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RSC Nanoscience & Nanotechnology

Soft Nanoparticles for

Biomedical Applications

Edited by José Callejas-Fernández,

Joan Estelrich, Manuel Quesada-Pérez
and Jacqueline Forcada
Soft Nanoparticles for Biomedical Applications
RSC Nanoscience & Nanotechnology

Editor-in-Chief:
Paul O’Brien FRS, University of Manchester, UK

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Applications
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Soft Nanoparticles for
Biomedical Applications

Edited by

José Callejas-Fernández
University of Granada, Granada, Spain
Email: jcalleja@ugr.es

Joan Estelrich
Univeristy of Barcelona, Barcelona, Spain
Email: joanestelrich@ub.edu

Manuel Quesada-Pérez
University of Jaén, Jaén, Spain
Email: mquesada@ujaen.es

Jacqueline Forcada
University of the Basque Country, San Sebastián, Spain
Email: jacqueline.forcada@ehu.es
RSC Nanoscience & Nanotechnology No. 34

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Preface

The book is focused on soft nanoparticles, a specific area in the field of

nanotechnology. The activity in this field has grown exponentially worldwide
during the past couple of decades, becoming a major interdisciplinary area
of research. This growth is driven to a great extent by the integration of
nanotechnology into medical science, where it is opening up many new
possibilities. For instance, nanoparticles can be potential or actually used in
diagnostic tests (detecting extremely low concentrations of pathogens and
analytes), non-invasive imaging techniques and gene and drug delivery.
Among the existing soft nanoparticles, we have chosen those useful for
actual or potential biomedical applications. More specifically, our choice is a
compromise between well-established nanoparticles and promising candi-
dates under testing. On the other hand, we have included some soft nano-
particles that have been used for decades (e.g. liposomes), which have been
extensively described elsewhere.
Chapter 1 introduces us to the realm of soft nanoparticles and tells us how
some of these nanoparticles began to be employed in pharmaceuticals. This
chapter also outlines some colloidal aspects of these particles and sum-
marizes the possibilities that computer simulations offer in this field.
Concerning the biomedical applications of soft nanoparticles, great em-
phasis has been placed to date on their chemistry. However, physical
properties may also have a considerable influence. In fact, some authors
suggest the importance of improving physics-derived solutions (including
simulations) for controlling processes in which nanoparticles are involved.
Trying to fill this gap, Chapter 2 focuses on key physical properties of
nanoparticles (such as size, shape, surface charge and internal structure)
and the methods and techniques used for their measurement.

RSC Nanoscience & Nanotechnology No. 34

Soft Nanoparticles for Biomedical Applications
Edited by José Callejas-Fernández, Joan Estelrich, Manuel Quesada-Pérez and
Jacqueline Forcada
r The Royal Society of Chemistry 2014
Published by the Royal Society of Chemistry, www.rsc.org

vii
viii Preface

Magnetoliposomes are hybrid systems formed by encapsulating iron oxide

nanoparticles within liposomes. They are a platform that presents the ad-
vantages of being non-toxic and biocompatible and displaying magneto-
phoretic mobility. In other words, they can carry a drug within them and
under the influence of an external magnetic field can be directed to a tar-
geted area (tissues, cells, etc.) of the human body. The rationale for mag-
netoliposome-based targeting lies in the potential to reduce or eliminate the
side effects concomitant with the administration of some drugs. Chapter 3 is
devoted to the synthesis, characterization and applications of these soft
nanoparticles.
Micro/nanogels are cross-linked colloidal particles that can swell by ab-
sorption (uptake) of large amounts of solvent, but they do not dissolve owing
to the structure of the polymeric network, physically or chemically cross-
linked. Nanogels are considered good candidates for gene and drug delivery.
In their design, however, we should keep in mind that the cornerstone that
controls the uptake and release of the load is the interaction between the
molecules of the substance and the nanogel. If the cargo is an ionic species,
the nanogel charge and the electrostatic interaction play an essential role. In
any case, the encapsulation of a given solute inside the nanogel is a complex
process that depends on many physical and chemical parameters. These are
analysed in Chapter 4, which also presents a survey on the interactions that
govern the colloidal stability of these nanoparticles.
Polymeric micelles can be considered a type of nanoparticles with a core–
shell structure. The inner core is the hydrophobic part of a block copolymer,
which usually contains the poorly water-soluble drug. The outer shell is the
hydrophilic part of the block copolymer and its mission is to protect the drug
from the aqueous environment. Polymeric micelles are interesting owing
to their proved effectiveness for the specific delivery of anticancer drugs to
tumours. Chapter 5 describes the synthesis of suitable block copolymers to
design the desired micelles, their conversion into micelles, the character-
ization of the micelles formed and actual and future applications of these
soft nanoparticles.
Gene therapy represents a new paradigm of therapy for diseases, in which
the disease is treated at the molecular level by restoring defective biological
functions or reconstituting homeostatic mechanisms within cells. Specific
and efficient delivery of genetic material to diseased sites and to particular
cell populations is a challenge that is being addressed using a variety of non-
viral delivery systems, all of which have distinct advantages and disadvan-
tages. Substantial progress has been made in binding DNA to nanoparticles
or encapsulating DNA in and controlling the behaviour of these complexes.
In Chapter 6, recent advances in the major colloidal delivery carriers are
reviewed. The structure, synthesis, biological properties and the cellular
transfection capabilities of the different colloidal systems are discussed.
Dendrimers are another class of highly branched polymers having low
polydispersity. Usually, they have a core, branched units and surface groups
that provide building blocks for the design of delivery systems for various
Preface ix

