being
permeabilized. Cutaneous and subcutaneous tumors can be effectively treated
by plate electrodes, whereas appropriate electric field distribution in deep-seated
tumors is assured by using needle electrodes (Miklavcic et al. 2014). The advan-
tage of the plate electrodes over needle electrodes is that they are noninvasive.
• Electric pulse parameters, amplitude, number, duration, and frequency of the
electric pulses are very important for antitumor effectiveness. Several studies
showed that for surface tumors appropriate amplitude over distance ratio for safe
and effective electrochemotherapy, when using plate electrodes, is between 1000
and 1500 V/cm. Amplitude over distance ratio below 1000 V/cm does not cause
tumor electroporation, whereas above 1500 V/cm normal tissue damage around
tumor occurs due to irreversible cell permeabilization (Sersa et al. 1995;
Miklavcic et al. 2014). Most of the studies demonstrated good antitumor effec-
tiveness without tissue damage at amplitude over distance ratio of 1300 V/cm.
Electric pulse parameters for other types of electrodes need to be numerically
calculated to ensure appropriate electric field distribution in the tumor tissue at
coverage of 400 V/cm or higher for 8 100 μs. Normally 8 electric pulses of
100 μs are used for electrochemotherapy, but minimum 4 pulses should be used
(Gothelf et al. 2003; Miklavcic et al. 2014). Repetition frequencies of the pulses
are either 1 Hz or 5 kHz. If the drug concentration in the tumor is sufficient, both
frequencies cause equal antitumor effect (Sersa et al. 2010).
In vivo experiments in animal models provided necessary data to prove that

electrochemotherapy with bleomycin or cisplatin is effective in the treatment of
solid tumors. Good antitumor effectiveness was obtained in all tumor models tested,
with drug concentrations that had no antitumor effectiveness by itself. Furthermore,
the application of electric pulses alone does not exert antitumor effectiveness but is
only the means for drug delivery to cells. Antitumor effectiveness can be achieved
with irreversible electroporation, where pulses only are responsible for the ablative
effect, but at higher amplitude and more pulses need to be delivered. Complete or
partial response is achieved after a single treatment, although repetitive treatment
was possible as well. The therapy was demonstrated to be effective on a variety of
tumor histotypes, based on the simple principle that with electroporation, as a
physical method, cells in tumors can be electroporated and that bleomycin or
cisplatin can thus exert cytotoxicity on any kind of the tumor type.
However, since early studies variable effectiveness of electrochemotherapy on
different tumor models has been observed. Though electrochemotherapy is effective
on most tumor types, underlying mechanisms of differential susceptibility of the
tumors has not been explored. The most plausible option is that different tumor cells
have different intrinsic susceptibility to electrochemotherapy as observed also in
in vitro studies. Cemazar et al. showed different IC50 for cisplatin and bleomycin in
several different tumor cells and also in endothelial cells (Cemazar et al. 2001a). The
difference in in vitro cell sensitivity could be due to intrinsic sensitivity of the cells to
different antitumor drugs. Mechanisms responsible for the tumor cell’s resistance to
bleomycin could be related to higher effectiveness of electrochemotherapy in sar-
coma cells SA-1 than in carcinoma cells EAT were already identified. Difference in
cell susceptibility to bleomycin could be due to increased DNA repair, decreased
drug accumulation, metabolic inactivation with hydrolase, or partially also due to
defects in membrane protein involved in bleomycin endocytosis. Furthermore, the
difference in susceptibility was observed also between the parental and
chemoresistant cell line (Cemazar et al. 2001b).
In vivo studies on murine models confirmed these in vitro observations. Beside
mechanisms already discussed in vitro in tumor cell models in vivo have also
additional mechanisms contributing to overall effectiveness. Thus, the histological
characteristics of the tumors have to be considered as well, predominantly the
vascularization of tumors and the composition of extracellular matrix. Further

studies are needed in the future also to identify mechanisms involved in differential
effect on tumor versus normal cells or tissue.
Mechanisms of Action
Electroporation of targeted cells is the principal mechanism of electrochemotherapy.

Drug concentration in the cells is significantly increased and thus can efficiently
reach the intracellular target. However, due to the permeabilization of other stromal
cells, like endothelial cells, also the vascular effects of electrochemotherapy are
present (Jarm et al. 2010). Furthermore, the nonthermal effects of electroporation
enable the preservation of tumor antigens, which are shed in the tumor environment,
and can elicit local immune response. All three mechanisms of action are involved in
overall antitumor effectiveness of electrochemotherapy.
Electroporation Mediated Drug Uptake in the Cells
Although there were several different chemotherapeutic drugs tested in vitro for
combination with electrochemotherapy, two most prominent and commonly used
drugs, bleomycin and cisplatin, will be discussed here.
Bleomycin is a nonpermeant drug that when internalized exerts high cytotoxicity.
It was demonstrated that on average, 200 internalized molecules of bleomycin is
sufficient to kill cells. Bleomycin induces single and double DNA strand breaks.
There are many theories, but the exact mechanism of DNA strand scission still
remains unresolved. The reaction between DNA and bleomycin is universal and will
occur in all cells regardless of tumor type, even in normal cells. Two types of cell
death were observed: mitotic cell death and pseudoapoptosis. In vivo slow death,
mimicking in vitro mitotic cell death, was observed. Mitotic cell death is specific for
dividing cells and in the context of higher dividing rate of tumor cells compared to
normal cells the differential susceptibility of tumor versus normal tissues is postu-
lated. In vivo studies also clearly demonstrated that exposure of tumors to electro-
poration significantly increases bleomycin accumulation in the tumors, with
prolonged retention in them (Mir et al. 1996). Recently, a new analytical method
for the determination of bleomycin in serum and tissue samples was developed that
may in further studies be used and help to explore the pharmacokinetics and
pharmacodynamics of bleomycin and provide further data that could be usefully
exploited in the clinics (Kosjek et al. 2016).
Unlike bleomycin cisplatin is not classified as nonpermeant, but its complexes
seem to be low permeant. Normally cisplatin enters the cells by passive diffusion and
through copper transporters (Dilruba and Kalayda 2016). After electropermeabi-
lization cisplatin is up to 70-fold more cytotoxic for the cells, proportional to the
higher uptake of cisplatin (Sersa et al. 1995). It interferes with DNA replication;
forms inter- and intrastrand DNA adducts; and thus halts proliferation of the cells
and induces apoptosis. Since enhanced proliferation is typical for tumor cells,
cisplatin enters tumor cells to a greater extent. Also increased cisplatin accumulation
in tumors after electroporation was demonstrated in experimental tumors. Further-
more, the increased DNA binding of cisplatin in tumors was demonstrated in the
range of a factor of 2–3 (Cemazar et al. 1999).
All these data indicate that electroporation of tumors increases drug accumulation
in tumors and undoubtedly in tumor cells. The drugs, bleomycin or cisplatin by their
DNA damaging effect, therefore exert the antitumor action and induce slow, mitotic
or apoptotic cell death of tumor cells.
Antivascular Effects
Early studies demonstrated that exposure of tumors to electroporation had tumor

blood flow modifying effect. The electroporated area becomes whitish, for a certain
period of time with restoration of blood flow after 10–15 min (Sersa et al. 1999).
These early studies demonstrated that the effect is not confined to tumor vasculature
but is expressed also in normal tissue, where it is however less pronounced and
shorter lived (Gehl et al. 2002). Later studies demonstrated also increased tumor
vascular permeabilization. Both phenomena contribute to better drug distribution
and longer retention of the drug in the tumors, known also as vascular lock.
Since all stromal cells in the electric field induced by electroporation can be
permeabilized, several studies have explored the vascular disrupting action of
electrochemotherapy. A body of evidence demonstrates that endothelial cells are
permeabilized and undergo apoptosis, thus hampering the perfusion of the tumor
vessels (Sersa et al. 2008b). The effect is confined predominantly to smaller vessels,
while bigger ones, due to the more robust scaffold, remain intact (Markelc
et al. 2013). The size of the vessels that are not affected by electroporation or
electrochemotherapy are not determined yet but need to be in further studies. Similar
phenomena are observed also after irreversible electroporation. This abrogation of
tumor perfusion then results in ischemia and induces a secondary cascade of tumor
cell death. Based on these findings, a model of vascular effects of electroporation and
electrochemotherapy was proposed (Jarm et al. 2010). Later findings also pointed to
differential susceptibility of the vasculature in the tumors to the normal vasculature
in the surrounding normal tissue (Markelc et al. 2013). This finding is specifically of
interest, since it supports the observations of normal tissue sparing effect of
Induction of Immune Response
Several ablative techniques such as electrochemotherapy induce a local immune

response. The induced immunologic cell death activates the immune response that
can contribute to the overall effectiveness of electrochemotherapy. This statement is
supported by several lines of evidence. First the response of tumors was less
pronounced in immunodeficient mice, and lower degree of complete responses was

observed (Sersa et al. 1997). Immunological cell death was observed after
electrochemotherapy with bleomycin (Calvet et al. 2014). The response of tumors
was to some degree dependent on the immunogenicity of tumors (Heller et al. 2000).
And, the response of tumors was boosted by adjuvant immunotherapy (Sedlar
et al. 2012; Cemazar et al. 2015). Detailed discussion about this phenomenon and
its implications on the development of electrochemotherapy are discussed in other
chapters in this book. In summary, the induced immune response is important for the
eradication of the last remaining tumor cells that are not directly or indirectly killed
by electrochemotherapy. The extent of its contribution to the overall response,
though, still needs to be determined.
These findings can be exploited for the “in situ” vaccination by electroche-
motherapy, both using bleomycin or possibly cisplatin and boosted in different
ways. One of the approaches would be by immunostimulation, with cytokines, and
the other one by inhibition of immune checkpoint inhibitors. These approaches are
currently under extensive investigation and are being tested also in clinical trials
(▶ Chap. 103, “Adjuvant Immunotherapy as a Tool to Boost Effectiveness of
Electrochemotherapy” by Kamensek, U., Kos, S., Sersa, G).
In summary, so far three mechanisms of electrochemotherapy have been deter-
mined: increased drug delivery to tumor cells, vascular disrupting effect, and the
involvement of immune response (Fig. 3). The degree of the contribution of each of
the mechanisms has not been determined but may vary according to the tumor type.
For example, better vascularized tumors may have greater contribution of the
vascular disrupting effect, while more immunogenic tumors induce a greater
immune response.
Perspectives
Based on the preclinical data and the knowledge of the basic mechanisms of action
of electrochemotherapy, it has been swiftly translated into the clinics.
Electrochemotherapy is being widely used as a treatment modality for local tumor
control but could also be used as a concomitant treatment to standard and newly
emerging therapies. The mechanistic studies that would shed some light on the subtle
variations of the cell sensitivity are needed, as well as studies on immune aspects and
the physiology of the tumors, all, that could affect the response rate of the tumors,
and help to refine electrochemotherapy in clinical setting.
One of the possibilities that still awaits clinical application is radiosensitizing
effectiveness of electrochemotherapy. Electroporation is a site-specific delivery
method and thus it could be used for effective tumor-specific delivery of radio-
sensitizing drugs. By more precise targeting of tumor tissue and less involvement of
surrounding normal tissue, the therapeutic index of tumor irradiation may be
increased. Previous studies of electrochemotherapy with bleomycin or cisplatin
combined with radiotherapy demonstrated potentiation of the radiation response in
sarcoma tumor: 1.9-fold for electrochemotherapy with bleomycin and 1.6-fold for
Fig. 3 The three mechanisms involved in antitumor effectiveness of electrochemotherapy. The

direct effect on tumor cells is due to the increased drug uptake. The indirect effect on tumor cells is
due to the vascular disrupting effect of electrochemotherapy. The involvement of the immune
response is due to the shedding of the tumor antigens and elicitation of local immune response
electrochemotherapy with cisplatin. The radiosensitizing effect of electroche-

motherapy with bleomycin was demonstrated in fractionated radiation regime in
murine sarcoma model and with cisplatin in breast cancer tumor model. Preclinical
studies on the fractionated radiation regime are good indicator for potential of the
combined therapy also in the clinic (▶ Chap. 89, “Combined Treatment of
Electrochemotherapy with Irradiation” by Kranjc, S., Kamensek, U., Sersa, G.).
Since electrochemotherapy reduces blood flow and lowers partial oxygen pres-
sure (pO2) in the tumors, it can activate bioreductive drugs to exhibit a cytotoxic
effect on hypoxic cells (Cemazar et al. 2001c). Tumor cells in suboptimal physio-
logical conditions, like hypoxia, are more sensitive to heat, thus electroche-
motherapy could also improve therapeutic conditions for the use of hyperthermia.
A more pronounced effect of electrochemotherapy could be gained by combining
it with gene electrotransfer of different plasmids coding for therapeutic genes, acting
either locally or systemically. For local effect electrochemotherapy could be com-
bined with antiangiogenic plasmids such as plasmid AMEP (antiangiogenic
metargidin peptide) or plasmids coding for shRNA against MCAM (melanoma
cell adhesion molecule) or endoglin. Furthermore, a systemic effect could be
added to electrochemotherapy by combining it with IL-2, IL-12, p53, or granulocyte
macrophage colony-stimulating factor (GM-CSF). Some of the combinations have

already been tested and results support this concept (▶ Chap. 103, “Adjuvant
Immunotherapy as a Tool to Boost Effectiveness of Electrochemotherapy” by
Kamensek, U., Kos, S., Sersa, G).
The emerging therapies with immune checkpoint inhibitors are gaining in clinical
importance, and electrochemotherapy could be an adjunct to them. In the clinical
setting, antitumor effectiveness after concomitant treatment with electroche-
motherapy and CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) inhibitor
has already been tested. The first clinical data are encouraging, but the preclinical
data on interaction of these treatment combinations are lacking. The same holds true
for targeted therapies.
Electrochemotherapy could also be combined with other targeted therapies such
as BRAF inhibitors targeting serine/threonine-protein kinase B-Raf (BRAF). First
preclinical study has already been conducted and showed synergistic action. Case
report of combining dabrafenib with electrochemotherapy also confirmed safety and
feasibility of treatment. All the accumulated preclinical data on these new combina-
tions could be further used in human clinics. The combined treatments could
strengthen the effect of electrochemotherapy, with additive or synergistic action.
Beside its great potential in human clinical use it can also be used in veterinary
medicine. Studies on dogs, cats, hamsters, rabbits, and horses demonstrated good
anticancer effectiveness for different tumors, such as fibrosarcoma, neurofibroma,
mast cell tumor, perianal tumors, mammary adenocarcinoma, hemangioma,
hemangiosarcoma, and sarcoids. Electrochemotherapy has also been demonstrated
to be effective in combination with other treatment, especially surgery (▶ Chap. 106,
“Electrochemotherapy and Gene Electrotransfer in Veterinary Oncology”: Tamzali,
Y., Cemazar, M., Tozon, N.).
Conclusions
In summary, after the important contribution of preclinical research for the develop-
ment of electrochemotherapy and for translation to the clinics, nowadays the
research is focused on the emerging questions that arise from the clinical use. The
main question is how to optimally combine electrochemotherapy with targeted
therapies, immunostimulators and immune checkpoint inhibitors. In this way
electrochemotherapy can be extended from local to systemic treatment or, from the
other point of view, use electrochemotherapy as adjuvant or in situ vaccination to
new immune therapies.
Cross-References

Electrochemotherapy
▶ Combined Treatment of Electrochemotherapy with Immunomodulators
▶ Combined Treatment of Electrochemotherapy with Irradiation

Tissue Blood Flow
▶ Electrochemotherapy and Gene Electrotransfer in Veterinary Oncology
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annurev-bioeng-071813-104622
Preclinical Studies on Irreversible
Electroporation 79
Suyashree Bhonsle, Robert N. Neal II, and Rafael V. Davalos
Abstract
Irreversible electroporation (IRE) employs a series of brief electric pulses to
destabilize cell membranes by altering the transmembrane voltage to create
nanoscale defects that induce cell death. Its nonthermal mechanism makes it an
ideal treatment modality for treatment of tumors near critical structures and
vasculature, which are contraindications for other thermal ablation modalities.
Since its conception, IRE has been investigated for applications in several organs
to determine its safety and efficacy. IRE ablation zones, thermal effects, potential
for real-time imaging, and cell death mechanism have been thoroughly investi-
gated in vivo, leading to its successful transition to the clinical environment. This
chapter describes IRE findings in the preclinical setting, with a focus on the
pancreas, kidney, liver, bone, brain, prostate, and lung, and general implications
with respect to successful therapy outcomes. These include consistency of abla-
tion zones with different treatment parameters, histology of ablation zones show-
ing tissue and tumor destruction, in vivo safety during and after treatment, and
efficacy in terms of tumor regression. In addition, the patency of critical structures
such as blood vessels, nerves, and ductal systems is briefly discussed. Newer
S. Bhonsle
Virginia Tech Wake Forest School of Biomedical Engineering and Sciences, Virginia Tech,
Blacksburg, VA, USA
The Bradley Department of Electrical and Computer Engineering, Virginia Tech, Blacksburg, VA,
USA
R.N. Neal II
Radiology Department, Alfred Hospital, Melbourne, VIC, Australia
R.V. Davalos (*)
Department of Biomedical Engineering and Mechanics, Virginia Tech – Wake Forest University
School of Biomedical Engineering and Sciences, Blacksburg, VA, USA
Department of Mechanical Engineering, Virginia Tech, Blacksburg, VA, USA
e-mail: davalos@vt.edu

DOI 10.1007/978-3-319-32886-7_46
1528 S. Bhonsle et al.
preclinical findings such as immune response activation, adjuvant therapies, and

modulating pulse regimes to improve treatment outcome and ease treatment
delivery are outlined.
Keywords
Irreversible electroporation • Pulsed electric fields • Preclinical studies • Non-
thermal mechanism • Critical vasculature
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1528
Considerations for Reducing Thermal Damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1529
Organ-Specific Ablations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1530
Pancreas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1531
Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1532
Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1533
Bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1534
Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1534
Prostate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1535
Lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1536
Preserved Patency of Sensitive Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1536
Blood Vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1536
Nerves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1537
Ductal Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1537
Advances in Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1538
Immune Response and Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1538
Adjuvant Augmentation of IRE Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1539
High-Frequency Irreversible Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1540
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1540
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1541
Introduction
Electroporation is a process in which short, high voltage electric pulses cause an

increase in the biological cell membrane permeability, due to the formation of
nanoscale defects, referred to as “pores.” For applications that involve cell drug
delivery, the goal is to limit the pulse amplitude and number so that the pores are
temporary, allowing transmission of small molecules across the membrane, and the
cell remains viable (Overview and History of Electrochemotherapy). Alternatively,
irreversible electroporation (IRE) utilizes a higher pulse amplitude and number to
induce cell death as a tissue ablation modality. With appropriately designed pulse
protocols, IRE creates lesions without causing significant thermal damage to the
tissue (Davalos et al. 2005). The mechanism of IRE is generally characterized by
irreversible structural defects, chemical imbalances due to the influx and efflux of
ions, and subsequent cell death (Electropore Energy and Thermodynamics) (Lee
et al. 1993).
79 Preclinical Studies on Irreversible Electroporation 1529
The use of IRE as a therapy for ablating tumors was first demonstrated in an
animal tumor model in 2007 (Al-Sakere et al. 2007). IREs clinical efficacy has since
been demonstrated in numerous organs for soft tissue targets, particularly tumors.
IREs ability to generate regions of dead cells while maintaining the critical extra-
cellular architecture allows IRE to be used in regions containing sensitive structures
that make such targets contraindicated for other focal therapies or surgical resection.
In this chapter, the wide array of preclinical IRE studies in numerous animals
(including animal tumor models) and organs are presented. These studies character-
ize IRE ablations in response to different treatment parameters and show its efficacy
in relation to preservation of extracellular components.
Considerations for Reducing Thermal Damage
IRE employs brief (~100 μs) but intense square wave electric pulses (50–400)
delivered via electrodes inserted into or adjacent to the targeted tissue. These pulses
induce an electric field distribution in the target and surrounding tissue, depending
on the electrode configuration, pulse amplitude, and tissue type. For a given tissue
and set of pulse parameters (pulse length, number of pulses, delivery rate), an electric
field threshold can be determined that induces cell death and forms a lesion.
Numerical models are often employed to predict the induced electric field distribu-
tion and ablation zone, using a priori knowledge of the tissue electrical properties.
The electric pulses used in IRE treatment delivery induce collateral heating due to
the resistive properties of biological tissue, known as Joule heating. In order to
maintain IRE’s distinction from thermally based therapies, pulse protocols must be
designed to limit Joule heating below thresholds that cause structural thermal
damage to the tissue and surrounding sensitive structures.
Numerous publications in the literature use numerical and experimental studies to
address consideration of sufficient IRE without the clinically limiting thermal
damage. These studies aim to delineate IRE cell death from the thermal damage
that occurs when tissues are exposed to temperatures higher than their physiological
norm for extended periods of time. While 50 C is typically chosen as the threshold
to begin introducing thermal damage at the cellular scale, and it has been noted that
prolonged exposure to mildly elevated temperatures as low as 43 C can lead to cell
death, what is considered instantaneous thermal damage occurs as high as 83.6 C
(prostate) or 74.7 C (liver) (Thomsen and Pearce 2010).
From an experimental perspective, in vivo studies have measured temperature
during pulse delivery to examine the potential for thermal damage. The increase in
temperature during treatment is proportional to the applied electric field and is
therefore dependent on the electrode configuration utilized in the study. Two con-
figurations, namely, the monopolar and bipolar electrode configurations, are com-
monly employed to deliver IRE treatment. The monopolar electrode configuration
contains the source and sink electrodes on two different needles and spacers need to
be used between the needles to maintain electrode distance. On the other hand, the
bipolar electrode configuration has the source and sink on the same needle. This is
accomplished using concentric cylinders inside the needle, with insulating material
between the two cylinders. For monopolar and bipolar needle electrodes, the electric
field is highest closest to the electrodes and decays away from the electrodes, and
therefore, the temperature is always the highest near the electrodes. For flat plate
electrodes, the electric field is uniform between the electrodes and so is the temper-
ature distribution.
In a clinical canine case using IRE to treat a soft tissue sarcoma, the temperature
immediately adjacent to the electrode was measured using a fiber optic temperature
probe. This study employed monopolar or bipolar electrodes of spacing 8–15 mm to
deliver 80 pulses of 70–100 μs width and 800–1250 V at a rate of 90 pulses/min or
immediately following the R-wave peak on the electrocardiogram. The maximum
temperature rise at this location during the IRE procedure was 2.4 C (Neal et al.
2011) with the study showing successful tumor remission. Furthermore, an in vivo
study in brain with thermal probes attached to the electrodes showed that it was
possible to generate substantial volumes of IRE tissue while max tissue temperature
rise was 1.15 C (Garcia et al. 2010). An additional in vivo study on porcine kidneys
employed a more aggressive IRE protocol of three to four monopolar electrodes with
electrode spacing and exposure of 15 mm to deliver three sets of 70 pulses, each
90 μs long, at a rate of 90 pulses/min. The peak temperature recorded at the center of
the three- and four-electrode configuration was 57 and 79 C, respectively (Wagstaff
et al. 2014). Consistent with the rapid decay in thermal effects with increasing
distance from the electrodes, max temperatures of 40 and 42 C were measured
1 cm outside the electrode geometries, which was still within the IRE ablation zone.
These studies demonstrate that while IRE ablation occurs primarily due to a
nonthermal mechanism, careful attention must be paid to heating effects to ensure
they remain below those to cause thermal damage to the critical structures and
surrounding vasculature. In current IRE protocols, the temperature rise close to
electrodes is inevitable and may lead to thermal damage near the electrodes
(▶ Chap. 54, “Treatment Planning for Electrochemotherapy and Irreversible
Electroporation of Deep-Seated Tumors”); however, majority of the ablation zone
away from electrodes is nonthermal, caused by electroporation-induced effects.
Several studies have suggested techniques to reduce temperature rise during IRE
treatment, including shorter pulse durations, reducing the pulse delivery to allow
convective and conductive cooling between pulses, as well as proactive interven-
tions to cool the electrode and/or tissue.
Organ-Specific Ablations
IRE’s potential for ablation has been studied in several organs, each showing
different implications in regard to successful therapy outcomes. This section
addresses some of the more common IRE therapeutic applications and the preclinical
data regarding the use of IRE for these targets.
Pancreas
Locally advanced pancreatic cancers comprise approximately 40% of the pancreatic

tumors. Although not metastasized, these tumors have innervated and surrounded
critical structures such as the pancreatic duct and the superior mesenteric artery.
Therefore, the tumors around this region cannot be safely resected without damage
to these critical structures, thereby, risking patient morbidity and mortality. Further-
more, the use of thermally ablative therapies in such environments has largely been
avoided due to the possibility of thermal injury-induced pancreatitis. This leaves a
large number of pancreatic cancers inoperable by the time of diagnosis. IRE’s ability
to spare the major vasculature and potentially reduce risk of pancreatitis offers an
approach to addressing the tumor in these regions. It could be used as either a stand-
alone treatment modality, in conjunction with drugs, or to augment the treatment
margin of surgical resection.
Preclinical data in four female swine showed IRE can be safely administered in
the pancreas (Charpentier et al. 2010). Treatment parameters involved the use of
monopolar electrodes at a spacing of 9–15 mm; 90 pulses of 1350–2250 V were
delivered to the healthy pancreas. All animals survived their designated times of 2 h,
2 days, and 2 weeks with no treatment-related complications. The tissues in and
around the ablation zones were sliced into sections of thickness 5 mm, perpendicular
to the electrode placement. The 5 mm section in the center of the ablation cavity had
heights ranging from 10 mm to 21 mm and widths ranging from 10 mm to 16 mm.
Pancreatic tissue at 2 weeks after IRE showed scarring in the ablation zone with
preservation of pancreatic ducts. This study was also the first to show that triphenyl-
tetrazolium chloride (TTC) can predict the IRE zone of ablation within 2 h of
treatment. TTC stains the live mitochondrial activity in cells. While IRE ablation
zones are detectable visually without staining, TTC staining enhances the contrast
between the ablated and non-ablated areas.
Another study in swine pancreas was performed with two electrode configu-
rations – monopolar (19 gauge, i.e., diameter 0.9 mm) and bipolar (16 gauge, i.e.,
diameter 1.2 mm) – and the animals were euthanized after 3, 7, and 14 days
(Bower et al. 2011). The monopolar electrode configuration consists of two needle
electrodes, with source on one needle and sink on the other. Alternately, the
bipolar probe contains both the source and sink on the same probe at a fixed
spacing of 8 mm. Treatment parameters included electrode spacing from 8 to
20 mm, pulse numbers of 50–100, and applied voltage ranging from 2300 to
3000 V. All animals survived the study, with transient increases in amylase and
lipase that normalized on third day. Amylase and lipase are key digestive
enzymes, produced by the pancreas, that help break down starch and digest fats.
High levels of these enzymes are indicative of pancreatitis. Gross analysis of
samples revealed ablation zones of median size of height 3 cm and width 2.8 cm.
Histological analysis revealed significant destruction of the pancreatic tissue with
patent vascular structures and no significant difference between the two types of
probes. Additionally, terminal deoxynucleotidyl transferase dUTP nick end
labeling (TUNEL) assays’ staining identified apoptotic markers in the IRE

ablation zone.
Furthermore, IRE was shown to be effective in treating pancreatic ductal adeno-
carcinoma, an aggressive form of pancreatic cancer, in orthotopic mouse models
(José et al. 2012). IRE treatment exhibited significant antitumor effects and extensive
tumor necrosis, reduced tumor cell proliferation, and disruption of microvessels.
Data in this study indicated that IRE leads to an increase in survival (from 42 days in
untreated mice to 88 days in the IRE-treated group) with 25% of mice showing
complete tumor regression.
Kidney
The increase of high-resolution diagnostic imaging is partially responsible for a

rapid rise in the number of diagnoses of asymptomatic small renal masses, with over
half of new cases detected incidentally. Frequent detection of low-risk renal tumors
has encouraged care toward less aggressive, nephron-sparing approaches. Preserva-
tion of renal function in the affected kidney is vital for patients with solitary kidneys,
comorbid conditions, multiple tumor sites, and genetic predisposition for recurrent
bilateral renal tumors (von Hippel-Lindau disease). Partial nephrectomy still requires
considerable invasiveness, while sensitivity of the ureter and high blood perfusion
rate mitigate the efficacy of thermal therapies. These factors provide considerable
value for IRE treatment of small renal masses.
Preclinical investigations into the ablation of kidneys investigating IRE effects on
macro- and microscopic healthy renal tissue show complete cell death of glomerular
and tubule cortex structures while sparing major vasculature. In an IRE study on eight
female Yorkshire pigs, laparoscopic ablations were performed with either monopolar
or bipolar electrodes (Tracy et al. 2011). The pigs were euthanized between 10 min and
14 days after IRE, and the kidneys were harvested for gross and histological analysis
for cellular viability. Monopolar ablations were performed at 2300 Vand 90 pulses at a
pulse length of 100 μs and 1.5 cm spacing, while bipolar ablation was performed at
2700 V and 90 pulses at a pulse length of 70 μs. The spacing between electrodes was
fixed at 0.8 cm. Overall lesion volume was smaller when the bipolar probe was used
than when the monopolar was used. In the bipolar group, the mean gross lesion size,
immediately and an hour after death, showed a volume of 1756 mm3 (assuming
ablation shape of a prolate spheroid). In the monopolar group, the mean volume was
4440 mm3. By 7 days, the ablations in both groups remained similar to their imme-
diately post-IRE size (10 min). However, by 14 days, there was substantial lesion
contraction, regardless of the modality applied. There was heterogeneous and
unpredictable involvement of the urothelium, ranging from no damage to complete
ulceration and necrosis. However, it was more common with the bipolar probe, which
was placed to traverse the collecting system as opposed to the monopolar probe
technique where the ablation zone traversed the collecting system without direct
puncture. The urothelium was partially spared in 60% of the cases, with further
evidence of repair/regrowth by 14 days despite direct involvement during ablation.
Another swine study evaluated the effect of IRE on the renal urine-collecting
system using intravenous urology and urinary cytology, which are routine urological
examinations, magnetic resonance imaging (MRI), and histology (Wendler et al.
2012). Each IRE procedure consisted of nine sets of ten IRE pulses, pulse length
70–100 μs, voltage 2300–2700 V. All animals were euthanized after a 28-day period.
MRI scans did not show substantial lesion immediately after IRE; however, the sizes
increased to an average diameter size of 16.5 mm at day 7 and shrunk to ~8 mm at
day 28. These sizes showed good correlation with the ones from gross macroscopic
analysis at day 28. Initial urothelial injury was noted, but the urinary system was
completely regenerated 28 days post-IRE with the preservation of urothelial base-
ment membranes and no urinoma evidence.
Similarly, other IRE studies showed that renal ablations showed a demarcation
between necrotic and normal tissue, 24 and 36 h after IRE treatment with evidence of
tubule degeneration (Deodhar et al. 2011). Ablations showed cortical fibrosis,
regenerating renal pelvic epithelium, and intact pelvic extracellular matrix. No
thermal injury, renal pelvis, or blood vessel injury was seen. Two to four weeks
later, there was evidence of regenerating tubules and pelvic epithelium with intact
extracellular matrix and ablations turning to contracted scars. The urine-collecting
system was essentially preserved with regenerated urothelial tissue. This study
therefore demonstrated the nonthermal and connective tissue-sparing mechanism
of action of IRE.
Liver
The size of the liver relative to typical tumors, coupled with its regenerative capacity,
makes the liver a common target for focal ablation of tumors. IRE has been clinically
applied in this environment, particularly for targeted regions near the inferior vena
cava, gallbladder, and external sensitive organs such as the bowel, pancreas, heart,
and spleen. Further, the relative homogeneity and isotropy of hepatic organization
make the liver a suitable model for investigation of various aspects for IRE therapy.
The greatest collection of preclinical investigations regarding the effects from IRE
therapy exists for liver ablations. Many of these studies are generalized to effects of
IRE as a whole, such as monitoring IRE ablation zones using different imaging
techniques, as well as studies into different pulse parameters and electrode designs.
In a study conducted on 16 pigs, IRE ablations were imaged with ultrasound
(US), magnetic resonance (MR), and computed tomography (CT) (Lee et al. 2010).
Treatments were delivered with two electrode configurations – a single 16-gauge
bipolar probe and two to three 18-gauge monopolar probes. Treatment parameters
include inter-electrode spacing between 1.5 and 3.0 cm and 90 IRE pulses, each
lasting 100 μsec at 2000–3000 V. At gross section examination, the mean diameter of
the ablation zones was 33.53.0 mm which was achieved in 6.9 min (mean total
procedure time per ablation), with a mean difference of 2.53.6 mm between US
and gross section measurements. No complications were seen in any of the 16 ani-
mals. IRE ablation zones were well characterized with US, CT, and MR imaging,
and real-time monitoring was feasible with US. Bile ducts and vessels were
completely preserved. Areas of complete cell death were stained positive for apo-
ptotic markers, suggesting involvement of the apoptotic process in the pathophysi-
ology of cell death caused by IRE.
Additional studies on the liver analyzed the effects of different treatment parameters
on IRE ablation sizes (Ben-David et al. 2012). An IRE study performed in 25 pig livers
using two 18-gauge (diameter 1.2 mm) monopolar electrodes evaluated the effect of
five treatment parameters including pulse number (20–90), pulse length (20–100 μs),
voltage (2250–3000 V), inter-electrode spacing (1.5–2.5 cm), and length of active
electrode exposure (1.0–3.0 cm). For all combinations of pulse parameters, the study
reported average ablation widths between 2.6 and 3.9 cm and depths between 1.3 and
5.0 cm. In the range tested, the study found that increasing voltage amplitude had a
statistically significant increase in ablation size. However, increasing pulse numbers or
pulse duration did not have a significant increase. Therefore, IRE is not strictly energy
dose dependent, i.e., increasing energy through pulse number or duration does not
have the same effect as increasing energy through voltage amplitude.
In addition, preclinical studies have shown that IRE can be used to effectively treat
liver tumors in small animal models (Guo et al. 2010). In this study, hepatomas were
established in an N1-S1 rodent model. Thirty rats were divided into treatment and
control groups. For treatment groups, IRE electrodes were inserted and eight 100 μs
and 2500 V pulses were applied to ablate the targeted tumor tissues. Magnetic
resonance images showed statistically significant tumor size reductions (p 0.05)
compared to the untreated tumors within 15 days post-therapy, with reduction in tumor
diameters of 3231%. Pathology correlation studies documented extensive tumor
necrosis and full regression in nine of ten treated rats in 7–15 days after treatment.
Bone
While IRE has primarily been evaluated for implications in soft tissue ablation, several
studies have examined its effects on regions of the bone. In a study performing IRE in
porcine lumbar vertebrae, it was shown that IRE produces localized regions of well-
demarcated necrosis with no detectable change in bone texture and limited neural
toxicity (Tam et al. 2014). This study was performed in the lumbar vertebrae of ten
pigs using bipolar electrodes. Treatment parameters utilized 20–90, 2700 V IRE pulses
of pulse width 70 μs. Well-delineated areas of necrosis of the bone, bone marrow, and
skeletal muscle adjacent to the vertebral body were found with gross ablation average
widths of 9 mm and lengths of 33 mm. This study showed IRE to be a safe and feasible
technique to be utilized in the spine, with preservation of nerves.
Brain
IRE utility in brain has been characterized with preclinical and clinical canine
models. The high vascularization and extreme sensitivity of adjacent neural tissue
require precise treatment plans and blunt-tip electrodes to deliver the IRE pulses.
Preclinical studies in canine patients have shown IRE to safely ablate pathologically
heterogeneous brain tissue while preserving vascular integrity and patient neurolog-
ical functions.
In a preliminary study on five dogs, focal IRE ablations were made in the
ectosylvian gyrus (Ellis et al. 2011). Three dogs were treated with recommended
IRE parameters, one dog was a control, and the other was treated with a higher
voltage to determine the upper safety limits of the procedure. For the three dogs that
were noncontrols, treatment parameters included electrode separations of 5–8 mm,
electrode exposures 5–7 mm, voltages from 500 to 1600 V, 50 μs pulse duration, and
nine sets of ten pulses. These resulted in average ablation volumes between 0.258
and 1.655 cm3, with smaller volumes for lower voltage. Histological analysis
showed submillimeter boundary between the necrotic and normal brain. The animals
tolerated the procedure with no apparent complications except for the animal that
was treated at the upper voltage limit.
Canine malignant gliomas exhibit similar clinical, biologic, pathologic, molecu-
lar, and genetic properties as their human counterparts providing a good translational
model for clinical investigations. In an early case study, IRE attained a 75%
reduction in tumor volume within the first week post-IRE (Garcia et al. 2011).
Treatment parameters utilized 40–80, 500 V IRE pulses of pulse width 50 μs. The
electrode spacing and exposure was 5 mm. Following adjuvant fractionated radio-
therapy, the tumor was determined to be in complete remission prior to the sugges-
tive onset of early-delayed radiation encephalopathy, though recurrent glioma could
not be excluded. In a recent medium-term examination of the treatment cohort, it was
found that for the patients that survived and were discharged after the procedure,
Karnofsky performance scores were improved in 6/6 patients over pretreatment
values, while seizure control improved in 5/6 (Rossmeisl et al. 2015).
Prostate
Optimal treatment strategies remain to be determined for low- to medium-risk organ-

confined prostate cancer. While radical prostatectomy offers strong efficacy to tumor
control of tumors that have not metastasized, it carries high rates of morbidity in regard
to incontinence and impotence post-prostatectomy, including with robot-assisted pro-
cedures. This morbidity results from damage to the urethra and sensitive neurovascular
bundles at the perimeter of the prostate. Thermal and other focal therapies also risk
such adverse effects, and efforts to mitigate these risks may jeopardize the efficacy of
the treatment. Where it remains to be well defined which prostate tumors require
intervention, IRE may serve as an ideal focal or regional ablation approach to address
identified tumor regions with significantly lower risk to potency and continence.
Preclinical studies, primarily in vivo canine prostate studies, have shown the
ability to safely create ablation lesions while preserving integrity and regular system
function. In an early study, IRE was shown to produce significant ablations in
prostate while preserving the urethra and neurovascular bundle (Onik et al. 2007).
A further study investigated the implications of metallic seed implants within the
prostate, identical to those used for brachytherapy (Scheffer et al. 2003). This study
found that the presence of the seeds did not significantly affect the ablation zone or
thermal effects from the electric pulses, though it does not consider tissue changes in
response to prior radiotherapy, such as fibrosis.
Lung
Lung tumors have been treated with thermal ablation; however, their application is
limited due to thermal sinks from blood vessels, air spaces, and potential thermal
injury to critical tissues, including the heart, making it less safe and efficacious. IRE’s
nonthermal mechanism of cell death and sparing of critical structures make it a
potential candidate to treat lung tumors. A preliminary study on swine lung evaluated
the safety of IRE in the lung (Dupuy et al. 2011). This study performed percutaneous
IRE on nine domestic swine using bipolar as well as monopolar electrodes delivering
90 and 70 μs pulses of 1700–3000 V with electrocardiogram synchronization. The
results showed that all swine successfully completed IRE treatment without any
cardiac arrhythmias. CT showed focal areas of spiculated high density ranging from
1.1 to 2.2 cm. Histological analysis revealed focal areas of diffuse alveolar damage
with fibrosis and inflammatory infiltration with sharp boundaries of the interlobular
septa. The bronchioles and blood vessels within the areas of IRE were intact and did
not show signs of tissue injury. However, optimization of IRE procedures for pulmo-
nary targets remains to be performed before this organ can be successfully targeted for
IRE treatments, due to the unique electrical and thermal characteristics caused by
dynamic air spaces within the lung and its unique parenchymal structure.
Preserved Patency of Sensitive Structures
There are numerous relevant critical structures that may be implicated within or
around targeted volumes for IRE therapy. The relative importance of each will vary
with the targeted organ and cancer variety.
Blood Vessels
Extensive work has examined the effects of IRE on different organ blood vessels,
some of which have been mentioned above. While IRE is shown to kill the
endothelial cells and disrupt capillary-level vasculature, preservation of the collagen
framework facilitates continued blood flow in large blood vessels.
In a pilot study, the long-term effects of IRE on a large blood vessel were
studied (Maor et al. 2007). Treatment parameters included a sequence of ten IRE
pulses of 3800 V/cm voltage-to-distance ratio, 100 μs each, applied at a frequency

of ten pulses per second directly to the carotid artery in six rats. All the animals
survived the procedure and showed no side effects. Histology performed 28 days
after the procedure showed that the connective matrix of the blood vessels
remained intact, and the number of vascular smooth muscle cells (VSMC) in the
arterial wall decreased with no evidence of aneurysm, thrombus formation, or
necrosis.
In another IRE study (Appelbaum et al. 2012), Doppler ultrasound showed
continued blood perfusion through a major vessel contained within a tissue region
immediately after IRE treatment. Further, longitudinal studies have demonstrated the
regeneration of endothelial cells within the affected regions of the blood vessels
within 7 days, permitting full recovery and long-term function of the vessel.
Nerves
In an early in vivo study, it was shown that canine prostate tissue could be ablated
while preserving the neurovascular bundles necessary for potency and continence
(Onik et al. 2007). Following this study, the possibility of preserved neural function
has since been explicitly investigated.
In addition to the peripheral nerve effects from the vertebral bone IRE studies, a
long-term in vivo IRE study was performed on a rat sciatic nerve (Li et al. 2011). A
sequence of ten pulses with voltage-to-distance ratio of 3800 V/cm, each 100 μs long,
was only applied directly on the sciatic nerve to produce a treated length of about
10 mm. Electrophysiological, histological, and functional studies performed immedi-
ately and up to 10 weeks following surgery showed that, despite an initial decrease in
functionality, the nerve attained full recovery approximately 2 months later.
Ductal Systems
In addition to vascularity and neurological implications that may be present for many
organs targeted for IRE ablation, there are also many organ-specific ductal systems
which also seem to have preserved function and patency while being contained
within or adjacent to ablation zones. These include bile duct preservation in pancre-
atic and liver ablations (Bower et al. 2011), collecting system and ureter in renal
ablations (Deodhar et al. 2011), and urethra in prostate ablations (Onik et al. 2007).
The action behind this preservation likely relates to the structural organization of
many ductal systems, which are composed of relatively low-permeability connective
tissue innervated with endothelial and epithelial cellularity. Although IRE will
initially kill the cells within the ductal system architecture, preservation of the
extracellular constituents permits continued function of these tissues and supports
the recellularization of the systems over time.
Advances in Technology
The main focus of the early preclinical studies described here is targeted toward
describing IRE’s ability to destroy a targeted volume of tissue, while preserving
critical structures in the vicinity, and describing the variation of observed effects in
numerous tissue varieties. Following the successful translation of IRE to the clinic,
newer advances to the technology are being explored at the benchtop and preclinical
settings. These studies explore secondary effects of IRE that may further expand the
treatment zone, improve selectivity, as well as improve ease of application. Some
preclinical investigations into a selected number of these effects are presented below.
Immune Response and Activation
Where IRE is typically implemented by delivering electric pulses through electrodes

inserted directly into the targeted tissue to induce the cell death, the affected tissue
remains in situ following treatment. This permits the release of tumor-specific and
tumor-associated antigens, as well as the various signals of cell distress into the
treated and peripheral volumes. These molecules may have implications in the
promotion of local and systemic immune responses, similar to those described
from other ablation modalities.
One study explored the immunologic response to tumor ablation with IRE using
a subcutaneous xenotransplanted osteosarcoma rat model (Li et al. 2012). The
animals were randomized into four groups: the control, sham operation, surgical
resection, and IRE group. Another set of rats without tumor cell implantation
served as the normal non-tumor-bearing group. In each of the groups,
anticoagulated venous whole blood samples were acquired before and days after
the operation to monitor for T lymphocyte activity, the major source of cellular
antitumor immunity in cancer patients. Specifically, the CD3+ T lymphocytes that
represent the major lymphocyte subset in peripheral blood, the CD4+ (T helper
cells) and the ratio of CD4+/CD8+ (T suppressor/cytotoxic cells), are generally used
as an indicator of antitumor immunity. Additionally, rats were killed independently
in each treatment group, and splenocytes were assayed for IFN-γ and IL-4 produc-
tion using intracellular cytokine staining. T-cells exert their effector functions
partly by producing and releasing cytokines and are characterized by their distinct
cytokine expression patterns. Th1 cells secrete IFN-γ and IL-2, whereas Th2 cells
produce IL-4, IL-5, and IL-10. The results of the study showed that the application
of 1500 V/cm voltage-to-distance ratio in nine trains of ten direct current IRE
pulses, each 100 μs long, could produce complete osteosarcoma cell ablation, while
also providing a substantial immune response. Seven days after treatment, the IRE
group had a significant increase in CD3+ and CD4+ cells and increased ratio of
CD4+/CD8+. Additionally, IRE group showed an increased percentage of IFN-γ
splenocytes.
A comparative in vivo murine study was conducted on immunocompetent
(IC) versus immunodeficient (ID) mice implanted with RENCA murine kidney
cancer cells to gauge if IRE invoked a systemic immune response in the tumor
environment (Neal et al. 2013). Two flat plate electrodes were utilized to apply IRE
treatment on the subcutaneous murine tumors with a highly conductive gel facilitat-
ing improved current delivery into the tumor. Treatment parameters included
voltage-to-distance ratio of 1500 V/cm, each 100 μs long, delivered at a rate of
1 pulse per second. A total of 100 pulses were delivered, reversing polarity after the
first 50. Following pulse delivery, the electrodes were reoriented 90 , and the pulsing
process was repeated, delivering a total of 200 pulses to the tumor. This protocol was
selected due to its ability to produce an observable treatment response relative, but
not strong enough to cause complete regressions in both strains of mice, which
would make it difficult to discriminate any differences in treatment outcome. The
treated IC group responded significantly better than the treated ID group, despite no
inherent difference in initial tumor susceptibility. Similar susceptibility was indi-
cated by tumors reaching a treatable size for both groups within the same number of
days and similar tumor growth response to sham treatments. This suggests that
although an immune response is not required for complete tumor regression, thera-
peutic response in immunocompetent patients may be better than predictions based
on experimental studies using ID cancer models.
Overall, the exploitation and encouragement of the immune response demon-
strated from IRE treatment of tumors remains a promising yet relatively unexplored
field for improving IRE therapy outcomes.
Adjuvant Augmentation of IRE Treatments
In addition to encouraging a more robust immune response to further encourage

effective oncologic outcomes from IRE therapy, numerous other promising adjuvant
approaches exist to increase ablation zone and oncologic outcome from a given pulse
protocol for IRE therapy. The most obvious of these is the inclusion of targeted
chemotherapeutics already shown for electrochemotherapy to selectively kill revers-
ibly electroporated cells in the zone surrounding the irreversibly electroporated cells.
In addition, the use of conventional therapies as adjuvants to IRE may also improve
patient outcomes, such as the inclusion of standard chemotherapy regimens to kill
any distant micro-metastases, while IRE kills the primary tumor, or the use of
transarterial chemoembolization to further stress and promote death of tumor cells.
Several studies have examined potential IRE and reversible electroporation
therapy augmentation by locally manipulating the properties of the cell membranes
to increase their susceptibility to electroporation and IRE. One approach that has
been suggested, with implications primarily likely for in vitro applications, is the
addition of dimethyl sulfoxide (DMSO) in the cell membrane to alter the structural
properties of the lipid bilayer (Jiang et al. 2014). This study along with the first IRE
tumor study (Al-Sakere et al. 2007) also explored the potential of modifying the
temporal mode of delivery of pulses on IRE outcome, where it was shown that
adding delays between trains of pulses can decrease the IRE electric field ablation
threshold.
An additional study examined the sensitization of cells to electroporation within a

given electric field by altering the electrochemical environment of cells in tissue
in vitro with cationic and anionic substances. This study found markedly promising
results with the inclusion of cationic anesthetics in the cellular environment, with
particular benefits exhibited from procaine and lidocaine, where a 50% reduction in
the strength of the electric field is required to induce cell death with IRE. While it
remains to be determined whether such a pronounced effect will translate to in vivo
environments, it offers a valuable potential adjuvant to safely augment the treatment
zone of IRE and other electroporation-based therapies (▶ Chap. 91, “Combining
Electrolysis and Electroporation for Tissue Ablation”).
High-Frequency Irreversible Electroporation
Apart from improving ablation sizes, preclinical studies have shown that the ease of
application of IRE treatment can be improved by manipulating pulse parameters,
such as pulse duration and polarity. Clinically, IRE requires the administration of
paralytic agents to prevent muscle contractions during treatment that are associated
with the delivery of unipolar pulses of longer durations (~>5 μs). By reducing the
pulse duration and applying bipolar IRE-inducing pulses, the threshold for nerve
stimulation is increased and the muscle contractions are eliminated, removing the
need for paralytic agents.
The first preclinical study on high-frequency irreversible electroporation
(H-FIRE) was conducted on rat brain (Arena et al. 2011), and muscle contractions
were quantified via accelerometers placed at the cervicothoracic junction. MRI and
histological evaluation was performed postoperatively to assess ablation character-
istics. Treatment parameters consisted of bipolar pulses of 1–2 μs duration and
100–400 V amplitude delivered in 200 μs long bursts at a frequency of 1 burst per
second. Blunt electrodes of 1 mm spacing and exposure were used for the treatment.
Results showed that, even in the highest energy H-FIRE protocol, no detectable
peaks in acceleration above the inherent noise of the system were observed. Histo-
pathologic examination of brain sections from all treatments demonstrated clear
areas of ablation and sharp delineation from adjacent normal brain. This study
showed H-FIRE to be a feasible technique for nonthermal tissue ablation that
eliminates muscle contractions.
Conclusions
IRE’s unique nonthermal modality of ablation without denaturing extracellular pro-

teins permits IRE treatment in sites containing critical structures and vasculature that
contraindicate them for thermal ablation or surgical resection. The advantages of
IRE have resulted in the treatment being performed in an array of applications and
anatomical target regions. This chapter discusses the preclinical findings of IRE in
different sites and its treatment outcome in terms of ablation size and preservation of
critical structures. Additionally, secondary effects that may foster exploitation for
enhancement of IRE treatment, such as anti-immune response, adjuvant therapies,
and advances to IRE pulse protocols, offer evidence suggesting improved clinical
outcomes for the technology and its clinical implications as the base of knowledge
continues to grow.
Cross-References
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Preclinical Studies on Nanosecond Pulses
80
Stephen J. Beebe
Abstract
Nanosecond pulses have been used for over a decade to treat tumors in preclin-
ical studies in mice and rats. The technology based on pulsed power refocuses
cancer treatment from chemistry and drugs to physics and high-intensity,
nonthermal electric fields (EFs) to tumors by administering pulses with dura-
tions in the nanosecond domain. This constitutes a high-power, low-energy
impact that is unique and distinct from other physical medical technologies,
such as radiofrequency ablation and ionizing radiation. Nanosecond pulsed
electric fields (nsPEFs) have successfully eliminated a variety of cancers in
subcutaneous ectopic models or allographs, orthotopic models in liver, induced
skin cancer models and human cancer xenographs in immunodeficient mice.
Recent data suggest, but do not yet prove, that nsPEFs induce immune
responses in addition to ablating primary tumors. This chapter will review
what has been done with nsPEF technology in preclinical cancer models, how
these studies have been conducted, what should be considered when
establishing new cancer models, what precautions need to be taken with high
EFs, what effects nsPEFs have on the skin and blood flow, and what is known
about how nsPEFs cause tumor cells to die. Finally, what is known about the
possible immune response induced by nsPEFs will be discussed. Overall, this
chapter will present what is presently known about nanosecond pulses for the
treatment of cancer in animal models.
Keywords
Pulsed power • Electropermeabilization • Ectopic/orthotopic • Allograph/
xenograph • Electric field breakdown • Regulated cell death (RCD) • Caspases •
TUNEL
S.J. Beebe (*)

e-mail: sbeebe@odu.edu

DOI 10.1007/978-3-319-32886-7_47
1544 S.J. Beebe
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1544
Paradigm Shifts in Cancer Treatment from Chemistry to Physics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1545
Nomenclature for nsPEF-Treated Tumor Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1546
nsPEF Conditions for Effectively Treating Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1546
Considering nsPEF Conditions When Establishing a Tumor Model . . . . . . . . . . . . . . . . . . . . . . . . . . 1551
Cautions Needed Using Nanosecond High EFs on Animal Skin and Tissues . . . . . . . . . . . . . . . . 1552
Effect of Nanosecond Pulsed EFs on the Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1552
Effect of Nanosecond Pulsed EFs on Blood Flow to the Skin and Liver . . . . . . . . . . . . . . . . . . . . . 1553
Mechanisms for nsPEF-Induced Tumor Elimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1554
Apoptosis and Regulated Cell Death (CD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1554
Do nsPEFs Induce DNA Damage In Vivo? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1557
Do nsPEFs Induce Immune Responses? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1557
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1559
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1560
Introduction
Pulsed power is the fundamental technology behind high-intensity electric field

(EF) pulses with sub-microsecond durations. Pulsed power has been used for
decades in high-powered physics and for military applications such as radar. Now
with the advent of nanosecond pulsed electric fields (nsPEFs) for modulating cell
functions and for eliminating cancer, pulsed power physical principles have been
intertwined with biology and medicine. Pulsed power technology accumulates
and compresses energy and immediately releases high power in nanosecond
durations. An example of pulsed power principles inherent in nsPEFs is to
compare storage of 1 J of energy released in 1 s (1 W) versus when released in
1 μs (1 MW, 106 W) or 1 ns (1 GW, 109 W). By using high-voltage capacitors with
fast-switching and discharge capabilities, nanosecond pulses are delivered with
high-intensity EFs into cells and tissues. At lower EFs, this mimics hormones and
neurotransmitters, and at higher EFs, this mimics chemotherapeutic drugs and
internal stress without molecules – the low-energy, high-power electric input
affects plasma membranes (PMs) as well as intracellular membranes on organelles
and maybe proteins. Biological cells have not seen any impact like this in the
history of life on earth. Therefore, how cells and tumor tissues respond to them as
either nonlethal or lethal stimuli is of considerable interest. This chapter will
discuss how tumors respond to nsPEFs under conditions that are intended to
eliminate them. Interestingly, because energy is relatively low, yet power is
high, normal cells and cancer cells do not immediately die by abrupt membrane
rupture or primary necrosis. They die by regulated cell death (RCD). More about
that later.
Pulsed power can produce pulses with durations in a billionth of a second (109 s,
nanosecond) and even trillionth of a second (1012 s, picosecond). Considered
another way, in a nanosecond, light travels (1.86 105 mi/s or 2.99 105 km/s)
80 Preclinical Studies on Nanosecond Pulses 1545
about 12 in or 30 cm; in a picosecond, light travels about 0.01 in or 0.3 mm. In

contrast, conventional electroporation or electropermeabilization (EP) used in gene
electro-transfer (GET), electrochemotherapy (ECT), and irreversible electroporation
(IRE) generates microsecond (1 106 s) and millisecond (1 103 s) pulses. In a
microsecond and a millisecond, light travels about 1,000 ft or 300 m and about
200 mi or nearly 300 km, respectively.
Millisecond and microsecond (>100 μs) pulses are generally considered to be
conventional EP conditions. These pulses produce defects in PMs, called aque-
ous pores (Weaver and Chizmadzhev 1996). While PMs spontaneously form
very small, unstable, rapidly resealing pores, EFs lower the energy required for
pore formation, making greater numbers of more stable pores. For both GET and
ECT, EFs are generally low (0.1–1.0 kV/cm), so these aqueous pores are
transient, allowing resealing and maintenance of cell viability. GET allows
DNA to enter cells for transcription to accomplish an intended function (see
▶ Chap. 19, “Nucleic Acid Electrotransfer in Mammalian Cells: Mechanistic
Description” by Golzio and Rols). In ECT, transient pores allow poorly perme-
able chemotherapeutic agents (bleomycin) to enter cells causing cell death
(CD) (see ▶ Chap. 78, “Preclinical Studies on Electrochemotherapy” by Serša
et al.). In contrast, IRE uses much higher EFs (1 kV/cm) such that permanent
pores are formed and cells lose PM integrity and die by primarily by primary
necrosis (see ▶ Chap. 40, “Tissue Ablation by Irreversible Electroporation” by
Relong et al.).
Paradigm Shifts in Cancer Treatment from Chemistry to Physics
Considerations for nsPEFs to eliminate cancer evolved from experiments showing

that they could lyse bacteria and stun brine shrimp (Schoenbach et al. 1997). Soon,
attention shifted from environmental decontamination to applications in biology and
medicine (Schoenbach et al. 2001; Beebe et al. 2002, 2003; Vernier et al. 2003).
Applications for pulsed power in cancer presented a new paradigm in cancer
treatment and has demonstrated significant efficacy in many preclinical studies as
will be discussed in detail in this chapter. In addition to eliminating tumors by RCD
mechanisms, including apoptosis (Schoenbach et al. 2001; Chen et al. 2010, 2012,
2014a; Nuccitelli et al. 2015), nsPEFs have been shown to induce a vaccine-like
effect in mice (Beebe et al. 2011; Chen et al. 2013) and rats (Chen et al. 2014a).
Other evidence also suggests nsPEF-induced immune responses (Nuccitelli et al.
2012a, 2015) (see section “Do nsPEFs Induce Immune Responses?”). During tumor
RCD, dying cells activate immune mechanisms that can prevent cancer recurrence
(Kroemer et al. 2013). Consequently, nsPEF treatment may become a unique
approach to immunotherapy that does not involve delivery of DNA or virus,
checkpoint inhibitors, engineered patient immune cells, or molecules of any kind.
Furthermore, this approach provides a shift from cancer treatments with chemistry to
cancer treatments with physics.
1546 S.J. Beebe
Nomenclature for nsPEF-Treated Tumor Models
In this section, nomenclature for various tumor models will be presented without
specific references to them, until discussed later (Table 1, section “NsPEF Condi-
tions for Effectively Treating Tumors”). Most preclinical studies with nsPEFs have
been carried out in mice with subcutaneously injected tumor cells. These formed
tumors are ectopic or in an abnormal position. Some studies have used “allographs”
with cells or tissues transplanted in a recipient from a genetically nonidentical donor
of the same species; they are non-syngeneic. Syngeneic models have also been used
when donor tumor cells and recipient are genetically identical. So, tumors can be
ectopic and non-syngeneic or syngeneic. Tumors may be more readily eliminated in
non-syngeneic models because animals may have some immune resistance to the
nongenetically identical tumor cells. In orthotopic models, tumors are implanted in
their natural tissues. All orthotopic models have been syngeneic in nsPEF studies.
Other nsPEF studies have been done with subcutaneous xenographs, when human
tumors or cells are implanted into immunodeficient mice. Other tumors have been
induced in mice that are susceptible to UV light or ionizing radiation. Finally,
nsPEFs have been used to treat tumors in humans. Thus, nsPEFs have demonstrated
efficacy as therapy for local cancers in melanoma, basal cell carcinoma (BCC),
hepatocellular carcinoma (HCC), pancreatic and breast cancer in mice and rats, and
BCC in humans.
nsPEF Conditions for Effectively Treating Tumors
In all, studies using nsPEFs to treat tumors have used pulse durations of 7, 14, 20, 30,
100, or 300 ns with EF amplitudes between 20 and 68 kV/cm and pulse repetition
rates (pulses/second) of 0.5–7 Hz. All pulse durations demonstrated EF-dependent
and/or pulse number-dependent decreases in tumor size without significant increases
in temperature. However, repetition rates of 5–7 Hz caused increases in temperature
but apparently did not produce thermal effects (<36 C; anesthetized mice, basal
tumor temperature ~28 C). Accordingly, at least for pulse durations with lower
repetition rates, pulses cause nonthermal, EF-specific effects. In general, conditions
with long pulse durations (100 and 300 ns) have been shown to be most effective and
most commonly used. Given that extremely high EFs are generally used, the number
of pulses required for tumor elimination is surprisingly high. This is apparently
because of the extremely short pulse duration and tumor resistances to stresses. This
section will present a number of tumor models and their responses to a variety of
nsPEF conditions.
The first tumor treatment in animals was an ectopic, syngeneic B10.2 fibrosar-
coma in mice. Treatment included twice over an 8-day period with five or seven
pulses with durations of 300 ns and EFs of 75 kV/cm. There is a 60% reduction in
growth compared to controls as the initial response to nsPEFs.
In ectopic, non-syngeneic models with B16f10 mouse melanoma (Chen et al.
2010; Nuccitelli et al. 2006, 2009) in SKH-1 immunocompetent, hairless albino
80
Table 1 Some tumor types treated with nsPEF and outcomes

Immuno-
compromised Pulse #/Hz/ns/kV
Tumor type Model Species (yes/no) per cm Result References
B10.2 fibrosarcoma Ectopic syngeneic C57/BI6 mice No 5–7/0.3/300/75 60% growth decrease Beebe et al. (2002)
B16f10, melanoma Ectopic SKH-1 mice No 300/1/300/40 Good efficacy Chen et al. (2010),
non-syngeneic (hairless, albino) Nuccitelli et al. (2006),
and Nuccitelli et al. (2009)
AsPC-1, human Xenograph Nude mice Yes 100–300/20/20/45 Regression Garon et al. (2007)
pancreatic
SCC Chemical induced SENCAR mice No 50–500/50/14/40 85% at 1 week Yin et al. (2012)
efficacy
Preclinical Studies on Nanosecond Pulses
Hepa1-6 Ectopic syngeneic C57/BI6 mice No 1,000/1/100/65 75% clearance Chen et al. (2012)
HCCLM, human Xenograph Balb/c nude mice Yes 300/1/100/40 Slow tumor growth Chen et al. (2014b)
HCC
B16f10-GFP, Ectopic Nu/nu mice Yes 2,000/5–7/100/30 Complete elimination Nuccitelli et al. (2010)
melanoma non-syngeneic
Melanoma UV light-induced C57/BL6-HGF/ No 2,000/5–7/100/ Complete elimination Nuccitelli et al. (2012a)
SF mice/ 30 kV/cm
transgenic
BCC Ionizing Ptch-1 No 2,700/5–7/100/30 Complete elimination Nuccitelli et al. (2012b)
radiation-induced K14-Cre-p53 fl/fl
mice/transgenic
Human pancreatic Xenograph Nu/nu and Yes 200–500/2–5/100/30 Complete elimination Nuccitelli et al. (2013)
NIH-III mice
(continued)
1547
1548
Table 1 (continued)
Immuno-
compromised Pulse #/Hz/ns/kV
Tumor type Model Species (yes/no) per cm Result References
MCF7, human Xenograph Nude mice Yes 720/5/100/30 79% efficacy Wu et al. (2014)
breast
N1-S1, HCC Orthotopic Sprague Dawley No 100/1/100/50 80–90% efficacy Chen et al. (2014a)
rats
McA7777 Orthotopic Buffalo rat No 400/2–4/100/12–15 Good efficacy in Nuccitelli et al. (2015)
ablation zone
S.J. Beebe
mice, using a five-needle electrode array for the delivery of 300 ns pulses, tumors
were essentially unaffected with as many as 100 pulses at 10 kV/cm. Tumor growth
was slowed, but growth continued with ten pulses at 20 kV/cm. However, with one
or two treatments using 100 pulses (0.5 Hz) with durations of 300 ns and EF
strengths of 20 kV/cm, tumors shrunk 90% in 2 weeks (Chen et al. 2010; Nuccitelli
et al. 2006). In this same study, using two parallel plate electrodes that pinched
tumors, regression required as many as seven 100-pulse treatments over several days
with durations of 300 ns and 40 kV/cm. In a later study with this same B16f10
melanoma model using 300 pulses, 300 ns durations, and 40 kV/cm, complete tumor
elimination occurred in 24%, 59%, and 18% of tumors with one, two, and three
treatments, respectively (Nuccitelli et al. 2009).
Two studies carried out using pulse duration 20 ns suggested that pulse
durations this brief were less effective than 100 ns or 300 ns pulses. In a small
study with AsPC-1 human pancreatic xenographs in immunodeficient mice, three
separate treatments using a five-needle electrode array with 20 ns durations,
40–45 kV/cm, three 75 pulse bursts at 20 Hz 60 s apart cleared half of the tumors;
however, efficacy was considered after only 4 days; only tumor regression, not
clearance, could be declared (Garon et al. 2007). Another study used 7 and 14 ns
durations, 31 and 40 kV/cm, and 50, 200, and 400 pulses at 50 Hz using a five-needle
array to treat cutaneous papillomas and squamous carcinomas in sensitivity to
carcinogenesis (SENCAR) mice. These mice developed tumors in 20–30 weeks
when treated with methyl-N0 -nitro-N-nitrosoguanidine and tumor promoter 12-O-
tetradecanoylphorbol-13-acetate (TPA) (Yin et al. 2012). These tumors were elim-
inated efficiently for at least 1 week using one treatment with 14 ns duration pulses
(85%), and all remaining lesions were eliminated after a second treatment (15%).
Seven ns pulse durations were considered ineffective. While treatment temperatures
were not noted, it is likely that 50 Hz treatments, especially at 400 pulses, resulted in
thermal effects.
Another study determined efficacy of shorter versus longer pulse durations in a
syngeneic, ectopic mouse Hepa1-6 HCC in C57Bl/6 mice using a five-needle array
electrode. Treatments compared pulses with 30 ns and 100 ns durations and EFs of
33, 50, and 68 kV/cm at 1 Hz (Chen et al. 2012). Tumor-free survival was
determined after 9 months. The study found that protocols of 300 pulses every
other day for 3 days (3 300) or one 900-pulse treatment (1 900) with durations
of 100 ns and EFs at 65–68 kV/cm eliminated about 75% of tumors equally well.
Surprisingly, survival was less than 40% with the 3 300-pulse treatment with 100 ns
duration, 50 kV/cm. Survival was only about 25% for 3 300 treatment with 30 ns
and 65 kV/cm and less than 20% for the same protocol except with 50 kV/cm.
Therefore, for this model, 100 ns duration were significantly better than 30 ns,
65 kV/cm was better than 50 kV/cm, and a single treatment with 65 kV/cm and
900 pulses was sufficient for reasonable efficacy.
The efficacy of treatment protocols with a single treatment versus split treatment
was analyzed using implanted human HCCLM3 (HCC) tumor tissues in nude mice
with 100 ns and 40 kV/cm pulses (Chen et al. 2014b). Using parallel plate
electrodes, three treatments every other day with 100 pulses stabilized tumors,
1550 S.J. Beebe
while a single treatment of 300 pulses only slowed tumor growth. A similar study
using this model and conditions also showed a split treatment was superior to a
single treatment using an electrode design with a needle (anode) in the tumor mass
and a semiring (cathode) on the periphery with treatments in two orientations (Yin
et al. 2014).
Other studies using GFP-expressing B16f10 melanoma-forming tumors in
immunodeficient nu/nu mice (Nuccitelli et al. 2010) and UV-induced murine
melanomas in C57⁄Bl6-HGF⁄SF transgenic mice (Nuccitelli et al. 2012a) required
2,000 pulses (5–7 Hz) with a protocol of 100 ns durations and 30 kV/cm to
completely eliminate tumors in a single treatment. Mouse (Cs137) ionizing
radiation-induced BCC in Ptch1+/K14-Cre-ER p53 fl/fl mice were effectively
eliminated with the same protocol using 2,700 pulses (Nuccitelli et al. 2012b).
nsPEFs (nanoelectroablation) successfully eliminated human pancreatic tumors in
nu/nu and NIH-III immunodeficient mice with a protocol using a suction electrode
with 250–500 pulses (2–5 Hz) with the duration of 100 ns and amplitudes of
30 kV/cm (Nuccitelli et al. 2013).
A human MCF-7 model was treated in immunodeficient nu/nu mice using a
clamp electrode similar to parallel plate electrodes using a soft sponge. Treatment
protocols included three treatments of 240 pulses each day (720 total) at 4 Hz with
100 ns pulse duration and EFs of 30 kV/cm. Tumor growth was inhibited
75% and 79% after the first and second week of treatment, respectively
(Wu et al. 2014).
nsPEF treatments are also effective in rats. Syngeneic rat orthotopic N1-S1
HCC tumors in Sprague Dawley rat liver grew slowly 2 weeks after treatment with
100 and 300 pulses at 1 Hz, durations of 100 ns, and EF amplitudes of 50 kV/cm
using a five-needle array electrode. However, 1,000 pulses eliminated 82–90% of
them (Chen et al. 2014a). Another syngeneic orthotopic model with McA-RH7777
HCC cells in the Buffalo rat liver was completely eliminate in the exposure region
with 400 pulses with durations of 100 ns and EFs of 12–15 kV/cm delivered from a
two-needle electrode (Nuccitelli et al. 2015).
The first human treated with nsPEFs was a single subject with BCC treated with
200 pulses (10 bursts every 20 s) with 20 ns durations and 35 kV/cm. A complete
pathologic response was achieved (Garon et al. 2007).
A human clinical trial effectively treated 10 BCC on three subjects using 100 ns
durations, 30 kV/cm EF amplitudes, and 100 pulses at 2 Hz with a single treatment
lasting 1 min. Seven of ten were completely free of basaloid cells when biopsied and
two partially regressed. Two of the seven exhibited seborrheic keratosis (benign skin
growth) in the absence of basaloid cells. One of the ten treated lesions recurred by
week 10 and histologically had the appearance of a squamous cell carcinoma
(Nuccitelli et al. 2014).
nsPEFs effectively eliminate melanoma, BCC, HCC, and pancreatic and breast
tumors with a variety of models in mice and rats and importantly in human
subjects.
Considering nsPEF Conditions When Establishing a Tumor Model
The first tumor model treated with nsPEFs was an ectopic, syngeneic mouse B10-2
fibrosarcoma (Beebe et al. 2002). In vitro studies at that time were using pulsers with
durations of 10, 60, and 300 ns. For all effects observed, including propidium iodide
uptake, calcium mobilization, phosphatidylserine externalization, caspase activa-
tion, cytochrome c release, and loss of cell viability, longer pulse durations required
lower EFs (Beebe et al. 2002, 2003). Therefore, 300 ns pulses were used because
they required lower EFs. That turned out to be a reasonable pulse duration, as
indicated above.
There are a number of different strategies that could be considered and tested
when establishing a new model. However, beginning a study with pulse durations
of 100 or 300 ns would be advisable. Choosing an EF strength requires several
considerations. Compact, solid-state designs will have the greatest applicability in
future settings and reaching EFs as high as 60 kV/cm is possible, although not
trivial. Thus, choosing an EF of 20–40 kV/cm (or higher if possible) and perhaps
considering a 300 ns duration requiring a lower EF would be pragmatic. A logical
approach would be to treat with the highest EF possible with increasing pulse
numbers and then treat again if tumors regrow. Using lower EFs in subsequent
studies could be better if breakdown or spark formation over the skin becomes a
problem – but this is resolvable (see section “Cautions Needed Using Nanosecond
High EFs on Animal Skin and Tissues”). Conditions should likely be attempted
with relatively low pulse repetition rates to prevent thermal effects. Treatments
with 1,000 pulses at 1 or 2 Hz takes about 17 and 8 min, respectively (Chen et al.
2014a). When “nonthermal” was defined as not increasing temperatures above
37–40 C nor causing thermal damage, 4–7 Hz may be alright (Yin et al. 2012;
Nuccitelli et al. 2010), but temperatures should be checked if EF-specific effects
are a goal. Temperatures less than 43 C for short time periods will not induce
thermal damage and could reduce requirements for higher EFs and/or pulse
numbers. Electric field-specific, nonthermal effects are best assured by low rep-
etition rates. However, clinically, time is an important factor.
Another approach to determine in vivo tumor treatment conditions is to use
available data comparing in vitro conditions for 80–90% lethality with in vivo
conditions eliminating 75–90% of tumors. Comparing in vitro cell suspension
and in vivo treatments using a five-needle array electrode, in vivo conditions
required a factor of 20 to 30 times greater than in vitro conditions when the
product of pulse duration, EF, and pulse number was used. This was observed for
ectopic, non-syngeneic B16f10 mouse melanomas (Chen et al. 2010); ectopic,
syngeneic mouse Hepa1-6 HCCs (Chen et al. 2012), and orthotopic rat N1-S1
HCCs (Chen et al. 2014a). This was also observed with human HCCLM3
xenographs as cells or tumor blocks using parallel plate electrodes (Chen et al.
2014b; Yin et al. 2014). Consequently, this factor range held for three different
tumor types, in two different species, with syngeneic and non-syngeneic ectopic
1552 S.J. Beebe
and orthotopic models as well as human xenographs in nude mice, using two
different electrode designs. Therefore, it may be helpful to use this 20–30 factor
in this way when designing in vivo experiments based on the capabilities of your
pulsing system.
Cautions Needed Using Nanosecond High EFs on Animal Skin

