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DRUG DELIVERY TRENDS
EXPECTATIONS AND REALITIES OF
MULTIFUNCTIONAL DRUG
DELIVERY SYSTEMS
VOLUME 3
Edited by

RANJITA SHEGOKAR, PhD

Capnomed GmbH, Zimmern, Germany
Elsevier
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 Contributors
Salvana Costa Universidade Tiradentes (UNIT),
Aracaju, Sergipe, Brazil
J. Dias-Ferreira Department of Pharmaceutical
Technology, Faculty of Pharmacy, University of
Coimbra (FFUC), P
olo das Ciências da Sa
ude,
Azinhaga de Santa Comba, Coimbra, Portugal
A.R. Fernandes Department of Pharmaceutical
Technology, Faculty of Pharmacy, University of
Coimbra (FFUC), P
olo das Ciências da Sa
ude,
Azinhaga de Santa Comba, Coimbra, Portugal
Rohit C. Ghan Aurobindo Pharmaceuticals USA
Inc., Dayton, NJ, United States
Randeep Kaur University Institute of Pharma-
ceutical Sciences, Panjab University, Chandigarh,
India
Matthias M. Knopp Department of Pharmacy,
University of Copenhagen, Copenhagen, Denmark
A. K€onigsrainer National Center for Pleura and
Peritoneum, University of T€
ubingen, T€ubingen,
Germany
Korbinian L€ obmann Department of Pharmacy,
University of Copenhagen, Copenhagen, Denmark
Conrado Marques Universidade Federal de Sergipe
(UFS), Campus Lagarto, Departamento de Medic-
ina, Sergipe, Largarto, Brazil
Muhaned Al-Hindawi OnTarget Pharma Consul-
tancy Limited, New Malden, Surrey, United
Kingdom
Luciana Nalone Universidade Tiradentes (UNIT),
Aracaju, Sergipe, Brazil; Instituto de Tecnologia e
Pesquisa (ITP), Aracaju, Sergipe, Brazil
Adam Procopio Merck & Co., Inc., Kenilworth, NJ,
United States
vii
Saeed A. Qureshi Pharmacomechanics, Ottawa,
ON, Canada
Vivek Ranjan Sinha University Institute of Pharma-
ceutical Sciences, Panjab University, Chandigarh,
India
M.A. Reymond National Center for Pleura and Peri-
toneum, University of T€
ubingen, T€
ubingen, Germany
Patrícia Severino Universidade Tiradentes (UNIT),
Aracaju, Sergipe, Brazil; Instituto de Tecnologia e
Pesquisa (ITP), Aracaju, Sergipe, Brazil
Ranjita Shegokar Capnomed GmbH, Zimmern,
Germany
A.A.M. Shimojo Department of Materials Engineer-
ing and Bioprocesses, School of Chemical Engineer-
ing, State University of Campinas (UNICAMP),
Cidade Universit
aria Zeferino Vaz e Bar~ ao
Geraldo, Campinas, S~ ao Paulo, Brazil
A.M. Silva Department of Biology and Environ-
ment, School of Life and Environmental Sciences,
University of Tr
as-os-Montes and Alto Douro,
Vila Real, Portugal; Centre for the Research and
Technology of Agro-Environmental and Biological
Sciences, University of Tr
as-os-Montes and Alto
Douro, Vila Real, Portugal
Eliana B. Souto Department of Pharmaceutical
Technology, Faculty of Pharmacy, University of
Coimbra (FFUC), P

olo das Ciências da Sa

ude, Azin-
haga de Santa Comba, Coimbra, Portugal; CEB e
Centre of Biological Engineering, University of
Minho, Campus de Gualtar, Braga, Portugal
M.C. Teixeira Department of Pharmaceutical Tech-
nology, Faculty of Pharmacy, University of Coim-
bra (FFUC), P
olo das Ciências da Sa
ude,
Azinhaga de Santa Comba, Coimbra, Portugal
viii Contributors

Divya Tewari Noramco Inc., Wilmington, DE, Akif Emre T€ €

United States
Pramil Tiwari Department of Pharmacy Practice,
National Institute of Pharmaceutical Education &
Research (NIPER), S.A.S. Nagar, Punjab, India
Nazende G€ unday T€ ureli MJR PharmJet GmbH,
€
Uberherrn, Saarland, Germany
ureli MJR PharmJet GmbH, Uberherrn,
Saarland, Germany
Danillo F.M.C. Veloso Department of Pharmacy,
University of Copenhagen, Copenhagen, Denmark
Venkat Tumuluri Novartis Healthcare Pvt. Ltd.,
Hyderbad, India
 Preface

The book series titled Expectations and
Realities of Multifunctional Drug Delivery
Systems covers several important topics on
drug-delivery systems, regulatory requirements,
clinical studies, intellectual properties trends,
new advances, manufacturing challenges, etc.
written by leading industry and academic
experts. Overall, the chapters published in this
series reflect the broadness of nanopharma-
ceuticals, microparticles, other drug carriers, and
the importance of respective quality, regulatory,
clinical, GMP scale-up, and regulatory
registration aspects.
This series is destined to fill the knowledge
gap through information sharing and with orga-
nized research compilation between diverse
areas of pharma, medicine, clinical, regulatory
practices, and academics.
Expectations and Realities of Multifunctional
Drug Delivery Systems is divided into four
volumes:
Volume 1: Nanopharmaceuticals
Volume 2: Delivery of Drugs
Volume 3: Drug Delivery Trends
Volume 4: Drug Delivery Aspects
The specific objectives of this book series
are to:
1. provide a platform to discuss opportunities
and challenges in development of nano
medicine and other drug-delivery systems;
2. discuss current and future market trends;
3. facilitate insight sharing within various areas
of expertise; and
4. establish collaborations between academic
scientists, and industrial and clinical
researchers.
Innovative cutting-edge developments in
micro-nanotechnology offer new ways of
preventing and treating diseases like cancer,
malaria, HIV/AIDS, tuberculosis, and many
more. The applications of micro-nanoparticles
in drug delivery, diagnostics, and imaging are
vast. Hence, Volume 3: Drug Delivery Trends
in the book series mainly reviews advances in
drug delivery areas via targeted therapy with
improved drug efficiency at a lower dose, trans-
portation of the drug across physiological
barriers, as well as reduced drug-related toxicity.
The contribution by Fernandes et al.
(Chapter 1) discusses new trends in drug
delivery areas via bioactive hybrid nanowires.
Nanowires offer multifunctionality and the pros-
pect of biofunctionalization, thereby reducing
toxicity and side effects. This synergistic approach
overcomes the challenges associated with conven-
tional nanomedicines and exhibits better perfor-
mance. The authors review the potential of
nanowires in this chapter.
The chapter by Procopio and Tewari (Chapter
2) highlights another industry trenddi.e., 3D
printing. This is a fascinating topic recently
adopted by industry. The Food and Drug
Administration has already approved the first
product: Spritam (levetiracetam), a 3D printed
tablet (Aprecia Pharmaceuticals). A thorough
overview of material requirements, types of

ix
x Preface
polymer, available 3D printing techniques, and
regulatory aspects is provided. Finally, the au-
thors present case studies from industry on
tunable release technology and paste gel
extrusion in tableting.
The contribution by Al-Hindawi (Chapter 3)
describes marketing authorization and licensing
of medicinal products in the European Union.
The main aim of this chapter is to provide
readers with a generalized overview of the steps
and criteria while applying licenses in the
European Union. The author also highlights
various directives, extension requests, protection
periods, and legal requirements guidance to
industry and researchers. On the other hand,
preclinical understanding is key to proposing
products for particular indications.
The chapter by Tiwari et al. (Chapter 4)
reviews preclinical considerations on micro-
and nanodrug delivery, which will lead to the
proper positioning of products for market
authorization.
The work by Ghan (Chapter 5) is aimed at dis-
cussing synergistic delivery of nanoparticles
using traditional approaches like tableting or
other forms. The author reviews various nano-
particulate treatments for oral delivery to
improve bioavailability, target specific regions
in the gastrointestinal tract, improve physiolog-
ical stability in the gut environment, and
modulate release when needed. Drug delivery
systems like solid lipid nanoparticles, polymeric
nanoparticles, nanomicelles, and nanosus-
pension are also reviewed.
The chapter by Tumuluri (Chapter 6)
highlights opportunities and challenges in
formulating minitablets. This is another trend
in oral drug delivery systems besides nano-oral
and 3D printed dosage forms. The author
describes in detail technological potential, indus-
trial advantages, technological availability, and
the limitations of minitablets.
The topic presented by Nalone et al.
(Chapter 7) describes the potential of liquid crys-
talline systems in drug delivery. This chapter
discusses in detail the mechanism of formation
of liquid crystalline systems, types of structure
formed, factors affecting formation of liquid crys-
talline systems, compositions, advantages, and
limitations of these forms of drug delivery
systems.
The chapter by Veloso et al. (Chapter 8)
reviews opportunities and challenges in amor-
phous drug stabilization using mesoporous
materials. The team of authors highlights key
points like the role of mesoporous materials,
structural characterizations of such systems,
physical forms of drug loading, and physical
performance of mesoporous particles. Mesopo-
rous particles have huge potential in pharmaceu-
ticals as a drug delivery system, in cosmetics, in
nutraceuticals, and in fast-moving consumer
goods (detergent, oral care), and is a highly
explored trend in the market.
Quality is an important aspect in regulatory
which makes sure patients receive “quality”
products. Newer trends in pharmaceuticals like
nanomedicines, drug device combinations,
nanoparticulate-based tablets, etc. require
special techniques and an understanding of
quality. The chapter by Qureshi (Chapter 9)
asks questions like “what is quality?”, “do we
evaluate quality as per a regulatory definition?”,
and “is there a need to change the definition of
quality when a product is altered?” The main
theme of this chapter is to underline basic
concepts of “quality” and discuss them with
regard to current practices. The author provides
his views on further modifications of current
testing methodologies to assure “real quality.”
The work by Reymond and K€ onigsrainer
(Chapter 10) is aimed at discussing the potential
of pressurized intraperitoneal aerosol chemo-
therapy (PIPAC). The authors review various
chemotherapeutic systems currently used
in vitro/ex vivo models and the success of
clinical trials to date. This generalized overview
of PIPAC technology provides readers with
updates from another innovative trend in
medical practice.
 Preface
The last contribution by T€ ureli and T€ ureli
(Chapter 11) describes industrial challenges of
upscaling and good manufacturing practice in
the production of pharmaceutical drug delivery
systems. The authors highlight the current regu-
latory status of approved nanomedicines and
manufacturing limitations and initiatives.
In summary, I am sure this book volume and
the complete book series will provide you great
xi
insights in areas of micro-nanomedicines, drug
delivery sciences, new trends, and regulatory
aspects.
All the efforts of experts, scientists, and au-
thors are highly acknowledged for sharing their
knowledge, ideas, and insights about the topic.
Ranjita Shegokar, PhD
Editor
 C H A P T E R

1
Bioactive hybrid nanowires: a new in
trend for site-specific drug delivery and
targeting
A.R. Fernandes1, J. Dias-Ferreira1, M.C. Teixeira1,
A.A.M. Shimojo2, Patrícia Severino3,4, A.M. Silva5,6,
Ranjita Shegokar7, Eliana B. Souto1,8
1
Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra (FFUC), P
olo
ude, Azinhaga de Santa Comba, Coimbra, Portugal; 2Department of Materials
das Ci^encias da Sa

Engineering and Bioprocesses, School of Chemical Engineering, State University of Campinas
(UNICAMP), Cidade Universit
aria Zeferino Vaz e Bar~ao Geraldo, Campinas, S~ao Paulo, Brazil;
3
Universidade Tiradentes (UNIT), Aracaju, Sergipe, Brazil; 4Instituto de Tecnologia e Pesquisa (ITP),
Aracaju, Sergipe, Brazil; 5Department of Biology and Environment, School of Life and Environmental
Sciences, University of Tr
as-os-Montes and Alto Douro, Vila Real, Portugal; 6Centre for the Research
and Technology of Agro-Environmental and Biological Sciences, University of Tr
as-os-Montes and Alto
Douro, Vila Real, Portugal; 7Capnomed GmbH, Zimmern, Germany; 8CEB e Centre of Biological
Engineering, University of Minho, Campus de Gualtar, Braga, Portugal

1. Introduction can be terminated at temperatures ranging

Hyperthermia (“hyper” and “therme”, mean-
ing “rise” and “heat”) is a therapeutic approach
to cancer treatment. Some researchers have
related that a sarcoma disappeared after a very
high fever. This finding is due to the reaction
of immune systems with bacterial infection [1].
Cancer cells are recognized as being vulnerable
to high temperatures. The growth of these cells
from 41 to 46
C or below 47
C for at least
20e60 min [2,3]. Hyperthermia is therefore
used locally to prevent disease by exposing the
whole body to high temperatures to overcome
adverse side effects and to increase treatment ef-
ficiency [4].
The introduction of magnetic nanoparticles
in cancer hyperthermia has been developed
and grown significantly during the last decade.