applications. The molecular architecture of dendrimers can be controlled

precisely through synthetic methods, resulting in well-defined nano-
structures. Chapter 7 is devoted to these soft nanoparticles.
Bicelles are emerging as promising membrane models and, because of
their attractive combination of lipid composition, small size and morpho-
logical versatility, they have become new targets in skin research. These
nanoparticles are formed by two kinds of phospholipids, one with a large
hydrocarbon chain that forms bilayers over a wide range of temperature and
water content, and the other with a short hydrocarbon chain that generally
forms micelles. Binary mixtures of these phospholipids at a suitable molar
ratio tend to form disc-shaped particles known as bicelles (or bilayered
micelles). The use of bicelles for skin applications is clearly advantageous:
bicelles are constituted exclusively of lipids and have the ability to penetrate
through the narrow intercellular spaces of the stratum corneum of the skin
to reinforce its lipid lamellae. In addition, the bicelle structure allows for the
incorporation of different molecules that can be carried through the skin
layers. Bicelles are discussed in Chapter 8.
Hybrid nanoparticles consist of an inner part (core) and an outer part
(shell) and both can be organic- or inorganic-based materials. Among the
available core–shell nanoparticles, we have focused on those with a soft shell
(hard nanoparticles such as quantum dots are not considered). Nowadays, a
plethora of soft hybrid nanoparticles have been developed to target specific
biomedical applications and to perform desirable diagnostic and ther-
apeutic functions. As the reader can guess, there are also many methods that
have been developed to synthesize such soft hybrid nanoparticles. The
most important ones are briefly outlined in Chapter 9. Their relevance in
biomedical applications such as cancer and gene therapy, diagnosis and
bioimaging is concisely discussed in this chapter.
To finish, Chapter 10 offers an illustrative example of how coarse-grained
simulations can provide valuable information about the behaviour of
nanoparticles: the complexation of DNA-like polyelectrolytes and oppositely
charged spherical nanoparticles. In the first part of the chapter, different
computer simulation techniques are briefly discussed. In the second part,
the effects of surface charge density, ionic strength, pH, ion valence and
chain flexibility are considered.
The Editors express their gratitude to all the authors who have kindly
contributed to this work. The editors also acknowledge the financial support
from ‘Ministerio de Economı́a y Competitividad, Plan Nacional de Investi-
gación, Desarrollo e Innovación Tecnológica (I þ D þ i)’, Projects MAT2012-
36270-C04-01, -02, -03 and -04.
Contents

Chapter 1 Introductory Aspects of Soft Nanoparticles 1

Joan Estelrich, Manuel Quesada-Pérez, Jacqueline Forcada
and José Callejas-Fernández

1.1 Nanoparticles 1
1.2 Soft Nanoparticles 5
1.3 Colloidal Aspects of Soft Nanoparticles 8
1.4 Measuring the Properties of Soft Nanoparticles 9
1.5 Computer Simulations and Soft Nanoparticles 12
Acknowledgements 16
References 17