and Tissues
There are several issues that should be considered when delivering extremely high
voltages to tissues. These include the tumor position, electrode design, “air pockets”
in the EF, and hair in the treatment zone.
For subcutaneous ectopic models, tumors should be lifted away from the body so
EFs are confined to the tumor and are not exposed to major body structures, such as
muscles or abdominal wall. Placing tumors away from hind limb muscles limits
muscle contractions. However, such contractions are relatively light compared to EP
or IRE conditions and paralytic medications are not necessary. It is also best to place
tumors nearer to the animal’s back and away from the abdomen. Generally, for
ectopic models, parallel plate or suction electrodes should be used. To isolate tumors
from the body, needle electrodes are very difficult to use in ectopic models. For
orthotopic liver tumors, needle electrodes are best.
Regardless of the design, electrodes have to be in complete contact with tumors,
and there can be no air within the EF, because a breakdown or a flashover spark will
occur across the tumor tissue causing extreme heat and skin burning. Thus, elec-
trodes must be covered with an insulating fluid such as K-Y jelly or olive, mineral, or
vegetable oil. Ultrasound gel can be used on subcutaneous tissues. These insulating
lubricants must cover all electrode surfaces not in contact with tumor tissue during
the entire treatment. This is especially important with higher repetition rates and or
long treatment times, which results in gel removal by flow and when using a suction
electrode, removing gel by suction. For needle electrodes, breakdown can occur
between the top of the tissue and electrode handle if not covered with gel. Teflon
sleeves can help where the needles leave the base of the handle. Stopping during
longer treatments to add more gel is advisable.
Another source of EF breakdown is hair follicles, which make excellent EF
conduits. The animal’s hair must be shaved very close to the skin. A depilatory
cream such as Nair can be applied to remove all hair shafts in the treatment area;
however, this treatment should be very brief, because Nair can cause skin irritation –
but not as badly as burning with EF breakdown.
Effect of Nanosecond Pulsed EFs on the Skin
Because tumors can be easily treated on body surfaces, most studies have evaluated
effects of nsPEFs subcutaneous tumors. These studies used various electrode designs
including a five-needle array (Chen et al. 2010, 2012; Nuccitelli et al. 2006), two
parallel plates that “pinch” tumors between them (Nuccitelli et al. 2006, 2009; Wu
et al. 2014), and suction devise with electrodes in a small cavity containing parallel
plate electrodes using a vacuum to pull the tumor away from the body (Nuccitelli
et al. 2010). Two other designs included a ring electrode with a center needle and a
coaxial ring applicator completely surrounded tumors (Chen et al. 2012) and needle
(anode) and a semiring (cathode) on the periphery (Yin et al. 2014).
In general, nsPEF conditions that are effective to eliminate tumors have transient
damaging effects on the skin that are resolved within 2 weeks (Chen et al. 2010,
2012; Nuccitelli et al. 2006, 2009, 2010). After treatment with the five-needle array,
small superficial erosions appeared on the skin as the stratum corneum shows signs
of necrosis and hemorrhage with superficial erosion of the epidermis. Damaged
subcutaneous tissue and skin recovered and inflammation resolved within
7–14 days. When parallel plate electrodes or suction electrodes were used, a hard,
black scab appeared on the stratum corneum and remained for about 2 weeks.
Treated skin has a different appearance than surrounding skin, but this discoloration
fades leaving no permanent scar. Notably, the mild skin-damaging effects are not
immediate. While this damage is often “necrosis,” it is not primary necrosis that
occurs with tissue burning. Damage appears later, indicating a slower process.
Although not specifically studied in nsPEF-exposed skin, this response is likely
RCD by cornification (Galluzzi et al. 2012). Thus, nsPEFs appear to effectively treat
skin cancer without scarring.
Effect of Nanosecond Pulsed EFs on Blood Flow to the Skin

and Liver
nsPEFs also have effects on blood flow to treated tissues. Pulsed skin undergoes an
acute edema with local bleeding evident 10 min after treating B16f10 melanoma
(Nuccitelli et al. 2006). Transillumination images and power Doppler reconstruction
before and after nsPEF treatment show that blood flow to tumors is significantly
reduced 15 min after treatment and vessels feeding these tumors disappear within a
day after pulsing. Local blood flow usually does not recover for about 2 weeks. The
number of counted primary and secondary vessels feeding sham-treated B16f10
tumors increased in a time-dependent manner while decreasing in treated tumors by
the end of the first week. By 14–21 days vessel numbers were less than 15% of
controls (Chen et al. 2010). After B16f10 melanoma (Chen et al. 2010) and Hepa1-6
HCC tumors (Chen et al. 2012) were treated with nsPEFs, microvascular density
markers CD34, CD31, and CD105 were significant decreased. Vascular endothelial
growth factor and platelet-derived endothelial cell growth factor (PD-ECGF) also
decreased. Decreases in CD34 (Nuccitelli et al. 2009) and VEGF, VEGFR, and
CD34 (Wu et al. 2014) were also observed. So, in addition to cytotoxic effects,
decreased blood flow to tumors contributes to regression as well as the cancer
hallmark of sustained angiogenesis and attenuates another cancer hallmark, evasion,
and metastasis. Returning blood flow to tumors promoted new growth, indicating
that nsPEF treatment must nearly be complete to prevent regrowth. However,
1554 S.J. Beebe
antitumor immune mechanisms are naturally active, but of limited efficacy if tumor
growth is significant. In addition, there is significant evidence that nsPEFs induce its
own immune response as will be discussed in section “Do nsPEFs Induce Immune
Responses?”
Effects of nsPEFs on tumor blood flow in the skin are different than in the liver,
which has a distinct architecture and circulation. In liver, hepatocytes are bathed in
open sinusoids. These sinusoids are like open wetlands that rivers or streams (hepatic
portal vein or hepatic artery) empty into before flowing into another stream (hepatic
vein). Upon treating a liver tumor, blood is emptied from the treatment area as it is
blanched and surrounding areas appear dark red and blood rich. Laser Doppler
indicates that 15–30 min after treatment, blood flow was decreased by 50–60% in
the treated liver area but tended to be greater than normal 7 days later (Chen et al.
2013). This suggests that in nsPEF-treated liver, anti-vascular effects or effects on
angiogenesis that occurred in ectopic tumors are not likely to be a factor in nsPEF-
treated HCC demise. This returned blood flow may be important for an immune
response discussed in section “Do nsPEFs Induce Immune Responses?”
Mechanisms for nsPEF-Induced Tumor Elimination
What do nsPEFs do to tumor cells in tissues? It is easier to investigate tumor cells

in vitro where cells can be analyzed in real time by microscopy or by flow cytometry
for PM potentials or pores and ion transport through them, reactive oxygen species,
mitochondria membrane potential (ΔΨm), cytochrome c release, and calcium mobi-
lization, among others. One way to evaluate effects in vivo is by modeling, for
example, with multicellular system models using irregular shaped liver cells (Esser
et al. 2009). These “in vivo” simulations are similar to in vitro results showing that
nsPEFs induce supra-electroporation including large numbers of nanometer-sized
pores in all cell membranes allowing higher conductance compared to smaller
numbers of larger pores than conventional EP and IRE (Esser et al. 2007). It was
proposed (Gowrishankar et al. 2006) and appears likely (Beebe et al. 2013) that
supra-electroporation was responsible, in part, for nsPEF-induced apoptosis.
Apoptosis and Regulated Cell Death (CD)
About the time studies began with nsPEFs on cancer cells and tumors, Hanahan and
Weinberg published a paper on Hallmarks of Cancer (Hanahan and Weinberg 2000)
and then expanded the concept later (Hanahan and Weinberg 2011). Realizing that
cancers exhibit hundreds of different genotypes defined by scores of mutations in a
wide variety of genes, they sought some simplification by defining eight major
cancer hallmarks that control normal cell homeostasis and proliferation. These
include self-sufficiency in growth signals, insensitivity to anti-growth signals, eva-
sion of apoptosis, limitless replicative potential, sustained angiogenesis, tissue
invasion/metastasis, reprogramming energy metabolism, and evasion of immune
surveillance. Unstable genomes and inflammation can also advance multiple cancer
hallmarks. The fact that nsPEFs induce CD in cancer cells in vitro and eliminate
tumors in vivo begs the question of what are mechanisms or targets that lead to
cancer cell and tumor elimination. These questions were first addressed in vitro and
found to include RCD by apoptosis as defined by the presence of active caspases,
calcium mobilization, cytochrome c release, loss of the mitochondria membrane
potential, PARP cleavage, and cell demise that is delayed in time after treatment
(see ▶ Chap. 30, “Regulated and Apoptotic Cell Death After Nanosecond Electro-
poration” by Beebe). This suggested that nsPEFs could alleviate the cancer hallmark
of evasion of apoptosis. However, as indicated in that chapter, it must be kept in
mind that not all cell types die by apoptosis in response to nsPEFs, indicating that
other RCD mechanisms occur depending on cell type.
Given that apoptosis and necrosis were the only known CD mechanisms when
nsPEF studies began and that cells did not die by immediate membrane rupture, or
primary necrosis, apoptosis was a likely candidate for CD induced by nsPEFs. In the
first studies with nsPEF-treated fibrosarcoma tumors, EF-dependent increases in
caspase-3/7 catalytic activity of DEVD-afc (Asp-Glu-Val-Asp-afc) were observed
in tumor extracts 3 h after tumor treatment with three or five pulses with 300 ns
durations and EF amplitudes from 18 to 60 kV/cm (Beebe et al. 2002, 2003). While
this caspase-3/7-specific catalytic assay is sensitive, it does not inform about the
percentage of cells that are actively caspase positive. This is also true for Western
blot analysis using antibodies specific for cleaved caspases.
Percentages of executioner caspases (-3, -6, and -7) in tissue slices of nsPEF-
treated B16f10 melanoma tumor (100 pulses, 300 ns 40 kV/cm) demonstrated a
time-dependent increase in active caspases peaking at 6 h in 45% of cells but was
less than 5% of cells 24 h later (Chen et al. 2010). However, large DNA fragments,
but not a 180 bp DNA ladder typical of apoptosis-mediated fragmentation, were
observed, suggesting incomplete apoptosis. A later study in Hepa1-6 HCC tumor
slices indicated levels of specific active executioner caspase-3 (~45%), caspase-6
(~15%), and caspase-7 (~30%) in a time-dependent manner, peaking at 3 h with
nsPEF conditions that eliminated ~75% of tumors (Chen et al. 2012). As would be
expected with a presence of active apoptosis, phosphorylated BAD was not observed
(see below).
In both of these studies, there were significant numbers of cells that were negative
for active caspases at all time points tested, indicating that other RCD mechanisms
were likely present. It was also found in orthotopic N1-S1 HCC tissue slices with
conditions that eliminated 80–90% of tumors that active caspase-3 and caspase-9,
but not caspase-8, were present, suggesting activation of an intrinsic apoptosis
pathway (Chen et al. 2014a), which is consistent with the concept that nsPEFs have
intracellular effects. These studies demonstrated that nsPEFs could bypass the cancer
hallmark of evasion of apoptosis and induce other RCD mechanism as well. The
presence of caspase-9 and caspase-3 was also demonstrated 2 h after orthotopic
McA-RH7777 HCC tumors were treated with nsPEFs (400 pulses, 100 ns 15 kV/cm)
using a two-needle electrode (Nuccitelli et al. 2015). Thus, nsPEFs activate caspases in
melanoma and HCC in mice and two different rat strains, in syngeneic and
1556 S.J. Beebe
non-syngeneic ectopic as well as orthotopic models through the intrinsic apoptosis

pathway, but other undefined CD mechanisms also are present.
A number of other papers report that nsPEFs induce apoptosis. However, not all
assays allow specific identification of apoptosis. For example, although terminal
deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) is often called and
used as an apoptosis marker, it is not specific for apoptotic DNA strand breaks
because it does not provide specificity for DNA cleavage around nucleosomes.
Consequently, DNA cleavage identified by TUNEL can be due to non-apoptotic
mechanisms. Likewise, pyknotic nuclei could be due to other forms of CD. Correctly
demonstrating apoptosis-mediated effects in DNA requires identification of
180–200 bp DNA fragments forming a DNA ladders on agarose gels. Caspase-
activated DNAse (CAD) cleaves DNA at nucleosomes wrapped around histone core
units where cleavage sites are present every ~180–200 bp.
Another set of markers sometimes used for apoptosis are the BCL2 (B-cell
CLL/lymphoma 2) family of anti-apoptotic proteins such as BCL2 and BCLxl,
proapoptotic proteins such as BCL2-associated X protein (BAX) or BCL antagonistic/
killer (BAK) and the BH-3 only proapoptotic BCL-associated agonist of cell death
(BAD) (Czabotar et al. 2014). Tumors often have high levels of anti-apoptotic
proteins that allow them to avoid apoptosis (Hanahan and Weinberg 2000, 2011).
BCL2/BCLxl protects mitochondria by dimerizing with BAX/BAK, preventing
pore formation in the outer mitochondrial membrane by the proapoptotic proteins.
Since it is the balance between pro- and anti-apoptotic proteins that determine
cytochrome c release, the absolute levels of either one does not sufficiently inform
about which one predominates. The time at which these proteins are determine
after treatment is also important. For example, decreases BCL2/BCLxl 24 or 48 h
after treatment could be due to elimination of BCL2-expressing cancer cells that
died by another mechanism.
BAD is another BCL2 family protein that has been used as an indicator of
apoptosis. When BAD is not phosphorylated, it binds to BCL2/BCLxl. This frees
BAX/BAK to induce apoptosis (Czabotar et al. 2014). So, it is the phosphorylation
state of BAD that determines its proapoptotic activity, not its absolute levels. One
study observed that BAD was not phosphorylated in the first 8 h after nsPEF
treatment (Chen et al. 2010). Thus, BAD could be binding to BCL2/BCLxl, freeing
BAX to induce cytochrome c release. Such a result is consistent with the presence of
apoptosis, but it does not specifically indicate it.
In general, demonstrating the presence of active caspases in vivo has been the
most commonly used marker for apoptosis induction in response to nsPEFs. It can
be done with catalytic assays using specific caspase substrates. Catalytic assays
with caspase-3, -8, and -9 using Jurkat cells that did not express FADD, caspase-
8 or APAF-1 demonstrated good caspase isozyme specificity (Beebe et al. 2013).
Likewise, antibodies specific for cleaved, active forms of caspases can be identi-
fied on Western blots from tumor extracts. However, using antibodies for specific,
active, cleaved initiator caspases (Chen et al. 2014a) or active executioner caspase
assays (Chen et al. 2012, 2014a) in tumor tissue slices allows an analysis of
relative cell numbers that express these active caspases. Other markers such as
absolute levels of BCL2 family proteins by themselves can be used to support an

apoptosis finding but specifically demonstrate apoptosis. Pyknosis, TUNEL, or
other assays for DNA damage can be due to apoptosis or other mechanisms of
RCD or primary necrosis.
Do nsPEFs Induce DNA Damage In Vivo?
Given that nsPEFs were expected to show effects on intracellular structures and that
the nucleus is the biggest structure in cancer cells, these were likely targets for
nsPEFs in cancer cells in vitro and in vivo. A number of in vitro studies indicated
that nsPEFs induced DNA damage including positive comet assays and upregulation
of DNA repair genes after nsPEFs (Stacey et al. 2003). nsPEFs was shown to induce
DNA damage using the TUNEL assay in fibrosarcoma tumors (Beebe et al. 2002,
2003), coincident time-dependent increases in cells positive for TUNEL (55% of
cells), and histone 2AX phosphorylation (85% of cells), in B16f10 melanoma
tumors (Chen et al. 2010).
The peak appearance of the earliest DNA damage marker (3 h) was before the
peak appearance of active caspases-3/-6/-7 (6 h), suggested that DNA damage was
caspase independent. Furthermore, appearances of all DNA damage markers were
not present in the first hour after treatment, indicating that DNA damage was not
immediate as might be expected if nsPEFs directly induced DNA damage. In Hepa1-
6 HCC tumors (Chen et al. 2012), DNA damage by TUNEL occurred in the first
hour and peaked with active caspases at 3 h.
Perhaps the best evidence for nsPEF-induced DNA damage is indicated in vitro
by a DNA smear on agarose gels immediately after nsPEF treatment (1 pulse, 60 ns,
60 kV/cm) (Stacey et al. 2003) and severely compromised telomere sequences,
regions that tether DNA to the nuclear membrane and stabilize it, immediately
after exposure to single 60 ns, 60 kV/cm pulses in cell suspensions (Stacey et al.
2011). However, the in vivo data for DNA damage due to nsPEFs is more evasive.
While DNA damage may be independent of apoptosis-related fragmentation or
degradation, the absence of significant levels of one of the earliest DNA damage
markers, γH2AX, within the first hour after treatment (Chen et al. 2010) suggests
that nsPEF-induced DNA damage in vivo is not directly related to the pulses
themselves but is more likely downstream events that are set in motion by nsPEFs.
Nevertheless, nsPEFs effects on DNA require additional studies.
Do nsPEFs Induce Immune Responses?
Immunogenic cell death (ICD) induces immune responses through activation of

dendritic cells (DCs) that activate specific T-cell responses to specific antigens
(Kroemer et al. 2013). While there is a controversy about which CD mechanisms
induce ICD, in chemotherapy-induced CD in mice, some (anthracyclines), but not all
(mitomycin C) caspase-inducing chemotherapeutic agents, initiate ICD. Apoptosis
1558 S.J. Beebe
has been shown to induce maturation of dendritic cells, antigen cross-presentation to

T-cells and immunity. Damage-associated molecular patterns (DAMPs), such as
externalized calreticulin and release of ATP and HMGB1, enhance DC engulfment
and antigen presentation to T-cells. Given that nsPEFs induce apoptosis and other
RCD programs, it is possible that nsPEF-induced dying cells may release these
DAMPs to induce an immune response.
The first suggestion that nsPEFs induced an immune response was in a small
study with mice that had been cleared of ectopic, syngeneic mouse Hepa1-6 HCC
tumors with 1,000 pulses with 100 ns durations and an EF amplitude of 65 kV/cm
(Beebe et al. 2011; Chen et al. 2013). When these tumor-free mice were challenged
with a second injection of the same cells, they did not grow tumors, demonstrating a
vaccine-like protection. A larger study followed this one using an orthotopic rat N1-S1
HCC model (Chen et al. 2014a). nsPEFs (1,000 pulses, 1 Hz, 100 ns, 50 kV/cm)
eliminated 84–90% of tumors that remained tumor-free for 5 months. When these
tumor-free rats were orthotopically challenged with the same cells, they too did not
grow tumors. This demonstrates a protective, vaccine-like effect after tumor elimina-
tion. The nsPEF treatment induced intrinsic apoptosis and the treated microenviron-
ment exhibited granzyme B, which is released by cytotoxic T-cells and natural killer
(NK) cells to induce apoptosis in tumor cells. Together, these findings suggest an
immune response and raise the possibility that nsPEFs could also address another
cancer hallmark by overriding evasion of immune surveillance. However, these data
do not show direct evidence for an immune response.
In another study using the same strategy with a rat McA-RH7777 HCC tumors
(Nuccitelli et al. 2015), challenge injections failed to grow tumors well and in two
instances not at all. In another strategy, a CD8 antibody was injected into animals
after a first tumor was ablated and 1 day before a second tumor was injected. CD8
was shown to be absent from blood samples 24 h after injection, and second tumors
grew much faster in CD8+-depleted rats with the second tumor size 54% of the first
tumor. Vaccinating mice with nsPEF-treated tumor cells stimulated a CD8+-
dependent immune response that inhibited growth of secondary tumors. CD8+
cells were present in the treated microenvironment, but activation phenotype was
not determined. Caspase-8 and -9 were shown to be activated and cells expressed
calreticulin on plasma membranes when treated in vitro. While these results indicate
that CD8+ cells are important for slowing tumor growth, they do not show direct
evidence for an immune response.
Another study demonstrated effective elimination of UV-induced mouse mela-
noma with nsPEF treatment (nanoelectroablation: 2,000 pulses, 5–7 Hz, 100 ns,
30 kV/cm) in C57/Bl6-HGF/SF transgenic mice (Nuccitelli et al. 2012a). The nsPEF
treatment was superior to surgical excision. An immune response was reported based
on the presence of CD4+ T-cell in the treated tumor and in other melanoma tumors
that were not treated. However, these cells could be CD4+ Foxp3 T-suppressor cells,
which are common in immunosuppressive tumor microenvironments. No further
T-cell phenotypes were determined.
Another study hinted that nsPEFs elimination of a local HCC tumor may prevent
lung metastasis suggesting an immune response (Yin et al. 2014). The study showed
that nsPEFs eliminated a highly metastatic human HCCLM3 HCC xenograft in nude
mice and that metastasis to the lung was reduced. While this is an encouraging result,
it cannot be excluded that the local treatment was sufficient to prevent cells from
metastasizing to the lung as opposed to inducing an immune response to prevent
metastasis.
This is a suggestive evidence nsPEFs can induce ICD and induce an immune
response, but there are no definitive data demonstrating an immune response.
For example, cytotoxic T-cells that express effector and central memory pheno-
type, especially to a specific tumor antigen, have not been shown in blood,
spleen, or tumor microenvironments. Also cytotoxic lymphocyte responses
toward tumor cells have not been demonstrated. Nor has it been shown that DC
cells exposed to nsPEF-treated cancer cells undergoing apoptosis, or some other
RCD mechanisms, expressing calreticulin and releasing HMGB and ATP, can
activate DC cells that cross present antigen to T-cells. In addition, it has not
been shown that the nsPEF-treated tumor microenvironment is depleted of
immunosuppressive immune cells such as myeloid-derived suppressor cells or
T-regulatory cells.
Conclusions
Overall, these studies demonstrate a clear potential for nsPEFs to provide an

effective anticancer local treatment strategy. This approach represents a paradigm
shift from medicine based on chemistry to medicine based on physics with novel use
of pulsed power technology. A wide range of tumor types, animal models, and
several effective electrode designs have been used to show notable efficacy. In
general, good results have been obtained with pulse durations of 100 and 300 ns
and EFs of 30–65 kV/cm and pulse numbers from 400 to 2,700 depending on the
tumor model and electrode design. Slower repetition rates take more time but allow
electric field, nonthermal effects. Treatments have transient effects on the skin but do
not result in scarring. However, because EFs are high, care must be taken to isolate
tumors from the body proper, and electrodes must be completely within tumor tissue
or covered with gels to exclude air and hair to prevent breakdown and skin burning.
Mechanisms that induce nsPEF tumor damage and death are still not completely
clear. While in vitro data for nsPEF-induced DNA damage is convincing, evidence
for direct DNA damage in vivo is less persuasive. However, data demonstrating
induction of intrinsic apoptosis in two orthotopic liver models suggests that nsPEFs
induce intracellular stresses that cannot be resolved, leading to self-induced CD by
apoptosis as well as by other RCD mechanisms. More recent evidence suggests that
nsPEFs can induce immune responses, but this has not yet been completely proven
and requires additional studies for validation. Nevertheless, the induction of ICD and
an immune response suggests that nsPEF cancer treatment may provide protection
from recurrent disease and perhaps metastatic, systemic disease. Ongoing studies are
addressing these important issues and pushing toward clinical trials for human
cancer therapy.
1560 S.J. Beebe
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▶ Preclinical Studies on Electrochemotherapy
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Principles of Electroporation for Gene
Therapy 81
Loree C. Heller
Abstract
Gene therapies can compensate for missing or mutated proteins, directly treat
cancer, act as cancer or infectious disease vaccines, or modulate protein expres-
sion. For any type of gene therapy, the dose of expressed protein should be
appropriate to correct the disease. Therapeutic development is often hampered
by the concept that an optimal therapy is one with the highest and longest
transgene expression coupled with the inability to predict the ultimate expression
levels and duration of the therapeutic protein.
Gene therapy methods can be loosely described as biological or nonbiological.
All gene therapies achieving regulatory approval to date are biological gene
therapies delivered by engineered viruses. Nonbiological transfer by chemical
and physical means removes the need for a biological vector and improves the
safety profile. Several nonbiological delivery methods, including electroporation,
have been developed to enhance gene delivery. In electroporation or electroper-
meabilization, controlled electric pulses produce temporary permeabilized areas
in a cell’s membrane to allow molecular transfer. Electrotransfer has been used to
transport a variety of molecules ranging from ions to drugs to nucleic acids across
the plasma membrane and into cells.
Electrotransfer also allows molecular transfer in vivo to many tissue types.
After satisfactory preclinical studies developing gene therapies using this delivery
method, several therapeutic applications have reached Phase II clinical trials. This
chapter overviews the steps necessary to develop a gene therapy using
electrotransfer, including vector design, electric pulse choice, and electrode
optimization. In addition, the complexity of the tissue target must be considered
for successful development of an electrotransfer-based gene therapy.
L.C. Heller (*)

Frank Reidy Research Center for Bioelectrics, Medical Diagnostic and Translational Sciences,
College of Health Sciences, Old Dominion University, Norfolk, VA, USA
e-mail: lheller@odu.edu

DOI 10.1007/978-3-319-32886-7_48
1564 L.C. Heller
Keywords
Gene therapy • Electrotransfer • Clinical Trials • Nucleic acids • Electrodes •
Tissues
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1564
Gene Delivery Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1566
Electroporation/Electropermeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1567
Nonviral Nucleic Acid Formats for In Vivo Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1570
Electric Pulses Used for In Vivo Electrotransfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1571
Electrode Design for In Vivo Electrotransfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1572
Tissue Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1574
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1575
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1576
Introduction
Gene therapies fall into five basic categories. First, in protein replacement, the
wild-type complementary Deoxyribonucleic Acid (cDNA) is delivered so the
expressed therapeutic protein compensates for a missing or mutated protein, as in
cystic fibrosis or hemophilia. Second, cancer therapies are generally designed to
induce cell death directly, to stimulate the immune system against the tumor and
its metastases, or to modify the tumor’s microenvironment. Third, to produce
classic infectious disease vaccines, the organism must be cultured and attenuated
or inactivated as the vaccine is designed. The use of the organism’s genes or
cDNAs for antigen expression allows the components of the vaccine to be
tailored for efficacy while avoiding the possibility of infection. For cancer
vaccines, tumor antigens are delivered to activate the patient’s immune response
to the antigen and induce a preventative effect. Fourth, cDNA can be delivered to
produce a protein that modulates an endogenous response, for example, to
support wound healing. Finally, disease-related proteins can be downregulated
or cells can be sensitized to specific therapies using several possible nucleic acid-
based mechanisms.
While delivery of the appropriate section of genomic DNA encoding the therapeu-
tic protein and its regulatory elements would produce physiologically appropriate
expression of the gene, mammalian genes and their regulator regions can be quite
large. For example, the gene mutated in the disease Duchenne muscular dystrophy is
more than two million base pairs. Mammalian genes contain introns, noncoding
sequences that are transcribed but removed before translation (Fig. 1, steps 1 and 2).
A DNA sequence must be minimized to be packaged for gene therapy; the initial step
is to artificially synthesize cDNA from processed messenger ribonucleic acid (mRNA)
(Fig. 1, step 3).
81 Principles of Electroporation for Gene Therapy 1565
Fig. 1 Production of cDNA

for gene therapies. Most
eukaryotic genes are composed
of exons (expressed DNA) and
introns (intragenic regions).
Genomic DNA (a) is
transcribed by RNA
polymerase II to produce
primary transcripts (b). In the
nucleus, primary transcripts are
capped at the 50 end with
7-methylguanosine and after 30
cleavage, a polyadenosine tail
is added. Introns are removed
by splicing (c). Alternative
splicing may produce multiple
mature mRNA transcripts. To
produce cDNA, RNA is
isolated from the cell and
mRNAs are reverse transcribed
into single stranded DNA.
Double stranded DNA is
produced by DNA polymerase
for insertion into a vector
Gene therapy development is often hampered by the concept that an optimal

therapy is one with the highest and longest transgene expression. This is not always
the case. As with drugs, the dose of expressed protein should be appropriate to
correct the disease. While a low dose may be ineffective, a high dose may be toxic or
may set off signaling cascades that regulate the response to or activity of the protein.
Transgene expression should also be tailored so the therapeutic protein is located in
the appropriate tissue and cellular location. For example, in the case of muscular
dystrophy, the corrected protein should be expressed in muscle cells. Factor VIII for
hemophilia should be adequately expressed in the circulation.
For any type of gene therapy, it is difficult to predict the expression levels and
duration of the therapeutic protein since production of the protein is dependent on
the cell or tissue that is transfected. Cell type, cell viability, and cell cycle status may
affect transcription, mRNA processing, and mRNA degradation. After translation,
posttranscriptional modifications such as proteolytic cleavage or glycosylation
must be appropriate for protein function. The expressed protein may not have the
same half-life as the natural protein. Because of these difficulties, the pharmacoki-
netics and pharmacodynamics variables of expressed therapeutic proteins, includ-
ing as rate of accumulation, peak levels, and rate of degradation have been poorly
explored.
1566 L.C. Heller
Gene Delivery Methods
Gene therapy methods can be loosely described as biological or nonbiological. Viral

gene therapy, a form of biological therapy, is probably the most advanced delivery
system. Viruses can have natural or pseudotyped tissue tropism and are capable of
long-term expression, although onset of expression can take weeks. Viral vectors are
usually engineered to be replication-defective, but the emergence of replication-
competent vectors is possible by recombination with wild-type virus. As of February
2016, viral delivery has accounted for at least 67% of clinical gene therapy trials
historically.
The primary viral vectors currently used are adenovirus, lentivirus, and adeno-
associated virus. Each of these viruses has advantages and limitations (Table 1). One
fundamental constraint of viral vectors is the limited insert size, since the packaging
capacity is dictated by the size of the viral capsid. Helper-dependent “empty”
adenoviral vectors have been developed, increasing capacity to >30 kb. However,
the primary limitation of adenoviral vectors is the strong immune response in the
presence of preexisting immunity. Lentiviral vectors based on human immunodefi-
ciency virus or nonhuman retroviruses produce long-lasting expression after inte-
gration into the host genome. However, genomic integration is associated with
insertional mutagenesis, which can be tumorigenic. Wild-type adeno-associated
viral vectors also integrate into the genome in a site-specific manner. Due to the
observation of tumorigenesis in preclinical models, vectors for adeno-associated
viral gene therapy are designed to remain within cells episomally.
The gene therapies achieving regulatory approval to date are viral gene therapies.
In 2004, the State Food and Drug Administration of China approved the first gene
therapy, Gendicine, a recombinant p53 therapy delivered by adenovirus for head and
neck squamous cell carcinoma. The European Medicines Agency (EMA) has now
approved two gene therapies, Glybera, an adeno-associated vector engineered to
produce lipoprotein lipase approved in 2012 for a rare inherited deficiency, and
Strimvelis, an ex vivo gene therapy delivered by gamma retrovirus approved in 2016
for the treatment of severe combined immunodeficiency due to adenosine deaminase
deficiency. In 2015, the United States Food and Drug Administration (FDA)
approved talimogene laherparepvec, an oncolytic herpesvirus expressing granulo-
cyte macrophage colony stimulating factor, for the treatment of unresectable mela-
noma recurrence.
Table 1 Common viral gene therapy vectors

Adenovirus Lentivirus Adeno-associated virus
Capsid size (nm) 70–120 80–100 20–25
Packaging capacity (kb) 7.5–10 6–10 4.5–5
Genomic integration No Yes Low
Immune response Yes (preexisting) Yes (transfected cells) Yes (preexisting)
Transgene expression Transient Long-lasting Long-lasting
nm nanometers, kb kilobases
Gene transfer using plasmid DNA is an attractive option because it removes the
need for a biological vector, which improves the safety profile compared to viral
delivery. Plasmid DNA is more easily prepared and more economical to produce
than viral vectors. Simple intramuscular plasmid injection produces gene expression
(Wolff et al. 1990). However, this expression is limited due in part to low transfec-
tion efficiency. Extracellularly, the presence of nucleases and the difficulty of transit
across the interstitial space between cells are barriers to delivery. Depending on the
delivery method, cell-related barriers may include the plasma membrane, cytoplas-
mic nucleases, transport through the cytoplasm, and transport across the nuclear
membrane.
Several nonbiological chemical and physical delivery methods have been devel-
oped to enhance the delivery of naked nucleic acids. Chemical delivery involves
packaging of the nucleic acid into particles such as nanoparticles formed primarily
from cationic lipids, polymers, or peptides. Physical methods include ultrasound-
induced sonoporation, hydrodynamic delivery, laser-induced stress waves,
magnetofection, jet injection with highly pressurized gas, jet injection with heavy
metal particles, and electroporation. Synthetic and biologically based transposase/
integrase systems including phage phiC31, PiggyBac, and Sleeping Beauty offer
nonviral integration into the genome for long-term expression. Although integration
is reasonably targeted, these delivery systems may have the potential to produce
insertional mutagenesis.
Several chemical and physical gene delivery methods have reached clinical trials.
The first nonviral gene therapy trial was initiated in 1992 (Nabel et al. 1993). A
DNA-liposome complex encoding a foreign major histocompatibility complex pro-
tein was injected into the patient’s melanoma nodules. Since then, nonviral DNA and
RNA delivery directly to patients by methods including simple injection, lipofection,
particle bombardment, and electroporation have averaged approximately 20 per year
over the last 15 years (Fig. 2). Clinical trials utilizing in vitro applications, in which
cells such as T lymphocytes or dendritic cells are modified nonvirally then injected
into the patient, have averaged 9 per year in that time.
The concept of gene therapy is not without controversy. Recently, the discovery
of the potential for biased inheritance of genes (“gene drive”) using the Clustered
Regularly Interspaced Short Palindromic Repeats/CRISPR associated (CRISPR/
Cas) technique has raised ethical concerns. These considerations, although they
constitute very important questions, are beyond the scope of this chapter.
Electroporation/Electropermeabilization
Controlled electric pulses produce temporary permeabilized areas in a cell’s mem-

brane that can be used to accomplish several functions (Fig. 3), which is referred to
as electroporation or electropermeablization. Irreversible electroporation, the most
extreme application of electroporation, is defined as cell destruction by electric
pulses to ablate undesirable tissues (Jiang et al. 2015). This is accomplished with
1568 L.C. Heller
Fig. 2 Number of nonviral

gene therapy clinical trials
worldwide by year. In vivo,
therapeutic DNA or RNA is
introduced directly into the
patient. In vitro, autologous or
allogeneic cells are modified
in vitro and introduced into
the patient (Source: Gene
Therapy Clinical Trials
Worldwide provided by the
Journal of Gene Medicine)
Fig. 3 Cell modification after electroporation. Depending on the pulse regimen, electroporation
can have several effects on a cell. Temporary pores are induced by reversible electroporation. The
cell survives but is intentionally modified in some way, i.e., by molecular delivery, fusion between
cells, or insertion of proteins or other molecules into the plasma membrane. Application of similar
but more numerous pulses produces irreversible pores and cell death
extended delivery of short, high-voltage pulses. Ablation with this technique for
multiple tumor types has reached clinical trials.
However, pulse application does not necessarily destroy the cell. In reversible
electroporation, the cell survives and several therapeutic applications are possible.
Membrane fusion can be induced by electric fields (Zimmermann 1982) and has
been used for the formation of hybridomas (Lo et al. 1984) and for in vivo cell-tissue
electrofusion (Grasso et al. 1989). Famously, Dolly the sheep, the first mammal
cloned from an adult somatic cell, was formed after electrically induced cell fusion
between a nuclear donor cell and an enucleated egg (Wilmut et al. 1997). Electric
pulses can also be used to insert proteins into a cell’s plasma membrane (Mouneimne
et al. 1989) although protein insertion may be short-term, limiting its therapeutic
value. The third reversible application, molecular delivery or gene electrotransfer,
will be described here.
In 1982, plasmid delivery using electric pulses was first demonstrated (see “First
Demonstration DNA Electrotransfer”) to cells in culture by the formation of stable
transformants (Neumann et al. 1982). In the first application of pulses to living
animal (Okino and Mohri 1987), tumor reduction was observed after electroporation
of bleomycin into hepatocellular carcinomas in a rat model. After additional pre-
clinical development, this therapy, electrochemotherapy, was tested in clinical trials
in the first application of pulses to human patients and is now included in several
guidelines for the treatment of cutaneous tumors in the European Union (Mir
et al. 2006). The demonstration of plasmid delivery and the clinical success of
drug delivery enabled the development of gene therapy by electrotransfer.
The first confirmation of gene delivery in vivo was the demonstration of gene
transfer to mouse skin (Titomirov et al. 1991). In this case, antibiotic resistance after
in vivo delivery was demonstrated in vitro. In vivo expression of the reporter genes
β-galactosidase in rat brain tumors (Nishi et al. 1996) and luciferase in healthy rat
liver (Heller et al. 1996) was subsequently demonstrated. In the first exploration of
therapeutic gene delivery, Nishi et al. demonstrated monocyte invasion into tumors
after delivery of the gene encoding monocyte chemoattractant protein-1 (Nishi
et al. 1996). Within a few years, several preclinical studies demonstrated therapeutic
efficacy. Intramuscular electrotransfer of a plasmid encoding erythropoietin
achieved therapeutic levels and increased the hematocrit (Kreiss et al. 1999; Rizzuto
et al. 1999). Intratumor delivery of a plasmid encoding dominant-negative Stat3
induced apoptosis in melanomas (Niu et al. 1999). Eventually, in the first therapeutic
gene electrotransfer clinical trial, the p35 and p40 cDNAs of interleukin 12 were
delivery intratumorally, inducing tumor regression and other antitumor effects in
both treated and untreated tumors (Daud et al. 2008). Gene therapy by electrotransfer
has reached clinical trials for several applications including cancer vaccines and
therapies and infectious disease vaccines. Overall, more than 80 in vivo clinical trials
have utilized electroporation for delivery, with seven of these trials reaching
Phase II.
Since it was first described, electroporation has been used to transport a variety of
molecules ranging from ions to drugs to nucleic acids across the plasma membrane
and into the cell. Small molecules such as chemotherapeutic agents diffuse freely
1570 L.C. Heller
through the plasma membrane during or after electrotransfer. Negatively charged

oligonucleotides such as small interfering RNAs are approximately 6 kDa in size and
freely enter a cell’s cytoplasm when present during electrotransfer, but do not enter
when added after pulse application. An average plasmid weighs >3000 kDa and the
mechanism by which it enters the cell is significantly more complex (Rosazza
et al. 2016), requiring a combination of membrane electroporation, DNA electro-
phoresis, endocytosis, and intracellular transport via the cytoskeleton.
Nonviral Nucleic Acid Formats for In Vivo Delivery
Plasmids are the most common format for electrotransfer-based gene therapies.
Sequences as large as 15 kilobases can be inserted into plasmid DNA although
manipulating the plasmid becomes more difficult with larger inserts. Along with
promoters and enhancers, a translation start site, or Kozak sequence, is necessary for
eukaryotic protein expression. A secretory sequence allows protein secretion if
necessary. An internal ribosome entry site allows polycistronic co-expression in
the same transfected cell. This is useful for therapeutic molecules encoded by
multiple mRNAs such as interleukin 12 and for co-expression of complimenting
therapeutic genes such as angiogenic factors. Finally, the sequence must a have a
polyadenylation signal to produce readable mRNA.
Gene expression is tightly regulated in nature. For example, housekeeping genes
are transcribed and translated at low levels to maintain basic cellular processes. The
expression of many genes is regulated in response to environmental or developmental
stimuli. Mammalian regulatory elements can be located proximal to a gene or hun-
dreds of thousands of base pairs distant. Similar to the coding sequences, these
sections must be minimized in gene therapy applications. In many clinical trials,
gene expression is driven by viral promoters such as the cytomegalovirus immediate
early enhancer and promoter or simian virus 40 promoter. These promoters are short
sequences that produce high transgene expression in most mammalian cell types but
are inactivated over time, limiting expression duration. Tissue-specific promoters,
which theoretically restrict expression to a specific tissue and therefore reduce
off-target effects, are also commonly used in clinical trials although expression is
generally much lower than that produced by viral promoters. Cell-specific targeting is
also possible via sequences controlling nuclear import (Dean 2013). Promoters that
can be controlled by an exogenous factor, referred to as regulatable promoters, also
have potential but should be designed for inducible rather than repressible expression
to minimize patient exposure to the inducing agent.
Along with the sequences necessary for expression of the therapeutic gene or
cDNA in mammalian cells, plasmids require prokaryotic sequences, including an
antibiotic resistance gene and origin of replication, for propagation in E. coli. Major
regulatory agencies have found these prokaryotic sequences to be problematic, but
disagree on the limitations that should be placed on the plasmids used for gene
therapy. The EMA has recommended the use of vectors without antibiotic resistance
gene or other selection element to avoid horizontal gene transfer to commensal
bacteria. In this case, these bacterial sequences can be removed by several methods
to produce minicircles, although low efficiency hampers their production and puri-
fication. Linear expression cassettes generated using PCR completely avoid the
presence of bacterial sequences. The FDA states that beta lactam antibiotics should
be avoided due to the risk of patient hypersensitivity to these antibiotics and that
antibiotics in clinical use should be avoided. The policy of the State Food and Drug
Administration of China corresponds with the FDA in that penicillin should be
avoided to prevent allergic reactions in some patient populations.
Messenger RNA does not require nuclear transport, so transgene expression after
delivery may be more reproducible than DNA delivery. The most common applica-
tion of mRNA electrotransfer is to engineer antigen presenting cells in vitro to
induce antitumor activity when these cells are injected into the patient. Gene
expression can be downregulated via RNA interference following direct delivery
of sequence targeted small RNAs. Double-stranded small interfering RNA and stem-
loop containing microRNA are 20–30 base pair oligonucleotides based on endoge-
nous RNAs. These RNAs are processed in the cytoplasm via similar pathways to
induce cleavage and degradation of specific target mRNAs. RNA-based gene ther-
apies may be difficult to administer because RNA is degraded by ubiquitous and
resilient ribonucleases. One alternative option for RNA interference is the delivery
of plasmid DNA encoding small hairpin RNAs driven by the RNA polymerase III
promoters U6 or H1. Another alternative is to stabilize the molecule by chemical
modification of the nucleic acid backbone, producing nuclease resistance.
Each individual mammalian cell possesses the ability to detect pathogen invasion
by binding of pattern recognition receptors. These receptors detect specific pathogen
components such as bacterial endotoxin as well as shared pathogen components such
as nucleic acids, producing an inflammatory response and/or cell death. Any gene
therapy, including viruses, plasmids, and oligonucleotides, can be detected by these
receptors and misinterpreted as pathogen invasion. For example, the endosomal toll-
like receptor 9, found in immune cells, detects a specific sequence motif, an
unmethylated CpG motif, found in artificially and bacterially synthesized. Multiple
cytosolic DNA-specific pattern recognition receptors are found in all cell types
(Desmet and Ishii 2012). Since plasmid DNA is also found in the cell’s cytosol
after electrotransfer (Rosazza et al. 2016), these receptors may also be bound and
activated (Znidar et al. 2016), producing unanticipated endogenous responses.
Electric Pulses Used for In Vivo Electrotransfer
Two basic pulse types can be used for nucleic acid delivery in vivo. Exponentially
decaying pulses are produced when a charged capacitor discharges into the tissue
(Fig. 4a). These are commonly used in bacterial transfection and were used in the
initial demonstrations of the in vivo delivery of chemotherapeutic agents (Okino and
Mohri 1987) and plasmid DNA (Titomirov et al. 1991). Square wave pulses allow
better control of pulse parameters necessary for the clinical development of in vivo
delivery. To create square wave pulses, the exponential pulse is truncated by fast
1572 L.C. Heller
Fig. 4 Electroporation pulse types. (a) Exponential decay or capacitor discharge pulse, (b)
square wave pulse produced by limiting the length of an exponential pulse. Pulse types used in
gene therapies include (c) short, high voltage pulses, (d) long, low voltage pulses, and (e) pulse
combinations
switching. Although these pulses are referred to as “square wave,” a small voltage
drop still occurs over the time of the pulse (Fig. 4b).
Pulse variables can be used to deliver nucleic acids and to tailor the consequent
protein expression levels and kinetics of a therapeutic protein. These variables
include pulse applied voltage, length, number, and frequency. For in vivo applica-
tions, the pulse voltage-to-distance ratio is generally <1500 V/cm. The individual
pulse length can vary between 100 μs and 100 ms. In general, the total pulse number
(<10) and pulse frequency (<10 Hz) are low. Short, high voltage pulses fall in the
microsecond range (Fig. 4c), while long, low voltage pulses generally fall in the
millisecond range (Fig. 4d). Combinations of these pulse types (Fig. 4e) can be used
to take advantage of the electroporation-electrophoresis mechanism of plasmid DNA
electrotransfer.
Electrode Design for In Vivo Electrotransfer
Cuvettes are widely available and commonly used for in vitro electroporation. It is
obvious that this electrode configuration will not function in vivo. Many noninvasive
and invasive configurations have been designed in research labs for preclinical
research. For example, flexible electrodes have been incorporated into microsystem
endoscopic devices for electrochemotherapy or gene therapy in the endoscopic
electroporation system (Soden et al. 2004). Delivery with this system has reached
Fig. 5 Commercially available electrode configurations. Electrodes for preclinical applications.

(a) caliper electrode (Reprinted with permission of Harvard Apparatus, Holliston MA, USA); (b)
tweezer electrode (Reprinted with permission of Nepa Gene Co., Ltd. Ichikawa-City, Chiba, Japan);
(c) needle electrode for muscle delivery (Reprinted with permission of Harvard Apparatus,
Holliston MA, USA); Electrodes for clinical applications (d) finger electrode reprinted with
permission of Igea Medical, Carpi, Italy)
clinical trials (clinicaltrials.gov identifier NCT01172860). To reduce the applied

voltage in skin-directed and other applications, a system consisting of an array of
independently addressable electrodes was developed (Heller et al. 2010). Delivery of
an influenza vaccine with an electrode array based on this design has also reached
clinical trials (NCT01403155). A numerical modeling system to optimize electrode
for deep-seated tumor nodules (Miklavcic et al. 2010) has reached phase II clinical
trials for electrochemotherapy (NCT02352259). A deployable needle electrode to
increase the geometrical tolerance and optimize delivery to soft tissues such as brain
(Mahmood and Gehl 2011) has reached clinical trials for electrochemotherapy
(NCT01322100). In an exquisitely tissue-specific gene delivery, a cochlear implant
electrode array was utilized for pulse delivery (Pinyon et al. 2014). Although several
of these electrodes reached clinical trial for drug delivery, it is clear that these
electrodes will also function for plasmid DNA delivery.
Commonly used commercially available preclinical and clinical electrodes are
shown in Fig. 5. Initially, electrode plates were mounted onto a caliper to form
caliper electrodes (Fig. 5a) that allow pulse application to surface tissues such as skin
and subcutaneous tumors. The electrode gap is directly measured and the voltage to
distance ratio can be calculated specifically for each use. In a similar approach, the
electrodes are mounted on tweezers (Fig. 5b). Plate electrodes produce a homoge-
nous field, but do not reach deeper tissues without surgical intervention. Therefore,
although the electric field produced is not as homogenous, invasive or needle
1574 L.C. Heller
electrodes are necessary for deeper tissues. Figure 5c shows a simple 2-needle array
for muscle delivery. Figure 5d shows a clinical fingertip electrode used to access
difficult tissues such as oral tumors.
Tissue Targets
Therapeutic gene electrotransfer has been achieved in a variety of tissue types

including skin, kidney, liver, testis, brain, cartilage, arteries, prostate, cornea, heart
and skeletal muscle, as well as many tumor types. Given that tissues and cells vary
significantly, this confirms the functionality of gene electrotransfer. Tumors, muscle
and skin are the most common targets; delivery to each of these tissues has reached
clinical trials.
The specific characteristics of the tissue and its cells must be considered for
successful in vivo electroporation. In general, cell types in vivo are not homogenous,
they may not have consistent shape or size, and they may be densely packed in the
tissue surrounded by other cells and extracellular matrix. These factors may influ-
ence the distribution of the nucleic acid injected into the tissue and the electric fields,
important considerations in pulse parameter and electrode choice.
Skeletal muscle is one of the most common targets for molecule electrotransfer.
Transgenes are expressed at high levels in this tissue, potentially with long expres-
sion kinetics. Since this tissue is highly vascularized, expressed proteins can be
secreted into the circulation at physiologically relevant levels. Therefore, muscle acts
as an excellent “bioreactor” for protein manufacture. To reach this deeper tissue,
needle electrodes of various configurations are generally used for muscle delivery in
humans. However, a higher body mass index increases muscle depth and may
require longer needles or surgical exposure (Kalams et al. 2013).
The skin is the largest and most accessible organ in the body and therefore is
the most versatile site for gene electrotransfer. It is an especially attractive site for
DNA vaccination applications because of the presence of immune cells. Epider-
mal cell renewal is age dependent but takes approximately 20–30 days, limiting
the duration of transgene expression. Depending on the depth of the injection, the
choice of applicator (plates or needles), and the pulse parameters, transgene
expression can be local, systemic, or both. Limited intradermal space is a draw-
back, as only small volumes of nucleic acid can be injected, but this can be
overcome by the application of multiple treatments or by increasing the treatment
area (Heller et al. 2010).
Tumors are heterogeneous tissues of variable viability that include tumor cells as
well as fibroblasts, endothelial cells, and immune cells such as macrophages and
lymphocytes (Fig. 6). The proteoglycan and collagen content, cell size, density, and
shape in the tissue all play a role in transfection efficiency (Mesojednik et al. 2007).
These factors increase the variability of expression after tumor transfection. Surface
tumors can be accessed by simple plate or needle electrodes, but deeper tumors
require more sophisticated approaches such as surgical exposure, long needle elec-
trodes, or endoscopic electrodes.
Fig. 6 The heterogeneous tumor microenvironment. Cancer cells within tumors are supported
by other cell types that maintain the tumor structure, supply it with nutrients, and suppress an
antitumor immune response, among other functions. These cells may also be targets of gene
electrotransfer
Conclusions
Several factors must be understood to develop a gene therapy delivered by electrotransfer

or any other method. The initial and most important step is to determine what level,
duration, and site of protein expression are necessary to treat the disease or produce the
vaccine effect. Factors such as the patient’s health and age will affect subsequent gene
expression. An understanding of the target tissue composition and viability is necessary
to predict subsequent gene expression. Some tissues are more easily transfected than
others and produce higher levels or longer duration gene expression.
Vector design can be used for tissue targeting and to tailor gene expression
appropriately. The wide variety of pulse regimens and electrodes described and
tested in the literature supply an initial idea of the expected gene expression for
the design of specific therapies. However, no single pulse regimen is appropriate for
all tissues. While commonly and commercially available electrodes may be adequate
for the application, specific designs may be necessary.
Electroporation can dramatically increase plasmid delivery and gene expression.
While many groups studying this technique focus on maximizing expression, it is
much more important to establish protocols that can accurately predict expression.
The design of electrotransfer gene therapies is a complex process requiring interdis-
ciplinary collaboration between biologists and engineers.
1576 L.C. Heller
Cross-References

Electrochemotherapy
▶ Clinical Use of DNA Vaccines
▶ Delivery of Cytokines Using Gene Electrotransfer
▶ Irreversible Electroporation in Treatment of Prostate Cancer
▶ Irreversible Electroporation Techniques in the Treatment of Locally Advanced
Liver and Pancreatic Cancer
▶ Preclinical Studies on Irreversible Electroporation
Deep-Seated Tumors
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Tissue Engineering with Electroporation
82
Mary Phillips Ho
Abstract
Organ failure and the demand for donor organs for transplantation has resulted in
recent progress within the field of tissue engineering and increased functionality
in the development of tissues and whole organs. This complex field brings
together many different techniques and technologies in an attempt to build a
tissue construct that can support cell growth and fully integrate into the body after
transplantation. Electroporation is one technology that has been applied towards
various applications within the field of tissue engineering. For example, nonther-
mal irreversible electroporation (NTIRE) has been shown as a potential method
for tissue decellularization, producing tissue- and organ-derived scaffolds by
removing the cellular content while preserving important structural and biochem-
ical features to support cell growth. This method has been applied both in vivo
and ex vivo with the use of active perfusion. In addition, nonthermal irreversible
electroporation (NTIRE) has been applied to develop natural scaffolds with
controlled porosity to enable the development of vasculature within the newly
developing tissue, allowing for nutrients to be delivered throughout. Reversible
electroporation (RE) has also shown potential for applications within the field of
tissue engineering. By creating temporary pores within the cell membrane,
reversible electroporation can be used for gene transfection, resulting in
transfected cells that express growth factors needed for vasculature development
and also having applications for stem cell differentiation. With further research,
these areas of electroporation may develop into impactful technologies for the
field of tissue engineering.
M.P. Ho (*)
Department of Engineering, Quinnipiac University, Hamden, CT, USA
e-mail: Mary.Ho@quinnipiac.edu

DOI 10.1007/978-3-319-32886-7_49
1580 M.P. Ho
Keywords
Electroporation • Tissue engineering • Nonthermal irreversible electroporation •
Reversible electroporation • Tissue decellularization • Tissue scaffold • Gene
electrotransfection
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1580
Scaffold Development for Tissue Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1583
Natural Scaffold by Decellularization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1584
The Extracellular Matrix (ECM) as a Scaffold for Tissue Engineering . . . . . . . . . . . . . . . . . . . 1584
Methods of Tissue Decellularization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1586
Tissue Decellularization by Nonthermal Irreversible Electroporation (NTIRE)
In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1588
Tissue Decellularization by Nonthermal Irreversible Electroporation (NTIRE)
Ex Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1590
Increasing the Porosity of Natural Polymer Scaffolds Using Nonthermal Irreversible
Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1591
Nonthermal Irreversible Electroporation for Controlled Pore Formation Within Bacterial
Cellulose Scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1592
Gene Electrotransfer for Tissue Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1593
Blood Vessel Formation In Vivo and Use in Tissue Engineering . . . . . . . . . . . . . . . . . . . . . . . . . 1594
Electroporation for Gene Therapy in Tissue Engineering Applications . . . . . . . . . . . . . . . . . . . 1596
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1597
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1598
Introduction
Tissue engineering attempts to replace diseased tissues of the body with functional,
engineered constructs. This approach to regenerative medicine is used to create
tissues or organs within the laboratory setting. The development of a tissue
engineered construct might be built based on one of several different technology
platforms that have seen recent promise for producing a transplantable bioartificial
organ. Technological platforms for tissue engineering include (Ren and Ott 2014):
(a) Whole-organ decellularization and regeneration based on using the organ’s

native extracellular scaffold
(b) Natural and synthetic scaffolds produced through a variety of methods, includ-
ing three-dimensional organ printing of a matrix scaffold and targeted position-
ing of desired cell types
(c) Self-organization of different cell types into specific tissue structures
These different approaches towards tissue engineering are illustrated in Fig. 1.

Electroporation is a technology that uses high-magnitude electric pulses to
permeabilize the cell membrane. Reversible pore formation caused by reversible
electroporation (RE) enables drugs and macromolecules to be passed through the
82 Tissue Engineering with Electroporation 1581
Fig. 1 Technological platforms applied towards tissue engineering. (a) Tissue decellularization
and regeneration. (b) Three-dimensional printing of the tissue scaffold, which may include direct
printing of cells within the scaffold matrix. (c) Organoid engineering (Image from Ren and Ott
(2014) with permission from Springer)
cell membrane. RE has been utilized for a variety of applications, including gene
transfection as well as electrochemotherapy for tumor ablation, enabling cancer
drugs to be introduced directly into tumor cells. Changing the electrical parameters
such as increasing the applied voltage or number of applied pulses can create
permanent cell membrane defects, resulting in cell death from irreversible
1582 M.P. Ho
electroporation (IRE). Though joule heating can occur from passing an electric
current through tissue, the electrical parameters of IRE can be controlled such that
thermal effects are minimal and thermal damage to the tissue is avoided. Thus, the
cell membrane is targeted directly, and cell death occurs due to nonthermal irrevers-
ible electroporation (NTIRE).
Electroporation has found applications in a variety of areas within tissue
engineering. For scaffold-based approaches, such as those that utilize
decellularization or additive manufacturing, a scaffold is produced using a syn-
thetic or natural extracellular biomaterial that provides a temporary physical
support to accommodate and encourage three-dimensional cell growth as the
engineered tissue develops. NTIRE has shown potential as a method for
decellularizing native tissue, producing an acellular extracellular matrix (ECM)
scaffold that still retains much of its vital structure and function to support cell
growth, migration, and differentiation. NTIRE can also be combined with
manufacturing methods to fabricate 3D scaffolds. One current challenge for the
field of tissue engineering is producing thick tissues and organs with a developed
vasculature system to support nutrient exchange for the cells below the tissue
surface. Proper metabolism of tissue implants with a volume of more than
2–3 mm3 cannot be supported by passive diffusion, and thus a perfusable vascular
network is necessitated to maintain cell viability (Ren and Ott 2014). NTIRE has
been shown as a promising method to create interconnected pores within tissue
scaffolds that are fabricated from bacterial cellulose (Baah-Dwomoh et al. 2015)
and may prove to have additional applications within the process of creating tissue
engineered scaffolds.
Since it takes time for a host’s own vasculature to grow into a transplanted
tissue, it is necessary to regenerate functional vasculature within tissue-
engineered constructs to maintain cell viability. Many approaches in tissue engi-
neering involve seeding a tissue scaffold in vitro and allowing the tissue to
develop prior to transplantation. The development of tissue vasculature is a
complicated process that relies on growth factors and other biological cues. In
order to encourage blood vessel formation, growth factors are often bonded to the
scaffold to manipulate cell behavior from an “outside-in” approach. Another
method utilizes gene therapy strategies to control the proliferation, migration,
and differentiation of cells through an “inside-out” approach. Nucleic acids are
delivered to the cell to trigger cell expression of various growth factors. Revers-
ible electroporation is a nonviral method that can be used to introduce these
nucleic acids to the cells, prompting the cells to produce the growth factors needed
for the development of a vasculature system and a heterogeneous and viable tissue
construct.
This chapter primarily focuses on the ability to use NTIRE for tissue scaffold
development. The use of reversible electroporation for gene transfection is also an
important tool within tissue engineering that is introduced here. A more detailed
review of the mechanisms and applications for electroporation for gene transfection
can be found in other chapters of this book (see “Cross-References”).
Scaffold Development for Tissue Engineering
Tissue engineered constructs can be categorized as scaffold-based and scaffold-free

approaches. This chapter focuses on scaffold-based approaches that have benefited
from the use of electroporation. Though the field of tissue engineering has advanced
greatly over the past three decades, major challenges remain in the growth of cells
with the right phenotypes and organization in suitable scaffolds, delivering and
sustaining adequate oxygen and nutrients in tissue constructs, determining optimal
engineering environments for tissue growth, and integration of the tissue into the
host (Patnaik et al. 2014). For example, tissues with a thickness of more than
100–200 μm have limited diffusional mass transfer and require vascularization to
deliver nutrients, oxygen, growth factors, and biochemical signaling factors through-
out the tissue (Sarker et al. 2015). In addition, these microvasculatures are necessary
for the removal of carbon dioxide and metabolic waste. The choice of biomaterial
used for the scaffold matrix has a profound impact on cell adhesion, migration, and
proliferation as well as vascularization. A tissue scaffold should facilitate cell
survival, proliferation, migration, and differentiation, have sufficient mechanical
strength to keep the scaffold architecture intact, and display controlled biodegrad-
ability such that the mechanical properties of the tissue are maintained as new tissue
forms and replaces the original scaffold material. Biopolymers for scaffold construc-
tion may be categorized as synthetic polymers, natural polymers, or tissue-derived
scaffolds.
Scaffolds can be developed as permanent scaffolds or temporary scaffolds which
degrade over a period of time. Temporary or degradable scaffolds can be built from
synthetic or natural materials. A variety of biodegradable, biocompatible, and
nontoxic synthetic polymers have shown potential as tissue engineering scaffold
material. Synthetic polymers allow for controlled fabrication and attainment of the
desired mechanical properties, porosity, and surface topography of the engineered
tissue. Limitations of this approach, however, include uncertainties regarding the
degradation rate and products as well as concerns related to cell compatibility.
Naturally derived polymers are often seen as advantageous over synthetic polymers
in respect to many features, but no one method is seen to be without drawbacks.
Protein-based polymers include collagen, fibrin, fibronectin, elastin, and silk fibroin.
Polysaccharide polymers have also been utilized, including hyaluronic acid (HA),
alginate, and chitosan (Sarker et al. 2015). Though these natural polymers often
possess intrinsic cell compatibility, they often exhibit weak mechanical properties,
making it difficult to manipulate them and for the scaffold to maintain its structure. A
vast array of techniques have been developed to form 3D scaffolds from a large
variety of synthetic and natural biomaterials. In addition, tissue-derived scaffolds can
be developed through the process of decellularization where cellular components are
removed from tissues and organs, resulting in an acellular ECM template that retains
structural and functional proteins to support cell growth.
The type of method used to develop the scaffold will depend on the specific organ
or tissue type and application. In each case, the following functional demands must
1584 M.P. Ho
be considered to develop a successful transplantable bioartificial organ (Khan

et al. 2014):
• Type of organ scaffold

• Sterilization of the scaffold
• Integrity of the vasculature and natural architecture within the scaffold
• Assessment of immunological barriers
• Type of cells/tissue to regenerate
• Functional aspects of the regenerated organ
• Type of cells for infusion
• Route of cell delivery
• Required induction factors
• Assessment of long-term cell survival and engraftment
Natural Scaffold by Decellularization
Tissue- or organ-derived biomaterials are developed through a process termed

“decellularization.” During the decellularization process, cellular content is
removed, leaving the native ECM behind. The resulting decellularized scaffold
can then be repopulated with suitable cells, creating a tissue-engineered construct
for tissue or organ transplantation, as illustrated in Fig. 2.
The removal of cellular content and antigens from the natural tissue results in a
scaffold with reduced risk for foreign body reaction, inflammation, and the potential
for immune rejection. Decellularization techniques seek to preserve the integrity of
the ECM as much as possible.
The Extracellular Matrix (ECM) as a Scaffold for Tissue Engineering
The extracellular matrix provides signals and cues for cell processes such as prolif-
eration, migration, differentiation, and apoptosis. The ECM is a complex structure
composed of fiber-forming protein chains (e.g., collagen, elastin), proteoglycans
(PGs) carrying glycosaminoglycans (GAGs), glycoproteins (e.g., fibronectin, lami-
nin), and soluble macromolecules (e.g., growth factors, cytokines, and chemokines)
(Sulaeva et al. 2015). The composition of the ECM varies between different tissue
types, and the ECM serves not only to provide structure and strength but also directly
affects the cells, influencing cell attachment, migration, and proliferation, and the
elasticity of the ECM can strongly influence the cell phenotype. Collagen is the most
abundant protein in the ECM, and each type of collagen contributes to the distinct
mechanical and physical properties of the ECM in each tissue and location through-
out the body (Van der Rest and Garrone 1991). For example, type I collagen is a
major structural protein, and type IV collagen is found especially in the basement
membrane of vascular structures. Fibronectin is also a very important and prominent
ECM protein. This dimeric molecule possesses ligands for many different cell
Fig. 2 The application of a decellularized scaffold for tissue engineering. Human or animal-
derived decellularized tissues or organs can be recellularized, producing a tissue or organ suitable
for transplantation (Image from Rana et al. (2015), # 2015 John Wiley & Sons, Ltd)
adhesions (Miyamoto et al. 1998) and is critical in the development of vascular

constructs. Laminin, an adhesive protein found within the basement membrane is
important in the formation and maintenance of the blood vessels. GAGs are also
found throughout the ECM and play a crucial role in the endothelial cell and smooth
muscle cell proliferation (Badylak 2004). Growth factors found within the ECM are
very diverse in structure and function and are used to modulate cell behavior. These
include vascular endothelial cell growth factor (VEGF), fibroblast growth factor
(FGF), transforming growth factor beta (TGF-β), and platelet-derived growth factor
(PDGF) (Badylak 2004). Proteogylcans are macromolecules that consist of GAGs
bound to a protein core and serve a variety of functions such as binding extracellular
matrix components, mediating the binding of cells to the matrix, and capturing
soluble molecules such as growth factors into the matrix and at the cell surfaces
(Ruoslahti 1989).
As can be seen, the ECM is a dynamic and complicated structure that strongly
influences the mechanical structure of the tissue, the individual functions of the cells,
and the overall tissue’s ability to function and remodel. To complicate this even
further, important components of the cell and tissue response are mediated by the
1586 M.P. Ho
Fig. 3 Complex interactions between cells and the ECM affect cell behavior and ECM
remodeling. The behavior of cells and their interactions with the ECM is regulated by intricate
interactions. Extracellular factors include signals within the ECM (both soluble and physically
bound), mechanical stimuli, and cell-cell interactions. These various factors converge to regulate
gene expression, affecting cell phenotype and cell fate processes (Adapted with permission from
Macmillan Publishers Ltd: Nat. Biotechnol. Lutolf and Hubbell (2005), copyright 2005)
products of ECM degradation. Cell behavior is strongly influenced by the compo-

nents of the ECM (Fig. 3), and thus the process of decellularization is crucial in
developing a tissue engineered construct for recellularization and transplantation.
Methods of Tissue Decellularization
Tissue decellularization can be accomplished through a combination of chemical,

enzymatic, and/or physical agents. The type of decellularization method chosen
will largely depend on the tissue’s size, thickness, density, cellularity, and lipid
content.
Chemical methods include the use of acid/base solutions, detergents, or ionic
solutions to remove the cellular components. For example, sodium dodecyl sulfate
(SDS) is an ionic detergent that is very widely used as a decellularization agent.
Limitations of the use of chemical agents, however, include removal of ECM growth
factors, damage to collagen and GAGs, calcification, and incomplete removal of the
chemical agents from the ECM after treatment (Patnaik et al. 2014; Sarker
et al. 2015). Enzymes such as nucleases, trypsin, or dispase serve to catalyze the
hydrolysis of RNA and DNA chains, providing high specificity for removal of
cellular residues. Complete removal of cellular contents by enzymatic methods,
however, is difficult, and enzyme residues may impair recellularization or evoke
an adverse immune response (Crapo et al. 2011). Physical methods such as agitation,
sonication, mechanical massage, pressure, and freezing and thawing are used to
disrupt the cell membrane and release the cellular content (Gilbert et al. 2006). While
concern of cytotoxic residue is alleviated, potential damage to the ECM ultrastruc-
ture remains. The optimal method for decellularization is dependent upon tissue
characteristics such as thickness and density as well as the intended clinical appli-
cation. Methods are often combined in order to develop protocols that are most
efficient and effective for specific decellularization applications and tissue types.
Nonthermal irreversible electroporation (NTIRE) is an additional physical
method for producing a decellularized ECM for use as a scaffold for tissue engi-
neering. NTIRE results in cell death due to the lipid bilayer destabilization and
permeabilization, and this cell ablation modality has been harnessed to selectively
target cells while retaining the structural integrity of blood vessels, nerves, and
extracellular matrix (Jourabchi et al. 2014). The ablative modality of NTIRE has
been shown to result in a quicker recovery of the biological tissues (Phillips
et al. 2012). Because the procedure targets the cell membrane, critical features of
the extracellular matrix are spared.
NTIRE has demonstrated promising features for developing a naturally
decellularized tissue scaffold that preserves many of the important structural and
functional aspects of the extracellular matrix. For example, NTIRE has been shown
to ablate vascular smooth muscle cells without causing damage to the extracellular
components (Maor et al. 2009). NTIRE does not rely on a thermal mechanism for
cell ablation, preserves the structures of the underlying extracellular matrix (Maor
et al. 2007), preserves the nerve conduits and bile ducts (Maor et al. 2007), and
should not create or deposit toxins during the cell ablation. Several possible methods
could be applied to derive a decellularized tissue scaffold using NTIRE, with the
most straightforward being to simply apply NTIRE to xenographic or human donor
tissue just prior to implantation. The simplicity of this method is substantially
advantageous. However, the immune response and other cellular and enzymatic
processes involved in the removal of the cellular debris by the host organisms may
prove detrimental to the host. Substantial further research on the immunological
response to NTIRE cell damage is needed before this method could be applied.
Another method involves applying NTIRE to the donor tissue, waiting for the
donor’s host response to depopulate the cells, and then harvesting the tissue scaffold.
Additional tissue processing steps would be required after the tissue is removed in
order to ensure all cellular components are fully removed from the tissue, and this
technique would only be applicable for noncritical tissues and organs. Finally, a
third method employs NTIRE to excised tissue or organs, possibly with the assis-
tance of organ perfusion and/or a bioreactor, employing cell removal and reseeding
ex vivo.
1588 M.P. Ho
Tissue Decellularization by Nonthermal Irreversible Electroporation

(NTIRE) In Vivo
The use of NTIRE for tissue decellularization has been applied in vivo for the
development of a decellularized arterial graft (Phillips et al. 2010, 2011).
Decellularized rat and rabbit arteries were developed using two different electrode
configurations: by applying plate electrodes across the artery and by applying the
electrical parameters in a minimally invasive fashion through the use of an
endovascular electrode device. The plate electrodes were used to apply 90 pulses
of 70 V and 100 μs at a frequency of 1 or 4 Hz across the 0.4 mm thick rat arterial
tissue, resulting in an electric field of approximately 1750 V/cm. Rabbit iliac arteries
were treated in an endovascular fashion, using 90 pulses with an applied voltage of
600 V, a pulse length of 100 μs, and a frequency of 4 Hz. Finite element modeling
indicated that the entire volume of arterial tissue experienced an electric field of
1000 V/cm or greater. Both methods showed that after treatment the artery becomes
decellularized, and there is a period of time before new cells begin to grow back. The
peak level of decellularization was achieved between 3 and 5 days after treatment
(Fig. 4). This is when the decellularized tissue could potentially be harvested.
Evidence that the ECM can retain its structure and ability to support cell migra-
tion and growth is very important in regard to developing a decellularized tissue
scaffold. After NTIRE treatment, the ECM appeared undamaged (Fig. 4). Within
7 days after treatment, a full endothelial layer was observed lining the lumen of the
treated arteries, indicating that the scaffold was left intact and was able to encourage
cell growth and that problems such as thrombogenicity experienced by many other
tissue engineered grafts may be avoided. This in vivo decellularization method
assumes a reliance on the immune response or some other cellular or enzymatic
mechanism to remove the ablated cells. This response could potentially cause
remodeling to the ECM, and thus further work is warranted to assess the effects of
the host response on the scaffold and to gain a deeper understanding of the mech-
anisms involved on the cellular level.
The potential for decellularization by NTIRE has also been demonstrated in vivo
on a rat small intestine model (Phillips et al. 2012). One day after treatment, the small
intestine saw complete cellular ablation throughout its entire circumference. Cellular
destruction to the tissue was complete, and a full loss of cellular architectural details
was seen. However, the extracellular matrix was not affected by NTIRE, and the
structural integrity of the small intestine remained intact. Despite the complete
obliteration of the cellular structure, the tissue showed signs of recovery, and tissue
repair was evident within 3 days posttreatment. Immature epithelial cells were able
to migrate from the edges of the treated zone, producing a new epithelial cell layer.
Within 7 days after treatment, normal repair and replacement of the mucosa,
submucosa, and muscularis was occurring, providing evidence that the small intes-
tine was able to go from complete cellular destruction to regeneration of the
intestinal layers and villi within 1 week. The ECM is still intact after NTIRE
Fig. 4 Smooth muscle cell removal and preservation of ECM. Top row: Arteries stained for
α-smooth muscle actin (shown as the darker staining in between the elastin fibers of the medial
layer) demonstrate a decrease in vascular smooth muscle cells at 3 days. Bottom row: Movat’s
pentachrome stain of the artery 3 days after NTIRE treatment shows undamaged elastin fibers
(black) as well as collagen and reticulum fibers (orange) and proteoglycans (blue and highlighted
by arrows) (Images from Phillips et al. (2010). # 2010 by ASME)
treatment, providing a framework for epithelialization and restoration of blood

supply.
This quick repair and recellularization is due to unique ablation mechanism of
NTIRE, selectively targeting the cell membranes, while leaving the ECM intact.
Though Phillips et al. (2012) did not demonstrate complete removal of cellular
debris and decellularization of the small intestine in vivo, these results point to the
promise of using this technique, likely as a decellularization technique ex vivo
coupled with perfusion or other methods to aid in the cellular removal process.
The unique ability of NTIRE to preserve blood vessels and the extracellular matrix
support its use as a mechanism for decellularization.
1590 M.P. Ho
Tissue Decellularization by Nonthermal Irreversible Electroporation

(NTIRE) Ex Vivo
Tissue decellularization by NTIRE has been demonstrated ex vivo by Sano

et al. (2010a). Machine perfused livers were treated with NTIRE using external
plate electrodes or needle electrode configurations. Plate electrodes were used to
apply 99 pulses of 1000 V/cm across the liver tissue with a pulse length of 100 μs
and a frequency of 0.25, 0.5, 1, or 4 Hz. Largest lesion sizes and largest percent
removal of cellular debris were obtained for the 1 Hz protocol. Histological evalu-
ation of the treatment area 24 h after treatment revealed the selective ablation
modality of NTIRE (Fig. 5). Despite cell ablation and degradation, major acinar
features were preserved, including connective tissue borders, blood vessels, and bile
ducts. After 24 h of active perfusion the cellular content within the tissue construct
was reduced, but cellular debris remained. This is likely due to adhesion of cellular
debris to the ECM, low physiological flow rates, pressures at the lobule level, and
possible damage to microvasculature by NTIRE. In addition, vascular occlusion
occurring after electroporation has been shown to decrease blood flow for 4–8 h after
electroporation for tumors in vivo. This effect could decrease the cellular debris
clearance mechanism while under perfusion.
Though complete cellular debris removal was not achieved by this initial study, it
does show promise for the use of this technology in obtaining decellularized tissue
constructs ex vivo. When comparing the plate electrode versus needle electrode
method for applying NTIRE, the plate electrodes gave the most promising results;
needle punctures damaged the tissue and provided an alternative path for fluid flow,
affecting the perfusion process. By optimizing the treatment parameters and perfu-
sion system, larger ablation volumes and efficient cellular debris removal could be
achieved. When applying irreversible electroporation for tissue decellularization,
Fig. 5 Effect of NTIRE and perfusion on liver tissue. (a) A section of untreated liver after 24 h
of perfusion. (b) The same liver treated with 100 pulses of 1500 V/cm, 100 μs in length at 1 Hz
using needle electrodes after 24 h of perfusion at 20 magnification. Cell density is reduced, but
cellular debris remains. Bile ducts and vascular structures remain intact (Image from Sano
et al. (2010a) (# Sano et al.; licensee BioMed Central Ltd. 2010) licensed under CC BY 2.0)
it is vital that thermal damage due to Joule heating is avoided, since any thermal
effects would also cause damage to the ECM. When the treatment parameters are
applied ex vivo, however, the tissue and perfusion fluid may be maintained at below
physiological temperatures. Thus, the applied voltage or treatment time may be
increased compared to in vivo applications while still avoiding thermal damage.
Optimization of NTIRE for tissue decellularization ex vivo may include the longer
applications of sub 1000 V/cm pulses or the use of increased voltages for
decellularizing thicker structures. In addition, longer perfusion times or perfusion
at higher than physiological pressures may help to optimize the removal of cellular
debris.
Previous work has shown that the most effective tissue decellularization methods
often consist of a combination of techniques. This may also be true for developing
NTIRE into an effective decellularization protocol. For example, cytotoxic agents,
enzymes, or detergents could be incorporated into the perfusion fluid to increase the
effectiveness of the cellular debris removal. This work demonstrated that NTIRE is
capable of decellularizing large volumes of tissue when performed in conjunction
with active organ perfusion. Further optimization and/or combination with other
decellularization protocols may result in an effective ex vivo method of tissue and
organ decellularization that preserves the ECM, promoting the cellular migration,
proliferation, and adhesion and resulting in a fully functional tissue engineered
construct for transplantation.
Increasing the Porosity of Natural Polymer Scaffolds Using

Nonthermal Irreversible Electroporation
Though decellularized scaffolds hold promise for tissue engineering applications,

there are additional advantages of fabricating a tissue scaffold from synthetic or
natural polymers, allowing for controlled manufacturing and eliminating the issue
of complete cellular removal. While fabricated scaffolds may not be achieved with
all the complexity and cell signaling features of the natural ECM, biodegradable
scaffolds may serve as an initial construct for cell growth. By providing the
necessary mechanical strength and features for cell seeding, these fabricated
scaffolds could then be replaced by a natural ECM that the cells themselves
produce over time. Scaffolds have been fabricated using methods such as gas
foaming, sintering, salt leeching, solvent casting, fiber bonding, electrospinning,
and phase separation as well as 3D manufacturing techniques. Many different
methods have been researched, holding varying levels of promise and each with
their own limitations. One of the major challenges seen in the field of tissue
engineering is delivering and sustaining adequate oxygen and nutrients in tissue
constructs, especially for thicker tissues and organs where diffusional mass
transfer cannot penetrate well below the tissue surface. Irreversible electropora-
tion has been recently applied as a method for increasing porosity in a naturally
derived scaffold matrix, allowing interconnected pores for cell migration and the
development of vasculature.
1592 M.P. Ho
Nonthermal Irreversible Electroporation for Controlled Pore

Formation Within Bacterial Cellulose Scaffolds
Many methods of scaffold creation limit the ability to incorporate mass transport
properties into the scaffold such as nutrient and gas exchange. These techniques
often allow for fairly uniform pore distribution, but pore interconnectivity and
microvasculature formation has proven a challenge. Cellulose, a natural polymer,
can be synthesized into nanofibrils by bacteria. Bacterial cellulose (BC) has high
tensile strength, flexibility, and water holding capacity and has shown a great
compatibility with living tissues (Sulaeva et al. 2015). BC has shown promise as a
biomaterial due to its good biocompatibility as well as low cytotoxicity and
genotoxicity (Sulaeva et al. 2015). BC is currently used as an antimicrobial wound
dressing and has been studied for use in tissue engineered bone grafts, cartilage, and
blood vessels (Baah-Dwomoh et al. 2015). BC has good strength and structural
properties, making it an appealing material to use as a tissue scaffold for treating
cartilage and bone defects. However, in creating BC, the bacteria produces a densely
packed network of cellulose nanofibrils, which can prove challenging for microvas-
culature formation for tissue engineering applications. Natural pore size within BC
of diameters of 20–100 nm is not advantageous for cell ingrowth and microvascu-
lature development, and penetration and migration into the BC substrate is limited
(Sulaeva et al. 2015). A variety of techniques have been applied to change the pore
size and porosity with BC scaffolds. For example, porogens of various sizes, such as
paraffin wax or starch microspheres, have been added to growing cultures during the
BC synthesis process (Sulaeva et al. 2015). Additionally foaming agents, gamma
irradiation, emulsion freeze-drying, and laser perforations have all been investigated
as methods for increasing perforation. Many of these methods, however, are limited
in the level of interconnected pores that can be achieved.
Nonthermal irreversible electroporation is another technique that can be applied
to introduce porosity into the BC scaffold. Baah-Dwomoh et al. (2015) used
Acetobacter xylinum to synthesize BC scaffolds, applying NTIRE during the
nanofibril synthesis process. This work demonstrated that when NTIRE is used to
kill bacteria at specified locations and times during the cellulose production process,
cellulose deposition is prevented at the treatment sites, resulting in the controllable
formation of pores and conduits within the final scaffold (Fig. 6). This method can be
used to generate 3D tissue constructs that mimic the native tissue microarchitecture.
The pore size can be controlled by varying the voltage delivered and the time
duration for each pulse.
This initial study shows the promise of using NTIRE as a method for developing a
porous network within tissue scaffolds that could be incorporated into current 3D
manufacturing technologies (Baah-Dwomoh et al. 2015). For example, a 3D tissue
scaffold could be “printed” using a layer-by-layer fabrication process. An array of
microelectrodes could deliver electrical pulses to the growing BC layer produced by
the bacteria at preprogrammed locations, resulting in a controllable pore distribution
on the XY surface. As each layer is completed, the print head would ascend in the
Z-direction, allowing precise and controllable placement of voids within each new
Fig. 6 SEM image of a lesion diameter of approximately 2 mm created in the cellulose sample after
being treated with 90 10-μs-long pulses of 1000 V, delivered at a 1 Hz frequency every 15 min for
72 h using a pair of needle electrodes spaced 1 mm apart (a). The electrode placement is visible in an
untreated sample, but no lesion is observed (b) (Image from Baah-Dwomoh et al. (2015) with
permission from Springer)
layer as it develops. Changing pore location and pore size would enable the creation
of a scaffold with controlled porosity and pore gradient. This technique for scaffold
construction using BC as a biomaterial could be combined with other methods. For
example, electrokinetic forces can be used to control the motion of Gluconace-
tobacter xylinus, allowing for control of the orientation and organization of the
cellulose fibers produced by the bacteria (Sano et al. 2010b). This technology can
be used to modulate the mechanical properties of the resulting BC scaffold. Elec-
trokinetic forces and NTIRE could in theory be combined to fabricate customizable
scaffolds. In addition to electrokinetic forces and other methods for manipulating the
mechanical response of the scaffold, cell adhesion molecules have been incorporated
onto BC scaffolds for improved biofunctionality and biocompatibility for desired
cell lines (Sulaeva et al. 2015). Growth factors have been immobilized onto BC
surfaces to promote initiation of cell growth, differentiation, and proliferation.
Combining NTIRE with other treatment methodologies during and after the BC
synthesis process can allow for the controlled production of tissue scaffolds that
promote cell growth and microvasculature development while mimicking the
mechanical properties of the tissue it serves to replace.
Gene Electrotransfer for Tissue Engineering
Larger tissue and whole organ tissue engineering is currently limited by the ability to
bring oxygen and nutrients throughout the entire tissue. For tissues with thicknesses
of more than 100–200 μm diffusional mass transfer is limited, resulting in ischemia
and cell death without a proper vasculature system in place (Sarker et al. 2015).
1594 M.P. Ho
Thus, the tissue engineering approach must promote the formation of blood vessels
throughout the engineered tissue construct. From a scaffold development perspec-
tive, this may include producing scaffolds with the required level of porosity to
induce cell migration and blood vessel development. Steps can also be taken during
the cell seeding phase to encourage the formation of blood vessels, thus enabling the
tissue to get required nutrients and remain viable.
Blood Vessel Formation In Vivo and Use in Tissue Engineering
A vascular network can be formed in vivo by either vasculogenesis or angiogenesis.