Drug Delivery Trends

https://doi.org/10.1016/B978-0-12-817870-6.00001-8 1 © 2020 Elsevier Inc. All rights reserved.
2 1. Bioactive hybrid nanowires: a new in trend for site-specific drug delivery and targeting
The special features of these particles are related
to their capacity to efficiently accumulate at the
tumor cells through the increased permeability
of the tumor vessels and by cancer-specific bind-
ing agents, making the treatment more selective
and effective [5]. The application of an alter-
nating magnetic field (AMF) with the introduc-
tion of magnetic nanoparticles generates local
heat in the tissues that contain these nanopar-
ticles due to magnetic relaxation and hysteresis
loss [6]. Particle characteristics such as size distri-
bution, shape, crystal structure, particle mag-
netic anisotropy and its temperature
dependence on magnetization, fluid viscosity,
amplitude and frequency of the AMF directly
affect the generation of heat, which in turn de-
pends on the absorption efficiency of the mag-
netic particles [1,7].
A significant number of magnetic nanopar-
ticles have been studied over the last few de-
cades. Examples of well-known hyperthermic
agents include iron oxide-based nanomaterials
such as magnetite (Fe3O4) and maghemite (g-
Fe2O), which continue attracting attention due
to their lack of toxicity and excellent biocompat-
ibility [8]. Ferrite nanoparticles (XFe2O4, where
X can be Co, Mn, Ni, Li, or mixes of these
metals), metallic nanoparticles, such as Mn, Co,
Ni, Zn, Gd, Mg, and their oxides, or metal alloys
(FeCo, CoPd, FePt, NiPd, NiPt, NiCu) have also
been studied as possible candidates for hyper-
thermia treatments [9e11].
There are new designs of magnetic nanomate-
rials based on a core/shell approach that have
started to gain prominence due to their versatility
to tailor properties of both core and shell and to
offer multifunctionality, such as core protection,
biofunctionalization platform, toxicity reduction,
and increase in biocompatibility. Examples of
these particles are gold- or silica-coated ferromag-
netic particles [12]. Magnetic nanoparticles also
hold great promise for drug delivery by heating
the tissues. The drug can be released using two
strategies. In the first approach, the drug mole-
cules are attached to the particles through a
linker, which breaks with the heat generated by
the presence of AMF, with the consequent release
of the drug. In the second approach, the release of
drugs takes place from a polymeric matrix with
magnetic material [5,13]. The heat created by
the magnetic field produces crevices or cracks in-
side the polymeric matrix, which releases the
encapsulated drugs [5].
Nanowires, nanowhiskers, nanofibers, nano-
tubes, and other one-dimensional nanostructures
have demonstrated huge abilities for improving
the electrical, optical, thermal, and mechanical
properties of a broad range of functional mate-
rials and composites [14]. These enhancements
substantially exceed those offered by micro- or
nanosized particles. Most of the methods used
for their synthesis are relatively expensive and
difficult to scale up [15]. The underlying princi-
ples for the synthesis of one-dimensional mate-
rials offer significant challenges in the control of
diameter, structure, and composition in the axial
and radial coordinates, which are essential for
the synthesis of materials with designed and
tunable functionality [16].
Nanowires, besides their magnetic perfor-
mance, also have aso an interest in developing
intrinsic mobility triggered by a photochemical
reaction. Examples of applications of magnetic
segmented nanowires are:
1. Magnetic alignment and wireless manipula-
tion (Au/polypyrrole/Ni) [17]
2. Magnetic field sensors and spintronic nano-
devices (Co/Cu and FeCoNi/Cu) [18]
3. Photochemical conversion and hydrogen
generation (Ag/ZnO) [19]
4. Detection of DNA molecules (CdTe/Au/
CdTe) [20]
5. Magnetic control of biomolecule desorption
(FeCo/Cu) [21]
6. Exchange-coupled patterned media (Ni/
CoPt) [22]
7. Nanosensors (Au/Co) [23]
8. Catalytic activities (Pt/Ni) [24]
9. Higher oxygen reduction reaction activity
(Co/Pt) [25,26]
10. Drug delivery
 3. Production methods
Magnetic nanoparticles (including nanowires)
are recognized as nanoparticles with unique
physicochemical properties and are mostly
different from those of conventional materials,
specifically the electromagnetic properties. Mag-
netic nanoparticles show good magnetic orienta-
tion, small size, biodegradability, and reactive
functional groups [27]. The biocompatibility of
magnetic nanoparticles can be improved by
combining them with a variety of functional
molecules such as enzymes, antibodies, cells,
DNA, or RNA. The coating of other materials
such as polyethylene glycol (PEG), chitosan,
lipids, and proteins with good biocompatibility
can stabilize magnetic nanoparticles in physio-
logical fluids and provide chemical functionality
for additional modifications [28].
2. Types of nanowires
In the last few years, magnetic hybrid nano-
wires have been intensively studied for many
applications, such as optics and medicine. There
are two types of morphologies in hybrid
nanowires:
1. Radial structures (core/shell type); and
2. Axial structures (segmented or layered type).
The nanowires that present a core/shell struc-
ture explain many physical characteristics in the
magnetism of the nanoparticles. The hard/soft
core/shell nanoparticles have been studied and
reveal interesting magnetic properties, i.e.,
reversible tuning of the blocking temperature
[29], improved microwave absorption [30], opti-
mized hyperthermia [31], and enhanced coer-
civity [32]. The magnetic segmented nanowires
have multifunctional and structural advantages
compared to their counterparts, single-
component nanowires. The literature reports
that magnetic segmented nanowires are
3
composed of alternating structures of ferromag-
netic/ferromagnetic or ferromagnetic/nonmag-
netic materials, such as Ni/Cu [33], Ni/Au [34],
Co/Cu [35], NiFe/Cu [36], CoNi/Cu [37],
FeCoNi/Cu [38], FeGa/Cu [39], Co/Pt [40],
NiFe/Pt [41], and NiCoCu/Cu [42], among
others.
3. Production methods
The properties of many systems are basically
dependent of the material type used in produc-
tion; however, in the case of nanowires the
material geometry is also important. Thus to
produce and maximize all the properties of
nanowires requires reliable and controlled syn-
theses. The synthesis methods can be grouped
into two categories: (1) top-down and (2)
bottom-up synthesis.
3.1 Top-down method
The most conventional top-down method in
the fabrication of nanowires is lithography.
Lithography is based on the deposition of a resis-
tant material, for example, poly(methylmetha-
crylate), that has the function to act as a
photographic film for the production of a pattern
after exposure and development using a
patterned mask. The resolution of this technique
is dependent on the wavelength of light used in
photolithography and sometimes is not suitable
for small nanowires [43]. To obtain patterns
with a higher resolution, normally electron-
beam lithography is the method used, which
does not use a mask and has direct-write expo-
sure [44]. Thus nanowires can be obtained by
etching the extraneous material from the wafer.
Resistance can be applied directly because the
etch mask can serve as the template for the depo-
sition of a much more stable mask material, for
example, gold. Material that can be used to
4 1. Bioactive hybrid nanowires: a new in trend for site-specific drug delivery and targeting
etch a pattern is, for example, potassium hydrox-
ide (a wet chemical etchant) or another electro-
chemical etchant. With these materials it is
possible to produce tapered cylindrical wires
once the etching is underneath the mask [45].
One way to obtain cylindrical vertical wires is
to change the wet chemical etch with a highly
anisotropic deep reactive ion etch [46]. Nano-
sphere lithography is another approach that
promises higher resolution by combining the
self-assembly of a monolayer of nanospheres of
polystyrene, for example, onto a substrate in a
close-packed lattice [47]. The nanospheres serve
as a model for the deposition of a metal or
another material and are removed after deposi-
tion. Nanoscale patterns can be produced by me-
chanical transfer using nanoimprint lithography
[48,49].
3.2 Bottom-up method
In contrast to top-down techniques, bottom-
up synthesis offers the opportunity to control
nanowire composition during growth.
In this technique of the production of nano-
wires, the anisotropic growth of nanowires is
normally done using nanoparticle catalysts and
gas-phase precursors. The most used method of
production is vapor/liquid/solid growth. In
this method, gaseous precursors are used to
obtain the desired nanowires and these precur-
sors are dissolved into a liquid-metal catalyst,
for example, in the case of silicon nanowires
the precursor used is SiCl4. After the catalyst is
supersaturated, solid nanowire crystallization
from the liquid catalyst begins [50,51]. In this
process, the metal should form a droplet in the
liquid state that will serve as the catalyst. This
droplet, in some cases, will melt at a lower tem-
perature when compared to pure metal, due to
its eutectic composition. In the case of the syn-
thesis of binary or ternary compounds, which
are metals with low melting points, the vapor/
liquid/solid system can be self-catalyzed [52].
The solution/liquid/solid method is another
technique of nanowire production similar to
the vapor/liquid/solid method; however, in
this case nanowire precursors are dissolved in
a high-boiling liquid and the catalysts are sus-
pended in this liquid [53]. Substrates, such as
anodic aluminum oxide, can be used as template
solution for nanowire growth, using electro-
chemical deposition and after filling the channels
in the template [54]. Control of growth along the
axes of nanowires is necessary for the introduc-
tion of surfactants capable of changing the sur-
face energy of crystal facets, for example,
hexadecyltrimethylammonium bromide.
Anisotropy of nanowires is easy to achieve by
the control of surface chemistry [55] (Tables 1.1
and 1.2 and Fig. 1.1).
4. Applications of nanowires
Nanowire biosensors consist of typical field-
effect transistor-based devices, made up of three
electrodes that are very sensitive to the variation
in the charge density that promotes changes in
the electric field at the external surface of the
nanowires [64].
Nanowires have a high surface-to-volume ra-
tio and well-defined geometry; they have high
sensitivity and short response time. These char-
acteristics offer applications in biology and
chemistry. Applications of nanowires can be
categorized into two methodologies: electrical
detection and optical detection [49].
4.1 Nanowires in bioanalytical chemistry
One of the bioapplications of nanowires is
biomolecule analysis. This application includes
the study of mechanical cell lysis. Cellular lysis
is a fundamental process in the study of intracel-
lular components. There are a number of well-
established methods that can analyze the cell
components, such as chemical, electrical, and
mechanical methods. In the case of chemical
cell lysis many steps are necessary and it is an
expensive process, consuming many reagents
aimed at the purification of biomolecule sam-
ples. Another disadvantage is the high probabil-
ity of the occurrence of harmful effects on
 4. Applications of nanowires 5

FIGURE 1.1 Scanning electron microscopy images of CuS nanowires: (A) array; (B) copper oxide (CuO) nanotubes;
(C) array; (D) fabrication using anodic aluminum oxide template. Image copied from Mu C, He J, Confined conversion of CuS nano-
wires to CuO nanotubes by annealing-induced diffusion in nanochannels. vol. 6. 2011. p. 150. Available via license: CC BY 2.0 (https://
creativecommons.org/licenses/by/2.0/).

TABLE 1.1 Advantages and disadvantages of top-down and bottom-up methods [56e58].

Methods Advantages Disadvantages

Top-down Easy to construct order arrays of nanowires. The applicability of the photolithography method decreases as
This order facilitates electrical contact with the the desired length scale diminishes, which requires the use of
nanowires and their integration into large-scale more advanced methods, for example, extreme ultraviolet
devices lithography, electron-beam and scanning probe lithographies

Compatibility of production methods with
standard microelectronics industry processes.
Easy scale-up
Bottom-up Provides the opportunity for the control of the
composition of nanowires during growth, which
permits the production of complex superlattice
structures
Nanowires formed by top-down methods frequently lack
complex electronic characteristics. All the codifications after
growth greatly increase the material cost of nanowire
The major challenge of these methods is their integration into
large-scale devices

microorganisms [65,66]. The solution to this low throughput [67]. The ultimate discovery
problems is the use of electrical cell lysis, which was the use of nanowires because of their small
is less harmful than the aforementioned method; size (smaller than the cells) and the critical advan-
however, it still an expensive method and has a tage is that the nanowire tip can penetrate and
6 1. Bioactive hybrid nanowires: a new in trend for site-specific drug delivery and targeting

TABLE 1.2 Drugs incorporated in nanowires.

Active pharmaceutical Production

ingredient (API) Type of system Aim methods References

Paclitaxel Cu nanowires To target the spleen Bottom up [59]

Dexamethasone Polypyrrole For ulcerations, deep bone injuries, Bottom up [60]
nanowires or tumors; avoids the side effects of
systemic treatment with
steroids or chemotherapy
Curcumin Silver nanowires Cancer treatment Bottom up [61]

Doxorubicin Silver nanowires Cancer treatment Bottom up [62]

Cerebrolysin Hydrogen titanate Reduction of brain edema Top down [63]
nanowires

disrupt the function of cellular membranes [68]. used for biomedical applications. Major chal-
Nanowires eliminate microorganisms in cells lenges include
much faster than the previously described
1. advanced techniques and easy methods
methods.
(needed to increase the sensitivity of nanowire-
In analytical and biological processes, the
based electrochemical cytosensors in signal
development of biomolecule separation and
amplification),
analysis is essential. In the separation of long
2. further research into nanowires to promote
DNA molecules, conventional gel electropho-
cell adhesion, sensitivity, and selectivity,
resis has a disadvantage: it is necessary to
3. more specialized coatings to decrease non-
analyze biomolecules for several hours. The
specific bonding,
combination of nanostructures produced by
4. protocols and further experiments to deter-
top-down approaches and microfluidic systems
mine the exact nature of the nanotoxicity of
is usually proposed to overcome the problem.
nanowires and their constituents,
However, difficulty in their production using
5. innovative solutions to reduce fabrication and
an electron-beam lithography process makes it
running costs of nanowire-based micro/nano-
a very expensive and sophisticated system
fluidic devices to make them economically
[69,70]. On the other hand, nanostructures pro-
viable,
duced by bottom-up approaches offer easy fabri-
6. with every emerging technology, standards to
cation and separation of biomolecules; however,
avoid doubts about the lack of reproducibility,
size limitation causes difficulty in their develop-
repeatability, and compatibility across plat-
ment. To overcome all these problems, self-
forms and laboratories, and
assembled nanowire structures of metal oxides
7. an opportunity for further advances and de-
have been investigated due to their rigidity and
velopments of cytosensing devices based on
the possibility of reusability [49] (Table 1.3).
electrochemical methods [73].
Circulating tumor cells play an essential role
4.2 Nanowires as biosensors in medical in cancer metastasis, and knowledge of their
diagnosis presence in blood samples of cancer patients is
There are many challenges ahead that must be needed to understand more about the type of
addressed before nanowires can be successfully cancer. Hosokawa et al. have shown an array
 4. Applications of nanowires 7
TABLE 1.3 Nanowires in bioanalytical analyses.

Nanowire type Production method Results References

Mechanical ZnO nanowires Method of low- Higher extraction efficiency for nucleic acids [71]
cell lysis (diameter: 100 nm) temperature and proteins than using chemical cell lysis
on the surface of a hydrothermal methods
pillar array in a reaction
microchannel
ZnO nanowires were Method of low- Easy and rapid mechanical cell lysis [72]
synthesized on the Si temperature Higher extraction efficiency for proteins and
membrane (average hydrothermal reaction nucleic acids than that obtained for
pore diameter: 75 nm) commercially available kits

Biomolecule SnO2 nanowires Photolithography Nanowire structure controlled the pore [49]
separation produced into process and vapor/ size (20e400 nm) by varying the number of
and filtration fused silica liquid/solid technique nanowire growth times
microchannels Highly dense nanowires, used as a
molecular filter, could provide high-throughput
filtration of DNA molecules