Chapter 2 Experimental Techniques Used for the Characterization

of Soft Nanoparticles 19
J. Callejas-Fernández, J. Ramos, O. Sanz, J. Forcada,
J. L. Ortega-Vinuesa, A. Martı́n-Molina, M. A. Rodrı́guez-
Valverde, M. Tirado-Miranda, A. Schmitt, B. Sierra-Martin,
A. Maldonado-Valdivia, A. Fernández-Barbero, R. Pons,
L. F. Capitán-Vallvey, A. Salinas-Castillo, A. Lapresta-Fernández,
B. Vázquez, M. R. Aguilar and J. San Román

2.1 Introduction 20
2.2 Imaging Techniques 21
2.2.1 Electron Microscopy 21
2.2.2 Transmission Electron Microscopy 21
2.2.3 Scanning Electron Microscopy 24
2.3 Techniques for the Determination of the Surface
Charge Density of Soft Nanoparticles 27

RSC Nanoscience & Nanotechnology No. 34

Soft Nanoparticles for Biomedical Applications
Edited by José Callejas-Fernández, Joan Estelrich, Manuel Quesada-Pérez and
Jacqueline Forcada
r The Royal Society of Chemistry 2014
Published by the Royal Society of Chemistry, www.rsc.org

xi
xii Contents
2.3.1 Foundations of Titration 27
2.3.2 Titration of Non-Penetrable Particles with
Only Strong Acid Groups on Their Surface 32
2.3.3 Titration of Non-Penetrable Particles with
Weak Groups on Their Surface 33
2.3.4 Titration of Non-Penetrable Particles with
Strong and Weak Acid Groups on Their
Surface 36
2.3.5 Dependence of Surface Charge Density on pH 37
2.3.6 Titration of Soft Colloidal Particles 39
2.4 Electrokinetic Techniques 42
2.4.1 Introduction 42
2.4.2 Electrophoretic Light Scattering 43
2.4.3 Charged Hard Sphere Limit 46
2.4.4 Charged Spherical Polyelectrolytes 48
2.5 Scattering Techniques 51
2.5.1 Introduction 51
2.5.2 Light Scattering 52
2.5.3 Neutron Scattering 67
2.5.4 X-Ray Scattering 74
2.6 Fluorescence 81
2.6.1 General Remarks on Luminescence and
Fluorescence 81
2.6.2 Fluorescence Techniques and Internal
Structure of Nanoparticles 83
2.7 NMR Spectroscopy 89
2.7.1 Introduction 89
2.7.2 Basic Principles of NMR 90
2.7.3 High-Resolution NMR Spectroscopy 91
2.7.4 NMR Spectroscopy in Solids 95
2.7.5 Magnetic Resonance Imaging 98
References 101

Chapter 3 The Original Magnetoliposomes: from the

Physicochemical Basics to Theranostic Nanomedicine 109
Marcel De Cuyper

3.1 Introduction 109

3.2 Magnetoliposomes 110
3.2.1 Magnetoliposomes: What Are They About? 110
3.2.2 Magnetizable Nanoparticles 112
3.2.3 Superparamagnetic Fluids 113
3.2.4 Preparation of Magnetoliposomes 113
3.2.5 Cationic Magnetoliposomes 114
3.2.6 Characterization of Magnetoliposomes 115
3.3 Magnetoliposome Uptake in Cells 117
Contents xiii
3.3.1Effect of Surface Charge 117
3.3.2Effect of Cationic Surfactant Content 117
3.3.3Incubation of Cationic Magnetoliposomes
with Various Cell Types 119
3.3.4 Uptake Mechanism 119
3.3.5 Cytotoxicity 120
3.4 Targeting of Magnetoliposomes 121
3.5 Magnetoliposomes as Theranostics 122
3.5.1 Diagnosis: Magnetoliposomes as MRI T2
Contrast Agents 123
3.5.2 Therapeutic Aspects 125
3.6 Conclusion 129
References 130

Chapter 4 Nanogels for Drug Delivery: the Key Role of Nanogel–Drug

Interactions 133
Jose Ramos, Miguel Pelaez-Fernandez, Jacqueline Forcada
and Arturo Moncho-Jorda