Vasculogenesis typically occurs during embryonic development, though the process
may also take place during tumor growth, revascularization following trauma, and
endometriosis. This process results in the formation of completely new blood
vessels. During vasculogenesis, endothelial progenitor cells (EPCs) assemble and
form capillaries and vascular networks in response to the presence of various local
cues and growth factors. During angiogenesis, new blood vessels sprout from
preexisting vessels due to specific local signals.
Vasculogenesis is a complicated process, and EPCs from bone marrow or blood
are mainly responsible for pre- and postnatal vasculogenesis (Sarker et al. 2015). A
host of growth factors and other signals are necessary through each step of the
process. For example, EPCs are recruited to the unvascularized tissue due to factors
including vascular endothelial growth factor (VEGF), basic fibroblast growth factor
(bFGF), and stromal cell-derived factor-1 (SDEF-1) (Sarker et al. 2015). EPCs attach
to the ECM and proliferate due to factors that include VEGF, immunoglobulin, and
epidermal growth factor (EGF) (Sarker et al. 2015). Differentiation occurs due to
factors such as SDF-1, VEGF, and platelet-derived growth factor (PDGF).
The process of angiogenesis is not without its own complexities and results in
response to many different biochemical signals, chemical gradients, and attractive or
repulsive signals. The spatial and temporal distribution of guidance molecules and
growth factors such as VEGF, fibroblast growth factor (FGF), EGF, interleukin-
8 (IL-8), and angioprotein-1 (Ang-1) are very crucial for guiding the growth of new
capillaries within the tissue (Sarker et al. 2015). Part of the angiogenesis process
includes the breakdown and degradation of the basement membrane and ECM,
which in turn can release additional growth factors and other signaling molecules.
Though great strides have been made in the field of tissue engineering, formation
of 3D vascular networks still remains a challenge due to the complex process of
vessel formation, as briefly illustrated here. Strategies to form complex vascular
networks can be scaffold based or scaffold free. The scaffold-based approach
involves the development of a 3D tissue scaffold from a variety of biofabrication
methods or through decellularization. Angiogenic factors that promote blood vessel
formation may be seeded directly to the scaffold itself. An additional method to
introduce these growth factors into the newly forming tissue is to transfect cells by
gene therapy. The use of gene therapy encourages the cells to release growth factors
Fig. 7 Methods of introducing growth factors into a tissue scaffold. (a) Various growth factors
are bonded to the tissue scaffold prior to cell seeding. (b) A gene transfection method such as
electroporation is used to introduce the gene of interest past the cell membrane and into the target
cells. These transfected cells are then seeded onto the tissue scaffold, where they then express the
desired growth factor
for angiogenesis after the cells are seeded in the scaffold matrix, thus promoting
blood vessel formation. Since a working vasculature system is vital for maintaining
cell function within thicker tissues and organs and angiogenic sprouting of host
vasculature into a tissue engineering scaffold requires time, capillary development is
often sought ex vivo prior to tissue or organ transplantation into the host. Gene
transfection is one method that can be used introduce the needed growth factors into
the tissue and promote vasculature formation (Fig. 7).
Many different growth factors are involved in blood vessel formation and stabi-
lization. A number of strategies have been implemented to load these growth factors
directly to the tissue scaffold. This includes techniques such as surface coating,
encapsulation, multilayer coating with controlled release rates, and incorporation of
spatial gradients of growth factors when attached to the scaffold. These growth
factors, however, have short protein half-lives in vivo, limiting their direct use in
tissue engineering applications (Sarker et al. 2015). Gene therapy can be utilized to
encourage cell production of growth factors and to prolong their use through the
transplantation of genetically engineered cells onto the tissue scaffold. Prior to
in vivo transplantation of a new tissue engineered construct, therapeutic cells are
isolated from their source, modified ex vivo with gene therapy, and seeded into the
scaffold. For example, Jabbarzadeh et al. (2008) transfected endothelial cells with
genes that increased the cell production of VEGF, which led to enhanced vascular
growth compared to that achieved with nontransfected cells. Gene transfection of
human mesenchymal stem cells (hMSCs) and human embryonic stem cell-derived
cells (hESdCs) also led to enhanced cellular VEGF production, increased cell
viability, and a higher blood vessel density (Yang et al. 2010).
1596 M.P. Ho
Electroporation for Gene Therapy in Tissue Engineering Applications
Viral and nonviral vectors can be used to deliver therapeutic genes to the cells. While
many different nonviral techniques have been developed, including physical and
chemical methods, this section focuses on the use of electroporation as a physical
method to introduce genes into cells. This process, termed gene electrotransfer, is
discussed in much more detail in other chapters of this book (see “Cross-Refer-
ences”). However, it is worth mentioning here in relation to its potential use in the
field of tissue engineering.
Electroporation uses high-magnitude electric pulses to permeabilize the cell
membrane. Reversible pore formation within the cell membrane has been developed
for a variety of applications in vitro including loading genes by DNA transfection
into cells, cell fusion, and inserting proteins into the cell membrane by
electroinsertion. Reversible electroporation has also been investigated for in vivo
uses such as transdermal drug delivery, electrochemotherapy, and gene
electrotransfer for applications such as localized gene therapy and delivering vacci-
nations by DNA transfer.
Gene delivery by electroporation must take into account distinct physical and
biological considerations to achieve maximum transfection efficiency. The size and
type of cell, cell orientation, nucleic acid concentration, pulse duration, pulse shape,
and electric field strength must all be considered (Mellott et al. 2013). Electropora-
tion has been successfully used to transfect both dividing and nondividing cells as
well as progenitor cells and stem cells (Mellott et al. 2013), making this technique a
very promising method for tissue engineering applications. In addition, electropo-
ration can be used to transfect a large number of cells at a time, giving it an
advantage over some other transfection methods. Cell transfection by electropora-
tion can be used to introduce genes into cells that promote the expression of growth
factors needed for vasculature development. Gene transfection by electroporation
can have other applications for cell differentiation within the field of tissue engi-
neering. For example, gene transfected human bone marrow-derived stem cells have
been used to form new bone tissue both in vitro and in vivo (Mellott et al. 2013).
Pluripotent stem cells were generated from transfecting mouse embryonic fibroblasts
and were then able to differentiate in vitro (Mellott et al. 2013).
Gene therapy strategies are used to control cellular behavior through an “inside-
out” approach, delivering nucleic acids directly into cells and promoting or
suppressing cell gene expression. These strategies are employed within the field of
tissue engineering, promoting cells to differentiate for heterogeneous tissue growth
and the development of vasculature throughout the tissue construct. Electroporation
is one method that can be used to introduce these nucleic acids into the cells,
allowing increased control in cell seeding and tissue growth on a tissue scaffold.
Here, the use of gene electrotransfer for tissue engineering applications is only
introduced to demonstrate its potential in this field. Further information on the
development and use of this technology can be found in other chapters of this
book (see “Cross-References”).
Conclusions
Electroporation is a versatile technique that has seen potential for multiple applica-
tions within the field of tissue engineering. Previous studies on the effects of
nonthermal irreversible electroporation on tissue structures have illustrated its ability
to target the cell membrane while preserving critical features such as blood vessels,
bile ducts, and the extracellular matrix. This molecular selectivity of nonthermal
irreversible electroporation (NTIRE) has made it a promising mechanism for achiev-
ing tissue decellularization and producing a decellularized tissue scaffold composed
of the natural extracellular matrix (ECM), including critical features and biological
cues necessary for cell growth and proliferation. NTIRE for tissue decellularization
has been demonstrated both in vivo and ex vivo. NTIRE has an advantage over many
tissue decellularization protocols since it does not rely on chemical or enzymatic
methods that could damage the ECM or leave behind residue after treatment. Future
development of NTIRE for this application, however, may show greatest efficiency
when combined with other decellularization methods.
The development of a tissue scaffold with interconnected pores is critical for the
growth of a vasculature system within the engineered tissue, allowing for the
delivery of nutrients. NTIRE has been demonstrated as a method for producing
controlled porosity within tissue engineered scaffolds produced from bacterial
cellulose and could be further developed into a 3D manufacturing method for
fabricating bacterial cellulose (BC) scaffolds with preprogrammed porosity and
mechanical strength.
Reversible electroporation has been developed for gene transfection, and this,
too, has seen applications within the field of tissue engineering. For example, the
presence of specific growth factors is critical for vasculogenesis and angiogenesis.
One method of delivering these growth factors to the tissue scaffold is through gene
transfection techniques, enabling increased expression of the growth factor from
transfected cells that are seeded within the tissue scaffold. Gene transfection by
reversible electroporation also has potential in areas of stem cell differentiation.
While further work is necessitated for the development of electroporation as a
tool within the tissue engineering field, this chapter gives examples of areas that have
shown promise. The development of a functional tissue engineered organ constructs
for transplantation is an incredibly complicated process that requires the combina-
tion of many different techniques and a further understanding of the cell migration,
proliferation, and differentiation as well as critical cell-ECM interactions. Electro-
poration is one technology that can prove beneficial in this field.
Cross-References

▶ Gene Electrotransfer for Ischemic Tissue
1598 M.P. Ho
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3317–3322
Predictable Animal Models
for Translational Electroporation-Based 83
Cancer Immunotherapy Studies
Mariangela De Robertis, Ursa Lampreht Tratar, Maja Čemažar,

and Emanuela Signori
Abstract
Emerging strategies in cancer treatments, such as gene electrotransfer and
electrochemotherapy, are very promising. However, preclinical studies of effec-
tive therapeutic agents delivered in animal models to translate into the clinic still
remain a crucial feature. To pass successfully from bench to bedside, attention on
in vivo models to employ in promising translational preventive and/or therapeutic
protocols must become of utmost importance. In this chapter, it has been called
attention to important aspects that can make a predictable animal model, such as
the possibility to mimic the onset and development of tumors occurring in
humans.
M. De Robertis (*)
Laboratory of Genetic and Clinical Pathology, University Campus Bio-Medico of Rome, Rome,
Italy
Laboratory of Molecular Pathology and Experimental Oncology, Institute of Translational
Pharmacology, National Research Council (CNR), Rome, Italy
e-mail: mariangela.derobertis@gmail.com
U.L. Tratar (*)
e-mail: ulampreht@onko-i.si
M. Čemažar (*)
E. Signori (*)
Laboratory of Molecular Pathology and Experimental Oncology, Institute of Translational
Pharmacology, National Research Council (CNR), Rome, Italy
e-mail: emanuela.signori@ift.cnr.it

DOI 10.1007/978-3-319-32886-7_50
1602 M. De Robertis et al.
The strengths, weakness, applicability, and predictability to human cancer of

these classes of preclinical models are discussed in this chapter, in the context of
immunotherapeutic studies based on electrotransfer.
Keywords
Animal models • Translational studies • Tumorigenesis • Cancer immunotherapy •
Electrotransfer • Preclinical protocols
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1602
Predictable Model: What (Is Predictable?), Why (Is It Predictable?),
When (Is It Predictable?) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1603
Translational Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1604
Mouse Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1610
Transplantable Tumor Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1610
Genetically Engineered Mouse Model (GEMM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1611
Humanized Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1612
Carcinogen-Induced Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1613
Naturally Occurring Pet Tumors: Canines and Felines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1613
Canines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1613
Felines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1615
Nonhuman Primate Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1615
Electroporation-Mediated Immunotherapy: From Preclinical Studies to Clinical Trials . . . . . 1615
Electroporation-Mediated Immunotherapy in Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1617
Electroporation-Mediated Immunotherapy in Pet Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1618
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1619
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1620
Introduction
The use of animal models to predict human response to drugs and disease is one of
the main focal points of the scientific community. The assumption that animal
models are predictive of human disease is introductory for much of their use in
biomedical research, especially in toxicology and pathophysiology.
Various animal models of cancer disease have been developed showing a relevant
ability to reproduce the correspondent human pathology in terms of peculiar tumor
hallmarks, such as molecular modifications, timing of tumor lesions appearance,
neoangiogenesis, metastatic process, etc.
However, considering what we have learned from fields such as evolutionary and
developmental biology, gene regulation and expression, epigenetics, and compara-
tive genomics, it should not be surprising that some animal models fall short of being
able to predict human disease in its entirety. For this reason, the definition of the
“predictability” of different classes of animal models is of paramount importance for
the selection of the best model in studies aimed to the chemopreventive/therapeutic
effect evaluation or to the assessment of mechanisms of carcinogenesis.
83 Predictable Animal Models for Translational Electroporation-Based. . . 1603
In the field of cancer treatment, new therapeutic approaches as gene

electrotransfer and electrochemotherapy are emerging as very promising strategies.
However, their current evaluation in preclinical immunotherapeutic studies is of
crucial importance; hence, it results fundamental to select a predictable animal model
to employ in translational preventive and/or therapeutic protocols.
Predictable Model: What (Is Predictable?), Why (Is It Predictable?),

When (Is It Predictable?)
Cancer is not a single disease state, but exhibits considerable intra- and inter-tumor
heterogeneity both genotypically and phenotypically that are both dynamic and
variable in nature, with most cancers utilizing multiple and redundant dysregulated
survival and growth-regulatory pathways in the course of their adaptive evolution.
Importantly, tumor evolution is the result of a complex network of relations between
neoplastic cells and microenvironment components. As a major example, the inter-
actions between the tumor and the immune system are a key feature of cancer, and
for some kinds of tumors, the presence of infiltrating immune cells in the tumor
microenvironment may have a primary influence on patients’ survival.
Various animal models have been established to investigate carcinogenic mech-
anisms and to assess the potential of novel antitumor agents in a preclinical setting.
Selection of appropriate preclinical models based on similarity to human biology and
disease genotype and phenotype carries considerable potential to ensure higher
reliability.
This brings into question the predictability of preclinical tumor models as well as
the method to analyze and translate the results of preclinical studies. In this regard,
good predictive models would hopefully permit a more effective evaluation of novel
cancer treatments, as cancer immunotherapy, as well as a more rational design of
potent anticancer drug combinations.
For many years, cell lines have been the elective experimental model in cancer
studies, and they are still a valuable tool for investigating many aspects of cancer
biology, such as genetic, epigenetic, and cellular pathway alteration, deregulation of
proliferation, and apoptosis, and for testing therapeutic drugs. Nevertheless, the
heterotypic interactions between tumors and the multiple distinct cell types in the
microenvironment, including immune cells, are missing in these in vitro studies. The
microenvironment evolves in response to tumor survival adaptation, thereby
enabling primary, invasive, and potentially metastatic growth. This dynamic reci-
procity between tumor cells and their environment sculpts the hallmarks of cancer
and poses additional challenges in the design of appropriate experimental models.
Therefore, in vivo animal models, rather than in vitro investigations, represent a
precious solution for cancer immunotherapy studies and for preclinical protocols
based on the administration of immunotherapeutic treatments by electrotransfer
(Sersa et al. 2015).
A predictable model should reliably exhibit analogy to the human disease show-
ing all the critical manifestations of the pathology relevant to the investigation, such
as the insensitivity to growth-inhibition signals, the ability to sustain new angiogen-

esis, the genetic instability, tissue invasion, metastasis, etc. (Fig. 1). Furthermore, a
predictable model should display homogeneity with the human disease studied.
Consistency with disease manifestation and development is also an important
parameter to ensure reliable evaluation of the potential for exacerbation of the
disease phenotype or target.
In this perspective, a preclinical model can be considered predictable when it
shows relative strengths in mimicking the human disease and in appropriately
reflecting its cellular and molecular pathology.
Translational Models
Although preclinical animal models have definitely contributed to our understanding

of molecular mechanisms of carcinogenesis and of the role of tumor immunology,
the use of any animal model will involve some degree of compromise relative to
studying the human cancer in its natural environment.
It is critical to recognize that each type of animal model of cancer has intrinsic
advantages and limitations, leading to practical considerations for their effective use
in drug discovery, in the elucidation of the mechanisms of tumor initiation and
progression, and in the responsiveness of a particular tumor type to specific
therapies.
Each of the particular classes of preclinical tumor models, and the data generated
with them, should not be viewed in isolation or as an absolute predictor of the human
response to a novel therapeutic agent. Rather, the specific classes of models detailed
in this chapter for their strengths, weaknesses, utility, and clinical predictive value in
oncology drug discovery should be considered as a portfolio of sophisticated
biological tools that can be utilized at various stages in the drug discovery process,
to answer specific experimental questions.
In the iterative drug discovery process, the decision of what types of models to
use, how many, and at what stages is directly dependent on the specific target to
investigate. It is important to recognize that classes of models can be used to varying
degrees at multiple stages in the drug discovery process and beyond, dependent upon
the molecular target or pathways to be explored.
The careful interpretation and extrapolation of data derived from these models to
human patients, and a correspondingly greater emphasis placed on translational
medical research in early-stage clinical trials, are essential to improve the current
clinical attrition rates for novel therapeutic agents in oncology.
Animal models of cancer encompass a wide spectrum and include:
1. Ectopic xenografts (subcutaneous, intraperitoneal, intravenous, intramuscular) of

tumor-derived cell lines or tissue explants implanted into syngeneic or immune
compromised hosts
2. Orthotopic models where the explants of tumor tissues or established tumor lines
are implanted within the proper organ or tissue, thus recapitulating the complex
Fig. 1 Cancer hallmarks and animal model predictability

cell-cell interactions of the local microenvironment in which a primary tumor

grows and disseminates
3. Germ-line transgenic and conditional transgenic models (GEMMs) in which
the expression patterns of specific oncogenes or tumor suppressor genes can be
regulated systemically or in a tissue- and temporal-specific manner,
respectively
4. Primary human tumor grafts maintaining the genotypic and phenotypic profile of
a primary tumor from which they are derived
5. Various carcinogen-induced models that recapitulate the time-dependent and
multistage progression of tumor pathogenesis in response to environmental
carcinogens and tumor-promoting agents
Besides the rodent models, which are the most investigated in the laboratories due
to their experimental feasibility and relatively low prices, a wide variety of other
animal species are also available for specific models of human diseases such as pet
animals. Spontaneous tumors in pet animals especially in canines present an excel-
lent model for cancer research. Tumors in canines share many similarities with
human tumors. One of the important features is that cancer in canines is histologi-
cally comparable to human cancer. Furthermore, the development of tumors in
canines can be observed over a long period of time, and the majority of them appear
in older subjects as it happens in humans. Moreover, canine tumors respond simi-
larly to human tumors to conventional therapies. The life span of canines is propor-
tionally more comparable to humans than the life span of rodents, which is a
valuable characteristic when the side effects that could develop over time need to
be evaluated. Due to the same environmental conditions shared between pets and
owners, factors influencing the pathogenesis of the diseases can be explored. Fur-
thermore, due to the lack of “gold standards,” new therapies can be assessed before
entering human clinical trials and thus provide valuable information for human
treatment (Ranieri et al. 2013).
The creation of different canine breeds has resulted in many founder mutations that
are associated with specific diseases. This is consistent with the fact that some breeds
are predisposed to a distinct set of diseases and one of them is cancer. Due to this trait,
single breeds such as golden retrievers, Boxers, and German shepherds have higher
incidence of cancer, thus representing a powerful tool as a translational model
(Rowell et al. 2011).
The incidence of neoplasms in canines and felines is difficult to measure since
population-based registries do not exist. But an estimate annual incidence of cancer in
canines is 2–4% (and in felines is 0.5%) with the mortality rate of 20% for canines.
The incidence and mortality rate differ in different breeds, with golden retrievers,
Boxers, and German shepherd having the highest percentage (Withrow and Vail
2001).
The strengths, weakness, applicability, and predictability to human disease
of these classes of preclinical models are discussed in this chapter and
highlighted in Table 1, in the context of immunotherapy field investigation
based on electrotransfer.
83
Table 1 Characteristics, applicability, and predictability to human disease of classes of preclinical models
Type of cancer
model Development strategy Advantages Weaknesses Predictability
Ectopic xenograft Subcutaneous, Reproducibility Loss of tumor Poor predictiveness for most cancers for
models intraperitoneal, intravenous, heterogeneity the limited phenotypic similarities to
or intramuscular ectopic primary cancers
implantation of tumor- Easy induction of various Limited histological and The study of immunotherapies is not
derived cell lines or tissue tumor types phenotypic similarities to possible in immunocompromised mice
fragments primary cancer
(in immunocompetent and Preservation of Loss of native tumor
immunodeficient rodents) immunocompetence microenvironment
(immunocompetent hosts)
Low cost Nonanatomically
appropriate growth
location
Orthotopic models Implantation of tumor- Correct tumor Not easy handling Limited/moderate predictiveness
derived cell lines or tissue microenvironment
fragments into the organ of High metastatic rates Difficult tumor growth Moderately predictive in the study of the
origin monitoring clinical course of metastatic disease
(in immunocompetent and Limited histological and Moderately predictive in the evaluation
Predictable Animal Models for Translational Electroporation-Based. . .
immunodeficient rodents) phenotypic similarities to of influence of immune surveillance and

primary cancer evasion in immunocompetent hosts
(continued)
1607
1608
Table 1 (continued)
Type of cancer
model Development strategy Advantages Weaknesses Predictability
Genetically Manipulation of the mouse Genetic and phenotypic Not easy achievement Predictive in therapeutic responsiveness
engineered mouse genome for the spontaneous similarities to primary and in development of therapeutic
model (GEMM) and autochthonous cancers resistance
development of tumors in Development of tumors in a Possibility of not complete Predictive in the study of the clinical
appropriate variety of organs that carry tumor penetrance course of metastatic disease and tumor
microenvironment the genetic signature of the angiogenesis
(in immunocompetent human malignancy
mice)
Metastatic patterns may Predictive in the evaluation of influence
not mimic human cancers of immune surveillance and evasion in
immunocompetent hosts
Humanized mice Insertion of human genes, Phenotypic similarities to Long latency for tumor Used for “clinical trials in a mouse”
cells, or tissue fragments primary cancer development
into immunodeficient mice Potential to metastasize High costs and labor- Highly predictive in the evaluation of the
intensive preparation interaction between human tumor and
Easy tumor measurement Access to freshly excised immune system
human tumors
Carcinogen- Induction of tumor Phenotypic similarities to Long latency for tumor Predictive models of developmental
induced models development through primary cancer development sequelae of human cancers
carcinogens and tumor- Reproduction of multistage High costs Highly predictive in testing therapeutic
promoting agents developmental process of and chemopreventive agents
cancer
Reproducibility Different mouse strains Highly predictive in the evaluation of the
Assess influences on tumor show varied sensitivity to interaction between human tumor and
immune surveillance/ carcinogens immune system
evasion
M. De Robertis et al.
83
Pets as animal Naturally occurring tumors Histologically comparable Recruitment depends on Some breeds prone to development of
models tumors to human tumors owner’s will for specific cancers
participating
Development of tumors in No official registry for Highly predictive for evaluating immune
older subjects epidemiological data on therapies and the translation into human
neoplasms medicine
Similar response of Predictive response to conventional
immune system to humans therapies
Longer life span than
rodents
The same environmental
factors as humans
Lack of “gold standard”
enables evaluation of new
therapies
Nonhuman Naturally occurring tumors Similarity of genetic Low incidence of Highly predictive for the social and
primate animal evolution spontaneous tumors neurophysiological studies
models Experimentally induced Up to 96% of genome High costs Predictive response to conventional
tumors similarity therapies
Predictable Animal Models for Translational Electroporation-Based. . .
1609
Mouse Models
In vivo mouse models may represent precious predictable models. The laboratory
mouse is one of the best model systems for cancer investigations, owing to various
factors including small size and propensity to breed in captivity, lifespan of 3 years,
extensive physiological and molecular similarities to humans, low cost, ease of
handling, and an entirely sequenced genome. Preclinical mouse models can be
divided into four main categories: transplantable tumors derived from tumor cell
lines or explants, genetically engineered/transgenic models, humanized mouse
models, and carcinogen-induced models.
Each approach has its own advantages and disadvantages, and it is important to
choose the most appropriate system with respect to the target of the studies to be
performed.
Transplantable Tumor Models
Syngeneic mouse models injected with murine cell lines have commonly been used
as a standard in vivo model for tumor immunology investigations (Berns and
Barbacid 2013). The advantages of this model are reproducibility, ability to easily
induce various tumor types, and immunocompetence. It also allows the study of
tumor growth and/or metastasis in a three-dimensional structure, thus preserving the
interactions between cancer cells and microenvironment stroma, including immune
cells. Although they probably represent only a fraction of the total heterogeneity of
the original cancer type, transplantable tumors are exploited to study tumor-
associated antigens (TAA) immunogenicity, to assess the generation of antitumor
response in the host and the mechanisms of immune-mediated tumor rejection. On
the other hand, this model often shows a different response in comparison to the
results from in vitro assays in human cancer cells.
To overcome this disadvantage, a battery of xenograft models was developed by
injection of human tumor cell lines into immunodeficient (severe combined immu-
nodeficient (SCID) or nude) mice. The development of xenograft models enabled the
rapid and facile in vivo assessment of tumor tissue and cell lines in immunocom-
promised mice. Indeed, patient-specific models have recently been proposed as a
means to utilize prospectively personalized treatment regimens.
Because of its ease and low cost, this approach has been used extensively in
academia and in the pharmaceutical industry during the past three decades for cancer
gene validation and testing of novel targeted compounds. Nevertheless, transplant-
able tumors progress very rapidly following inoculation, and spontaneous metastatic
dissemination is often missing, losing the slow step-wise progression that charac-
terizes human tumors. Moreover, several crucial differences exist when comparing
tumor xenografts with patient-derived specimens or autochthonous murine tumors,
including the derangement of the normal tumor architecture and diminished genetic
heterogeneity that is inherent to autochthonous tumors. Crucial features of the tumor
microenvironment that are altered or lost in xenograft tumors include nearby normal
tissues, stromal cells, vasculature and lymphatic circulation, and immune cells.
Moreover, they do not grow in the anatomically appropriate site, thus not mimicking
the organ-specific features of the original tumor and its interaction with the immune
system. Additionally, due to the immunocompromised state of transplanted mice, the
study of immunotherapies is not possible in the xenograft model.
Orthotopic models can represent an evolution with some advantages (Bibby
2004), but are not easy to handle, as monitoring tumor growth is often tricky.
It should be noted that despite their flaws, xenograft studies can have predictive
value, and they have helped to prioritize compound testing. Indeed, nearly every
successful cancer therapy developed in the modern era has undergone xenograft
testing.
Genetically Engineered Mouse Model (GEMM)
Various methods for generating mouse models have been established to overcome
the limitations associated with transplantable models. One model is the genetically
engineered mouse model (GEMM), which is an experimental system that mimics
human malignancy, employed for evaluating carcinogenesis mechanisms and drug
resistance (Singh et al. 2012).
Thanks to the advances in genetic engineering, tumor biology, and biomedical
technology, it has become possible to reproduce human cancer in GEMM models
through the expression of known oncogenes, specific genetic mutations, or inactiva-
tion of known tumor suppressor genes in all cells or only in a specific tissue
compartment and/or developmental stage of interest. Manipulations of the mouse
genome may thus result in the development of tumors in a variety of organs that carry
the genetic signature of the human malignancy and spontaneously progress through
premalignant and malignant stages, resembling human carcinogenesis (de Jong and
Maina 2010).
These models that more accurately phenocopy human cancer are useful for
elucidating biological processes and investigating tumor cells and their
microenvironment.
In GEMM models, the tumors develop slowly, allowing a long-lasting interaction
with the host immune system, and occasionally there is a natural occurrence of
invasion and development of metastasis (de Jong and Maina 2010).
These similarities with the human cancer represent the appealing features of
GEMM that can be successfully employed to identify cooperating tumor-associated
antigens (TAA) critical for tumor initiation, survival, or progression and thus serve
as potentially effective targets for the design of translational cancer immunother-
apies. Moreover, they would enable investigators to better test the effectiveness of
cancer immunotherapy and model mechanisms of action and therapeutic resistance.
In addition, they could aid in the identification of optimal dosing regimens, both
alone and in combination with other agents.
Nevertheless, one limitation of this system is that it may be difficult to elicit the
exquisite control necessary to express oncogenes at physiological levels. This
represents an important consideration as many oncogenes cause toxic effects,

including senescence or apoptosis, when overexpressed in vivo. Additionally,
tumor penetrance is not always complete, and transgene expression is usually
under the control of a heterologous promoter, leading to nonphysiological levels
of the transgene product that may influence the tumor microenvironment and the
immune response to the transgene product itself.
Conditional GEMM, in which the expression of TAA can be timely and spatially
regulated allowing tumor onset when the host has a mature immune system, as
frequently happens in humans, are a further evolution of preclinical cancer models
(Berns and Barbacid 2013).
Humanized Mice
One of the major criticisms of modeling cancer in mice is that the biology of humans
and mice is too disparate, and mouse models cannot recapitulate aspects of human
disease.
Although orthologous genes are often highly conserved, significant differences do
exist between the mouse and human genomes that might limit the ability to model
human cancer. Therefore, one mechanism of humanization is the insertion of human
genes into the mouse genome (Shultz et al. 2012). This approach is frequently
addressed through recombinase-mediated genomic replacement method, which con-
sists of the knock in of a human cDNA into the corresponding murine genomic locus.
Different approaches have been performed on humanized mice which have been
accurately extrapolated to humans, as the use of humanized M. musculus telomeres
or drug-metabolizing enzymes, but humanized mice represent particularly important
in vivo preclinical model to evaluate the interaction between human tumors and
human immune system.
To date, humanizing the murine immune system has consisted of ablating the
endogenous immune system followed by engraftment with human immune cells;
however, species-specific differences in both the adaptive and innate immune
systems can confound interpretations. Immunodeficient mice transplanted with
human hematopoietic stem cells are indeed a powerful tool for the study of human
hematopoiesis and immune function in vivo. An important step in this direction
has been achieved by the introduction of transgene coding for various human
cytokines or human leukocyte antigen (HLA) class I and class II molecules. The
engrafted human immune system is competent in generating T- and B-cell
immune responses, making this mouse model appealing for cancer immunother-
apy translational studies.
Patient-derived xenografts in these humanized mice are an evolution of trans-
plantable tumor models and allow the study of the interactions between the human
immune system and the progressive stages of the tumor, an important step toward the
personalized immune-oncology medicine.
Carcinogen-Induced Models
Certain mouse strains are prone to develop cancer either spontaneously or after
exposure to a given carcinogen or cancer-causing agent. The agent could be chemical,
radiation, or even physical impacts that result in alterations and mutations that lead to
uncontrolled cell growth. Combined with particular strains or genetically modified
mouse models, carcinogen-induced models can be immensely informative.
Abundant examples of chemically induced model systems exist.
Many murine models of sporadic and inflammation-related colon carcinogenesis
have been developed in the last decade. Among the chemically induced colorectal
cancer (CRC) models, the azoxymethane (AOM)/dextran sulfate sodium (DSS)
model has become an outstanding model for studying colon carcinogenesis and a
powerful platform for chemopreventive intervention studies (De Robertis et al. 2011,
2015). Breast cancer studies have long relied on spontaneous and induced models
(Steele and Lubet 2010). Oral and lung cancers can be modeled with tobacco-
specific carcinogenic compounds (Steele and Lubet 2010). Additionally, some
chemical agents may have broad-spectrum effects in mouse models. For example,
cadmium or arsenic have been shown to induce a range of cancers in animals.
Carcinogen-induced models have collectively been useful for various preclinical
studies, including the identification of oncogenes and tumor suppressor genes, for
the study of tumor susceptibility traits, and the assessment of the carcinogenic or
chemopreventive effects of various compounds. However, as with all models, these
may vary somewhat from the related human cancer, and therefore they should be
analyzed with awareness and understanding of the differences. Additionally, such
models are able to reproduce a restricted subset of tumor types and grades with
incomplete penetrance and variable latency.
Naturally Occurring Pet Tumors: Canines and Felines
Canines
The most relevant spontaneously occurring tumors in canines for translational

oncology are soft tissue sarcoma, malignant melanoma, osteosarcoma, and
lymphomas.
Soft Tissue Sarcoma (STS)

STS are genetically complex cancers which include hemangiosarcomas
(angiosarcomas in humans), fibrosarcomas, and histiocytomas. Because canines
have a similar genetic complexity to that of humans, they present an exceptional
model for STS. More specific, breeds like golden retrievers, German shepherds,
and Boxers have a breed predisposition for the development of hemangiosarcoma,
which account for 5–7% of all observed tumors in canines (Rowell et al. 2011).
Combining all the characteristics of STS in canines listed above, canine model
represents a more relevant model than other animal models for direct human STS
application.
Malignant Melanoma
One of the most lethal skin cancer in humans is malignant melanoma, due to the
rising incidence and mortality rates. In canines, malignant melanomas represent
7% of all malignant neoplasms and the most common tumor of oral cavity with
less commonly affected sites as lips, the skin, and digits. Human and canine oral
malignant melanoma share many characteristics at molecular level, having
similar molecular and signaling pathways. Furthermore, they share the pheno-
typic characteristics as both tumors show aggressive behavior and are highly
metastatic with poor prognosis (Atherton et al. 2016). Breeds that are more
prone to develop malignant melanomas are poodles, Beauce Shepherd, and
Rottweiler. Interestingly, poodles present 77% of oral melanoma, and Beauce
Shepherd and Rottweiler present 80% and 69% of cutaneous melanoma. There
is an overrepresentation of tumors in black-coated breeds and an underrepresen-
tation of pale-coated or even hairless canines. This indicates that the UV
radiations do not have the dominant role in the development of canine melano-
mas (Gillard et al. 2014).
Osteosarcoma
Osteosarcoma (OSA) is the most commonly diagnosed primary tumor in the bone
and represents over 56% of all bone tumors in humans and account for 85% of all
bone tumors in large and giant breeds of canines. OSA characteristics are similar in
humans and canines. The sites of the tumor are comparable and are usually located
on the distal radius, proximal humerus, distal femur, and proximal tibia. In addition,
OSA is biologically and histologically similar in humans and canines with related
responses to the treatment. The main cause of death in both human and canines is
pulmonary metastasis (Rowell et al. 2011).
Lymphoma
Lymphomas are 5% of all human cancers and account for 24% of all cancers in
canines. Accordingly, 90% of all lymphomas are non-Hodgkin lymphomas
(NHL). NHL in canines and humans have similar biology, behavior, and genetic
aberrations (Rowell et al. 2011). The majority of canine NHL (60–80%) is a
source of malignant B cells, which is also the case in humans (Ranieri et al. 2013).
Compelling correlation between the occurrence of human and canine NHL and
environmental factors, such as radioactive waste and polluted sites, has been
observed. Due to this similarity, canines represent a good model for translational
research studying lymphomas. An additional advantage of the canine model is the
increased prevalence of lymphoma in certain canine breeds. Approximately one in
four Boxers and one in eight golden retrievers develop lymphomas (Rowell
et al. 2011).
Felines
Squamous cell carcinoma (SCC) is the most common oral tumor in felines
(60–80%). The risk of feline oral SCC (FOSCC) is associated with environmental
factors, as it increases with the use of flea collar and high intake of canned foods or
the exposure to household tobacco smoke. FOSCC frequently invades bone, and the
degree of invasion is usually severe (Withrow and Vail 2001). Head and neck SCC
(HNSCC) in humans comprise approximately 90% of all head and neck cancers.
FOSCC and HNSCC are clinically similar in the disease progression and biological
behavior. Both cancers are locally invasive with late metastasis mostly to regional
lymph nodes and lungs. Importantly, felines are the only species, other than humans,
that develops SCC of the tongue (Wypij 2013).
Nonhuman Primate Models
Nonhuman primates (NHP) could be the most appropriate animal model for human
research, rather than rodent models, due to the similarity of the genetic evolution,
anatomy, biochemistry, immune system, physiology, and metabolism. Another
aspect of the suitability of NHP as a good model for human cancer is the fact that
almost all human cancer genes are highly conserved between humans and NHP.
The NHP can be employed in cancer research studies with spontaneous or
experimentally induced tumors, although cancer incidence in NHP is very low.
The incidence of spontaneous tumors is approximately 5% in all age groups in
NHPs, but it increases in frequency with age – similar to humans (Xia and Chen
2011). The most common spontaneous tumors in NHP develop in the urogenital
system of females, followed by skin papilloma and neoplasia of the endocrine and
alimentary systems in both sexes.
Due to the low incidence of spontaneous tumors in NHP, experimentally induced
tumors are a necessity for the use of NHP in human cancer research. Different
chemical, biological (hormones, bacteria, and viruses), and physical carcinogens
have been used to induce a wide range of tumors in different NHPs. NHP have two
major limitations: cost and lack of efficacy. Due to the huge expenses associated with
these animals, research institutes rarely perform studies using NHP. Another impor-
tant aspect of NHP is that this model cannot help determining the therapeutic effect
and impact on tumor-induced immunosuppression, since spontaneous tumors rarely
occur even in large NHP colonies (Xia and Chen 2011).
Electroporation-Mediated Immunotherapy: From Preclinical

Studies to Clinical Trials
By means of immunotherapy, it is possible to prompt a person’s immune system to

fight diseases such as cancer. In the last few decades, immunotherapy has become an
important part of treating some types of cancer. Newer kinds of immune treatments
are now being studied, and they include treatments that work in different ways. Some
boost the body’s immune system in a very general way. Others help by stimulating
the immune system to attack cancer cells specifically.
The main types of immunotherapy currently used to treat cancer include anti-
bodies, immune checkpoint inhibitors, adjuvants, immunochemotherapy, and
vaccines.
Cancer vaccines cause the immune system to attack cells with one or more
specific antigens. Some cancer vaccines are made up of cancer cells, parts of cells,
vector-based vaccines, or pure antigens administered as peptides or inserted in
plasmid DNA. Vaccines are often combined with other substances called adjuvants
that help boost the immune response even further.
Monoclonal antibodies (mAbs) can be very useful in treating cancer because they
are designed to attack a very specific part of a cancer cell. They have already become
an important part of the treatment for many cancers. Many strategies have been
adopted for making monoclonal antibodies safer and more effective as new forms of
mAbs which are less likely to cause immune reactions (mAbs fragments, diabody,
etc.). Newer forms of mAbs have also been developed attaching them to drugs or
other substances to make them more powerful or combining parts of two antibodies
together (known as a bispecific antibody).
Immune checkpoint inhibitors basically take the “brakes” off the immune
system, which helps it recognize and attack cancer cells. Targeting these check-
points is quickly becoming an important part of the treatment for some cancers,
such as melanoma and non-small cell lung cancer. Unlike most other cancer drugs,
these checkpoint inhibitors seem to be helpful against many different types of
cancer.
Immunochemotherapy (also called chemoimmunotherapy) combines two power-
ful treatments: immunotherapy and chemotherapy. A number of mechanisms have
been proposed for the chemotherapy-triggered enhancement of immunotherapy
response. Thus, chemotherapy may favor tumor cell death and by that enhance
tumor-antigen cross presentation in vivo. Furthermore, the recently reported synergy
between monoclonal antibodies and chemotherapy or peptide vaccination is based
upon the induction of endogenous humoral and cellular immune responses. This
would suggest that mAbs may not only provide passive immunotherapy but can also
promote tumor-specific active immunity.
Numerous strategies have been adopted for optimizing the immunological
response elicited by the disparate immunotherapeutic treatments described above.
Particularly, many efforts have been made to improve cancer vaccines efficacy.
Many tumor-associated antigens are excellent targets for immune therapy and
vaccine design. Optimally designed cancer vaccines should combine the best
tumor antigens with the most effective immunotherapy agents and/or delivery
strategies to achieve positive clinical results.
Various vaccine delivery systems such as different routes of immunization and
physical/chemical delivery methods have been used in cancer therapy, with
electroporation (EP) one of the most promising for DNA delivery (Kraynyak
et al. 2015).
EP is a safe and reproducible methodology that efficiently and effectively delivers

DNA vaccines to the target cells with the least amount of cellular damage and the
appropriate levels and kinetics of expression, both in small and large animal models
(Kraynyak et al. 2015).
The promising potential of EP has been increasingly recognized in the field of
cancer immunotherapy research. The selection of the model that mimics as closely as
possible the human cancer for which the therapy or vaccine is designed, and the best
strategy to deliver it, is found to be a potent translational tool to assess the safety and
the efficacy of EP-mediated immunotherapies, hugely accelerating new drug devel-
opment in the human clinical setting.
The first uses of this concept were in the skin. Since then, this technique has been
applied to many other tissues and animal models including the skeletal muscle,
kidney, testes, brain, cartilage, arteries, prostate, cornea, heart and many tumor types
in rodents, guinea pigs, canines, rabbits, and nonhuman primates.
To date, EP-based therapies with innovative electroporation devices have been
introduced into the clinical practice, performing a new treatment called electroche-
motherapy (ECT) (Miklavčič et al. 2014). After the publication of European Stan-
dard Operating Procedures on Electrochemotherapy (ESOPE) study, ECT-based
approaches are exponentially expanding (Marty et al. 2006). ECT is becoming a
therapeutic procedure adopted in several European cancer centers for the treatment
of cutaneous and subcutaneous tumors. Recently, it has been introduced into the
treatment of deep-seated and endoluminal tumors as well. Clinical trials have been
performed to test ECT on multiple tumor types including melanoma, squamous cell
carcinoma, liver metastases, and basal cell carcinoma. Many treatments demonstrate
promising early efficacy results, making this therapeutic strategy closer to the
clinical reality. Moreover, ECT has been proposed in combination with immuno-
therapy, using ECT boosted with immunogene electrotransfer as in situ vaccination
for successful tumor treatment (Sersa et al. 2015).
Following the First World Congress on Electroporation (https://wc2015.electro
poration.net), several recommendations for improving the quality of reporting clin-
ical electrotransfer studies have been detailed. These recommendations can improve
the quality of the reports of the clinical data and help to the broader recognition and
dissemination of ECT (Campana et al. 2016).
Electroporation-Mediated Immunotherapy in Mice
In several reports, EP-mediated DNA delivery is described as effective tool in

eliciting antigen-specific immune response in small and large animal models,
being responsible for the generation of an inflammatory environment with immune
cell infiltration.
Numerous studies performed in mice models provide evidence that this technique
induces a moderate tissue injury and the generation of a pro-inflammatory context with
cytokine release promoting the conditions for immune response. For this, EP has also
exhibited adjuvant-like effects useful in vaccination protocols (Chiarella et al. 2013a, b).
Strategies of cancer immunotherapy based on genetic vaccination in association

to EP have been tested on different mouse models. It should be referred to other
chapters of this book for a more specific information.
Electroporation-Mediated Immunotherapy in Pet Animals
The purpose of EP-mediated immunotherapy in pet animals differs from those in

mice. The majority of them are headed toward therapy of different diseases in pet
animals and therefore present a unique way to research different therapies that could
also be applied to humans. Again, pet animals represent a valuable model for cancer
research, due to the characteristics listed above. Canines are especially very useful in
immunotherapy translational studies as compared to rodents or other pet animals for
the similarity of the immune system existing between canines and humans (Rowell
et al. 2011).
Several different molecules have shown to be of great importance in immunother-
apy of canines. Interleukin-12 (IL-12) was observed to be a potent cytokine in the
antitumor effectiveness of the immune system as it enhances the production of
interferon-γ (IFN-γ) which increases the activity of cytotoxic cells and antigen-
presenting cells (Trinchieri 2003). Many clinical studies based on electroporation
using plasmid that encodes interleukin-12 (IL-12) have been already performed in
mice, and there are also several human clinical studies using electrotransfer of
plasmid IL-12. However for more convincing translation of this new treatment, it is
essential for clinical studies to be performed on canines with spontaneous solid
tumors. In the last two decades, many clinical studies on canines have been performed
with different routes of administration. With intratumoral application, the reduction of
the tumors was observed with confirmed systemic effect and without any side effects.
When used intramuscularly in spontaneous tumors in canines, elevated concentration
of IL-12 and IFN-γ was observed, leading to a prolonged disease-free survival (Pavlin
et al. 2012). Another delivery route is the peritumoral administration, where the skin
in the proximity of the tumor is electroporated to achieve maximal systemic effect as
observed in mice (Pavlin et al. 2009). Apart from gene electrotransfer used as a
primary treatment, it was also used as an adjuvant treatment to ECT. One of the latest
study showed that EGT combined with ECT successfully achieved up to 72% of
complete responses at the end of observation period (median 40 months) in 18 canines
with mast cell tumors (Cemazar et al. 2016).
Another molecule used in vaccination studies was vascular endothelial growth
factor-2 (VEGFR-2) which is overexpressed on tumor blood vessel. Therefore,
VEGFR-2 represents an important target which can result not only in antiangiogenic
treatment but it can also elicit antitumor effect. Additionally, VEGFR-2 is also
expressed on regulatory T cells and myeloid-derived suppressor cells which can
have an immune-stimulating effect, as these cells suppress antitumor immune
response. Plasmid-encoding VEGFR-2 was introduced into the skin by
electroporation, and the results showed that this treatment is capable to elicit a
cellular and humoral immune response in canines after the administration, with no
adverse side effects (Denies et al. 2016).
Telomerase reverse transcriptase (TERT) and HER-2/neu molecules represent
suitable targets for translational cancer immunotherapy in canines as well. TERT
activity is largely confined to tumor tissue and absent in normal tissue, making TERT
a tumor-associated antigen (Peruzzi et al. 2010a). HER-2/neu is an oncoprotein
overexpressed in canine mammary carcinoma and is an optimal target for therapeutic
intervention. dTERT is a vaccine against canine telomerase reverse transcriptase
administered by adenoviral vector using electroporation. This vaccine showed great
promise for treatment of malignant lymphoma with the combination of standard
chemotherapy. The combined treatment induced the immune response and signifi-
cantly increased survival time after therapy (Peruzzi et al. 2010a). Another study was
published where a combined vaccine with dTERT and HER-2/neu delivered by
electroporation was used (Peruzzi et al. 2010b). Results of this study showed that
the vaccine was safe and effective and able to induce a strong immune response to
those antigens by cytotoxic CD8+ cell response.
Recently, studies with chondroitin sulfate proteoglycan-4 (CSPG4), which is an
early cell surface progression marker involved in tumor cell proliferation, migration,
and invasion, were initiated. hCSPG4 DNA-based vaccine in combination with
electroporation was used in the treatment of canine malignant melanoma which,
due to its similarity to human malignant melanoma, represents a valuable transla-
tional model. Vaccination resulted in prolonged disease-free survival with lower
recurrence of lung metastatic rate (Piras et al. 2016).
Conclusions
In conclusion, it can be affirmed that predictable models are translational animals

which allow to reliably transfer to bedside the findings obtained in preclinical
studies. According to these properties, they are valuable for basic carcinogenesis
studies and to study basic mechanisms of interaction between cancer cells and the
tumor microenvironment, to study the basic mechanisms of metastasis development,
as well as immune response to tumors. They also help in drug discovery and in the
optimization of therapeutic strategies in a defined and flexible setting which is often
not feasible within individual patients.
Cross-References

▶ Gene Transfer to the Skin
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Antiangiogenic Gene Therapy
84
Maja Čemažar, Tanja Dolinsek, Masa Bosnjak, and Gregor Serša
Abstract
Angiogenesis is one of the crucial steps in tumor development and progression, as
the formation of a blood vessel network within the tumor enables the tumor to
grow and allows tumor cells to enter the blood stream and form distant metasta-
ses. Signaling molecules consist mainly of soluble ligands and their receptors on
endothelial cells, and the most representative activators are vascular endothelial
growth factor (VEGF), transforming growth factor beta (TGF-β), platelet-derived
growth factor (PDGF), and basic fibroblast growth factor (bFGF/FGF2).
Targeting of angiogenesis activators or the delivery of inhibitors by gene
electrotransfer is a promising approach for cancer therapy. Gene electrotransfer
is based on electroporation, a physical method, which causes a transient increase
in cell membrane permeability due to the application of electric pulses and thus
enables the transport of large molecules into cells. Gene electrotransfer was
already used to deliver antiangiogenic plasmids or small interfering molecules
(siRNA) into cells, targeting different molecular targets involved in angiogenesis,
including VEGF pathway, TGF-β and endoglin pathway, integrins, FGF2, and
others. Gene electrotransfer of plasmids encoding different antiangiogenic mol-
ecules has been proven to be safe, feasible, and effective. Various in vitro and
in vivo studies demonstrated its great potential for further research. This approach
could easily be implemented into everyday clinical practice. For ensuring safer
gene electrotransfer in clinical practice, the use of tissue-specific plasmids,
without antibiotic resistance gene, would be preferred.
M. Čemažar (*)
T. Dolinsek • M. Bosnjak • G. Serša
e-mail: tdolinsek@onko-i.si; mbosnjak@onko-i.si; gsersa@onko-i.si

DOI 10.1007/978-3-319-32886-7_51
1624 M. Čemažar et al.
Keywords
Gene electrotransfer • Antiangiogenic • Gene therapy • Electroporation
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1624
Vascular Endothelial Growth Factor Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1625
Endoglin and Transforming Growth Factor-Beta Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1627
Interleukin-12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1631
Integrins and Plasmid Antiangiogenic Metargidin Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1631
Fibroblast Growth Factor 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1633
Melanoma Cell Adhesion Molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1633
Endostatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1634
Plasminogen Fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1634
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1635
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1635
Introduction
One of the most important events in tumor formation and progression is the
formation of a blood vessel network within the tumor, which is formed by the
process of angiogenesis. It is initiated when the tumor size reaches 1–3 mm and
the simple diffusion of oxygen and nutrients is not sufficient enough to fill the
nutritional needs of all cells constituting the tumor mass. The interface of this
avascular and vascular phase is called angiogenic switch, which marks the initiation
of angiogenesis and ensures exponential tumor growth (Folkman 2007). The newly
formed tumor blood vessels supply tumor cells with oxygen and nutrients and also
remove metabolites from the tumor. Furthermore, tumor blood vessels also enable
tumor cells to enter the bloodstream and form distant metastasis. Therefore, angio-
genesis is a crucial event and hallmark in tumor growth and progression (Figg 2008).
The growth of the tumor vasculature is triggered by signals that are released from
the tumor, which cause sprouting and growth of new blood vessels into the tumor,
from existing blood vessels surrounding the tumor. This process, angiogenesis, is
regulated by a variety of signaling molecules, which act as activators or inhibitors.
When angiogenesis in the tissue is not needed, the concentrations of inhibitors are
high and vice versa; when angiogenesis is needed (during embryogenesis, wound
healing, tumor growth, etc.), the concentrations of angiogenesis activators are
higher. Signaling molecules consist mainly of soluble ligands and their receptors
on endothelial cells, and the most representative activators are vascular endothelial
growth factor (VEGF), transforming growth factor beta (TGF-β), platelet-derived
growth factor (PDGF), and basic fibroblast growth factor (bFGF/FGF2). Among
inhibitors, the most representing ones are angiopoietins 1 and 2, endostatin,
vasostatin, interleukin-12, and others. Integrins and adhesion receptors are also
essential in the regulation of angiogenesis. They are overexpressed on endothelial
cell surface and maintain the viability of endothelial cells during detachment from
84 Antiangiogenic Gene Therapy 1625
extracellular matrix and migration toward the tumor during sprouting of new blood
vessels (Avraamides et al. 2008). Furthermore, matrix metalloproteinases are also
important players in angiogenesis regulation as they are released from stimulated
endothelial cells and cleave the pro-angiogenic factors, which are stored in the
extracellular matrix (Folkman 2007).
Angiogenesis is an attractive target for cancer therapy. The antiangiogenic
approach is based on the fact that it prevents the growth of tumor blood vessels
and thus deprives cancer cells of oxygen and nutrients, hence causing a cascade of
cell death. In addition, it is also known that antiangiogenic therapies can also cause
disruption of existing tumor blood vessels. Antiangiogenic therapy targets main
activators of angiogenesis with different approaches. Several antiangiogenic inhib-
itors were developed, which were used in clinical trials, and many have been
approved for treatment of cancers of different histotypes, alone or in combination
with other cytotoxic therapeutic modalities. VEGF is the main target for
antiangiogenic therapy, and three major classes were developed, monoclonal anti-
bodies, VEGF decoy receptors, and small-molecule tyrosine kinase inhibitors
(TKIs), which are being used in clinical setting (Al-Husein et al. 2012).
In preclinical studies, other approaches to target tumor blood vessels are being
developed. One of them is the use of gene electrotransfer. Gene electrotransfer is
based on electroporation, a physical method, which causes a transient increase in cell
membrane permeability due to the application of electric pulses and thus enables the
transport of large molecules into cells. Gene electrotransfer has already been used to
deliver antiangiogenic plasmids or small interfering molecules (siRNA) into cells,
targeting different molecular targets involved in angiogenesis, including VEGF
pathway, TGF-β and endoglin pathway, integrins, and others, which will be
addressed in this chapter.
Vascular Endothelial Growth Factor Pathway
VEGF, also known as vascular permeability factor (VPF), is a potent mitogen for
vascular endothelial cells. It induces angiogenesis, stimulates growth and prolifera-
tion of endothelial cells, increases survival and prevents apoptosis of endothelial
cells, and regulates vascular permeability. VEGF is a secreted glycoprotein, whose
expression is induced predominantly by the decrease of oxygen in the tissues or
hypoxia, which causes the activation of transcription factors named hypoxia-
inducible factors-1 (HIF-1). Other VEGF-inducing agents are soluble mitogens
like TGF-α and TGF-β, epidermal growth factor (EGF), FGF, PDGF, and insulin-
like growth factor 1 (IGF-1). VEGF family members include VEGF A, VEGF B,
VEGF C, VEGF D, VEGF E, and placental growth factor (PIGF), with VEGF A
showing predominant activity on vascular endothelial cells and therefore being
involved in tumor angiogenesis. VEGF receptors are tyrosine kinase receptors
VEGF R1, VEGF R2, and VEGF R3, which are transmembrane proteins. They
can be membrane-bound (mbVEGFR) or soluble (sVEGFR) receptors. VEGF R2 is
located on endothelial cells and is the main receptor for the angiogenic effects of
VEGF (Yadav et al. 2015).
Gene electrotransfer was used to silence VEGF with plasmid vectors encoding
short hairpin RNA (shRNA) molecules against mouse VEGF A and VEGF C in
mouse mammary cancer model. ShRNA molecules are artificial RNA molecules
containing a hairpin turn. These molecules can be used to silence target gene
expression by destruction of mRNA molecules via RNA interference. In this pro-
cess, an effector siRNA molecule is generated from shRNA by the cleavage of the
hairpin structure, and this siRNA then binds to target complementary mRNA and
causes enzyme-directed cleavage of mRNA. In this way, VEGF A and VEGF C
synthesis was prevented in the study of Shibata et al. (2008) using gene
electrotransfer of plasmids encoding shRNA against VEGF A and VEGF
C. VEGF A exerts a crucial role in tumor angiogenesis by stimulating endothelial
cells to proliferate. VEGF C plays a central part in lymphangiogenesis, and tumor
cell-derived VEGF C is believed to increase metastasis to lymph nodes. Vectors
encoding short hairpin RNA (shRNA) molecules against mouse VEGF A, VEGF C,
or the combination of the two were injected intratumorally once a week for 8 weeks
in the amount of 50–75 μg, depending on the tumor size. After injection, gene
electrotransfer was performed by forceps platinum electrodes with electrical param-
eters 8 square-wave pulses, 100 V, and 20 ms. The treatment was started on small
tumors at the time of angiogenic switch. This is the appropriate time of treatment for
testing the antiangiogenic action, since angiogenesis and lymphangiogenesis are in
full swing at that time. Furthermore, a repetitive approach was chosen to overcome
the weaknesses of RNA interference mechanism, which is its transient nature of
action. Gene electrotransfer of the shRNA expression vectors against VEGF A,
VEGF C, or the combination of the two effectively reduced growth of tumors. Either
of the therapies, anti-VEGF A or anti-VEGF C alone, reduced the occurrence of the
lymph node metastases, while a combination therapy was needed to inhibit lung
metastases. Therefore, gene electrotransfer using RNA interference demonstrated to
be a novel and safe treatment modality to target VEGF-driven tumor angiogenesis
and lymphangiogenesis.
RNA interference mechanism was utilized also for antiangiogenic gene
electrotransfer in the study of Vader et al. targeting Rac1 (Vader et al. 2011). Rac
1 is the Rho GTPase, which functions as molecular switch regulating VEGF-
mediated angiogenesis. It cycles between a guanine diphosphate (GDP)-bound
inactive state and guanine triphosphate (GTP) active state. In the active GTP state,
it binds the effector proteins and transduces the signal downstream from VEGF
receptors toward the nucleus by activating transcription factors and in this manner
regulates several cellular processes including endothelial cell proliferation, migra-
tion cell cycle control, and lumen formation. Gene electrotransfer was used to silence
Rac1 expression by siRNA in vitro and in vivo. Gene electrotransfer in vitro was
performed on human umbilical vein endothelial cells (HUVEC) in 4 mm gap
electroporation cuvettes at 180 V and 70 ms with 700 pmol of siRNA. In vivo,
subcutaneous mouse brain neuroblastoma tumors Neuro2a were electroporated
(200 V/cm, 100 ms, 2 2 pulses at perpendicular directions) in the presence of
10 μg of anti-Rac1 siRNA. In this study, slightly larger tumors were treated