of microcavities to perform size-selective capture
of circulating tumor cells [74]. Another study re-
ported that a herringbone chip captured and iso-
lated clusters of circulating tumor cells from the
patient’s blood, which had a capture efficiency of
more than 80% [75]. Tseng et al. developed sili-
con nanowires, which they called a NanoVelcro
chip, to capture and release circulating tumor
cells from blood samples with high selectivity
[76,77]. Si nanowires were produced based on
substrates by a standard photolithography and
chemical wet etching process, and they were
then bonded to a chaotic mixture of microfluidic
channels to fabricate the NanoVelcro chip. This
procedure of surface modification with cell sur-
face markers of anti-EpCAM increased the
capturing efficiency of circulating tumor cells
or of anti-CD45-depleted white blood cells on
the nanowires [78,79]. The NanoVelcro chip
with nanowires has been developed for single-
circulating tumor cell isolation by depositing
thermoresponsive polymer brushes, poly(N-iso-
propylacrylamide), on silicon nanowires [78].
NanoVelcro chips are promising tools in
diagnosis, because they capture and purify circu-
lating tumor cells rapidly prior to circulating tu-
mor cell molecular analysis [49,76].
A silicon nanowire-based electrical cell
impedance sensor has been developed for the
detection of cancerous cultured living lung cells
by monitoring their spreading state at which
the cells stretched and became extended on
nanowires [80]. The diagnosis was carried out
by penetration into the extended membrane of
malignant cells with respect to healthy cells.
Silicon nanowire biosensors have advantages
in molecular detection because of their high
sensitivity and fast response. A polycrystalline
silicon nanowire field-effect transistor device
was developed to achieve specific and ultrasen-
sitive detection of microRNAs without labeling
and amplification, showing that the diagnostic
and prognostic value of microRNAs in a variety
of diseases is promising. Thus the polysilicon
nanowire biosensor device is promising for
microRNA detection [81].
In short, semiconductor nanowires are
emerging as promising biosensors enabling
8 1. Bioactive hybrid nanowires: a new in trend for site-specific drug delivery and targeting
TABLE 1.4 List of biosensors in the literature based on nanowires.
Type of biosensor
Silicon nanowire field-effect transistors
NanoVelcro chip with nanowires
Silicon nanowire-based electrical cell impedance
sensor
Nanowire-based field-effect sensor devices (which
can be modified with specific surface receptors)
direct electrical detection of various biomole-
cules. A comparative analysis of bio-
functionalization strategies needs to be
discussed to design and develop optimum mem-
ristive biosensors to be implemented in label-free
sensing applications. The surface of the device is
modified with a specific antigeneantibody via:
(1) direct adsorption on the device surface, (2) a
bioaffinity approach using the appropriate
combination, and (3) the optimum memristive
biosensor, which is defined via the calibration
and comparative study of biosensors’ electrical
response under controlled environmental condi-
tions, such as humidity and temperature, aiming
to maximize the performance of the biosensor.
This modified system shows potential for gen-
eral application in molecular diagnostics, and,
in particular, for the early detection of cancer,
namely, prostate [82] (Table 1.4).
Some investigators of the University of San
Diego have been developing nanowires with
the purpose of recording the electrical activity
of neurons in fine detail. The ambition of the
group is that one day this new nanowire technol-
ogy could serve as a method to screen drugs
used specifically in neurological diseases, which
could help researchers to understand the mecha-
nism of how single cells can communicate in
complex neuronal networks. The main objective
is to allow the scientific community to delve
deeper into how the brain works. In the future,
the goal of researchers is to implant this new
nanowire technology into the brain [85].
Aim References
Detection of proteins, DNA sequences, small molecules, [83]
cancer biomarkers, and viruses
Developed for single-circulating tumor cell isolation [78]
Detection of cancerous cultured living lung cells [80]
Used as a powerful detection device for a broad range of [84]
biological and chemical species in solution
4.3 Nanowires for delivery of
chemotherapeutics
Sharma et al. developed noncytotoxic, mag-
netic, Arg-Gly-Asp (RGD)-functionalized nickel
nanowires (RGD nanowires) that could trigger
specific cellular responses via integrin trans-
membrane receptors, resulting in the dispersal
of nanowires [86]. Their results showed that
dispersal of 3 mm long nanowires increased
considerably with functionalization by RGD
when compared to PEG, through integrin-
specific binding, internalization, and prolifera-
tion in osteosarcoma cells. Additional results
showed that a 35.5% increase in cell density
was observed in the presence of RGD nanowires
when compared to an increase of only 15.6%
with PEG nanowires. These results are very
promising to advance applications of magnetic
nanoparticles in drug delivery, hyperthermia,
and cell separation where uniformity and high
efficiency in cell targeting are desirable.
Contreras et al. showed that magnetic nano-
wires with weak magnetic fields and low
frequencies could induce cell death via a mecha-
nism that does not involve heat production [87].
The low-power field exerted a force on the mag-
netic nanowires, causing a mechanical distur-
bance to the cells. In their results, cell viability
studies showed that the magnetic field and the
nanowires had separately decreased deleterious
effects on the cells. On the other hand, when
combined, the magnetic field and nanowires
 4. Applications of nanowires
caused cell viability values to drop by up to 39%,
depending on the strength of the magnetic field
and the concentration of the nanowires. Cell
membrane leakage experiments showed mem-
brane leakage of 20%, which proved that cell
death mechanisms induced by nanowires and
magnetic fields involve cell membrane rupture.
Thus these results suggested that magnetic nano-
wires can kill cancer cells. The advantages of this
process are the use of simple and low-cost equip-
ment with exposure to only very weak magnetic
fields for brief time periods.
Another alternative is ultrasound-powered
nanowire motors based on nanoporous gold seg-
ments that are developed for increasing drug
loading capacity. These nanowire porous motors
are characterized by a tunable pore size, high
surface area, and high capacity for the drug
payload. These highly porous nanomotors are
prepared by template membrane deposition of
a silver/gold alloy segment followed by dealloy-
ing the silver component. The chemotherapeutic
drug doxorubicin was loaded within the nano-
pores via electrostatic interactions with an
anionic polymeric coating. The nanoporous
gold structure facilitates near-infrared light-
controlled release of the drug through photo-
thermal effects, which is a great advantage.
Incorporation of the nanoporous gold segment
leads to a nearly 20-fold increase in the active
surface area compared to common gold nano-
wire motors [88].
The latter work offers very important infor-
mation for the treatment of cancer patients at a
patient-specific level based on specific drug re-
sponses of circulating tumor cells. So, platforms
for high capture efficiency of circulating tumor
cells are essential for clinical evaluation of
patient-specific drug responses of circulating tu-
mor cells. Recently, nanostructure-based plat-
forms have been developed. In the Kim et al.
study, the breast carcinoma cell-line with an ul-
tralow abundance range was captured by
streptavidin-functionalized silicon nanowire
platforms for evaluation of capture efficiency
9
[89]. In this case, a capture efficiency of more
than 90% was achieved. Specific drug responses
of breast carcinoma cell-line cells captured on
these platforms were analyzed using tamoxifen
or docetaxel as a function of incubation time
and dose. In addition, circulating tumor cells
were successfully captured, and this study sug-
gests that this platform is adaptable for clinical
use in the evaluation of circulating tumor cells
and drug response tests.
Magnetic silica core/shell nanovehicles pre-
senting atherosclerotic plaque-specific peptide-1
as a targeting ligand have been prepared
through a double-emulsion method and surface
modification with magnetic iron oxide (Fe3O4)
nanoparticles. The results demonstrated that un-
der a high-frequency magnetic field, magnetic
carriers incorporating the anticancer drug doxo-
rubicin collapsed, releasing approximately 80%
of the drug payload, due to the heat generated
by the rapidly rotating Fe3O4 nanoparticles,
thereby realizing rapid and accurate controlled
drug release. At the same time, the magnetic
Fe3O4 could also kill the tumor cells through a
hyperthermia effect, i.e., inductive heating. The
combination of remote control, targeted dosing,
drug-loading flexibility, and thermotherapy
and chemotherapy suggests that these magnetic
nanovehicles have great potential for application
in cancer therapy [90].
Another study showed that an electrorespon-
sive drug release system based on polypyrrole
nanowires was developed to induce the local de-
livery of the anticancer drug doxorubicin, ac-
cording to the applied electric field. These
nanowires were initially prepared by electro-
chemical deposition of a mixture of pyrrole
monomers and biotin as dopants in the anodic
alumina oxide membrane as a sacrificial tem-
plate. Additionally, the antitumor efficacy of
doxorubicin released from these nanowires in
response to the external electric field using two
kinds of cancer cell lines, human oral squamous
carcinoma cells and human breast cancer cells,
was investigated. An advantage of these
10 1. Bioactive hybrid nanowires: a new in trend for site-specific drug delivery and targeting
particles is the strong photothermal effect as a
result of the near-infrared absorbing ability of
polypyrrole synergistically that, as a conse-
quence, maximizes chemotherapeutic efficacy,
which is very promising for many therapeutic
applications, including cancer [91].
To detect specific mRNA sequences, essential
in the treatment of cancer, molecular beacons
have been widely employed as sensing probes.
Kim et al. developed a nanowire-incorporated
and pneumatic pressure-driven microdevice for
rapid, high-throughput, and direct molecular
beacon delivery to human breast cancer MCF-7
cells to monitor survivin mRNA expression
[92]. This microdevice is composed of three
layers: (1) a pump-associated glass manifold
layer, (2) a monolithic polydimethylsiloxane
membrane, and (3) a ZnO nanowire-patterned
microchannel layer. The molecular beacons are
immobilized using the ZnO nanowires by disul-
fide bonding, and the glass manifold and mono-
lithic polydimethylsiloxane membrane serve as a
microvalve. The cellular attachment and detach-
ment on the molecular beacon-coated nanowire
array can be easily manipulated. All these pro-
cedures enable the transfer of molecular beacons
into the cells in a controllable way with high cell
viability and are useful to detect survivin mRNA
expression quantitatively after docetaxel treat-
ment [92].
Combination therapy is a promising cancer
treatment strategy that is usually based on the
utilization of complex nanostructures with mul-
tiple components. Ultrathin tungsten oxide
nanowires (W18O49) were synthesized using a
solvothermal approach and were examined as
a multifunctional theragnostic nanoplatform
[93]. In vitro and in vivo analyses demonstrated
that these nanowires could induce extensive
heat- and singlet oxygen-mediated damage to
cancer cells under 980 nm near-infrared laser
excitation. The comparison of near infrared-
induced photothermal therapy/photodynamic
therapy and radiation therapy alone showed
that W18O49-based synergistic trimodal therapy
eradicated xenograft tumors, and no recurrence
was observed. In conclusion, these nanowires
have shown significant potential for cancer ther-
apy with inherent image guidance and synergis-
tic effects from phototherapy and radiation
therapy, which warrants further investigation
[94].
5. Conclusions
This chapter summarizes the critical results ob-
tained using nanowire structures as a platform
useful in bioanalytical chemistry and medical di-
agnostics. Nowadays, there are various technical
approaches to develop nanowires for bio-
applications in molecular to cellular levels. Nano-
wires have been integrated with microchannels,
providing a novel pathway from the macroscale
to the nanoscale that will allow researchers to
observe and analyze target molecules such as
DNA, RNA, proteins, and circulating tumor cells.
Another benefit of nanowires is their very small
diameter size with high aspect ratio; this can
allow researchers to use nanowires as a probe
tip to stimulate and record changes in electrical
signals in living cells. Nanowires were also used
as biological optical sensors. These improvements
in nanowire structures will allow the develop-
ment of new bioanalytical chemistry and medical
diagnostics tools that will open a new age of
nanotechnology with the widespread use of
nanowires for bioapplications.
Acknowledgments
The authors acknowledge the financial support received from
the Portuguese Science and Technology Foundation (FCT/
MCT) and from European Funds (PRODER/COMPETE) un-
der the project reference M-ERA-NET/0004/2015-PAIRED,
cofinanced by FEDER, under the Partnership Agreement
PT2020.
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 C H A P T E R
2
Opportunities and challenges of
3D-printed pharmaceutical dosage forms
Adam Procopio1, Divya Tewari2
1
Merck & Co., Inc., Kenilworth, NJ, United States; 2Noramco Inc., Wilmington, DE, United States
1. Introduction
Drug product development can be a long and
complex process. On average, it is estimated
that it takes about 10 years and costs
US$2.5e5 billion for a new drug product to
get to the market [1,1a]. Given this significant
investment, and the knowledge that any delay
in getting the drug product to the market re-
duces exclusivity, there is a desire to reduce
this development timeline providing an overall
benefit to patients and the industry. 3D printing
(3DP) can allow for a robust, flexible, and cost-
effective approach to drug development in
which drug release profiles may be tailored to
a particular outcome using a single
manufacturing method. Moreover, 3DP allows
for custom designs and dosing amounts such
that the dosage forms may be tailored to a spe-
cific patient population. Due to longer and com-
plex formulation processes, development of
delayed or extended release formulations is
typically even more prolonged as well as
requiring expensive and propriety drug release
technology. To date there is only one approved
Drug Delivery Trends
https://doi.org/10.1016/B978-0-12-817870-6.00002-X 15
product (Spritam) that uses a 3DP technology
based on powder layering launched by Aprecia
Pharmaceuticals. There are existing examples of
implementing 3DP technology to rapidly proto-
type release rates using different strategies,
largely focused on maintaining a similar mate-
rial feedstock and using creative printing pa-
rameters to generate various releases. With
these examples, at least one solid filament mate-
rial is preprocessed to contain active pharma-
ceutical ingredients (APIs). Modifications to
what is called the “infill” parameters of the
printed tablet can manipulate release rates
[1be3]. During a fused deposition modeling
(FDM) printing process, the print head will
print an outer shell in the shape of the part,
and the inside of the shell is largely hollow.
The material that is printed on the inside of
the shell is called the infill and can be controlled
through software by taking into account
what percentage of the shell is hollow and the
geometric design of the infill (honeycomb,
rectilinear, etc.). Other published work involves
the changing of the active dosage form’s overall
shape, size, and surface area, which has
© 2020 Elsevier Inc. All rights reserved.
16 2. Opportunities and challenges of 3D-printed pharmaceutical dosage forms
shown to modify the release rate [4e7].
Manufacturing of drug product dosage forms
that combine a shell-based approach to be
described in detail in a later section have
demonstrated a unique ability to generate
distinct release rates. Such core/shell tablets
have been manufactured by using a second
API-containing material [8] or a placebo mate-
rial with the intent to mimic enteric-coated tab-
lets [9,10] and have demonstrated the agility of
3DP to change the onset of the release of the core
of the dosage form.
While these approaches have demonstrated
an ability to use software for tuning drug
release rate while maintaining a constant mate-
rial feedstock, they are reliant on a successful
hot melt extrusion (HME) formulation of a
printable filament for each API. Developing
process conditions to incorporate API into an
excipient-based solid filament is not trivial
[11e14], and these filament processing develop-
ments add to the product development burden,
reducing the rapid prototyping advantage 3DP
brings to the table for early drug screenings. A
major hurdle the pharmaceutical 3DP field has
yet to overcome is providing a wide, distinct
range of dosage forms using a universal set of
starting print-ready materials to accommodate
any API without filament formulation burden,
and has even a greater hurdle on aligning
manufacturing partners to generate good
manufacturing-processed pharmaceutical ma-
terials that are printer ready.
2. Materials
Pharmaceutical dosage form design begins
with material selection. Because the materials
are altered during the 3DP process, it is
imperative to understand the source, purity
and associated material chemistry changes of
the chosen material. Material properties have
wide-ranging impact, from influencing the
preferred route of manufacturing to the phys-
ical properties of the dosage form to its pharma-
codynamic fate in the body. A wide range of
materials are used as substrates in 3DP; howev-
er, because of their origin in industrial prototyp-
ing, most 3DP techniques lack availability of
suitable developed materials [15].
The successful design and printability of the
3D-printed dosage forms is dictated by the phys-
ical, chemical, thermal, and mechanical proper-
ties of the chosen material. Additional
considerations should be given to ease of avail-
ability and the regulatory status of the materials.
In the absence of the standard test methods a
specifically designed method to characterize
the material properties of the additives can be
used, and we have compiled current test proced-
ures employed by various researchers and high-
lighted some of the standard utilized ASTM
methods.
The range of polymers used in 3DP include
thermoplastics, thermosets, elastomers, hydro-
gels, functional polymers, polymer blends, com-
posites, and biomaterials [16]. Polymeric
materialsdpolymersdconstitute the majority
of materials used in 3DP due to several
advantages such as low cost, biocompatibility,
availability, ease of processing, and physico-
chemical properties. Material selection is
dictated by the choice of the 3DP technology,
e.g., polymeric filaments used by FDM must
have a constant diameter of 1.75 mm, an ideal
melt viscosity to facilitate viscous melt forma-
tion preextrusion and solidification postextru-
sion, and a sufficient elastic modulus-to-melt
viscosity ratio to prevent filament buckling and
shear thinning tendencies in liquid form [17].
Commonly used polymers include aliphatic
polyesters (poly(lactide) [PLA], poly(glycolide),
poly(caprolactone) [PCL]), cellulosic derivatives
 2. Materials
(hydroxypropylcellulose [HPC], hypromellose
[HPMC], HPMC acetate succinate [HPMCAS],
cellulose acetate, and cellulose acetate phthalate),
vinyl polymers (polyvinylpyrrolidone [PVP] and
copovidone), polyethylene oxide, polyethylene
glycol (PEG), and acrylic polymers (Eudragit).
Table 2.1 provides an overview of 3DP tech-
nologies and desired material properties
required for successful development of 3D-
printed dosage forms.
17
2.1 Aliphatic polyesters
Aliphatic polyesters are synthetic homopoly-
mers or copolymers of lactic acid, glycolic acid,
lactide, glycolide, and 6-hydroxycaproic acid.
Typically, the molecular weights of homopoly-
mers and copolymers range from 2000 to
>100,000 Da. The representative chemical struc-
tures are provided in Fig. 2.1 and a brief sum-
mary of their physical, chemical, and
mechanical properties is outlined in Tables 2.1
and 2.2.

TABLE 2.1 Summary of material properties and test methods commonly employed.

Material properties Key properties Testing methods commonly employed

Powder physical properties
Mechanical properties
Thermal properties
Optical properties
Rheological properties
Particle shape, particle size distribution,
bulk and tap densities, crystallinity,
moisture content
Yield strength, elasticity, modulus,
elongation at break
Melting point, glass transition temperature,
degradation temperature
Ultraviolet absorption, laser power
Viscosity of the solution, binderepowder
interaction, melt viscosity, melt index,
surface tension
Laser light diffraction, densitometry,
powder X-ray diffraction, differential
scanning
calorimetry, Karl Fischer, flow index
ASTM D638, D3039, D882, ISO 527-2,
three-point bend test
Thermogravimetric analysis
AERS-G2 rheometers, viscometers (USP
911), ASTM D1238

FIGURE 2.1 Representative chemical structures of the aliphatic polyesters.

18 2. Opportunities and challenges of 3D-printed pharmaceutical dosage forms

TABLE 2.2 Typical chemical names and trade names of the representative aliphatic polyesters.

Composition

Generic name Lactide Glycolide Caprolactone Trade name Manufacturer

Poly(L-lactide) 100 0 0 Lactel L-PLA Durect

100L Lakeshore
Resomer L206 S, 207S, Boehringer
209 S, 210 and 201 S Ingelheim
Poly(DL-lactide) 100 0 0 Lactel DL-PLA Durect
Purasorb PDL 02A, 02, 04, 05 Purac
Resomer R 202 S, 202 H, 203 S, Boehringer
203 H Ingelheim

Poly(L-lactide-co-glycolide) 85 15 0 Resomer LG 855 S, 857 S Lakeshore

Boehringer
Ingelheim
Poly-ε-caprolactone 0 0 100 Lactel PCL Durect
100 PCL Lakeshore
Poly-(DL-lactide-co- 85 0 15 8515 L/PCL Lakeshore
ε-caprolactone)

Adapted from Handbook of Pharmaceutical Excipients.