4.1 Introduction 133

4.2 Design of Nanogels for Drug Delivery 135
4.2.1 Basics Concepts of the Synthesis of Nanogels 135
4.2.2 Thermo-Responsive Nanogels 136
4.2.3 Multi-Responsive Nanogels 138
4.3 Interactions Between Nanogels and Biomolecules
or Drugs 138
4.3.1 Nanogel–Solute Electrosteric Interaction 140
4.3.2 Nanogel–Solute Specific Interaction 145
4.4 Stability of Nanogel Suspensions: Modelling the
Nanogel–Nanogel Interactions 147
4.4.1 London–van der Waals Interaction 148
4.4.2 Depletion Interaction 149
4.4.3 Elastic Repulsion 150
4.4.4 Electrostatic Interaction 151
4.5 Conclusion and Perspectives 152
References 153

Chapter 5 Polymeric Micelles 157

P. Taboada, S. Barbosa, A. Concheiro and C. Alvarez-Lorenzo

5.1 Introduction 157

5.2 Block Copolymer Synthesis and Characterization 158
5.2.1 Polymerization Methods 158
5.2.2 Block Copolymers with Complex
Architectures 162
xiv Contents
5.2.3 Polymer Characterization 163
5.3 Micelle Formation and Characterization 165
5.3.1 Thermodynamics of Micelle Formation and
Critical Micelle Concentration 165
5.3.2 Micellization of Block Copolymers 167
5.3.3 Characterization of the Micellization Process
and the Micelle Structure 172
5.3.4 Micelle Structure 179
5.3.5 Safety Evaluation 182
5.4 Pharmaceutical Applications 183
5.4.1 Drug Solubilization 183
5.4.2 Passive and Active Targeting 189
5.4.3 Stimuli-Responsive Drug Release 191
5.4.4 Biological Response Modulators 197
5.5 Conclusion 201
Acknowledgements 202
References 202

Chapter 6 DNA Particles 216

M. Carmen Morán

6.1 Introduction 216

6.2 Colloidal Delivery Systems for DNA 219
6.2.1 Polymeric Nanoparticles 219
6.2.2 Polymeric Micelles 221
6.2.3 Dendrimers 223
6.2.4 Liposomes 227
6.2.5 Solid Lipid Nanoparticles 232
6.2.6 Hydrogels 234
6.3 Prospects 237
Acknowledgements 238
References 238

Chapter 7 Dendrimers 246

A. J. Perisé-Barrios, D. Sepúlveda-Crespo, D. Shcharbin,
B. Rasines, R. Gómez, B. Klajnert-Maculewicz,
M. Bryszewska, F. J. de la Mata and M. A. Muñoz-Fernández

7.1 Introduction 246

7.2 Dendritic Structures 247
7.2.1 Synthesis of Dendrimers 249
7.2.2 Types of Dendritic Systems 250
7.2.3 Structural Characterization of Dendrimers
and Dendriplexes 258
7.3 Applications of Dendrimers in Nanomedicine 261
Contents xv
7.3.1 Dendrimers as Vectors for Gene Therapy 262
7.3.2 Dendrimers as Vectors for Targeted Drug
Delivery 264
7.3.3 Dendrimers as Therapeutic Agents 265
7.3.4 Dendrimers in Diagnostics 268
References 272

Chapter 8 Bicellar Systems: Characterization and Skin

Applications 280
Gelen Rodrı́guez, Lucyanna Barbosa-Barros,
Mercedes Cócera, Laia Rubio, Carmen López-Iglesias,
Alfons de la Maza and Olga López

8.1 Introduction 280

8.2 Bicelles and Bicellar Systems: Key Parameters 282
8.3 Morphological Transformations 284
8.3.1 DMPC–DHPC Bicelles 284
8.3.2 DPPC–DHPC Bicelles 294
8.4 Bicelle Suitability for Skin-Related Applications 299
8.4.1 Application of Bicelles to the Skin 301
8.5 Conclusion 306
References 307

Chapter 9 Soft Hybrid Nanoparticles: from Preparation to

Biomedical Applications 312
Talha Jamshaid, Mohamed Eissa, Nadia Zine,
Abdelhamid Errachid El-Salhi, Nasir M. Ahmad and
Abdelhamid Elaissari