(100 mm3), and repetitive approach was also chosen with treatment repeated four
times every second day (on days 0, 2, 4, and 6). The treatment of HUVEC reduced
proliferation, migration, and invasion of cells and also inhibited tube formation,
indicating an antiangiogenic action of anti-VEGF gene electrotransfer approach.
This was confirmed also in vivo where antiangiogenic and antitumor effectiveness
was demonstrated by Matrigel plug assay, tumor growth delay, and histological
analysis. Once again, gene electrotransfer as delivery system demonstrated promis-
ing local therapeutic strategy for antiangiogenic therapy of tumors.
Besides RNA interference strategy, another therapeutic approach for VEGF
targeting was established using gene electrotransfer. Soluble variant of VEGF R1
(known as sFlt1) was used as VEGF trap (Verrax et al. 2011). Flt1 is considered to be
a decoy receptor for VEGF due to its weak tyrosine kinase activity (and therefore
inability to activate the receptor complex) and higher affinity for VEGF binding
compared to VEGF R2. Plasmid encoding sFlt1 protein was transfected into skeletal
muscle in mice using gene electrotransfer to enable the production and consequent
release of the therapeutic protein into the blood stream of tumor-bearing mice. The
sFlt-1 encoding plasmid was injected into the mouse tibial cranial muscle of the
posterior leg in the amount of 40 μg, and thereafter gene electrotransfer was
performed using two parallel stainless-steel plate electrodes with 4 mm gap, and
8 square-wave electric pulses were delivered (200 V/cm, 20 ms, 2 Hz). The treatment
produced high amounts of functional protein in muscles and significantly reduced
growth of transplantable liver tumor (TLT) hepatocarcinoma grown in contralateral
muscle. Therefore, gene electrotransfer can be also used for interesting treatment
modality that is based on the overexpression of negative regulators of angiogenesis.
In summary, antiangiogenic gene electrotransfer targeting VEGF was demon-
strated to be feasible and effective and may be of high clinical significance for the
treatment of cancer. Additionally, it can also represent an alternative to anti-VEGF
therapies currently used in the clinic.
Endoglin and Transforming Growth Factor-Beta Pathway
Endoglin is a transmembrane homodimeric glycoprotein present in the cell mem-

brane. Endoglin expression is increased in proliferating vascular endothelial cells in
tissues undergoing angiogenesis; therefore, it is expressed during embryogenesis,
inflammation, and wound healing and also within as well as surrounding the tumors.
It is a TGF-β co-receptor, which participates in the activation of a multifaceted
signaling pathway, which mediates endothelial cell proliferation, migration, and
adhesion. It is categorized as TGF-β type III receptor, which, in contrast to type I
and II receptors, lacks kinase activity and is therefore a regulator of binding and
signaling through type I and II receptors. Endoglin signaling is initiated by the
binding of TGF-β ligand and results in a series of phosphorylation steps leading to
transcriptional activity that causes activation of endothelial cells and thus promoting
angiogenesis (Nassiri et al. 2011). Systemic anti-endoglin treatment with antibodies
was demonstrated effective in several preclinical studies, and subsequently anti-

endoglin antibody TRC105 is undergoing several clinical trials for the treatment of
advanced cancer as single agent or in combination with VEGF inhibitors (Rosen
et al. 2014).
Gene electrotransfer provided local approach for endoglin targeting with the
delivery of siRNA or plasmids encoding shRNA against endoglin. For the siRNA
gene electrotransfer, 800 pmol of siRNA molecules was injected intratumorally, and
immediately thereafter 8 square-wave electric pulses were delivered through two
parallel stainless-steel electrodes (600 V/cm, 5 ms, 1 Hz). The treatment was
conducted on smaller TS/A mouse adenocarcinoma tumors with a diameter of
3 mm, which do not express endoglin; therefore, anti-vascular effect of the therapy
could be followed due to the effect of the therapy only on vascular endothelial cells.
Gene electrotransfer was performed once or three times, and antitumor effectiveness
of these approaches were compared. Repetitive approach was more effective; a
significant tumor growth delay was observed in the couple of days after the begin-
ning of the therapy, and also there was a reduction in the number of blood vessels
(Dolinsek et al. 2013). The results were encouraging however; the treatment was not
very effective due to instability and short half-life of siRNA molecules inside the
cells.
Therefore, to provide a longer-lasting effect and to increase the effectiveness of
the treatment, a plasmid encoding shRNA with the same sequence as siRNA under
the control of constitutive U6 promoter was constructed (Dolinsek et al. 2015a). For
gene electrotransfer in vitro, 10 μg of plasmid was electroporated into 2H11 mouse
endothelial cells (2 mm plate stainless-steel electrodes, 8 square-wave pulses,
600 V/cm, 5 ms, 1 Hz). The treatment reduced proliferation, migration, invasion,
and tube formation but increased adhesion of endothelial cells, demonstrating
altogether the antiangiogenic effect of the gene electrotransfer against endoglin
in vitro. The treatment was further investigated in vivo on TS/A tumor model (the
same as with siRNA molecules) as repetitive treatment. Gene electrotransfer was
conducted three times on days 0, 2, and 4 with intratumoral injection of 50 μg of the
plasmid followed by the application of electric pulses (plate stainless-steel elec-
trodes, 8 square-wave pulses, 600 V/cm, 5 ms, 1 Hz) 10 min after the injection. After
the treatment, a significant antitumor effectiveness was demonstrated by tumor
growth delay, decreased number of blood vessels, and increased necrosis of tumors,
confirming the antiangiogenic effect of the treatment. The treatment of tumors was
more effective in the case of plasmid compared to siRNA molecules; therefore, gene
electrotransfer of plasmid should be utilized for antiangiogenic gene therapy rather
than siRNA molecules.
The most intriguing results of anti-endoglin gene electrotransfer came from
intravital microscopy of tumors growing in dorsal window chamber model in
mice, where the evidence of both antiangiogenic and vascular disrupting effective-
ness of the treatment was demonstrated. Dorsal window chamber or dorsal skinfold
chamber is an experimental technique performed in living animals, which enables
the visualization and assessment of blood vessel network by intravital microscopy.
For this purpose, two titanium frames are secured to the double layers of the dorsal
skinfold in mouse with stainless-steel screws and silk sutures, and thereafter one
layer of the skin with the fat and connective tissues is excised, exposing the
vasculature of the remaining skin layer. The TS/A tumors were induced in the
remaining skin layer by injection of dense cell suspension. Tumors were treated
with gene electrotransfer of 20 μg of plasmid encoding shRNA against endoglin
(4 mm plate electrodes, 8 square-wave pulses, 600 V/cm, 5 ms, 1 Hz) at the time of
angiogenic switch or later when the blood vessels were already formed. In control
groups, where the tumors were treated by injection of water, the blood vessels were
growing into the tumor forming chaotic vascular network. However, the treatment
showed a quick onset of the vascular effects beginning 1 day after gene
electrotransfer. No new tumor blood vessels were formed after gene electrotransfer
of plasmid encoding shRNA against endoglin, when the tumors were treated at the
onset of angiogenic switch. The destruction of blood vessels was observed with
hemorrhagic areas at sites where the capillaries with activated endothelial cells were
damaged. These vascular disrupting effects were observed when the treatment was
performed on tumors with established blood vessel network. Therefore, both
antiangiogenic and vascular disrupting action of gene electrotransfer of anti-
endoglin plasmid were demonstrated (Fig. 1).
These studies were all conducted in endoglin non-expressing tumor model TS/A,
which enabled determination of vascular targeted action of anti-endoglin gene
electrotransfer. In endoglin-expressing mouse melanoma models B16F1 and
B16F10, increased therapeutic effect of anti-endoglin gene electrotransfer was
observed compared to TS/A tumor model, which was contributed to simultaneous
action of vascular targeted effect and direct effect on tumor cells (Dolinsek
et al. 2015b).
To achieve higher specificity and safety of the anti-endoglin gene electrotransfer
treatment, a plasmid with tissue-specific promoter for endothelial cells endothelin-1
(ET) was constructed (Tesic et al. 2015). Changes in the properties of endothelial
cells involved in angiogenesis were confirmed in vitro as gene electrotransfer of
plasmid (10 μg of plasmid, 2 mm plate stainless-steel electrodes, 8 square-wave
pulses, 500 and 600 V/cm, 5 ms, 1 Hz), reduced cell proliferation, migration,
invasion of mouse endothelial cells SVEC4-10, and tube formation of SVEC4-10
and 2H11 endothelial cells (Stimac et al. 2015; Tesic et al. 2015). The antitumor
effectiveness of repetitive intratumoral gene electrotransfer (three times of 50 μg of
plasmid, 8 square-wave pulses, 600 V/cm, 5 ms, 1 Hz) was confirmed on TS/A
mouse mammary adenocarcinoma model in both small avascular tumors and bigger
well-vascularized tumors with better effectiveness on smaller tumors. Furthermore, a
decreased number of blood vessels were observed, confirming the antiangiogenic
effect of gene electrotransfer (Stimac et al. 2015). The antiangiogenic action of gene
electrotransfer of the same plasmid was also confirmed in another tumor model of
B16F10-luc mouse melanoma (two times of 50 μg, 8 square-wave pulses, 600 V/cm,
5 ms, 1 Hz). In that study decreased number of blood vessels and prolonged tumor
growth delay was achieved after repetitive treatment (Tesic et al. 2015).
Fig. 1 Antiangiogenic and vascular disrupting effect of gene electrotransfer of plasmid encoding
shRNA against endoglin on TS/A mouse mammary adenocarcinoma model in dorsal window
chamber model at the time of angiogenic switch (antiangiogenic effect) and when the blood vessels
were already formed (vascular disrupting effect)
The plasmid with tissue-specific promoter for endothelin-1 was also tested in
combination with radiotherapy on TS/A tumors. Gene electrotransfer was performed
three times (50 μg of plasmid, 8 square-wave pulses, 600 V/cm, 5 ms, 1 Hz), and
tumors were irradiated with single dose of 15 Gy or split dose (3 5 Gy). The
pronounced radiosensitization was demonstrated, which was mainly attributed to
anti-vascular effect of gene electrotransfer, which was also confirmed by histological
analysis of blood vessel number (Stimac et al. 2016).
In summary, gene electrotransfer of plasmids encoding shRNA against endoglin
demonstrated to be a safe and efficient approach to target tumor vasculature, which
could be used instead of or in combination with current anti-VEGF therapies.
Interleukin-12
Interleukin-12 (IL-12) is a soluble cytokine, which is naturally produced by phago-

cytes and dendritic cells in response to antigenic stimulation. It is a heterodimeric
protein, composed of two subunits, which are covalently linked by a disulfide bridge.
Interleukin-12 induces the production of interferon-gamma (IFN-γ), increases activ-
ity of natural killer cells and cytotoxic T lymphocytes, and drives the differentiation
of naïve helper T cells to Th1 cells. This is one of the main mechanisms of IL-12
antitumor effectiveness. Furthermore, IL-12 has also antiangiogenic action, which is
mediated by the production of IFN-γ that increases the production of interferon-
inducible protein-10 (IP-10) and monokine Mig that both mediate the antiangiogenic
effect (Cemazar et al. 2010).
Gene electrotransfer was used to deliver a plasmid encoding IL-12 for the
treatment of melanoma B16F10 tumors in mice. Intratumoral and intramuscular
gene electrotransfer was compared. For intratumoral administration a circular appli-
cator containing six penetrating electrodes was used to deliver 50 μg of plasmid (six
rotating pulses, 1500 V/cm, 99 μs, 1 Hz), and for intramuscular administration an
applicator containing four penetrating electrodes in rectangular pattern was used to
deliver 50 μg of plasmid (12 pulses, 100 V/cm, 20 ms, 1 Hz). Based on immuno-
histochemical staining of CD31 antigen, only i.t. gene electrotransfer significantly
reduced blood vessel number, thus demonstrating antiangiogenic effect of
intratumoral IL-12 gene electrotransfer (Lucas et al. 2002).
Another study was conducted comparing intratumoral and intramuscular gene
electrotransfer of plasmid encoding IL-12 in murine squamous cell carcinoma
SCCVII (Li et al. 2005). Repetitive gene electrotransfer of 10 μg of plasmid was
performed with two pulses of 450 V/cm, 20 ms for intratumoral administration, and
two pulses of 350 V/cm, 20 ms for intramuscular administration. Blood vessel
density was reduced in both types of applications to similar levels, and morpholog-
ical changes were observed in blood vessels, demonstrating antiangiogenic effec-
tiveness of both types of IL-12 gene electrotransfer.
Therefore, the mechanism of antitumor effectiveness of gene electrotransfer of
IL-12 includes antiangiogenic component, which probably contributes extensively
to the success of the treatment.
Integrins and Plasmid Antiangiogenic Metargidin Peptide
Other regulators of tumor angiogenesis are integrins, transmembrane cell receptors

regulating intracellular signaling, and expression of genes involved in cell differen-
tiation, proliferation, adhesion, migration, and survival. Among integrins, integrin
αvβ3 plays a very important role in the process of angiogenesis. It is barely
expressed on normal, resting blood vessels but significantly upregulated in human
tumor vessels (Brooks et al. 1994). Another very important integrin which was found
to be overexpressed in blood vessels of human and mouse tumors is α5β1 integrin.
Cross-talk between different integrins has already been shown, and also for integrin
α5β, it was proven, in vitro and in vivo, that it regulates the functions of integrin
αvβ3.
Plasmid AMEPTM (Onxeo) is a plasmid DNA devoid of any antibiotic resistance
gene, encoding disintegrin domain of ADAM-15 (antiangiogenic metargidin
peptide – AMEP). Besides αvβ3 it binds also α5β1 integrins and thus has dual,
antiangiogenic, and antitumor effect. Properties of endothelial cells involved in
angiogenesis after gene electrotransfer of plasmid AMEP in vitro (two parallel
stainless-steel electrodes, 8 square-wave pulses, 600 V/cm, 5 ms, 1 Hz) were tested
in two different human endothelial cell lines, human microvascular endothelial cells
(HMEC-1) and human umbilical vein endothelial cells (HUVEC). The results of the
study showed that gene electrotransfer of plasmid AMEP reduced cell proliferation,
migration, and invasion of both cell lines. Furthermore, the effect on biological
response of endothelial cells correlated well with the integrin quantity on the cells,
but not with the expression level of AMEP mRNA. This finding indicates that
quantity of integrins is the limiting factor for antiproliferative and antimigratory
effects of plasmid AMEP therapy. The adhesion of those cells was not affected at all
after plasmid AMEP gene electrotransfer indicating that probably some other
integrins took over the αvβ3 anchoring role. Furthermore, the formation of
capillary-like structures in vitro was more abrogated in HMEC-1 than in HUVEC,
most probably due to the fact that HMEC-1 are better approximation to tumor
endothelial cells than HUVEC. In contrast to antiproliferative and antimigratory
effect, decrease in the formation of capillary-like structure depends on AMEP
mRNA levels in cell lines. This indicates another mechanism which might be
involved in the process of integrin-mediated angiogenesis (Bosnjak et al. 2013).
The antiangiogenic potential of gene electrotransfer with plasmid AMEP was
further characterized in vivo in murine melanoma B16F10 model. Plasmid AMEP
(100 μg on day 0 and day 2) was injected intratumorally, and 10 min after 8 square-
wave pulses, with amplitude over distance ratio 600 V/cm and pulse length 5 ms,
were applied. First, the antiangiogenic effects were proven on immunohistologically
stained tumor sections. Staining for CD31 endothelial marker demonstrated a strong
reduction of the number and the size of tumor blood vessels. Intravital microscopy
demonstrated complete inhibition of tumor blood vessel formation in melanoma
tumors, growing in dorsal window chamber model, after gene electrotransfer of
plasmid AMEP. In contrary, the control tumors continuously formed a chaotic
vascular network which enabled tumor growth. FITC-labeled dextrans were used
to display functional blood vessels in untreated tumors and abrogated blood flow in
treated tumors (Bosnjak et al. 2015).
Noncommercial equivalent of plasmid AMEP, a plasmid encoding recombinant
disintegrin domain (pRDD), was tested also in combination with radiotherapy. Five
minutes after intratumoral injection of 200 μg pRDD, 1 high voltage pulse
(1250 V/cm, 100 μs) and 1 low voltage pulse (400 V/cm, 400 ms) were applied
with 1000 ms lag in between. Gene electrotransfer of pRDD increased oxygenation
of melanoma tumors by decreasing local oxygen consumption. Therefore, at the time
of maximal oxygenation, combined therapy with irradiation showed improved
effects on tumor growth delay (Crokart et al. 2013). Previous study confirmed that
gene electrotransfer of pRDD in murine melanoma B16F10 model has
antiangiogenic effectiveness and thus can also reduce metastasis development
(Daugimont et al. 2011).
Finally, a clinical trial testing plasmid AMEP was conducted, showing safety of
intratumoral gene electrotransfer with plasmid AMEP in advanced melanoma
patients. Due to difficulties with the recruitment of the patients because of the
availability of new, more efficient drugs, the study was prematurely terminated
(Spanggaard et al. 2013).
Fibroblast Growth Factor 2
Fibroblast growth factor 2 (FGF-2) is one of the most important factors among the
growth factors involved in the process of angiogenesis. It can act as an endogenous
factor, through cell surface receptors and their signaling pathways, or as an exoge-
nous factor, through translocation to the nucleus in late G1, where it can play a role
in replication and/or in transcription of rDNA (Bossard et al. 2004).
Based on previous knowledge, Bossard and colleagues conducted a study with an
aim to counteract the angiogenic properties of FGF-2 (Bossard et al. 2004). They
first isolated recombinant fragment containing the type III domains 12–14 of fibro-
nectin and called it fibstatin. It is an endogenous human basement membrane-
derived inhibitor of angiogenesis. To prove its antiangiogenic activity, gene
electrotransfer of fibstatin-encoding plasmid DNA was performed into quadriceps
of C57Bl/6 mice, previously inoculated with B16F10 cells. Gene electrotransfer of
plasmid (50 μg) was performed with 10-mm-diameter caliper electrodes set 4 mm
apart, delivering 8 pulses of 80 V and 20 ms. Five days later gene electrotransfer was
repeated.
Gene electrotransfer of fibstatin-encoding plasmid DNA inhibited tumor growth
by 50%. Immunohistological staining of tumor sections with CD31 antibodies
demonstrated significant decrease in CD31-positive cells in treated group. Taken
together, the study demonstrated antiangiogenic effect of fibstatin-encoding plasmid
DNA after gene electrotransfer in B16F10 melanoma immunocompetent mouse
model.
Melanoma Cell Adhesion Molecule
Melanoma cell adhesion molecule (MCAM/CD146/MUC18) is a multifunctional

transmembrane glycoprotein that is involved in the development of melanoma and
also angiogenesis. It is highly expressed in cells, which constitute the blood vessel
wall, including endothelial cells, smooth muscle cells, and pericytes, and promotes
the formation of new blood vessels.
To explore the antiangiogenic action of MCAM silencing, gene electrotransfer

was used to deliver a plasmid encoding shRNA against mouse MCAM. The
antiangiogenic action of the treatment was demonstrated in vitro, as proliferation
and tube formation of 2H11 mouse endothelial cells were reduced after gene
electrotransfer (8 pulses, 600 V/cm, 5 ms, 1 Hz); furthermore, significant tumor
growth delay of B16F10 mouse melanoma was observed after intratumoral injection
of 15 μg of plasmid in 40 μl of water and repeated (three times) gene electrotransfer
(stainless-steel plate electrodes, 8 square-wave pulses, 600 V/cm, 5 ms, 1 Hz) in vivo
(Prosen et al. 2014).
Endostatin
Endostatin is a product of proteolytic cleavage of collagen XVIII. It inhibits the

proliferation and organization of endothelial cells into new blood vessels. Endostatin
may also interfere with fibroblast growth factor and vascular endothelial growth
factor.
Antiangiogenic activity of a plasmid encoding endostatin after gene
electrotransfer was demonstrated in renal adenocarcinoma tumors (Renca cells).
Gene electrotransfer was performed on tumors 5–6 mm in diameter, every second
day, from day 10 until day 30 with 10 μg of plasmid injected intratumorally. The
electrodes used were 3 mm caliper electrodes and the parameters of electric pulses
were 200 V and 100 ms. A strong antitumor activity was observed in the group
treated with endostatin-encoding plasmid after gene electrotransfer. Furthermore,
antimetastatic activity of the same plasmid was proven after muscle gene
electrotransfer. After intravenous injection of B16F10 melanoma cells into the tail
vein, gene electrotransfer into the muscle was performed on day 4 and 7. Three
weeks later, the mice were sacrificed and the lungs examined for the metastatic foci.
There was more than 50% decrease in the number of lung metastases in treated mice.
Endostatin levels in peripheral blood were markedly higher (Cichon et al. 2002).
Plasminogen Fragments
Plasminogen is a zymogen from which an active protein plasmin is generated by

proteolysis. Proteolytic fragment derived from plasminogen is called KI-5, which
suppresses endothelial cell growth, angiogenesis, and tumor growth. Antiangiogenic
action of KI-5 was demonstrated by gene electrotransfer (stainless-steel plate
electrodes, 8 square-wave pulses, 270 V/cm, 20 ms, 1 Hz) of KI-5 encoding
plasmid into skeletal muscle in mice with the systemic release of the KI-5 into
the blood stream. The treatment inhibited the growth of MDA-MB-231 human
mammary adenocarcinoma tumor and reduced the microvessel density in the
tumor tissue immunohistochemically stained with anti-CD31 antibodies, demon-
strating antiangiogenic effect of gene electrotransfer of KI-5 expressing plasmid
(Martel-Renoir et al. 2003).
Conclusions
Gene electrotransfer of plasmids encoding different antiangiogenic molecules has

already been proven to be safe, feasible, and effective. Various in vitro and in vivo
studies demonstrated its potential for further research. This approach could easily be
implemented into everyday clinical practice. For ensuring safer gene electrotransfer
in clinical practice, the use of tissue-specific plasmids, without antibiotic resistance
gene, would be preferred.
Cross-References

Electrochemotherapy
Tissue Blood Flow
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New Drugs for Electrochemotherapy
with Emphasis on Calcium Electroporation 85
Stine Krog Frandsen, Hanne Falk Hansen, and Julie Gehl
Abstract
Electrochemotherapy is an anticancer treatment inducing uptake of chemothera-
peutics by electroporation. This treatment is increasingly being used clinically
and further investigations for improvement of the treatment and discovery of new
drugs are ongoing. The most commonly used drugs in electrochemotherapy are
bleomycin and cisplatin, but many other chemotherapeutic drugs have been tested
in vitro and in vivo. Drugs with an anticancer effect such as radioactive com-
pounds and calcium have also been tested in combination with electroporation.
Different aspects have to be considered when investigating possible new drugs in
combination with electroporation, such as the properties of the drug and whether
electroporation can increase the uptake of the drug, as well as the effect of the
treatment on normal cells and tissue. In this chapter some of the drugs tested in
combination with electroporation are described followed by a broader description
of a new drug that has recently been investigated, calcium. Here we describe the
process from idea over in vitro and in vivo experiments to clinical trial. The
results showed that calcium electroporation is effective in inducing cell death
in vitro and tumor necrosis in vivo, and very interestingly it has also been shown
that normal cells are less sensitive to the treatment than cancer cells. The effect of
this potential novel anticancer treatment is also investigated in a clinical trial for
treatment of cutaneous metastases, and calcium could be a new drug for
electroporation-based therapy.
S.K. Frandsen • H.F. Hansen • J. Gehl (*)

Center for Experimental Drug and Gene Electrotransfer (C*EDGE), Department of Oncology,
Herlev and Gentofte Hospital, University of Copenhagen, Herlev, Denmark
e-mail: Stine.Krog.Frandsen@regionh.dk; hanne.falk.hansen01@regionh.dk; Karen.Julie.
Gehl@regionh.dk

DOI 10.1007/978-3-319-32886-7_53
1638 S.K. Frandsen et al.
Keywords
Electrochemotherapy • Cytotoxicity • Hydrophilic • Lipophilic • Calcium elec-
troporation • In vitro • In vivo • Clinical trial
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1638
Drugs Investigated for Use with Electroporation in Cancer Treatment . . . . . . . . . . . . . . . . . . . . . . . 1639
Calcium Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1641
Effect In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1642
Effect In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1642
Effect on Normal Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1643
Mechanisms of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1644
Clinical Trial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1647
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1648
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1648
Introduction
Electrochemotherapy, where a chemotherapeutic drug is internalized into cells by

electroporation, is used clinically as an anticancer treatment in more than 140 cen-
ters in Europe. The chemotherapeutic drugs bleomycin and cisplatin are the most
commonly used drugs for electrochemotherapy but new drugs are being tested.
When testing new drugs for electrochemotherapy, some aspects should be consid-
ered, including the properties of the drug, whether it is lipophilic, amphiphilic, or
hydrophilic. Lipophilic drugs can easily migrate across the membrane due to the
properties of the plasma membrane, which is why the increased permeability of the
plasma membrane induced by electroporation does not further increase the uptake
of these drugs. Thus, the cytotoxicity of lipophilic drugs is often not increased
when combined with electroporation. Amphiphilic drugs, which possess both
lipophilic and hydrophilic properties, can also migrate across the membrane but
equilibrium is reached slower than with lipophilic drugs. A study on uptake of
daunorubicin and doxorubicin showed equilibrium after 30 and 60 min, respec-
tively. When testing amphiphilic drugs, the time of incubation before electropora-
tion affects if, or how much, drug uptake is increased. Thus, at long incubation
time, the drugs will cross the membrane before electroporation, and the uptake will
not increase further by electroporation. It is of course important to choose an
incubation time that relates to the clinical use of these drugs (the half-life of the
drugs). The highest increased uptake and thereby cytotoxicity are often seen when
testing hydrophilic drugs in combination with electroporation. Hydrophilic drugs
only cross the membrane limitedly; thus, an increase in membrane permeability
induced by electroporation will often increase the uptake and thereby the cytotox-
icity of these drugs.
85 New Drugs for Electrochemotherapy with Emphasis on Calcium Electroporation 1639
Drugs Investigated for Use with Electroporation in Cancer

Treatment
Several drugs have been tested in combination with electroporation, both lipophilic,
amphiphilic, and hydrophilic drugs. When testing new drugs in combination with
electroporation, one of the most important aspects is the increase in cytotoxicity
caused by the increased uptake of the drug. Thus, the properties of the drugs tested
are important since uptake of drugs that easily migrate across the membrane is not
further increased after electroporation unlike drugs with a reduced ability to cross the
plasma membrane. Below, some of these drugs and their ratio of increased cytotox-
icity are described as well as in Table 1.
Several chemotherapeutic drugs have been tested in combination with electropo-
ration including the lipophilic chemotherapeutic drugs etoposide and paclitaxel.
These drugs can easily cross the plasma membrane; thus, when tested in combina-
tion with electroporation, the cytotoxicity (measured on IC50 – inhibitory concen-
tration 50%) of these drugs was similar with and without electroporation (Gehl
et al. 1998; Jaroszeski et al. 2000). Amphiphilic chemotherapeutic drugs, that also
cross the plasma membrane although slower than lipophilic drugs, have also been
tested in combination with electroporation. Mitomycin C was tested in vitro with no
or limited increase in cytotoxicity when combined with electroporation (Jaroszeski
Table 1 Change in cytotoxicity of drugs in cells treated with or without electroporation

in vitro. Shown as ratio of IC50 (inhibitory concentration 50%) for drug alone to IC50 for drug in
combination with electroporation. Drugs are divided in lipophilic, amphiphilic, and hydrophilic.
ND, not determined, because IC50 for drug alone as well as for drug combined with electroporation
was not reached with the concentrations tested. >, IC50 for drug alone was not reached with the
concentrations tested, but IC50 for the drug in combination with electroporation was found
Lipophilic Amphiphilic Hydrophilic References
Etoposide, (Gehl et al. 1998; Jaroszeski et al. 2000)
ND-1
Paclitaxel, (Gehl et al. 1998; Jaroszeski et al. 2000)
ND-1.3 Mitomycin C, (Jaroszeski et al. 2000; Vasquez et al. 2015)
ND-1.4
Doxorubicin, (Gehl et al. 1998; Jaroszeski et al. 2000)
0.67–2
Bleomycin, (Orlowski et al. 1988; Gehl et al. 1998;
100–5000 Jaroszeski et al. 2000)
Cisplatin, (Gehl et al. 1998; Jaroszeski et al. 2000;
1–12.2 Vasquez et al. 2015)
Carboplatin, (Gehl et al. 1998; Jaroszeski et al. 2000)
ND-13
Calcium, (Frandsen et al. 2012)
>10.6
et al. 2000; Vasquez et al. 2015); however, a reduction in tumor size in vivo was
observed after treatment with mitomycin C and electroporation, compared with
mitomycin C alone (Vasquez et al. 2015). Another amphiphilic chemotherapeutic
drug, doxorubicin, has also been tested in vitro in combination with electroporation
showing no increased effect in one study (Gehl et al. 1998) but increased effect in
another study (Meschini et al. 2012). When doxorubicin was tested in vivo in
combination with electroporation, an increased effect was seen when compared
with the drug alone (Meschini et al. 2012). This inconsistency in data of the effect
of amphiphilic drugs combined with electroporation might be due to the lipophilic
and hydrophilic properties of these drugs as well as the different cell lines used.
Furthermore, a vascular effect of electroporation with these drugs would of course
not be present in the in vitro setting, only in vivo ▶ Chap. 39, “Blood Flow
Modifying and Vascular-Disrupting Effects of Electroporation and Electroche-
motherapy”. Hydrophilic chemotherapeutic drugs, such as bleomycin, cisplatin,
and carboplatin only, limitedly cross the plasma membrane to reach their effective
site. These drugs showed the highest factors of increased cytotoxicity when tested in
combination with electroporation. The cytotoxicity increased with 100–5000-fold
for bleomycin (Orlowski et al. 1988; Gehl et al. 1998; Jaroszeski et al. 2000),
1–12.2-fold for cisplatin (Sersa et al. 1995; Gehl et al. 1998; Jaroszeski
et al. 2000), and 3–13-fold for carboplatin (Gehl et al. 1998; Jaroszeski
et al. 2000). Bleomycin and cisplatin have also been shown effective when combined
with electroporation in clinical trials (Electrochemotherapy and Its Clinical
Applications). Indeed, electrochemotherapy with these drugs is now an accepted
standard.
Other drugs have also been tested in combination with electroporation. Calcium,
which is also hydrophilic, was tested in combination with electroporation showing
that a high uptake of calcium induced cell death in vitro with an increased cytotox-
icity of calcium of more than tenfold. IC50 was not reached for calcium alone with
the tested concentrations which is why the exact increase in cytotoxicity is unknown.
Calcium electroporation is described further below. The hypoxic cytotoxin
tirapazamine, a drug that is toxic in hypoxic conditions such as in solid tumors,
has also been tested in combination with electroporation. Since the blood flow is
temporarily reduced by electroporation (Effects of Electroporation on Blood Flow),
this might enhance the efficacy of hypoxic cytotoxins. An increased effect has been
shown when combining tirapazamine with electroporation and radiotherapy in vivo
(Maxim et al. 2004). The ruthenium(III) compound KP1339 is a drug that mimics
iron in the binding to biological molecules such as transferrin and is activated by
reduction in the hypoxic tumor tissue. The cytotoxicity of this drug is increased 1.5-
fold when combined with electroporation in vitro, and the effect also increased
in vivo; however, the increase in cytotoxicity was not as high as for bleomycin
and cisplatin, possibly due to binding of the ruthenium(III) compound to serum
proteins (Hudej et al. 2010). Another approach is to combine radioactive compounds
such as iodide with electroporation showing a clear increase in the uptake of the drug
(Gopal et al. 2003). Electroporation can also increase the uptake of the
nonradioactive isotope boron used for boron neutron capture therapy where the
tumor with increased concentrations of boron is irradiated afterward activating the

cytotoxicity of the drug (Ceberg et al. 1994; Cemazar et al. 2000).
As described above, it seems that the cytotoxicity is increased the most for non- or
low-permeant drugs in combination with electroporation. Thus, several factors
should be considered before testing a new drug in combination with electroporation
as an anticancer treatment: electroporation increases the uptake of non- or
low-permeant drugs the most, a drug with low effect when not internalized in the
cell will likely cause fewer side effects, and drugs that are already clinically
approved will likely be easier and faster to get approved for use in a clinical trial
when combined with electroporation. When testing a new drug in combination with
electroporation for use as a possible new anticancer treatment, the treatment should
of course be tested in vitro as well as in vivo and in different cell lines. If the
treatment is effective, further questions need to be answered such as what doses of
the drug are most effective, how the normal tissue is affected, and what the
mechanisms of action are. Finally, the drug needs to be tested in a clinical trial to
investigate the efficacy and possible side effects of the new treatment.
In the following section, the investigation of a new drug, calcium, in combination
with electroporation will be described, from in vitro experiments to in vivo exper-
iments to clinical trial.
Calcium Electroporation
Calcium is one of the new drugs that have been tested in combination with electro-
poration as an anticancer treatment. Calcium is a very important second messenger
involved in numerous intracellular processes from fertilization through develop-
ment, differentiation, and proliferation of cells to cell death (Berridge et al. 1998).
It is known that high intracellular concentrations of calcium are cell cytotoxic, but
calcium cannot freely cross the plasma membrane which is why the idea of inter-
nalizing high concentrations of calcium into cancer cells using electroporation
originated. What if calcium, a ubiquitous ion that induces cell death in cells, could
be used as a new drug in combination with electroporation?
Calcium is a tightly regulated second messenger. There is a 10–20,000-fold
concentration gradient of calcium across the plasma membrane. Thus, the cell has
to chelate, compartmentalize, or extrude calcium to keep the ion homeostasis
(Clapham 2007). Due to the high concentration gradient, even a small change in
the permeability of the plasma membrane creates a significant increase in the
intracellular calcium concentration. Calcium can enter the cell through calcium
channels in the plasma membrane when not permeabilized. Once inside the cell,
calcium binds to different proteins and is transported into different organelles (such
as the endoplasmic reticulum (ER) and mitochondria) or transported out of the cell
by the plasma membrane calcium ATPase or Na+/Ca2+ exchanger (Clapham 2007).
In 2003, it was described in vitro that Ca2+ enters cells after electroporation in a
calcium-containing buffer and high extracellular calcium concentrations during
electroporation caused a decrease in viability (Golzio et al. 2003). And in 2012

calcium electroporation was described as a possible novel anticancer treatment
(Frandsen et al. 2012).
Effect In Vitro
Calcium electroporation has been tested in vitro in 11 different cell lines (eight
cancer cell lines, one transformed cell line, and two normal cell lines). In all cell
lines, calcium electroporation clearly induced cell death in a dose-dependent manner,
and in the experiments finding IC50, it ranged from 0.26 to 1.25 mM calcium. Even
though a clear effect of calcium electroporation was observed, there was no effect of
treatment with calcium alone in concentrations up to 5 mM and only small effects of
treatment with electroporation alone; see Fig. 1 (Frandsen et al. 2012, 2014; Hansen
et al. 2015; Zielichowska et al. 2016). In three of the cell lines, the effect of calcium
electroporation and bleomycin electroporation (electrochemotherapy) was compared
showing a similar effect of both treatments. When treating with bleomycin alone, a
slight decrease in viability was observed, and since treatment with calcium alone had
no effect on the cells, this could indicate that calcium electroporation will cause few
side effects like treatment with bleomycin electroporation; however, this needs to be
tested in vivo and clinically. The effect of treatment with calcium and bleomycin
together in combination with electroporation has also been tested, and this showed
no synergistic effect, but there seemed to be an additive effect of the two drugs
Effect In Vivo
Due to the results of the in vitro experiments showing that calcium electroporation
induced cell death in several tested cell lines, the next step was to test the
treatment in vivo. Calcium electroporation has been tested in vivo in human
small cell lung cancer tumors grown subcutaneously on immunocompromised
mice. The treatment induced significant reduction of tumor size, and eight out of
nine treated tumors were completely eliminated. Two days after treatment, tumors
were 88% necrotic and completely necrotic 6 days after treatment; see Fig. 2
Thus, calcium electroporation has been shown effective in inducing cell death
both in vitro and in vivo but with a difference in sensitivity between different cancer
cell lines, and this might also be the case when testing different tumor types in vivo.
Calcium electroporation was shown to induce tumor necrosis in vivo, and this could
mean that tumors resistant to apoptosis will likely still be affected by this treatment.
Since treatment with calcium alone both in vitro and in vivo caused no effect, it
could be believed that calcium electroporation will cause few side effects like
Fig. 1 Effect of calcium electroporation in vitro. Survival of three different cell lines (DC-3 F,
transformed Chinese hamster lung fibroblasts; K-562, human leukemia cells; and murine Lewis
lung carcinoma cells) treated with increasing calcium concentrations and electroporation (red,
closed) or not (blue, open). Viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay 1 day after treatment and depicted as percentages of controls
(electroporated or not at 0 mM calcium). Mean +/ s.d.; n 6; EP, electroporation (8 pulses of
100 μs, 1 Hz, and 1000 V/cm); and no cells, measured background level of the medium without
added cells (From Frandsen et al. 2012)
Effect on Normal Cells
The effect of calcium electroporation on normal human dermal fibroblasts has been
investigated in vitro, where cells were treated in suspension and in spheroids, a 3D
tumorlike model. Even though the normal dermal fibroblasts treated in suspension
were affected by calcium electroporation, a clear difference was seen when treating
these normal cells as a spheroid. The effect of calcium electroporation and
bleomycin electroporation was tested on three different cancer cell spheroids (breast,
bladder, and colon cancer) and normal dermal fibroblast spheroids. Very interest-
ingly, the study clearly showed that calcium electroporation and bleomycin
a b
2 hours after CaCl2 + EP 6 days after CaCl2 + EP
100
% necrosis in tissue sections
80
60
HE, original magnification 20x HE, original magnification 20x

40
20
0
0 2h 1d 2d 6d
Time after treatment HE, original magnification 400x HE, original magnification 400x
Fig. 2 Calcium electroporation induces tumor necrosis in vivo. Small cell lung cancer tumors
were treated with calcium electroporation and removed at different time after treatment. (a) Fraction
of necrosis in tumors was estimated by stereological point counting, n = 4 for treated tumors and
n = 2 for untreated tumors. (b) Light microscope images of hematoxylin and eosin (H&E) sections
of tumors 2 h and 6 days after treatment (From Frandsen et al. 2012)
electroporation induced spheroid size reduction and cell death in all three cancer cell
spheroids but not in the normal cell spheroids (Frandsen et al. 2015). These results
were confirmed with normal muscle cells treated in suspension in vitro where clearly
lower cell death was seen in normal muscle cells compared with cancer muscle cells
when treating with low concentrations of calcium electroporation (0.5 mM calcium)
(Zielichowska et al. 2016). Thus, normal cells seem less sensitive to calcium
electroporation than cancer cells, like seen when treating with electrochemotherapy
using bleomycin (Landstrom et al. 2015) and when permeabilizing cells using
nanosecond pulse electric fields (Yang et al. 2011). This could indicate that normal
surrounding tissue is spared when treating tumors with calcium electroporation like
seen when treating tumors with electrochemotherapy.
Mechanisms of Action
The mechanism behind calcium electroporation has been studied but is still not fully
elucidated. It is likely that several mechanisms contribute to the tumor cell kill
observed, as well as to the lower sensitivity in normal cells.
The energy level of cells after calcium electroporation was investigated, and it has
been shown in vitro that the intracellular adenosine triphosphate (ATP) level
decreases significantly after calcium electroporation using increasing calcium con-
centrations and/or increasing electric field (Frandsen et al. 2012; Hansen et al. 2015).
Fig. 3 Proposed mechanisms of action of calcium electroporation. (1) Electroporation induces

reversible pores in the plasma membrane allowing influx of calcium and sodium and efflux of ATP
and potassium. The change in intracellular ion concentrations leads to (2) increased ATP consump-
tion by Ca2+ ATPases and Na+/K+ ATPases. High intracellular calcium concentration may lead to
(3) opening of permeability transition pores (PTP) in the mitochondria membranes leading to loss of
ATP production. This results in ATP depletion that, together with (4) other cellular effects, might
cause necrosis (From Frandsen et al. 2012)
The ATP level has also been shown to be decreased in spheroids both in normal
fibroblast spheroids and cancer cell spheroid after treatment with calcium electro-
poration (Frandsen et al. 2015). Since the decrease in ATP level is also seen in the
normal cell spheroid, this does not explain the difference in sensitivity between
normal cells and cancer cells observed in vitro; however, the normal cells seem able
to survive this ATP depletion.
The loss of intracellular ATP level has been proposed to be due to (1) a direct loss
of ATP after permeabilization of the membrane by electroporation (Rols and Teissie
1990), (2) increased ATP consumption by ATPases that actively pump calcium and
sodium into storages or out of the cell, and (3) loss of ATP production by disruption
of the mitochondrial metabolism (see Fig. 3 (Cerella et al. 2010)).
The observed difference in sensitivity to calcium electroporation between normal
and malignant cells is not explained by differences in ATP level but might partly be
due to differences in membrane repair. The capability of membrane repair has been
tested in seven different cancer cell lines, two immortalized cell lines, and normal
dermal fibroblasts by disruption of the membrane using a laser followed by moni-
toring dye entry over time (Frandsen et al. 2016). Of all cell lines tested, the normal
cell line showed the slowest rate of dye uptake suggesting more effective membrane
repair capability in these cells. This could be part of the explanation for the observed
difference in sensitivity between normal and malignant cells both when treating with
electrochemotherapy and calcium electroporation. Since normal cells seem to repair
more effectively than cancer cells, the normal cells could be able to begin
reestablishing homeostasis sooner than cancer cells, and this might increase the
chance of survival.
Cells tightly regulate the intracellular calcium concentration to control the cal-
cium homeostasis. Calcium binds to proteins, is transported to intracellular com-
partments for storage, or is transported out of the cell. Calcium is transported into the
endoplasmic reticulum for storage by the sarco-endoplasmic reticulum calcium
ATPase (SERCA) and transported out of the cell by the plasma membrane calcium
ATPase (PMCA). Calcium overloading by calcium electroporation is expected to
lead to increased transportation of calcium that requires ATP. But due to the
depletion of ATP during permeabilization of the membrane induced by electropora-
tion together with the less effective membrane repair in cancer cells, these cells
might not be able to pump all calcium out of the cell by using the ATP-dependent
PMCA. Thus, cancer cells might not be able to reestablish calcium homeostasis
which leads to cell death. But other differences between cancer and normal cells
would also contribute to the difference in sensitivity to calcium electroporation
between these two cell types.
The plasma membrane calcium ATPase (PMCA) is a calcium pump that
exchanges protons for one Ca2+ per ATP hydrolyzed. PMCA is encoded by four
genes (PMCA1–4) with many different splice variants (Clapham 2007). PMCA1
and 4 are expressed in all cells unlike the two other PMCA isoforms. In different
colon and gastric cancer cell lines, an increased expression of PMCA4 with increas-
ing differentiation has been observed. Thus, lower PMCA4 levels were observed in
cancer cell lines compared with normal cell lines (Aung et al. 2007; Ribiczey
et al. 2007). In line with this, a reduction in PMCA4 expression has been observed
from normal colon mucosa to adenoma over adenocarcinoma to lymph node metas-
tases (Ruschoff et al. 2012). This shows that the expression of the PMCA4 is reduced
with increasing stage of the disease. The dissociation constant (Kd) for ATP of
PMCA1 is lower than the Kd for PMCA4; thus, PMCA1 is regulating the cytosolic
calcium concentration at low calcium concentrations, whereas PMCA4 can cope
with higher calcium concentrations (Guerini et al. 1998). This has been shown after
silencing either PMCA1 or PMCA4 in the MDA-MB231 breast cancer cell line to
see the role of the two PMCA isoforms when increasing the cytosolic calcium
concentration (Curry et al. 2012). PMCA4 seems to be reduced in cancer cells
compared with normal cells, as described above, and this PMCA isoform plays an
important role in pumping calcium out of the cells at high cytosolic calcium
concentrations. Thus, cancer cells could have reduced the ability to decrease the
high cytosolic calcium concentrations induced by calcium electroporation, and this
might also contribute to the observed difference in sensitivity to the treatment
between cancer and normal cells.
Clinical Trial
The investigation of calcium electroporation as a potential novel anticancer treatment

has shown that calcium electroporation effectively induces cell death in vitro and
tumor necrosis in vivo, and there seems to be a difference in sensitivity between
normal and malignant cells. The mechanisms of action are still not fully uncovered
but it has been shown that the intracellular ATP level is depleted after calcium
electroporation. The mechanism behind the difference in sensitivity is not understood
but might be affected by differences in membrane repair capability as well as
differences in plasma membrane calcium ATPase expression which has been
shown to be lower in some cancer types. An ongoing clinical trial was opened in
2013 to investigate the effect of calcium electroporation on cutaneous metastases of
any histology (ClinicalTrials.gov ID- NCT01941901). In this randomized double-
blinded phase II trial, tumor response of small cutaneous metastases of any histology,
and below 3 cm in diameter, is compared after treatment with calcium electroporation
and electrochemotherapy using intratumoral injection of calcium as well as
bleomycin. The effect is evaluated by change in tumor size (RECIST-like criteria)
and side effects are registered. The metastases are randomized individually to
treatment with either calcium electroporation or electrochemotherapy. Thus, patients
receive both treatments allowing them to be their own control. Up to six metastases
per patient are randomized and evaluated on tumor response, and if the patient has
more than six metastases, biopsies will be collected for histology. Tumors are
injected with volumes according to European Standard Operating Procedures of
Electrochemotherapy (ESOPE). Metastases 0.5 cm3 are injected with a volume
equivalent to tumor volume, and metastases 0.5 cm3 are injected with a volume
equivalent to 50% of tumor volume. Based on experience from in vivo studies,
calcium concentration is set to 220 mM, taking the larger extracellular volume in
humans into account. It is a once-only treatment and the patients are followed
regularly with tumor measurements and recording of adverse events. Six months
after treatment, the randomization code is revealed. This is a non-inferiority study
including 34 evaluable metastases, and preliminary results indicate that calcium
electroporation is efficient on patients with smaller cutaneous metastases with min-
imal side effects. The trial is expected to close in the end of 2016, and the results of
this trial will provide further evidence for electroporation with calcium as a potential
novel anticancer treatment. As calcium electroporation is a gentle treatment, it could
also be relevant in treatment of benign lesions such as keloids (hypertrophic scars),
and a phase I trial is initiated, investigating safety and efficacy of calcium electro-
poration in treatment of keloids (ClinicalTrials.gov ID- NCT01941914). Calcium
electroporation is a simple and inexpensive potential anticancer treatment without the
use of chemotherapeutic drugs, so less preparation is needed, and calcium, given as
calcium chloride, has a long shelf life and is more heat stable than chemotherapeutic
drugs. Thus, this treatment also has a high potential in less developed countries
where almost 60% of all new cancer cases in the world is (WHO 2012).
Conclusions
Investigations in new drugs in electrochemotherapy have shown that more drugs

than the most commonly used drugs, cisplatin and bleomycin, are potential candi-
dates for use in electrochemotherapy. Several chemotherapeutic drugs have been
tested as well as other drugs such as radioactive drugs and calcium. New potential
drugs should preferably be found among non- or low-permeant drugs where the drug
uptake and thereby cytotoxicity will increase the most when combined with elec-
troporation. The process from idea to clinical trial has been described for one of the
new drugs tested, calcium. This new treatment is a potential novel anticancer
treatment since investigations have shown that calcium electroporation induces
cell death in vitro and tumor necrosis in vivo while sparing normal cells. The first
clinical trial using calcium electroporation opened in 2013 investigating the effect of
this new treatment on cutaneous metastases.
Electrochemotherapy is an efficient and simple treatment with a great potential of
discovering new drugs to be used in combination with electroporation as anticancer
treatment.
Cross-References

Electrochemotherapy
Tissue Blood Flow
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Gene Transfer to the Skin
86
Anita Gothelf
Abstract
The skin holds a number of advantages useful in gene therapy and is a highly
interesting organ for gene transfection using electroporation. It is accessible and
can be easily evaluated, not only clinically but also histologically. Furthermore, it
has been shown that the skin is a very active metabolic organ and is able to
produce proteins, which can act in the local environment as well as at distant
target sites. Most importantly is that the skin is a very competent organ in terms of
immunological activity. It is part of the body’s immune surveillance and contains
antigen presenting cells, which make it suitable for transfection of DNA vaccines
with both prophylactic and therapeutic purpose.
Work is ongoing, primarily in the field of improving immunological responses
after gene transfection to skin with DNA vaccines targeting infectious and
malignant diseases, and focus is on bringing this treatment modality into the
clinical setting.
However, regarding plasmid doses, type of electrode, electrical pulse param-
eters, and spatial and temporal settings, there exists no gold standard, and the
optimal transfection can probably be achieved in many ways.
This chapter aims at giving a brief introduction to gene electrotransfer to skin
and hopefully inspires new and interesting research.
Keywords
Electroporation • Skin • Gene transfection • Animal models • DNA vaccination •
Wound healing
A. Gothelf (*)
Department of Oncology, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark
e-mail: Anita.Birgitte.Gothelf@regionh.dk

DOI 10.1007/978-3-319-32886-7_57
1652 A. Gothelf
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1652
The Skin and Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1654
The Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1654
Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1657
Reporter Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1657
Therapeutic Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1657
Choice of Electrodes and Electrical Parameters for Gene Transfection . . . . . . . . . . . . . . . . . . . . . . . 1657
Which Cells Are Transfected? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1658
Evaluation of the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1659
Clinical Use and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1659
DNA Vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1661
Wound Healing and Peripheral Artery Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1662
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1662
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1663
Introduction
In the search for new ways of treating both chronic and life-threatening diseases,
the idea of gene therapy has been increasingly interesting (Gothelf and Gehl
2010).
Since the sequencing of the human genome, focus has been on the therapeutic use
of DNA and other nucleotides, but the initial optimism was moderated due to
negative results and side effects observed in clinical studies (Alexander et al.
2007). However, more than 10 years of research has provided a large amount of
knowledge and has proven that gene therapy can be a possibility in the future (Daud
et al. 2008).
The effect of gene transfection is determined by the chosen plasmid and the
encoded transgene which can result in expression of a protein that can overcome a
deficiency due to metabolic disease or a molecule with therapeutic effect. The
purpose of gene therapy can thus theoretically be divided in three therapeutic groups
(Fig. 1) (Gothelf and Gehl 2010).
The first is the single-cell approach (Fig. 1a) in which it is possible with gene
therapy to correct or silence a mutated gene in single cells. In theory, this
approach is attractive, e.g., the idea of correcting premalignant or malignant
cells in a population of cells. In vivo, it is nearly impossible to achieve successful
transfection of every single cell; in vitro it could nonetheless be applicable. A
scenario could be transfection of stem cells in order to correct disease-related
genes in patients with single-gene disorders with subsequent transplantation to
the patient.
The next column (Fig. 1b) depicts the paracrine approach. The transfection takes
place in vivo, but not every single cell needs to be transfected to achieve the intended
outcome. The transgene protein will be produced and work locally either by stim-
ulating protein pathways or activating an immune response.
86 Gene Transfer to the Skin 1653
Fig. 1 The three approaches. (a) Single-cell approach, (b) paracrine approach, (c) systemic
approach (See text for further details; from Gothelf and Gehl 2010)
The final approach is the systemic approach (Fig. 1c), in which the transfected cells
act as “protein factories” where the transgene protein can be produced. It subsequently
enters the blood circulation and is able to act systemically. Examples are hormones
(Hojman et al. 2007) or proteins with antineoplastic effect (Trochon-Joseph et al. 2004).
Electroporation is accepted as an efficient and safe means of transporting small
and large molecules across the cell membrane. A wide variety of tissues and organs
have been evaluated for gene transfection, including the skin, which is readily
available, accessible, and easy to evaluate.
The first study regarding electrotransfer to skin was reported in 1991 by
Titomirov (Titomirov et al. 1991), and since then, more than 40 studies have been
published (Gothelf and Gehl 2010). It has been shown that the skin is an excellent
target for gene transfection and all three approaches mentioned above could be
applied. The single-cell transfection is probably most efficiently carried out
in vitro by electroporation of, e.g., harvested fibroblasts or keratinocytes, but the
paracrine and the systemic approach can be performed in vivo and in clinical studies.
The distinction between paracrine effect and systemic effect is however arbitrary and
will depend on the product of the transfection; some transgene proteins are capable
of creating both effects (Pavlin et al. 2009).
1654 A. Gothelf
It is possible, as will be described below, to achieve successful transfection after

gene electrotransfer to the skin, which can be monitored either locally or
systemically.
The Skin and Animal Models
The skin is the largest organ in the body and holds a number of physical and
biochemical properties. It serves as a protective layer, shields the body from micro-
organisms and irritants, and maintains body temperature and fluid homeostasis. It is
furthermore metabolically active and capable of both secretion and absorption. The
structure consists of three layers: the epidermis, the dermis, and the subcutaneous
layer (Fig. 2); each layer contains different cell types.
The most superficial layer, the epidermis, contains mainly keratinocytes,
which originate from stem cells connected to the basal membrane. As they
during their maturation move more superficially, they lose the properties of a
living cell and create the cornified layer, the stratum corneum, as flat thickened
and keratin-rich membrane shells. Located among the keratinocytes are the
melanocytes responsible for the melanin production and the antigen-presenting
Langerhans cells.
Beneath the basal membrane is the dermis, which mainly contains connective
tissue, fibroblasts, and antigen-presenting dendritic cells, mast cells, and macro-
phages, and further below is the subcutaneous layer containing fatty tissue, connec-
tive tissue, and in some animal models smooth muscle cells.
In the in vivo setting for gene transfection to the skin, rodents, primarily mice,
are the animal model most often used. This is due to availability and costs. For
skin more similar to human, porcine skin or hairless guinea pigs are
recommended.
There are no constraints to the choice of animal model, but even though skin
structure is similar across the species, differences exist. This is mainly in terms
of number of hair follicles and other appendages, which can vary between
animal types. The fact that, e.g., mice have smooth muscle cells (the panniculus
carnosus) in the subcutaneous layer may influence the outcome of the transfec-
tion. The impact of the differences can be visible when the effect of the
transfection is evaluated; worst case scenario is the lack of positive result in
human skin due to lack of features, which were successfully transfected in the
animal model.
The Procedure
Gene transfection can be performed quickly and easily (Gothelf and Gehl 2014).
The challenge lies in making the procedure more tolerable in the clinical setting.
Work is ongoing concerning needleless injection of plasmid and optimization of
Fig. 2 Simplified cartoon of the cells in the skin. Cells in the epidermis are (a) keratinocytes, (b)
melanocytes, and (c) Langerhans cells. The stratum corneum (d) is the superficial cornified layer. In
the dermis are (e) fibroblasts, (f) dendritic cells, and (g) mast cells located. (h) Is the border between
the dermal and the subcutaneous layer
the electric pulses rendering them less uncomfortable (Guo et al. 2013; McCoy
et al. 2015). An important perspective for gene transfection to the skin is DNA
vaccination, and in situations where a large number of healthy individuals are
going to get skin electroporation, it is central that the discomfort is at a
minimum.
The elements of the gene transfection procedure are (a) intradermal injection of
the plasmid, (b) application of the electrodes, and (c) delivery of the electric
pulses.
It is important that the electric field covers the area of the skin containing the
plasmid. If the injection is not administered with caution, the risk of the plasmid
1656 A. Gothelf
Fig. 3 Examples of noninvasive and invasive electrodes for gene transfection. (a) Injection of the
plasmid; (b) a bleb is visible in the skin, if the injection was located intradermally; (c) plate
electrode (IGEA, Carpi, Italy); (d) placement of the electrode around the injected volume; (e)
needle electrode; (f) penetration of the needles into the skin (From Gothelf et al. 2011)
being delivered subcutaneously instead of intradermally is high, particularly, if the

skin is thin like mouse skin.
If the plasmid is delivered in the dermis, it will be visible as a “bleb,” and the
placement of the electrodes should be uncomplicated (illustrated in Fig. 3b on page
xx, from Gothelf et al. (2011)).
Plasmids
The plasmids that have been transfected into the skin can be divided in two groups:
reporter genes, which mainly serve for proof of concept, investigating level and
duration of expression, kinetics, etc., and therapeutic genes, which apart from the
qualities mentioned above have the possibility of treating or preventing a disorder.
Reporter Genes
The reporter genes most commonly used are luciferase, green fluorescent protein
(GFP), and to a minor extent beta-galactosidase. Plasmids encoding a protein in the
red fluorescent spectrum (red fluorescent protein (RFP) and Katushka) have been
transfected as well (Gothelf and Gehl 2010). It is possible to evaluate the expression
either directly in vivo or ex vivo by examination of tissue samples. The drawbacks
with the reporter genes being used today are large variability in level of expression,
sensibility to pigmentation, oxygen or hemoglobin (luciferase), autofluorescence
(GFP), or false positive results (beta-galactosidase). Results must therefore, as
always, be interpreted with caution and compared with relevant controls.
Therapeutic Genes
A number of therapeutic genes have been investigated in gene transfection to the

skin (Gothelf and Gehl 2012), both in animal models and may be more important in
clinical studies. The list consists of plasmids such as growth factors with the purpose
of increasing wound healing (vascular endothelial growth factor, VEGF,
keratinocyte growth factor, KGF), hormone (erythropoietin, EPO) molecules with
antineoplastic potential (interleukin-12), and DNA vaccines against a variety of
infectious diseases (hepatitis, human immunodeficiency virus (HIV), malaria).
The promoter most often used for gene transfection to the skin has been the
constitutive cytomegalovirus (CMV) promoter. Investigations are ongoing
concerning improving the efficacy of gene transfection using tissue-specific pro-
moters (Vandermeulen et al. 2009; Kos et al. 2015).
Choice of Electrodes and Electrical Parameters for Gene

Transfection
Electrodes for electroporation can be divided in two groups: noninvasive and

invasive electrodes, and both are used for skin transfection (Fig. 3). The noninvasive
electrodes are plates and patches, which are placed on the skin surface, whereas the
invasive electrodes consist of needles penetrating the skin. Different plate types,
patches, and needle configurations exist. The plate electrodes consist of two or four
plates with adjustable distance, which are placed around the injected volume.
1658 A. Gothelf
Fig. 4 Pulse parameters for gene transfection to the skin. The graph depicts the different electrical
pulse combinations that have been applied in the skin (From Gothelf and Gehl 2012)
Patches are placed upon the injection site and can have a configuration consisting of,
e.g., 16 pins on a multielectrode array (MEA electrode) (Heller et al. 2010). Needle
electrodes can have different configurations (e.g., triangular, hexagonal, rows) and
are penetrated into the skin surrounding the injected volume.
In mice skin noninvasive electrodes are preferred due to the thinness of the skin;
however in porcine skin needle electrodes seem to have an advantage (Gothelf et al.
2011).
The pulse parameters used for gene transfection to the skin vary from many short
pulses (μs) to few long pulses (ms) and a number of combinations in between (Fig. 4,
(Gothelf and Gehl 2012)). Likewise, there is a significant variety in the voltage
applied from 10 to 1,500 V/cm. There exist no standard electrical parameters, and
many pulse combinations have been reported successful, which is probably due to
the differences in electrodes and animal models.
Which Cells Are Transfected?
It is important that the electric field is covering the area of the plasmid, in order to
obtain transfection. Up to now, it has not been clearly established which cells are
transfected. Different cell types have been reported in the literature, keratinocytes,
fibroblasts, and mononuclear cells (Gothelf and Gehl 2010), and this is probably due
to the injection technique and variety of electrodes used. Surface electrodes tend to
achieve transfection in more superficial layers than penetrating needles. The skin in
animal models is often very thin, and one has to be very careful or the injection will
be subcutaneous rather than intradermal.
Evaluation of the Results
The effect of the gene transfection procedure can be evaluated according to the
plasmid transfected. In summary, it can be measurement of luciferase activity in
tissue homogenates; in vivo measurement of luciferase activity after injection of
luciferin in live animals; detection of fluorescence by bioimaging; immunohisto-
chemistry of tissue containing the transgene protein; enzyme-linked immunosorbent
assay (ELISA) measurements of levels of transgene protein in, e.g., blood samples;
real-time polymerase chain reaction (qPCR) analysis of tissue; or indirect measure-
ment of the effect of the transgene protein, e.g., detection of hemoglobin levels.
The diversity of cell types in the skin probably has significance for the variability of
the gained expression that can be observed after gene transfection. Different cell types
and more importantly different cell shapes will not react with the same response to
electroporation and hence level of permeabilization. However the cell variety can be
used in a beneficial way. The effect of the transfection can in theory be either protein
production, (stable) transfection of stem cell, or initiation of an immune response
dependent of which cell types are transfected. Furthermore, the duration of the trans-
fection can in theory be determined by the transfected cell type; keratinocytes are short-
lived, whereas stem cells and, e.g., fibroblasts have a longer life span.
The duration of the expression after gene transfection to skin has been predom-
inately reported as short compared with the duration observed after gene transfection
to muscle. The level of expression peaks after 2–9 days, Fig. 5 (Gothelf et al. 2010;
Gothelf and Gehl 2012) and Fig. 6 (Gothelf et al. 2010), and remains for weeks,
whereas a duration of several months can occur in muscles (Hojman et al. 2007).
The optimal plasmid dose for achieving the most efficient transfection is probably
dependent on injection technique, plasmid type, promoter, etc. Increasing the plas-
mid dose will secure that the maximum level of expression will be reached. At some
point, however, the plasmid dose will be too high or too concentrated, and the effect
will be reduced by the toxic effect.
Clinical Use and Perspectives
There is no consensus concerning which is the optimal plasmid dose, electrode,

pulse combination, or promoter to achieve the most efficient transfection. The
transgene expression is a combination of a long list of variables as depicted in
Fig. 7 (Gothelf and Gehl 2012).
Therapeutic perspectives for gene therapy can be divided into systemic treatment,
local treatment, and vaccinations. Regarding systemic treatment, e.g., production of
a protein with therapeutic effect, the muscle is a far more efficient organ to transfect;
1660 A. Gothelf
Fig. 5 Duration of expression after gene electrotransfer to the skin with plasmids encoding
luciferase, Katushka, and erythropoietin, in (Gothelf and Gehl (2012) modified from (Gothelf
et al. 2010)
Fig. 6 In vivo bioimaging of skin after gene electrotransfer with Katushka to skin. With this
modality, it is possible to perform consecutive scans and thus continuous monitoring of the same
animal. The expression peaks 9 days after the transfection and is declined substantially after 21 days
(From Gothelf et al. 2010)
less plasmid is needed, the level of expression is much higher, and the duration is
often long term (Hojman et al. 2007). However gene transfection to the skin has a
number of possibilities for other clinical applications. The skin can be an advantage
when short-term transfection is wanted and particularly in cases of vaccination
where a long-term transfection is unnecessary since the focus is on creating an
immune response by transfecting immune cells (Brave et al. 2011).
Several clinical trials are or have been ongoing trying to elucidate the effect of
gene electrotransfer to the skin (www.clinicaltrials.gov). The aim of the studies is
prophylactic (preventing infectious diseases) or therapeutic (often treating cancer).
Cell type
Age of Animal
skin model
Electrode –
Kinetics
skin contact
Transgene
expression Plasmid
Electric pulses
Promoter
Delivery of
pulses at the Dose
Electrodes location of
the plasmid Injected
volume
Fig. 7 Efficacy of the gene transfection and the amount of transgene expression are dependent on a
number of variables. Consistency in performing preclinical and clinical studies is important in order
to obtain valid results (From Gothelf and Gehl 2012)
The use of transgene proteins for therapeutic applications is mainly possible in

cases where only local treatment is needed; however, local gene transfection to a
tumor in combination with systemic antineoplastic treatment is a treatment modality
that merits further investigation.
Furthermore, electroporation to the skin can be applicable not only for plasmid
DNA but also oligonucleotides and siRNA with the purpose, e.g., to knockdown a
gene signal (Broderick et al. 2012).
DNA Vaccination
DNA vaccination can crudely be divided into two groups; the prophylactic vaccines,
which aim at treating infectious diseases, and the therapeutic vaccines, which aim at
treating, e.g., a malignant disorder.
A number of important viral diseases have been the target for DNA vaccination.
Mainly human immunodeficiency virus (HIV) and influenza (Amante et al. 2015)
have been described in the preclinical setting but focus is also on, e.g., smallpox,
human papilloma virus (HPV), and malaria.
1662 A. Gothelf
Martinon et al. achieved both humoral and cellular immune responses after gene
transfection to the skin with a DNA vaccine coding for HIV-1 viral proteins
(Martinon et al. 2009). Particularly the cellular immune response was long lasting
and shows that this is a promising approach for delivery of vaccines.
If prophylactic DNA vaccination is applied to healthy individuals, it is important
that the procedure is quick and with a minimum of pain. McCoy et al. tested
multiheaded device for intradermal electroporation that allows for administration
of several vaccines at the same time (McCoy et al. 2015). Intradermal injection
volume cannot be higher than 100–150 μl before it creates discomfort for the
individual, and they found that the device could be an option for delivering sufficient
amounts of plasmid.
With therapeutic vaccines it could be possible to treat malignant diseases such as
prostate cancer or breast cancer.
Roos et al. sought to optimize the immunological response after transfection of a
DNA vaccine coding for prostate-specific antigen (PSA) (Roos et al. 2006). They
found that immunity could be induced but the extent was dependent on the electric
pulse parameters.
Wound Healing and Peripheral Artery Disease
Gene transfection with growth factors is highly interesting due to the potential
impact on known diseases and conditions such as diabetes, arteriosclerosis, and
ischemic heart disease. Impaired blood supply to the heart and brain can result in
life-threatening situations, and pharmacological treatments, change of life-style, and
surgical interventions are not always sufficient. A controlled formation of new blood
vessels (angiogenesis) can be a supplement to the existent treatment modalities.
Ferraro et al. showed that gene transfection with a plasmid encoding fibroblast
growth factor-2 (FGF-2) can increase angiogenesis and perfusion in ischemic limbs
in a rat model evaluated with a laser Doppler perfusion imager and immunohisto-
chemical staining of muscle samples (Ferraro et al. 2010).
Treatment of ischemic and chronic wounds can be improved with gene transfec-
tion of vascular endothelial growth factor (VEGF) to the skin as demonstrated by
Basu et al. (2014). They treated skin flaps in a rat model and found significant
reduction of necrosis in the gene-transfected skin flaps. Furthermore, they evaluated
the concentration of plasmid, number of treatment sites, and the optimal time for
treating the skin postsurgery to obtain the highest probability for flap survival.
Conclusions
Gene electrotransfer to the skin is an efficient and easy approach to achieve gene
transfection and has a large number of potential possibilities. It is applicable both in
the preclinical and in the clinical setting and could easily be applied in, e.g., in
wound healing, infectious, and hopefully, also malignant diseases. Future work will
clarify the most optimal conditions for the transfection procedure.
References
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(part 1). Gene Ther 14:1439–1447
Amante DH, Smith TRF, Mendoza JM et al (2015) Skin transfection patterns and expression
kinetics of electroporation-enhanced plasmid delivery using the CELLECTRA-3P, a portable
next-generation dermal electroporation device. Hum Gene Ther Methods 26:134–146.
doi:10.1089/hgtb.2015.020
Basu G, Downey H, Guo S et al (2014) Prevention of distal flap necrosis in a rat random skin flap
model by gene electro transfer delivering VEGF(165) plasmid. J Gene Med 16:55–65
Brave A, Nystrom S, Roos AK, Applequist SE (2011) Plasmid DNA vaccination using skin
electroporation promotes poly-functional CD4 T-cell responses. Immunol Cell Biol 89:492–496
Broderick KE, Chan A, Lin F et al (2012) Optimized in vivo transfer of small interfering RNA
targeting dermal tissue using in vivo surface electroporation. Mol Ther Nucleic Acids 1:e11.
doi:10.1038/mtna.2012.1
Daud AI, DeConti RC, Andrews S et al (2008) Phase I trial of interleukin-12 plasmid electropo-
ration in patients with metastatic melanoma. J Clin Oncol 26:5896–5903
Ferraro B, Cruz YL, Baldwin M et al (2010) Increased perfusion and angiogenesis in a hindlimb
ischemia model with plasmid FGF-2 delivered by noninvasive electroporation. Gene Ther
17:763–769. doi:10.1038/gt.2010.43
Gothelf A, Gehl J (2010) Gene electrotransfer to skin; review of existing literature and clinical
perspectives. Curr Gene Ther 10:287–299
vaccines. Hum Vaccines Immunother 8:1694–1702. doi:10.4161/hv.22062
Gothelf A, Gehl J (2014) Electroporation-based DNA delivery technology: methods for gene
electrotransfer to skin. In: Rinaldi M, Fioretti D, Iurescia S (eds) Methods in molecular biology,
3rd edn. Humana Press, Clifton, pp 21–31
Gothelf A, Eriksen J, Hojman P, Gehl J (2010) Duration and level of transgene expression after gene
electrotransfer to skin in mice. Gene Ther 17:839–845. doi:10.1038/gt.2010.35
Gothelf A, Mahmood F, Dagnaes-Hansen F, Gehl J (2011) Efficacy of transgene expression in
porcine skin as a function of electrode choice. Bioelectrochemistry 82:95–102. doi:10.1016/j.
Guo S, Israel AL, Basu G et al (2013) Topical gene electrotransfer to the epidermis of hairless
Guinea pig by non-invasive multielectrode array. PLoS One 8:e73423. doi:10.1371/journal.
pone.0073423
Heller R, Cruz Y, Heller LC et al (2010) Electrically mediated delivery of plasmid DNA to the skin,
using a multielectrode array. Hum Gene Ther 21:357–362
Hojman P, Gissel H, Gehl J (2007) Sensitive and precise regulation of haemoglobin after gene
transfer of erythropoietin to muscle tissue using electroporation. Gene Ther 14:950–959
Kos S, Tesic N, Kamensek U et al (2015) Improved specificity of Gene electrotransfer to skin using
pDNA under the control of collagen tissue-specific promoter. J Membr Biol 248:919–928.
doi:10.1007/s00232-015-9799-4
Martinon F, Kaldma K, Sikut R et al (2009) Persistent immune responses induced by a human
immunodeficiency virus DNA vaccine delivered in association with electroporation in the skin
of nonhuman primates. Hum Gene Ther 20:1291–1307
McCoy JR, Mendoza JM, Spik KW et al (2015) A multi-head intradermal electroporation device
allows for tailored and increased dose DNA vaccine delivery to the skin. Hum Vaccines
Immunother 11:746–754. doi:10.4161/21645515.2014.978223
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Pavlin D, Cemazar M, Kamensek U et al (2009) Local and systemic antitumor effect of intratumoral
and peritumoral IL-12 electrogene therapy on murine sarcoma. Cancer Biol Ther 8:2114–2122
Roos AK, Moreno S, Leder C et al (2006) Enhancement of cellular immune response to a prostate
cancer DNA vaccine by intradermal electroporation. Mol Ther 13:320–327
Titomirov AV, Sukharev S, Kistanova E (1991) In vivo electroporation and stable transformation of
skin cells of newborn mice by plasmid DNA. Biochim Biophys Acta 1088:131–134
Trochon-Joseph V, Martel-Renoir D, Mir LM et al (2004) Evidence of antiangiogenic and anti-
metastatic activities of the recombinant disintegrin domain of metargidin. Cancer Res
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immunisation by DNA electroporation. Vaccine 27:4272–4277
Gene Electrotransfer for Ischemic Tissue
87
Abstract
Angiogenesis, vasculogenesis, and arteriogenesis are endogenous responses to
obstructive arterial disease or trauma causing tissue ischemia such as atheroscle-
rotic plaques obstructing perfusion of the lower limbs or myocardial ischemia.
Sufficient growth of collateral vessels is often impaired in patients due to factors
such as the underlying atherosclerosis, age, or diabetes. There are many preclin-
ical and clinical studies seeking exogenous angiogenesis induction to alleviate
tissue ischemia. Translation of these therapies into clinical applications could
address a multitude of ailments including wound healing, tissue grafting, bone
and muscle regeneration, and myocardial repair. Gene therapy approaches deliv-
ering angiogenic factors have been attempted using various gene delivery
methods. This chapter will focus on evaluating preclinical studies investigating
application of gene electrotransfer approaches for ischemic tissues, in particular
for wound healing, peripheral artery disease, and myocardial infarction. Gene
electrotransfer has been utilized to deliver proangiogenic factors such as vascular
endothelial growth factor, insulin-like growth factor, basic fibroblast growth
factor, angiopoietin, etc. Delivery and expression have been demonstrated min-
imally invasively and invasively to the skin, to skeletal muscle, and to cardiac
muscle. Notable improvement in tissue perfusion was consistently reported in
skin flap wound healing models, in hind limb ischemia models, and in cardiac
muscle ischemia model.
Keywords
Gene electrotransfer • Electroporation • Angiogenesis • Gene therapy • Ischemia •
Peripheral artery disease • Coronary artery disease
A.A. Bulysheva (*) • R. Heller

e-mail: abulyshe@odu.edu; rheller@odu.edu

DOI 10.1007/978-3-319-32886-7_58
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1666
Lower Limb Ischemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1666
Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1671
Delivery to Nonischemic and Ischemic Left Ventricular Myocardium . . . . . . . . . . . . . . . . . . . . . . . 1672
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1675
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1675
Introduction
Tissue ischemia is often caused by blockage of blood flow as a result from trauma or
blood vessel disease such as atherosclerosis. Advanced atherosclerosis can lead to
peripheral artery disease (PAD) or coronary artery disease (CAD) often with
comorbidities depending on which arteries are affected (Ross 1993). Significant
tissue ischemia results in diseased tissue and subsequent remodeling of the normal
tissue structure, altering function. Patient quality of life is highly dependent on
preserving as much of the original tissue function and tissue repair as possible.
Clinical treatment options for patients with severely ischemic tissues focus on
re-vascularizing either by circumventing blocked vessels with a bypass graft vessel
or attempting to remove the blockage of the existing vessel and maintaining wall
integrity with stents (Go et al. 2013). Multiple clinical studies investigating angio-
genic therapies aiming to induce collateral vessel development have been reported
with conflicting results (Wolfram and Donahue 2013). Main gene therapy
approaches involve gene delivery of an angiogenic cytokine or growth factor,
primarily vascular endothelial growth factor A (VEGF) or basic fibroblast growth
factor (bFGF). The most commonly utilized delivery methods utilize viral-based
methods. Adeno-associated virus (AAV) has been used extensively with high effi-
ciency of gene transfer. However, there are considerable immunogenicity and
toxicity considerations associated with this delivery method (Wolfram and Donahue
2013). Naked plasmid DNA delivery poses fewer side effects; however, lower gene
transfer efficiency is expected (Wolfram and Donahue 2013). Gene electrotransfer
(GET) in vivo has been shown to enhance naked DNA delivery, improving expres-
sion levels (Ferraro et al. 2009). In this chapter, GET delivery of angiogenic factors
to ischemic tissues for wound healing, peripheral artery disease, and coronary artery
disease will be evaluated.
Lower Limb Ischemia
Peripheral artery disease (PAD) generally refers to atherosclerosis of the arteries

supplying the legs. Depending on the extent of the disease, PAD symptoms include
patients experiencing pain, weakness, or numbness during walking; sores on toes,
feet, or legs; change in leg color; and weak pulse. Severe PAD leads to tissue injury,
possible open sore infections in feet and legs as well as tissue death sometimes
87 Gene Electrotransfer for Ischemic Tissue 1667
requiring limb amputation. Severe PAD is often treated surgically with bypass
grafting or percutaneous interventions (Collinson and Donnelly 2004). Patients
with mild to moderate disease have few treatment options. Those having recurrent
symptoms after already exhausting surgical interventions also have few available
treatments (Collinson and Donnelly 2004). Due to the atherosclerosis, endogenous
angiogenesis in response to hypoxic/ischemic conditions is impaired (Collinson and
Donnelly 2004). Developing collateral circulation may improve patient symptoms;
thus, many preclinical therapies are focused on angiogenesis induction. The standard
preclinical in vivo model for PAD is the hind limb ischemia model in a small animal
like mouse or rat, where a portion of the femoral artery is either ligated or even
excised to induce ischemia to the downstream limb. Here, preclinical gene therapy
approaches to PAD with gene electrotransfer-enhanced gene delivery in the hind
limb ischemia model will be discussed.
Rabinovsky et al. (2004) reported the first gene electrotrasfer of an effector gene
for repair of ischemic skeletal muscle. Plasmid DNA encoding insulin-like growth
factor-I (IGF-I) was used for its angiogenic properties and potential therapeutic
benefits for skeletal muscle repair postischemic damage. An intramuscular injection
of DNA was performed to the anterior tibialis muscle. Parallel plate electrodes were
placed directly over the injection site of the muscle to deliver pulsed electric fields.
Two disease models were utilized. A crushed sciatic nerve model was used to cause
muscle atrophy. In a different set of experiments, the femoral artery was ligated in
diabetic mice to cause hind limb ischemia. In the case of sciatic nerve damage and
subsequent muscle atrophy, increased levels of endogenous VEGF and VEGF
receptors were observed after IGF gene transfer. While in the hind limb ischemia
model, increased blood flow to the ischemic limb was confirmed with laser Doppler
imaging, indicating angiogenesis had occurred (Rabinovsky and Draghia-Akli
2004).
A similar approach was reported by Nishikage et al. (2004). Gene electrotransfer
of basic fibroblast growth factor (bFGF) was performed in a rabbit hind limb
ischemia model. Ischemia of the hind limb was induced by surgically excising a
portion of the femoral artery. GET was performed directly on the muscle 10 days
after excision of a portion of the femoral artery. Plasmid DNA encoding bFGF was
injected into the ischemic muscle followed by insertion of needle electrodes 1 cm
apart. Pulsed electric field was delivered with six pulses of 50 ms, and alternating
polarity, 75 V, was applied. Angiogenesis was confirmed with angiography imaging.
The vascular tree contained new blood vessels implicating angiogenesis induction.
Limb survival compared to controls was also observed, implicating functional blood
vessel growth and resolution of ischemia (Nishikage et al. 2004).
A common concern associated with angiogenesis inducing therapies is develop-
ing leaky, unstable vessels that never mature to functional vasculature. Immature
blood vessels are inefficient at reducing ischemia and promoting tissue repair
postischemic damage. Jiang et al. (2006) attempted to address this concern with
co-delivery of VEGF and angiopoietin-I (Ang-I) encoding plasmid DNA via electro-
transfer. Experiments were conducted in a rat hind limb ischemia model. A portion
of the femoral artery was excised to establish distal limb ischemia. GET was
performed on the muscle tissue distal to the excision site. Pulses were delivered with
a two-needle electrode with a 5 mm gap between the needles. One hundred volts
were applied across the needles. Pulse duration was 40 ms. Six pulses were applied.
Microvessel density measurements and angiography were performed to evaluate
angiogenesis induction. A modified Evans blue assay was performed to indirectly
evaluate blood vessel maturation and integrity of the vessel walls. Leakage through
the blood vessels was assessed by measurement of the Evans blue in tissue interstitial
space. Co-expression of Ang-I and VEGF resulted in increased perfusion relative to
single expression controls of either Ang-I or VEGF. Evans blue dye analysis
indicated more presence of dye in excised muscle tissue from single expression
groups than the co-expression group, indicating leaky vessels, although it is unclear
how it was determined that extracted dye from muscle tissue came from interstitial
fluid and not blood vessel lumens located within the excised tissue. It was concluded
that Ang-I and VEGF have a synergistic effect on angiogenesis and blood vessel
maturation, resulting in functional vasculature development in the ischemic muscle
(Jiang et al. 2006).
An alternative strategy for inducing angiogenesis and blood vessel maturation,
thus reducing leaky, receding vessels, reported co-delivering phage phiC31 integrase
with mVEGF164. A mouse hind limb ischemic model was used for these co-delivery
experiments. Portlock et al. (2006) hypothesized that transient expression of angio-
genic factors resulted in transient perfusion to the ischemic limbs and long-term
expression of angiogenic factor may lead to angiogenesis, blood vessel maturation,
and stable blood vessel supply to the ischemic tissue. Therefore, site-specific
integration of plasmid DNA encoding mVEGF164 was proposed. The phiC31
integrase system with VEGF was injected into the ischemic muscle and was
followed by pulsed electric field application to achieve electrotransfer and site-
specific delivery. Levels of VEGF expression were measured over a 40 day period.
Persistently high VEGF levels were observed throughout the duration of the study
up to 40 days. Site-specific integration of plasmid DNA was confirmed, implicating
constitutive expression of VEGF (Portlock et al. 2006). While therapeutic merit was
demonstrated over 40 days, and further long-term benefits are strongly implicated as
the benefits of permanent integration and constitutive expression of VEGF, there are
considerable concerns with permanent overexpression of a potent angiogenic factor.
In atherosclerotic disease, established blood vessels harden and become blocked
over time, reducing blood flow to distal regions. In patients affected by atheroscle-
rosis, normal angiogenesis in response to hypoxia/ischemia may be impaired.
Therefore, preclinical models demonstrating angiogenesis in non-atherosclerotic
ischemic disease models may not be representative of clinical outcomes in patients
with impaired angiogenesis. In order to investigate angiogenesis in an ischemia
model closely resembling atherosclerotic disease, hind limb ischemia was induced in
ecNOS-KO mice. These mice have impaired angiogenesis. NOS1177D encoding
plasmid DNA was injected into the ischemic skeletal muscle after induction of
ischemia via femoral artery occlusion. Caliper electrodes were used to deliver
eight pulses of 200 V/cm. Caliper electrodes require pinching the tissue of interest
between the plates of the caliper and thus are difficult to translate to clinical
applications especially for gene delivery to skeletal muscle. The pulse duration was
20 ms. Qian et al. (2006) reported reversal of hind limb ischemia in angiogenesis
impaired mice (Qian et al. 2006).
Another co-expression study was reported by Sacramento et al. (2010) delivering
VEGF and granulocyte colony-stimulating factor (G-CSF) encoding DNA in a
mouse model of hind limb ischemia. The authors hypothesized that local transient
expression of VEGF and stem cell mobilizer G-CSF genes may complement activ-
ities in repair of ischemic limbs. Hind limb ischemia was surgically induced in a
mouse model. Three pulses were delivered with needle electrodes. An electric field
of 75 V/cm was applied with a 20 ms pulse length. Necrosis, capillary density,
muscle mass, muscle force, and hematopoietic cell mobilization were measured and
analyzed 3–4 weeks post electrotransfer. Animals that were treated with both VEGF
and G-CSF genes presented with reduced incidence of limb necrosis. Muscle mass
recovery was significantly higher than in animals treated with either VEGF or
G-CSF genes individually. The animals in the double transfection group also had
higher muscle force than those in single transfection groups, up to 60% of muscle
force measured in the nonischemic group. Capillary density was found to be higher
in the double transfection group, with reduced degree of fibrosis. Infiltration of
Sca1 + cells and neutrophils was also higher in the double transfected group,
indicating a possible mechanism for reduction of necrosis. Therefore, a synergistic
effect between promoting blood vessel formation and tissue repair upregulation was
observed (Sacramento et al. 2010).
Ferraro et al. (2010) reported a noninvasive gene delivery approach to improving
hind limb ischemia in a rat model. This noninvasive approach delivers plasmid DNA
to the skin overlaying ischemic tissue minimizing the need for surgery to expose
ischemic muscle and potentially minimizing the need for general anesthesia. Hind
limb ischemia was established in a rat model with excision of a portion of the
femoral artery. Fibroblast growth factor-2 (FGF-2) encoding plasmid DNA was
delivered intradermally to the skin overlaying the ischemic muscle. A 16-pin,
multi-electrode array (MEA) was used to deliver eight pulses at 250 V/cm, with a
150 ms pulse duration. The pins are located 2 mm apart, in a 4x4 array. Additionally,
this electrode design does not require pinching tissue between the electrodes, thus
enhancing translation to clinical applications. Laser Doppler imaging was performed
to monitor perfusion to the ischemic limbs in animals over time. Improved perfusion
was observed in the gene transfer group compared to no gene transfer controls. Limb
survival was also dramatically improved in the FGF-2 gene transfer group compared
to appropriate controls (Ferraro et al. 2010). This report was the first instance of a
noninvasive electrotransfer delivery approach to the skin that enhanced translatabil-
ity of electrotransfer-mediated gene delivery for peripheral artery disease.
Co-expression of VEGF and hepatocyte growth factor (HGF) in a mouse hind
limb ischemia model was reported by Makarevich et al. (2012). Hind limb ischemia
was established by occluding the femoral artery. Plasmid DNA encoding VEGF
and/or plasmid DNA encoding HGF were injected into the ischemic muscle. Twee-
zer electrodes were used to apply electric pulses directly to the ischemic muscle after
plasmid DNA injection. Immunohistochemistry for the endothelial cell marker
CD31 revealed higher microvasculature density in the co-expression group compared