Aliphatic polyesters are United States Food
and Drug Administration (FDA) and European
Medicine Agency approved, versatile thermo-
plastic polymers that are used in a number of
3DP technologies such as FDM, selective laser
sintering (SLS), pressure-assisted microsyringes
(PAMs), etc. due to their biocompatibility,
biodegradability, high mechanical strength
and modulus, and processability [18]. Ease of
availability and cost effectiveness make
aliphatic polyesters highly desirable polymers
for 3DP, whereas the main disadvantages are
the appearance of rough surfaces and low
resolution.
PLA is by far the most widely used material
for FDM printing. PLA and its derivatives are
poorly water soluble but have good solubility
in dioxane, acetonitrile, chloroform, methylene
chloride, 1,1,2-trichloroethane, and dichloroace-
tic acid. The thermal and mechanical properties
of PLA are influenced by small amounts of
enantiomeric impurities. Amorphous grades
were reported to have better processability
and a wider processability window but lower
mechanical properties [19] (Table 2.2).
PCL is a hydrophobic polymer with excellent
blend compatibility with many other polymers
such as polyvinyl(acetate), poly(vinylchloride),
poly(styrene-acrylonitrile), and poly(acryloni-
trile butadiene styrene). Its blend compatibility,
biodegradability, low melting point, and solubi-
lity make this polymer suitable for precise
extrusion deposition and FDM techniques. The
only disadvantage of PCL is its hydrophobicity,
which might adversely impact drug dissolution
characteristics.
2.2 Cellulose ethers and esters
Cellulose is the most abundant naturally
occurring polysaccharide. Each polysaccharide
unit is linked by b-1,4-glycosidic bonds. Each
glucose unit has three hydroxyl groups that
can be derivatized and the average substitution
grade cannot exceed three. Alkalization of
 2. Materials
cellulose, followed by etherification reaction at
elevated temperatures and pressures, is used to
convert cellulose molecules into their corre-
sponding ether, such as HPC, HPMC, and
many other semisynthetic cellulosics. Esterifica-
tion of the cellulose ethers could be used to
derive molecules such as HPMCAS. At the basic
level, cellulose derivatives are characterized by
their average molecular weight distribution
and average composition. Compositionally,
these polymers are defined by the percent
weight of the functional group attached to the
backbone, the degree of substitution per anhy-
droglucose, or the total molar substitution per
anhydroglucose residue [20]. The representative
chemical structures are provided in Fig. 2.2 and a
summary of their physical, chemical, and
mechanical properties is provided in Table 2.3.
FIGURE 2.2 Representative chemical structures of cellulose ethers and esters.
19
Thermoplastic polymers are typically mate-
rials of choice in 3DP coupled with extrusion
because they can be processed at suitable tem-
peratures without affecting the stability of the
APIs [21]. Cellulose esters and ethers have been
tested as carriers or matrices for drugs in FDM
technology with HME [22]. HPMC in either solu-
tion, dispersion, or paste forms has also been
used in PAMs printing technology [23].
2.3 Acrylic polymers
Polymethacrylates are synthetic cationic and
anionic polymers of dimethylaminoethyl meth-
acrylates, methacrylic acid, and methacrylic
acid esters in varying ratios [24]. Eudragit poly-
mers are copolymers derived from esters of
acrylic and methacrylic acid whose
20 2. Opportunities and challenges of 3D-printed pharmaceutical dosage forms

TABLE 2.3 Typical physical and mechanical properties of the aliphatic polyesters.

8515
Properties L-PLAa DL-PLAa PGAa PCLa 85/15 DL-PLGa L/PCLb

Molecular weight (Da) 40,000e100,000 40,000e100,000 >100,000 80,000e150,000 40,000e100,000

Melting point (
C) 173e178 Amorphous 225e230 58e63 Amorphous Amorphous
Glass transition 60e65 50e60 35e40 65 to 60 50e55 20e25b
temperature (
C)
Color White White Light tan White White to light gold
Tensile strength (psi) 8,000e12,000 4,000e6,000 10,000þ 3,000e5,000 6,000e8,000 3,254
Elongation (%) 5e10 3e10 15e20 300e500 3e10 >6.4

Modulus (psi) 4e6  10 5

2e4  10 5
1  10 6
3e5  10 4
2e4  10 5
8.4  104

PCL, Polycaprolactone; PGA, poly(glycolide); PLA, poly(lactide); PLG, poly(lactide-co-glycerol).

a
Specifications from Durect, b Specifications from Lakeshore Biomaterials and process temperature range 140e160
C.
Adapted from Handbook of Pharmaceutical Excipients.

physicochemical properties are determined by members of this product family. PVA is a

functional groups. Several compositional copol-
ymer variants are derived from esters of acrylic
and methacrylic acid, whose physical, chemical,
mechanical, and thermal properties are deter-
mined by the functional groups. The representa-
tive chemical structures and summary of typical
trade names and suppliers is provided in Fig. 2.3
and Tables 2.4e2.6, respectively.
Acrylic polymers have been used for FDM
[11,12], binder jetting additive manufacturing,
and SLS printing technologies [25e28].
2.4 Vinyl polymers
Polyvinyl alcohol (PVA) polymers and PVP
polymers and copolymers are important
FIGURE 2.3 Representative chemical structures of acrylic
polymers.
water-soluble synthetic polymer represented
by the formula (C2H4O)n. It is a synthetic, linear,
semicrystalline polymer produced via the
hydrolysis of polyvinyl acetate in methanol,
ethanol, or a mixture of alcohol and methyl
acetate, using alkalis or mineral acids as cata-
lysts. Unlike other vinyl polymers, it is not pro-
duced via the polymerization of repeating units
of vinyl alcohol because it cannot be obtained in
the quantities and purities required for poly-
merization purposes. It is manufactured by
hydrolysis of polyvinyl acetate and the removal
of acetate groups. It has low solubility in
ethanol and is insoluble in many organic sol-
vents. Its physical properties are dictated by
the degree of polymerization and the degree
of hydrolysis. The pharmaceutical grades are
partially hydrolyzed and available in different
viscosity types.
PVPs or povidone are water-soluble linear
synthetic polymers, manufactured by free
radical polymerization of N-vinylpyrrolidone.
Vinylpyrrolidone-vinylacetate copolymer or
copovidone (PVP/VA) are water-soluble copoly-
mers of the two components in the ratio of 6:4. It
is also produced by free radical polymerization
 2. Materials 21
TABLE 2.4 Typical chemical names and trade names of the representative cellulosic polymers.

Generic name Assay Trade name (grades) Manufacturer

Hydroxypropylcellulose % Hydroxypropoxy Klucel HPC (Klucel EL, LF, GF) Ashland

53.4e80.5a Nisso HPC (Nisso-L) Nippon Soda
Nisso (Seppic)
Hydroxypropylmethylcellulose Type 2910 Methocel HPMC (Methocel E3, DuPont Specialty Solutions
% Hydroxypropoxy E6, E10, K100LV, K4M) (previously Dow Wolff
7.0e12.0 Klucel HPMC (Benecel K100LV Cellulosics)
% Methoxyl PH PRM, Benecel K4M) Ashland
28.0e30.0 ShinEtsu
Type 2208 Lotte Fine Chemical
% Hydroxypropoxy
4.0e12.0
% Methoxyl- 19.0e24.0
Hydroxypropylmethylcellulose % Hydroxypropoxy Aqoat HPMCAS ShinEtsu
acetate succinate 4.0e23.0 (LG, MG, HG) DuPont Specialty
% Methoxyl Affinisol HPMCAS Solutions
12.0e28.0 (LG, MG, HG) Ashland
% Acetate-2.0 16.0 Aquasolve HPMCAS
% Succinate 4.0e28.0 (LG, MG, HG)

Ethylcellulose % Ethoxyl 44.0e51.0 Aqualon ethylcellulose DuPont Specialty

(N types) Solutions (previously Dow
Ethocel ethylcellulose Wolff Cellulosics)
(N types) Ashland
a
Specifications from USP 41-NF 36.

reaction in an organic solvent such as ethanol or
2-propanol [29].
The representative chemical structures, com-
mercial supplier information, and polymer prop-
erties are given in Fig. 2.4 and Tables 2.7 and 2.8,
respectively.
PVA is a thermoplastic, water-soluble excip-
ient that is commonly employed as polymeric
support material for FDM-based 3DP
[5,6,8,30,43,44]. The degree of hydrolysis impacts
the physicochemical, thermal, and mechanical
properties of the resultant PVA grade. Besides
FDM printing, PVA has also been used in inkjet
printing [31].
PVP and PVP/VA polymers are known for
their application in the solubility enhancement
of poorly water-soluble drugs via HME. Due to
the complementarity of HME technology with
FDM, polymers used in HME are frequently
adapted for use in 3DP. Additives, such as plas-
ticizers and fillers, are usually employed to
reduce the Tg of PVP polymers and render
them suitable for FDM printing coupled with
HME. Major et al. examined the material proper-
ties of PVP/VA copovidone copolymers in hot
melt extrusion-based 3DP and encountered diffi-
culties in printing due to brittleness and high
stiffness of the copolymer. Melt blending with
a carrier polymer such as PCL improved flexi-
bility and ductility thereby resolving the print-
ability issue. Polyethylene oxide was also
added to the formulation to reduce the negative
impact of PCL on drug release profiles [32]. Melt
blending PVP/VA with hydrophilic polymers
such as HPMC and HPMCAS resulted in imme-
diate release formulations [33].
22 2. Opportunities and challenges of 3D-printed pharmaceutical dosage forms

TABLE 2.5 Typical physical, mechanical, and thermal properties of the cellulose polymers.

Aqualon
Properties Klucel HPCa Benecel HPMC AquaSolve HPMCAS ethylcellulose

Molecular weight (Da) 40,000e1,150,000 20,000e1,200,000 55,000e93,000d 75,000e215,000f

Melting point (
C) Softens at 130 190e200 156f
Glass transition 0 and 120b 170e180 120e125 129e133e
temperature (
C)
Color White to slightly White to off-white White to off-white White to light
yellow colored powder powder or granuled tan-colored
powdere
Tensile strength (psi) 1450 (Klucel HPC EF) 6816 (Benecel HPMC E6) 5076 (HPMCAS L) 6899
(ASTM D882) 5366 (HPMCAS M)
5802 (HPMCAS H)g
Elongation (%) 12 (Klucel HPC EF) 4 (Benecel HPMC E6) 11 (HPMCAS L) 9
(ASTM D882) 19 (HPMCAS M)
16 (HPMCAS H)g
Modulus (psi) 200,000e630,000c 367,090 (Benecel HPMC E6) 1574 (L) 302,403
(ASTM D882) (grade dependent) 1523 (M)
1494 (H)g,h
a
Adapted from Klucel HPC Physical and Chemical Properties Book (https://www.ashland.com/file_source/Ashland/Product/Documents/Pharmaceutical/
PC_11229_Klucel_HPC.pdf), b Klucel HPC is a special polymer that can show dual Tg because it has a beta transition, c Reference [21], d Handbook of
Pharmaceutical Excipients, Sixth Edition, 330e332, e Handbook of Pharmaceutical Excipients, Sixth Edition, 262e267, f Aqualon Ethylcellulose EC
Physical and Chemical Properties, Product Brochure-PRO 250-42a, g PC-12624 AquaSolve HPMCAS Handbook, h Handbook of Pharmaceutical
Excipients, Sixth Edition, 326e329.

2.4.1 Novel polymers in the market
Melfil is a water-soluble filament of butane-
diol vinyl alcohol copolymer specifically
designed for FDM 3DP. It offers superior water
solubility and printability along with the flexi-
bility to use as a support material or a water-
soluble model (see the Nippon GohseidMelfil
Product Brochure [53]).
Thermoplastic polyurethanes (TPUs) are
elastic and melt processable linear-segmented
block copolymers. TPUs offer a unique advan-
tage over other thermoplastic polymers because
of their extreme material adaptability. This is
due to the flexibility in modifying the molecular
weight, ratio, and chemical composition of soft
(polyether or polyester based) and hard seg-
ments (aliphatic or aromatic based) of the
TPUs [34].
Polyether ether ketone (PEEK) and polyether
imide (PEI, brand name ULTEM) are newer ther-
moplastic semicrystalline materials from the pol-
yaryletherketone (PAEK) family of polymers
currently used in FDM printers [35]. PAEK poly-
mers can withstand high temperatures while
maintaining mechanical strength [36]. PEEK is
a superhigh-performance, biocompatible, chemi-
cally stable, semicrystalline plastic that offers the
advantages of high-temperature resistance
(melting point of 334
C, Tg of 143
C) and excel-
lent mechanical properties, including high
 2. Materials 23
TABLE 2.6 Typical chemical names and trade names of the representative acrylic polymers.

Polymer dry
Generic name weight content (%) Trade name (supply form) Manufacturer

Poly(butyl methacrylate, 98% Eudragit E 100 (granules) Evonik Industries

(2-dimethylaminoethyl) 12.5% Eudragit E 12.5 (organic solution)
methacrylate, methyl methacrylate) 1:2:1 98% Eudragit E PO (powder)
Poly(ethyl acrylate, methyl 97% Eudragit RL 100 (granules) Evonik Industries
methacrylate, trimethylammonioethyl 97% Eudragit RL PO (powder)
methacrylate chloride) 1:2:0.2 30% Eudragit RL 30 D
(aqueous dispersion)
Poly(methacrylic acid, ethyl acrylate) 1:1 95% Acryl-EZE (powder) Colorcon
30% Eudragit L 30 D-55 Evonik Industries
95% (aqueous dispersion) Eastman Chemical
30% Eudragit L 100-55 (powder) BASF Fine
30% Eastacryl 30 D (aqueous dispersion) Chemicals
95% Kollicoat MAE 30 DP
(aqueous dispersion)
Kollicoat MAE 100 P (powder)

Adapted from Handbook of Pharmaceutical Excipients.

FIGURE 2.4 Representative chemical structures of vinyl polymers.

TABLE 2.7 Typical chemical names and trade names of the representative vinyl polymers.

Polymer dry weight

Generic name content (%) Trade name (grades) Manufacturer

Polyvinyl alcohol Degree of hydrolysis Goshenol EG Granules/Powder (EG-03P, Nippon Synthetic

(PVOH/PVA) 86.5e89.0 EG-05P, EG-18P, EG-22P, EG-30P, EG-40P) Chemical Company
Polyvinylpyrrolidones K value- 25-90 Kollidon povidone BASF
(PVP, Povidone) Plasdone povidone Ashland
Polyvinylpyrrolidone: K value - 25.4-34.2a,b Kollidon VA 64 copovidone BASF
vinylacetate 6:4 Plasdone S630 copovidone Ashland
(PVP/VA, Copovidone)
a
http://www.nichigo.co.jp/english/lifechemical/pharma/index.html, b From the Ashland brochure. Adapted from Handbook of Pharmaceutical Excipients.
24 2. Opportunities and challenges of 3D-printed pharmaceutical dosage forms

TABLE 2.8 Typical physical, mechanical, and thermal properties of the vinyl polymers.