9.1 Introduction 312

9.2 Development of Soft Hybrid Core-Shell
Nanoparticles 314
9.2.1 Polymer Immobilization on Preformed
Particles 314
9.2.2 Adsorption of Polymers on Colloidal Particles 315
9.2.3 Adsorption of Polymers via Layer-by-Layer
Self-Assembly 316
9.2.4 Adsorption of Nanoparticles on Colloidal
Particles 316
9.2.5 Chemical Grafting of Preformed Polymers 318
9.2.6 Polymerization from and on to Colloidal
Particles 319
9.2.7 Click Chemistry 322
9.2.8 Atom-Transfer Radical Polymerization
(ATRP) 323
xvi Contents
9.2.9Reversible Addition–Fragmentation
Chain-Transfer Radical (RAFT)
Polymerization 325
9.2.10 Nitroxide-Mediated Polymerization (NMP) 327
9.2.11 Conventional Seed Radical Polymerization 329
9.3 Biomedical Applications of Soft Hybrid Particles 330
9.3.1 Extraction of Nucleic Acids 330
9.3.2 Extraction of Proteins 332
9.3.3 Extraction of Viruses 334
9.4 Conclusion 335
References 336

Chapter 10 Computer Simulations of Soft Nanoparticles and

Their Interactions with DNA-Like Polyelectrolytes 342
Serge Stoll

10.1 Introduction 342

10.2 Computer Simulation Techniques 344
10.2.1 Molecular Dynamics 346
10.2.2 Brownian Dynamics 346
10.2.3 Monte Carlo Simulations 347
10.2.4 Coarse-Grain Models 347
10.2.5 Coarse-Grain Monte Carlo Model of
Polyelectrolyte and Nanoparticle Complex
Formation 349
10.3 Monte Carlo Simulations of Complex Formation
Between Nanoparticles and Polyelectrolyte Chains 352
10.3.1 Complexation Between a Strong Negatively
Charged Polyelectrolyte Chain and an
Oppositely Charged Nanoparticle 352
10.3.2 Complex Formation Between
Nanoparticles and Weak Polyelectrolyte
Chains 359
10.3.3 Effect of Solution Chemistry (Presence of
Di- and Trivalent Ions) on Complex
Formation 361
10.4 Conclusions and Outlook 367
Acknowledgements 368
References 368

Subject Index 372

CHAPTER 1

Introductory Aspects of Soft

Nanoparticles
JOAN ESTELRICH,*a MANUEL QUESADA-PÉREZ,b
JACQUELINE FORCADAc AND JOSÉ CALLEJAS-FERNÁNDEZd
a
Departament de Fisicoquı́mica, Facultat de Farmàcia, Universitat de
Barcelona, 08028 Barcelona, Spain; b Departamento de Fı́sica, Escuela
Politécnica Superior de Linares, Universidad de Jaén, 23700 Linares, Jaén,
Spain; c POLYMAT, Bionanoparticles Group, Departamento de Quı́mica
Aplicada, UFI 11/56, Facultad de Ciencias Quı́micas, Universidad del Paı́s
Vasco UP/EHU, Apdo. 1072, 20080 Donostia-San Sebastián, Spain; d Grupo
de Fı́sica de Fluidos y Biocoloides, Departamento de Fı́sica Aplicada,
Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain
*Email: joanestelrich@ub.edu

1.1 Nanoparticles
Nanotechnology is the science that deals with matter at the scale of 1 bil-
lionth of a metre (i.e. 109 m ¼ 1 nm) and is also the study of manipulating
matter at the atomic and molecular scale. A nanoparticle is the most fun-
damental component in the fabrication of a nanostructure and is far smaller
than the world of everyday objects that are described by Newton’s laws of
motion, but larger than an atom or a simple molecule that are governed by
quantum mechanics.
According to the definition of the International Organization for Stan-
dardization (ISO), a nanoparticle is a particle whose size spans the range
between 1 and 100 nm.1 Metallic nanoparticles have different physical and

RSC Nanoscience & Nanotechnology No. 34

Soft Nanoparticles for Biomedical Applications
Edited by José Callejas-Fernández, Joan Estelrich, Manuel Quesada-Pérez and
Jacqueline Forcada
r The Royal Society of Chemistry 2014
Published by the Royal Society of Chemistry, www.rsc.org