to single expression control groups. Laser Doppler revealed improved perfusion in
VEGF and HGF co-expression group compared to the control groups, indicating an
advantage of the co-expression strategy over single expression gene electrotransfer
(Makarevich et al. 2012). The advantage of this approach to prior co-expression
strategies is unclear; however, this study is consistent with other co-expression studies
indicating an advantage over delivery of the VEGF alone for PAD.
Another angiogenic gene therapy mediated with GET delivered a constitutively
expressed hypoxia-inducible factor 1-alpha (HIF-1α) in a mouse hind limb ischemia
model. Hypoxia was induced in mice assigned to three experimental groups by
ligating the femoral artery. HIF-1α with GET, HIF-1α without electrotransfer, and
empty vector with GET were the three groups. Plasmid DNA was injected directly
into the ischemic muscle. Electrotransfer was performed with a three-electrode array.
Two pulses were applied around 0.1 AMP. Limb perfusion was monitored with laser
Doppler imaging over 21 days. Muscle movement was scored for muscle function
recovery postischemic induction and gene therapy. Adductor muscle was evaluated
for necrosis. Capillary density was evaluated by immunohistochemical staining for
the endothelial cell marker, CD31. Improved limb perfusion and survival, angio-
genesis, muscle function, and necrosis reduction were reported for the HIF-1α GET
group compared to control groups (Ouma et al. 2014). While this method was
reported as a noninvasive gene delivery strategy, the needles used to inject plasmid
DNA into the muscle tissue and electrode needles used to deliver electric pulses still
pierce the skin and muscle tissue; therefore, a minimally invasive designation might
be more accurate.
Parallel plate electrodes have recently been used for minimally invasive intrader-
mal gene transfer of hCAP-18/LL37 in a mouse hind limb ischemia model. The
femoral artery and vein were resected to establish hind limb ischemia. Plasmid DNA
encoding hCAP-18/LL37 was injected intradermally into the skin overlaying the
ischemic muscle. One high-voltage pulse (700 V/cm, 100 μs) followed by one
low-voltage (200 V/cm, 400 ms) pulse were applied to the skin overlaying ischemic
muscle after intradermal injection of plasmid DNA. Laser Doppler imaging was used
to monitor tissue perfusion. Muscle atrophy was evaluated to assess treatment
benefits. Angiogenic chemokine levels were measured for VEGF, SDF-1a, as well
as their receptors VEGF-R and CXCR-4. Steinstresser et al. (2014) reported
improved limb perfusion. Muscle atrophy was reduced in the gene transfer group
compared to control groups. Angiogenic chemokine levels were increased as well as
presence of their respective receptors. Therefore, gene transfer of plasmid encoding
hCAP-18/LL37 may be a promising approach for treatment of ischemic disease
(Steinstraesser et al. 2014). While this is also a minimally invasive approach, it is
difficult to translate application of parallel plates to human skin for clinical therapies.
In summary, multiple strategies have utilized electrotransfer as a delivery modal-
ity inducing angiogenesis in preclinical animal models of hind limb ischemia.
Multiple genes, combinations of genes, multiple pulsing protocols, and invasive
and minimally invasive delivery sites have been evaluated. All have shown
improved perfusion and limb rescue. No resulting clinical studies investigating
electrotransfer-mediated gene therapy for peripheral artery disease have been

reported. It is not clear what confounding factors are limiting clinical translation;
since toxicity of the therapy is reportedly low, the expression profiles indicate
temporary gene expression; and gene integration is unlikely due to the plasmid
design and delivery methods. Clinical studies with other delivery methods or with
naked plasmid DNA delivery of VEGF and bFGF have been reported with
conflicting results (Collinson and Donnelly 2004). Gene electrotransfer may resolve
some of those problems by enhancing delivery efficiency and allowing for modu-
lating protein levels to achieve a therapeutic dose of the effector protein.
Wound Healing
Skin grafts and other forms of wound healing require an abundant blood supply.
Providing adequate perfusion to grafted tissue is often challenging and is a common
limiting factor for successful tissue repair and generation. Angiogenesis stimulating
therapies are emerging as promising strategies for improving wound repair and
regeneration therapies. Gene electrotransfer, as a physical method for gene expres-
sion enhancement with minimal side effects, has gained interest for enhancing
transient expression of angiogenic factors for wound healing applications.
GET-mediated angiogenic therapies have been evaluated in the flap model by
multiple groups with encouraging results. A review of GET for wound healing
applications in vivo is presented in this chapter section.
Fujihara et al. (2005) reported using bFGF gene therapy mediated by GET for
wound healing in a small animal model. A full-thickness skin flap wound was
created surgically on the backs of Sprague-Dawley rats. The dorsal muscle under-
lying the skin flap was designated as the recipient bed. The recipient bed received an
injection of plasmid DNA encoding bFGF or LacZ. Electrotransfer groups received
six pulses of 50 ms with an amplitude of 50 V delivered directly to the muscle with a
two-needle electrode. The gap between the electrode needles was 1 cm. Three sites
received treatment per flap. Micro-angiography was performed to evaluate tissue
perfusion. Macroscopic evaluation of necrosis was also performed. GET was
performed 2 days post flap surgery. Increased blood vessel density was observed
in the electrotransfer of bFGF group compared to control groups. Gene expression of
bFGF in the dorsal muscle was confirmed. Increased distal vascularization of the
skin flap was also observed in the bFGF-GET group compared to control groups.
Necrosis evaluation showed reduced necrosis of distal regions of the skin flaps in the
bFGF-GET groups. Together, the study suggests that bFGF gene therapy mediated
by electrotransfer yielded a therapeutic benefit for wound healing applications
(Fujihara et al. 2005).
A noninvasive electrotransfer-mediated gene therapy approach to wound healing
was reported by Ferraro et al. (2009). Plasmid DNA encoding VEGF was delivered
intradermally in the flap wound healing model in a rat. A full-thickness skin flap
wound was surgically created on a flank of a Sprague-Dawley rat. Four equally
spaced sites were identified for treatment on each flank. Plasmid DNA was injected
intradermally at each site, and animals randomly assigned to the GET group received
electric pulses at each injection site, while control groups received only plasmid
DNA injections, without electric pulses. A four-plate electrode was utilized with a
6 mm gap. Eight pulses were delivered per site with a 20 ms pulse duration and a
200 V/cm electric field. Laser Doppler imaging was performed to quantitatively
assess perfusion and angiogenesis at different time points during the study. Plasmid
VEGF delivery enhanced by electrotransfer resulted in increased perfusion of the
skin flap and reduced necrosis of tissue distal to the flap attachment point compared
to perfusion and necrosis measures in plasmid DNA injection’s only control group.
Intradermal delivery of pVEGF was sufficient to promote healing, induce angiogen-
esis, increase perfusion, and reduce distal necrosis (Ferraro et al. 2009). The advan-
tage of this approach compared to delivery to the recipient bed is the noninvasive
nature, as well as timing of the therapy, which does not require angiogenic therapy
prior to injury, and thus has a higher potential for translation to clinical wound
healing applications.
Basu et al. (2014) reported a variation of the noninvasive method of plasmid
DNA encoding VEGF delivery. The flap wound in a rat model was created as in the
previous studies. A multi-electrode array (MEA) composed of 16 blunt tipped pins
with a 2 mm gap was utilized in the rat wound healing model. Plasmid DNA was
injected intradermally into the skin of the flap at two or four equally space sites.
Animals designated to the GET group received pulses of 50 V with a 150 ms pulse
width. Each of the nine four-pin regions within the MEA electrode received eight
pulses per site. The control groups received plasmid DNA injections without applied
pulses. Laser Doppler was used to monitor perfusion through the flap, and necrosis
evaluation was performed. Timing of the gene transfer treatment was also studied to
determine the optimal window for when treatment could be effective after injury
onset. As in the previous study, GET-enhanced VEGF delivery groups had higher
perfusion, than corresponding control groups. Two treatment sites per flank were
found to be equivalent to four treatment sites in terms of therapeutic benefit.
Additionally, it was determined that there is a 2 day delivery window post injury
that allowed for increased perfusion and prevention of necrosis (Basu et al. 2014).
Wound healing preclinical studies consistently suggest that GET-mediated angio-
genic treatments are feasible for translation to clinical applications and are poten-
tially therapeutically effective. Ischemic skin can recover from injury with increased
blood perfusion and reduced necrosis with gene delivery of angiogenic factors such
as bFGF or VEGF enhanced by gene electrotransfer. Additionally, these therapies
can be administered noninvasively (Ferraro et al. 2009; Basu et al. 2014).
Delivery to Nonischemic and Ischemic Left Ventricular

Myocardium
Heart disease accounted for one quarter of all deaths in the United States and is the
leading cause of all deaths (Go et al. 2013). The 5-year survival rate for patients
diagnosed with heart failure is 50% (Go et al. 2013; Krumholz et al. 2009). Coronary
arteries are susceptible to chronic or acute plagues causing blockage and triggering
downstream tissue ischemia in advanced states of atherosclerotic disease (Ross
1993). Improving direct revascularization of ischemic tissue distal to myocardial
infarction is therefore the main focus of current clinical therapies (Mukherjee et al.
1999). Angiogenesis induction is another strategy currently pursued in clinical and
preclinical studies. Gene therapy approaches with exogenous expression of angio-
genic agents such as VEGF for collateral vessel development in hypoxic myocar-
dium have been studied extensively with many viral delivery methods (Wolfram and
Donahue 2013). These viral delivery methods include adenoviral vectors and adeno-
associated vectors with major possible side effects including toxicity and immuno-
genicity. There are fewer side effects associated with naked plasmid DNA delivery
including low immunogenicity, low toxicity, and reduced cost, at the expense of
lower gene transfer efficiency (Wolfram and Donahue 2013). GET delivery
approaches directly to the ischemic and nonischemic ventricular myocardium have
been demonstrated in large and small animal models, respectively. Short-term
preclinical studies were conducted for evaluation of feasibility, safety, and efficiency
of reporter and effector gene delivery and expression. Long-term preclinical studies
were conducted for the evaluation of the therapeutic potential of VEGF to induce
angiogenesis and arteriogenesis and repair ischemic myocardium (Marshall et al.
2010; Ayuni et al. 2010; Eigeldinger-Berthou et al. 2012; Hargrave et al. 2013, 2014;
Bulysheva 2016).
The first report of successful gene delivery with gene electrotransfer directly to a
beating heart was performed in a swine cardiac model (Marshall et al. 2010). In this
study, Marshall et al. (2010) reported delivering a reporter gene (luciferase encoding
plasmid DNA) and an effector gene (VEGF encoding) plasmid DNA directly to the
ventricular myocardium. A sternotomy was performed and the pericardium was
surgically removed to expose the epicardial surface of the ventricular myocardium.
Plasmid DNA was injected at a predetermined depth into the myocardium. A
penetrating four-needle electrode was inserted into the myocardium at the site of
DNA injection, and pulses were delivered to enhance gene transfer to the myocar-
dium. The key to making delivery possible and avoiding ventricular fibrillation,
while applying electric pulses to the heart, was synchronizing the timing of applied
pulses with the absolute refractory period. Additional electrical stimulation of
cardiac cells during the absolute refractory period resulted in no additional contrac-
tion of those cells; thus, arrhythmias can be avoided. The electrocardiogram (ECG)
was used to determine the timing of the absolute refractory period, which occurs
during the QRS-wave. Each pulse was electronically synchronized to the rise of the
R-wave and was completed before the beginning of the T-wave. Custom pulsing
software and hardware were developed to accomplish synchronization of applied
pulses and the ECG. Several applied voltages were tested with clear differences
between expression levels and pulsing conditions, suggesting ability to modulate
expression levels to an optimal level with changing pulse conditions. No damage
from electrotransfer was observed (Marshall et al. 2010). This study was followed up
with a separate study thoroughly evaluating different electrode designs and different
applied voltages to optimize delivery to swine myocardium as evident by reporter
gene delivery encoding luciferase and green fluorescence protein (Hargrave et al.
2013).
The first report of GET-mediated gene delivery to the ischemic myocardium in a
swine model of acute myocardial infarction detailed variable pulsing conditions to
achieve optimal expression profiles and kinetics in ischemic and nonischemic swine
myocardium (Hargrave et al. 2014). Acute myocardial infarction was surgically
induced by permanently ligating the left anterior descending (LAD) coronary artery.
The optimal pulsing conditions were defined by enhanced expression of VEGF
documented in both ischemic and nonischemic regions. These pulsing conditions
were empirically determined to be 60 V applied, eight pulses, with 20 ms pulse
duration. Each applied pulse was synchronized with the rise of the R-wave of the
ECG. The optimal gene delivery pulsing electrode had a four-needle array. The gap
between the four penetrating needles was 5 mm. The needles were 7 mm long, with
DNA injection port injecting DNA at a depth of 3.5 mm from the epicardium. VEGF
expression was evaluated over time in ischemic and nonischemic treatment sites via
ELISA quantitation of harvested myocardial tissue homogenate. This study also
reported evaluating perfusion through the heart 2 weeks after onset of acute myo-
cardial infarction and gene transfer. Animals assigned to the GET groups received
four treatment sites with plasmid DNA injections followed by electrotransfer appli-
cation at those sites. The control animals received plasmid DNA injections, without
electrotransfer. Fluorescence perfusion imaging was performed to quantitatively
evaluate blood perfusion through the ischemic myocardium post myocardial infarc-
tion onset and gene transfer treatment. Perfusion was significantly improved in the
GET-enhanced VEGF delivery groups at the 2-week time point compared to
corresponding controls (Hargrave et al. 2014).
Bulysheva et al. reported a long-term study evaluating safety and effectiveness of
pVEGF GET-mediated delivery in a swine myocardial infarction model (Bulysheva
2016). Acute myocardial infarction was induced surgically with a permanent occlu-
sion of the LAD coronary artery in swine. Animals were randomly assigned to either
the GET group delivering plasmid DNA encoding VEGF or a control group with
plasmid DNA injection without electrotransfer application. Angiography, echocar-
diography, dobutamine stress test, and infarct size measures were assessed to
evaluate cardiac repair 7 weeks post onset of myocardial infarction (MI) and gene
transfer treatment. A smaller infarct size and arteriogenesis were observed relative to
controls at the treatment sites 7 weeks post MI. Also there was a notable improve-
ment of contractility and relaxation of the left ventricle as evident from the
dobutamine stress test. Together, this study suggests repair of myocardium is
possible due to delivery of pVEGF via GET in a swine model of acute myocardial
infarction (Bulysheva 2016).
Shortly after the first report of GET to swine nonischemic myocardium (Marshall
et al. 2010), Ayuni et al. (2010) reported GET plasmid DNA encoding GFP or
luciferase to rat myocardium (Ayuni et al. 2010). Electroporation was achieved
without synchronization with the ECG; however, no fibrillation was observed.
Plasmid DNA was injected into temporarily occluded coronary sinus, while the
entire heart was pulsed by fitting the organ between parallel plate electrodes.
Histological analysis for damage and GFP expression revealed minimal damage and
higher expression of GFP and luciferase compared to controls; therefore, GET
enhanced GFP expression (Ayuni et al. 2010). This was followed up by a similar
study also in nonischemic rat myocardium but with direct injection of plasmid DNA
into the myocardium (Eigeldinger-Berthou et al. 2012). GFP expression was eval-
uated to assess delivery efficiency, which was significantly higher than respective
controls (Eigeldinger-Berthou et al. 2012).
In summary, application of GET to cardiac muscle can be consistently accom-
plished in large and small animal models (Marshall et al. 2010; Ayuni et al. 2010;
Eigeldinger-Berthou et al. 2012; Bulysheva, 2016). Reporter gene delivery studies
indicate that changing gene transfer conditions such as applied voltage or electrode
design can modulate gene expression (Hargrave et al. 2013, 2014). It was also
determined that ischemic myocardial repair is similar to ischemic skeletal muscle
repair with GET-mediated VEGF gene delivery; tissue perfusion was improved and
some repair of function was observed in both tissues post VEGF delivery and
expression.
Conclusions
All studies of gene electrotransfer for tissue ischemia report significant reversal of
ischemia in animal models. GET of particular proangiogenic growth factors: bFGF
and VEGF consistently alleviated ischemia in the skin, skeletal muscle, and cardiac
muscle, as reported by multiple groups in multiple preclinical in vivo studies in small
and large animal models. GET can be performed noninvasively to the skin for PAD
and wound healing without loss of efficacy compared to intramuscular delivery.
Gene delivery to a beating heart mediated by electrotransfer has been shown in small
and large animal models. Effector protein levels can be modulated with pulsing
parameters; therefore, therapeutic window of expression can be determined. In
conclusion, gene electrotransfer for ischemic disease has been shown to be effective
in preclinical studies and is a promising gene delivery therapy with clinical applica-
tions to wound healing, peripheral artery disease, and even myocardial ischemia.
There are currently no clinical studies reporting GET for ischemia; however, all
preclinical studies show consistent reversal of ischemia and minimal known safety
concerns; therefore, clinical applications are the next logical steps.
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Cochlear Implant Close-Field
Electroporation 88
Gary D. Housley, Cherylea J. Browne, Edward N. Crawford,
Matthias Klugmann, Nigel H. Lovell, and Jeremy L. Pinyon
Abstract
Applications for gene therapy that depend upon localization and control of gene
expression are challenged by the mode for gene delivery. Viral vectors inherently
suffer spread of the viral particles, as does liposome-based gene transfer. Similarly
conventional “open-field” electroporation (OFE) achieves gene electrotransfer of
naked plasmid DNA injected generally into the target tissue by means of a brief train
of high-voltage electrical pulses creating a quasi-uniform electric field. The targeting
Conflict of Interest: Three patent filings are associated with this research: (i) Title: Method of
providing agents to the cochlea. Inventor: GD Housley. Filing status: National Phase Examination –
Europe (application no. 10799287.7; filing date 5 July 2010), United States (application
no. 14/145,673; approved 26th August 2016). Assignee: NewSouth Innovations Pty Limited.
(ii) Title: Method and apparatus for close-field electroporation. Inventor: GD Housley, M
Klugmann, J Pinyon. Filing status: Provisional, Australia (application no. 2013902263; filed
21 June 2013). Assignee: NewSouth Innovations Pty Limited. (iii) Title: Electroporation system for
controlled localized therapeutics delivery. Inventor: GD Housley, NH Lovell. Filing status:
Australian Provisional Patent filing (application no. 2015902456; filing date 25 June 2015).
Assignee: NewSouth Innovations Pty Limited.
G.D. Housley (*) • E.N. Crawford • M. Klugmann • J.L. Pinyon
Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences,
UNSW Australia, Sydney, NSW, Australia
e-mail: g.housley@unsw.edu.au; E.Crawford@unsw.edu.au; m.klugmann@unsw.edu.au;
j.pinyon@unsw.edu.au
C.J. Browne
Translational Neuroscience Facility and Department of Physiology, School of Medical Sciences,
UNSW Australia, Sydney, NSW, Australia
School of Science and Health, Western Sydney University, Sydney, NSW, Australia
e-mail: C.Browne@westernsydney.edu.au
N.H. Lovell
Graduate School of Biomedical Engineering, UNSW Australia, Sydney, NSW, Australia
e-mail: n.lovell@unsw.edu.au

DOI 10.1007/978-3-319-32886-7_59
1680 G.D. Housley et al.
of the gene transfer with OFE can be controlled to some extent by the placement of the
electrodes in the tissue and the volume of injected DNA. In this chapter the application
of “close-field” electroporation (CFE) is described, where a gene delivery probe is
used for highly localized and controlled gene electrotransfer. CFE arose from use of
cochlear implant electrode arrays, designed for spatially constrained electrical stim-
ulation of the primary auditory neurons in the cochlea, where the array was
reconfigured to provide electric field focusing that produced electric field strengths
sufficient for gene delivery in the domain close to the bionic array using applied
voltages considerably below that normally required for conventional electroporation.
CFE has proved to be a highly robust and efficient means for targeting small tissue
regions (hundreds of um2 – to several mm2) for efficient gene delivery, where the
shape of the region of the tissue targeted for gene delivery can be controlled by varying
the electric field shape around the bionic array and where near “dial-up” control of the
delivery of the DNA payload can be achieved by varying the pulse parameters. The
CFE gene electrotransfer platform has been successful in establishing a proof of
principle for local neurotrophin gene therapy in the cochlea that regenerates primary
auditory neuron dendrites in a deafened animal model, improving cochlear implant
performance for hearing. The ability to control the distribution of the electric field
around the bionic electrode array to deliver titrated local gene electrotransfer suggests
that CFE could have broad application for gene therapy.
Keywords
Gene electrotransfer • Bionic array • Electric field focusing • Naked DNA •
Hearing therapeutics • Neurotrophin • Brain-derived neurotrophic factor •
HEK293 cells • Gene therapy
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1680
Close-Field Electroporation Ex Vivo Reveals the Significance of Electrode Configuration
in Determining the Shape and Size of Transduced Fields of Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1682
Experimental Details for CFE: Ex Vivo with Organotypic Tissue Culture . . . . . . . . . . . . . . . . . . . 1682
Close-Field Electroporation for Cochlear Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1685
In Vitro Close-Field Electroporation Using HEK293 Cell Monolayers . . . . . . . . . . . . . . . . . . . . . . . 1687
Electric Field Focusing Through Variable Configuration of Cochlear Implant Electrode
Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1691
Extension of Close-Field Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1693
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1696
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1697
Introduction
A major challenge for gene therapy is the targeted delivery of the expression
cassettes. The development of “close-field” electroporation (CFE)-based gene
electrotransfer through bionic arrays provides a means for localized dose-controlled
88 Cochlear Implant Close-Field Electroporation 1681
delivery of naked plasmid DNA. Development of this platform technology is likely

to have application across a range of experimental paradigms for manipulating gene
expression and lend considerable utility for gene therapy where precise control of the
population of transduced cells is beneficial. This chapter describes the basis and
practical application for CFE, which stems from local electric field focusing that can
be achieved using bionic array electrodes.
The proof of principle of CFE gene delivery was established using cochlear implant
electrode arrays (Browne et al. 2016; Pinyon et al. 2014a). These “bionic ear” arrays
are the neural interface that enables restoration of hearing to people with profound
sensorineural hearing loss. The operation of the cochlear implant device consists of an
external speech processor that provides digital coding in real time to a control unit
incorporating an inductive power and data pickup permanently implanted behind the
ear. The array consists of a linear series of submillimeter platinum ring electrodes that
are inserted into the spiraling perilymphatic fluid compartment of the cochlea (up to
25 mm). While cochlear implant devices have been continually refined over more than
30 years (Clark et al. 2013; O’Leary et al. 2009), the design of the cochlear implant
arrays themselves has changed very little, with the number of electrodes typically
limited to 22 or fewer. The primary reason for this limitation in array density is
“current spread,” as the cochlear implant array is designed to electrically stimulate
the atrophied spiral ganglion neuron (SGN) somata located a few millimeters distant
from the electrodes, within the central spiraling bony core of the cochlea (Rosenthal’s
canal of the modiolus). This current flow is relatively divergent as it travels from
individual electrodes on the cochlear array to a distant return electrode. The current
pulses passing through Rosenthal’s canal causes groups of SGNs to fire action
potentials which travel to the cochlear nucleus in the brainstem via the centrally
projecting axons. Plasticity of central auditory processing centers in the brain then
enable this crude caricature of normal sound coding to be understood as speech. The
limitation of 22 electrodes reflects the fact that there is no functional benefit gained by
using higher electrode densities. The target neuron groups cannot be differentially
recruited using closer spacing because the electric field broadens across the millimeter
distances between the electrodes and the target cells. If the SGN neurites can be
regenerated and migrate close to the cochlear implant electrodes, then the current pulse
threshold amplitudes for recruitment of the SGNs would be lower and more selective
recruitment of groups of SGNs would be possible. With natural hearing, approxi-
mately a dozen SGNs synapse with each sound transducer inner hair cell and each of
the ~4,000 hair cells from the base to the apex of the cochlea encodes successively
lower frequencies of sound. It is therefore remarkable that bionic-based hearing is so
effective, although pitch perception is inherently poor (Zeng et al. 2014). The devel-
opment of CFE gene therapy evolved from the utility of the focal electric fields
produced by the cochlear implant array. In a proof of principle CFE gene therapy
application, naked plasmid DNA incorporating an expression cassette encoding brain-
derived neurotrophic factor (BDNF) was used. Endogenous BDNF expression that
normally supports SGN synapses with the inner hair cells is greatly reduced following
sensorineural hearing loss. Exogenous BDNF causes extension of neurites from the
atrophied SGN somata in deafened animal models (Pinyon et al. 2014a, b). Using
CFE-based BDNF gene therapy treatment, the mesenchymal cells in closest proximity
to the cochlear implant electrodes were transduced with the BDNF-encoding DNA,
using electrical pulses delivered by the cochlear implant electrodes. As a result of this,
tens to hundreds of mesenchymal cells next to the cochlear implant electrodes started
to secrete recombinant BDNF. This established a molecular gradient sufficient to
recruit SGN neurite regeneration and outgrowth toward the transformed mesenchymal
cells and hence toward the cochlear implant electrodes. As predicted, SGN recruitment
thresholds were lowered and hence cochlear implant-based hearing performance
improved (Pinyon et al. 2014a).
The development of CFE arose from the limited success of conventional “open-
field” electroporation for gene electrotransfer. This was observed in an in vitro adult
guinea pig cochlea model, where voltage pulses were passed across the tissue (the
complete hearing organ) using silver needle electrodes inserted into the base and
apex of the cochlea after perfusion of a saline carrier solution with a naked mam-
malian expression vector plasmid DNA incorporating an expression cassette
encoding the DsRed2 fluorescence reporter driven by the immediate early promoter
of cytomegalovirus (CMV) (pIRES-DsRed2) or a plasmid incorporating a nuclear-
localized green fluorescent protein (GFP) reporter (Housley 2016). Very few trans-
duced (red/green fluorescence) mesenchymal cells were observed lining the
perilymphatic compartment after several days in organotypic culture, despite the
use of long-duration high-voltage pulses (e.g., 100 V, 50 ms) – Fig. 1 insert.
However, these experiments indicated that internally generated high field strengths
would likely prove to be highly effective. This was subsequently verified when an
eight-node cochlear implant was used in the guinea pig cochlea for CFE.
Close-Field Electroporation Ex Vivo Reveals the Significance

of Electrode Configuration in Determining the Shape and Size
of Transduced Fields of Cells
The wiring of the bionic array plays a key role in determining which cells are
transduced. Figure 2 shows the configuration of an experimental eight-node cochlear
implant array with an external connector which facilitates rewiring to gang particular
electrodes. Guinea pig experiments utilized all eight nodes, configured either in an
“alternating” (+/ /+/ /+/ /+/ ) arrangement or “tandem” (+/+/+/+/ / / / )
arrangement.
Experimental Details for CFE: Ex Vivo with Organotypic Tissue

Culture
Using the ex vivo guinea pig Cochlea vs. cochlear model, CFE-based gene
electrotransfer was performed to assess a range of parameters known to generally
affect gene electrotransfer (Burgain-Chain and Scherman 2013; Golzio and Rols
2016; Heller et al. 2011; Satkauskas et al. 2002). This included pulse amplitude
Fig. 1 Comparison of conventional open-field electroporation of the guinea pig cochlea (inset)
using two silver needle electrodes spanning the hearing organ, with close-field electroporation
(CFE) where a cochlear implant electrode array is inserted into the basal turn of the guinea pig
cochlea via the round window. In these examples, electroporation was undertaken in an ex vivo
model where the isolated cochlea was perfused with the naked DNA solution, and in the case of
CFE, the cochlear implant array was inserted into the base of the cochlea and voltage pulses were
delivered. A key element of close-field electroporation is the wiring of the electrode configuration,
where multiple electrodes (nodes) are ganged together. Legend: Scala Tympani (ST)
Fig. 2 Image of cochlear implant array incorporating eight platinum ring electrodes. This is an array
developed for use in animal models, although the dimensions of the electrodes (~350 μm diameter and
~300 μm gaps separated with silicone insulting rings) are similar to those used for the clinical arrays
(part no. z60274, Cochlear Ltd, Sydney Australia; these electrodes can be obtained by contacting the
manufacturer). The electrodes span ~5 mm, which approximates the extent of the basal turn of the
guinea pig cochlea. The lower image provides detail of the array and indicates the “tandem”
configuration that gangs four adjacent electrodes as cathodes and the next four as anodes (+/+/+/+/
/ / / ). This configuration was found to provide significantly higher gene electrotransfer effi-
ciency than the alternating electrode configuration due to focusing of the electric field
(typically 4–100 V), pulse duration (50 ms range, 1 per second), and number of
pulses (typically 5–50) (Pinyon et al. 2014a, b). Briefly, after euthanasia following a
protocol approved by the institutional animal ethics committee, the temporal bones
containing the cochleae are removed and placed into Dulbecco’s Modified Eagle
Medium (DMEM), and then the cochlea bulla was dissected to expose the cochlea.
The cochlea with the round window exposed was then placed onto tissue dampened
with phosphate-buffered saline (PBS) (Fig. 1). The naked DNA, consisting of
circular plasmid DNA (Fig. 3) (2 μg/μl) in Tris-buffered saline (TBS), was perfused
through the perilymphatic compartment by matching a cannula to the round window
face and using a syringe pump. The perfusion of the DNA solution at ~30 μl/min
(20 μl total) flushed the entire perilymphatic compartment within both scala tympani
and scala vestibuli, exiting via a perforation in the oval window.
The cochlear implant array was then inserted into the basal turn of the cochlea,
and electroporation of the mesenchymal cells lining the scalae was achieved using a
square wave electroporator. As noted above, the electrode array was externally wired
into either a tandem or alternating electrode configuration. Following CFE electro-
poration, where, for example, 5 50 ms pulses at 20–40 V were typically utilized,
the cochleae were maintained in a humidity chamber for an additional 20 min (total
time ~30 min), and then the bony lateral (exterior) was resected to facilitate survival
of the interior tissues with organotypic culture. The pieces of cochlea were placed
into DMEM supplemented with 10% fetal bovine serum and 100 units/ml strepto-
mycin and penicillin and placed into an incubator for up to 4 days (37 C, 5% CO2).
The tissue was then fixed (4% paraformaldehyde (PFA) in 0.1 M phosphate buffer
(pH 7.4); 30 min) before transferring to PBS. Imaging of the nuclear-localized GFP
expression across the cochlea tissue was documented using confocal imaging
Fig. 3 Schematic of a plasmid (~7.7 kb) utilized for CFE. The bicistronic vector included a FLAG-
tagged brain-derived neurotrophic factor (BDNF), with an internal ribosome entry site (IRES)
element enabling coupled expression of a humanized renilla green fluorescent protein reporter
that incorporated a nuclear localization signal (GFPnls). Expression was under the cytomegalovirus
promoter (CMVp). Legend: rat brain-derived neurotrophic factor (rBDNF) and aminoglycoside
phosphotransferase (aph), poly-A tail (pA), E. coli origin of replication (OriR)
(488 nm excitation; 493–598 nm emission) (Fig. 4). In addition, direct evidence of

the translation of the BDNF therapeutic gene construct was achieved using anti-
FLAG immunofluorescence. Control experiments that included loading of the plas-
mid DNA into the cochlea and insertion of the bionic array, but no CFE, failed to
produce GFP positive cells.
Three key findings arose from these experiments. Firstly, the expression was
limited to the basal turn of the cochlea, despite the fact that the naked DNA solution
was present in all four turns. This indicated that proximity to the electrode array was
a critical factor. Secondly, there were significantly greater numbers of GFP-positive
mesenchymal cells when the tandem configuration was used compared with when
the alternating configuration was used (Pinyon et al. 2014a). This was associated
with the unanticipated finding that the tandem configuration could achieve efficient
gene electrotransfer with fewer pulses and lower voltages than the alternating
configuration, despite the fact that apart from this wiring, other parameters, including
electrode surface area, were identical (Fig. 2c, d of Pinyon et al. 2014a). In combi-
nation with subsequent analysis using a high-throughput human embryonic kidney
(HEK293) cell monolayer model, these findings reflect the inherent nature of bionic
array-based CFE, namely, that the linear array wired in the tandem configuration
provides focusing of the local current path and hence very high strength, but spatially
constrained, electric fields (see below).
Close-Field Electroporation for Cochlear Gene Therapy
CFE-based gene electrotransfer for spatially constrained gene therapy was validated
in vivo in a chemically deafened guinea pig model (Pinyon et al. 2014a). Gene
electrotransfer with the BDNF-encoding plasmid was performed 2 weeks after a
combination treatment with the diuretic frusemide (furosemide) and the
aminoglycoside antibiotic kanamycin, which causes loss of the cochlear sensory
hair cells and the peripheral neurite processes of the SGNs within the osseous spiral
lamina (after Richardson et al. 2009). Regrowth of the SGN peripheral neurites was
confirmed at 7 days post- CFE in the cochlea receiving the gene therapy, while the
other cochlea in each animal provided a control. Figure 5 shows the regeneration of
cochlear nerve within the (left) treated cochlea, where the SGN somata were larger in
size than the atrophied SGN somata of the untreated cochlea, and the neurites
extended back out through the osseous spiral lamina and down into scala tympani
while developing terminal fine ectopic branching reminiscent of neurite extension
during cochlear development (Greenwood et al. 2007). This SGN neurite outgrowth
was constrained to the basal half of the cochlea, suggesting that the basal turn gene
delivery causes limited functionally effective diffusion of the recombinant BDNF
with ex vivo and in vivo analysis of the CMVp-BDNF-IRES-GFPnls plasmid gene
electrotransfer data. This contrasts with the outcome of viral vector-based
neurotrophin gene therapy studies where the growth activity is throughout the
cochlea and the extent of neurite outgrowth is considerably greater and results in
disruption of the place-dependent neurite localization (Wise et al. 2010).
Fig. 4 Examples of nuclear-localized GFP expression by mesenchymal cells on the modiolar wall
of guinea pig cochleae after 72 h of tissue culture following ex vivo close-field electroporation using
an eight-node cochlear implant wired in the tandem configuration (+/+/+/+/ / / / ). ST scala
tympani, SV scala vestibuli. Note the restriction of gene expression to the basal turn, which was the
location of the array insertion. The more apical turns lacked GFP expression (only the basal and
second turn regions for the four cochlear turns are shown). (a) 40 V pulses (20) at 50 ms duration;
(b) 20 V pulses (10) at 50 ms duration; (c) 20 V pulses (5) at 50 ms duration
The in vivo cochlear CFE gene electrotransfer followed the protocol as for
ex vivo studies, with the use in some experiments of plasmid gutted of the BDNF-
coding element, alongside chronic implantation of the cochlear implant array. Those
control experiments lacked regeneration of the peripheral neurites in the treated
cochlea and failed to exhibit functional improvement of cochlear implant perfor-
mance measured using electrically evoked auditory brainstem responses (Fig. 6)
(Pinyon et al. 2014a).
Fig. 5 Images of the spiral ganglion neurons from the left (CFE-based BDNF gene therapy treated)
and right (control) cochleae of a deafened guinea pig. CFE-based gene electrotransfer of a BDNF
gene cassette in a naked plasmid under the CMV promoter causes regeneration of the peripheral
neurites of spiral ganglion neurons in the basal turn region of the left cochlea (arrow heads indicate
the extent of regrowth of the peripheral neurites). The same region of the right cochlea from this
animal reveals the absence of the peripheral neurites and atrophy of the somata. Deafening was
undertaken using a combination treatment of furosemide and kanamycin 2 weeks prior to CFE gene
electrotransfer of the BDNF-encoding plasmid to mesenchymal cells in the scala tympani (ST)
perilymphatic compartment. The innervation, shown here 2 weeks post-gene delivery, is imaged
using β-tubulin immunofluorescence (green), with the structure of the cochlea outlined by DAPI
nuclear labeling. Previously published in The Scientist (Yandell 2014). Legend: Spiral Ganglion
Neuron (SGN), Osseous Spiral Lamina (osl) Scala Media (SM)
In Vitro Close-Field Electroporation Using HEK293 Cell

Monolayers
The principal parameters underlying bionic array-based CFE have been character-
ized using a high-throughput human embryonic kidney (HEK293) cell monolayer
bioassay (Browne et al. 2016). Such bioassays have been effective in the past for
analysis of electric fields for gene electrotransfer (Rebersek et al. 2007). The
HEK293 cells were grown on coverslips to ~80% confluence in DMEM with 10%
heat-inactivated fetal bovine serum (HI-FBS), 1% penicillin/streptomycin mix, 1%
non essential amino acids. The mammalian expression vector was a similar plasmid
to that shown in Fig. 3. As well as the tandem and alternating electrode configura-
tions, other electrode wiring patterns were also studied, including using just two
electrodes with a range of separations (1 + 2, 300 μm separation; 1 + 5, 2.5 mm
separation; 1 + 8, 5 mm separation). In addition to the cochlear implant configura-
tion, pulse parameters that were characterized for gene transfer based on numbers of
HEK293 cells with nuclear-localized GFP fluorescence were voltage, pulse duration,
pulse number, pulse separation, and plasmid DNA concentration in TBS. The CFE
was undertaken by removing the coverslips with the HEK293 cell monolayers from
the incubator, removing them from the media and placing them on a microscope
stage, adding 20 μl of the plasmid DNA (as above), then overlaying the cochlear
Fig. 6 Assessment of functional recovery of cochlear nerve-derived activity in central auditory

pathways following CFE-based BDNF gene electrotransfer into the deafened guinea pig cochlea. The
GFP only column (control animal) shows the electrically evoked auditory brainstem response (eABR)
when 100 μs constant current pulses are delivered with increasing amplitude via the tandem configured
chronically implanted cochlear implant 2 weeks after CFE gene electrotransfer of a control plasmid
lacking the BDNF-coding element in the bicistronic expression cassette. The gene therapy was provided
2 weeks following ototoxic deafening as shown in Fig. 5. The blue arrow demarks the response
threshold (~353 μA). The right column shows the eABR measurements made following CFE-based
BDNF gene therapy treatment of a separate deafened guinea pig. These eABR recordings show a
substantially lower eABR threshold (140 μA) and, critically, showed progressive recruitment of the
spiral ganglion neuronal population with increasing stimulus intensity. These data confirm that the
BDNF treatment closed the neural gap between the cochlear implant array and the primary auditory
neurons. The eABR traces are averaged from 512 repetitions of the stimulus and are recorded using
subdermal platinum electrodes placed at the skull vertex and close to the temporal bone on the side of
the ear receiving the gene therapy. The differential in the cochlear implant-driven eABR responses
extended beyond a month
implant array onto the coverslip using a micromanipulator, with positioning aligned
to a mark on the coverslip for orientation of the GFP fluorescence readout. Imme-
diately after CFE, the coverslips were placed in a humidity chamber for 2 min and
then returned to pre-warmed culture media in culture plates in the incubator (37 C,
5% CO2). GFP fluorescence was evident within 18 h, and the cells were maintained
in culture for up to 48 h prior to imaging at low power using a confocal microscope.
Such experiments illuminated a key finding around CFE with bionic arrays, namely,
that the shape of the region of GFP reporter-positive cells was dependent upon the
Fig. 7 Grayscale images of HEK293 cell monolayers exhibiting fluorescence arising from expres-
sion of the GFPnls reporter following “close-field” electroporation of plasmid DNA with Tris-
buffered saline. Tandem configuration eight electrode cochlear implant array (From Pinyon 2016).
A feature of the tandem wired electrode configuration (+/+/+/+/ / / / ) is that the GFP-positive
cells define a spherical region. In addition, fewer (2) pulses of longer duration pulses (100 ms)
achieve similar transduction efficiency to more (5) shorter (50 ms) pulses, so overall the charge
transfer is less if longer pulses are used. (a) 40 V pulses (5) at 50 ms duration; (b) 40 V pulses (2) at
100 ms duration; (c) 20 V pulses (5) at 50 ms duration; (d) 20 V pulses (2) at 100 ms duration
cochlear implant array configuration. Coverslips using the tandem configuration had
circular-shaped regions of transduced cells centered around the middle of the array,
whereas the alternating configuration caused linear-shaped regions of fluorescing
cells aligned along the length of the array (Figs. 7 and 8). This reflects the bioreporter
representation of the underlying shape of the different electric fields around the
cochlear implant array created by the tandem and alternating electrode configura-
tions (below). Both these array configurations produced substantially greater gene
Fig. 8 Grayscale images of HEK293 cell monolayers exhibiting fluorescence arising from expres-
sion of the GFPnls reporter following close-field electroporation of plasmid DNA with Tris-buffered
saline. Alternating configuration eight electrode cochlear implant array (From Pinyon 2016). A
feature of the alternating electrode configuration (+/ /+/ /+/ /+/ ) is that the region of GFP
positive cells is aligned with the array. As for the tandem configuration, fewer (2) pulses of longer
duration pulses (100 ms) achieve similar transduction efficiency to more (5) shorter (50 ms) pulses;
therefore charge transfer is less with longer pulse durations for the same gene electrotransfer.
(a) 40 V pulses (5) at 50 ms duration; (b) 40 V pulses (2) at 100 ms duration; (c) 20 V pulses
(5) at 50 ms duration; (d) 20 V pulses (2) at 100 ms duration
Fig. 9 Near dial-up precision

in gene electrotransfer can be
achieved using the tandem
bionic array configuration
(40 V, 5 pulses) alongside
increasing pulse duration.
Bioassay using numbers of
GFP-positive HEK293 cells.
Mean s.e.m., n = 3–9.
Note the high correlation
coefficient for the single
exponential curve fit, showing
that 100 ms duration
approaches optimal for 2 μg/μl
plasmid DNA in Tris-buffered
saline
electrotransfer than the single pairs of electrodes with the minimum (300 μm
separation), with further rapid attenuation of gene transfer efficiency with increasing
separation of the pairs of electrodes (Browne et al. 2016). Such studies, particularly
those using the higher efficiency tandem configuration, demonstrated that transduction
efficiency improved as pulse duration was extended, with maximum transduction
efficiency around 100 ms (Figs. 7, 8, and 9). This effect is consistent with previous
reports using conventional open-field electroporation (Heller et al. 2011). High volt-
ages (e.g., 40 V) resulted in a considerable increase in the area of transformed cells
compared with lower voltages (Figs. 7 and 8), although increasing pulse numbers
likely exacerbated gassing from the platinum electrodes and caused disruption of the
cells under the array. This physical and potential toxicity effect likely accounts for the
reported falloff in numbers of GFP-positive cells with larger numbers of pulses (>20)
and longer pulse durations (>100 ms); inter-pulse interval had no effect on numbers of
transduced cells (separation 50 ms–1 s); using 100 ms pulses enabled translation of
HEK293 cell monolayer findings to reduced voltage dependence (to 10 V) for in vivo
guinea pig cochlea gene electrotransfer (Browne et al. 2016).
Electric Field Focusing Through Variable Configuration

of Cochlear Implant Electrode Arrays
Sampling of the voltage around the eight-node cochlear implant array as voltage
pulses were delivered to it, using platinum probe and reference electrodes isolated
from the voltage pulses, enabled reconstruction of the electric fields for the range of
wiring configurations used for GFP readout of gene electrotransfer in HEK293 cell
monolayers (Browne et al. 2016). This revealed, as predicted, that in the tandem
configuration, a null extended orthogonally from the point between electrodes 4 and
5, which is the boundary between the four ganged anodes and the four ganged
cathodes. The highest magnitude-sampled voltages were at the ends of the array, but
Fig. 10 Point sampling of voltage within the electric field alongside a 5 mm eight electrode
cochlear implant array wired in the tandem configuration (+/+/+/+/ / / / ). Voltage in the field
(Vf) is shown in response to 5 V applied to the array as shown in inset. The steepest change in
voltage over distance (electric field strength) occurs in the mid-region of the array. Upper image is a
video montage showing the sequential placement of the probe used to measure Vf relative to the
eight-node cochlear implant array
the region of greatest field strength (change in voltage over distance) was in the area
surrounding the mid-position of the array, which closely matched the distribution of
the GFP reporter cells. Thus the CFE-based gene electrotransfer is not dependent
upon the magnitude of the voltage transient for the pulses but rather to the local
transcellular field strength in a place-specific manner. Figure 10 shows an example of
the measurement of the electric field along the length of the cochlear implant
electrode array using an isolated Pt electrode sensor on a translational stage with
5 V applied to the array. This generated a maximum field strength for the tandem
array configuration of ~2.6 V/cm. Allowing for the polarizing potential of the
platinum electrodes (~2.5 V), an estimate of the maximum field strength adjacent
to the cochlear implant array at 20 V applied is ~21 V/cm, hence at a cellular level,
pancellular field gradients providing efficient gene electrotransfer are ~2.1 mV/μm,
generated in a highly nonlinear state within millimeters of the bionic array. In
comparison, conventional open-field electroporation typically requires hundreds of
volts to create a broadly linear field of equivalent intensity, with consequent toxicity
and limited spatial control of the gene electrotransfer, even when using arrays of
needle electrodes in applications such as gene therapy based on stimulation of
immune responses in cancers (Heller 2016; Heller and Heller 2010).
Extension of Close-Field Electroporation
Measurement of the electric fields around the cochlear implant array across various
wiring configurations of the electrodes facilitated the understanding of minimum
parameters to achieve efficient electric field focusing. To test the concept that the
tandem configuration provides an optimum compression of the current path along
the axis of the array, this was modeled using two electrodes, each equivalent in
dimensions to the four ganged electrodes forming either the anode or cathode in the
tandem eight-node array. In this prototype, the gap between the electrodes was
insulated with epoxy, and a heat shrunk plastic sheath enabled delivery of the
DNA through the initial electrode port (Fig. 11). This “gene delivery probe” was
tested using the HEK293 cell monolayer, with standard CFE electroporation param-
eters (20 V, 5 50 ms pulses) and produced regions of GFP-positive cells equiv-
alent to that achieved using the bionic array-based CFE (Fig. 12).
Fig. 11 Prototype gene delivery probe developed using the submillimeter dimensions of the cochlear
implant array but comprising two electrodes, each equivalent to the four ganged electrodes used as an
anode or cathode for the tandem configuration of the cochlear implant array that has this highest gene
electrotransfer efficiency with CFE. Inset shows the emission of the plasmid DNA solution from a
port located between the two electrodes. This ensures control of the DNA concentration in the vicinity
of the electrodes, which is a critical feature of close-field electroporation
Fig. 12 HEK293 cell monolayer showing nuclear-localized green fluorescence protein (nlsGFP)
following close-field electroporation (CFE) with a prototype two-electrode configuration of CFE
gene delivery probe (see Fig. 11). (a, b) Show spherical fields of transformed cells, as anticipated
based on field focusing akin to that achieved using the cochlear implant array wired in the tandem
configuration. (c) control where the DNA solution was placed onto the coverslip of HEK293 cells
on the microscope stage, and the gene delivery probe was lowered onto the cells, but no electrical
pulses were delivered. Arrows define the perimeter of the field of cells expressing GFP
Modeling of the electric fields generated by CFE-based gene electrotransfer is a

valuable tool in refining the design of both electrode geometries as well as electrode
return path and stimulus configurations. Figure 13 demonstrates a basic model of the
cochlear array placed linearly in a saline bath with electroporation waveforms
applied in both alternating and tandem electrode configurations. This modeling
approach has the potential to be extended to incorporate a distant monopolar ground
return configuration along with the alternating and tandem configurations to create
more refined field shapes (Khalili Moghadam et al. 2013).
0.6
0.4
0.2
0
-5 -5
b Multislice : log10(ec.normE)
5
4.5
4
3.5
3
2.5
2
1.5
1
y 0.5
x 0
y
C Multislice : log10(ec.normE)
5
4.5
4
3.5
3
2.5
2
1.5
1
y
0.5
x 0
Fig. 13 A finite element model (a) of the eight electrode cochlear implant array placed in a saline
bath. The electric field generated by the model for a tandem CFE array configuration (b) (+/+/+/+/ / /
/ ) has a spherical distribution about the center. The same model but for an alternating CFE array
configuration (c) (+/ /+/ /+/ /+/ ) shows a reduced field intensity that has a linear dispersion. Models
were generated and solved using the COMSOL software (COMSOL Multiphysics, Burlington USA)
Conclusions
Close-field electroporation, which is defined as the application of a nonlinear electric

field projected into a target tissue, arose from the application of the cochlear implant
electrode arrays for delivery of voltage pulses into the small cylindrical chambers of
the cochlea. This perilymphatic chamber has dimensions closely matched to the
submillimeter diameter of the linear bionic array. The electrical pulses which were
applied were outside the operational capacity of the cochlear implant controller used
for hearing and were driven using a conventional isolated constant voltage
electroporator which could be wired to drive the array of electrodes in varying
configurations. Delivery of a neurotrophin gene therapy cassette in a naked plasmid
by CFE resulted in local transduction of the mesenchymal cells lining the
perilymphatic compartment of the cochlea, but only in the region where the cochlear
implant array was closely positioned to the target cells, as evident from GFP reporter
fluorescence. The cells produced the BDNF, and in a deafened guinea pig model, this
recombinant BDNF was sufficient to locally drive regrowth of the peripheral neurite
processes of the primary auditory neurons into the vicinity of the cochlear implant
array. This improved the neural interface by closing the gap between the SGNs and
electrodes, which was supported functionally by significant reductions in the stim-
ulus thresholds and expansion of the recruitment bandwidth, measured by electri-
cally evoked ABRs. These animal studies determined that a particular configuration
referred to as the tandem array had unanticipated efficiency in gene electrotransfer,
which was found, though CFE studies with HEK293 cell monolayers, to be due to
nonlinear compression of the electric field with respect to the linear electrode array.
Control of the electrode configuration enabled the shape of the region of cells that
were transduced to be manipulated, while selection of voltage pulse parameters
enabled dial-up precision in gene delivery in an HEK293 cell monolayer model.
These factors combine to make CFE a highly flexible gene electrotransfer platform
which shows promise for use as a gene therapy adjuvant to improve hearing with
cochlear implants. The CFE platform also has the potential for extension of appli-
cation to other tissues and experimental applications, where it could provide robust
and highly controlled delivery of naked DNA payloads.
Acknowledgments Dr. Amr Al Abed is thanked for the electric field modeling shown in Fig. 12.
Funded by an Australian Research Council Discovery Grant (DP151014754). All data derived from
animal experiments followed protocols approved by the UNSW Animal Care and Ethics
Committee.
Cross-References

▶ Tissue Engineering with Electroporation
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Combined Treatment of
Electrochemotherapy with Irradiation 89
Simona Kranjc, Urska Kamensek, Maja Čemažar, and Gregor Serša
Abstract
Radiotherapy is the use of ionizing radiation in the treatment of malignant tumors.
It is one of the main treatment modalities for many forms of cancer, with more
than half of all cancer patients receiving radiation therapy at some point in their
treatment. Despite the technical progress in targeting ability of radiotherapy, poor
therapeutic window remains a problem; therefore in clinical practice radiotherapy
is commonly combined with chemotherapy. By using electrochemotherapy, the
uptake of the non or poorly permeable chemotherapeutic drugs, bleomycin and
cisplatin, into the tumors can be increased and thus also their radiosensitizing
effect. In combined treatment of electrochemotherapy preceding irradiation, an
increased radio-response was demonstrated with an enhancement factor of up to
4.6, regardless of using radiomimetic (bleomycin) or radiosensitizing (cisplatin)
drug. The improved antitumor effectiveness is mainly enabled by increased drug
accumulation in the tumors. Radiosensitization was demonstrated in vitro and
in vivo in variable tumor models irradiated in a single dose and a fractionated
regime. Low and acceptable radiation damage of normal tissue in the irradiation
field was observed. All the data provided a starting point for translation of
combined electrochemotherapy and tumor irradiation into the clinic, which has
already begun.
S. Kranjc (*) • U. Kamensek • G. Serša

e-mail: skranjc@onko-i.si; ukamensek@onko-i.si; gsersa@onko-i.si
M. Čemažar

DOI 10.1007/978-3-319-32886-7_61
1700 S. Kranjc et al.
Keywords
Electrochemotherapy • Cisplatin • Bleomycin • Irradiation • Radiosensitization •
Radio-response
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1700
Electrochemotherapy and Why Combine It with Irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1702
In Vitro Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1703
In Vivo and Clinical Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1705
Side Effects of Electrochemotherapy and Irradiation Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . 1712
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1713
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1713
Introduction
Radiation is one of the oldest and most common treatments of cancer and over 50%
of patients receive it at some time during their course of treatment (Begg et al. 2011).
It is a localized treatment, valuable and successful in confining cancer locally.
Radiation can be used with the purpose of cure or as palliative treatment to alleviate
the symptoms of cancer. Although the technological improvements in the delivery of
radiation that reduce side effects on normal tissues in irradiated field, while enhanc-
ing the effect on the tumor, were made in the last decade, new approaches for
widening of the therapeutic index are still being sought (Kamrava et al. 2009).
Understanding of cancer as a multigenetic and multicellular disease led to the
development of multimodal treatment approaches of cancer. Radiation can be used
in combination with surgery, immunotherapy, hormonal therapy, chemotherapy, or
targeted therapies (Begg et al. 2011; Joiner and Kogel 2009; Kamrava et al. 2009).
Treatment that combines chemotherapy with radiation therapy has been termed
chemoradiotherapy. Systemic treatments with chemotherapeutic drugs given in
different regimens in combination with radiotherapy have been shown to greatly
improve the overall survival of cancer patients. In fact, certain chemotherapeutic
drugs can sensitize tumors to irradiation and provide a systemic effect in addition to
the local effect of the radiation; thus an additive or even synergistic effect in local
tumor control has been demonstrated (Begg et al. 1994; Seiwert et al. 2007; Strojan
et al. 2016).
Enhanced radiation sensitivity occurs through different mechanisms, i.e.,
increased DNA damage and decreased DNA repair, interference with the cell
cycle, hypoxic sensitization and damage of tumor vasculature, immunological
effects, and others (Joiner and Kogel 2009). In combined modality regimen in the
clinic, the radiosensitization of tumor cells was demonstrated when systemic drug
administration preceded tumor irradiation (Joiner and Kogel 2009; Seiwert
et al. 2007). However, besides potentiation of the radiation response, systemic
chemotherapy also contributes to local or systemic toxicity (Joiner and Kogel 2009).
89 Combined Treatment of Electrochemotherapy with Irradiation 1701
Among chemotherapeutic drugs in combined modality, mostly cisplatin and other

platinum-based agents are used in chemotherapeutic schemes. Cisplatin binding to
DNA causes single and double DNA strand cross-links that differ from single and
double DNA strand breaks caused by radiation, which are reflected in decreasing
efficiency of repair mechanisms in sublethal and potentially lethal DNA damages
after combined treatment modality (Joiner and Kogel 2009). Furthermore, cisplatin
exhibits radiosensitization through cell cycle arrest, suppression of tumor neovascu-
larization, and especially in hypoxic cells through the formation of OH (hydroxil)
radicals by scavenging of hydrated electrons (Joiner and Kogel 2009; Seiwert
et al. 2007).
That cisplatin radiosensitizes tumor cells by complex interaction, which was
shown in several in vitro and in vivo studies (Begg et al. 1994; Gorodetsky
et al. 1998). Results of preclinical studies using different schedules and dosages of
each therapeutic ranged from not significant interaction to pure additive effect and
also synergistic interaction. Synergistic effect was exhibited if cisplatin was given
shortly before or immediately after irradiation in a single dose or fractionated
regime, confined mostly to hypoxic cell population (Begg et al. 1994; Gorodetsky
et al. 1998; Lagrange et al. 1993). On the contrary, when the time interval between
cisplatin administration and irradiation was longer than 2 h, only an additive effect
was observed (Joiner and Kogel 2009; Rantanen et al. 1995; Seiwert et al. 2007).
Several clinical studies have shown synergy of combined treatment, which leads to
significant improvement of radio-response, resulting in better local tumor control
and overall survival. Clinical data of concurrent chemoradiotherapy confirmed the
radiosensitization with cisplatin in various cancers (head and neck, gynecological,
lung, breast, glioma) (Joiner and Kogel 2009; Strojan et al. 2016), and
chemoradiotherapy with cisplatin is nowadays standard therapy in management of
head and neck cancer, significantly improving locoregional failure-free survival at
5 years (Strojan et al. 2016).
Bleomycin is another chemotherapeutic drug frequently used in chemotherapeu-
tic schemes in combination with radiotherapy, mainly in the treatment of head and
neck tumors (Zakotnik et al. 2007). Bleomycin belongs to a group of antitumor
glycopeptide antibiotics and can induce single and double DNA breaks (Dorr 1992).
The major mechanism of bleomycin action is the formation of pseudoenzyme due to
the chelation of metal ions, which by reacting with oxygen free radicals causes the
DNA breaks. Since bleomycin predominantly acts well on oxygenated cells, it is
necessary to combine it with other chemotherapeutic drugs to hit the wide specter of
biological targets in tumors (Joiner and Kogel 2009).
By mode of action, bleomycin is classified as radiomimetic and not radio-
sensitizer (Joiner and Kogel 2009). In combined modality of bleomycin and irradi-
ation, a potentiation of radiation response was demonstrated, mainly due to
pronounced enhancement of bleomycin-induced DNA damages by radiation (Joiner
and Kogel 2009). Preclinical studies demonstrated that bleomycin has to be admin-
istered up to 20 min prior to a single-dose or fractionated irradiation schedule in
order to achieve the best potentiation of radiotherapy (Jiang et al. 1989; Joiner and
Kogel 2009). Chemoradiotherapy with bleomycin alone or in chemotherapy regime
with other drugs concurrently with a fractionated irradiation regime was also proven
effective in the clinical setting, resulting in improvement of locoregional control and
survival of patients. Furthermore, radiosensitization of head and neck tumors with
bleomycin has become an established treatment approach (Zakotnik et al. 2007).
Taken together, combined modality treatment of cancer by radiation and chemo-
therapy, i.e., chemoradiotherapy, improves locoregional control, disease free and
overall survival, and is now standard of care for several types of tumors. However,
unwanted side effects and high percentage of mortality still remain a great challenge
in disease control. Therefore, better understanding of biological properties of cancer
cells, accurate planning, and technological development of combined treatment is
needed.
Electrochemotherapy and Why Combine It with Irradiation
The major limitation of chemoradiotherapy is the cumulative normal tissue toxicity

and insufficient effectiveness of the available drug in most of the tumors. Therefore,
research is aimed towards the innovative approaches to target cancer cells more
specifically and locally, in order to improve treatment and prevent side effects. A
promising approach to increased delivery of chemotherapeutic drugs to cells and
tumors is by various delivery systems, using tumor-specific antibodies, magnetic
particles, liposomes, or other vehicles, or by selectively increasing permeability of
the plasma membrane by chemical or physical methods (Tiwari et al. 2012). Among
them electroporation could be used to increase intracellular accumulation of drugs,
which consequently increase radiosensitization of tumor cells (Yarmush et al. 2014).
The phenomenon of electroporation by using short high-voltage electric pulses,
which induces membrane permeabilization, allowing drug uptake into the cells, was
discovered in the early 1980s of the last century (Yarmush et al. 2014). Local
application of electric pulses (electroporation) to the tumor combined with local or
systemic administration of poorly or nonpermeable chemotherapeutic drugs was
named electrochemotherapy (see ▶ Chap. 93, “Electrochemotherapy and Its Clinical
Applications”) (Mir et al. 1991; Yarmush et al. 2014).
Among chemotherapeutic drugs used in the treatment of cancer, bleomycin and
cisplatin were demonstrated as the most potent in electrochemotherapy. By using
electrochemotherapy, the potentiation (several fold) of cytotoxicity and antitumor
effectiveness of bleomycin and cisplatin has been proven in several preclinical
studies. An increase in drug delivery to the cells and tumors was demonstrated to
be the main underlying mechanism for improved cytotoxicity in vitro and antitumor
effect in vivo (Yarmush et al. 2014). Furthermore, human (see ▶ Chaps. 93,
“Electrochemotherapy and Its Clinical Applications” ▶ 96, “Electrochemotherapy
of Cutaneous Metastases,” and ▶ 102, “Electrochemotherapy of Head and Neck
Cancer”) and veterinary (see ▶ Chap. 105, “Electrochemotherapy in Veterinary
Oncology”) clinical studies dealing with cutaneous and subcutaneous tumor nodules
of different histology have shown similar response rate, approximately 80%
objective responses and approximately 70% long-lasting complete responses (Mali

et al. 2013). Electrochemotherapy is mainly used as adjuvant or palliative therapy for
solid cutaneous melanoma, carcinoma, and sarcoma metastases (see ▶ Chaps. 93,
“Electrochemotherapy and Its Clinical Applications,” ▶ 96, “Electrochemotherapy
of Cutaneous Metastases,” and ▶ 102, “Electrochemotherapy of Head and Neck
Cancer”) and with technological improvement became feasible also for deep-seated
liver metastases (see ▶ Chap. 93, “Electrochemotherapy and Its Clinical Applica-
tions”) (Yarmush et al. 2014).
The effectiveness of combined electrochemotherapy with cisplatin or bleomycin
and radiotherapy was tested in several preclinical studies, which demonstrated that
electrochemotherapy acts synergistically with radiotherapy, exerting the radio-
sensitizing effects on different types of cells in vitro and tumors in vivo. The
encouraging results led to a few clinical reports.
In Vitro Data
Only a few in vitro studies evaluated combined treatment of electrochemotherapy

with irradiation. In the first study, cisplatin-sensitive SCK mammary carcinoma cells
turned out to be more radiosensitive than cisplatin-resistant EAT-E (Ehrlich-Lettre
ascites carcinoma) cells (Kranjc et al. 2003a). In fact, intrinsic radiosensitivity
determined at D0 (D0- a dose that reduces survival from 0.1 to 0.037) was 1.2 Gy
for SCK cells and 2.0 Gy for EAT-E cells. Exposure of cells to cisplatin, prior to
irradiation with 4 Gy, significantly increased radiation response of cells. Moreover,
concomitant exposure of cells to cisplatin and application of electric pulses, i.e.,
electrochemotherapy, before irradiation additionally decreased cell survival in both
cell lines to the same level (IC50 = 0.9 0.2 μg/ml for SCK cells and IC50 = 0.9
0.3 μg/ml for EAT-E cells). Thus electrochemotherapy of cells with cisplatin
combined with irradiation was proven to radiosensitize the cells with different
intrinsic chemo- and radiosensitivity, resulting in decreased cell survival by 3.7-
fold in SCK cells and by 2.4-fold in EAT-E cells compared to electrochemotherapy-
treated cells only. Similar results were obtained on moderately radiosensitive LPB
sarcoma cells (D0 = 1.6), demonstrated by two fold decrease of cell survival (Fig. 1)
(Kranjc et al. 2003b). Moreover, radiosensitizing effect (1.4-fold) of cisplatin
electrochemotherapy in combination with graded doses of irradiation from 2 to
8 Gy was shown. However, radiation response was enhanced to some extent also
by electroporation only (1.1-fold), while cisplatin alone treatment at low concentra-
tion had no effect.
Another drug used in electrochemotherapy is bleomycin (see ▶ Chap. 93,
“Electrochemotherapy and Its Clinical Applications”), which is cytotoxic if only
few hundred molecules are present inside the cells (Yarmush et al. 2014). Thus, by
increasing its uptake after electropermeabilization of cells, the radio-response of
cells could be potentiated. In fact, the electroporation of cells with low doses of
bleomycin was shown to have a good radiosensitizing effect (Kranjc et al. 2005).
Fig. 1 Survival curves of LPB sarcoma cells irradiated with graded doses only (IR) or irradiated
after combined treatment with cisplatin (CDDP) or bleomycin (BLM) and application of electric
pulses (electrochemotherapy, ECT) (ECT BLM + IR, ECT CDDP + IR) (Adapted from Kranjc
et al. (2003b, 2005))
Treatment of cells either with bleomycin or electroporation alone had some radio-
sensitizing effect (around 1.2-fold), whereas after bleomycin electrochemotherapy
the radiation response was increased for a factor of 1.5 (Fig. 1).
In summary, it was determined from in vitro experiments, radiosensitization by
electrochemotherapy with bleomycin was more pronounced compared to electroche-
motherapy with cisplatin, (1.4-fold for cisplatin and 1.5-fold for bleomycin) (Kranjc
et al. 2003b). However, the exposure of cells to electric pulses alone prior to
irradiation potentiated radiation response as well (up to 1.3-fold). Therefore,
obtained radiosensitization of cells exposed to electrochemotherapy might not be
ascribed just to the increased drug concentration after electroporation but also to the
effect of exposure of cells to short high-voltage electric pulses (see ▶ Chap. 28,
“Involvement of Reactive Oxygen Species in Membrane Electropermeabilization”)
(up to 1.2-fold) (Kranjc et al. 2003b) (Fig. 1). Namely, reversible electroporation (see
▶ Chap. 28, “Involvement of Reactive Oxygen Species in Membrane Electroper-
meabilization”) induces the generation of free reactive oxygen species, which are
present only for a short period of time after electroporation and can damage proteins,
lipids, and DNA (Shil et al. 2005). Reactive oxygen species are known to contribute
to radiation damage of the cells. Therefore, predisposition of cells to irradiation by
electroporation is also expected and was demonstrated (West 1992; Shil et al. 2005).
In fact, a significant increase in production of free reactive oxygen species after
combined treatment of Ehrlich ascites carcinoma cells with radiation and electropo-
ration was obtained, resulting in significant enhancement of radio-response after
electroporation (Shil et al. 2005). Thus, presumably DNA damages due to bleomycin
and free reactive oxygen species sensitize cells to radiation, and, consequently,
decrease cell survival.
Taken together, these data indicate that electroporation of tumor cells with
cisplatin or bleomycin, irrespective of the varying degrees of chemo- or radiosensi-
tivity of cells, potentiates radiosensitizing effect of either drug used.
In Vivo and Clinical Data
Until now, there have only been a few preclinical in vivo studies evaluating
electrochemotherapy and tumor irradiation. Combination of electrochemotherapy
with cisplatin or bleomycin and single or fractionated irradiation regime were tested
in different mouse experimental tumor models (EAT carcinoma; LPB and SA-1
sarcoma; and CaNT mammary adenocarcinoma) and one spontaneous mammary
carcinoma model (Sersa et al. 2000; Kranjc et al. 2003a, 2005; Raeisi et al. 2012).
A proof of principle, that electroporation can increase radiosensitizing effect of
cisplatin, was first reported by Sersa et al. 2000. Single treatment of induced EAT
subcutaneous tumors, either with intravenous injection of cisplatin (4 mg/kg) or
application of electric pulses only, delayed tumor growth moderately, without any
tumor cure, while electrochemotherapy alone resulted in 12% tumor cures and
irradiation with 15 Gy alone in 27% tumor cures. Administration of cisplatin alone
or application of electric pulses alone 20 min prior to irradiation increased the radio-
response of tumors to 73% and 54% tumor cures, respectively. Consistently,
electrochemotherapy given 20 min prior to tumor irradiation increased the radio-
response of tumors even more, resulting in 92% tumor cures (Sersa et al. 2000).
Therefore, due to increased delivery of cisplatin into cells by electroporation, the
radiosensitization of tumors with cisplatin was demonstrated. The observed radio-
sensitization of electroporation was hypothesized to be, on one hand, due to the
effect of electroporation on intracellular targets or to the perturbation of the plasma
membrane, and, on the other hand, possible mechanisms could also involve the
effects of electric pulses on modification of tumor blood flow and oxygenation
(Fig. 2).
Next, combined electrochemotherapy treatment with cisplatin and tumor irradi-
ation with graded doses was evaluated on LPB sarcoma tumors in order to achieve
tumor curability dose of radiation (TCD50) (Fig. 3) (Kranjc et al. 2003b). In this
study attempts were made to elucidate underlying mechanism of the combined
modality treatment, i.e., tumor blood flow changes and platinum uptake determina-
tion. Local treatment of animals by application of electric pulses to the tumors
20 min prior to tumor irradiation did not affect the response of tumors to radiation,
while the intravenous injection of cisplatin (4 mg/kg) 20 min prior to local tumor
irradiation resulted in a statistically significant enhanced radio-response of tumors.
TCD50 of combined modality treatment in relation to irradiated tumors only was
lowered from 22.1 to 19.6 Gy. However, by electrochemotherapy 20 min prior to
irradiation, a significant enhancement of tumor radio-response was achieved
Fig. 2 Scheme of a tumor nodule treated with electrochemotherapy and irradiation. (a) Three
minutes after systemic administration of cisplatin or bleomycin tumor nodule was treated with
electric pulses (electrochemotherapy) and 20 min after irradiated with single dose. (b) Scheme of
(TCD50 = 14.2 Gy). These data proved that the radio-response of tumors after
electrochemotherapy was significantly increased in relation to only irradiated tumors
(enhancement factor; EF = 1.6) and to tumors treated with cisplatin and irradiation
(EF = 1.4) (Fig. 3) (Kranjc et al. 2003b).
Furthermore, based on the promising radiosensitization results obtained in these
two studies (Sersa et al. 2000; Kranjc et al. 2003b), it was necessary to clarify the
underlying mechanisms of combined treatment modality. Firstly, the increase in
platinum content in the tumors due to electroporation was evaluated as the underly-
ing mechanism of enhanced tumor radio-response (Kranjc et al. 2003b). The plati-
num content in tumors treated with electroporation with or without tumor irradiation
was approximately twice as high as in tumors treated with cisplatin or cisplatin and
irradiation. The observed difference in platinum content occurred already a few
minutes after the treatment and lasted up to 24 h after application of electric pulses to
the tumors (Kranjc et al. 2003b). These results indicated that increased delivery of
cisplatin to tumors by application of electric pulses is probably the main mechanism
Fig. 3 Local tumor control curves of LPB sarcoma tumors treated with combination of systemic
administration of cisplatin (CDDP) or bleomycin (BLM) and application of electric pulses
(electrochemotherapy, ECT) 20 min prior to irradiation (IR) (ECT BLM + IR or ECT CDDP + IR);
enhancement factor (EF); irradiation dose (D) (Adapted from Kranjc et al. (2003b, 2005))
Fig. 2 (continued) tumor nodule after treatment with electrochemotherapy and radiation. Forma-
tion of reactive oxygen species and toxic effect of chemotherapeutic drug due to increased drug
uptake in tumor cells overcame the depletion in oxygen (hypoxia) caused by reduced blood flow
after application of electric pulses. Tumor cells are dying of apoptotic and necrotic cell death,
resulting in good local tumor response
of the observed increase in cisplatin radiosensitization of tumors. Worth mentioning

is also that irradiation of tumors had no influence on the electroporation-mediated
increase in platinum content and the kinetics of platinum washout from the tumors.
As a proposed second mechanism involved in tumor radio-response after
electrochemotherapy are the effects of electrochemotherapy on tumor blood flow.
Namely, the blood flow is strongly associated with oxygen levels in tumors that play
an important role in radiation response (Fig. 2b). Measurements of tumor blood flow
(see ▶ Chaps. 35, “Effects of Reversible and Irreversible Electroporation on Endo-
thelial Cells and Tissue Blood Flow,” and ▶ 39, “Blood Flow Modifying and
Vascular-Disrupting Effects of Electroporation and Electrochemotherapy”) demon-
strated that cisplatin or irradiation monotherapy, as well as combination of both did
not affect perfusion level in the first 24 h after treatment (Kranjc et al. 2003b).
However, the application of electric pulses to the tumors alone, and all treatment
combinations with electroporation, significantly reduced perfusion in tumors imme-
diately after electric pulses were applied, returning to 50% of the pretreatment value
at 24 h posttreatment. The reduced blood flow at the time of irradiation could lead to
radiobiologically relevant hypoxia. Thus, the increased radiation response of LPB
tumors treated with electrochemotherapy was the result of higher platinum content
and cisplatin radiosensitization of hypoxic cells, which has exceeded the reduction in
tumor blood flow and oxygenation. The increased radio-response of solid tumors
treated by application of electric pulses only seems to be tumor type dependent.
Namely, the application of electric pulses potentiated the radio-response in EAT
carcinoma (Sersa et al. 2000; Shil et al. 2005), but not in LPB sarcoma. Proposed
mechanisms could be that EAT carcinoma tumors are more sensitive to radiation
because application of electric pulses generates more free reactive oxygen species
(see ▶ Chap. 28, “Involvement of Reactive Oxygen Species in Membrane
Electropermeabilization”), as shown in vitro (Shil et al. 2005), which prevail over
the effect of reduced tumor blood flow and oxygenation, also caused by application
of electric pulses. Nevertheless, the radio-response of LPB sarcoma tumors could
presumably be ascribed to outweigh the oxygen reduction over the other effects
(Fig. 3).
Further efforts were focused on electrochemotherapy with bleomycin as radio-
sensitizing therapy. To clarify the underlying mechanisms for radiosensitization by
bleomycin electrochemotherapy, tumor oxygenation after combined treatment was
measured in LPB sarcoma tumors (Kranjc et al. 2005). Tumors were treated by
electrochemotherapy with bleomycin (0.5 mg/kg; intravenous injection) 20 min
prior to single-graded doses of tumor irradiation (5–50 Gy). Treatment of animals
with intravenous injection of bleomycin alone or application of electric pulses to the
tumors prior to irradiation of tumors had no effect on local tumor control.
Electrochemotherapy with bleomycin significantly increased radio-response of
tumors compared to irradiation treatment only. The TCD50 value was lowered
from 23.1 Gy, obtained after irradiation alone, to 12.4 Gy if electrochemotherapy
preceded irradiation of tumors (Fig. 3). A great potentiation of radio-response
(EF = 1.9) indicated that electroporation of tumors significantly contributed to
radiosensitization of tumors with bleomycin, specifically because combined
treatment of electrochemotherapy with irradiation was more effective than irradia-

tion in combination with electroporation or bleomycin alone (Fig. 3). Obtained
potentiation of radio-response by using bleomycin in electrochemotherapy protocol
was even higher compared to cisplatin, presumably due to lack of transport mech-
anisms for bleomycin (Kranjc et al. 2003b, 2005).
In order to further support the presumption of correlation between immediate
effects of electroporation or electrochemotherapy on tumor blood flow (see ▶ Chaps.
35, “Effects of Reversible and Irreversible Electroporation on Endothelial Cells and
Tissue Blood Flow,” and ▶ 39, “Blood Flow Modifying and Vascular-Disrupting
Effects of Electroporation and Electrochemotherapy”) and oxygen level in tumors, in
this study the partial tumor oxygen tension (pO2) in the LPB tumors was evaluated
(Kranjc et al. 2005). As expected, decreased partial tumor oxygen tension (pO2) in the
LPB tumors (Kranjc et al. 2005) correlated with changes in tumor perfusion. Five
minutes after reversible electroporation of the tumors, pO2 was significantly reduced,
in the center by 75% and in the periphery by 50%. Afterwards tumors started to
reoxygenate, but the reoxygenation was not complete; even 24 h after electroporation
oxygen levels were ~90% of the pretreatment level. Specifically, at the time of tumor
irradiation, i.e., 20 min after electroporation of tumors, oxygen levels in the tumor
were still reduced to the level of radiobiological relevant hypoxia, which could have
an influence on the tumor radio-response (Kranjc et al. 2005). However, radio-
response of tumors after electroporation was the same as by irradiated tumors only,
as demonstrated in the previous study (Kranjc et al. 2003b). Again, due to achieved
enhanced radio-response of electroporated tumor cells in vitro, some other mecha-
nisms were assumingly involved in radio-response in vivo, i.e., reactive oxygen
species formed after electroporation of cells (Shil et al. 2005; West 1992). Overall,
both effects of electroporation, i.e., induced radiobiological relevant hypoxia and the
formation of free reactive oxygen species, are counteracted. However, it seems that
tumor type plays an important role in response of tumors to combined treatment
modality of electroporation and irradiation (Sersa et al. 2000; Shil et al. 2005).
Another chemotherapeutic that was used in combination with electroporation and
irradiation was doxorubicin. Similar to cisplatin and bleomycin, a study in mouse
EAT carcinoma tumors demonstrated that electroporation significantly enhanced
antitumor effectiveness of a moderate irradiation dose and low concentration of
doxorubicin; a significant tumor growth delay was obtained, 1.2 days when com-
bined irradiation and electroporation, 1.5 days when combined doxorubicin and
electroporation, and by all treatment combinations 1.7 days (Shil et al. 2006).
Promising results of tumor radiosensitization with electrochemotherapy using
bleomycin have driven further studies to evaluate the interaction of this treatment
with single-dose or fractionated irradiation regime and to make this treatment a
feasible approach in the clinic (Kranjc et al. 2009). The interaction was evaluated in
two tumor models with different histology and radiosensitivity: radiosensitive SA-1
sarcoma and radioresistant CaNT adenocarcinoma tumors. In both tumor models,
low irradiation doses and low dose (0.5 mg/kg) of bleomycin with a minor antitumor
effect in electrochemotherapy were used in order to evaluate interaction in combined
treatment. Radiosensitive SA-1 tumor with TCD50 value 25 Gy was irradiated with a
Fig. 4 Treatment schedules of tumors with electrochemotherapy and tumor irradiation with single
dose or fractionated regime
cumulative dose of 10 Gy in a single or fractionated regime (2 Gy/day, 5 days/week).