Properties PVA Povidone Copovidonea

Molecular weight (Da) 20,000e200,000b 28,000e1,150,000 45,000e75,000

Melting point (
C) 180e190 for partially Softens at 150
Cc 140
hydrolyzed grades
228 for fully hydrolyzed
grades

Glass transition 85 120e175d 106

temperature (
C)

Color White to cream-colored White to creamy white powderc White to off-white

granular powder free-flowing powder
Tensile strength (psi) 44,961e Films brittle; difficult
e to assess pure film properties
Elongation (%) 2
Modulus (psi) 20,305e

PVA, Polyvinyl alcohol. a Specifications from the Ashland brochure, b Handbook of Pharmaceutical Excipients, Sixth Edition, 564e565, c Handbook of
Pharmaceutical Excipients, Sixth Edition, 581e585, d Ashland Literature PTR-092 Plasticizer compatibility and thermal and theological properties of
Plasdone povidone and copovidone polymers for hot-melt extrusion applications, e Hamied, S.F.A; Abd El-Kader, K.A.M. Preparation of poly (vinyl alcohol)
films with promising physical properties in comparison with commercial polyethylene film.

strength, elastic modulus, and fracture tough- 3. Technology details

ness [37]. PEI was developed by General Elec-
tric’s plastics division in the 1980s (later
acquired by SABIC) and demonstrates superior
thermal properties and mechanical strength
characteristics of the family.
2.4.2 Additives
Plasticizers, fillers, and lubricants are com-
mon additives used to improve printability by
either modifying the melt and mechanical prop-
erties [33] or reducing the friction between the
filament and walls of the printing extruder [10].
Commonly used additives include fillers such
as talc, lactose, microcrystalline cellulose, mag-
nesium stearate, and tricalcium phosphate, or
plasticizers, such as triethyl citrate, triacetin,
PEG 400, Tween 80, etc.
3DP has been gathering significant attention
from both industry and academicians. Research
efforts have spanned the applications of both
novel drug delivery to replacement/supplement
of traditional manufacturing approaches. To
accommodate these various manufacturing
modes, different 3DP approaches are employed.
This section reviews the available technologies
along with their advantages and disadvantages
from a technical point of view. In general, the
technology is built on the principle that matter
is converted from either liquid to solid or un-
dergoes a transition from solid to liquid back to
solid in a layer-by-layer approach either through
chemical means or thermal energy. The contrib-
uting limitations to either print resolution or
print speed are a result of the fundamental
 3. Technology details
mode of the physics used in the print process.
ASTM ascribes seven different 3DP technologies
(ASTM F2792). We discuss here the top three
technologies that are most relevant for the phar-
maceutical industry:
1. Vat photopolymerization
2. Powder-based processes
3. Material extrusion
The aim here is not to describe all of these
technologies in detail but to cover the 3DP
modes that have contemporaneous or direct im-
mediate impact on the biopharmaceutical indus-
try in their application of drug product
prototyping and at-scale manufacture.
3.1 Vat photopolymerization
Vat photopolymerization carries alternative
names with concurrent differing underlying
technologies such as stereolithography appa-
ratus (SLA), digital light processing (DLP), and
continuous liquid interface production (CLIP).
The fundamental principle of operation is that
a liquid photopolymer resin formulation
comprising a monomer, oligomer, and photoini-
tiator is cured through selective exposure to light
using a specified light source (most typically a
450 nm laser) either in a raster mode or as a pro-
jected 2D image (e.g., DLP). The light source is
Vat
SLA Photo
resin
FIGURE 2.5 Principal components of a stereolithography apparatus (SLA) printer.
25
controlled both in terms of energy supplied
and in the amount of time that drives the photo-
polymerization reaction to cross-link the liquid
formulation and convert it to solid polymer pre-
cisely at the regions where the light source is
focused. Vat photopolymerization has the high-
est lateral and vertical print resolution in the
range of 1e10 mm. Lateral resolution is defined
by the positional control of the light source,
whereas vertical resolution is controlled by the
penetration depth of the light source and any
light-absorbing additives that are added to
photochemical resin to control any unwanted
light-scattering events.
3.1.1 Stereolithography apparatus
Fig. 2.5 depicts the principal components of a
typical SLA printer. The platform is precisely
controlled in concert with the position of the
focused laser source and any mirrors used to
direct the light source to sequentially scan or
project the laser light source within a plane on
the surface of the photosensitive resin formula-
tion. The time spent on any individual 2D layer
depends on the chemistry of the resin formula-
tion to successfully complete the cross-linking re-
action and convert the liquid formulation to
solid polymer resin. The lateral (xey) position
of the laser is typically controlled with a pair of
mirrors within servo-controlled galvanometers,
Laser
3D Printed Y plaorm
Object
26 2. Opportunities and challenges of 3D-printed pharmaceutical dosage forms
which are electromechanical instruments used to
precisely control the position of the mirror and
hence the position of the laser spot location.
This process is conducted layer by layer whereby
the slicing software converts the 3D image to be
printed into a series of control statements that
ascribe not only the position of the platform po-
sition, but also the laser energy pulse and well as
the tilt angle of the mirrors used to position the
xey position of the spot. A unique feature of
photopolymerization 3D printers is the ability
to resolve fine details by the application of galva-
nometer dithering (or high-frequency move-
ments) to effectively process grayscale images
that prescribe laser energy states between full
on or full off. This technique allows for the crea-
tion of highly resolved surfaces. In general, pho-
topolymerization techniques allow for highly
resolved features and surfaces on the order of
1 mm. Their main disadvantages are extremely
limited for use as a biopharmaceutically accept-
able process in that they are using both toxic
monomer and oligomer materials and usually
have lengthy postprocessing steps to remove
any unreacted monomer and oligomer as well
as completely consume any unreacted free radi-
cals as a result of photochemistry. The materials
available for creating 3DP drug products from
photochemical reactions are limited but research
in this area is evolving.
3.1.2 Digital light processing
DLP is analogous to SLA because both pro-
cesses use a controlled wavelength light source
to selectively drive a photochemical reaction of
a resin formulation. The main difference be-
tween SLA and DLP is that the light source in
SLA acts as an XeY rastering, whereas in DLP
the entire layer to be cured is projected onto
the focal plane at one time. The technology
used with DLP is the same technology used in
overhead projectors, which allows for dithering
as described in the SLA section earlier and for
grayscale image processing and hence higher
resolution features and smoother printed
surfaces. Unlike SLA where the photochemical
reaction is near the liquid/air interface and sub-
ject to oxygen inhibition less direct control of the
photochemical cross-linking reaction, DLP 3D
printers are controlled in the reverse direction
where the reaction layer occurs at a plane
immersed well below the liquid/air interface.
An example of the application of DLP in 3DP is
the work by Kim et al. with precision bioprinting
of silk fibroin bioink for applications in building
complex organ structures [38].
3.1.3 Continuous liquid interface production
The latest variety of vat photopolymerization
is CLIP [39,40]. This technology addresses the
major time-limiting step of both SLA and DLP,
which is the required mechanical separation of
the just-cured material from the vat of unpoly-
merized material. This 3DP technique uses an
oxygen permeable membrane to inhibit poly-
merization at the interface nearest to the ultravi-
olet (UV) light source. This region creates a
10e100 mm “dead zone” where free radical
polymerization does not occur. Just above this
zone, light-catalyzed free radical polymeriza-
tion occurs on the focal plane of the projected
light. This innovation is key to facilitate a faster
3DP process because the need to refresh or
recoat the region between the printed part and
the light source using a mechanically activated
platform is not needed. Using CLIP, this region
is continuously present with uncured formula-
tion and the 3D-printed part appears to
“grow” out of the resin. Resolution of the part
in the vertical direction is improved by
increasing the concentration of the passive light
absorber. This slows down the production
speed because light penetration is in a smaller
volume of the resin. By lowering the concentra-
tion of this additive, deeper penetration of light
can be realized and hence faster production
speeds. Part quality is also improved by
removing the need to mechanically separate
the part from the resin bath. This mechanical
separation that is typical in most SLA 3D
 3. Technology details
printers causes undue stress on the part and can
lead to feature distortion or even failure. CLIP
allows for both high print quality and speeds
and can produce parts with features below
100 mm at growth rates in the range of a vertical
support plate speed of 1000e3000 mm/h.
3.2 Powder bed fusion processes
3.2.1 Selective laser sintering process/laser
sintering process
Generally, laser sintering 3DP allows for
many more materials over other 3DP techniques
from high-performance thermoplastic polymers
to even metal powders.
The operating principle behind powder-based
SLS consists of powder deposition from the feed
chamber to the build chamber by powder trans-
fer and consists of build surface preparation by
rolling and leveling with a scraper, laser raster-
ing and particle melting and sintering, cooling
and solidification, followed by the build cham-
ber being lowered by one-layer thickness to
repeat with a recoating of fresh material from
the feed chamber. During the printing process,
the laser, in most applications a 2 W blue diode
laser (445 nm) light source, is rastered
(w100 mm/s) to match the geometry of the layer
[27]. The light energy from the laser source is
absorbed by the particles at the site of the laser
focal spot, which in turn heats the material
beyond the thermal transition (Tg or Tm) allow-
ing for interparticle contact diffusion and bind-
ing. After removal of the light source the
energy dissipates, and the newly formed
coherent body solidifies. The unprinted material
surrounding the printed material serves as an
intrinsic support material. The fact that the 3D-
printed parts are constantly surrounded by
unprinted support material means that parts
can be effectively stacked and printed together
to make efficient use of the build volume but it
27
also implies that a lengthy and “dirty” postpro-
cessing is required to remove the bulk powder
from the build chamber as well as the powder
that is loosely adhered to the final printed part.
Because of the impact of the heat-affected zone
powder, not all of this unprinted powder can
be reused, and it is good practice to blend virgin
powder with this recycled material.
One key to this technique is for the process to
proceed so that enough thermal mass is present,
such that not only are the particles bonded
within the as-printed 2D layer, but this layer
also softens/melts and binds through the same
diffusional process to the layer(s) just below to
form our 3D-printed part. For thermoplastic ma-
terials, liquid-phase sintering drives capillary in-
teractions between neighboring particles
resulting in bonds due to the diffusion of poly-
mer chains or chemical cross-linking.
One method that ensures a well-formed 3D-
printed part is to keep the entire 3DP chamber
at a temperature just below the softening or
melting point of the material to decrease the pro-
cessing time and reduce thermal gradients
within the part, which can lead to part distor-
tions caused by the relatively large volume
changes in semicrystalline or amorphous poly-
mers. In this method, maintaining an elevated
environment is a key consideration in the pro-
cessing of thermally sensitive materials (e.g.,
oxidation) and would need careful evaluation
depending on the material that is used for print-
ing. To provide the best part quality and mini-
mal part warpage, the build volume is left to
cool gradually over 24e48 h for both safety in
handling and to avoid distortion caused by pre-
mature handling while the parts are in a softened
condition.
One of the more critical criteria for this pro-
cess to be effective is the flow and particle pack-
ing properties of the starting material. Because
the powder deposition process between adja-
cent layers is done by depositing material
28 2. Opportunities and challenges of 3D-printed pharmaceutical dosage forms
through a blade and roller recoating process,
the distribution of the particles across the 2D
plane to be printed directly impacts part qual-
ity. If the particles do not flow and fill in the re-
gion in a uniform manner there will be voids in
the final printed part. The size and sphericity of
the particle properties also directly influence the
surface roughness and spatial print resolution
of the manufactured parts. Part resolution on
the order of 100 mm is typical for a printed
part. Compressibility, or volume reduction, un-
der the roller assembly can aid in powder bed
uniformity and this predensification can enable
the printing of higher-density final parts.
Many SLS 3D-printed parts undergo a series
of postprocess finishing operations to provide
more elegant surface properties. In addition,
intrinsic to the type of 3DP, because of the use
of powder as the starting material, final parts
will be porous in nature, which may be consid-
ered as defects from a mechanical strength point
of view or could aid in the disintegration of oral
dosage forms as in traditional compressed tab-
lets. For biomedical applications the porosity
present in these 3D-printed parts could also
serve as a scaffold for cell growth.
Regardless of the material used, the parts
obtained by the powder bed fusion processes
will typically exhibit a certain level of porosity.
The amount of free volume is dependent on par-
ticle size distribution, material choice, and pro-
cess parameters. The pores remaining within a
green part after the additive manufacturing
process represent potential weak points in
models subjected to mechanical load. If high
mechanical strength is required for a given
application, it is therefore common practice to
improve mechanical properties by means of
isostatic pressing, infiltration with suitable
resins, or sintering. On the positive side, SLS-
fabricated parts are light and porosity can be
advantageous in other applications that require
large surface areas, for example, scaffolds for
cell growth in tissue engineering. SLS is appli-
cable to materials with vastly different bulk
properties. Moreover, SLS powders for the
same bulk material can also vary in their
morphology, sintering, and melting behavior.
3.2.2 Powder binding technology
Like the SLS processes, in the first step of
powder binding 3DP a powder layer is depos-
ited using a roller/scraper assembly from a
feeder chamber to the build chamber. Unlike
SLS, which used a thermal method of binding
powder, in liquid-phase powder binding 3DP,
the powder is bound together with the use of a
liquid that is dispensed using an inkjet printing
head. The inkjet head will either contain a sol-
vent (e.g., water) or a solventebinder solution.
In the former, the binder is contained within
the powder formulation whereas if the inject
print head has a solventebinder solution the
binder is dispensed from the print head. The
finished 3D-printed part is then cleaned of any
residual powder using a combination of a vibra-
tory plate and airflow. Much like SLS, the parti-
cle properties drive product quality and final
part resolution, but unlike SLS the inkjet print
head spatial resolution is lower than that of a
laser spot size. This technology offers the ability
to print several materials because of two reasons:
either the powder loaded into the feed chamber
is a blend of multiple materials or the inkjet print
head could also contain a different material such
as an active ingredient or a colorant. This tech-
nology is likely the closest analogy to a tradi-
tional wet granulation process because of the
similarities in materials that are used. In fact,
the powder binding technology is the same
core process that is used by Aprecia Pharmaceu-
ticals to manufacture the Spritam tablet. The
porous nature of the powder bed process creates
a dosage form that instantaneously dissolves
because of the formulation and the intrinsic
capillary wicking action of the dosage form.
 3. Technology details 29

3.3 3D material extrusiondfused extrusion head or build platform moves in z

filament fabrication
3DP extrusion-based processes have seen a
bolster of activity in recent years and cover a
wider range of materials, including thermo-
plastic polymers, pastes, and thermo or UV
curable gels. In this process a nozzle or piston
(e.g., syringe) is fixed to a gantry that moves in
xey space. After a single layer is deposited the
space to complete the next layer. The most com-
mon extrusion-based 3DP is known as fused fila-
ment fabrication (FFF), also known as FDM.
The operating principle is shown in
Figs. 2.6e2.8, where the thermoplastic polymer
filament with a round cross-section of 1.75 or
3.00 mm in diameter is mechanically fed using
a gear-based extruder to a cartridge-heated

Connuous elevaon
Build Support Plate

Photo Dead zone

3D Printed
Resin
Object

Imaging Unit
O2 permeable
window

Mirror

FIGURE 2.6 Schematic of continuous liquid interface production.

Mirror Laser

Roller and scraper

3D printed object Thermoplasc

powder

Build plaorm

Build Chamber Feeder Chamber

FIGURE 2.7 Schematic of selective laser sintering process.

30 2. Opportunities and challenges of 3D-printed pharmaceutical dosage forms
Filament
Extruder Gears
3D printed object
Build plaorm
FIGURE 2.8 Operating principle of extrusion-based 3D printing process.
nozzle assembly. Process temperature is
defined by the thermal properties of the poly-
mer filament, which for amorphous polymers
is above the glass transition temperature and
for semicrystalline polymers is above the
melting point. The melted filament forms a
molten bead upon exit from the small nozzle
orifice (0.1e1 mm diameter) and begins solidifi-
cation at the location from which it was
extruded. The resolution of an FFF-printed
part is often defined by the diameter of the
nozzle. Generally, a smaller nozzle results in a
surface that more closely follows the profile of
the 3D geometry; however, this results in an in-
crease in print time because of the requirements
of more nozzle traces to completely define the
geometry as well as often slowing the print
speed because of the increase in nozzle melt
pressure as a result of the smaller diameter.
The rheology of viscous thermoplastic polymer
Material
Spool
Heater
Element
Nozzle
Molten Bead
is often the limiting factor for this 3DP tech-
nique. Processing not only needs to consider
the speed for appropriate bead deposition but
also the temperature and time required for
fusion of the deposited beads onto the adjacent
layers that were previously printed.
Often in 3DP for a new polymer the impact of
several parameters is often experimentally
derived such as filament extrusion feed rate,
temperature and thermal gradients, nozzle
design, die swelling, polymer melt rheology,
quench rate using convective air cooling, nozzle
path direction, and part orientation. These
parameters are optimized to improve 3DP
efficiency, surface roughness, dimensional
accuracy, mechanical properties, and isotropy.
Many common thermoplastic materials (e.g.,
PLA, acrylonitrile-butadiene-styrene copoly-
mers, polycarbonate, and polyamides), have
been optimized for fused filament fabrication
 4. Regulatory and quality considerations
3DP, while other more nascent thermoplastic
polymers relevant for the pharmaceutical
industry are still being experimented.
3.3.1 Postprocessing
Across most all 3DP technologies, the final
part requires additional processing steps after
completion of 3DP. Depending on the printing
process these steps involve either mechanical
removal of material used to improve adhesion
to the printing plates, removal of support
material used in the printing process, chemical
and/or thermal treatment of unreacted surface
material, removal of unbound surface powder,
or heat treatment to reduce unwanted part
residual stresses. Careful consideration and
execution of the postprocessing steps is crucial
to ensure that the part does not suffer undue
damage.
4. Regulatory and quality considerations
The FDA recently issued a guidance for
industry entitled “Technical Considerations for
Additive Manufactured Medical Devices.”
Even though medical device and combination
products are regulated by the Center for
Devices and Radiological Health, many ele-
ments discussed in this document highlight
key considerations for additive manufactured
drug products that are regulated by the Center
for Drug Evaluation and Research. This guid-
ance like most offers supplementary regulatory
guidance that covers 3DP-specific recommen-
dations. The device-specific 3DP guidance
document covers (1) design for 3DP, (2)
patient-matched device design, (3) software
workflows, (4) controls over materials used for
3DP, (5) postprocessing considerations, (6) pro-
cess validation and product acceptance testing,
(7) quality, and (8) device testing consider-
ations. It is not the intention of this section to
recount this guidance document but to direct
31
the reader to key recommendations that are
common for drug products. Generally, regu-
lated products must fulfill standard Quality
System requirements. Specific to 3DP of drug
products, manufacturers must validate their
process and establish and maintain procedures
for monitoring and controlling processing
parameters to ensure that the specifications of
the drug product can be met with a high degree
of confidence and the product performs as
intended. There are numerous 3DP technologies
described that can be used to manufacture drug
products and hence there are different process-
ing steps that are implemented to manufacture
a quality drug product. Because of the relative
novelty of 3DP, a higher level of scrutiny should
be expected due to the integration of a novel
manufacturing technique with traditional or
novel materials that have been reprocessed or
adapted to be enabled by 3DP. One unique reg-
ulatory consideration that is atypical from tradi-
tional pharmaceutical manufacturing is the
utilization of software in both the design of
the final product and in the control of the pro-
cess to manufacture the final drug product.
3DP involves a multistep software process that
is used to design and convert 3D dosage form
shapes ranging from simple/traditional to com-
plex (more on these designs will be highlighted
in the sections that follow), into sliced 2D layers
(using “slicer” software). This geometrical 2D
information is then used as input into control
software that then translates this information
into print commands. To enable consistency
across the industry, the FDA guidance proposes
the utilization of a specific file format for addi-
tive manufacturing (ISO/ASTM 52915 “Stan-
dard specification for additive manufacturing
file format”). The intention of this standard is
to create a well-controlled and integrated file
that describes the printed volume, material
information, and the print controls and print
location within the print volume. As will be
highlighted later, and perhaps more unlike
32 2. Opportunities and challenges of 3D-printed pharmaceutical dosage forms

other traditional pharmaceutical applications, Table 2.9 highlights some of the critical 3DP
software processes are as critical as and, in processing variables and failure modes that
some instances, more critical of final product need to be considered when establishing the
quality compared to traditional pharmaceutical quality system for a drug product.
process hardware/critical process parameters.
Quality that is governed by software is not
only process control over material being 5. Pharmaceutical applications for drug
handled by the printer in terms of temperature delivery
and time as was pointed out earlier, but just as
important is the structure and path for the print- The current advantages of using 3DP for
ing tool that drives the printing of the drug pharmaceutical dosage forms are targeted at
product. dosage form design and patient customization.
Just like other pharmaceutical processes, 3DP Here we detail several published accounts of
often uses environmentally sensitive material as applying 3DP in the production of more tradi-
a matrix and therefore needs to be handled and tional oral dosage forms, customized oral
evaluated similarly. In addition, many physical dosage forms that highlight the ability to tailor
and chemical attributes that govern product doses and release rates to meet patient needs,
quality in traditional processes are just as impor- as well as nonoral dosage forms that aim to pro-
tant in 3DP. For example, particle size for SLS is a vide more patient complaint dosage forms
critical material attribute that defines not only through longer acting drug delivery. Another
final dosage form elegance but also mechanical advantage of 3DP in drug product development
strength and dissolution variability due to defect is the ability to circumvent the long and com-
populations because of broad particle size distri- plex clinical R&D process. This is especially
bution and insufficient sintering at specified true when the work requires: increasing drug
locations because of voids. solubility by converting the active ingredient
from crystalline to amorphous using processes
such as spray drying or HME, protecting the
TABLE 2.9 Examples of fused filament fabrication active ingredient from a specific region of the
process critical process variables and gut, or altering the drug release profile to over-
failure modes. come pharmacokinetic-related adverse events.
3DP can allow for production of dosage forms
Process variables Failure modes
that overcome these common R&D challenges
Hardware/software Voids between layers in a cost-effective and rapid approach in a
input/output Incomplete layer print single-step process.
Extrusion rate Interlayer adhesion
Retraction settings Plateepill adhesion
Nozzle temperature Stringing
Nozzle size Temperature excursions 5.1 Tunable release technologies
Nozzleeplatform gap Thermal degradation
Platform surface type
Currently, there are limited pharmaceutically
Platform surface roughness acceptable materials available in filament form,
Platform temperature which is the raw material feedstock for FDM
Flow rate printers. Many traditional polymer excipients
Print Speed do not have the appropriate thermal and
 5. Pharmaceutical applications for drug delivery
mechanical properties for filament processing
or the physical properties are altered when
drug is incorporated in the filaments. To have
a robust filament the polymer must be suffi-
ciently rigid to maintain its form as it is pushed
from the compression gear through the hot end
nozzle orifice of the printer. The polymer
should be sufficiently tough so the extruder
gear of the FDM printer can gently depress
and grip the filament to generate an extrusion
force greater than the resistance from the
molten polymer flow out of the nozzle. In addi-
tion, the melting temperature or glass transition
temperature must be significantly higher than
the temperature inside the printing enclosure
to allow forced air cooling to rapidly quench
the extrudate. The melting temperature should
also be below 250
C, which is the maximum
temperature allowed in most commercially
available FDM printers. Finally, to maintain
proper molten flow, the thermoplastic material
must not degrade while it is held at elevated
temperatures during the printing process for
extended periods of time, usually on the order
of minutes. Once a filament is extruded, X-ray
computed tomography can be used as a quality
check for surface or volumetric defects
[40a,40b]. Diameter variations in the filament
tend to strongly correlate with the quality of
the final print as most commercial printers do
not dynamically change the extrusion rate
based on the filament’s instantaneous diameter.
Typically, pharmaceutically acceptable poly-
mers have been experimented with varying suc-
cess. HPC has been used to print drug-free
capsules that are manually filled and assembled
postprinting [41]. Additional work has high-
lighted the difficulties and limitations of using
FDM for printing PVA capsules where the
dosage forms were printed for hand filling
with placebo liquids, followed by manual as-
sembly and sealing, and external and internal
33
surface roughness of the printed capsule walls
were investigated. Typically, however, the fila-
ment that is loaded into the FDM 3D printer is
preprocessed using extrusion to incorporate
active ingredients, which are then used to print
the final dosage form. PVP [10,41a,41b] mixed
with drug in an HME process has been used
with FDM to construct oral dosage forms.
PVA has been most commonly used due to its
beneficial mechanical and thermal properties
aiding the FDM process [6,8,30,40b,42e44].
Recent work on manufacturing filaments for
3DP examined the use of Eudragit EPO, a
cationic acrylic polymer with dimethylamine-
containing side chains, which is a polymer typi-
cally unsuitable for FDM due to its brittle prop-
erties [14]. This study showed that Eudragit
EPO could be compounded with a plasticizer,
triethyl citrate, and a nonmelting filler, trical-
cium phosphate, to optimize the hardness and
flexibility properties to enable printing of the
filament. The same research group
demonstrated the feasibility of printing an
enteric-coated 3D-printed caplet using the
same plasticizer and filler approach with PVP
and drug-free Eudragit EPO [10]. Other
extruded materials, where quinine was mixed
individually with Eudragit RS, PCL, PLA, and
ethyl cellulose at a 5 wt% drug loading, demon-
strated viability for use as FDM filaments for
preparing 3D-printed implants [44a]. It has
also been demonstrated that soaking filaments
in a poor or nonsolvent solution containing
drug can result in diffusion of drug into the
filament, although drug loading is intrinsically
lower than extrusion methods [42]. To date,
there are limited successful piloting examples
of pharmaceutically acceptable filaments, and
the surface quality of these printed dosage
forms indicates more optimization is required
before widespread adoption can be realized.
34 2. Opportunities and challenges of 3D-printed pharmaceutical dosage forms

As of 2019, there are numerous examples of
applying 3DP to produce dosage forms in a rapid
prototyping method with variable and tunable
release rates within the same manufacturing
step. Several examples rely on incorporating an
API into the filament used in extrusion-based
3DP using a typical HME process
[1,2,5,9,10,41,43,45e48] or solvent-based diffu-
sional processes [26,42]. The primary limitation
with these approaches is the limited amount of
drug that can be incorporated into the filament
and hence the final 3D-printed dosage form
with typical drug loadings in the 1e30 wt%
range. The release rates of dosage forms made
by this approach are governed by either diffusion
or erosion for which the volume and geometry of
the final dosage form play are key role. An advan-
tage of this approach of manufacturing dosage
forms directly from drug-loaded filament is that
the final dosage forms are generally robust
enough to be used immediately after printing.
Figs. 2.9 and 2.10 highlights a well-known study
by Ref. [5,8,43,44] where the dosage form surface
area, surface area/volume, or mass were
discretely controlled and thereby directly
impacted the dissolution rate without changes
to the formulation.
Another degree of freedom in 3DP is the
modification of the infill parameters of the
printed tablet, which can manipulate diffu-
sional length scales as well as the dosage form
buoyancy and hence the release rates [1e3].

FIGURE 2.9 3D-printed dosage forms of various geometries using poly(lactide) as the primary matrix for tunable release
rates at constant (A) surface area, (B) surface area/volume ratio, and (C) mass (scale bar in cm). Adapted from Goyanes A, Chang
H, Sedoug HD, Hatton GB, Wang J, Buanz A, Gaisford S, Basit AW. Fabrication of controlled-release budesonide tablets via desktop
(FDM) 3D printing. Int J Pharm 2015b;496:414e420; Goyanes A, Martinez PR, Buanz A, Basit AW, Gaisford S. Effect of geometry
on drug release from 3D printed tablets. Int J Pharm 2015c;494:657e663; Goyanes A, Buanz AB, Hatton GB, Gaisford S, Basit AW.
3D printing of modified-release aminosalicylate (4-ASA and 5-ASA) tablets. Eur J Pharm Biopharm 2015a;89:157e162; Goyanes A,
Wang J, Buanz A, Martínez-Pacheco R, Telford R, Gaisford S, Basit AW. 3D printing of medicines: engineering novel oral devices
with unique design and drug release characteristics. Mol Pharm 2015d;12:4077e4084.
 5. Pharmaceutical applications for drug delivery 35

FIGURE 2.10 Paracetamol dissolution profiles from 3DP solid dosage with surface area/volume ratio 1 in phosphate
buffer (pH 6.8). Adapted from Goyanes A, Chang H, Sedoug HD, Hatton GB, Wang J, Buanz A, Gaisford S, Basit AW. Fabrication
of controlled-release budesonide tablets via desktop (FDM) 3D printing. Int J Pharm 2015b;496:414e420; Goyanes A, Martinez PR,
Buanz A, Basit AW, Gaisford S. Effect of geometry on drug release from 3D printed tablets. Int J Pharm 2015c;494:657e663; Goyanes
A, Buanz AB, Hatton GB, Gaisford S, Basit AW. 3D printing of modified-release aminosalicylate (4-ASA and 5-ASA) tablets. Eur J Pharm
Biopharm 2015a;89:157e162; Goyanes A, Wang J, Buanz A, Martínez-Pacheco R, Telford R, Gaisford S, Basit AW. 3D printing of
medicines: engineering novel oral devices with unique design and drug release characteristics. Mol Pharm 2015d;12:4077e4084.

Infill is the process by which the print head will
print an outer shell of the shape of the part and
the inside of this shell is filled in with a partic-
ular pattern to accommodate a predefined vol-
ume percentage. The material that is printing
between the outer shell is termed “infill” and
can be controlled through software to define
both the geometry of the infill material as well
as the amount of infill.
In addition to their use in solid filament as the
starting material for extrusion-based 3DP,
viscous pastes and UV curable polymers have
been shown to be viable feedstocks for
extrusion-based 3DP of active dosage forms
[46,47,49]. Preparing these starting materials as
shown in Fig. 2.11 requires less process develop-
ment as compared to an extrusion-based
approach; however, these dosage forms typically
require postprocessing, such as drying or active
thermal curing, and the mechanical properties
for the resulting dosage product have not been
investigated thoroughly. With both of these ap-
proaches, API chemical and/or physical stability
may be compromised. These paste formulations
have been printed using similar equipment to an
FDM printer, except the hot end is replaced with
a closed shot canister. Using this approach,
HPMC and polyacrylic acid (Carbopol 974P)
[46], HPMC and lactose [47], and HPMC hydro-
alcoholic gels [49] have been printed. Notably,
this approach has been used for polypills
[47,49], which are single oral dosage forms that
contain three or more isolated volumes each con-
taining a different active ingredient. While paste
formulations open doors to more material
choices, this approach typically requires
36 2. Opportunities and challenges of 3D-printed pharmaceutical dosage forms

FIGURE 2.11 (i) Schematic diagrams of (A) the dispersion technique of hypromellose (HPMC) 2910 powder and
(B) formulation of HPMC hydroalcoholic gel [46]. (ii) Photograph of a RegenHU 3D printer (left) RegenHU Switzerland
(regnhu,com), and image of a multiactive tablet (right) (10.45 mm [height], 6 mm [radius]) composed of a captopril osmotic
pump compartment (bottom), and nifedipine (hole I) and glipizide (hole II) sustained release compartments (top) and joining
layer (middle).

subsequent steps such as overnight drying of the
print to remove any solvent or water from the
dosage form for long-term physical stability,
and it is unclear at this time how the mechanical
robustness of paste-printed dosage forms will
endure secondary packaging and user handling.
These are existing examples of implementing
3DP technology into rapid prototype release
rates using different strategies, largely focused
on maintaining a similar material feedstock
and using creative printing parameters to
generate various releases. With all the following
examples, at least one solid filament material is
preprocessed to contain API. The extrusion of
filaments or pastes has been used to manufac-
ture what are termed core/shell tablets, where
 5. Pharmaceutical applications for drug delivery
the outer shell’s thickness is varied, and has
demonstrated the generation of distinct release
rates. Core/shell tablets have been manufac-
tured by using a second API-containing mate-
rial [8] or a placebo material with the intent to
mimic enteric-coated tablets [9,10], and have
demonstrated the agility of 3DP to change the
onset of the release of the core of the dosage
form by as much as 2 h in vitro using the same
material feedstock. While these strategies have
been demonstrated to provide a software tun-
ing knob for release rate manipulation while
maintaining a constant material feedstock, all
of these strategies rely on HME formulation of
a printable filament for each API. Developing
process conditions to incorporate API into an
excipient-based solid filament is usually not
trivial [11e14], and these filament processing
developments add to the product development
burden, reducing the rapid prototyping advan-
tage 3DP brings to the table for early drug
screenings.
Fina et al. [52] presented the first published
work to utilize SLS for the production of oral
dosage forms using two thermoplastic
pharmaceutical-grade polymers, Kollicoat IR
(75% polyvinyl alcohol and 25% PEG
copolymer) and Eudragit L100-55 (50% metha-
crylic acid and 50% ethyl acrylate copolymer),
with immediate and modified release char-
acteristics. For this process to achieve print-
ability and aid in the sintering process,
pharmaceutical-grade silicate and oxide-based
pigments are added to improve laser energy
absorption and dissipation. In general, SLS-
printed dosage forms have poorer surface qual-
ity and higher porosity as shown in the example
from [52] (Fig. 2.12A). Control of the release rate
of the SLS dosage forms based on the research
thus far is governed more by the thermoplastic
material than by the printing conditions as
seen in Fig. 2.12B.
37
5.2 Paste/gel extrusion-based
technologies
A unique approach that attempts to incorpo-
rate both filament-based and paste/gel
extrusion-based technologies has been devel-
oped by Smith et al. [50,51]. The aim of their
work was to take advantage of the printability
of pharmaceutically acceptable polymers like
PLA and PVA while limiting the processing of
API to similar approaches shown earlier for
paste, liquids, and gel-based formulations.
They developed a single-step FDM 3DP process
to manufacture thin-walled drug-free capsules,
which can be filled with dry or liquid drug prod-
uct formulations. Drug release from these sys-
tems is governed by the combined dissolution
of the FDM capsule “shell” and the dosage
form encapsulated in these shells. To prepare
the shells, the 3D printer files (extension
“.gcode”) were modified by creating discrete
zones, so-called “zoning process,” with individ-
ual print parameters. Their work clearly shows
several unique aspects of the difficulty in 3DP
quality dosage forms that are elegant and water
tight. The geometry of the dosage form requires
a design that is specific to the 3DP process,
breaking from the more traditional shapes to
account for the physics of 3DP. In their work
they highlight the need to redesign the shape
with different angles (so-called zoning) using
software that is commonly available to the pub-
lic. Fig. 2.13 shows different colors within the
dosage form to show where FDM thermal and
mechanical process conditions are purposely
changed to improve the quality of the final
printed dosage form.
It is well known that the speed of FDM 3D
printers is not particularly fast as compared to
traditional dosage form manufacturing where a
rotary tablet press can accommodate on the
order of 1,000,000 tablets per hour. A 3D printer
38 2. Opportunities and challenges of 3D-printed pharmaceutical dosage forms

FIGURE 2.12 (A): Scanning electron microscopy images of the selective laser sintering of printlet vertical sections. On the
top from left to right, Kollicoat IR K5, K20, and K35. On the bottom from left to right, Eudragit L100-55 E5, E20, and E35, where
5, 20, and 35 represent the wt% of paracetamol used as a model active pharmaceutical ingredient. (B) Drug dissolution from
Eudragit SLS printlets. Adapted from Fina F, Goyanes A, Gaisford S, Basit AW. Selective laser sintering (SLS) 3D printing of medicines.
Int J Pharm 2017;529:285e293.
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 CHAPTER I.