Radioresistant CaNT tumors with TCD50 value 70 Gy were irradiated with a
cumulative dose of 20 Gy in a single or fractionated regime (2 Gy/day, 5 days/
week, 2 weeks). Electrochemotherapy was performed once, 20 min prior to the first
fraction in the fractionated regime or tumor irradiation with a single dose (Fig. 4)
(Kranjc et al. 2009).
Radio-response of radiosensitive SA-1 and radioresistant CaNT tumors after
electrochemotherapy with bleomycin was pronounced in both single-dose or frac-
tionated irradiation regimen. The resulting tumor growth delay was synergistic, since
it was more than the sum of the effects of bleomycin or application of electric pulses
to the tumors combined either with single-dose or fractionated tumor irradiation. On
the contrary, the radiosensitization of both tumor models was not significantly
improved when combined with intravenous injection of bleomycin alone or appli-
cation of electric pulses alone. In general, tumor radiosensitization effect of
electrochemotherapy proved to be more pronounced in fractionated irradiation
regime than in single-dose irradiation. In fact, in SA-1 tumors the radio-response
after electrochemotherapy was enhanced by 2.7-fold in single-dose irradiation and
by 4.6-fold in the fractionated regime (Fig. 5). In CaNT tumors, a 2.3-fold enhance-
ment of radio-response in single-dose irradiation and a 3.1-fold in the fractionated
regime was obtained. Despite obvious radio-response obtained in both tumors, the
potentiation of radio-response was more evident in the radiosensitive sarcoma SA-1
tumors than in the radioresistant adenocarcinoma CaNT tumors (Kranjc et al. 2009).
Furthermore, to make radiosensitization of experimental tumors with electroche-
motherapy more comparable and valuable for use in clinical setting, large sponta-
neous mouse invasive ductal carcinoma tumors were used in the study by Raeisi
et al. (2012). Tumors were irradiated in suboptimal doses of 3 Gy or 5 Gy 30 min
after treatment with electrochemotherapy. Compared to other studies (Sersa
et al. 2000 and Kranjc et al. 2003b), a high dose of cisplatin, 8 mg/kg, injected
intratumorally, was used in electrochemotherapy protocol. Electrochemotherapy
alone significantly prolonged tumor growth delay (up to 15.5 days). Local irradiation
of tumors with single suboptimal doses alone or combined with cisplatin adminis-
tration prior to irradiation delayed tumor growth approximately to the same extent
(up to 21 days). However, electrochemotherapy with cisplatin significantly increased
radio-response of tumors at both irradiation doses used. The antitumor effectiveness
in combined treatment was dose dependent, resulting in tumor growth delay of
25.7 days at a dose of 3 Gy and 38.6 days at a dose of 5 Gy, with two complete
responses. Compared to previously obtained radio-response of variable tumors at
Fig. 5 Antitumor effectiveness of electrochemotherapy (ECT) with bleomycin (BLM) combined

with tumor irradiation (IR), either with the single dose or fractionated regime, in a SA-1 sarcoma
and EAT carcinoma tumors. Data represent the mean of tumor doubling time and their standard
error, which were pooled from at least eight animals per treatment group. Enhancement factor (EF)
of radio-response was calculated based on the tumor doubling time of the compared experimental
groups (Adapted from Kranjc et al. (2009))
higher irradiation doses (Sersa et al. 2000 and Kranjc et al. 2003b), these data
indicate that the chosen tumor model is quite radiosensitive and not chemosensitive.
With respect to clinical use, the first report of radiosensitization by electroche-
motherapy with cisplatin was in a patient with tubal dedifferentiated papillary
adenocarcinoma skin metastases (Sersa et al. 1999). Combined treatment,
electrochemotherapy with cisplatin injected intratumorally and single-dose irradia-
tion, was used for palliative treatment of skin metastases. In a very short observation
time (11 days) after electrochemotherapy a complete reduction of tumor size was
achieved, with no palpable viable tumor tissue under a scab. On contrary, no
reduction in the size of skin metastases was obtained after single-dose irradiation
alone or its combination with cisplatin treatment alone and application of electric
pulses alone. However, treatment of skin metastases with electrochemotherapy prior
to irradiation increased radio-response of metastases, resulting in a complete
decrease in the size of the treated metastases, which occurred much faster than by
treatment with electrochemotherapy alone (Sersa et al. 1999). In addition, a few
years ago combined treatment of electrochemotherapy with bleomycin and external
beam radiotherapy or brachytherapy for treatment of patients with very large,
previously multitreated or in advanced stage tumors of different type was used

(Skarlatos et al. 2011). Again, a great enhancement of radio-response by electroche-
motherapy with bleomycin was observed, resulting in complete response up to
2 years. Furthermore, electrochemotherapy with bleomycin and irradiation with
fractionated regime was successfully used on aggressive hidradenocarcinoma, a
complete remission of this malignant lesion was achieved, and lasted up to 7 months
(Kyrgias et al. 2013).
Side Effects of Electrochemotherapy and Irradiation Treatment
Ongoing advances in the techniques of radiation, finding of new approaches to

treat cancer in combination with irradiation, and progress made in understanding
the biology of cancer cell responses to combined treatments endeavor the improve-
ment in the survival and reduce treatment side effects for cancer patients. Thus,
determination of side effects of new treatment combinations is necessary, espe-
cially to provide final therapeutic index (normal tissue damage compared to local
tumor control at the dose curing 50% of tumors) as indicator of successful therapy.
Across the studies, combining electrochemotherapy and irradiation, done so far,
the low doses of irradiation and chemotherapeutics (cisplatin, bleomycin) were
chosen for the proper evaluation of the effects of combined treatment. As expected,
treatment with electrochemotherapy prior to irradiation showed minimal or no
local side effects in the irradiation field as well as no systemic side effects.
Precisely, animal body weight changes during and after the treatment as well as
the skin reactions around the tumor in the irradiation field after single dose or
fractionated regimen were assessed (Kranjc et al. 2009). In general, single-dose
irradiation alone or combined with bleomycin, application of electric pulses, and
electrochemotherapy induced more serious normal skin damage and animal weight
loss compared to irradiation in fractionated regime. Animals mostly lost weight at
the beginning of the treatment and during the fractionated irradiation regime.
However, the animal weight and physical condition were stabilized approximately
2 weeks after the treatment. Single dose irradiation alone or in combination with
electrochemotherapy provoked much more serious normal skin damage a week
earlier, compared to the fractionated radiotherapy regime, inducing edema, ery-
thema, and moist desquamation with moderate ulceration. On contrary, irradiation
in fractionated regime, alone or combined with electrochemotherapy, induced skin
damage only after 30–40 days after the first tumor irradiation and was around four
times lower, compared to single-dose irradiation, irrespective of the treatment
combinations (Kranjc et al. 2009). In addition, special attention to side effects
should be paid when using electrochemotherapy as adjuvant therapy in the previ-
ously irradiated field, because electrochemotherapy can induce radiation recall
(Spugnini et al. 2008). However, side effects after treatment with electroche-
motherapy prior to irradiation indicate this treatment as safe and feasible to provide
an appropriate therapeutic index for further development of this combined treat-
ment approach.
Conclusions
Potentiation of the radiation response is needed in clinical situations when patients

present accessible tumor masses, and good therapeutic effect is not expected in
conventional irradiation treatment. In such cases electrochemotherapy can be used as
radiosensitizer, using either cisplatin or bleomycin. The preclinical data provide
solid evidence that either with cisplatin or bleomycin good potentiation of radio-
response can be expected in the range of enhancement factor of 1.7–4.6. The
enhancement is tumor type dependent; however, radioresistant tumors are also
well responsive. Only one electrochemotherapy session is needed for the response
preceding the single dose or fractionated regimen. The first clinical data also
indicated the potential use of combined electrochemotherapy and radiation, provid-
ing evidence for broader clinical application of this combined treatment.
Cross-References

Electrochemotherapy
Tissue Blood Flow
▶ Electrochemotherapy in Veterinary Oncology
▶ Electrochemotherapy of Cutaneous Metastases
▶ Electrochemotherapy of Head and Neck Cancer
▶ Involvement of Reactive Oxygen Species in Membrane Electropermeabilization
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Combined Treatment of
Electrochemotherapy 90
with Immunomodulators
Vesna Todorovic and Maja Čemažar
Abstract
Electrochemotherapy is a nonthermal tumor ablation modality. The local delivery of
electric pulses directly to the tumor transiently increases permeabilization of the cell
membrane and significantly increases the uptake of chemotherapeutic drugs into
cells. Several mechanisms, including active immune response, contribute to the
antitumor effectiveness of electrochemotherapy. After electrochemotherapy, the
tumor-associated antigens are released in situ; however, they are not presented to
the cells of the immune system efficiently to stimulate also a distant antitumor
response. Although electrochemotherapy alone can stimulate the immune system,
the induced immune response is not sufficient to also obtain a systemic effect.
Combining electrochemotherapy with adjuvant immunomodulatory molecules is a
promising approach to stimulate the immune system more effectively and thus
increase antitumor effectiveness of electrochemotherapy. This approach has been
evaluated in preclinical studies on experimental and spontaneous tumor models.
Electrochemotherapy can be combined with different immunomodulatory mole-
cules, including cytokines, such as interleukin-2 and interleukin-12, CpG oligodeox-
ynucleotides, monoclonal antibodies, and plasmids coding immunomodulatory
molecules. Results of the preclinical studies combining electrochemotherapy with
immunomodulatory molecules are promising. In addition to potentiation of local
antitumor effectiveness, a systemic response can be obtained which affects also
distant untreated tumors, namely, reducing the number of metastasis and cures of
untreated tumors. The induced antitumor response is tumor specific, generating
V. Todorovic (*)
e-mail: vtodorovic@onko-i.si
M. Čemažar (*)

DOI 10.1007/978-3-319-32886-7_62
1718 V. Todorovic and M. Čemažar
tumor-specific cytotoxic T cells and protection against the development of the same
tumor type. Based on these results, combination of electrochemotherapy and immu-
nomodulatory molecules bears a great potential for cancer therapy, specifically by
adding a systemic component to localized electrochemotherapy treatment.
Keywords
Cytokines • Electrochemotherapy • Electroporation • Immune response •
Immunomodulation • Immunomodulators
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1718
ECT-Induced Cell Death and Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1719
Immunomodulatory Molecules, Networks, and Pathways of the Immune Response . . . . . . . . 1719
Immunomodulation in Cancer Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1720
Immunomodulators Used for Stimulation of Immune Response with ECT . . . . . . . . . . . . . . . . . . 1721
Interleukin 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1722
Tumor Necrosis Factor α . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1723
Interferon α . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1725
CpG Oligodeoxynucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1725
Ipilimumab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1726
Gene Electrotransfer of Plasmids Coding Immunomodulatory Molecules . . . . . . . . . . . . . . . . 1728
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1729
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1730
Introduction
Electrochemotherapy (ECT) is a nonthermal tumor ablation modality. It is based on

the local delivery of electric pulses combined with specific chemotherapeutic drugs
directly to the target tissue. Application of electric pulses affects permeability of the
cell membrane. This transient change in cell membrane permeability allows
non-permeant or low-permeant anticancer drugs to enter the cell. In this way, more
anticancer drug molecules enter cells, resulting in higher intracellular anticancer
drug concentration and augmented cytotoxicity of anticancer drugs.
The most suitable candidates for use in a combination with electric pulses are
hydrophilic drugs which lack or have a limited transport system in the cell mem-
brane. Many anticancer drugs were evaluated for potential use in combination with
electroporation, including 5-fluorouracil, actinomycin D, bleomycin, carboplatin,
cisplatin, cyclophosphamide, daunorubicin, doxorubicin, etoposide, gemcitabine,
mitomycin C, paclitaxel, vinblastine, and vincristine (Miklavcic et al. 2014). How-
ever, a significant cytotoxicity potentiation up to several 1000-fold was observed
only for bleomycin (Orlowski et al. 1988; Gehl et al. 1998; Jaroszeski et al. 2000)
and approximately 70-fold for cisplatin (Sersa et al. 1995); therefore, they were
designated as the most suitable anticancer drugs for clinical use of ECT.
Several mechanisms contributed to the effectiveness of ECT. Application of con-
trolled electric pulses transiently increases cell membrane permeability and results in
90 Combined Treatment of Electrochemotherapy with Immunomodulators 1719
increased intracellular drug concentrations and cytotoxicity. Application of controlled

electric pulses transiently reduces blood perfusion in tumors and causes a vascular
lock; therefore, tumor cells are exposed to cytotoxic drug for a prolonged period of
time. Electrochemotherapy causes cell death of not only tumor but also endothelial
cells and thus generates a vascular disruption effect. In addition, the immune system is
activated by tumor antigen shedding into the bloodstream after ECT and is also
involved in the antitumor effectiveness of ECT. The mechanisms associated with
ECT antitumor effectiveness include increased drug cytotoxicity, vascular disruptive
effect, and vasoconstriction and immune response which are discussed in more details
in ▶ Chap. 78, “Preclinical Studies on Electrochemotherapy.”
ECT-Induced Cell Death and Immune Response
ECT with bleomycin or cisplatin induces massive cell death in situ within 24 h after
treatment. Different types of cell death, such as apoptosis, necrosis, mitotic cell
death, or immunogenic cell death, can be observed after ECT, depending on the
anticancer drug used, its concentrations, the parameters of electric pulses, and the
overall metabolic condition of the cell.
After ECT, dying cells and tumor-associated antigens remain in situ, where they
can be presented to the cells of the immune system. After ECT-induced cell death,
different cells of the immune system are recruited to the tumor site, including
potential antigen-presenting cells, different subsets of dendritic cells, and mononu-
clear leukocytes (Mekid et al. 2003; Roux et al. 2008; Gerlini et al. 2013). The extent
of involvement of the host’s immune system in response to antitumor therapy
depends on tumor immunogenicity and immunocompetence of the host. Tumors
differ in their ability to elicit immune response, which is essentially mediated by
functional T cells. This was confirmed by evaluation of antitumor response on
immunocompetent and immunodeficient mice bearing the same tumor type, where
tumor cures were obtained only in immunocompetent mice. The effect of ECT on the
treated tumors is usually manifested as a local delay in tumor growth and tumor
cures; however, there is no effective systemic response. In addition, the tumor-
associated antigens released after ECT do not seem to be presented efficiently to
the cells of the immune system as no significant distant antitumor effect can be
observed. In an attempt to further increase local responses and obtain also a systemic
antitumor effectiveness, ECT can be combined with immunomodulatory molecules
as adjuvant treatment.
Immunomodulatory Molecules, Networks, and Pathways

of the Immune Response
The immune system is a complex and dynamic network of organs, cells, molecules,
and pathways working together in a tightly regulated and interconnected fashion
to provide an efficient defense mechanism against harmful or possibly harmful
substances and microorganisms. Organs of the immune system, such as the bone
marrow and thymus, regulate the development of immune cells, while other organs,
such as the spleen and lymph nodes, coordinate the encounter of antigens with antigen-
specific lymphocytes and their development into effector and memory cells. Cells of
the immune system originate from a single cell type, the hematopoietic stem cell,
which differentiates into two precursor cell types: myeloid progenitor cell, which in the
process of hematopoiesis gives rise to different types of mature myeloid cells (i.e., mast
cells, phagocytes, granulocytes, natural killer cells), and lymphoid progenitor cell,
which differentiates into lymphoid cells (i.e., B cells, T cells, and natural killer T cells).
These different cell types are involved in innate and adaptive immune response.
Innate response is a nonspecific defense system and responds very quickly to the
presence of an antigen. It forms the first line of defense and includes specific cell
types, such as phagocytes, dendritic cells, and natural killer cells. Pathogens are
recognized by pattern recognition receptors through specific molecular patterns,
such as unmethylated CpG motifs. On the other hand, adaptive immune response
is normally quiescent and gets activated by the presence of specific antigens that are
presented to the cells of adaptive response by antigen-presenting cells, such as
dendritic cells, which are a member of phagocytes of the innate immune response.
Cells of the adaptive immune response adapt to the specific antigen through activa-
tion of potent mechanisms for eliminating the specific antigen. The adaptive immune
response can be mediated by T cells (cell-mediated immunity composed of three
types of cells: helper, cytotoxic, and suppressor T cells) or by antibodies produced by
B cells (humoral immunity). In comparison to innate response, adaptive response
takes more time to develop. The combined action of innate and adaptive response is
needed to provide efficient defense mechanism.
Components of the immune system communicate through elaborate networks of
chemical messengers, i.e., cytokines. Cytokines are principally produced by helper T
cells and macrophages, but can also be transiently induced and secreted by all
nucleated cells. Cytokines regulate the immune response by binding to their
corresponding receptors on target cells and initiation of a cascade of intracellular
events, stimulating or inhibiting activation, proliferation, and differentiation of
various cells. Cytokines act in an autocrine, paracrine, or endocrine fashion. They
affect different cells in a pleiotropic, redundant, synergistic, antagonistic, or cascade
action. An effective and functional immune system is maintained by a delicate
balance of cytokines, which is often disrupted in cancer.
Immunomodulation in Cancer Therapy
Immunomodulation is a treatment aimed at modulating the activity of the immune

system. By immunomodulation, the immune system activity can be either induced or
enhanced (immunostimulation) or inhibited (immunosuppression). The objective of
immunomodulation in cancer therapy is to modulate the existing immune response
toward the desired antitumor effect that is clearance of tumor cells by the immune system.
The immune system has a dual role in cancer development and progression. It can
protect against and promote tumor growth through a combination of tumor-inhibiting
and tumor-enhancing processes mediated by the immune system. The so-called

cancer immunoediting consists of three distinct and sequential phases, elimination,
equilibrium, and escape (3Es of cancer immunology). During protection against
tumor cells, in the elimination phase, both innate and adaptive immunities work
together and are responsible for recognizing and eliminating the vast majority of
mutated and possibly harmful cells, including tumor cells. The mechanism involved
in the identification and eradication of tumor cells is called immunosurveillance. In
the second phase, equilibrium, the rare tumor cell clone, which is not destroyed by
the immune system, persists; however, the outgrowth of the tumors is prevented by
the action of adaptive immunity. T cells, IL-12, and IFN-γ are required to maintain
tumor dormancy. In this phase, editing of tumor immunogenicity occurs, which can
result in control of the outgrowth of the cancer for the lifetime, or due to the constant
pressure of the immune system, other clones of tumor cells may evolve. These
evolved (selected) tumor cell clones are not visible to the immune system due to the
loss of antigens, or they become insensitive to immune effector mechanism or they
are able to induce a tumor-suppressive microenvironment. In each of these cases, the
tumor cells enter into the escape phase, in which the progression of cancer is not
controlled (blocked) by the immunity. The onset of these clinically apparent tumors
is clearly an indication that individual tumor cells managed to avoid detection by the
immune system or were able to limit the extent of immunological killing, thereby
evading eradication by the immune system (Schreiber et al. 2011). The ability of
cancer cells to evade elimination by the immune system is now also accepted as one
of the emerging hallmarks of cancer. Overcoming regulation of the immune system
by tumor cells and restoring immune system activity to normal can improve the
management of cancer disease.
Immunomodulators are a group of diverse agents modulating the activity of the
immune system. They can be either immunostimulatory or immunosuppressive. In
cancer treatment, the general approach for immunomodulating treatment is to
enhance the antitumor response through stimulation of different cells of the immune
system. This can be achieved through the addition of different stimulatory cytokines
(i.e., interferon α, tumor necrosis factor α, interleukin 2, interleukin 12) and activa-
tion of T cells and/or antigen-presenting dendritic cells. In addition, stimulation of
antitumor immunity can be achieved through the promotion of antigen presentation
function of dendritic cells or production of protective T cell response. Recently,
immune therapies overcoming immunosuppression that exists in the tumor showed
great antitumor effectiveness and are currently under rapid development.
Immunomodulators Used for Stimulation of Immune Response

with ECT
The concept of combining ECT with immunomodulatory molecules is discussed in

more detail in ▶ Chap. 103, “Adjuvant Immunotherapy as a Tool to Boost Effec-
tiveness of Electrochemotherapy.” Briefly, combining ECT with immunomodulatory
molecules takes advantage of tumor destruction by ECT and exposure of different
tumor-associated antigens in order to increase tumor immunogenicity. Albeit ECT
alone can stimulate the immune system and at least in part induce an immune
response, it is not sufficient to achieve a strong immunogenic environment allowing
efficient priming and cross-presentation of tumor-associated antigens to the immune
system. Introduction of immunomodulatory molecules in the form of recombinant
proteins, oligonucleotides, monoclonal antibodies, or plasmids coding immunomod-
ulatory molecules can be applied in combination with ECT to stimulate the immune
system more actively and possibly achieve a systemic response.
The high local effectiveness of ECT can be mostly contributed to the direct cell
killing due to increased cytotoxicity of chemotherapeutic agents. However, cell
killing mediated by the cells of the activated immune system is also involved and
could be further exploited to increase the antitumor effectiveness of ECT not just
locally but also systemically. Namely, ECT treatment in solid tumors can be limited
by insufficient drug distribution due to irregular vascular organization in tumors,
high interstitial pressure, and composition of the extracellular matrix confining drug
diffusion. In addition, the inhomogeneity of electric field distribution, leading to
areas of tumors covered with electric field below the threshold value for permeabi-
lization, can also limit the effectiveness of ECT. Combining local ECT with different
immunomodulatory molecules in a tightly regulated and controlled fashion can
improve the overall antitumor effectiveness and obtain also a noticeable therapeutic
effect on distant untreated metastasis.
Interleukin 2
Interleukin 2 (IL-2) is a well-characterized cytokine produced by and secreted from

activated helper and cytotoxic T cells, natural killer cells, and natural killer T cells.
IL-2 is the most widely used drug in immunotherapy of cancer (Andersen
et al. 2003). IL-2 has no direct effects on cancer cells but rather mediates its
antitumor activity by altering host immune reactions. IL-2 exerts a myriad of
pleiotropic effects on different target cells. IL-2 acts as a growth factor and stimulates
proliferation and activation of natural killer cells and increases their cytolytic
capacity, induces proliferation and differentiation of T cells, and is involved in the
expansion of immunosuppressive regulatory T cells. In addition, IL-2 can also affect
cellular metabolism and glycolysis.
IL-2 is a powerful biological response modifier and has been extensively used for
modulation of antitumor immune responses. IL-2 has been combined with ECT
either indirectly through the addition of IL-2 secreting cells or directly as a recom-
binant protein. The first reports of ECT in a combination with IL-2 immunomodu-
latory therapy utilized specific histoincompatible cells engineered to produce and
secrete IL-2 (Mir et al. 1995; Ramirez et al. 1998). Preestablished murine LPB
sarcoma were treated with ECT with intravenous bleomycin application followed by
peritumoral injection of histoincompatible IL-2-secreting cells. As a result of this
combined therapy, 90% of the tumors were cured. Furthermore, this combined
therapy also led to the cures of more than half of the contralateral untreated tumors.
A systemic antitumor immune response was established through the generation of

helper and cytotoxic T cells and provided protection also against new tumor devel-
opment (Mir et al. 1995). Tumors treated with IL-2-secreting cells alone demon-
strated a slower growth and increased generation of cytotoxic T cells; however, no
cures were observed. The induced antitumor response is tumor specific since there
was no effect on distant untreated tumors of different origin indicating a generation
of tumor antigen-specific cytotoxic T cells.
This approach was evaluated also on larger VX2 carcinoma in rabbit livers. IL-2-
secreting cells were injected intratumorally after ECT with intravenous bleomycin
application. A combination of ECT and IL-2-secreting cells resulted in increased
regression of the primary liver tumor and largely reduced the number of visceral
metastases with no massive metastatic spreading in the lungs (Ramirez et al. 1998).
Tumors injected with IL-2-secreting cells only demonstrated a slow tumor evolution,
indicating that IL-2 alone was able to activate an immune response, however not
sufficiently to completely inhibit tumor growth. Taken together, the addition of IL-2-
secreting cells after ECT resulted in increased local antitumor effectiveness and
exerted also a systemic effect as manifested by the reduced number of visceral and
lung metastases (Ramirez et al. 1998) and protection from rechallenge with tumor
cells (Mir et al. 1995).
Another approach combining ECT and IL-2 immunomodulatory therapy was
based on the addition of low-dose recombinant IL-2. Nodules of advanced malignant
melanoma were first treated with ECT with intratumoral bleomycin application
followed by peritumoral injections of recombinant IL-2. During and after the
combined ECT and IL-2 treatment, specific tumor-associated antigen cytotoxic T
lymphocyte responses were monitored. In all patients, a decreased amount of tumor-
specific cytotoxic T cells in the blood was observed during treatment. When IL-2
administration was interrupted, the cytotoxic T cells reappeared in the blood. Further
studies revealed that the recurring cytotoxic T cell responses were due to the
induction of new reactive cytotoxic T cells and not to the return of the same cytotoxic
T cells into the circulation (Andersen et al. 2003). In addition, the decreased amount
of antitumor cytotoxic T cells in the blood can be attributed to the migration of
antitumor cytotoxic T cells to the tumor area where these cytotoxic T cells exert their
action. Taken together, this data supports the hypothesis that the extensive tumor cell
death elicited by ECT may have attracted dendritic cells that captured the newly
exposed tumor antigens and initiated cytotoxic T cell responses against them.
Tumor Necrosis Factor a
Tumor necrosis factor α (TNF-α) is a well-known proinflammatory cytokine with a

wide range of biological functions. TNF-α is produced and secreted by activated
macrophages, monocytes, neutrophils, natural killer cells, and T cells. It affects
growth, differentiation, survival, and function of different cells, including cells
outside the immune system. TNF-α is an interesting immunomodulatory agent for
adjuvant cancer therapy. It is weakly cytotoxic to most tumor cells but affects cells of
the immune system and has an antivascular action in tumors due to its specificity for
endothelial cells of tumor blood vessels.
In preclinical studies, TNF-α has been combined with ECT with suboptimal
bleomycin or cisplatin concentration to evaluate its potential to improve antitumor
effectiveness of ECT. After combining TNF-α injected either intratumorally or
peritumorally with ECT with bleomycin, increased antitumor effectiveness on
SA-1 sarcoma was observed (Sersa et al. 1997). Similarly, significant tumor growth
delays and tumor cures were observed also when SA-1 sarcoma was treated with
combination of ECT with intravenous cisplatin application and adjuvant
intratumoral TNF-α therapy (Cemazar et al. 2015). Since TNF-α is not directly
cytotoxic to tumor cells, the observed tumor growth delays, prolonged survival
times, and tumor cures are attributed to the vascular action of TNF-α. When
TNF-α is applied in a combination with ECT, the observed synergistic effects are
attributed to the dual role of TNF-α in the potentiation of antitumor effectiveness of
ECT. On the one hand, early TNF-α effects are manifested in the first 24 h
posttreatment and are aimed directly at the tumor vasculature, affecting the number
of functional tumor blood vessels, reducing interstitial pressure, and increasing
intratumoral concentration of chemotherapeutic agents (Fig. 1). On the other hand,
TNF-α-related late effects are immunomodulatory and add a systemic component to
the local ECT treatment. Namely, TNF-α has a relevant and critical role at multiple
levels of T cell immune response, such as activation of antigen-presenting cells, T
cell priming, proliferation, function, and recruitment. Most importantly, TNF-α is
involved in promotion of T cell proliferation to cross-presented tumor antigens.
The use of TNF-α is specifically significant for ECT of large tumors where a
limited drug delivery and/or penetration of chemotherapeutic drug within tumors is
expected. It is of key importance to apply TNF-α intra- or peritumorally to evade its
Fig. 1 Immunomodulation of ECT-treated tumor with TNF-α. Addition of TNF-α to ECT (c)
potentiates antitumor effectiveness of ECT and results in increased vascular permeability and
activates different immune cells in comparison to untreated (a) or ECT-treated tumor (b)
systemic toxicity. The potential of combined ECT and TNF-α immunomodulatory

therapy lies also in its role in T cell response, specifically adding a systemic
component to the localized ECT.
Interferon a
Interferon α (IFN-α) is an immunostimulatory cytokine. IFN-α stimulates natural

killer cells and induces expression of major histocompatibility complex class I
antigens. IFN-α is approved by the FDA as adjuvant treatment for patients with
high-risk melanoma. IFN-α polarizes immune response of helper T cells toward Th1,
enhances cytotoxicity and survival of natural killer cells, induces generation and
survival of cytotoxic T cells and memory CD8-positive T cells, positively regulates
antibody production, and promotes dendritic cell maturation, chemotaxis, and
CD8-positive T cell priming against tumor antigens. In addition, IFN-α has direct
antitumor activity. It upregulates major histocompatibility complex class I surface
molecules, promotes caspase-dependent apoptosis, and has antiangiogenic effects on
tumor vasculature.
Preclinical studies combining ECT with IFN-α have not been performed; how-
ever, the effect of adjuvant IFN-α prior to ECT was evaluated in a retrospective study
on 5 patients, eligible for inclusion in the study (Hribernik et al. 2016). All patients
had a primary melanoma surgically removed and had a high risk of recurrence;
therefore, they received adjuvant IFN-α therapy postsurgery. Recurrent metastases
were effectively treated with ECT. Taken together, the observed response rate of
ECT after adjuvant IFN-α therapy on melanoma metastases was higher than reported
for malignant melanoma treatment with ECT alone (Mali et al. 2013). This differ-
ence is possible due to the application of IFN-α prior to ECT and its stimulation of
the immune system. However, a progressed disease was observed at different time
intervals after ECT treatment. Although the study of adjuvant IFN-α therapy prior to
ECT was small, a potential for combining ECT with immunostimulatory therapies
was shown.
CpG Oligodeoxynucleotides
Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODNs) are well-

characterized immunostimulatory molecules. The unmethylated CpG motifs can be
found in high amounts in bacterial and viral DNA. These CpG motifs are recognized
by the vertebrate immune system through pattern recognition receptors as foreign,
and a protective immune response is triggered. Specifically, CpG ODNs are recog-
nized by endosomal toll-like receptor 9 (TLR-9) which is in turn activated and
triggers a signaling cascade which subsequently leads to the transcription of
proinflammatory chemokines and cytokines. This response can be achieved also
with synthetic CpG ODNs which can be exploited as vaccine adjuvants and immu-
notherapeutics for cancer treatment.
ECT in combination with CpG ODNs has been evaluated in three different
murine tumor models, namely, LPB sarcoma, B16F10 melanoma, and B16-OVA
melanoma (Roux et al. 2008). CpG ODNs have been selected as immunomodula-
tory agents based on the increased mRNA expression level of its corresponding
receptor, TLR-9, after ECT alone. In all three tumor models, the combination of
ECT with immunomodulatory CpG ODNs efficiently reduced local tumor growth
of the treated tumors and resulted also in a systemic antitumor effect in the distant
untreated contralateral tumors. Specific immune memory was induced, as the mice
were protected against a rechallenge with the same tumor cells, but not with
unrelated tumor cells. The combined ECT and CpG ODNs treatment induced
tumor-specific T-cell effectors in the tumor-draining lymph nodes and in the
spleen. Significantly more IFN-γ was secreted upon activation than with ECT or
CPG ODN alone (Roux et al. 2008). A synergistic interaction between ECT and
CpG ODNs was observed.
Ipilimumab
Ipilimumab is a fully humanized monoclonal antibody, approved by the FDA for the
treatment of advanced melanoma as monotherapy or as adjuvant therapy and is
directed against cytotoxic T lymphocyte antigen-4 (CTLA-4). CTLA-4 is one of the
immune checkpoint molecules and is a negative regulator of T lymphocyte activa-
tion and proliferation. CTLA-4 is present on activated T cells and binds to
costimulatory molecules CD80 or CD86 expressed on antigen-presenting cells.
This CTLA-4 ligation to CD80 or CD86 receptors inhibits activation and expansion
of T cells, controls immune response, and attenuates chances of autoimmune inflam-
mation. Inhibiting the interaction between CTLA-4 and its ligands by blocking
antibodies terminates negative regulation of T cell activation (Fig. 2). The role of
CTLA-4 in cancer development, specifically in melanoma, is to exploit this inhib-
itory mechanism to prevent clearance of cancer cells by the immune system. The
rationale for using ipilimumab as immunomodulatory agent lies in blocking the
inhibitory interaction of CTLA-4 with its B7 receptor complex on antigen-presenting
cells and thus augmenting T cell activation and proliferation.
Combination of ECT and ipilimumab has not been tested in preclinical studies;
however, the combination of ipilimumab as immunomodulatory agent and ECT has
been evaluated in a pilot study involving ten patients with advanced melanoma
(Mozzillo et al. 2015). An improved overall survival was observed after a combi-
nation of ipilimumab followed by ECT with systemic bleomycin in comparison to
ECT treatment alone in patients with advanced melanoma. Further immunological
analysis revealed that the amount of regulatory T cells was reduced in patients
responding to the treatment and was significantly lower than in the
non-responding patients (Mozzillo et al. 2015). In addition to the abovementioned
pilot study, a case of advanced melanoma treated with a combination of ECT and
ipilimumab was reported. A patient with advanced melanoma in-transit metastasis
was first treated with ECT with systemic bleomycin application and had a partial
Fig. 2 Immunomodulation of ECT-treated tumor with addition of ipilimumab. Ligation of CTLA-

4 to its corresponding receptors CD80 or CD86 inhibits activation and expansion of T cells
(a). Ipilimumab binds to CTLA-4 and blocks its interaction with CD80 or CD86 (b). Interaction
of CD28 with receptors CD80 or CD86 leads to activation and proliferation of T cells
response. After the last ECT session, multiple visceral metastases were discovered
and ipilimumab treatment was administered. After ipilimumab treatment, a complete
regression of visceral and cutaneous metastases was observed with loss of pigmen-
tation at the site of cutaneous metastatic lesions (Brizio et al. 2015).
Gene Electrotransfer of Plasmids Coding Immunomodulatory

Molecules
One of the challenges when using recombinant immunomodulatory proteins is to

achieve safe concentrations for effective immunotherapy. Often multiple applica-
tions of recombinant immunomodulatory proteins are needed to achieve long-term
stimulation of the immune system. However, repetitive applications of immuno-
modulatory molecules can also cause systemic toxicity. With the progress of gene
therapy, this challenge was successfully overcome, and long-term levels of immu-
nomodulatory molecules can now be sustained in the nontoxic concentration
range. Gene electrotransfer (GET) is a nonviral method for the delivery of genetic
material into cells based on the application of electric pulses. GET of plasmids
constructed to carry a code for different immunostimulatory molecules, such as
interleukin 12 (IL-12), IL-2, and granulocyte-monocyte colony-stimulating factor,
has been used for immunomodulation in a combination with ECT in preclinical
studies.
IL-12 is a promising immunomodulatory molecule for use in a combination with
ECT mostly due to its antitumor and antimetastatic activity. It is produced by
macrophages and dendritic cells and acts as a stimulatory factor for natural killer
cells and as maturation factor for cytotoxic T cells. Through induced proliferation of
natural killer cells and cytotoxic T cells, IL-12 induces production of IFN-γ,
increases activity of cytotoxic T cells, and drives the differentiation of naïve helper
T cells to Th1 cells.
GET of IL-12 plasmids has been tested in combination with ECT with either
bleomycin or cisplatin on different murine tumors, such as head and neck
carcinomas, fibrosarcoma, and mammary carcinomas. Intratumoral coapplication
of bleomycin and IL-12 plasmid followed by electroporation effectively reduced
the growth of preestablished tumors, resulted in tumor cures, prevented meta-
static progression and tumor redevelopment through increased IL-12 and IFN-γ
expression in the tumors, and amplified cytotoxic T lymphocyte activity (Torrero
et al. 2006). Intramuscular GET of IL-12 plasmid in combination with ECT with
cisplatin potentiated specific tumor growth delay and increased tumor cure rate in
immunogenic SA-1 sarcoma. In less immunogenic TS/A carcinoma, the
observed tumor growth delay was similar in immunocompetent and immunode-
ficient mice; however, tumor cures were obtained only in immunocompetent
mice. Clearly, an involvement of functional immune system is required to
achieve the desired antitumor effect (Sedlar et al. 2012). The observed antitumor
effect of adjuvant IL-12 GET can be attributed to the observed systemic release of
IL-12 and the concomitant increase of IFN-γ after intramuscular (Tevz
et al. 2009), intratumoral (Pavlin et al. 2011), or peritumoral GET (Pavlin
et al. 2009).
In addition, combination of ECT and GET of IL-12 plasmid has been successfully
used in veterinary medicine for the treatment of spontaneous canine tumors of
different histologies, including head and neck tumors, mast cell tumors, melanoma,
fibrosarcoma, and ameloblastoma (Cutrera et al. 2008, 2015; Reed et al. 2010;
Cemazar et al. 2016). Similar to murine experimental tumor models, intratumoral
or peritumoral GET of IL-12 plasmid in combination with ECT had a high antitumor
efficacy on histologically different spontaneous tumors. In addition, IL-12 contrib-
uted also to the prevention of tumor recurrence or distant metastases through the
activation of antitumor immune response (Reed et al. 2010; Cutrera et al. 2015;
Cemazar et al. 2016). In conclusion, the combination of ECT and GET of IL-12
plasmid can be used on histologically different tumors and can induce also a
systemic response, and the treatment is safe and effective.
Granulocyte-monocyte colony-stimulating factor (GM-CSF) is an important
hematopoietic cytokine. It can be secreted by different cells of the immune system,
such as T cells and macrophages. GM-CSF is involved in the production of
granulocytes and monocytes and in the maturation of macrophages and dendritic
cells. GM-CSF is used in immunotherapy of melanoma as monotherapy or in
combination with other approaches. It is specifically an interesting immunomodu-
latory cytokine for cancer therapy as it is able to increase recruitment of dendritic
cells at the tumor site and enhance their ability to capture tumor-associated antigens.
GET of plasmid-coding GM-CSF has been tested in experimental murine B16
melanoma alone or in combination with ECT. GET of GM-CSF plasmid alone had
no significant effect on tumor growth. However, in combination with ECT, in
addition to improved survival by the combined therapy, long-term immunity to
recurrence and resistance to challenge were induced in up to 12.5% of mice (Heller
et al. 2000). Similarly, peritumoral GET of IL-2 plasmid in combination with ECT
significantly increased tumor doubling time. Tumor-free mice after peritumoral GET
of IL-2 plasmid in combination with ECT were also protected from rechallenge
(Heller et al. 2000).
Conclusions
Based on the abovementioned preclinical and pilot clinical studies evaluating the
combination of ECT with different immunomodulatory molecules, such as cytokines
(IL-2, IL-12, TNF-α, IFN-α, GM-CSF), oligonucleotides with CpG motifs, and
antibodies targeting immune system’s cells (ipilimumab), the local antitumor effect
of ECT can be potentiated by the addition of immunomodulatory molecules to a
different extent. However, it is evident that in order to achieve a substantial and long-
term local and systemic effect, the application of immunomodulatory molecules in
combination with ECT has to be accurately planned. Specifically, preclinical studies
on temporal and spatial application of immunomodulatory molecules in combination
with ECT, which will lead to an optimized immunomodulatory and long-term
antitumor effect, are needed. Based on the knowledge gathered from preclinical
studies, clinical studies should be carefully planned and performed to achieve the
desired fine-tuned immunomodulatory response and long-term cancer control in
cancer patients.
Cross-References
▶ Adjuvant Immunotherapy as a Tool to Boost Effectiveness of Electrochemotherapy

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0432.CCR-05-1514
Combining Electrolysis
and Electroporation for Tissue Ablation 91
Mary Phillips Ho
Abstract
Electrolysis and electroporation technologies have been utilized to provide sev-
eral valuable nonthermal tissue ablation modalities. The synergistic combination
of electroporation and electrolysis (SEE) has produced a new method of tissue
ablation that has distinct advantages over electrolysis or electroporation alone.
Electrolysis uses a low-magnitude direct electric current to create chemical
species at the electrode-tissue interface which then diffuse through the tissue,
resulting in extreme pH changes and cell death. Electroporation, on the other
hand, is used to create permeabilizations in the cell membrane that can be used to
induce cell death by several different mechanisms: through electrochemotherapy,
cytotoxic drugs are introduced to the cell interior, and irreversible electroporation
results in cell death by loss of cell homeostasis. When electrolysis is combined
with electroporation, a new mode of tissue ablation is achieved that results in a
very effective method of cell death. This mechanism of action is likely due to the
ability of the electrolytic products to penetrate the cell membrane through the
permeabilizations created by electroporation. Here, fundamental principles of
electrolysis and electroporation are presented, the mechanism of ablation by
SEE is discussed, and different types of SEE protocols are examined with respect
to their effect on the tissue. This chapter hopes to serve as a foundation and
starting point for further research into this ablation modality as well as for the
development of new types of SEE protocols that may be used to address specific
clinical needs.
M.P. Ho (*)
Department of Engineering, Quinnipiac University, Hamden, CT, USA
e-mail: Mary.Ho@quinnipiac.edu

DOI 10.1007/978-3-319-32886-7_63
1734 M.P. Ho
Keywords
Synergistic combination of electroporation and electrolysis • Electroporation •
Reversible electroporation • Irreversible electroporation • Electrolytic ablation •
Tissue ablation
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1734
Fundamental Principles of Electrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1736
Mechanism of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1736
Pathology Findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1738
Advantages and Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1738
Fundamental Principles of Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1738
Mechanism of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1738
Advantages and Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1740
The Combination of Electrolysis with Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1741
Presence of Electrolytic Phenomenon During Traditional Electroporation . . . . . . . . . . . . . . . . 1741
Combining Electroporation and Electrolysis: Proposed Mechanism of Increased
Tissue Ablation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1743
Methods for Applying Synergistic Electroporation and Electrolysis (SEE) for
Tissue Ablation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1743
Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1751
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1751
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1752
Introduction
Electroporation is a technique which employs pulsed electric fields to create

permeabilized cell membranes. This phenomenon occurs when an electric field is
applied across the cell, destabilizing the electric potential maintained by the cell
membrane and resulting in the formation of nanoscale defects in the lipid bilayer.
Reversible pore formation caused by reversible electroporation (RE) has been utilized
as an effective method for introducing molecules such as genes and drugs into the cell
while maintaining the cell viability. Changing the electrical parameters can, however,
result in cell death by irreversible electroporation (IRE) due to extensive loss of
metabolites and loss of homeostasis. Electrical parameters for IRE can be employed
such that any Joule heating effects are minimized, resulting in a nonthermal tissue
ablation modality that spares important tissue components such as major blood
vessels, nerves, and the extracellular matrix. Electrochemotherapy (ECT) uses revers-
ible electroporation protocols to produce temporary pores in the cell membrane,
allowing cancer drugs such as bleomycin to be introduced directly into the tumor
cells. Protocols have been developed for treatment of metastases to liver, lung, brain,
bone, and head and neck cancers. ECT is increasingly becoming integrated with
other treatments, guidelines for treatment are beginning to list ECT as an option, and
91 Combining Electrolysis and Electroporation for Tissue Ablation 1735
in some locations, ECT is becoming available for treatment on a hospital level.

Nonthermal irreversible electroporation (NTIRE) for tumor ablation has also made
great strides and is currently seen as a promising nonthermal, minimally invasive
tissue ablation modality.
The high voltages employed with irreversible electroporation may induce muscle
contractions, requiring general anesthesia and neuroblocking agents to mitigate these
effects during the procedure. Such spasms may in turn cause treatment electrodes to
shift in location, resulting in incomplete ablation in the target site or damage to
nearby vital structures. Managing these effects adds a significant level of complexity
to the surgical procedure, resulting in increased surgical time and cost of
implementing this type of treatment method. Reversible electroporation does not
require as high of voltages and/or number of pulses as irreversible electroporation
and thus does not have the same issues with muscle contractions seen in irreversible
electroporation. RE has been used with success by combining it with cytotoxic
drugs, resulting in tissue ablation by electrochemotherapy. Nonetheless, there
would be a significant advantage in being able to obtain tissue ablation without the
requirement of introducing the cytotoxic drugs into the body as required for
Tissue ablation by electrolysis (E) (also known as electrochemical therapy) uses a
direct electric current to create tumor ablation due to the creation of chemical species at
the electrode-tissue interface as well as extreme pH changes throughout the ablation
volume. Electrolytic treatment has been reported to be a simple, safe, and low-cost
technique for ablating solid tumors. At the electrode-anode interface with the tissue,
electrochemical reactions consist mainly of water decomposition and chloride oxida-
tion. At the cathode surface, hydroxide ions and hydrogen are produced from water
decomposition. Chemical species generated at the interfaces of the electrodes diffuse
away into the tissue in a process driven by differences in electrochemical potential.
These electrolytic reactions yield changes in pH, resulting in an acidic region near the
anode and a basic region near the cathode. The changes in pH as well as the presence
of the chemical species formed during the electrolytic reactions result in a cytotoxic
environment. Electrolytic ablation requires very low direct currents (tens of mA) and
very low voltages (single to low tens of volts), thus requiring less expensive instru-
mentation than needed for tissue ablation by irreversible electroporation. However, for
traditional electrolytic ablation, a high level of electrolytic products must diffuse
through the tissue to create a cellular environment that is cytotoxic enough to produce
cell death. This requires a longer application time (tens of minutes to hours) for the
electrical current, increasing the length of the surgical procedure.
When electrolysis is combined with electroporation, a new method of tissue
ablation is achieved. It is hypothesized that the pores formed during electroporation
allow the electrolytic products to pass through the permeabilized cell membrane and
contribute to cell death at low levels of exposure to electrolytic chemical species. This
technique combines several advantages of electroporation and electrolysis alone and
can enable different modes of tissue ablation. For example, when reversible electro-
poration is combined with electrolysis, it applies the low current and low voltage
required to produce electrolysis along with the high speed of treatment time associated
1736 M.P. Ho
with electroporation. In addition, since the electrolytic products are at low concentra-
tions and only penetrate the electroporated cells, this new method allows for accurate
treatment planning and tight margins between the ablated and untreated tissue zones.
The primary goal of this chapter is to supply readers with the understanding
necessary to develop synergistic electroporation and electrolysis (SEE) protocols for
use in tissue ablation. Fundamental principles of electrolysis and electroporation
when applied separately are first presented. The proposed mechanism of ablation by
combining electrolysis with electroporation is discussed, and different types of SEE
protocols are examined with respect to their effect on the tissue. With this back-
ground and knowledge of how SEE can be applied toward tissue ablation, it is hoped
that the reader can gain a basic understanding of this new field and apply this
foundation toward investigating and developing new SEE protocols.
Fundamental Principles of Electrolysis
Mechanism of Action
Electrolysis is an electrochemical process that takes place at the electrode surface

and occurs during the passage of an electric current between two electrodes
immersed in an ionic solution. New chemical species generated at the interface of
the electrodes diffuse away from the electrodes into the tissue. If the anode is
electrically soluble, metal dissolution will be a major part of the resulting chemical
reaction occurring at the electrodes. The use of passivated metal electrodes can make
this effect negligible. During electrolysis, protons are produced at the anode,
according to the following reaction:
2H2 O Ð O2 þ 4Hþ þ 4e (1)
The major chemical reaction occurring at the cathode is the decomposition of water
into molecular hydrogen and hydroxyl ions:
2H2 O þ 2e Ð H2 þ 2OH (2)
Sodium chloride is ionized into Na+ and Cl ions, which move toward the cathode
and the anode, respectively. The concentration of Cl ions is higher at the anode, and
additional reactions result in the production of chlorine gas:
2Cl Ð Cl2 þ 2e (3)
The chlorine gas produced can go on to react with water to produce additional
chemical substances:
Cl2 þ H2 O Ð HClO þ Hþ þ Cl (4)
These chemical reactions are illustrated in Fig. 1.

Fig. 1 Essential chemical reactions occurring at the anode and cathode during the electrolytic
process. NaCl sodium chloride, NaOH sodium hydroxide, Cl2 chlorine (gas), HCl hydrochloric
acid, HClO hypochlorous acid, H2O water, OH hydroxide ion, O2 oxygen (gas), H2 hydrogen
(gas) (Reprinted from Gravante et al. (2011), copyright 2009, with permission from Elsevier)
Species produced at the anode and cathode are transported to the surrounding
tissue due to diffusion from a concentration gradient and migration due to the
potential gradient. Negatively charged ions become attracted to the anode, and
positively charged ions move toward the cathode. At the anode, destructive species
may include metal ions, hydrogen ions, and various chemical species containing
oxygen and chlorine (Nilsson et al. 2000). Smaller, more mobile, and abundant ions
initially alter the tissue’s local chemistry. Reactions at the anode attract protons (H+),
resulting in the formation of an acidic region. At the cathode, hydroxyl ions and
molecular hydrogen act as destructive reaction products (Nilsson et al. 2000).
Hydrogen is given off as a gas, and a more alkaline region forms at the cathode
due to electrochemical reactions and a repulsion of protons (Finch et al. 2002a).
Electrolytic cell death is driven by the presence of a cytotoxic environment that
develops due to local changes in pH as well as the presence of the new chemical
species formed during electrolysis. Cell necrosis occurs when a pH of less than 6 or
greater than 9 is reached (Finch et al. 2002b). The lethal effects of electrolysis on the
tissue is a function of the concentration of electrochemical products produced and
the time of exposure to these products. The area of necrosis has been shown to
depend on the coulomb dosage applied (Colombo et al. 2007; Czymek et al. 2011),
although this dependency may be contingent upon the experimental conditions.
1738 M.P. Ho
Pathology Findings
Electrolytic ablation is characterized by a lesion that begins at the electrodes and

propagates outward with increased delivered charge, as expected for a process that is
driven by diffusion mechanisms. The resulting lesion has an irregular shape, with a
greater volume affected near the tissue surfaces that were in contact with the
electrodes. The cell morphology is different at the cathode side of the lesion in
comparison to the anode side. Around the anode, the lobular architecture tends to be
retained, and the cells become more amorphous and eosinophilic, whereas, at the
cathode side, the tissue is more disrupted (Finch et al. 2002a). This dissimilar
appearance is caused by different products of electrolysis near the cathode and the
anode, resulting in different ablative reactions. Near the anode, the pH is low, and
many of the toxic chemical species are related to the various pH-dependent compo-
nents of Cl. Near the cathode, on the other hand, the pH is high and the toxicity is
primarily caused by various components of OH. In addition, electroosmotic migra-
tion of water from the anode to the cathode results in desiccation near the anode and
edema at the anode. It has been observed during electrolysis that the resulting lesion
near the anode surface is larger than that near the cathode. This may be attributed to
the formation of chlorine at the anode, which acts as a more destructive chemical
than those produced at the cathode (Finch et al. 2002a).
Electrolysis has been harnessed for tissue ablation in medicine since the early 1800s
(Amory 1886). During the last two decades, this field has seen substantial research
advances. From an operational standpoint, electrolysis requires very low voltages
and currents, providing advantages relative to other ablation techniques. Low volt-
ages can help avoid muscle contractions during application, and these low voltage
and current parameters reduce the need for instrument complexity. One major
limitation of electrolytic ablation, however, is that it is a lengthy procedure (requiring
current applications for tens of minutes to hours). The time scale for application is
controlled by the process of diffusion of the toxic chemical species from the
electrodes into the tissue. High concentrations of electrically produced ablative
chemical species are required, thus limiting the speed of the procedure.
Fundamental Principles of Electroporation
Mechanism of Action
Electroporation is characterized by the permeabilization of the cell membrane’s lipid

bilayer through the application of very brief (nanosecond to millisecond), high-field
(in the range of MV/m) electric pulses across the cell. The effects of electroporation
depend on the magnitude and duration of the pulsed electric field as well as other
factors such as cell size and shape and number of electrical pulses applied. The
electric field magnitude triggers pore formation, whereas electric field strength
and/or pulses with longer duration and/or number affect the pore expansion process
(Rems and Miklavcic 2016).
Though a comprehensive theory has not yet been developed to fully explain the
mechanism of electroporation, extensive experimental work and proposed models
have developed a strong foundation that has allowed for the development of
electroporation for a wide array of applications and is currently being built upon to
produce a more detailed understanding of the phenomenon. The essential features of
the electroporation process are known and can be briefly summed up as follows:
1. Electroporation utilizes short (on the order of ns to ms) electrical pulses, applying
an elevated transmembrane potential. The cell membrane requires a certain
charging time before the transmembrane potential reaches a critical threshold.
This charging time depends on electrical properties of the cell and solution as well
as cell size and shape (Rems and Miklavcic 2016) and may vary from an order of
100 ns to several microseconds.
2. The membrane conductivity increases immediately, and a time-dependent mem-
brane transition occurs as long as the externally applied electric field is held at
over above the critical value.
3. Depending on the electrical parameters utilized, once the external electric field is
removed, either membrane stabilization and resealing occur for reversible elec-
troporation or loss of cell homeostasis leads to cell death by irreversible
electroporation.
For reversible electroporation, once the electric field is lowered below the critical
value, a stabilization process occurs over a few microseconds. The transmembrane
potential drops quickly to near zero, and the membrane dramatically recovers to a
level in which it is permeable to only small molecules. The membrane reseals slowly
over seconds or even minutes. For irreversible electroporation, cell death occurs via
a number of potential mechanisms such as continued pore growth and membrane
rupture, membrane rupture due to colloidal-osmotic swelling, changes in ionic
concentrations, and loss of cellular content. This process of pore formation is
illustrated in Fig. 2, based on the theory of aqueous pore formation.
The family of electrical pulses that cause electroporation are divided into three
types: in reversible electroporation (RE), the cells survive the permeabilization
process, nonthermal irreversible electroporation (NTIRE) results in cell death due
to the lipid bilayer destabilization and permeabilization, and irreversible electropo-
ration coupled with thermal effects results when sufficiently strong fields are applied,
causing a temperature increase sufficiently high enough for thermal damage to occur.
Electroporation is becoming extensively utilized in biotechnology and medicine. In
the reversible mode, electroporation has become a central technology for cell
manipulation, and, in combination with chemicals, fundamental research and clinical
trials have demonstrated its promise for gene therapy (Lambricht et al. 2016), and
it is used clinically to target cancerous tissue through electrochemotherapy.
1740 M.P. Ho
Fig. 2 Membrane pore formation during electroporation. An idealized schematic of the lipid
bilayer membrane of electroporation based on the aqueous pore formation theory. (a) Thermal
fluctuations occur in the lipid bilayer membrane. (b) After the membrane has charged to threshold
value, a hydrophobic pore is created, allowing water molecules to start penetrating the bilayer. (c)
The lipids start reorienting to form a more stable hydrophilic pore, enabling more water and other
polar molecules and ions to pass through the membrane. (d) The pore will shrink and eventually
reseal after the external electric field has been removed. For irreversible electroporation, extensive
transport through the cell membrane and slow pore resealing result in cell death
Irreversible electroporation has been harnessed to ablate tissue while retaining the
structural integrity of blood vessels, nerves, and extracellular matrix (Jourabchi
et al. 2014). The ablative modality of NTIRE has been shown to result in a quicker
recovery of the biological tissues (Phillips et al. 2012). The ability to apply NTIRE in
a minimally invasive manner and the safety of this procedure have led to a recent
surge in its clinical use.
Pathology Findings
NTIRE results in focused apoptotic cell death and the creation of sharp boundaries
between the ablated cells and the adjacent, normal tissue. NTIRE has been shown to
spare the surrounding extracellular tissue structure. Blood vessels, bile ducts, the
urethra, and nerves are left intact and continue to function normally (Jourabchi
et al. 2014). It is believed that these structures remain undamaged because their
high collagenous connective tissue and elastic fiber content lack a cellular membrane
that could be targeted by NTIRE. This preserving feature could also be due to the gap
junction found within the cellular structure, allowing the electric current produced by
NTIRE to travel through the gap junctions without affecting the cell membrane
(Jourabchi et al. 2014).
A major advantage of tissue ablation by electroporation is the relative speed of the

procedure in comparison to any other ablation technique. In addition, because the
procedure targets the cell membrane, critical features of the extracellular matrix are
spared. However, irreversible electroporation usually employs up to one hundred

pulses of microsecond length and electric fields up to the several kV/cm range which
produce muscle contractions and require the use of paralyzing drugs and may affect
the electrical function of the heart. Reversible electroporation for electroche-
motherapy employs lower electric fields in the range of 300 V/cm to 1 kV/cm,
typically applied over eight pulses. Tissue ablation by RE, however, requires the
injection of chemo toxic drugs, such as bleomycin or cisplatin. Finding a way to
harness electroporation for tissue ablation without the need for additional drugs,
including total-body paralysis agents, would provide great strides in simplifying the
use of electroporation for medical applications and increase its ability for use within
developing countries.
The Combination of Electrolysis with Electroporation
Presence of Electrolytic Phenomenon During Traditional

Electroporation
Electroporation pulses in tissue have been shown to produce some level of electrol-
ysis at the electrodes, generating products of electrolysis that could ablate cells. This
phenomenon has been demonstrated through agar gel models (Turjanski et al. 2011),
ex vivo experimental work on excised tissue (Maglietti et al. 2013), and in vivo and
mathematical models (Olaiz et al. 2014). The resulting cytotoxic environment could
prove detrimental to some reversible electroporation processes that depend on the
cell to stay alive. For example, cell damage during gene electrotransfer would
decrease the overall efficiency of the procedure. Thus, initial efforts sought to reduce
and minimize this effect (Olaiz et al. 2014).
Electrolytic effects have been demonstrated to contribute to cell death in appli-
cations that were previously attributed solely to irreversible electroporation.
Rubinsky et al. (2015) demonstrated that the cytotoxic effects previously attributed
to IRE could actually be caused by at least three different mechanisms: reversible
electroporation combined with electrolysis, irreversible electroporation combined
with electrolysis, or irreversible electroporation without electrolysis. These different
mechanisms of cell death may result in distinct effects on the cell and tissue ablation.
A schematic to summarize which mode may become relevant, based on electric field
magnitude and electric current delivery time, is illustrated in Fig. 3.
The presence of electrolysis during electroporation procedures may also explain a
phenomenon that has been observed clinically. During clinical use of NTIRE as well
as occasionally during the clinical use of reversible electroporation, loud explosion-
like sounds may be produced. These noises may be caused by electric breakdown
across gases near the electrodes produced by electrolysis. During NTIRE studies, a
bright spot is often produced near the electrodes on ultrasound that increases in size
with the number of applied pulses. A bright hyperechoic appearance has also been
noted in the region adjacent and between the electrodes when performing electrol-
ysis experiments under the guide of ultrasound (Stehling et al. 2016). As noted by
1742 M.P. Ho
Fig. 3 Tissue ablation domains as a function of electric field and exposure time. E indicates
electrolysis ablation; R + E represents a combination of reversible electroporation and electrolysis
ablation; IRE + E represents a combination of irreversible electroporation and electrolysis
(Reprinted from Rubinsky et al. (2015) # the Author(s) 2015, permission from Sage Publishing)
Stehling et al. (2016), it is likely that this bright spot in both cases is caused by the
presence of gases produced due to electrolytic reactions at the electrodes. The
ultrasonic waves are reflected from the interface between the tissue and gases,
resulting in the observed hyperechoic appearance. Guenther et al. (2015) demon-
strated that the increase in electrolytic gases around the electrode, as observed on
ultrasound, corresponds to an increased electric discharge. This electric discharge
occurs primarily at the cathode and follows a pattern typical of electrical breakdown
across ionized gases at atmospheric pressures. The electrolytic products produced at
the cathode contribute toward the electrons at discharge. When the electric field
across the gas layer increases above the breakdown voltage, electric discharge
occurs, accompanied by light from the ionized gas and producing the violent
sound often observed (Guenther et al. 2015). The electric discharge phenomenon
generates high-pressure waves and could be detrimental to the organ being treated
(Stehling et al. 2016). A further understanding of the presence of electrolysis during
electroporation may help to devise electroporation protocols that minimize this
electric breakdown effect.
Overall, these studies indicate that some level of electrolysis may occur in all
electroporation protocols and must be considered and taken into account. However,
the combination of electrolysis and electroporation is not a detrimental in all cases
and indeed may be harnessed as new modality for tissue ablation that is seen to meet
many of the shortcomings of electroporation or electrolysis alone.
Combining Electroporation and Electrolysis: Proposed Mechanism

of Increased Tissue Ablation
It has been proposed that combining the cell permeabilization effect of electropora-
tion with the effects of the electrolytic products generated from electrostimulation
could result in a new ablation modality with an efficiency that is dramatically
increased compared to electrolysis alone. Though additional studies are warranted
to study this phenomenon, one explanation for this effect is that the electrolytically
produced chemicals can pass through the pores formed in the cell membrane during
electroporation, as illustrated in Fig. 4.
This interaction could be leveraged to induce cell death using a lower applied
electric charge than required for traditional electrolytic ablation. This mechanism of
tissue ablation could be harnessed through a variety of methods. New methods that
provide support for the proposed mechanism of action and take advantage of this
combined effect are being developed. Recent investigation into new synergistic
electroporation and electrolysis (SEE) protocols is described in the next section.
Methods for Applying Synergistic Electroporation and Electrolysis

(SEE) for Tissue Ablation
New methods and protocols are currently being developed to investigate the effect of
SEE and develop methods for tissue ablation that may be used clinically. Though this
field is new and much more work is needed for understanding the mechanisms at
play and optimizing treatment parameters for clinical use, it is beneficial to look at
different methods that have been used thus far to illustrate the SEE effect. These
experimental results give new insight into the technology and help develop a
foundation for future work. The types of SEE parameters that have been applied
experimentally can be divided into four different groups, each of which illustrates a
unique way to achieve tissue ablation by enabling electrolytically produced chemical
products’ access to the cell interior through electroporation. Each type of SEE
protocols may be more applicable for specific applications.
1. SEE by reversible electroporation and low-charge electrolysis in series

2. SEE by irreversible electroporation and low-charge electrolysis in series
3. SEE applied through multiple reversible electroporation-type pulses
4. SEE achieved through the use of a single decay pulse
1744 M.P. Ho
Fig. 4 Proposed mechanisms of combining electrolysis with electroporation. Low levels of

electrolysis may not be strong enough to cause cell death on their own, and reversible electropo-
ration on its own causes cell to become temporarily permeabilized followed by recovery. Combin-
ing these two phenomena, however, would allow the electrolytic products to pass through the
permeabilized cell and cause cell death at much lower concentrations
Each of these methods illustrates a unique way to achieve tissue ablation by

enabling electrolytically produced chemical products’ access to the cell interior
through electroporation. Overall, they help to gain a stronger foundation of under-
standing around the technology that can be used for further development in this
field.
SEE by Reversible Electroporation and Low-Dose Electrolysis in Series

An ablation protocol that combines reversible electroporation with low-charge elec-
trolysis would allow electrolytic products’ direct access to the cell interior, requiring a
much lower electric field than irreversible electroporation, reducing the treatment time
and level of electrolytic products required during electrolysis, and avoiding the need
for drug injection as practiced with electrochemotherapy. The synergistic electropora-
tion and electrolysis effect has been demonstrated as a new tissue ablation modality by
combing reversible electroporation and low-dose electrolysis together in series. This
was first proven using a simple parallel-plate electrode configuration to apply electrical
parameters across the rat liver in vivo (Fig. 5f) (Phillips et al. 2015a). Though the
experimental setup is less clinically relevant, the simple experimental design allows
for results that effectively illustrate the result of SEE. Hematoxylin and eosin (H&E)
cross sections of the treated livers are shown in Fig. 5. Parameters of typical electro-
lytic ablation (8 mA applied for 60 s) resulted in transverse ablation across the liver
thickness, as expected (Fig. 5a). A set of low-charge electrolysis parameters were
developed by reducing the current and applied time (4 mA, 30 s), resulting in a
decreased charge delivered to the tissue and a much smaller ablation volume, centered
around the electrode surfaces (Fig. 5b). Reversible electroporation parameters (electric
field of 250 V/cm, 100 μs pulse length, and 8 pulses applied at a 1 Hz frequency) were
also shown to result in minimal tissue ablation, with the volume of ablation located
near the electrode surfaces only (Fig. 5c). When the low-charge electrolysis was
Fig. 5 Demonstration of the effectiveness of combining electrolysis and electroporation for

increased ablation volume. Electrical parameters are applied across the liver lobe thickness using
plate electrodes (f). The anode is adjacent to the top surface of the liver and the cathode is adjacent
to the bottom surface. H&E staining is used to visualize the volume of ablation, and the region of
ablation is outlined using dashed lines. Typical parameters used for electrolysis (E) result in
transverse ablation across the liver cross section (a), delivering a total charge per unit area of
1.24 C/cm2. Decreasing the delivered charge to 0.31 C/cm2 results in non-transverse electrolytic
ablation located near the electrode surfaces (b). Typical parameters used for reversible electropo-
ration (RE) deliver a charge per unit area of 0.0001 C/cm2 and result in some minimal tissue ablation
near the electrode surfaces due to electrolytic effects (c). When electrolysis and reversible electro-
poration are combined in series, however, transverse ablation is achieved (d, e)
combined with reversible electroporation, however, transverse tissue ablation was

achieved (Fig. 5d, e).
During electrolytic ablation, the delivered charge can be used as a quantitative
measure of the amount of products of electrolysis generated at the electrodes. Since
these products of electrolysis propagate inward into the tissue through diffusion, the
extent of tissue ablation depends on the amount of charge delivered at the electrodes
as well as the distance of that point from the electrodes. Thus, when comparing SEE
protocol results with those of electrolysis and reversible electroporation alone, the
delivered charge can be used as a measure of the electrolytic products produced. The
delivered charge by reversible electroporation alone was orders of magnitude lower
than either electrolysis protocol and thus should have an infinitesimal effect on pure
electrolysis. Nonetheless, the results shown in Fig. 5 show that a substantial increase
in tissue volume when electrolysis and reversible electroporation are combined.
This hypothesis has been further supported by the use of a more clinically
relevant experimental setup, using titanium needle electrodes to apply the electrical
parameters to an in vivo porcine liver model (Stehling et al. 2016). When the
reversible electroporation parameters were followed by the low-charge electrolysis
protocol, the ablated area significantly increased. The treated volume was increased
1746 M.P. Ho
even further by applying reversible electroporation parameters, followed by the

low-charge electrolysis protocol, followed by another set of reversible electropora-
tion parameters. The mechanisms at play likely involve the use of conventional
electrolysis as the central tissue ablation modality while utilizing electroporation
pulses to permeabilize the cell membrane, allowing for cell death to occur at lower
amounts of electrolytic products.
SEE Applied as a Combination of Electrolysis and Irreversible

Electroporation
The combined use of electrolysis and electroporation has the potential application in
extending the ablated tissue volume in conventional NTIRE without increasing the
voltage on the electrodes. This would take advantage of the region that develops
outside of the irreversible electric field zone in which a lower electric field, typical of
reversible electroporation protocols, is present. The presence of even low-level
electrolytic products in this region could enable the ablation zone to be extended
to the edge of the reversible electroporation threshold of the electric field. Survival of
the tissue between electrodes is a critical concern during the use of NTIRE clinically,
and developing methods to increase the ablation zone would be greatly beneficial to
the field. SEE has been shown to increase this ablation zone (Stehling et al. 2016),
using an NTIRE-electrolysis-NTIRE treatment sequence. These results fit the overall
hypothesis of SEE. When NTIRE treatment parameters are applied by needle
electrodes, the strength of the electric field decreases with distance from the elec-
trodes. Beyond the volume of tissue treated by irreversible electroporation lies a
region where electric fields are still strong enough to produce reversible electropo-
ration effects (Fig. 6). It is expected that by combining electrolysis with NTIRE,
ablation can now be achieved within the reversible electroporation region via
electrolytically produced products.
SEE Applied Through Multiple Reversible Electroporation-Type Pulses

Reversible electroporation on its own can produce a limited electrolytic ablation
effect, typically confined to the tissue volume immediately adjacent to the electrode
surfaces. A third type of SEE parameters uses a single protocol of multiple reversible
electroporation-type pulses to increase the electrolytic effect, enabling tissue abla-
tion by applied electric fields below the threshold for irreversible electroporation
(Phillips et al. 2015b). By using delivered charge as a comparative measure, the
multiple-pulse SEE protocol can be designed to produce a much more effective
method of electrolytic ablation. The SEE protocol is able to produce damage
comparable to that of conventional electrolytic protocols while delivering orders of
magnitude less charge to the target tissue over much shorter periods of time. This
effect is illustrated in Fig. 7, using a simple parallel-plate electrode configuration
(same as shown in Fig. 5f) to apply the electrical parameters to liver tissue.
The ablation zone achieved by applying SEE through multiple low-voltage
electroporation pulses extends throughout the reversible electroporation zone sur-
rounding electrodes when using a clinically relevant two-needle electrode setup
Fig. 6 A mathematical model of two 1-mm-diameter needle electrodes in a volume of tissue.