HISTORY AND LITERATURE.

The commencement of the history of Ichthyology

Aristotle. coincides with that of Zoology generally. Aristotle
(384–322 b.c.) had a perfect knowledge of the
general structure of fishes, which he clearly discriminates from the
Aquatic animals with lungs and mammæ, i.e. Cetaceans, and from
the various groups of Aquatic Invertebrates. He says that “the
special characteristics of the true fishes consist in the branchiæ and
fins, the majority having four fins, but those of an elongate form, as
the eels, having two only. Some, as the Muræna, lack the fins
altogether. The Rays swim with their whole body, which is spread
out. The branchiæ are sometimes furnished with an opercle,
sometimes without one, as is the case in the cartilaginous fishes....
No fish has hairs or feathers; most are covered with scales, but
some have a rough or smooth skin. The tongue is hard, often
toothed; and sometimes so much adherent that it seems to be
wanting. The eyes have no lids; nor are any ears or nostrils visible,
for what takes the place of nostrils is a blind cavity. Nevertheless
they have the senses of tasting, smelling, and hearing. All have
blood. All scaly fishes are oviparous, but the cartilaginous fishes
(with the exception of the Sea-devil, which Aristotle places along with
them) are viviparous. All have a heart, liver, and gall-bladder; but
kidneys and urinary bladder are absent. They vary much in the
structure of their intestines: for whilst the mullet has a fleshy stomach
like a bird, others have no stomachic dilatation. Pyloric coeca are
close to the stomach, variable in number; there are even some, like
the majority of the cartilaginous fishes, which have none whatever.
Two bodies are situated along the spine, which have the function of
testicles, and open towards the vent, and which are much enlarged
in the spawning season. The scales become harder with age. Not
being provided with lungs, they have no voice, but several can emit
grunting sounds. They sleep like other animals. In the majority the
females exceed the males in size; and in the Rays and Sharks the
male is distinguished by an appendage on each side of the vent.”
 Aristotle’s information on the habits of fishes, their migrations,
mode and time of propagation, utility, is, as far as it has been tested,
surprisingly correct. Unfortunately, only too often we lack the means
of recognising the species of which he gives a description. His ideas
of specific distinction were as vague as those of the fishermen
whose nomenclature he adopted; it never occurred to him that such
popular names are subject to change, or may be entirely lost with
time, and the difficulty of deciphering his species is further increased
by the circumstance that popular names are often applied by him to
the same fish, or that different stages of growth are designated by
distinct names. The number of fishes known to Aristotle seems to
have been about 115, all of which are inhabitants of the Ægean Sea.
That one man should have discovered so many truths, and
formed so sure a base for Zoology, is less surprising than the fact
that for about eighteen centuries a science which seemed to offer
particular attractions to men gifted with power of observation, was no
farther advanced. Yet this is the case. Aristotle’s disciples, as well as
his successors, remained satisfied to be his copiers or
commentators, and to collect fabulous stories or vague notions. With
very few exceptions (such as Ausonius, who wrote a small poem, in
which he describes from his own observations the fishes of the
Mosel) authors entirely abandoned original research. And it was not
until about the middle of the sixteenth century that Ichthyology made
a new step in advance by the appearance of Belon, Rondelet, and
Salviani, who almost simultaneously published their grand works, by
which the idea of species was established definitely and for all times.

P. Belon travelled in the countries bordering on

Belon. the eastern part of the Mediterranean, in the years
1547–50; he collected rich stores of positive
knowledge, which he deposited in several works. The one most
important for the progress of Ichthyology is that entitled “De
aquatilibus libri duo” (Paris 1553; small 4to.) Belon knows about 110
fishes, of which he gives rude, but generally recognisable, figures. In
his descriptions he pays regard to the classical as well as vernacular
nomenclature, and states the outward characteristics, sometimes
even the number of fin-rays, frequently also the most conspicuous
anatomical peculiarities.
Although Belon but rarely gives definitions of the terms used by
him, it is generally not very difficult to ascertain the limits which he
intended to assign to each division of aquatic animals. He very
properly divides them into such as are provided with blood, and into
those without it: two divisions, called in modern language Vertebrate
and Invertebrate aquatic animals. The former are classified by him
according to sizes, the further subdivisions being based on the
structure of the skeleton, mode of propagation, number of limbs,
form of the body, and on the physical character of the localities
inhabited by fishes. This classification is as follows:—
I.The larger fishes or Cetaceans.
A. Viviparous Cetaceans with bony skeletons (= Cetacea).
B. Viviparous Amphibians.
1. “With four limbs: Seals, Hippopotamus, Beaver, Otter, and other
aquatic Mammalia.
2. With two limbs: Mermaids, etc.
C. Oviparous Amphibians (= Reptiles and Frogs).
D. Viviparous Cartilaginous fishes.
1. Of an oblong form (= Sharks).
2. Of a flat form (= Rays and Lophius).
E. Oviparous Cartilaginous fishes (= Sturgeons and Silurus).
F. Oviparous Cetaceans, with spines instead of bones (= large marine
fishes, like the Thunny, Sword-fish, Sciænoids, Bass, Gadoids,
Trachypterus).
II.Spinous Oviparous fishes of a flat form (= Pleuronectidæ).
III.Fishes of a high form, like Zeus.
IV.Fishes of a snake like form (= Eels, Belone, Sphyræna).
V.Small Oviparous, spinous, scaly, marine fishes.
1. Pelagic kinds.
2. Littoral kinds.
3. Kinds inhabiting rocky localities.
VI.Fluviatile and Lacustrine fishes.
 The work of the Roman ichthyologist, H. Salviani
Salviani. (1514–72), is characteristic of the high social
position which the author held as the physician of
three popes. Its title is “Aquatilium animalium historia” (Rom. 1554–
57, fol.) It treats exclusively of the fishes of Italy. Ninety-two species
are figured on seventy-six plates which, as regards artistic execution,
are masterpieces of that period, although those specific
characteristics which nowadays constitute the value of a zoological
drawing, were entirely overlooked by the author or artist. No attempt
is made at a natural classification, but the allied forms generally are
placed in close proximity. The descriptions are quite equal to those
given by Belon, entering much into the details of the economy and
usefulness of the several species, and were evidently composed
with the view of collecting in a readable form all that might prove of
interest to the class of society in which the author moved. Salviani’s
work is of a high standard, most remarkable for the age in which he
lived. It could not fail to convey valuable instruction, and to render
Ichthyology popular in the country to the fauna of which it was
devoted, but it would not have advanced Ichthyology as science
generally; and in this respect Salviani is not to be compared with
Rondelet or Belon.

G. Rondelet (1507–1557) had the great advantage

Rondelet. over Belon in having received a medical education at
Paris, and more especially in having gone through a
complete course of instruction in anatomy as a pupil of Guentherus
of Andernach. This is conspicuous throughout his works—“Libri de
Piscibus marinis” (Lugd. 1554, fol.); and “Universæ aquatilium
historiæ pars altera” (Lugd. 1555, fol.) Nevertheless they cannot be
regarded as more than considerably enlarged editions of Belon’s
work. For although he worked independently of the latter, and differs
from him in numerous details, the system adopted by him is
characterised by the same absence of the true principles of
classification. Rondelet had a much more extensive knowledge of
details. His work is almost entirely limited to European, and chiefly
Mediterranean, forms, and comprises not less than 197 marine and
47 freshwater fishes. His descriptions are more complete and his
figures much more accurate than those of Belon; and the specific
account is preceded by introductory chapters in which he treats in a
general manner on the distinctions, the external and internal parts,
and on the economy of fishes. Like Belon, he had no conception of
the various categories of classification—for instance, confounding
throughout his work the terms “genus” and “species;” but he had
intuitively a notion of what his successors called a “species,” and his
principal object was to collect and give as much information as
possible of such species.
For nearly a century the works of Belon and Rondelet remained
the standard works of Ichthyology; but this science did not remain
stationary during this period. The attention of naturalists was now
directed to the products of foreign countries, especially the Spanish
and Dutch possessions in the New World; and in Europe the
establishment of anatomical schools and academies led to the
careful investigation of the internal anatomy of the most remarkable
European forms. Limited as these efforts were as to their scope,
being directed either only to the fauna of some district, or to the
dissection of a single species, they were sufficiently numerous to
enlarge the views of naturalists, and to destroy that fatal dependency
on preceding authorities which had continued to keep in bonds the
minds of even such men as Rondelet and Belon.
The most noteworthy of those who were active in
W. Piso. G. tropical countries are W. Piso and G. Margrav.
Margrav. They accompanied as physicians the Dutch
Governor, Prince Moritz of Nassau, to Brazil (1637–
44). Margrav especially studied the fauna of the country, and
although he died before his return to Europe, his observations were
published by his colleague, and embodied in a work “Historia
naturalis Braziliæ” (Lugd. 1648, fol.), in which the fourth book treats
of the fishes. He describes about 100 species, all of which had been
previously unknown, in a manner far superior to that of his
predecessors. The accompanying figures are not good, but nearly
always recognisable, and giving a fair idea of the form of the fish.
Margrav himself, with the aid of an artist, had made a most valuable
collection of coloured drawings of the objects observed and
described by him, but many years were allowed to pass before it was
scientifically utilised by Bloch and others.
Of the men who left records of their anatomical
Anatomists, researches, we may mention Borelli (1608–79),
1600–1700. who wrote a work “De motu animalium” (Rom. 1680,
4to), in which he explained the mechanism of
swimming, and the function of the air-bladder; M. Malpighi (1628–
94), who examined the optic nerve of the sword-fish; the celebrated
J. Swammerdam (1637–80), who described the intestines of
numerous fishes; and J. Duverney (1648–1730), who entered into
detailed researches of the organs of respiration.

A new era in the history of Ichthyology commences with Ray,

Willughby, and Artedi, who were the first to recognise the true
principles by which the natural affinities of animals should be
determined. Their labours stand in so intimate a connection with
each other that they represent only one stride in the progress of this
science.

J. Ray (born 1628 in Essex, died 1705), was the

Ray and friend and guide of F. Willughby (1635–72). They
Willughby had recognised that a thorough reform of the
treatment of the vegetable and animal kingdoms had
become necessary; that the only way of bringing order into the
existing chaos was that of arranging the various forms with regard to
their structure; that they must cease to be burdened with inapplicable
passages and quotations of the ancient writers, and to perpetuate
the erroneous or vague notions of their predecessors. They
abandoned speculation, and adhered to facts only. One of the first
results, and perhaps the most important, of their method was, that
having recognised the “species” as such, they defined this term, and
fixed it as the base, from which all sound zoological knowledge has
to start.
Although they had divided their work thus that Ray attended to
the plants principally, and Willughby to the animals, the “Historia
piscium” (Oxford, 1686, fol.), which bears Willughby’s name on the
titlepage, and was edited by Ray, is clearly their joint production. A
great part of the observations contained in it were collected during
their common journeys in Great Britain and on the Continent, and it
is no exaggeration to say that at that time these two Englishmen
knew the fishes of the Continent, especially those of Germany, better
than any other Continental zoologist.
By the definition of fishes as animals with blood, breathing by
gills, provided with a single ventricle of the heart, covered with scales
or naked; the Cetaceans are excluded. Yet, at a later period Ray
appears to have been afraid of so great an innovation as the
separation of whales from fishes, and, therefore, he invented a
definition of fish which comprises both. The fishes proper are then
arranged in the first place according to the cartilaginous or osseous
nature of the skeleton; further subdivisions being formed with regard
to the general form of the body, the presence or absence of ventral
fins, the soft or spinous structure of the dorsal rays, the number of
dorsal fins, etc. Not less than 420 species are thus arranged and
described, of which about 180 were known to the authors from
autopsy: a comparatively small proportion, descriptions and figures
still forming at that time in a great measure a substitute for
collections and museums. With the increasing accumulation of forms
the want of a fixed nomenclature is now more and more felt.

Peter Artedi would have been a great ichthyologist

P. Artedi. if Ray or Willughby had never preceded him. But he
was fully conscious of the fact that both had
prepared the way for him, and therefore he derived all possible
advantages from their works. Born in 1705 in Sweden, he studied
with Linnæus at Upsala; from an early period he devoted himself
entirely to the study of fishes, and was engaged in the arrangement
and description of the ichthyological collection of Seba, a wealthy
Dutchman who had formed the then perhaps richest museum, when
he was accidentally drowned in one of the canals of Amsterdam in
the year 1734, at an age of twenty-nine years. His manuscripts were
fortunately rescued by an Englishman, Cliffort, and edited by his
early friend Linnæus.
The work is divided into the following parts:—
1. In the “Bibliotheca Ichthyologica” Artedi gives a very complete
list of all preceding authors who have written on fishes, with a critical
analysis of their works.
2. The “Philosophia Ichthyologica” is devoted to a description of
the external and internal parts of fishes; Artedi fixes a precise
terminology of all the various modifications of the organs,
distinguishes between those characters which determine a genus
and such as indicate a species or merely a variety; in fact he
establishes the method and principles which subsequently have
guided every systematic ichthyologist.
3. The “Genera Piscium” contains well-defined diagnoses of forty-
five genera, for which he fixes an unchangeable nomenclature.
4. In the “Species Piscium” descriptions of seventy-two species,
examined by himself, are given; descriptions which even now are
models of exactitude and method.
5. Finally, in the “Synonymia Piscium” references to all previous
authors are arranged for every species, very much in the same
manner which is adopted in the systematic works of the present day.
Artedi has been justly called the Father of
Linnæus. Ichthyology. So perfect was his treatment of the
subject, that even Linnæus could no more improve
it, only modify and add to it; and as far as Ichthyology is concerned,
Linnæus has scarcely done anything beyond applying binominal
terms to the species properly described and classified by Artedi.
Artedi had divided the fishes proper into four orders, viz.
Malacopterygii, Acanthopterygii, Branchiostegi, and Chondropterygii,
of which the third only, according to our present knowledge, appears
to be singularly heterogeneous, as it comprises Balistes, Ostracion,
Cyclopterus, and Lophius. Linnæus, besides separating the
Cetaceans entirely from the class of fishes (at least since the 10th
edition of the “Systema Naturæ”) abandoned Artedi’s order of
Branchiostegi, but substituted a scarcely more natural combination
by joining it with Artedi’s Chondropterygians, under the name of
“Amphibia nantes.”
His classification of the genera appears in the 12th edition of the
“Systema,” thus—
Amphibia Nantes.
Spiraculis compositis.
Petromyzon.
Raia.
Squalus.
Chimæra.

Spiraculis solitariis.
Lophius.
Acipenser.
Cyclopterus.
Balistes.
Ostracion.
Tetrodon.
Diodon.
Centriscus.
Syngnathus.
Pegasus.

Pisces Apodes.
 Muræna.
Gymnotus.
Trichiurus.
Anarhichas.
Ammodytes.
Ophidium.
Stromateus.
Xiphias.

Pisces Jugulares.
Callionymus.
Uranoscopus.
Trachinus.
Gadus.
Blennius.

Pisces Thoracici.
Cepola.
Echeneis.
Coryphæna.
Gobius.
Cottus.
Scorpæna.
Zeus.
Pleuronectes.
Chæetodon.
Sparus.
Labrus.
Sciæna.
Perca.
Gasterosteus.
Scomber.
Mullus.
Trigla.

Pisces Abdominales.
 Cobitis.
Amia.
Silurus.
Teuthis.
Loricaria.
Salmo.
Fistularia.
Esox.
Elops.
Argentina.
Atherina.
Mugil.
Mormyrus.
Exocœtus.
Polynemus.
Clupea.
Cyprinus.

Two contemporaries of Linnæus attempted a

Gronow and systematic arrangement of fishes; both had
Klein. considerable opportunities for their study, especially
in possessing extensive collections; but neither
exercised any influence on the progress of Ichthyology. The one, L.
T. Gronow, a German who resided in Holland, closely followed the
arrangements proposed by Artedi and Linnæus, and increased the
number of genera and species from the contents of his own
museum. He published two works, “Museum Ichthyologicum” (Lugd.
1754–6, fol.), and “Zoophylacium” (Lugd. 1763–81, fol.); a
posthumous work, containing numerous excellent descriptions of
new forms was published by J. E. Gray in 1854 under the title of
“Systema Ichthyologicum.” To Gronow also is due the invention of
preparing flat skins of fishes in a dry state, and preserving them in
the manner of a herbarium. The specimens thus prepared by him
belong to the oldest which have been preserved down to our time.
Much less important are the ichthyological labours of J. T. Klein
(1685–1759). They are embodied in five parts (Missus) of a work
entitled “Historia naturalis piscium” (Sedæ, 1740–9, 4to.) He
regarded a system merely as the means of recognising the various
forms of animals, not as the expression of their natural affinities; and
that method seemed to him to be the most perfect by which an
animal could be most readily determined. He eschewed all reference
to minute or anatomical characters. Hence his system is a series of
the most unnatural combinations, and we cannot be surprised that
Linnæus passed in silence over Klein’s labours.

The works of Artedi and Linnæus excited fresh

Pupils and activity, more especially in Scandinavia, Holland,
Successors of Germany, and England, such as has not been
Linnæus equalled in the history of biological science either
before or after. Whilst some of the pupils and
followers of Linnæus devoted themselves to an examination and
study of the fauna of their native countries, others proceeded on
voyages of discovery to foreign and distant countries. Of these latter
the following may be specially mentioned:—O. Fabricius worked out
the Fauna of Greenland, Kalm collected in North America,
Hasselquist in Egypt and Palestine, Brünnich in the Mediterranean,
Osbeck in Java and China, Thurnberg in Japan; Forskål examined
and described the fishes of the Red Sea; Steller, Pallas, S. T.
Gmelin, and Güldenstedt traversed nearly the whole of the Russian
Empire in Europe and Asia. Others attached themselves as
naturalists to the celebrated circumnavigators of the last century, like
the two Forsters (father and son), and Solander, who accompanied
Cook; Commerson, who travelled with Bougainville; and Sonnerat.
Numerous new and startling forms were discovered by those men,
and the foundation was laid of the knowledge of the geographical
distribution of animals.
Of those who studied the fishes of their native country the most
celebrated are Pennant (Great Britain), O. F. Müller (Denmark),
Duhamel (France), Meidinger (Austria), Cornide (Spain), Parra
(Cuba).
The materials brought together by those and other zoologists
were so numerous that, not long after the death of Linnæus, the
necessity was felt of collecting them in a compendious form. Several
compilators undertook this task; they embodied the recent
discoveries in new editions of Artedi’s and Linné’s classical works,
but not possessing either a knowledge of the subject or any critical
discernment, they only succeeded in covering those noble
monuments under a mass of confused rubbish. For Ichthyology it
was fortunate that two men at least, Bloch and Lacépède, made it a
subject of long and original research.

Mark Eliezer Bloch, born in the year 1723 at

M. E. Bloch. Anspach in Germany, practised as a physician in
Berlin; he had reached an age of fifty-six years when
he commenced to write on ichthyological subjects. To commence at
his age a work in which he intended not only to give full descriptions
of the species known to him from specimens or drawings, but also to
illustrate every species in a style truly magnificent for his time, was
an undertaking of the execution of which an ordinary man would
have despaired. Yet he accomplished not only this task, but even
more, as we shall see hereafter.
His work consists of two divisions:—
1. “Oeconomische Naturgeschichte der Fische Deutschlands”
(Berl. 1782–4, 4to. Plates in fol.)
2. “Naturgeschichte der auslændischen Fische” (Berl. 1785–95,
4to. Plates in fol.)
Bloch’s work is unique, and probably will for ever remain so.
Although Cuvier fifty years later undertook a similar general work on
fishes, the subject had then become too extensive to allow of an
attempt of giving illustrations of all the species, or illustrations of a
similar size and costliness.
The first division of the work, which is devoted to a description of
the fishes of Germany, is entirely original, and based upon Bloch’s
own observations. His descriptions as well as figures were made
from nature, and are, with but few exceptions, still serviceable; many
continue to be the best existing in literature.
Bloch was less fortunate and is much less reliable in his natural
history of foreign fishes. For many of the species he had to rely on
more or less incorrect drawings and descriptions of travellers;
frequently, also, he was deceived as to the origin of specimens
which he acquired by purchase. Hence his accounts contain
numerous confusing errors which it would have been difficult to
correct, if not nearly the whole of the materials on which his work is
based had been preserved in the collections at Berlin.
After the completion of his Ichthyology Bloch occupied himself
with systematic work. He prepared a general system of fishes, in
which he arranged not only those described in his great work, but
also those with which he had become acquainted afterwards from
the descriptions of others. The work was ably edited and published
after Bloch’s death by a philologist, J. G. Schneider, under the title
“M. E. Blochii Systema ichthyologiæ iconibus ex. illustratum” (Berl.
1801, 8vo.) The number of species enumerated in it amounts to
1519. The system is based upon the number of the fins, the various
orders being termed Hendecapterygii, Decapterygii, etc. We need
not add that an artificial method like this led to the most unnatural
combinations or severances.
Bloch’s Ichthyology remained for many years the
Lacépède. standard work, and, by the great number of excellent
illustrations, proved a most useful guide to the
student. But as regards originality of thought, Bloch was far
surpassed by his contemporary, B. G. E. de Lacépède, born at
Agen, in France, in 1756, a man of great and general erudition, who
died as Professor of the Museum of Natural History of Paris in 1826.
Lacépède had to contend with great difficulties in the preparation
of his “Histoire des Poissons” (Paris, 1798–1803, 4to, in 5 vols.),
which was written during the most disturbed period of the French
Revolution. A great part of it was composed whilst the author was
separated from collections and books, and had to rely on his notes
and manuscripts only. Even the works of Bloch and other
contemporaneous authors remained unknown, or at least
inaccessible, to him for a long time. Therefore we cannot be
surprised that his work abounds in all those errors to which a
compiler is subject. The same species not only appears under two
and more distinct specific names, but it sometimes happens that the
author understands so little the source from which he derives his
information that the description is referred to one genus and the
accompanying figure to another. The names of genera are unduly
multiplied; and the figures with which the work is illustrated are far
inferior to those of Bloch. Thus the influence of Lacépède on the
progress of Ichthyology was infinitely less than that of his fellow-
labourer; and the labour caused to his successors by correcting the
numerous errors into which he has fallen, probably outweighs the
assistance which they derived from his work.
The work of the principal cultivators of Ichthyology in
Anatomists. the period between Ray and Lacépède was chiefly
systematic and descriptive, but also the internal
organisation of fishes received attention from more than one great
anatomist. Haller, Camper, and Hunter, examined the nervous
system and organs of sense; and more especially Alexander Monro
(the son) published a classical work, “The Structure and Physiology
of Fishes explained and compared with those of Man and other
Animals” (Edinb. 1785, fol.) The electric organs of fishes (Torpedo
and Gymnotus) were examined by Réaumur, Allamand, Bancroft,
Walsh, and still more exactly by J. Hunter. The mystery of the
propagation of the Eel called forth a large number of essays, and
even the artificial propagation of Salmonidæ was known and
practised by Gleditsch (1764).
Bloch and Lacépède’s works were almost
Faunists. immediately succeeded by the labours of Cuvier, but
his early publications were of necessity tentative,
preliminary, and fragmentary, so that a short period elapsed before
the spirit infused by this great anatomist into Ichthyology could
exercise its influence on all workers in this field. Several of such
antecuvierian works must be mentioned on account of their
importance to our knowledge of certain Faunas: the “Descriptions
and Figures of Two Hundred Fishes collected at Vizagapatam on the
coast of Coromandel” (Lond. 1803; 2 vols. in fol.), by Patrick Russel;
and “An Account of the Fishes found in the River Ganges and its
branches” (Edinb. 1822; 2 vols. in 4to), by F. Hamilton (formerly
Buchanan)—works distinguished by a greater accuracy of their
drawings (especially in the latter), than was ever attained before. A
“Natural History of British Fishes” was published by E. Donovan
(Lond. 8vo, 1802–8); and the Mediterranean Fauna formed the study
of the lifetime of A. Risso (“Ichthyologie de Nice.” Paris, 1810, 8vo;
and “Histoire naturelle de l’Europe Meridionale.” Paris, 1827, 8vo). A
slight beginning in the description of the fishes of the United States
was made by S. L. Mitchell, who published, besides various papers,
a “Memoir on the Ichthyology of New York,” in 1815.[2]

G. Cuvier did not occupy himself with the study of

G. Cuvier. fishes merely because this class formed part of the
“Règne animal,” but he devoted himself to it with
particular predilection. The investigation of their anatomy, and
especially of their skeleton, was taken up by him at an early period,
and continued until he had succeeded in completing so perfect a
framework of the system of the whole class that his immediate
successors could content themselves with filling up those details for
which their master had no leisure. Indefatigable in examining all the
external and internal characters of the fishes of a rich collection, he
ascertained the natural affinities of the infinite variety of fishes, and
accurately defined the divisions, orders, families, and genera of the
class, as they appear in the various editions of the “Règne animal.”
His industry equalled his genius: he opened connections with almost
every accessible part of the globe; not only French travellers and
naturalists, but also Germans, Englishmen, Americans, rivalled one
another to assist him with collections; and for many years the
Muséum of the Jardin des Plantes was the centre where all
ichthyological treasures were deposited. Thus Cuvier brought
together a collection the like of which had never been seen before,
and which, as it contains all the materials on which his labours were
based, must still be considered to be the most important. Soon after
the year 1820, Cuvier, assisted by one of his pupils, A.
Valenciennes, commenced his great work on fishes, “Histoire
naturelles des Poissons,” of which the first volume appeared in 1828.
The earlier volumes, in which Cuvier himself took his share, bear
evidence of the freshness and love with which both authors devoted
themselves to their task. After Cuvier’s death in 1832 the work was
left entirely in the hands of Valenciennes, whose energy and interest
gradually slackened, to rise to the old standard in some parts only,
as, for instance, in the treatise on the Herring. He left the work
unfinished with the twenty-second volume (1848), which treats of the
Salmonoids. Yet, incomplete as it is, it is indispensable to the
student.
There exist several editions of the work, which, however, have
the same text. One, printed in 8vo, with coloured or plain figures, is
the one in common use among ichthyologists. A more luxurious
edition in 4to has a different pagination, and therefore is most
inconvenient to use.
As mentioned above, the various parts of the work are very
unequally worked out. Many of the species are described in so
masterly a manner that a greater excellency of method can hardly be
conceived. The history of the literature of these species is entered
into with minuteness and critical discernment; but in the later
volumes, numerous species are introduced into the system without
any description, or with a few words only, comparing a species with
one or more of its congeners. Cuvier himself, at a late period of his
life, seems to have grown indifferent as to the exact definition of his
species: a failing commonly observed among Zoologists when
attention to descriptive details becomes to them a tedious task. What
is more surprising is, that a man of his anatomical and physiological
knowledge should have overlooked the fact that secondary sexual
characters are developed in fishes as in any other class of animals,
and that fishes undergo great changes during growth; and,
consequently, that he described almost all such sexual forms and
different stages of growth under distinct specific and even generic
names.
The system finally adopted by Cuvier is the following:—
A. Poissons Osseux.
I.—a branchies en peignes ou en lames.
1. a mâchoire supérieure libre.
a. Acanthoptérygiens.
Percoïdes.
Polynèmes.
Mulles.
Joues cuirassées.
Scienoïdes.
Sparoïdes.
Chétodonoïdes.
Scomberoïdes.
Muges.
Branchies labyrinthiques.
Lophioïdes.
Gobioïdes.
Labroïdes.

b. Malacoptérygiens.
Abdominaux.
Cyprinoïdes.
Siluroïdes.
Salmonoïdes.
Clupeoïdes.
Lucioïdes.

Subbrachiens.
Sparoïdes.
Pleuronectes.
Discoboles.

Apodes.
 Murenoïdes.

2. a mâchoire supérieure fixée.

Sclérodermes.
Gymnodontes.

II. a branchies en forme de houppes.

Lophobranches.

B. Cartilagineux ou Chondroptérygiens.
Sturioniens.
Plagiostomes.
Cyclostomes.

We have to compare this system with that of Linnæus if we wish

to measure the gigantic stride Ichthyology has made during the
intervening period of seventy years. The various characters
employed for classification have been examined throughout the
whole class, and their relative importance has been duly weighed
and understood. Though Linnæus had formed a category of
“Amphibia nantes” for fishes with a cartilaginous skeleton, which
should coincide with Cuvier’s “Poissons Cartilagineux,” he had failed
to understand the very nature of cartilage, apparently comprising by
this term any skeletal framework of less firmity than ordinary bone.
Hence he considered Lophius, Cyclopterus, Syngnathus to be
cartilaginous fishes. Adopting the position and development of the
ventral fins as a highly important character, he was obliged to
associate fishes with rudimentary and inconspicuous ventral fins, like
Trichiurus, Xiphias, etc., with the true Eels. The important category of
a “family” appears now in Cuvier’s system fully established as that
intermediate between genus and order. Important changes in
Cuvier’s system have been made and proposed by his successors,
but in the main it is still that of the present day.
Cuvier had extended his researches beyond the living forms, into
the field of palæontology; he was the first to observe the close
resemblance of the scales of the fossil Palæoniscus to those of the
living Polypterus and Lepidosteus, the prolongation and identity of
structure of the upper caudal lobe in Palæoniscus and the
Sturgeons, the presence of peculiar “fulcra” on the anterior margin of
the dorsal fin in Palæoniscus and Lepidosteus: inferring from these
facts that that fossil genus was allied either to the Sturgeons or to
Lepidosteus. But it did not occur to him that there was a close
relationship between those recent fishes. Lepidosteus and, with it,
the fossil genus remained in his system a member of the order of
Malacopterygii abdominales.
It was left to L. Agassiz (born 1807, died 1873) to point out the
importance of the character of the structure of the scales, and to
open a path towards the knowledge of a whole new sub-class of
fishes, the Ganoidei.
Impressed with the fact that the peculiar scales of Polypterus and
Lepidosteus are common to all fossil osseous fishes down to the
chalk, he takes the structure of the scales generally as the base for
an ichthyological system, and distinguishes four orders:—
1. Placoids.—Without scales proper, but with scales of enamel,
sometimes large, sometimes small and reduced to mere points
(Rays, Sharks, and Cyclostomi, with the fossil Hybodontes).
2. Ganoids.—With angular bony scales, covered with a thick
stratum of enamel: to this order belong the fossil Lepidoides,
Sauroides, Pycnodontes, and Coelacanthi; the recent Polypterus,
Lepidosteus, Sclerodermi, Gymnodontes, Lophobranches, and
Siluroides; also the Sturgeons.
3. Ctenoids.—With rough scales, which have their free margins
denticulated: Chætodontidæ, Pleuronectidæ, Percidæ, Polyacanthi,
Sciænidæ, Sparidæ, Scorpænidæ, Aulostomi.
4. Cycloids.—With smooth scales, the hind margin of which lacks
denticulation: Labridæ, Mugilidæ, Scombridæ, Gadoidei, Gobiidæ,
Murænidæ, Lucioidei, Salmonidæ, Clupeidæ, Cyprinidæ.
We have no hesitation in affirming that if Agassiz had had an
opportunity of acquiring a more extensive and intimate knowledge of