Regions that experience an electric field magnitude over a given threshold (region within inner
contour line) experience irreversible electroporation, resulting in tissue ablation. Reversible elec-
troporation is achieved for lower electric fields (region between outer and inner contour line).
Results were obtained for an 800-μs, 1331-V pulse. By inducing cell death within the reversible
electroporation zone, the volume of treated tissue can be increased (Reprinted from Davalos
et al. (2005), # 2005, with permission of Springer)
(Stehling et al. 2016). This further supports the mechanistic explanation for how
SEE increases ablation efficiency and can be used to increase the treated volume. In
Fig. 8, calculated isoelectric field lines are superimposed on histology images of the
ablated zone. When a low number of pulses are applied, the ablation zone extends to
electric field lines greater than 500 V/cm corresponding to ablation by IRE and
suggesting that the combined effect of electrolysis and electroporation did not occur.
However, when the number of pulses is increased, increasing the total charge applied
to the tissue, the region of ablation extends to electric field lines of approximately
200 V/cm, indicating that cell death is occurring in the surrounding reversible
electroporation zone due to the SEE effect.
A possible application of this type of SEE parameters is a method of tissue
ablation that uses reversible electroporation strength fields to permeabilize the cell
membrane to products of electrolysis, thus inducing cell death. These SEE param-
eters result in cell death without requiring the high voltages of irreversible electro-
poration while relying on a drastically decreased level of electrolytic product
production than required for traditional electrolytic ablation.
1748 M.P. Ho
Fig. 7 SEE delivered by multiple reversible electroporation-type pulses. Electrical parameters

are applied across the liver lobe thickness using plate electrodes (Fig. 5f). The anode is adjacent to
the top surface of the liver and the cathode is adjacent to the bottom surface. Masson trichrome
staining is used to visualize the volume of ablation, and the region of ablation is outlined using
dashed lines. Typical parameters used for electrolysis (E) illustrate the pattern of tissue ablation with
increasing charge delivered (a–c). Reversible electroporation-type parameters were chosen to
increase the charge delivered (d–f), resulting in a pattern of ablation that is markedly similar to
that obtained by typical electrolysis. The volume of ablation achieved with SEE, however, occurs at
orders of magnitude less delivered charge than required for typical electrolysis
A Single Exponential Decay Pulse for Achieving SEE Ablation

A single exponential decay pulse is another type of SEE parameter that can produce
ablation with the tissue. A discharge capacitor can deliver a pulse that decays
exponentially and is defined by the initial voltage and the time constant. The applied
voltage for the exponential decay pulse can be given as:
t
V ðt Þ ¼ V o e τ (5)
where Vo is the initial voltage, t is time, and τ is the time constant. A set of capacitor
discharge parameters can be chosen such that the initial high voltage at the beginning
of the pulse serves to generate electroporation, while the rapid decay toward a
trailing low voltage generates sufficient charge for the generation of electrolytic
products. When the exponential decay pulse delivers a relatively low level of charge,
no tissue ablation is observed. Increasing the delivered charge by applying a higher
initial voltage and/or longer time constant can be used to increase the ablation
volume (Phillips et al. 2016). It should be noted, however, that continued increase
in applied voltage will eventually lead to tissue ablation by irreversible electropora-
tion effects rather than SEE.
These single exponential decay SEE parameters indicate a method for tissue
ablation that allows for reduced instrument complexity in comparison to that used
by irreversible electroporation while requiring shorter application time than
Fig. 8 Extent of tissue ablation achieved by eight electrochemotherapy-type pulses compared

to a high number of electrochemotherapy-type pulses. Eight typical electrochemotherapy
magnitude-type pulses (1000 V, 100 μs, 1 Hz) result in a minimal level of ablation, as shown by
gross macroscopic section (a) and trichromatic staining (b). This is compared to increased level of
ablation achieved by increasing the number of pulses to 297 (c) and (d). Calculated isoelectric field
lines are superimposed on the bottom row, showing ablation extents to an electric field of
approximately 200 V/cm. These isoelectric field lines are also applicable for the top row as well
and would indicate that ablation extents to an electric field of greater than 500 V/cm for the 8-pulse
protocol (Reprinted from Stehling et al. (2016) (# Stehling et al. 2016) licensed under CC BY 2.0)
traditional electrolysis. In addition, the single exponential decay pulse causes a

substantial reduction in muscle contractions in comparison to the same level of
ablation achieved by a typical multiple-pulse electroporation protocol. This may
pave the way to the development of protocols that do not require the use of general
anesthesia and neuroblocking agents during clinical use, an area which is currently
seen as a major limitation of current irreversible electroporation protocols.
Pathology Findings
Histological analysis of the lesion produced by SEE ablation reveals that multiple
modes of cell death may be present, depending on the type of SEE parameters
applied, the electrode configuration, and the location within the tissue relative to the
electrodes. The mechanisms of damage can be divided into at least five different
categories (Stehling et al. 2016):
1750 M.P. Ho
1. Dominant anodic ablation

2. Dominant cathodic ablation
3. Combination reversible electroporation and anodic compounds
4. Combination reversible electroporation and cathodic compounds
5. Irreversible electroporation
SEE ablation protocols result in a sharp transition between the untreated cells and
the ablated region, marked by the presence of congestion of the sinusoids. The
appearance of the ablated cells differs throughout the ablated region, and marked
differences are apparent when comparing cell morphology near the cathode to that
near the anode. The treated zone is larger near the electrodes and grows outward
toward the center tissue, resulting in either two separate lesions or a single dumbbell-
shaped lesion. Large blood vessels remain intact and patent (Stehling et al. 2016), a
feature that has been also observed for both electrolysis and electroporation when
applied in separate protocols.
When SEE was applied across the liver tissue using parallel-plate electrodes both
by the method of reversible electroporation and low-charge electrolysis in series
(Phillips et al. 2015a) and by the method of using multiple reversible
electroporation-type pulses (Phillips et al. 2015b), histological analysis indicated
that the mode of tissue ablation maintained key characteristics of electrolytic abla-
tion. The appearance of the treated tissue at the anode, at the cathode, and in the core
of the treated tissue was markedly different, caused by different products of elec-
trolysis near the anode versus the cathode that result in different ablative reactions.
The general mode of damage is consistent with an electrolysis mechanism of
damage; the tissue ablation begins from the vicinity of the electrodes, is different
at the anode side in comparison to the cathode side, and increases to encompass the
entire tissue between the electrodes with an increased delivered charge. Thus, it is
likely that dominant anodic ablation and cathodic ablation occurred immediately
adjacent to the electrode surfaces. However, the ablation seen throughout the
majority of the liver thickness in Figs. 5 and 6 is likely due to the third and fourth
mechanisms of damage, combining reversible electroporation with either electroly-
sis products produced from the anode or from the cathode. These electrolytic
products are able to penetrate the electroporated cells, resulting in electrolytic cell
death even when only low levels of electrolytic product are present.
An irreversible electroporation zone, however, can also be present, depending on
the parameters used. When applying irreversible electroporation with electrolysis to
increase the ablation volume, a volume of the tissue will experience histological
effects that can be primarily attributed to cell death by NTIRE. An NTIRE-
dominated ablation zone has also been shown to be present in histology analyzed
from multiple low-voltage electroporation pulses applied through needle electrodes
(Stehling et al. 2016).
Histological analysis for a single decay pulse also demonstrates an ablation
pattern similar to that achieved by electrolytic ablation. By increasing the applied
charge, the volume of ablation also increases, starting at the electrode surfaces and
moving in toward the center of the tissue volume. For the parallel-plate electrode
experimental model described by Phillips et al. (2016), initial electric field of

1000 V/cm and 500 V/cm appears to apply a mechanism of tissue ablation that
combines electroporation effects with electrolysis (damage modes 1–4). Increasing
the electric field to 1500 V/cm, however, results in a mode of ablation is likely
dominated by irreversible electroporation effects rather than the combination of
electrolysis and electroporation.
Imaging
Imaging methods for combined electrolysis and electroporation protocols may be

developed based on imaging success seen when each ablation modality is applied
separately. For example, ultrasound monitoring, magnetic resonance imaging, and
non-contrast and contrast-enhanced CT have been used to assess the volume of
ablation zone after electroporation (Yarmush et al. 2014). In addition, electropora-
tion leads to an immediately detectable impedance decrease, and electrical imped-
ance tomography is a method that is under development, providing a
two-dimensional reconstruction of tissue impedance that allows for near-real-time
monitoring of tissue electroporation (Yarmush et al. 2014). Local changes in pH can
be used to reliably monitor the extent of tissue ablation achieved by electrolytic
ablation (Finch et al. 2002b). Discrete pH-measuring devices, however, cannot
provide a continuous, spatial image of the pH distribution, and thus an alternative
method for real-time monitoring of lesion size is desired. Bubbles of gas produced
during electrolysis have made the use of ultrasound monitoring of lesions difficult
(Finch et al. 2002a). A technology that can detect pH changes in both time and space
is directly applicable for research and clinical applications of electrolysis. Electrical
impendence tomography has been shown as one possible mechanism for monitoring
lesions created during electrolysis in real time (Meir and Rubinsky 2015). This
method takes advantage of the relatively small size of proton ions and hydroxyl
ions compared to other physiological ions produced during the electrochemical
reaction. These smaller ions not only result in local pH changes but also lead to
increased local mobility, which in turn results in increased conductivity around the
anode and the cathode. This local change in tissue conductivity can be monitored in
real time using principles of electrical impedance tomography. Magnetic resonance
imaging (MRI) can also be used to image the process of electrolysis by detecting pH
fronts (Meir et al. 2015), resulting in another promising method for monitoring and
advancing the use of this technology. Future work will develop these imaging
techniques for real-time monitoring of tissue ablation by SEE.
Conclusions
The synergistic combination of electroporation and electrolysis can be used as a new

tissue ablation modality, resulting in cell death at much lower levels of electrolytic
product than required by traditional electrolysis alone. By combining electrolysis
1752 M.P. Ho
and electroporation, a new method may be achieved that addresses some of the
clinical drawbacks of each technology when applied individually. SEE may be
applied through a variety of different types of ablation protocols. This new type of
treatment method is still in the early stages of development, and substantial addi-
tional research and quantitative analysis are necessary to advance this field. Though
initial studies appear to support the proposed mechanism of SEE ablation, further
research is necessary to gain an in-depth knowledge of this phenomenon. There are
various parameter configurations that can be chosen to apply SEE, and the method of
choice may depend strongly on the individual application. These different types of
SEE protocols need to be further examined and refined in order to choose protocols
that are most relevant clinically. In addition, new types of SEE protocols may be
developed that allow for increased ablative efficiency, reduced instrument complex-
ity, or serve to meet other specific clinical needs.
Cross-References

▶ Mass Transfer of Electrolytic Species During Electric Field-Based Tumor
Treatments
▶ Medical Imaging of Electroporation
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Yarmush ML, Goldberg A, Sersa G, Kotnik T, Miklavcic D (2014) Electroporation-based technol-
Delivery of Cytokines Using Gene
Electrotransfer 92
Shawna A. Shirley
Abstract
Gene electrotransfer is a safe, inexpensive, effective, and reliable method of
delivering nucleic acids to individual cells or tissues in vivo. It is a physical
method of introducing external genetic material into a cell by application of a
controlled electrical field. Cytokine gene therapy shows great potential for the
treatment of a variety of diseases including cancers, infectious diseases, and
autoimmune disorders as it seeks to modulate immune function in order to resolve
disease. Electrotransfer of cytokine genes provides an attractive alternative
approach to therapy that circumvents many of the concerns associated with
delivery of cytokines as recombinant proteins or using viral vectors. Using
electrotransfer genes can be delivered as a single therapeutic agent or in combi-
nation with other therapeutic agents to targeted sites avoiding involvement of
surrounding tissue. Cytokines can be used as an adjuvant in DNA vaccines, to
restore homeostasis and reduce inflammation in autoimmune disorders or stimu-
late host inflammatory responses and immunity to cancers. Decades of preclinical
research has culminated in clinical studies investigating electrotransfer of cyto-
kine genes for treatment of cancers and infectious diseases. This chapter gives an
overview of how gene electrotransfer is being used clinically to deliver cytokine
genes for disease therapeutics.
Keywords
Cytokines • Interleukin-12 • Gene electrotransfer • Cancer • DNA vaccine •
Immunotherapy
S.A. Shirley (*)

Frank Reidy Center for Bioelectrics, Norfolk, VA, USA
OncoSec Medical, San Diego, CA, USA
e-mail: sashirle2@gmail.com; sshirley@oncosec.com

DOI 10.1007/978-3-319-32886-7_189
1756 S.A. Shirley
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1756
The Basics of Gene Electrotransfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1757
Cytokine Gene Electrotransfer for Cancer Immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1759
Cytokine Gene Electrotransfer for Autoimmune Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1760
Cytokine Gene Electrotransfer for Infectious and Other Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1761
Cytokine Gene Delivery in Clinical Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1761
DNA Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1763
Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1764
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1766
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1766
Introduction
Cytokines are a family of ligands that regulate immunologic cell functions ranging
from homeostasis to inflammatory processes. They act by binding to their specific
receptors, which can be either soluble or cell membrane bound, which initiates
intracellular signaling pathways that modulate inflammatory processes. Some cyto-
kines have direct immediate effects on their target, while others have indirect effects,
either locally or at distant sites. Cytokines function as immune system mediators,
stimulating or inhibiting growth and maturation of immune cells, inhibiting or enhanc-
ing the function of other cytokines. They regulate normal cell function and cytokine
receptor expression and modulate the balance between innate and adaptive immunity.
The objective of cytokine therapy is to either increase or blunt the expression of
specific cytokines or their signaling cascades in order to modulate immune function in
order to halt disease progression or prevent disease acquisition in the case of some
vaccines. In the case of cancer immunotherapy, it seeks to make a nonimmunogenic
environment, such as the tumor itself, and immunogenic by augmenting antigen
presentation, effector cell activation, and proliferation and by alleviating immune
suppression. Delivery of cytokines for anticancer therapy should provide just enough
stimulus to “tickle” host immunity, which once established will generate a robust,
effective, and lasting antitumor response. In the case of DNA vaccines, delivery of
cytokines acts as an adjuvant to stimulate host immunity to generate a lasting response
to the antigens being immunized against. Cytokine therapy can be useful in treating
autoimmune disorders as well. Here, the objective of the therapy is achieving and
maintaining homeostatic rather than stimulating immunity. Targeting certain inflamma-
tory cytokines associated with disease progression is a reasonable approach to treatment
and can be achieved by a variety of approaches. Generation of antibodies specific for
inflammatory cytokines, gene silencing using siRNA or shRNA, or even vaccination
against autologous cytokines are all approaches that have been considered.
Gene electrotransfer, also referred to as electroporation (EP), is a safe and
effective physical method of introducing molecules into cells using electric pulses.
It was first introduced in the 1980s as a method of transfecting cells in vitro. Since
then the technology has evolved, and it is now being used in clinical applications
92 Delivery of Cytokines Using Gene Electrotransfer 1757
ranging from gene therapy to DNA vaccine delivery. Studies have shown gene
transfer using electroporation can enhance expression up to 1000-fold higher than
injection of DNA alone (Mir et al. 1998). Using electroporation as the method for
cytokine delivery is a promising approach as efficient and targeted delivery can be
achieved. It has tremendous potential for therapeutic applications and will avoid
some of the potential safety concerns associated with delivery of cytokines as
recombinant proteins or using viral vectors. These issues include but are not limited
to toxicity, short half-life in circulation, and rapid degradation in the case of systemic
delivery of recombinant proteins and insertion into the genomic sequence, mutagen-
esis, and carcinogenesis in the case of viral vector delivery. Careful selection of
delivery parameters and route can enable the user to select the appropriate level and
duration of expression of the desired cytokine.
This chapter will give the reader a general understanding of how electroporation
has been used to deliver genetic material encoding cytokines for disease therapeu-
tics. It will provide a brief overview of preclinical studies but focus mainly on
clinical studies that are either currently underway or have been completed using
electroporation as a platform for cytokine delivery.
The Basics of Gene Electrotransfer
Electroporation is used to deliver macromolecules, proteins, and nucleic acids such

as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) into cells by breaching
the cellular membrane using electrical pulses. The method is versatile and can be
used to deliver material to a range of tissues including the skin, tumors, muscle, the
liver, the lung, the heart, and even the eye (Shirley et al. 2014). The parameters of
electroporation can be adjusted to suit the needs of the user. Thought should be given
to the desired level of gene expression, the duration of expression, and the charac-
teristics of the target tissue. Pulse length, duration, and frequency as well as the
voltage and waveform of the pulses can all be configured to achieve the desired level
of gene expression. The therapeutic purpose of gene electrotransfer should be
carefully considered when selecting the parameters and whether low or high levels
of expression of the transferred gene are required. These considerations are impor-
tant if the objective is to transfer cytokine genes or gene encoding proteins you want
to vaccinate against.
Pulse generators that can modify exponential pulses into square wave pulses can
be obtained commercially for clinical as well as experimental use. The end user can
configure settings such as voltage, number of pulses, and pulse duration. Pulse
duration can vary from microseconds to milliseconds, and the field strength
generated can range from below 200 volts per centimeter (V/cm) to over
1000 V/cm. Field strengths greater than 700 V/cm are generally considered high
field strength. Pulses that are below 700 V/cm are considered low field strength. As
mentioned earlier, manipulation of field strength, pulse number, and duration can
result in different amounts of gene expression. High field strength pulses of short
1758 S.A. Shirley
duration will result in lower gene expression than low field strength pulses of
longer duration. Pulse protocols have been established for various applications. In
some cases, a single pulse may be effective, while on others a chain of 8–10 pulses
may be most effective for gene transfer. In many cases parameters of gene transfer
are tissue specific. Other way to increase efficiency of gene electrotransfer pro-
tocols is to change the polarity, duration, and orientation of pulses or even
combining two or more pulse protocols.
Another important consideration is the selection of electrode. Electrodes can be
either invasive, consisting of needles, or noninvasive consisting of plates, pins, or
paddles. Needle and pin electrodes can be configured in a variety of patterns:
triangular, circular, square, rectangular, hexagonal, and even grids consisting of
more than nine electrodes. Noninvasive paddle and plate electrodes usually consist
of a pair of parallel electrodes. Some electrodes have more unusual designs: flexible
endoscopic electrodes for endo-luminal delivery, gutter-like electrodes for delivery
to blood vessels, and retractable electrodes that can be deployed into tissues that are
not readily accessible. The selection of an appropriate electrode is important because
it determines the distribution of the generated electric field. The selection of invasive
or noninvasive electrode is dependent on the characteristics and accessibility of the
target tissue. A variety of electrodes, including some mentioned above, are commer-
cially available for clinical as well as preclinical use.
Cytokine genes are commonly delivered by electroporation to muscle tissue for
DNA vaccines. The nondividing, fibrous vascularized nature of the tissue allows for
long-term expression. In clinical studies, needles are used to deliver to the deltoid
muscle and usually do not need surgical interventions. Cytokines genes are also
delivered to the skin for DNA vaccines. The skin is an attractive target for delivery as
it is the largest most accessible organ which has resident immune cells. Depending
on the application, delivery can either be local or systemic. For anticancer therapies,
cytokine genes are commonly directly delivered to the tumor, though some
researchers have used intramuscular delivery to achieve systemic levels of therapeu-
tic protein. Cytokine genes can be delivered to inhibit angiogenesis and increase
antitumor immunity without affecting surrounding healthy tissue or causing adverse
systemic effects.
Electroporation can be used to deliver nucleic acids such as DNA, messenger
RNA (mRNA), small interfering RNA (siRNA), and microRNA (miRNA). While it
is unclear if transfer of the smaller RNA molecules occurs in the same manner as the
transfer of larger DNA molecules, it has been suggested that the transfer of RNA is
more efficient and associated with reduced toxicity. The advantage to transfecting
cells with mRNA rather than DNA lies in allowing the host cell to translate and
process the protein by its own cellular pathways. Using this approach to deliver
mRNA encoding antigen, cytokine or even immune receptor ligands can program
immune cell behavior and function, which results in generation of antigen-
presenting cells and activated immune cells. Electroporation of mRNA into dendritic
cells is regarded by some as the method of choice for generating dendritic cell (DC)-
based vaccines for cancer immunotherapy.
Cytokine Gene Electrotransfer for Cancer Immunotherapy
Cytokine gene therapy has tremendous potential for use in the treatment of a range of
diseases. It is particularly of interest in the treatment of cancer. Delivery of cytokine
genes can elicit strong inflammatory reactions in the host that impair the growth of
tumor cells or kill them altogether. Another benefit is its potential for generating
long-lasting immune memory that goes along with tumor elimination. Therapeutic
cytokines that have been studied for use in cancer usually include type I and type II
interferons (IFN); interleukins (IL) such as interleikin-2 (IL-2), interleukin-12
(IL-12), interleukin-15 (IL-15), interleukin-10 (IL-10), and more recently
interleukin-17 (IL-17), interleukin-21 (IL-21), and interleukin-33 (IL-33); the hema-
topoietic growth factors granulocyte-macrophage colony-stimulating factor
(GM-CSF) and granulocyte colony-stimulating factor (G-CSF); and members of
the tumor necrosis factor (TNF) family, such as TNF-alpha, and members of the
transforming growth factor (TGF) family such as TGF-beta.
In order to develop a successful and effective vaccine or anticancer therapeutic,
certain limitations must be overcome. The therapy must enable the patient to
overcome the immune evasion and suppression mechanisms displayed by tumors.
To this end, the therapy must be able to recognize or target specific antigens
associated with the tumor, recruit antigen-presenting cells, augment antigen presen-
tation, and stimulate T-cell activation and proliferation. Electroporation provides a
unique platform for development of this kind of therapeutic. Electroporation is an
important tool that can be utilized in cytokine gene therapy as it allows for the safe
and efficient transient delivery of target genes directly to tissues of interest while
avoiding the involvement of surrounding tissues, consumption by competing sup-
pressor cells, and systemic toxicity that results from frequent high-dose administra-
tion of recombinant cytokines. Delivery of cytokine genes directly to tumor cells by
electrotransfer allows tumors to essentially become factories that, for a limited time,
can sustainably release the cytokine of interest directly into the tumor microenvi-
ronment. There they can act to stimulate dormant intratumoral immune cells and
recruit immune cells into the tumor. This “laboratory” provides the optimal condi-
tions for generating a robust immune response which involves the destruction of
tumor cells, subsequent presentation of a broad range of tumor antigens, and
generation of a tumor-specific adaptive response that may result in systemic specific
long-term immune memory.
The next generation of cytokine gene electrotransfer for cancer immunotherapy is
prophylactic anticancer vaccines. Efforts have been made toward accomplishing this
by delivering cytokines in combination with other molecules such as tumor antigens
and immune system activators. This will be discussed in greater detail in the context
of clinical studies later on in this chapter. IL-33, an alarmin-like molecule that is
released during tissue damage, mediates pro-inflammatory responses ranging from
activation and recruitment of antigen-presenting cells to enhancing adaptive immu-
nity. Investigators hypothesize that it could serve as a novel adjuvant for human
papilloma virus (HPV)-specific immunotherapy by augmenting type 1 helper (Th1)
1760 S.A. Shirley
and cluster of differentiation 8 (CD8) T-cell responses to induce antitumor immunity.

Using TC1, a B-cell tumor model that expresses HPV, they demonstrate amplifica-
tion of effector-memory CD8 T cells and protection from challenge upon IL-33
overexpression (Villarreal et al. 2014). They conclude that this cytokine is a strong
candidate for antitumor immunotherapy.
Electroporation can be used to transfer cytokines to immune cells, which are
subsequently adoptively transferred to the host. Weinstein-Marom et al. have shown
cytokine mRNA delivered by electroporation effectively enhances T-cell prolifera-
tion and function in vitro (Weinstein-Marom et al. 2016). While in vivo studies have
not yet been conducted, their findings may have potential for use as an adoptive cell
cancer therapy. Bonehill et al. showed enhanced DC maturation and T-cell stimula-
tory capacity when CD40 ligand (CD40L) mRNA is delivered to dendritic cells in
combination with CD70 and toll-like receptor 4 (TLR4) mRNA. They also show that
when tumor-associated antigen mRNA is also electroporated, they are able to
produce tumor-specific CD8 T cells (Bonehill et al. 2008). This research has led to
a phase I/II clinical trial in stage IV melanoma patients.
Cytokine Gene Electrotransfer for Autoimmune Diseases
Cytokine gene therapy for autoimmune diseases focuses on restoring immune

homeostasis by attempting to reduce inflammatory responses at the sites of inflam-
mation by delivering immune regulatory cytokines. Genes encoding cytokines can
be delivered by electrotransfer directly to immune cells which are then adoptively
transferred to patients, locally to autoimmune lesions or to muscle for sustained
systemic delivery. Cytokine gene therapy has been studied for a range of diseases
including autoimmune diabetes, experimental encephalomyelitis, lupus, and arthri-
tis. The approach to cytokine gene therapy can be multipronged: delivery of neu-
tralizing cytokine receptor genes, delivery of anti-inflammatory cytokine genes, or
vaccination with an autoantigen/cytokine combination.
DNA encoding cytokine inhibitors usually in the form of soluble receptors have
been delivered and show protection against autoimmune diseases. IFN-gamma
receptor, IL-1 receptor alpha, and TGF receptor II are some of the molecules
under investigation. DNA encoding IFN-gamma receptor delivered by electropora-
tion has been studied preclinically for the treatment of lupus. The investigators
demonstrate arrest of disease progression and improved overall survival when the
treatment was initiated in its early stages (Lawson et al. 2000). Electrotransfer of
soluble TNF receptors in ciliary muscles for the treatment of experimental autoim-
mune uveoretinitis resulted in reduction of retinal damage and reduced and delayed
uveitis (Kowalczuk et al. 2009; Touchard et al. 2009).
Anti-inflammatory cytokines produced mainly by regulatory T (Treg) cells and
type 2 helper (Th2) T cells TGF-β, IL-4, IL-10, and IL-13 have been the focus of
many studies on anti-cytokine gene therapy. IL-18 delivered intramuscularly by
electroporation was found to promote a pro-inflammatory immune response in
diabetic mice (Oikawa et al. 2003). Dendritic cells electroporated with IL-4
mRNA were investigated for the treatment of autoimmune diabetes (Creusot et al.
2003). An increase in regulatory T-cell function and maintenance of stable glycemia
was reported. Other studies examined the effect of electrotransfer of plasmid
encoding IL-4 for experimental allergic encephalomyelitis (Ho et al. 2006) and
collagen-induced arthritis (Ho et al. 2004); both reported disease modulation. The
findings of these studies are not always positive. A study of high-level expression of
IL-4 following electroporation in nonobese diabetic (NOD) mice resulted in disease
progression rather than amelioration (Horiki et al. 2003). Electroporation of plas-
mids encoding IL-10 has been investigated for the treatment of autoimmune myo-
carditis (Watanabe et al. 2001) and autoimmune optic neuritis (Matsuda et al. 2012).
In both cases, the treatment was effective at disease suppression.
Overall delivery of cytokine gene therapy for autoimmune diseases has improved
the efficacy of treatment compared to injection of DNA alone. With this in mind,
gene electrotransfer shows promise for the development of effective novel therapeu-
tics for wide range of autoimmune and chronic inflammatory disorders.
Cytokine Gene Electrotransfer for Infectious and Other Diseases
Cytokines and DNA vaccines delivered by electroporation hold great potential for
the treatment and eradication of some human diseases. It has been investigated
preclinically and clinically for the treatment of a variety of diseases including
human immunodeficiency virus (HIV), hepatitis, and other diseases. Electrotransfer
of cytokines for DNA vaccines is discussed in more detail below as it relates to
clinical studies and will not be covered in much detail here. It is noteworthy to
highlight in this section that researchers are investigating the potential of this therapy
beyond HIV and cancers. IL-5 delivered by electroporation for the treatment of
leishmaniasis was shown to be effective at slowing the development of infection.
Electroporation of cytokine genes has also been investigated in Alzheimer’s disease.
Immunization with DNA encoding an Aβ IL-4 fusion protein delivered by electro-
poration was able to induce anti-Aβ antibodies and elicited a Th2-polarized immune
response.
Cytokine Gene Delivery in Clinical Studies
Over the years, the interest in using electroporation in the clinic for gene delivery has
increased. Currently there are more than a dozen registered trials using electropora-
tion to deliver cytokine genes (Table 1). Gene electrotransfer has been found to be
safe and well tolerated. Most of the clinical studies listed in Table 1 are focused on
delivering cytokine genes for cancer immunotherapy or as an adjuvant in DNA
vaccines for cancer therapy or viral infections. There are two trials that use cytokine
electrotransfer to deliver genes to immune cells that are then adoptively transferred
to the patient for cell-based immunotherapy of cancer.
1762 S.A. Shirley
Table 1 Clinical trials involving the electrotransfer of cytokine genes registered with ClinicalTrials.
gov
Material
Cytokine Usage delivered Phase Condition Identifier
TGFB2 Combination DNA I Advanced NCT00684294
antisense/ metastatic
GMCSF carcinoma
GM-CSF Combination DNA I colorectal cancer NCT01064375
IL-2 Single agent DNA I Metastatic NCT00223899
melanoma
HPV FLT3L Combination DNA I Cervical NCT02411019
fusion intraepithelial
neoplasia 3
CD40L/CD70/ Combination mRNA I/II Melanoma NCT01530698
TLR4
IL12 Single agent DNA II Cutaneous NCT01579318
lymphoma (CTCL)/
mycosis fungoides
(MF)
IL12 Single agent DNA II Merkel cell NCT01440816
carcinoma (MCC)
IL12 Single agent DNA II Malignant NCT01502293
melanoma
IL12 Single agent DNA II Squamous cell NCT02345330
carcinoma of the
head and neck
IL12 Single agent DNA I Triple negative NCT02531425
breast
IL12 Single agent DNA I Malignant NCT00323206
melanoma
IL12/HIV- Combination DNA I Healthy individuals NCT01578889
MAG (for HIV)
IL12/HIV- Combination DNA 1 Healthy individuals NCT01496989
MAG (for HIV)
IL12/ Combination DNA I Healthy individuals NCT02431767
PENNVAX-GP (for HIV)
IL12/ Combination DNA I HIV-infected adults NCT01266616
HIV-MAG
IL12/ Combination DNA I Healthy individuals NCT00991354
PENNVAX-B (for HIV)
IL12/hTERT Combination DNA I Breast, lung, NCT02327468
pancreatic, and
other cancers
IL12 Combination DNA I HIV-infected adults NCT01859325
IL12/HBsAg/ Combination DNA I Chronic hepatitis C NCT02772003
HBcAg (HCV)
IL12/HBsAg/ Combination DNA I Chronic hepatitis B NCT02431312
HBcAg (HBV)
As mentioned previously, cytokine genes are most commonly delivered by

electrotransfer to the muscle, skin, or directly to the tumor. The route of delivery
for DNA vaccines delivered by electroporation is intramuscular or intradermal.
Intratumoral delivery is the most common route for the cancer therapy trials.
Interleukin-12 is the most extensively studied cytokine transferred by electropora-
tion in clinical trials to date. It has been studied as a single-agent intratumoral
therapeutic for a variety of cutaneous cancers or as an adjuvant in DNA vaccines
for the treatment of viral infections. CD40L, IL-2, Fms-like tyrosine kinase-3 ligand
(Flt3L), and GM-CSF are cytokines also being investigated, mainly as immunother-
apeutic interventions for cancer.
DNA Vaccines
DNA vaccines consist of plasmid DNA encoding genes associated with the condi-
tion being immunized against. This could be viral proteins or tumor antigens. The
route of administration can be intradermal, intramuscular, or even intranasal. Though
advances in the field over the years have improved vaccine efficacy, there is still
room for improvement as poor immunogenicity remains a concern. Advances in
molecular biology have provided us with a new generation of molecular and genetic
adjuvants that can be used as an alternative to conventional approaches such as
Freund’s adjuvant or aluminum salts. Using genetic adjuvants to enhance the
immunogenicity of DNA vaccines has been explored as well as using electroporation
as a delivery method.
Electroporation is a promising DNA delivery method that provides enhanced
expression of the transgene compared to DNA injection alone. Intradermal or
intramuscular delivery of an antigen/cytokine combination by electroporation
could improve the immunogenicity of DNA vaccines compared to injection of the
vaccine alone. The addition of a pro-inflammatory cytokine such as IL-12 is a logical
choice as a molecular adjuvant because it promotes cell-mediated immunity and
incudes adaptive immune responses. IL-15 and its receptor alpha is another likely
candidate that can be considered for its ability to promote development of memory
cells.
A number of clinical studies have been initiated to investigate the adjuvant effect
of IL-12 in combination with various DNA vaccines in both healthy and non-healthy
patients (Table 1). Some report superior immunogenicity with the addition of IL-12
compared to vaccination without the addition of IL-12 (Kalams et al. 2013; Jacobson
et al. 2016). The intensity of the immune response varies from study to study. Some
studies show the addition of IL-12 augments CD4 and/or CD8 T-cell responses,
while others show no increase in immunogenicity (Mpendo et al. 2015).
Kalams et al. administered PENNVAX-B (PV) DNA vaccine, which consists of
three plasmids encoding HIV proteins Env, Gag, and Pol, along with plasmid IL-12
to healthy HIV-uninfected adults in a phase I study resulting in superior immuno-
genicity compared to PV alone. The dose escalation study conducted by Jacobson
et al. administered plasmid DNA encoding multi-antigenic HIV (HIVMAG) and
1764 S.A. Shirley
IL-12 to HIV-infected adults. The HIVMAG DNA vaccine contains two plasmid
vectors; one encodes HIV-1 gag/Pol fusion and the other, a dual-promoter vector,
encodes a Nef/Tat/Vif fusion and envelope. The authors conclude this approach is
unlikely to be useful as a viral eradication strategy as no enhancement of antiviral
CD8+ T cells and only mild elevations in CD4 T cells were observed only at low
doses of plasmid IL-12. They hypothesize that a more robust response could be
elicited using a vaccine strategy in which viral vector delivered HIV-DNA is used to
boost a strategy that was used in macaques with positive results. It is noteworthy to
mention that the study by Mpendo et al. utilized a similar strategy, vaccinating with
HIVMAG and IL12 plasmid DNA delivered by EP followed by a viral vector boost.
They report no adjuvant effect of coadministration of IL12.
Other phase I studies are ongoing using IL-12 as an adjuvant for DNA vaccines
targeting chronic hepatitis B and C, HIV, and a variety of cancers. Based on the
clinical data published so far, it is clear that DNA vaccines are no longer in infancy
but have not yet realized their full potential. While safe and generally well tolerated,
the optimal dose of cytokine adjuvants such as IL-12 and the optimal treatment
regimen, prime boost with DNA EP vs. viral vectors, have not yet been determined.
Cancer
Cancer immunotherapy is centered around generating an immune response within

the host that is able to destroy tumor cells while allowing the host to generate and
retain lasting memory to the tumor antigens. Delivery of cytokine genes directly to
tumors using electroporation is an effective way of stimulating the host immunity to
tumor antigens which may result in reduction of tumor burden and lasting immunity.
IL-12 is currently the most studied cytokine delivered by EP as either DNA or RNA
to patients for cancer therapy, either as a single agent or in combination, in clinical
studies. It has been studied for treatment of melanoma, cutaneous lymphoma,
Merkel cell carcinoma, head and neck carcinomas, lung cancer, triple negative breast
cancer, and pancreatic cancer. EP delivery of other cytokines such as TGFB2,
GM-CSF, IL-33, and IL-2 has also been investigated.
The first in human phase I dose escalation study of intratumoral IL-12 electro-
poration in patients with metastatic melanoma showed cytokine electroporation is an
effective, reproducible, and titratable method of anticancer immunotherapy (Daud
et al. 2008). Two of nineteen patients showed complete regression of all metastases
including those that were untreated over a period of 6–18 months. Researchers
reported no new evidence of distant disease up to the date of publication. An
additional eight of nineteen patients showed stable disease lasting from 4 to
20 months at distant sites with no new lesions or a partial response to therapy.
Since then several studies have been initiated to further investigate the efficacy of
IL-12 electroporation for treatment of cutaneous lymphoma, Merkel cell carcinoma,
triple negative breast cancer, and squamous carcinoma of the head and neck. A phase
II study has been initiated and is currently ongoing to study the safety and effec-
tiveness of IL-12 electroporation in patients with metastatic melanoma (Daud et al.
2014). The investigators report three of 27 enrolled patients show complete response
and 13 patients show regression of untreated lesions suggesting the induction of a
systemic antitumor response up to the time of abstract publication. The investigators
also reported an increase in frequency of activated intratumoral and circulating
natural killer (NK) cells, in accordance with the expected effects of IL-12 delivery.
The success of these studies and the data from the preclinical studies that
preceded them demonstrate the potential for the inclusion of IL-12 in combination
with other anticancer agents, using the electroporation DNA transfer platform. There
is an ongoing clinical study utilizing IL-12 as an adjuvant in combination with the
human catalytic reverse transcriptase subunit of telomerase (hTERT). The investi-
gators hypothesize co-delivery of these agents using electroporation will generate a
robust immune response that will reduce the risk of relapse in high-risk breast, lung,
and pancreatic cancer (Vonderheide 2015).
Recognition and subsequent presentation of cancer-associated antigens is often a
problem that allows the persistence of malignancy. An ongoing clinical study seeks
to address this concern. A TGFB2 antisense GM-CSF combination vector will be
delivered to autologous cancer cells harvested from patients with advanced meta-
static carcinoma by electroporation. The cells are then irradiated and transferred to
the patient. The investigators hypothesize the GM-CSF will increase presentation of
tumor antigen as well as dendritic cell recruitment. At the same time, the TGFB2
antisense will reduce immune inhibition. While a promising approach, T-cell recruit-
ment and activation is not addressed in this study. A similar approach is undertaken
in another study that delivers GM-CSF as part of a combination vaccine to patients
with colorectal cancer. The vaccine consists of a plasmid vector encoding wild-type
human carcinoembryonic antigen (CEA) fused to a tetanus toxoid. The investigators
hypothesize GM-CSF will serve as an adjuvant for the priming of the immune
system against the CEA.
Delivery of IL-2 via electroporation has been shown to be safe and well tolerated
in patients. In a small phase I study in patients with metastatic renal cancer,
colorectal cancer, and lymphoma, patient natural killer-like T cells from patient
peripheral blood were obtained and transfected by electroporation with IL-2 and
infused back into the patient (Schmidt-Wolf et al. 1999). The investigators report an
increase in serum levels of IFNγ, GM-CSF, and TGF-β as well as CD3 lymphocytes
in the blood during treatment. All ten patients in this study had progressive disease
when they entered the protocol; after treatment, six continued to have progressive
disease, three patients showed no change by the treatment (no progression), and one
patient with follicular lymphoma showed a complete clinical response. These results
represent a promising first step toward future trials. Another phase I study has been
completed that investigated intratumoral delivery by electroporation of VCL-IM01,
a plasmid encoding IL-2 in patients with recurring metastatic melanoma. The results
of this study have not been published to date.
There are studies ongoing using electroporation to transfer other cytokine family
members including Flt3L and CD40L in combination with other agents to treat
cancer. Researchers are using electroporation to deliver an HPV DNA vaccine called
GX-188E which is comprised of the E6/E7 fusion protein of human papilloma virus
1766 S.A. Shirley
and the cytokine Flt3L to patients with cervical intraepithelial neoplasia 3 (Kim et al.
2014). Once again, in this vaccine the cytokine serves as an adjuvant, to stimulate the
proliferation of hematopoietic progenitor cells. In this study, eight out of the nine
patients showed an enhanced polyfunctional CD8 T-cell response, and seven out of
nine patients had complete regression of their lesions within 36 weeks of follow-up.
A study to investigate the safety and immunological effects of a TLR dendritic cell
vaccine and TriMix dendritic cell (DC) vaccine was completed in melanoma patients
(Bonehill 2009). TriMix-DC is an autologous monocyte-derived dendritic cell that is
electroporated with mRNA encoding gp100 and tyrosinase, TLR4, and CD70. The
investigators conclude electroporated TriMix mRNA can stimulate antigen-specific
T cells both in vitro and through vaccination.
Conclusions
Taken together, the clinical studies discussed in this chapter (Table 1.) provide
evidence that electroporation of cytokines, either as a single agent or in combination
with other interventions, is able to stimulate the immune system by promoting cell
proliferation or activation, antigen presentation, and pro-inflammatory cytokine
production. The procedures were well tolerated with no adverse events reported.
Delivery using electroporation greatly increases protein expression compared to
injecting DNA alone, which allows researchers to overcome one of the main
limitations of successfully using DNA-based cytokine gene therapy. The findings
of these studies provide insight for the design of future therapeutic vaccines that will
effectively treat a range persistent and resistant cancers and infections in patients.
Cross-References

Electrochemotherapy
▶ Clinical Use of DNA Vaccines
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CJI.0000000000000109
Part VIII
Biomedical Applications of Electroporation
Gregor Serša and Richard Heller

Electrochemotherapy and Its Clinical
Applications 93
Julie Gehl and Gregor Serša
Abstract
Electrochemotherapy has grown out of simple cell experiments through preclin-
ical studies to experimental clinical studies, to an accepted treatment modality for
cutaneous metastases for any type of cancer. Electrochemotherapy has had a
dynamic spread from few initial centers using custom-made electrodes to the
current situation where standard operating procedures and clinically approved
equipment are being used in many cancer centers. Treatment results have been
remarkably consistent, possibly due to a robustness of the technology, and helped
by the availability of clinically approved equipment and the early definition and
adoption of standard operating procedures. Based on successful treatment of
cutaneous metastases, a number of efforts have been initiated to expand treatment
to tumors in internal organs. This requires development of specialized equipment,
as well as clinical trials. The first of these trials are now published showing very
promising results, and many further studies are ongoing. Finally a number of
other avenues are being explored, including new drugs to be used, advanced use
of imaging techniques, as well as combination with other treatments.
Keywords
Electrochemotherapy • Clinical studies • SOP • Clinical indications • Technical
development
J. Gehl (*)
e-mail: Karen.Julie.Gehl@regionh.dk
G. Serša

DOI 10.1007/978-3-319-32886-7_91
1772 J. Gehl and G. Serša
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1772
Electrochemotherapy as a Cancer Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1775
What Types of Cancer Can Be Treated? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1775
The Effect on Normal Tissues Relative to Malignant Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1775
Principles of Electrochemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1776
Importance of the ESOPE Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1777
Recent Developments in Electrochemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1778
Prognostic Factors and Clinical Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1780
Tumor Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1780
Tumor Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1780
Previous Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1781
Current Developments for Treatment of Deep-Seated Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1782
Preparation of New Standard Operating Procedures and Introduction of
Electrochemotherapy into Standard Clinical Practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1783
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1784
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1784
Introduction
The first mention of the use of electric pulses to permeabilize cell membranes in
biology dates back to the late 1970s (Kinosita and Tsong 1977), followed by a
groundbreaking paper describing gene transfer by the use of electroporation in 1982
(Neumann et al. 1982). A second wave of the use of electric pulses for permeabi-
lization came with the first papers describing increased uptake of chemotherapeutic
agents (Preclinical Studies on Electrochemotherapy: Sersa, G., Bosnjak, M.,
Cemazar, M., Heller, R.) in cells exposed to permeabilizing electric pulses (Okino
and Mohri 1987; Orlowski et al. 1988), showing that in particular the effect of the
drug bleomycin could be enhanced several hundred fold. Bleomycin is a known
chemotherapeutic agent, which has been shown to act as an enzyme creating 5–10
double-strand breaks in DNA per molecule internalized into the cell, yet this drug is
hydrophilic and charged, with limited internalization into the cells without permeabi-
lization of the membrane (Gothelf et al. 2003). The further expansion of this work
into animal models confirmed the activity of bleomycin combined with electropo-
ration. Simultaneously, the work trying to help patients with this new treatment
entity ensued, and in 1993 (Belehradek et al. 1993), the first results on patients with
cutaneous metastases of head and neck cancer were published, using a standard
intravenous dose of bleomycin combined with electric pulses to the cutaneous
metastases. Also around this time, electrochemotherapy using bleomycin by
intratumoral delivery was being developed for the treatment of primary skin cancers
(Glass et al. 1996), as well as cutaneous metastases of malignant melanoma (Heller
et al. 1996; Gehl and Geertsen 2000) (Fig. 1).
Along with the use of bleomycin, also cisplatin was demonstrated to be effective
in electrochemotherapy, based on extensive preclinical studies demonstrating its
effectiveness, either by intravenous or intratumoral administration. The clinical
data obtained demonstrated its effectiveness on melanoma metastases as well as on
various other tumor types (Sersa et al. 2008).
93 Electrochemotherapy and Its Clinical Applications 1773
Fig. 1 Course of treatment for patient with malignant melanoma metastases, treated with intrave-
nous bleomycin and type III electrodes, having an array of needle electrodes fixed on a holder in a
hexagonal pattern, under general anesthesia. One of eight treated metastases is shown. Before
treatment the metastasis was ulcerated and caused hemorrhage, pain, and discomfort. One month
after treatment, the lesion was covered by a crust. Around the crust, needle marks in normal tissue
are visible, because the tumor was covered including a margin of normal tissue. Whereas the tumor
area became necrotic, the normal tissue was very little affected, indicating a therapeutic window. Six
months after treatment, the treated nodule was in CR. The crust fell off after 10 weeks, revealing
normal skin that had healed underneath the nodule (From Gehl, Ugeskrift for Laeger 2005, with
permission)
At this time it became evident that (1) electrochemotherapy was highly efficient
in cutaneous tumors, but also (2) that there was a need for commercialization to
allow purchasable clinically approved equipment, and (3) that the different methods
used in the initial studies needed to be tested in a larger setting in order to obtain
standard operating procedures. In other words, electrochemotherapy needed to move
from custom-made equipment in small investigator-initiated trials to a technology-
driven clinical acceptance of the technology.
To this end, the ESOPE (European Standard Operating Procedures on Electro-
chemotherapy) was formed as a consortium supported by the European Commis-
sion. Here, four European cancer centers gathered and defined a way to examine
standard operating procedures through experiences obtained by a common clinical
trial (Marty et al. 2006), where different electrodes (hexagonal, linear array, plate
electrodes), two drugs (bleomycin and cisplatin), and different methods of anesthesia
(general anesthesia, local anesthesia, surface analgesia) were tested and results in
terms of tumor response and patient-reported toxicity were measured. At the end of
this study, the first set of standard operating procedures were defined (Mir et al.
2006). This first manual for electrochemotherapy is extensively quoted and used and
made a very important contribution to the dissemination of electrochemotherapy.
In 2013, the National Institute of Health and Care Excellence (NICE) of the UK
released a guidance (National Institute for Health and Care Excellence 2013) stating
that “There is sufficient evidence of efficacy of electrochemotherapy for treating
metastases in the skin from tumors of non-skin origin and melanoma to support its
use as a palliative treatment. There are no major safety concerns.” This 2013 paper
marked the 20-year anniversary since the publication of the first clinical trial. In this
period, electrochemotherapy has progressed from a purely investigational treatment
performed with home-designed and produced equipment to a treatment that
was recognized as important for patients with cutaneous metastases (Spratt et al. 2014).
Fig. 2 Large recurrence with

varying depth and
inflammation on the left chest
wall of a 68-year-old female.
Left column shows from the
top clinical presentation,
CT-scan, PET scan at 60 min
p.i., PET scan at 120 min p.i.,
and PET scan 180 min
p.i. Right column. Same
patients after one treatment.
Change in SUVmax in target
lesion at baseline compared to
follow-up was 29.7% at
60 min p.i., 71.2% at 120 min
p.i., and 83.1% at 180 min
p.i. (From Matthiessen,
Radiol Oncol, 2013, with
permission)
Further developments have focused on transferring the success obtained in the

treatment of cutaneous metastases to tumors in internal organs (Miklavcic et al.
2012). And this is a challenge in different ways; on the one hand, there is great
benefit from the development of clinical-grade equipment, as well as extensive
clinical experience; on the other hand, all tissues and all presentations of metastatic
lesions are different, and this poses new challenges. To give an example, the
challenge in treating bone metastases is the rigidity of the tissue, whereas
soft tissue metastases in the liver may be accessed, but is situated just below the
heart.
Other new developments include new drugs, such as calcium electroporation
(▶ Chap. 85, “New Drugs for Electrochemotherapy with Emphasis on Calcium
Electroporation”: Frandsen, S., Gehl, J.), as discussed elsewhere in this book.
Important advances also include an understanding of the use of imaging modalities
such as MRI (▶ Chap. 41, “Diffusion Weighted Magnetic Resonance Imaging for
Detection of Tissue Electroporation In Vivo”: Mahmood, F.), and PET/CT Fig. 2,
and CT-guided placement of electrodes (▶ Chap. 54, “Treatment Planning for
Electrochemotherapy and Irreversible Electroporation of Deep-Seated Tumors”:
Kos, B.). Furthermore, combination with other treatments, such as immunotherapy
(▶ Chap. 103, “Adjuvant Immunotherapy as a Tool to Boost Effectiveness of
Electrochemotherapy”: Kamensek, U., Kos, S., Sersa, G.), is currently being
investigated.
This chapter describes the many aspects of clinical use of electrochemotherapy, as
well as future avenues.
Electrochemotherapy as a Cancer Treatment
What Types of Cancer Can Be Treated?
Due to the dramatic enhancement of cytotoxicity that can be achieved, electro-

chemotherapy has been shown to work across a wide range of tumor histologies
including malignant melanoma, breast cancer, colorectal cancer, planocellular car-
cinoma, basocellular carcinoma, and transitocellular carcinoma (Campana et al.
2014a). As electrochemotherapy is so highly efficient, most treatments are being
given as a once-only treatment. A concern in chemotherapy is the buildup of drug
resistance over time, but with electrochemotherapy there really is not much of
chance of building secondary resistance as a function of treatment exposure.
There is however a different challenge; in order to treat the tumor efficiently, the
whole tumor must be covered by the electric field, and the drug must be available in
sufficient quantities. In other words, although all types of solid cancers could be
treated, there is an anatomical restriction as the tumor in its entirety needs to be
covered by the electric field. In this way, electrochemotherapy carries similarities to
surgery – although electrochemotherapy may be used also in cases of inoperable
cancer or metastases.
The Effect on Normal Tissues Relative to Malignant Tumors
Interestingly, there is a striking difference between normal tissue and tumor sensi-
tivity to electrochemotherapy. As evidenced in Fig. 1, there is typically a rapid and
extensive effect on tumors, while normal tissue treated with the same drugs and
electrical parameters in the perimeter of the tumor shows very limited response.
The scientific background for this difference is currently being investigated, and
there are likely several factors involved in this differential response. Important
factors include differential drug uptake in malignant and normal tissues as in the
case of bleomycin, slower resealing of membranes in malignant than in normal cells
allowing higher uptake in malignant cells, differences in sensitivity to in vivo
electroporation, and differences in response of normal endothelial vasculature and
tumor endothelial vasculature (▶ Chap. 39, “Blood Flow Modifying and Vascular-
Disrupting Effects of Electroporation and Electrochemotherapy”: Cemazar, M.,
Markelc, B.; ▶ Chap. 78, “Preclinical Studies on Electrochemotherapy”: Sersa, G.,
Bosnjak, M., Cemazar, M., Heller, R.).
Some of these effects would be specific to electrochemotherapy, but most would
be relevant for different electroporation technologies. Thus, selectivity in treatment
of malignant tumors is also described for irreversible electroporation.
Principles of Electrochemotherapy
Electrochemotherapy is based on the principle that chemotherapeutic drug cytotox-

icity is increased by exposure of cells to externally applied electric field, which
permeabilizes the cell membrane and allows diffusion of the drug into the cells. The
whole procedure is based on a few basic principles:
• The first principle is that with electrochemotherapy, only drugs that have impaired
transport through the cell membrane can be used, such as bleomycin, cisplatin,
and as recently introduced calcium (▶ Chap. 85, “New Drugs for Electro-
chemotherapy with Emphasis on Calcium Electroporation”: Frandsen, S., Gehl,
J.). Several preclinical studies confirm this principle at least for the clinically
approved chemotherapeutics. These preclinical studies also unequivocally show
that lipophilic drugs, which have unhampered access to cytosol, are not suitable
for electrochemotherapy, and lack of its potentiation by electroporation was
confirmed. The potentiation of bleomycin and cisplatin was demonstrated by
the increased drug uptake in cells and tumors, as well as by the increased DNA
binding, and several fold potentiation of cytotoxicity was demonstrated.
• The second overall principle is that the drug needs to be present in the tumor when
electric pulses are being applied to the tumors. In the clinics two routes of drug
administration are being used, intravenous and intratumoral, and in a few studies
also the combination of both. The combination of both routes of drug adminis-
tration is not well established and needs to be used with great care not to overdose
the tumors. The time for drug to reach the entire tumor depends on the route of its
administration, after intravenous approximately 8 min, whereas after intratumoral
only after a few minutes. Due to the differences in the vascularization of different
tumors, the drug uptake and distribution vary. Predominantly tumors in heavily
pretreated areas, by surgery, or radiotherapy, may have lower drug uptake, which
may affect tumor response.
• The third principle is the necessity of coverage of the tumor with the adequate
electric field in order to permeabilize most of the tumor cells (Miklavcic et al.
2006). In principle with adequate electric field distribution and the optimal drug
distribution, complete response of the treated tumors is obtained. In principle
based on numerical modeling of the electric field and based on the clinical
experience for the commercially available electrodes with fixed geometry, the
electric field is sufficient to permeabilize the area of the electrode used. When the
tumors are bigger than the geometry of the electrodes, a repetitive application of
electric pulses, with repositioning of the electrodes to different places of the
tumor, can also achieve adequate coverage of the electric field. As will be
discussed later on, for the single electrode positioning, a treatment plan is needed
with electric field calculation. The choice of the electrode is predominantly
dependent on the anatomical location of the tumors to be treated. Different sets
of electrodes are available for the treatment of superficial tumors. These tumors
may differ both in size and depth, and other considerations may also affect
electrode choice including the anatomical position of the tumor on the body,
e.g., in sensitive areas, and in the face, smaller electrodes should be chosen
(Campana et al. 2014a).
Importance of the ESOPE Study
In 2006, standard operating procedures for electrochemotherapy (SOP) were

published (Mir et al. 2006). The SOP were derived from the experience of the
groups that have had the experience with electrochemotherapy and were adopted
to at that time the only clinically certified electric pulse generator with electrodes
with fixed geometry.
The SOP standardized electrochemotherapy concerning the drug dosage, admin-
istration route, and selection of the electrodes according to the size and the number of
the tumor nodules to be treated. The SOP also took into account whether local or
general anesthesia is needed. It provided an algorithm what to do in specific
situations and how to proceed throughout the treatment, including also the necessary
equipment and consumables.
With the generator, the clinicians had access to an instrument that helped perform
the treatment. In the ESOPE study, a center without previous experience in electro-
chemotherapy took part and in the study provided the same success rate as the other
already experienced centers. Furthermore, the clinical results that have followed the
ESOPE study have provided very similar tumor control rate as the ESOPE study and
the previous ones. This supports the view that electrochemotherapy is a robust
technology, with a high likelihood of success in different settings.
The clinical study ESOPE published alongside the SOP was possible by the
availability of the electric pulses generator that was designed and then produced and
certified for clinical use. The availability of this generator, in fact, enabled the fast
spread of electrochemotherapy throughout Europe.
The ESOPE study was conducted in order to develop the new SOP using
electric pulse generator and electrodes developed within Cliniporator project
(QLK-1999-00484). It had an ambitious goal to compare the effectiveness of
electrochemotherapy based on the different drugs used (bleomycin, cisplatin), on
different cutaneous metastases, with different drug administrations (intravenous,
intratumoral), with the use of different sets of electrodes. In spite of the different
variables in the study, the ESOPE study has demonstrated that regardless of the
tumor type treated or the drug or its administration route, the local tumor control
probability was 73% (complete response rate) (Marty et al. 2006). This was the
first nonrandomized, prospective multicenter study on electrochemotherapy with
the standardized treatment protocol. Its value is that it was so convincing that
many cancer centers throughout Europe started to use electrochemotherapy.
Furthermore, they have obtained similar results as the ESOPE study and the
studies before that (Sersa 2006). All these studies provided solid evidence on
effectiveness and safety of electrochemotherapy of cutaneous tumors of different
histology.
Recent Developments in Electrochemotherapy
The technology of electrochemotherapy has spread out through the Europe into more
than 140 centers. This was possible based on the SOP that showed safety of the
procedure, with no reported serious adverse effects.
After the ESOPE study, several case controlled studies demonstrated effective-
ness of electrochemotherapy on different tumor histologies. The studies confirmed
effectiveness of electrochemotherapy in breast cancer (Matthiessen et al. 2012;
Campana et al. 2012) and malignant melanoma other than skin cancers, including
basal cell carcinoma, squamous cell carcinoma of the skin, Merkel cell carcinoma,
Kaposi’s sarcoma as well as soft tissue sarcoma, squamous cell carcinoma of the
head and neck, cutaneous metastases of adenocarcinoma from, e.g., colorectal
cancer, as well as bladder cancer and renal cancer. The data provided evidence on
the effectiveness of electrochemotherapy on different superficial tumor types. How-
ever prospective, controlled studies were needed in order to explore more in depth
the clinical value of electrochemotherapy.
Such studies were possible by pooled data from different cancer centers. In order
to do that, an InSPECT database (▶ Chap. 95, “International Network for Sharing
Practices on Electrochemotherapy (InspECT): An Integrative Patients Treatment
Consortium”: Qualigno, P., Gehl, J.) was created. The database provided the basis
of pooling the data on different tumor types treated in different cancer centers. The
number of centers is steadily increasing and the number of patients in the database as
well. The InSPECT consortium was then able to provide clinical evidence on the
treatment of different patients in larger cohort. The first paper provided evidence on
the effectiveness of the different tumor types after ESOPE study (Matthiessen et al.
2012). However later on there were papers published on specific tumor types, for
example, on chest wall breast cancer (▶ Chap. 111, “Electrochemotherapy for Breast
Cancer”: Campana, L., Matthissen, L.) recurrences and lately on cutaneous head and
neck tumors (▶ Chap. 102, “Electrochemotherapy of Head and Neck Cancer”:
Benazzo, M., Bertino, G., Groselj, A.). Further details are given in respective
chapters of this book (▶ Chap. 96, “Electrochemotherapy of Cutaneous Metastases”:
Snoj, M., Farricha, V., Matthissen, L.; ▶ Chap. 97, “Electrochemotherapy of Basal
Cell Carcinoma”: Clover, J.; ▶ Chap. 98, “Electrochemotherapy of Liver Tumors:
Colorectal Liver Metastasis”: Edhemovic, I.; ▶ Chap. 99, “Electrochemotherapy of
Primary Liver Tumors”: Trotovsek, B., Đokić, M.; ▶ Chap. 100, “Electro-
chemotherapy of Locally Advanced Pancreatic Cancer”: Granata, V., Leongito,
M., Fusco, R., Piccirillo, M., Palaia, R., Lastoria, S., Petrillo, A., Izzo, F.; ▶ Chap.
102, “Electrochemotherapy of Head and Neck Cancer”: Benazzo, M., Bertino, G.,
Groselj, A.).
The other repository of the clinical data was established in Italy. More than
30 centers share the data and have published some interesting clinical reports. The
first one was predominantly on melanoma and pointed out on the effectiveness of
electrochemotherapy in repetitive treatment (Campana et al. 2009); the second one
has included a variety of tumor types and represents currently the largest number of
patients evaluated in one series (Campana et al. 2012). There is also a comprehensive
report on sarcoma tumors, which were bigger in size and were a challenge for
treatment with electrochemotherapy (Campana et al. 2014b).
Most of the papers have also touched upon the side effects and the quality of life
of electrochemotherapy-treated patients. In a series from InSPECT database, it was
reported that tumor especially preexisting pain before treatment, but also previous
irradiation, tumor size, and location, can predict the posttreatment pain related to
regression of tumors (Quaglino et al. 2015). Most of the recent studies have,
however, also evaluated the patients’ improvement of quality of life. It is a general
conclusion that electrochemotherapy is well accepted by the patients and that the
quality of life of these patients is significantly improved. Taken together, since
electrochemotherapy is currently applied predominantly in patients with palliative
intent, the benefit of the quality of life increase in combination with the effective
local tumor control provides significant benefit to these patients.
Based on this evidence, the National Institute for Health and Care Excellence,
NICE, recommended electrochemotherapy in treatment of cutaneous tumors and
also primary basal cell carcinoma in specific clinical situations. Since comparative
studies are currently underway and not yet published, a meta-analysis that was
published recently has compared the effectiveness of electrochemotherapy to other
local therapies of skin tumors. Based on the analysis, it is evident that electro-
chemotherapy is comparable to other local ablative techniques and radiotherapy,
although the data did not allow for direct comparison (Spratt et al. 2014).
Prognostic Factors and Clinical Features
Due to its principles of action, electrochemotherapy should be effective on all tumor

types. The use of powerful drugs like bleomycin and cisplatin that are effective on
most types of tumors and the physical drug delivery system that should not be
dependent on the type of the tumor cells enable that.
Tumor Type
The first studies, due to the low number of patients, could not detect a difference in
response between different tumor types. However, through the meta-analysis and
systematic review of all the data, some differences in responsiveness of tumors have
been discerned (Mali et al. 2013a). Basal cell carcinoma tumors have been identified
as the most responsive ones, with response rate close to 100%. Less responsive are
the sarcomas and carcinomas, with 70% response rate, and even lower are melanoma
metastases. Therefore when treating different types of tumors, one has to bear in
mind that tumor type is a predictive factor and that tumors with lower response rate
need to be retreated and more closely monitored for recurrences. Several underlying
mechanisms may play a role, but notably basal cell carcinomas also respond better
to, e.g., radiotherapy, than do melanomas. Recent data indicate that mutational status
or adaptation to previous treatments like radiotherapy can be the intrinsic tumor
cells’ factors that are involved. Other factors that might be involved are the structure
of tumor stroma and tumor vascularization, which might affect the drug distribution
in the tumors and hamper adequate drug quantity and distribution in the tumors. And
the third one is the electric field distribution that can be dependent on the size and
also the tissue structure of the tumor, as well as the surrounding tissue. All these
factors need to be explored in the future to refine electrochemotherapy for specific
tumors.
Tumor Size
With the size of the treated tumor, the parameters needed for the treatment change.
First the perfusion of the tumors is impaired and therefore also the drug distribution
to all the tumor cells. With intermittent and chronic hypoxia, this is well explained.
This holds true for intravenous drug administration. When the drug is administered
into the tumors directly, bigger tumors are difficult to infiltrate entirely as well. So
both routes have a drawback in tumors larger than 3 cm in diameter. The electric
field distribution adequately throughout the bigger tumors is difficult as well.
Either the electrodes are placed several times in order to cover the whole tumor,
including the safety margin, or long needle electrodes need to be placed, which
require the planning of the electric field distribution. All these factors are involved
in the treatment outcome of bigger tumors. In the ESOPE trial, tumors of up to 3 cm
in diameter were treated, however, due to the impressive results physicians stated
to treat also bigger tumors. Of course the response as measured in cutaneous

tumors reflects both the treatment efficacy and the time of healing of the treated
area which may be prolonged in large lesions, especially in previously irradiated
areas.
The first analysis on the responsiveness of the tumors depending on the tumor
size indicated that tumors bigger than 3 cm in diameter have lower response rate
(Matthiessen et al. 2011) which was later confirmed/elaborated (Mali et al. 2013b).
All the following studies have confirmed that tumors that are bigger than 3 cm have
lower response rate, the latest study was on head and neck tumors (▶ Chap. 102,
“Electrochemotherapy of Head and Neck Cancer”: Benazzo, M., Bertino, G.,
Groselj, A.) (Bertino et al. 2016).
The question is how to approach this problem? First of all, this may be a reporting
problem in that complete responses take a long time to achieve if the lesions have to
heal completely and the lesions are large. But also factors involving the treatment
can be discussed, including repeated treatment. The other approach would be to
combine local and systemic drug administration, which has already been explored,
but more evidence is needed for the appropriate drug dosage. The third approach
would be to combine electrochemotherapy with other local or systemic treatments.
This approach is being already explored with the new immunomodulatory systemic
treatments.
Previous Treatments
As discussed above, previous treatments can affect the tumor response to electro-
chemotherapy. The recent study by Bertino et al. has clearly indicated a higher
responsiveness of naïve tumors than ones that were previously treated by radiother-
apy, systemic treatment, or both (Bertino et al. 2016). The difference is substantial;
the naive tumors had a complete response rate of 70%, whereas the previously
treated tumors had a complete response rate of 55%. Again, several reasons can
contribute to the observed difference.
The first one is the immunological status of the organism. It is now known that
with the progression of tumors, immunoediting selects the resistant clones of tumors
that might have additional mutations than the naïve tumor cells. In addition, the
immune response is modulated, and since it is blocked at several immune check-
points, the immune response of the organism that is needed for the eradication of the
last tumor cells for complete response is lacking.
The second is that the previous treatments have induced drug resistance or
radioresistance in the remaining tumor cells, which also play a role in treatment
with electrochemotherapy. The molecular mechanism involved in such cells that
affect response to electrochemotherapy is just in the early phases of exploration. The
first reports indicate that BRAF-mutated cells are also sensitive to electro-
chemotherapy and that the recurrent tumors after therapy with BRAF inhibitors as
well (Valpione et al. 2015). In vitro data also indicate that concomitant electro-
chemotherapy and BRAF inhibitors treatment on BRAF-mutated cells may even
have a synergistic effect. Studies exploring the responsiveness of, for example,
radioresistant cells or HPV-infected cells are already underway.
The third aspect is the modulation of tumor perfusion by previous treatments. It is
well known the tumor bed effect of radiotherapy, where radiation therapy affects also
the normal tissue perfusion, therefore recurrent tumors in pre-irradiated areas are less
perfused, and in such case it seems to be more appropriate to give the drug
intratumorally than intravenously.
Current Developments for Treatment of Deep-Seated Tumors
Electrochemotherapy is now well established in treatment of cutaneous tumors, but

needs to be introduced also into the treatment of tumors located deeper in the body in
order to be universal and to compete with other ablative techniques, such as
radiofrequency ablation and irreversible electroporation.
In order to translate electrochemotherapy from cutaneous to deep-seated tumors,
technological development needs to provide equipment first. Usually, deep-seated
tumors are bigger, and therefore the current electrodes do not suffice. In order to
approach this, e.g., single needle electrodes are used that are individually placed into
the tumor and around it and then connected to the new pulse generator that is able to
produce larger electric fields, due to the bigger distance between the electrodes. For
certain indications, it is important to have synchronization of the pulse with the
heartbeat to be within the refractory phase of the heartbeat in order to avoid heart
fibrillation. This is important predominantly in treatment of tumors that are close to
the heart, such as liver tumors (Edhemovic et al. 2014; Mali et al. 2015).
Besides this technological approach, there is also development of endoluminal
electrodes for the treatment of colorectal tumors and esophageal tumors. The clinical
studies in this domain are not yet published, but the first patients have already been
treated. Therefore, for specific clinical situations also specific electrodes are needed,
such as umbrellalike, for the cranial tumors, which have already been developed.
Obviously the translation of electrochemotherapy into deep-seated tumors will have
to go hand in hand with technological developments (Miklavcic et al. 2012).
Nevertheless some clinical studies have already reported successful and safe
treatment of deep-seated tumors, like liver tumors, pancreatic tumors, bone metas-
tases, and a case of deep-seated head and neck tumor.
The first clinical study in treatment of colorectal liver metastases has already been
published. In the first 16 patients, it proved to be effective and safe. The procedure is
based on long needle electrode insertion into and around the tumor and delivery of
pulse in between the pairs of electrodes consecutively in order to cover the whole
tumor and the safety margin. For the appropriate electrode placement and the electric
field strength, treatment planning (▶ Chap. 54, “Treatment Planning for Electro-
chemotherapy and Irreversible Electroporation of Deep-Seated Tumors”: Kos, B.) is
used. The results of this study are described in detail in another chapter; in brief, the
study indicates that electrochemotherapy as nonthermal ablative technique is safe
and effective even when close or between the major hepatic vessels, where
radiofrequency ablation is contraindicated. The study is continuing into phase II, where
basically the same observations as in the phase I study are confirmed, but also on
tumors that are bigger than 3 cm in diameter, up to 6–7 cm. Another study is ongoing
on primary hepatocellular carcinoma tumors (▶ Chap. 99, “Electrochemotherapy of
Primary Liver Tumors”: Trotovsek, B., Đokić, M.), with also promising results.
Pancreatic cancer is difficult to treat with currently available treatments and with
low success rate. An effective ablative technique is needed, and electrochemotherapy
may provide that. The first clinical study has been published, which indicated
predominantly on safety of the use of electrochemotherapy, with partial responses
in tumors. The details of the study are elaborated in separate chapter (▶ Chap. 100,
“Electrochemotherapy of Locally Advanced Pancreatic Cancer”: Granata, V.,
Leongito, M., Fusco, R., Piccirillo, M., Palaia, R., Lastoria, S., Petrillo, A., Izzo, F.).
The long needle electrode approach could be useful also in treatment of bigger
head and neck tumors (▶ Chap. 102, “Electrochemotherapy of Head and Neck
Cancer”: Benazzo, M., Bertino, G., Groselj, A.). The first report on that is published
(Groselj et al. 2015). However in this body region, due to many sensitive structures,
exact placement of electrodes is a prerequisite; therefore the navigation of the
electrodes according to the treatment plan is needed. The technology is there and
can be applied in the future studies.
Preparation of New Standard Operating Procedures

and Introduction of Electrochemotherapy into Standard Clinical
Practice
The SOP that were published were prepared with the first experience that the first
centers had with electrochemotherapy. They were prepared for the treatment of
tumors that are less than 3 cm in diameter and for single treatment. Nowadays,
with accumulated knowledge and experience from many cancer centers, the tech-
nology has developed, and updated SOP is needed.
The centers with the most experience with electrochemotherapy have gathered,
and the experts have discussed the new findings, in order to organize them into new
SOP. The intent was to prepare more general guidelines, for treatment of also bigger
tumors, and repetitive treatment as well. Issues like pain control, anesthesia, and
drug administration are pointed out as well. In this respect, the new SOP are being
prepared and will hopefully enable further spread of electrochemotherapy also
outside of Europe, where it is already well established.
The introduction of electrochemotherapy is already underway; since with NICE
guidelines, it is being introduced also into many national guidelines and disease-
specific international guidelines. Further progress in the use of electrochemotherapy
is expected pending randomized studies on the specific indications. In order to guide
the authors, thorough reporting of data recommendations for the data reporting have
already been published (Campana et al. 2016).
Another aspect is that electrochemotherapy needs to be introduced along the other
standard treatments. This can be expected in several ways. Ongoing is the use of
electrochemotherapy as adjuvant treatment to other treatments, like neoadjuvant as

tumor debulking, or as adjuvant to emerging immunotherapies. In this respect,
electrochemotherapy can be seen as in situ vaccination (▶ Chap. 103, “Adjuvant
Immunotherapy as a Tool to Boost Effectiveness of Electrochemotherapy”:
Kamensek, U., Kos, S., Sersa, G.) where immune checkpoint inhibitors would
enhance the immune response elicited by electrochemotherapy. We can also look
at it the other way around; electrochemotherapy might enhance or help targeted
drugs such as the described recurrences after the treatment with BRAF inhibitors.
Other avenues seem to be feasible as well. The use of electroporation technology is
used also for gene therapy. In this way, both drug and plasmids encoding immuno-
modulatory molecules can be introduced into and around the tumors. The approach
seem to work well; the concept is sound and is proved in preclinical study in
spontaneous tumors in dogs (▶ Chap. 106, “Electrochemotherapy and Gene Electro-
transfer in Veterinary Oncology”: Tamzali, Y., Cemazar, M., Tozon, N.). However, also
other immunostimulatory approaches in combination with electrochemotherapy can
be used.
Conclusions
Electroporation-based technology provides an effective and safe approach for drug

or gene delivery. Electrochemotherapy is by far the most advanced one. In the two
decades of its development, it has reached the point to be accepted as standard
treatment modality, an ablative approach for cutaneous tumors. Continued refine-
ment of the standard operating procedures and the technological development will
help its spread, and also translation into the treatment of deep-seated tumors.
However, there are some detailed data still missing that will explain subtle differ-
ences in tumor responses and bring the approach on higher level of evidence-based
approach.
The new direction is to explore electrochemotherapy with the standard treatments
either targeted therapies, immune checkpoint inhibitors, or immunostimulants.
Another combined modality approach that also awaits clinical testing is radio-
sensitization of tumors (▶ Chap. 89, “Combined Treatment of Electrochemotherapy
with Irradiation”: Kranjc, S., Kamensek, U., Cemazar, M., Sersa, G.), where solid
preclinical data already exist.
The development is relatively slow, but it has to be so, due to the necessity of
solid preclinical and clinical data, extensive data analysis, and for the most of all, for
the safety of its use in patients.
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49:71–74
Tissue Ablation Using Nanosecond Electric
Pulses 94
Richard Nuccitelli
Abstract
Nanosecond electric pulses were first applied to mice for tumor ablation in 2002.
Since that time this technology has been used in dozens of laboratories in the
USA, Europe, and Asia (Batista et al. 2016). Rather than ablating by applying
heat or introducing a poison to physically damage the tumor, these nanosecond
pulses penetrate into the interior of cells and trigger the cell’s own apoptosis
pathway to initiate programmed cell death which occurs naturally in most cells in
our bodies once they have served their purpose. Therefore, the nanosecond pulses
have a cellular effect by sending the appropriate signal to the exposed cells,
leading to the name, Nano-Pulse Electro-Signaling. If the treated cells are tumors,
they will have neoantigens that the body’s immune system has not seen before,
and this immunogenic apoptosis pathway will then enlist the immune system to
target and destroy any cells with those same unique antigens on their surface. This
Nano-Pulse Electro-Signaling therapy both triggers apoptosis in treated tumors
and essentially transforms tumors into vaccines to direct the immune system to
destroy them. This therapy is drug-free and nonthermal so has advantages over
other tumor ablation therapies that will be discussed here. The most important of
these is the ability to enlist the immune system to provide a systemic attack on
similar tumor cells elsewhere in the body.
Keywords
Nanosecond pulsed electric field • Nano-pulse • Electro-signaling • Immune
response • Fibrosarcoma • Melanoma • Cytotoxic T cell • Vaccine • Vaccination •
Apoptosis • DNA fragmentation
R. Nuccitelli (*)
Pulse Biosciences Inc., Burlingame, CA, USA
e-mail: rich.nuccitelli@gmail.com; rnuccitelli@pulsebiosciences.com

DOI 10.1007/978-3-319-32886-7_93
1788 R. Nuccitelli
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1788
Initial Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1789
Melanoma In Vivo Tumor Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1789
Endogenous Tumor Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1791
Nano-Pulse Electro-Signaling Triggers an Immune Response to Nonself Antigens . . . . . . 1792
Clinical Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1793
Comparison with Thermal Ablation Modalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1794
Comparison with Other Nonthermal Ablation Modalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1795
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1796
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1796
Introduction
The field of Nano-Pulse Electro-Signaling (NPES) is fairly new compared to the

classical electroporation modalities that use longer pulses in the microsecond or
millisecond domain. These classical modalities have been used for over 40 years to
permeabilize cell membranes, but nanosecond pulse effects on cells have only been
studied for 15 years. The nanosecond domain is unique in three ways: (1) the very
short pulse length allows the electric field to penetrate into the cell and organelles;
(2) the short pulse length allows much larger fields to be applied; and (3) the short
pulse length generates transient pores in the cellular membranes that are only 1 nm
wide. Let’s consider each of these unique properties separately:
Cell penetration: Longer pulses that are a microsecond or more in duration
generally do not penetrate into the cell interior because of their slower risetime
and longer duration compared to the cellular ion redistribution time (Kotnik and
Miklavcic 2006). When an electric field is applied to a cell, ions in the conductive
cytoplasm rapidly redistribute with positive ions moving to the membrane facing the
negative pole of the field and negative ions moving to the membrane facing the
positive pole. This ion redistribution will charge the capacitance of the plasma
membrane generating an equal and opposite field to that applied so that the net
field within the cytoplasm is zero. However, this charge redistribution is not instan-
taneous. It takes a certain amount of time on the order of a microsecond. Pulses in the
nanosecond domain are therefore faster than this ion redistribution time so the field
goes right through the cell before the ions have time to redistribute.
Larger fields are applied: It takes about 500 mV to electroporate biological
membranes. So to electroporate an organelle that is 1 μm wide would require
500 mV across each membrane totaling 1 V across the 1 μm organelle. 1 V/μm is
equivalent to 10 kV/cm. This field strength only delivers fractions of a joule with
nanosecond pulses resulting in no significant heating. However, with 1000 times
longer microsecond pulses, the heat would be 1000 times greater and would cause
death by hyperthermia. Therefore, the main characteristic that sets nanosecond
pulsed fields apart from the microsecond domain is their ability to nonthermally
electroporate internal membranes (Schoenbach et al. 2001).
94 Tissue Ablation Using Nanosecond Electric Pulses 1789
One nanometer wide pores: These pulses are so short that the pore diameter
generated in membranes is only 1 nm as has been determined by the size of
molecules passing through them (Vernier et al. 2006). Such a small opening will
allow water and ions to pass through it but not proteins or DNA. It is this selectivity
that leads to the signaling component of these pulsed fields. Specifically, the key
signaling ion, Ca2+, can pass through these nanopores to trigger cellular responses
(Vernier et al. 2003).
These three critical characteristics of nanosecond pulses reveal why we refer to
their effects as Nano-Pulse Electro-Signaling. Nearly all signaling pathways in cells
involve ion flow (usually calcium ions) across either organelle or outer membranes.
A great deal is known about cell signaling pathways and most involve changes in
intracellular calcium levels. This is because cells spend a great deal of energy
maintaining a very low free calcium level in the cell cytoplasm that is typically on
the order of 0.1 μM. The external [Ca2+] is 10,000-fold higher, and this steep
concentration gradient will drive Ca2+ in through the nanopores. Small increases
in the local Ca2+ concentration have been demonstrated to have very strong effects
on a variety of cellular functions such as secretion, cell division, and apoptosis. This
chapter will concentrate on using NPES to trigger immunogenic apoptosis to ablate
tissue.
Initial Discovery
The first indication that nanosecond pulsed electric fields (nsPEFs) could be used to
reduce tumor growth came from the pioneering work of Beebe and Schoenbach
(Beebe et al. 2002) in which they reported the slowing of growth of subdermal
murine fibrosarcoma allografts following nsPEF treatment. That work started the
field of NPES-induced tumor ablation, and more than 60 papers have been published
since then describing various aspects of the nanoelectroablation of tumors. Much of
this work has been done in vitro studying the effect of NPES on a wide variety of
both human and murine tumor cell lines (Beebe et al. 2003; Hall et al. 2007; Ford
et al. 2010; Ren et al. 2012; Beebe et al. 2013), and many in vivo murine animal
tumor models have been studied as well and will be considered in more detail here.
Melanoma In Vivo Tumor Model
B16-F10 malignant melanoma cells were originally derived from C57Bl/6 mice so
are syngeneic with that mouse strain and recognized as “self” by the immune system.
Injecting about a million of these cells into or beneath mouse skin as an allograft
results in the formation of melanoma tumors that can be treated in situ. C57Bl/6 have
highly pigmented black skin that makes the tumors hard to photograph. However,
when the B6 albino or hairless SKH-1 strain is used, the tumor can be easily imaged
by shining light through the skin (Fig. 1b). The drawback for using the SKH-1 strain
1790 R. Nuccitelli
Fig. 1 Treating murine

melanoma with a “clothespin”
electrode. (a) Photo of mouse
being treated with a closeup
image of the electrode plates
in the inset; (b)
transillumination (left) and
surface view (rt) of a mouse
melanoma before treatment;
(c) 1 day after treatment; (d)
6 days after treatment; (e)
15 days; (f) 29 days
is that it is not syngeneic with B16-F10 cells, so there will be some immune response
against the tumor. A better choice is the B6 albino that has the same genetic
background as C57Bl/6 from which B16-F10 melanoma cells were derived.
Treating such subdermal tumors requires placing electrodes around them in such
a way that the entire tumor is exposed to the pulsed electric field. One of the first
approaches to this was the development of a “clothespin” electrode in which the skin
containing the tumor was pulled away from the body and the tumor was sandwiched
between two disc-shaped electrodes (Fig. 1a). For the very flaccid mouse skin, that
approach worked very well, and the subdermal allograft tumors began to shrink
within a day of treatment and typically were gone within 3 weeks (Fig. 1b–d). A
range of field strengths were tested. Very little inhibition of tumor growth was
observed when 10 kV/cm was applied using 10–100 pulses 300 ns long. However,
increasing the field to 20 kV/cm began inhibiting growth when 10 pulses were
applied and 100–200 pulses resulted in shrinkage of the tumor. Total remission could
be observed when 100 pulses of 40 kV/cm were applied 3 days in a row or 2000
pulses of 30 kV/cm in one treatment (Nuccitelli et al. 2010). A reduction in blood
flow to the tumor was observed (Nuccitelli et al. 2006), and pyknosis of nuclei
within the tumor was evident by histology carried out by Xinhua Chen (Chen
et al. 2009). Measurement of the tumor temperature during pulsing by Uwe Pliquett
indicated only a 2–3 C increase (Nuccitelli et al. 2006).
During this early work, several other observations were made regarding the
cellular responses to NPES. Patch-clamp measurements indicated a strong increase
in inward current upon a single, 600 ns pulse that recovered after several minutes
(Pakhomov et al. 2009), yet propidium iodide was too large to pass through any
pores generated by these pulses. These observations suggested that the NPES-
induced permeabilization involved very small pores in the nanometer range. Ca2+
was able to pass through these nanopores, and several measurements of the intra-
cellular Ca2+ increase were made and indicated both influx from outside the cell and
efflux from the endoplasmic reticulum (ER) (Vernier et al. 2003; White et al. 2004).
This ER permeabilization was another indicator that the electric field was penetrating
into the cell interior. Other observations included the rapid reduction of blood flow to
the tumor following NPES along with the fragmentation of DNA (Nuccitelli
et al. 2009) and initiation of apoptosis (Beebe et al. 2002; Hall et al. 2007).
Endogenous Tumor Models
In order to determine if NPES was also effective ablating tumors that develop from
the mouse’s own skin cells, two other tumor models were studied. One of these was a
basal cell carcinoma (BCC) model of autochthonous tumors in Ptch1+/ K14-Cre-
ER p53 fl/fl mice that develop BCCs several months after the pups are irradiated with
ionizing radiation (Nuccitelli et al. 2012a), and the second was a melanoma model
using C57⁄BL6-HGF⁄SF transgenic mice that were exposed to UV radiation as pups
and developed melanomas 6 months later (Nuccitelli et al. 2012b). NPES was found
to be very effective at ablating both of these endogenous tumor types.
1792 R. Nuccitelli
Nano-Pulse Electro-Signaling Triggers an Immune Response to

Nonself Antigens
In the process of treating melanomas with NPES, it became evident that once a tumor
was nanoelectroablated in a mouse, no subsequent injected melanomas would grow.
This observation suggested that the first ablation had alerted the immune system to
the tumor antigens so that when they appeared again their growth would be inhibited
by the mouse’s own immune system. This was initially confirmed by showing that
immunosuppressed mice had no problem growing second tumors. Moreover, immu-
nohistochemical studies indicated that enhanced levels of CD4+ T cells were present
in untreated tumors removed from mice in which another tumor had been treated
with nsPEF at either 19 or 32 days before removal (Nuccitelli et al. 2012b). Since
then more evidence for an immune response has appeared (Chen et al. 2014a, b; Yin
et al. 2014). In the strongest of these, Chen et al. used an orthotopic rat liver tumor
and observed that in 23 rats the secondary tumor failed to grow after the first was
nanoelectroablated. They also observed the presence of granzyme B-secreting cells
which could implicate either T cells and the adaptive immune response or natural
killer cells and the innate immune response.
This inhibition of second tumor growth was replicated in another rat orthotopic
liver tumor model (Nuccitelli et al. 2015). Rather than wait 7 weeks before injecting
the second tumor, it was injected after 3 weeks. One week later, the liver lobe was
removed so that the secondary tumor size could be determined. The secondary tumor
was still present after that time but was much smaller than the primary was after the
same growth period. However, when CD8+ cytotoxic T cells were depleted prior to
injecting the second tumor, it grew much faster, suggesting that it was the cytotoxic
T cells that were inhibiting tumor growth. This is the first evidence for the stimula-
tion of an adaptive immune response to nanoelectroablated tumors.
Another good test for the generation of an immune response is to determine if
NPES-treated tumor cells can be used to vaccinate a mouse against future tumor cell
exposure. This was done using mouse MCA205 fibrosarcoma cells that are synge-
neic with C57Bl/6 and B6 albino mice. One million of these cells were exposed to
NPES in an electroporation cuvette and then injected subdermally to vaccinate a
mouse. At the same time, control mice were vaccinated with either necrotic cells or
saline. Three weeks later, healthy MCA205 cells were injected into those same mice,
and the tumor growth was monitored. In both controls, the tumors grew rapidly
(Fig. 2a), but in mice that were vaccinated with NPES-treated cells, the tumor growth
was not only inhibited but the tumor was completely consumed by the immune
system within 13 days. In order to test for the involvement of the CD8+ cytotoxic T
cells, in six mice, we injected an antibody to CD8 IP to pull CD8+ T cells out of
circulation. In all of these mice, the challenge tumor grew as fast as the control
vaccinated with saline alone (Fig. 2b). These studies strongly support the stimulation
of an immune response by NPES treatment of the tumor. This NPES therapy
both triggers apoptosis in treated tumors and essentially transforms tumors into
vaccines to direct the immune system to destroy them wherever they are found in
the body.
Fig. 2 Growth of injected a

challenge tumor in mice that 100
were previously vaccinated as C57BL/6 mice
indicated. (a) Tumors in Vaccinated
with
Normalized tumor area

control vaccinated with either saline
necrotic cells or saline grew 10
n=3
very rapidly. Tumors in mice Vaccinated
with necrotic
vaccinated with Nano-Pulse cells (n = 3)
Electro-signaling-treated cells
did not grow. Each data point
is the average of the tumor 1
areas in three mice. (b) Vaccinated
Second experiment in which with
NPES-treated
an antibody to CD8 was cells (n = 3)
injected at the same time as 0.1
tumor cells showing that those 0 2 4 6 8 10 12 14
tumors grew as well as the Days after injecting tumor cells
controls. Each data point is an
B6 albino mice
average of the tumor surface b 10
areas in six mice. Error bars Vaccinated
indicate the SEM with
Normalized tumor area
saline Vaccinated
n=6 with
NPES-treated
cells (n=6) plus
anti-CD8 antibody
1
Vaccinated
with
NPES-treated
cells (n = 6)
0.1
1 2 3 4 5 6 7 8 9 10
Days after injecting tumor cells
Clinical Studies
There have only been two published reports of the nanoelectroablation of human
skin lesions. The first was the ablation of a single basal cell carcinoma (BCC) by
200 pulses 20 ns in duration (Garon et al. 2007), and the second described the
treatment of 10 BCCs on 3 patients with between 100 and 1000 pulses (100 ns,
30 kV/cm, 2 pps) (Nuccitelli et al. 2014). An electrode consisting of two parallel
rows of six needles each (0.1 mm wide at base, 1.2 mm-long acupuncture needle)
(Fig. 3) with an ablation zone of 5 mm 6 mm in size. A single treatment of
100 pulses was sufficient to cause most of these lesions to disappear during the
ensuing several weeks without leaving a scar. Edema was present following treat-
ment, and a crust appeared over the treatment region within 2 days and began to fall
off in 2 weeks. The skin was initially slightly pink in color and after a few weeks
appeared normal such that some treated lesions could not be located several weeks
after treatment without reference to a pretreatment map. Since the standard of care
for BCC removal, surgery or curettage, usually leaves a scar, the absence of scarring
1794 R. Nuccitelli
Fig. 3 Examples of NPES treatment of human skin lesions. (a and b) Two basal cell carcinomas
treated with 100 pulses 30 kV/cm using the needle electrode picture below. At 14-week
posttreatment, the skin was biopsied, and histological section is shown below. (c–e) Human cherry
angiomas treated with indicated number of pulses and photographed at 2-week intervals. Scale bars
all indicate 5 mm
following nanoelectroablation was a surprising result. About 14 weeks after treat-

ment, we removed the skin from the treated region and collected serial thin sections
every 100 μm through the entire region. These H&E-stained sections were subjected
to independent histological analysis by a dermatopathologist. Seven of the 10 lesions
were completely free of basaloid cells and two were partially ablated (Nuccitelli
et al. 2014).
Cherry angiomas have also been ablated using a range of pulse numbers in order
to determine the proper treatment parameters for human lesions. Pulse numbers of
10 or 50 have little effect on these lesions other than some hyperpigmentation, but
100 pulses and above always result in ablation (Fig. 3).
Comparison with Thermal Ablation Modalities
The most popular thermal ablation modalities are radio-frequency ablation, micro-
wave ablation, cryoablation, and highly focused ultrasound. All of these destroy
tissue by heating beyond hyperthermia temperatures or freezing. They are com-

monly used to ablate tumors in the liver and other organs and have been used
successfully on quite large tumors of 3–5 cm. The main differences when compared
to Nano-Pulse Electro-Signaling are greater collateral damage due to the diffusion of
heat to neighboring healthy tissue and heat-sink problems due to vessels in the
ablation zone. Heat diffuses quite readily through tissue so it is difficult to establish a
well-defined ablation zone when using thermal ablation methods. Moreover, if there
is a vessel or duct in the ablation zone, fluid movements will carry heat away from
that region and disrupt the ablation. In contrast, the ablation zone boundary for
NPES is very sharp, making it quite easy to avoid ablating neighboring tissue.
Moreover, the presence of vessels has no influence on the ablation zone since the
electric field will penetrate the vessels and the ablation mechanism is nonthermal.
Comparison with Other Nonthermal Ablation Modalities
Two other pulsed electric field ablation modalities currently in use are electroche-
motherapy (ECT) and irreversible electroporation (IRE). Both of these apply much
longer pulses on the order of 100 μs which generate larger pores in the plasma
membrane. Electrochemotherapy applies fields on the order of 1 kV/cm to generate
transient pores that are large enough to allow normally impermeant molecules such
as bleomycin to enter the exposed cells. Bleomycin then induces DNA strand breaks
and results in cell death. ECT has been approved for the treatment of many tumor
types in European countries but is not yet approved for human use in the USA. It has
been quite successful ablating quite large tumors, whereas NPES has thus far been
limited to small tumors on the order of 1 cm in diameter because of the high fields
required. There is a practical limitation on the delivered voltage due to insulation
requirements as well as the ionization of gases leading to corona formation and
ozone production. Therefore, treating tumors larger than 1 cm must be done with
multiple treatments involving repositioning the electrode or with multipole electrode
configurations. Therefore, pulse modalities using lower field strengths have the
advantage of being able to treat larger tumors more easily than NPES. Please see
the chapter describing electrochemotherapy by Lluis Mir for a detailed description of
this tumor ablation modality.
Irreversible electroporation (IRE) results from the application of 8–90 pulses of
larger pulsed fields 100 μs in duration at 1.5 kV/cm. The pores that form remain open
permanently and this results in cellular necrosis rather than apoptosis. Consequently,
the stimulation of the immune response observed following NPES is absent follow-
ing IRE. Irreversible electroporation has been approved for human use in the USA
for soft tissue ablation and is described at length in the chapter by Rafael Davalos.
The main drawback of this therapy is that the long pulses stimulate muscle contrac-
tion so that muscle blockade must be used. This is not necessary when using NPES
due to the much shorter pulse width. Another challenge of the technique is the
insertion of the electrodes around the tumor. They must be inserted as parallel as
possible, and the resulting field distribution must span the entire tumor in order to
1796 R. Nuccitelli
obtain complete ablation. Again, the low voltages required make it possible to more
easily treat larger tumors with IRE than with NPES.
Conclusions
Nano-Pulse Electro-Signaling can be used to trigger a variety of cellular responses

depending on signal strength and pulse number. It has been used for 15 years to
trigger immunogenic apoptosis when a sufficient number of pulses are applied at
field strengths greater than 20 kV/cm. The most common pulse lengths that have
been studied are in the 100–600 ns range, and these give comparable results when
the same amount of energy is delivered. NPES has been shown to be effective at
triggering apoptosis in every tissue and cell type to which it has been applied, and a
single treatment lasting a few minutes is sufficient for complete ablation without
recurrence. The first clinical trial treating basal cell carcinoma indicated a 78%
success rate without scarring. This lack of scarring is another important advantage
over competing technologies, but by far the most important advantage is the
initiation of an adaptive immune response in which the immune system is recruited
to eliminate any untreated tumor cells or metastases in mice and rats. If this
immunogenic apoptosis is also triggered following human therapy, this modality
will become very potent.
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International Network for Sharing Practices
on Electrochemotherapy (InspECT): An 95
Integrative Patients Treatment Consortium
Matteo Brizio, Simone Ribero, Luca Giovanni Campana, A. James P.

Clover, Julie Gehl, Christian Kunte, Francesca De Terlizzi, and Pietro
Quaglino
Abstract
In this chapter we will present the International Network for Sharing Practices on
Electrochemotherapy group, founded to answer the need of discussing issues and
share experience on the use of the procedure, its history, and the objective for
what it was established. Furthermore, on 2008 the online InspECT database was
created as a tool for the Network to record data regarding clinical general
examination of patients, measurement of tumor size to be treated, photographic
M. Brizio (*) • S. Ribero • P. Quaglino

Dermatologic Clinic, Department of Medical Sciences, University of Turin, Turin, Italy
e-mail: matteobrizio@virgilio.it; simone.ribero@unito.it; pietro.quaglino@unito.it
L.G. Campana
Surgical Oncology Unit, Veneto Institute of Oncology IOV-IRCCS and Department of Surgery
Oncology and Gastroenterology (DISCOG), University of Padova, Padova, Italy
e-mail: luca.campana@ioveneto.it; luca.campana@unipd.it; maximizing@hotmail.com
A.J.P. Clover
Department of Plastic Surgery, Cork University Hospital, Cork and Cork Cancer Research Centre,
Western Gateway Building, University College Cork, Cork, Ireland
e-mail: j.clover@ucc.ie
J. Gehl
e-mail: Karen.Julie.Gehl@regionh.dk
C. Kunte
Artemed Fachklinik München GmbH & Co., KG. München, Germany
e-mail: Christian.Kunte@artemed.de
F. De Terlizzi
IGEA Biophysics Lab, Via Parmenide, Carpi, Italy
e-mail: f.deterlizzi@igeamedical.com

DOI 10.1007/978-3-319-32886-7_95
1800 M. Brizio et al.
documentation, type of anaesthesia, data on electrochemotherapy procedure,

toxicity, side effects, pain, and quality of life. The amount of information stored
is recorded to produce collaborative publications since all the data are entered
according to the same parameters and continuously monitored according to good
clinical procedures.
Thanks to the collaborative work between the members of the Network it was
possible to realize the publication of papers focused on specific electro-
chemotherapy issues. The first of them was about the management of cutaneous
metastases from various solid tumors and allowed to state that electro-
chemotherapy offers a minimally invasive local treatment, provides local disease
symptoms control, and improves patient’s quality of life at the cost of only minor
side effects.
The second work published with the aim to evaluate which patients’
category could be at risk for pain associated with electrochemotherapy iden-
tified factors related with an higher risk of postprocedural pain as diagnosis of
breast cancer, treatment of preirradiated areas, largest diameter, and repetitive
treatments demonstrating, however, that pain levels and pain medication
overall decrease after the procedure. Finally, Bertino and Colleagues focused
their attention on melanoma and nonmelanoma skin cancers of the head and
neck treated with bleomycin electrochemotherapy. Electrochemotherapy
resulted an effective treatment option for skin tumors of this specific area
and in particular for basal cell carcinoma. The response rate in small, primary,
and treatment-naive tumors is high and the functional, anatomical, and aes-
thetic preservation of the HN structures can be excellent at cost of well-
tolerated side effects.
Keywords
INSPECT (International Network for Sharing Practices on Electrochemotherapy
group) • INSPECT database • Copenhagen agreement • INSPECT papers • Pro-
jects and perspectives
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1801
Purposes and History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1801
The Copenhagen Agreement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1802
The Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1802
Audit Program for InspECT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1804
The Standardized Informed Consent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1804
InspECT Website . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1805
Structure of the Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1806
InspECT: Results of the Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1806
Published Papers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1806
Projects and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1812
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1814
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1815
95 International Network for Sharing Practices on Electrochemotherapy. . . 1801
Introduction
After the first evidences on the biologic potential of electroporation to facilitate

transport of normally nonpermeant molecules into cell, on 1991 a first human trial
has been conducted after successful experiments in vitro and on animals. For the first
time it was demonstrated the efficacy and safety of the procedure in the treatment of
cutaneous and subcutaneous metastases, regardless of the origin of the primary
tumor (Mir et al. 1991). Then the field has rapidly developed since electro-
chemotherapy has been recognized as an effective and safe method for local
treatment of cutaneous, subcutaneous, and mucosal tumors (Mir et al. 1998; Sersa
et al. 2000, 2008). In the pre-ESOPE Era, clinical studies were performed with
variable treatment protocols, different electrodes, and different electric pulse gener-
ators. Thus, there was a need for a prospective nonrandomized multi-institutional
study that was conducted by a consortium of four cancer centers gathered in the
ESOPE (European Standard Operating Procedures of Electrochemotherapy) project.
After the publication of Standard Operating Procedures (Mir et al. 2006) arose the
need to establish a network of medical teams from different countries and with
different areas of interest sharing experiences with the aim of a continuous devel-
opment of the technology and a validation of the procedure in new fields of
application.
Purposes and History
The International Network for Sharing Practices on Electrochemotherapy (InspECT)

was founded in 2008 with the goal of facilitating networking between medical teams
working with electrochemotherapy, and in particular for meeting and discussing
issues related to the use of electrochemotherapy in order to strive for best practice
and ensure optimized patient care. It was recognized early that a collaborative effort
would speed up the incorporation of this emerging treatment into the mainstream.
The founders of the Network were Julie Gehl and Louise Matthiessen, from Herlev
Hospital (Copenhagen, Denmark), and Tobian Muir, from James Cook Hospital
(Middlesbrough, UK). At the end of 2015, there were 25 participating centers from
all over Europe.
As well as sharing best practice, InspECT hosts a database to record outcome
from electrochemotherapy treatments from the different centers. Each center has the
control of the data obtained, but one of the objective of the network is to produce
collaborative publications since all the data are entered according to the same
parameters (with high reproducibility and standardization) and continuously moni-
tored according to good clinical procedures. Each participant center’s monitoring is
invited to sign a written agreement, called “The Copenhagen Agreement,” where
rules applied among centers and conduction of the activity of data entering in the
database are reported.
InspECT is supported by IGEA (Carpi, Italy) in terms of upkeep of the database

and support for meetings, but the forum is formed and maintained by active
clinicians.
The National Institute for Health and Care Excellence (NICE), in the UK, has
reported in its guidance the importance of an International registry to record data on
all the electrochemotherapy sessions performed (https://www.nice.org.uk/guidance/
ipg446/chapter/1-guidance). NICE stimulates all the UK centers to enter data on
treatment into the InspECT registry, as reported below.
The Copenhagen Agreement
The Copenhagen agreement specifies that the Core Benefit of InspECT is to gather,
share, and publish clinical data and experience in order to evaluate, improve, and
develop the treatment with electrochemotherapy. Furthermore, it outlines that mem-
bers of InspECT have the intention to follow the Good Clinical Practice Guidelines
(GCP) when performing electrochemotherapy and creating data for the database. In
particular: all clinical data should be uploaded in the database and all patients treated
should be registered; all clinical information should be recorded, handled, and stored
allowing its accurate reporting, interpretation, and verification. Serious Adverse
Events should be reported in the specific section. Confidentiality of records that
could identify subjects should be protected, respecting the privacy rules in accor-
dance with the applicable regulatory requirement(s). Each individual involved in
conducting electrochemotherapy should be qualified by education, training, and
experience to perform his or her respective task(s); the rights, safety, and well-
being of the subjects to be treated are the most important considerations and should
prevail over interests of science and society; an audit assessor should always be
allowed to inspect the data at the site where the data is generated to ensure
verification.
As above mentioned one of the main aims of INSPECT is to bring faster and
better treatment to patients by publishing high quality research and by networking
for continuous training in the field.
The Database
The database is stored in a remote independent server located in Italy, and it is

accessible by a dedicated HyperText Transfer Protocol over Secure Socket Layer
webpage (https://insp-ect.eu). This ensures authentication of the website, privacy
protection, and integrity of data exchanged between the centers. The access is
allowed only to InspECT participants and each center has a unique username and
password to enter the database.
Fig. 1 Layout of the main screen of the InspECT database
The data requested to be uploaded for each patient/treatment are based on: clinical
general examination, characterization, and measurement of size of the tumor/s to be
treated, photographic documentation, type of anaesthesia or other drugs used, data
on electrochemotherapy procedure (drug, dose, administration modality, electrodes,
number of applications, duration of treatment), toxicity, side effects, pain, and
quality of life. Furthermore, an evaluation of general health, characterization and
measurement of size of the tumor/s, evaluation of the response to treatment, and
clinical photos should be collected every 4–6 weeks from treatment during the
follow-up period, up to at least 16 weeks. The database layout is shown in Figs. 1
and 2.
Fig. 2 Treatment data layout
Audit Program for InspECT
In accordance to the International Conference on Harmonization of Good Clinical

Practice (ICH-GCP) guidelines and as indicated by the “Copenhagen agreement,”
inspECT data collection is regularly inspected by an internal quality assurance
function at each center, where the data is generated to ensure verification. The
objectives of the InspECT audit program include authenticity and accuracy of
uploaded data verification, to document evidence of the signed informed consent
form given by the patients, data validation for research and dissemination intents,
and identification of training requirements for data collectors. For each inspECT
center, the audit sample is randomly selected from all patients that have undergone
electrochemotherapy and that have been registered to the inspECT database. Based
on the audit outcomes, for each center, recommendations are formed by the
INSPECT board.
The Standardized Informed Consent
Another key point of InspECT is the use of a common informed consent form for the
patient in all centers.
This document has been translated in all languages of the participants’ countries
(English, Italian, German, Slovenian, Hungarian, Polish, Danish). Specifically, it
reports exhaustive information on: type and duration of procedures, potential
Fig. 3 InspECT website home screen
benefits and risks, side effects and possible complications, and detailed description
of the procedure and of the device (Cliniporator, IGEA, Italy). Further information
regarding InspECT database, patient data collection, confidentiality, and the poten-
tial benefits of using the patients’ data for studying innovative treatments, develop-
ing new alternatives to conventional or existing therapies against cancer.
The standardized informed consent has been published on the official InspECT
website.
InspECT Website
InspECT website (home screen on Fig. 3) can be found at the following address:
http://www.insp-ect.org.
The website contains, among other information, the list of InspECT centers
(at “inspect centers” section), the rules for joining the group (at “rules for center
enrolment” section), the standardized informed consent documents in different
languages, the link to the database, updated analysis on the data, and sub-study
groups regarding specific issues.
You can also find information on congresses or events where InspECT is present
and details regarding the date and location of the past and next annual InspECT
meetings.
A forum, reserved to participants only, is included to discuss scientific issues or
interesting cases.
Structure of the Group
The group elects every 2 years the InspECT board, which is composed by the Chair,
the past-Chairs, a Vice-chair, a Secretary, and two Members. The board has the
responsibility to oversee that every center follows the rules of the group and to
accept the new centers asking to join the group, verifying that all conditions for
inclusion are met.
The InspECT group meets every year at the Annual Meeting.
InspECT: Results of the Database
The International Network for Sharing Practices on Electrochemotherapy (InspECT)

was founded in 2008 and consists of European medical teams working with electro-
chemotherapy. In order to achieve the purpose of the Group and thus to collect and
make data available a database has been set up on 2010.
The treatment data collection and their inclusion is a continuing effort that
involves the 25 European centers to date (Dec 2015) enrolled in InspECT (Fig. 4).
From 2010 up to December 2015 the number of treated patients in the database
increased progressively reaching 688 units, 316 males, and 372 females with a
median age of 74 years (Fig. 5).
As shown in “Fig. 6a, b” the most common disease treated with electro-
chemotherapy was metastatic melanoma followed by nonmelanoma skin cancers
(basal cell carcinoma and squamous cell carcinoma) and cutaneous metastases from
breast cancer (MA).
Published Papers
Up to now three papers have been published by this network.

The first one was published in 2008 and reported the results of a two-centers trial
concerning the management of cutaneous metastases from various solid tumors
(Matthiessen et al. 2011). Cutaneous metastases account for 0.7–9% of all metasta-
ses; breast cancer accounts for 51% of the total cases of cutaneous metastases, while
malignant melanoma accounts for 18% (Rolz-Cruz and Kim 2008). The manage-
ment of cutaneous metastases is challenging; they may be widespread and usually
recur after surgery, radiotherapy, or chemotherapy. In addition the presence of odor,
Site Medical center

HERLEV, DENMARK University Hospital Herlev, Copenhagen
MIDDLESBROUGH, UNITED KINGDOM James Cook University Hospital, Middlesbrough
TORINO, ITALY University of Turin
ROMA, ITALY University La Sapienza, Rome
LONDON, UNITED KINGDOM St. Georges Hospital, London
MUNCHEN, GERMANY Artemed Klinik, Munchen
PAVIA, ITALY University of Pavia
WELS, AUSTRIA Wels Hospital, Wels
WARSAW, POLAND Maria Sklodowska-Curie Memorial Cancer Centre, Warsaw
LIVERPOOL, UNITED KINGDOM Whiston Hospital, Prescot, Merceyside
CORK, IRELAND Cork Cancer Centre, Cork
LONDON, UNITED KINGDOM The Royal Free Hospital, London
TUEBINGEN, GERMANY Universitäts-Frauenklinik, Tübingen
BRADFORD, UNITED KINGDOM Bradford Royal Infirmary, Bradford
LONDON, UNITED KINGDOM Bart's Hospital, London
LEEDS, UNITED KINGDOM Leeds General Infirmary, Leeds
CASTLE HILL, UNITED KINGDOM Castle Hill Hospital, Castle Hill
PADOVA, ITALY Veneto Institute of Oncology, University of Padova
MANCHESTER, UNITED KINGDOM Christie Hospital, Manchester
BRISTOL, UNITED KINGDOM Frenchay Hospital, Bristol
LJUBLJANA, SLOVENIA Institute of Oncology, Ljubljana
ATHENS, GREECE St. Savvas Anticancer Hospital, Athens
BOCHUM, GERMANY Ruhr-Universitat Bochum
EXETER, UK Royal Devon and Exeter NHS foundation trust
SZEGED, HUNGARY Department of Dermatology and Allergology University of Szeged
Fig. 4 InspECT medical centers
exudate, and bleeding require a treatment to relieve the discomfort. The uncontrolled
cutaneous metastases can, in many ways, adversely affect body image and may
impact patient’s quality of life.
For this trial patients with cutaneous metastases, not eligible for conventional
treatments, were consecutively recruited at two institutions: Copenhagen University
Hospital Herlev, Denmark and James Cook University Hospital, Middlesbrough,
UK. The primary endpoint was the response rate. Secondary endpoints included the
safety of the procedure and the response rate according to size. Follow-up was
Fig. 5 Number of patients enrolled in the InspECT database
planned for up to 6 months. Patients who had been started on systemic antineoplastic
treatment after ECT were excluded from the study at that time.
The ECT sessions were performed based on the standard operating procedures for
electrochemotherapy (Mir et al. 2006). Bleomycin was administrated either
intratumorally (i.t.) or intravenously (i.v.). The decision to treat either i.t. or
i.v. was based on the number of the lesions to be treated and the size of the same.
Treatment was carried out both under general and local anesthesia. The response rate
was performed by measurement of two major diameters of the treated lesions. A
maximum of seven cutaneous metastases per patient were registered as target lesions
and were documented during subsequent visits. The onset of new cutaneous metas-
tases was not considered as response rate but these could be considered eligible for a
second ECT session. The response rate was evaluated using the Response Evaluation
Criteria in Solid Tumors (Therasse et al. 2000): complete response (CR) was defined
as disappearance of the target lesion; partial response (PR) as a minimal 30%
decrease in the diameter; progressive disease (PD) with at least a 20% increase in
the diameter; and stable disease (SD) with neither sufficient dimension reduction or
sufficient increase to qualify for PR or PD. Fifty-two patients with cutaneous
metastases from primarily malignant melanoma or breast cancer were enrolled
between June 2007 and April 2010. The median diameter of the cutaneous metas-
tases was 12 mm (range 1–200 mm). The 55% of the treatments was performed
under general anaesthesia, while the local anaesthesia was used in the remaining
cases. The median duration of a treatment session since the injection of the
bleomycin was 20 min for local anaesthesia and 25 min for general anaesthesia.
Only 47% of patients achieved a follow-up period higher than 60 days: Authors
attributed this to the fact that the large majority of patients travel long distances to
reach the center offering electrochemotherapy which hindered follow-up, and some
b
DIAGNOSIS N %
Melanoma 222 35%
Squamous cell carcinoma 144 23%
Breast cancer 126 20%
Basal cell carcinoma 112 18%
Kaposi sarcoma 16 3%
Skin mets from other
adenocarcinomas 20 3%
Other sarcomas 9 1%
Keloids 5 1%
Lymphoma 2 0%
Merkel cell carcinoma 3 0%
OTHER 29 5%
Fig. 6 (a, b) ECT treated diseases based on InspECT database
patients had systemic progression during follow-up and were offered other antineo-
plastic treatment.
In this study, objective response rate (OR) for patients with a follow-up period
higher than 60 days was 86% for cutaneous metastases smaller than 3 cm while for
cutaneous metastases larger than 3 cm OR was predictably lower reaching only the
31%. However, in patients with large disease volume, SD can be the aim of
electrochemotherapy treatment obtaining palliative relief in terms of decreased
odor, exudate, and bleeding. In the management of small lesions, control and
disappearance of cutaneous metastases can be the goal of treatment.
Reported adverse events were flu-like symptoms after treatment (10%), pain in
the treated area (10%), ulceration of treated area (4%), cough (2%), allergic skin
reaction (2%), and anxiety (2%). No serious adverse events were observed related to
the procedures. Due to the low incidence of complications, treatment can be repeated
several times in order to maintain local control or obtain control if not achieved by
the first treatment.
In conclusion, these results allowed to state that electrochemotherapy offers a
minimally invasive local treatment, provide local disease, symptoms control, and
improves patient’s quality of life at cost of only minor side effects. Finally, this trial
was conducted in two different centers (a department of plastic surgery and a
department of oncology), confirming that the introduction of the Standard Operating
Procedures allows to obtain similar results in medical teams with different
background.
In a second paper the InspECT group evaluated which patients’ category could be
at risk for pain associated with electrochemotherapy (Quaglino et al. 2015).
Patients referred for this procedure present with different tumor size and mor-
phology: from patients with multiple small cutaneous metastases (for example, from
a melanoma) to patients with a single lesion up to 40 cm (in most cases expression of
epithelial carcinoma like basal cell carcinoma, squamous cell carcinoma, or cutane-
ous recurrence of breast cancer).
Impressive clinical activity has been reported, with high response rates in the
treated lesions. Besides the clinical activity in terms of response rates, when it has
been applied to patients with larger cutaneous metastases, pain has been reported as a
more frequent side effect (Matthiessen et al. 2012).
Data from the INSPECT database were used to evaluate pain scores before and
after the procedure and to investigate if a subset of patients at risk of pain could be
identified. A total of 121 consecutive patients were included, treated at seven
institutions in the InspECT network. Electrochemotherapy was performed according
to the standard operating procedures published in 2006 (Mir et al. 2006). Local
anesthesia was used for small or few metastases, whereas general anaesthesia was
preferred for multiple metastases, metastases larger than 3 cm, and metastases
attached to the periosteum or situated in sensitive regions. Tumor response was
evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST
version 1.0) (Therasse et al. 2000) and a maximum of seven cutaneous metastases
per patient was registered as target lesion.
Pain assessment was performed at four time points: before treatment, within 24 h
after treatment, thereafter within 45 days from treatment, and after more than 45 days
from treatment. Pain intensity was evaluated using the 11-point Numeric Rating
Scale (NRS) for pain. We used a previously published cut-off on NRS score
(Gerbershagen et al. 2011): 0–2 mild pain, 3–4 moderate pain, and 5–10 severe
pain. Pain medication was registered as “none,” “sometimes,” “controlled by non-
opioid drugs,” “controlled by opioid drugs,” “uncontrolled,” or “unknown.”
Out of the 121 patients, the large majority did not experience any pain before
treatment (60% with NRS score “0”), this percentage dropped to 43% at within 24 h
after the procedure, and rose again to 57% and 66%, respectively, within 45 days
after treatment and after more than 45 days from treatment. Data clearly showed that
a high pain score before treatment is highly significantly correlated with pain scores
after treatment; thus a very obvious recommendation would be to assess patients for
pain at the first visit and institute an immediate, as well as a posttreatment and
follow-up, pain management plan.
Other factors related with increased risk for postelectrochemotherapy pain are:
diagnosis of breast cancer, previous irradiation to the treatment area, and largest
diameter. Probably, as found in another study, repetitive treatments increase risk
of pain.
A high current value, typically seen when tissue is highly conductive due to
ulceration or necrosis, was statistically correlated to pain within 24 h from treatment.
It remains to ascertain if current by itself has an effect on pain or whether high
current levels are only reflective of large and/or ulcerated metastases, which could be
painful. In conclusion, the data from the study allowed to state that pain levels and
pain medication overall decrease after electrochemotherapy.
In a more recent paper a European multi-institutional cooperation was established
for the European Research on Electrochemotherapy in Head and Neck Cancer
(EURECA) trial (Bertino et al. 2016). The purpose of this observational prospective
longitudinal study was to investigate the efficacy of electrochemotherapy in local
tumor control of skin cancer of head and neck (HN) area where standard treatment
modalities have failed or have been considered increasing the risk of serious
morbidity or of functional impairment.
In fact neoplasms of the head and neck area are often a challenge for clinician
specially if recurrent, locally advanced, or when the proximity of important anatom-
ical structures may lead to disfigurement or unacceptable functional outcome with
standard treatments. Clinical reports underlying the feasibility with minimal damage
to normal tissue in treatment of head and neck cancers are emerged in the last years
(Scelsi et al. 2013; Mevio et al. 2012). In addition to limited side effects and minimal
damage to normal tissue, as previously demonstrated, electrochemotherapy could
lead to objective response rate of skin tumors in 70–80% with good cosmetic results
(Cadossi et al. 2014; Spratt et al. 2014).
For the first time a study was designed to evaluate prospectively safety, overall
and disease free survival of electrochemotherapy in treatment of head and neck skin
cancers. Patients were enrolled and treated at six European institutions (Pavia,
Ljubljana, Barcelona, Bradford, Amsterdam, Copenhagen) working as part of the
International Network for Sharing Practice in Electrochemotherapy (INSPECT). All
the subjects eligible for the study were affected by recurrent, metastatic, or primary
cancer of the head and neck area not suitable for surgery or chemo/radiotherapy
because of patient comorbidity, anticipated negative outcome of major surgical
intervention, and previous treatment or patient preference. The study also included
patients with primary tumors who refused any other standard treatment.
Between November 2011 and January 2015, 105 patients with tumors of the skin
in the HN area were consecutively included, but 6 of them were not considered in the
outcome analysis. Electrochemotherapy was performed according to the standard
operating procedures published in 2006 (Mir et al. 2006) using bleomycin either
intravenously or intratumorally. Treatment was carried out both under general and
local anesthesia. Tumor response of target lesions was made according to RECIST
criteria version 1.1 (Eisenhauer et al. 2009) in two follow-up visit planned at 1 and
2 months after the procedure.
Fifty of the 105 patients treated were affected by squamous cell carcinoma, 34 by
basal cell carcinoma, 10 by melanoma metastases, 3 by undifferentiated carcinoma,
3 by adenocarcinoma, 1 by renal carcinoma, 1 by leiomyosarcoma, 1 by lentigo
maligna, 1 by syringoma, and 1 by sarcomatous tumor. Nineteen of them were
retreated with a second session.
Minor side effects observed were skin ulceration, skin hyperpigmentation, sup-
puration, headache, nausea, skin odor, dysphagia, and maculopapular rash. Only one
major adverse event was reported: a septic shock in the second day after the
procedure led the patient to death.
As observed in others studies the response rate was dependent on tumor type and
in particular the higher response rate was observed with BCC (Testori et al. 2010;
Kis et al. 2012) (objective response of 97% with a 91% of complete response). As
the histopathological details of basal cell carcinoma were not recorded, the study did
not provide information about response rate on different basal cell carcinoma sub-
types. Overall, 48 (74%) of the 65 other tumor types showed a response to treatment.
At 1-year follow-up the percentages of overall and disease-free survival were
76% and 89%, respectively.
According to previous experiences response to treatment resulted related with a
small tumor size (<3 cm) and was also dependent on previous treatments (Quaglino
et al. 2015). In fact a previous chemo- or radiotherapy treatment significantly
decreased tumor response rate probably as a consequence of changes in tissue,
such as disruption of the vasculature, scarring, or necrosis that impair drug distribu-
tion (Campana et al. 2014). Naïve patients showed the best responses.
The analysis of specific questionnaires showed that quality of life improved after
electrochemotherapy with long-lasting positive effects on pain control, perception of
well-being, and physical functioning. The findings on the positive effects of electro-
chemotherapy on pain published by the INSPECT group (Quaglino et al. 2015) were
confirmed by Authors: pain was usually observed to increase in the following
45 days after treatment and then decrease. Furthermore, patients need significantly
less analgesic medication after treatment.
In conclusion, this paper stated that electrochemotherapy is an effective option for
skin cancers of the HN area and can be considered a feasible alternative to standard
treatments when such an alternative is appropriate.
Other papers are in preparation and will be focused on the analysis of specific
patient subsets and issues.
Projects and Perspectives
InspECT centers are actively engaged in different fields concerning clinical electro-
chemotherapy. In fact, they are working together to refine indications for treatment,
to frame procedural aspects to be further elucidated, to undertake research studies,
and to disseminate their findings.
From 2008 to date, the achievement of trial based evidences allowed to clarify
important aspects regarding patients’ selection (i.e., prevention of side effects,
individuation of more reliable predictors of response). However, the acquisition of
a more consolidated know how has also highlighted the presence of several new and
unexplored issues. For this reason, one important goal for the InspECT group will be
to provide a more solid, evidence-based, support to clinical practice by conducting
good-quality clinical research and looking for a large, expert-based consensus
among clinicians of different specialties.
As the aim of the network is to provide a validation of operating procedures and a
common evidence based point of view on hot topics emerging from time to time it
were identified some unanswered questions for which the Group is committed to
give an answer. The main instrument to reach this goal is represented by INSPECT
database due to large patient numbers (more than 890 at the time of writing) and
amount of data recorded on it. The following summarizes some of these open
questions.
It remains to ascertain if some histological tumor subtypes are more responsive to
the technique compared to other. For example, it has been nowadays stated a high
rate of objective responses on the treatment of basal cell carcinoma so that it could be
evaluated for the electrochemotherapy, a possible role as a first-line curative
approach (Bertino et al. 2016). Other studies are required to obtain information on
tumor response with different subtypes as nodular or morphemic.
Another open question is if there are differences in terms of response between the
ways of drug administration. Intratumoral way minimizes the risk of side effects but
in some kind of lesion or in specific histological types it could not allow to obtain an
adequate drug concentration in the site of electroporation. By the way in case of
small tumoral lesions an intratumoral bleomycin administration might be preferred
but to date there is a lack of randomized trials supporting this experience based point
of view.
In previous studies, it has been noticed that a preirradiation decreases the
response rate (Bertino et al. 2016; Campana et al. 2014) but also this finding has
to be investigated. Similarly, it should be explored how or in which situations the
number of electrochemotherapy performed sessions influences the response.
There is also the need to determine how the response rate is influenced by specific
characteristics of tumoral lesions as for example the size of nodules (less than
1, between 1 and 2, between 2 and 3, or superior to 3 cm), tumor volume, or
anatomical site (head and neck, scalp, chest, abdomen, and limbs).
The consensus has to be reached regarding the need to a lateral margins treatment
and, if yes, on the amount of these margins. Similar to guidelines for surgical
treatment and radiotherapy planning (Telfer et al. 2008), it has been observed that
a rim of normal tissue surrounding the lesion of at least 5 or 10 mm must be treated to
maximize tumor response (Bertino et al. 2016).
Together with the clinical experience has emerged the necessity of testing the
different results, both functional and aesthetic, related with the electrode type used.
According to the Standard Operating Procedures plate electrodes (Type I) are often
chosen for superficial tumor nodules and needle electrodes for deeper seated tumors
or for the treatment of large superficial nodules: needle row (Type II) for small tumor
nodules and hexagonal centered configuration (Type III) for the large nodules (Mir
et al. 2006). It remains to ascertain if using needle instead of plate electrodes
determines a higher rate of skin side effects (ulceration, hyperpigmentation).
In order to fill these gaps, the Group promoted projects focused on specific issues
and diseases. In particular, an analysis of data concerning breast cancer and mela-
noma treatments recorded on database to promote and expand the use of the
technique in two of the firsts and more studied electrochemotherapy fields of
application will be provided. The Group is also involved in recording and analyzing
data about other oncologic diseases: basal cell carcinoma, squamous cell carcinoma,
as well as angiosarcoma.
The efforts will be addressed also to a revision of the Standard Operating Pro-
cedures, published by now 10 years ago (Mir et al. 2006). In this period of time,
inspired by the new findings, has emerged the needs to redefine technical aspects
(e.g., the treatment window or the intravenous bleomycin dose) or underline specific
recommendations concerning the electrodes selection and the management of post-
procedural pain and lesions.
The study of drugs pharmacokinetic could provide the scientific basis for
reviewing their recommended dose. A newly developed method was employed for
bleomycin pharmacokinetic studies in serum and tumors of laboratory animals. The
method was employed for determination of bleomycin pharmacokinetic parameters
in elderly patients (Kosjek et al. 2016). On this base arose the proposal for a trial to
evaluate the serum concentration also in younger patients treated with the procedure
in order to determine the effective therapeutic window of electrochemotherapy with
bleomycin.
Concerning the possibility of a drug dose reduction, the aim of the Group is to
analyze data of electrochemotherapy treatments performed using low dose
bleomycin recorded on database. A reduced dose of 10 mg/m2 bleomycin or even
less could lead to a reduced risk of toxicity (skin reactions, nausea, lung fibrosis,
pain) without compromising the effectiveness.
Finally, in order to obtain a consensus on selected aspects where there is a lack of
concordance among clinicians the members of the Network had identified the Delphi
method as a useful instrument (Nair et al. 2011). Developed in the 1960s, it takes its
name from the Delphic oracle. The team undertaking the Delphi invites experts to
provide opinions on a specific matter and participate in iterative questionnaire
rounds. In order to achieve a reliable consensus, the panel of participating panelists
should include experts from different specialties, such as medical oncologists,
surgeons, dermatologists, radiotherapists, etc.
Conclusions
The collaborative effort of InspECT centers has allowed to clarify some crucial
aspects in the clinical application of electrochemotherapy and quality of life (for
example, pain).
In fact it is now accepted that electrochemotherapy offers a minimally invasive

treatment, provide local disease, symptoms control at cost of only minor side effects
and the Standard Operating Procedures allow to obtain similar results in different
medical teams. Pain levels and pain medication overall decrease after electro-
chemotherapy and now we can identify at the pretreatment visit the patients at risk
for postprocedure pain. Furthermore, this procedure is an effective option for skin
cancers in challenging anatomical sites like head and neck area and can be consid-
ered a feasible alternative to standard treatments.
However, during the last 10 years, the fields of application of electro-
chemotherapy in the clinic have been continuously increasing. This, in turn, has
raised several issues concerning better patient selection, more effective treatment
administration, postoperative patient management, data reporting, and production of
high-evidence guidelines (Campana et al. 2016).
In future, this network can further improve the clinical practice by promoting a
continuous update for clinicians and by the conduction of multicenter clinical trials.
References
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92:205–216
Electrochemotherapy of Cutaneous
Metastases 96
Marko Snoj and Louise Wichmann Matthiessen
Abstract
Cutaneous metastases are fairly frequent manifestation of advanced malignant
disease, comprising about 0.7–9% of all cancer patients. The cutaneous metasta-
ses may present itself as an isolated disease or may present itself as a part of
multiple organ spread. Treatment of cutaneous metastases could reduce or elim-
inate symptoms and improve quality of life. There are various sorts of local
therapies and among them electrochemotherapy is gaining in importance. The
first clinical report of electrochemotherapy for cutaneous metastases came in
1991 which reported good clinical results in treatment of head and neck tumors.
The cumulative results of all studies till 2006 established that there has been
642 cutanoues melanoma metastases treated with a complete response of 67%
and an objective repsonse of 85%. The results of the first multicentric study were
published the same year showing objective response in 85% of treated metastases,
regardless of tumor histology, drug used, or route of drug administration. With
this multicenter study, the Standard Operating Procedures for electroche-
motherapy were established. Today electrochemotherapy is a feasible and desir-
able treatment option for cutaneous metastases of different malignant histologies.
It is more effective in cutaneous metastases smaller than 3 cm. The side effects
and adverse events of electrochemotherapy are minimal, with pain being the most
frequent. Its advantages over other local treatments are that it can be used in
heavily pretreated patients, may be repeated several times, and usually does not
produce the ulceration of treated lesion.
M. Snoj (*)
Institute of Oncology, Ljubljana, Slovenia
Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
e-mail: msnoj@onko-i.si
L.W. Matthissen (*)
Department of Oncology, Copenhagen University Hospital Herlev and Gentofte, Herlev, Denmark
e-mail: louisewichmannmatthiessen@gmail.com; louise.wichemann.matthiessen@regionh.dk

DOI 10.1007/978-3-319-32886-7_96
1818 M. Snoj and L.W. Matthiessen
Keywords
Electrochemotherapy • Cutaneous metastases • Bleomycin • Cisplatin
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1818
Treatment of Cutaneous Metastases with Electrochemotherapy Before the ESOPE Trial . . . 1819
Electrochemotherapy for Cutaneous Metastases After the ESOPE Trial . . . . . . . . . . . . . . . . . . . . . 1821
Special Situations for Applications of ECT in Cutaneous Metastases . . . . . . . . . . . . . . . . . . . . . . . . 1823
Description of the Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1824
Chemotherapy and Administration Route of Chemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1824
Choice of Electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1824
Anaesthesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1826
Dressings After Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1826
Side Effects and Adverse Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1827
The Electrochemotherapy of Cutaneous Metastases in Gudelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1827
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1828
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1828
Introduction
Cutaneous metastases are fairly frequent manifestation of advanced malignant

disease. Although its frequency is difficult to establish, older studies have shown
that 0.7–9% of all cancer patients develops cutaneous metastases (Lookingbill
et al. 1993). There are some reports dealing with this question (Krathen
et al. 2003; Siegel et al. 2013). It was estimated that in 2013, 77166 of the
1445960 newly diagnosed cancers in the United States (not including skin or
hematologic malignancies) will develop cutaneous metastases. The cutaneous
metastases may present itself as an isolated disease or may present itself as a part
of multiple organs spread.
Cutaneous metastases are a visible part of metastatic disease and may therefore
burden patients psychologically and cause suffering. Cutaneous metastases can
cause bleeding, oozing, disfigurement, or pain and might be serving as a nidus for
infection. Treatment of cutaneous metastases would reduce or eliminate these
symptoms problems and improve quality of life.
There are many different treatments for cutaneous metastases. If cutaneous
metastases are presented as a part of multiorgan disease, the treatment is usually
systemic antineoplastic treatment. If there is no response or they progress, the
therapy should be local.
There are various sorts of local therapies and among them electrochemotherapy
(ECT) is gaining in importance. The other possibilities are radiotherapy, which
delivers ionizing radiation to cutaneous metastases, intralesional therapy which
uses the administration of antineoplastic agent directly or in the vicinity of cutaneous
metastases. Topical therapy is the application of antineoplastic agent directly on
96 Electrochemotherapy of Cutaneous Metastases 1819
cutaneous metastases. Photodynamic therapy uses nontoxic light to activate

photosenzitizer which is applied topically or intravenously. Isolated limb infusion
and perfusion are the methods of vascular exclusion of a limb and using very high
concentrations of drug which would otherwise cause serious adverse events. Some-
times, when disease is bulky, numerous or hemoraghing profusely, even amputation
is performed.
Spratt et al. (2014) conducted a metaanalysis, comparing all different local
treatments for cutaneous metastases. There were 47 studies included for analysis.
The median age of included patients was 61 years; 306 patients were males,
565 were females, which reported 4313 cutaneous metastases. Histologies for the
cutaneous metastases were 83,3% metastatic melanoma, 13,5% breast cancer, 0,2%
unspecified sarcoma, 0,09% Kaposi sarcoma, 0,05% angiosarcoma, and 2,8% were
unspecified histologies. Objective response (OR), which is defined as complete
response (CR) + partial response (PR), was for all studies 60,2%. Complete
response was 35,5% and overall responce (OR) was estimated to be 9,2%. Grade
3 or more toxicity was reported in less than 6% of patients. Because the majority of
studies were prospective clinical trials and only few have been randomized, there
might have been inherent bias in patient selection. Therefore, no comparison
between the treatment options could have been produced. Nevertheless, OR in any
of the local treatment still surpass the OR with systemic treatment, which is around
25% (Richtig et al. 2005).
The treatment of cutaneous metastases with electrochemotherapy are the first
treatments of then new and unproven method, what electrochemotherapy (ECT) was
at time being, on patients (Mir et al. 1991). It has been performed by using
bleomycin or cisplatin. The basic principles of action and application of ECT are
explained elsewhere in the text. Those principles still stand today; in fact they were
standardized and put into Standard Operating Procedures (Marty et al. 2006). Fur-
thermore, with increasing knowledge of ECT in treatment of cutaneous metastases,
and approving its efficacy, they were included in national guidelines in Germany,
Spain, and UK.
Treatment of Cutaneous Metastases with Electrochemotherapy

Before the ESOPE Trial
The first clinical report of treatment of cutaneous metastases comes from Mir et al. in
1991 at Institute Gustav Roussy who reported good clinical results of ECT treatment
of head and neck tumors. These results were in fact not only the first treatment results
of treatment of cutaneous metastases, but these were the first results of ECT
treatments in a general clinical setting. The new promising treatment has been
promptly seized by groups of investigators in Institute of Oncology, Ljubljana,
Slovenia (Serša G et al.), the group from University in Tampa, California, USA
(Richard Heller et al.), and the group at Institute of Pharmacology and Structural
Biology and Institute Claudius Regaud In Toulouse. Later some other institutions
from Europe, USA and Japan joined (Serša 2006). These trials have not been
standardized and by 2003 the necessity to standarize them, in order to make compar-
ison of the results possible, emerged. Therefore, European Standard Operating Pro-
cedures of Electrochemotherapy (ESOPE) trial commenced, encompassing four
centers from Europe. The results were presented in 2006 (Marty et al. 2006) and
Standard Operating Procedures for electrochemotherapy were established (Mir et al.
2006).
The overview of trials before ESOPE trial is reported elsewhere in the handbook.
Here we would like to present only the highlights. After the first clinical report by
Mir et al.(1991), who treated 40 SCC nodules in 8 head and neck patients with
electroporation with i.v. bleomycin, with complete response of 57% and objective
response of 72% and no side effects, some further clinical studies emerged on
different histologies and using either i.v., or intratumoral bleomycin or cisplatin.
The results of these studies were summarized by Gothelf et al. (2003) and Serša
(2006). There have been 642 melanoma nodules treated with 67% of CR and 85% of
OR. The remaining 367 nodules treated so far were of other histologies (head and
neck squamocellular carcinoma (SCC), breast carcinoma, hypernephroma, salivary,
gland adenocarcinoma, bladder transitional cell carcinoma, chondrosarcoma, Kaposi
s sarcoma), and an OR of 79% was obtained in treated metastases and of them 58%
were in CR. The side effects of ECT were minimal; the treatment was quick and easy
to perform under systemic or local anesthesia. Patients could be offered repetitive
treatment if new cutaneous metastases emerged. Another advantage of ECT was
shown, namely, that it could be used in heavily pretreated areas. This was particu-
larly important because majority of patients was given ECT when all other thera-
peutic options were exhausted (Serša 2006).
Another question emerged from previous studies: which drug is superior
bleomycin or cisplatin and which way of delivery – either intravenously (i.v.) or
intratumoral (i.t) was superior. Since there were no studies designed to compare
these parameters, the data were deduced from existing studies (Serša 2006). It has
been shown that i.v. bleomycin gives OR of 78% and when applied intratumorally
87% OR. Furthermore, the CR was higher with bleomycin i.t. (77%), compared to
bleomycin i.v. (45%). When using cisplatin it was shown that when given i.t, it
results in much higher OR (83%) compared to i.v. administration (48%). Nowadays
cisplatin is only used with i.t. administration (Serša 2006).
The ESOPE trial was the first multicentric trial, encompassing four European
centers. It was designed to evaluate and confirm efficacy and safety of ECT with
bleomycin or cisplatin on cutaneous and subcutaneous metastases less than 3 cm in
diameter in patients with malignant disease. It was a 2-year long prospective
nonrandomized study on 41 patients evaluable for treatment response and 61 patients
evaluable for toxicity. The treatment was performed using either i.t. or i.v. applica-
tion of cisplatin or bleomycin, followed by application of electric pulses generated
by an electroporator (Italy) using plate or needle electrodes. An OR of 85% was
obtained on treated metastases, regardless of tumor histology, drug used, or route of
drug administration. At 150 days after treatment, local tumor control rate was 88%
with bleomycin given i.v. 73% with bleomycin given i.t., and 75% with cisplatin given
i.t., demonstrating that all three approaches were similarly effective. Moreover, ECT
was equally effective regardless of the tumor type and size of the metastases treated.
Observed side effects were minor and acceptable. Therefore, it has been demonstrated
that ECT is effective, safe, and cost-effective treatment for cutaneous and subcutane-
ous metastases of different malignant histologies (Marty et al. 2006).
All these facts were important in designing Standard operation procedures for
ECT (Mir et al. 2006). These procedures were developed in order to safely and
conveniently treat patients with cutaneous and subcutaneous metastases. They
recommend the electroporation system approved for clinical use and capable of
delivering 8 pulses of 100 ms at appropriate voltage. Three different sets of elec-
trodes for delivering pulses were recommended (See Fig. 2). Either bleomycin or
cisplatin can be used for treatment; however, only bleomycin is recommended for
i.v. administration. Details of the ECT procedure were described and decision tree
based on number and size of metastases and its size was presented.
The ESOPE study and Standard operating procedures for ECT were a milestone
in perception of ECT, which, then, became established and standardized treatment
for cutaneous metastases.
Electrochemotherapy for Cutaneous Metastases After the ESOPE

Trial
There were some articles written subsequently which added further layers of knowl-
edge on ECT.
Gaudy et al.(2006) evaluated tolerance of treatment in patients with cutaneous
metastases from malignant melanoma treated with i.t. bleomycin in local anesthesia
with 1% lidocaine solution with epinephrine. They have not observed any drug-
related toxicity. Although CR was 74%, in line with ESOPE protocol, all patients
reported discomfort on treated site during the ECT procedure, 75% the pain on the
treated site, and 25% muscle spasms secondary to electric pulses.
Quaglino et al. (2008) observed 74,5% local tumor control at 2 years. They
treated 233 cutaneous metastases from malignant melanoma ECT with bleomycin
i.v. in 14 patients. In seven patients CR was obtained with single treatment, seven
patients underwent a second and three patients a third treatment due to recurrence in
the treated area or of new cutaneous metastases outside the treated area. The patients
have been treated according to ESOPE recommendations.
More robust data were presented by Campana et al. (2009), who treated
52 patients, with 608 cutaneous metastases of different histologies. Mainly they
were melanoma and breast cancer patients. Patients were treated according to
ESOPE guidelines, except of tumor size, which was in 27% of patients larger than
3 cm. Treatment was well tolerated especially under general anesthesia. After a mean
follow-up of 9 months, only two patients experienced relapse within treatment field.
Nearly half of patients received repetitive treatment to achieve superficial tumor
control. Patients reported through the questionnaire a benefit in local-disease com-
plaints and in activity in daily living.
The size of the lesions and its effect on ECT results came into the limelight with
the work of Kis et al. (2011). Namely, 158 cutaneous and subcutaneous mestatases of
melanoma have been treated by ECT with intravenous application of bleomycin, and
there have been metastases up to 5,6 cm in diameter. CR was 23% and PR was 39%
giving an OR of 62% which is worse than reported in the ESOPE trial. This could be
explained by larger size of the tumor (8,5% of tumors were larger than 3 cm), and the
patients were subjected to treatment only once, which is another factor which may
explain those modest results.
These results have been supported by Mattheissen et al. (2011), who treated
52 patients with cutaneous and subcutaneous metasases of tumors of different
histologies with ECT using bleomycin, i.v. or i.t. It was shown that CR and PR
were 68% and 18%, respectively, for cutaneous metastases less than 3 cm in diameter
and 8% and 23% for cutaneous metastases more than 3 cm in diameter.
By 2012, there have been some mature data on using ECT in cutaneous and
subcutaneous metastases from breast cancer. This topic is covered comprehen-
sively elsewhere in this handbook. In short Benevento et al. (2012) showed that
one ECT with bleomycin i.v. is effective in breast cancer cutaneous metastases
with obtained CR in 75% and PR in 17% in 112 metastases. Moreover, Campana
et al. (2012) showed more elaborate results with patients having breast cancer.
They have treated 516 metastases in 37 breast cancer patients, with median size
of 20 mm, some of them up to 220 mm. Twenty-one patients received more that
one treatment. In 2 months, CR was 54,3% and PR 37,1%. Twenty-three patients
developed new lesions and were subsequently retreated by ECT. In 3 years time
local control rate was 81%. Related morbidity was mild, consisted primarily of
pain and dermatologic toxicity – ulceration appearing in up to 33% of patients.
There were enough data to conduct first metaanalysis on antitumor effectiveness
of ECT in cutaneous and subcutaneous tumors (Mali et al. 2013a). In this work,
44 studies involving 1894 tumors were included. The great majority of studies were
on cutaneous metastases, although in eight of them have been performed on primary
batocellular carcinoma or SCC of the skin. Data analysis confirmed that ECT had
significantly higher effectiveness than bleomycin or cisplatin alone. The effective-
ness was significantly higher for intratumoral than for intravenous administration of
bleomycin. Bleomycin and cisplatin administered intratumorally resulted in equal
effectiveness of ECT. Furthermore, it was shown that ECT was more effective in
sarcoma than in melanoma or carcinoma tumors.
Previous reports have suggested that response depends on tumor size and
response seems to be declining with increasing size. Larkin et al. (2007) observed
that metastases larger than 3 cm were less likely to respond with ORR in 8 out of
21 (38%) compared to a total ORR of 82%. Campana et al. (2009) and Quaglino
et al. (2008) likewise observed inverse correlation between response rate and size of
the cutaneous metastases.
Mali et al. (2013b) study confirmed these observations. In this study, author made
metaanalysis of nine eligible studies performed on CM in which data about the
tumor size might be extracted. Therefore, 1466 tumors of any histiotype were
analyzed over tumor response in 4 weeks after treatment by single application of

ECT. The results showed that there was significantly lower effectiveness of ECT on
tumors with maximal diameter equal to or larger than 3 cm (CR of 33,3%, OR of
68,2%) in comparison to smaller tumors (CR of 59,5% and OR of 85,7%). The
results of metaanalysis indicated that ECT performed on tumors smaller than 3 cm
significantly increase the probability of CR by 31% and OR by 24,9% on average in
comparison to larger tumors. Furthermore, the data showed significant decrease in
effectiveness of ECT progressively with increasing tumor diameter. Therefore, it was
concluded that standard operating procedures for tumors larger than 3 cm should be
reexamined and refined.
The usefulness of ECT in treating cutaneous metastases was further proved in six
German dermatology departments which had no previous experience in ECT
(Kreuter et al. 2015). They have treated 56 patients, 15 of them had primary SCC
and the rest have cutaneous metastases. They concluded that ECT was effective i

Handbook of Electroporation

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Handbook of Electroporation

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Damijan Miklavčič

With 932 Figures and 125 Tables

ISBN 978-3-319-32885-0 ISBN 978-3-319-32886-7 (eBook)

# Springer International Publishing AG 2017

Printed on acid-free paper

This Springer imprint is published by Springer Nature

In the familiar sense, a Foreword is to be a short informative introduction to a book

communication was extended to student education and technical instruction in

polyionic macromolecules that usually do not penetrate the unporated membrane

Department of Physical and

Electroporation is a word most often used to describe the phenomenon of membrane

which ﬁnally resulted in this Handbook of Electroporation. The list of chapters is

Faculty of Electrical Engineering Damijan Miklavčič

COST (European Cooperation in Science and Technology) is a pan-European

Part I Fundamental Mechanisms of Electroporation: Models and

10 Electropermeabilization and Electrostimulation by Picosecond

Part II Basic Description of Cell Electroporation in Vitro . . . . . . . . 253

23 Gene Delivery by Electroporation In Vitro: Mechanisms ....... 387

24 Electroporation of Bioﬁlms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403

25 Bacterial Cell Envelopes: Composition, Architecture, and

26 3D Culture Models to Assess Tissue Responses to

27 Mechanistic Description of Membrane

28 Involvement of Reactive Oxygen Species in Membrane

29 Plant Cell Wall: Description, Role in Transport, and Effect

30 Regulated and Apoptotic Cell Death After Nanosecond

Part III Electroporation Phenomena on Tissue Level and

31 Electroporation in Scars/Wound Healing and Skin Response . . . . 531

32 Principles and Use of Magnetic Resonance Electrical Impedance

33 Current Density Imaging as Means to Follow Tissue

34 Mass Transfer of Electrolytic Species During Electric

35 Effects of Reversible and Irreversible Electroporation on

Part IV Hardware Used in Electroporation Experiments and

47 Photoconductive Switching for Pulsed High-Voltage

Part V Modeling of Electroporation and Related Phenomena . . . 999

61 Dual-Porosity Model of Liquid Extraction by Pressing from

Part VI Laboratory Techniques Used in Electroporation

73 Different Cell Viability Assays Following Electroporation

74 Patch Clamp in Use of Electroporation Mechanisms Studies . . . . 1425

75 Parameters Affecting Cell Viability Following Electroporation

76 Techniques to Detect Electroporation in Food Tissues . . . . . . . . . . 1467

77 Determination of Pulsed Electric Fields Effects on the Structure

Part VII Preclinical Development for Biomedical Application of

78 Preclinical Studies on Electrochemotherapy . . . . . . . . . . . . . . . . . . 1511

79 Preclinical Studies on Irreversible Electroporation . . . . . . . . . . . . 1527

80 Preclinical Studies on Nanosecond Pulses . . . . . . . . . . . . . . . . . . . . 1543

81 Principles of Electroporation for Gene Therapy . . . . . . . . . . . . . . 1563

82 Tissue Engineering with Electroporation . . . . . . . . . . . . . . . . . . . . 1579

83 Predictable Animal Models for Translational

84 Antiangiogenic Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1623

85 New Drugs for Electrochemotherapy with Emphasis on

Part VIII Biomedical Applications of Electroporation . . . . . . . . . . . 1769

98 Electrochemotherapy of Liver Tumors: Colorectal Liver

111 Electrochemotherapy for Breast Cancer . . . . . . . . . . . . . . . . . . . . 2075

Part IX Electroporation in Biotechnology . . . . . . . . . . . . . . . . . . . . 2113

121 Pulsed Electric Fields-Assisted Extraction of Molecules from

122 Pulsed Electric Fields for Pasteurization: Deﬁning Processing

123 Pulsed Electric Fields-Assisted Acetylation of Starch . . . . . . . . . . 2297

124 Pulsed Electric Fields for Algal Extraction and Predator

Part X Electroporation in Food Science and Technology ....... 2333

125 Pulsed Electric Fields for Food Industry: Historical

126 Advantages of Pulsed Electric Field Use for Treatment

127 Impact of Pulsed Electric Fields on Enzymes . . . . . . . . . . . . . . . . 2369

128 Impact of Pulsed Electric Field Treatment on Must and Wine

129 Pulsed Electric Fields for Extraction of Secondary Metabolites