1
2 Chapter 1

chemical properties from bulk metals (e.g. lower melting points, higher
specific surface areas, specific optical properties, mechanical strengths and
magnetizations), properties that might prove attractive in various industrial
applications. However, how a nanoparticle is viewed and is defined depends
very much on the specific application. In this regard, for biomedical appli-
cations, structures and objects up to 1000 nm in size are included as
nanostructured materials used in medicine.2
Of particular importance, optical properties are among the fundamental
attractions and characteristics of a nanoparticle. For example, a 20 nm gold
nanoparticle has a characteristic wine-red colour, a silver nanoparticle is yel-
lowish grey and platinum and palladium nanoparticles are black. Not sur-
prisingly, the optical characteristics of nanoparticles have been used for
centuries in sculptures and paintings even before the fourth century AD. The
most famous example is the Lycurgus cup (fourth century AD). This cup, at
present in the British Museum in London, is the only complete historical ex-
ample of a special type of glass, known as dichroic glass, that changes colour
when held up to the light. When it is looked at in reflected light or daylight, it
appears green. However, when light is shone into the cup and transmitted
through the glass, it changes colour to red. This property puzzled scientists for
decades and the mystery was not solved until 1990, when researchers in
England scrutinized broken fragments under a microscope and discovered that
Roman artisans were nanotechnology pioneers: they had impregnated the glass
with a very small quantity of minute (B70 nm) colloidal silver and gold in an
approximate molar ratio of 14:1, which gives it these unusual optical properties.
Gold suspensions were familiar to alchemists in the Middle Ages and the
reputation of soluble gold was based mostly on its fabulous curative powers
against various diseases, for example, heart and venereal diseases, dysentery,
epilepsy and tumours. Metallic nanoparticles were used in mediaeval
stained glasses. The mediaeval artisans trapped gold nanoparticles in the
glass matrix in order to generate ruby-red colour in windows. They also
trapped silver nanoparticles, which gave the glass a deep-yellow colour.
Beautiful examples of these applications can be found in glass windows of
many Gothic European cathedrals.
In the seventeenth century, the so-called Purple of Cassius was highly
popular. It was a colloid made by reducing a soluble gold salt with stannous
chloride. It was used as a colorant and to determine the presence of gold as a
chemical test. The first scientific study of gold particles was carried out by
Faraday in 1857.3 He observed that gold suspensions with a ruby-coloured
appearance, made by reducing an aqueous solution of chloroaurate (AuCl4)
with phosphorus in CS2 (a two-phase system), changed their colour from red
to blue upon heating or addition of salt. Faraday correctly attributed the
colour change to an increase in the effective particle size caused by aggre-
gation. Since that pioneering work, thousands of scientific papers have been
published on the synthesis, modification, properties and assembly of metal
nanoparticles, using a wide variety of solvents and other substrates. Now-
adays, the most widely used nanotechnology product in the field of in vitro
Introductory Aspects of Soft Nanoparticles 3

diagnostics is colloidal gold in lateral flow assays, which is used in rapid

tests for pregnancy, ovulation, human immunodeficiency virus (HIV) and
other indications. Gold nanoparticles were introduced into these tests in the
late 1980s because gold conjugates have particularly high stability, which is
critical for avoiding false positives.
In 1959, Richard Feynman gave a talk entitled ‘There’s plenty of room at
the bottom’, where he predicted the new things and new opportunities that
one could expect in the very small world.4 Norio Taniguchi of Tokyo Uni-
versity of Science was the first to propose in 1974 the term ‘nanotechnology’.
The age of nanotechnology had begun.
The activity in the field of the nanotechnology has grown exponentially
worldwide during the past three decades, becoming a major interdisciplinary
area of research. This growth has been driven to a great extent by the inte-
gration of nanotechnology into the field of medical science, since nano-
structured materials have unique medical effects. The control of materials in
the nanometric range not only results in new medical effects but also requires
novel, scientifically demanding chemistry and manufacturing techniques.
This definition does not include traditional small-molecule drugs as they are
not specifically engineered on the nanoscale to achieve therapeutic effects
that relate to their nanosize dimensions. Nanoparticles have numerous
functional moieties on their surfaces capable of multivalent conjugation for
diagnostic, targeting, imaging and delivery of therapeutic agents (Figure 1.1).

Figure 1.1 Biomedical applications of nanoparticles.

4 Chapter 1

Owing to their unique characteristics, including large surface area,

structural properties and long circulation time in blood compared with
small molecules, nanoparticles have emerged as attractive candidates for
optimized therapy through personalized medicine. Potential advantages of
engineered therapeutic nanoparticles are the ability to convert unfavourable
physicochemical properties of bioactive molecules to desirable biopharma-
cological profiles, to improve the delivery of therapeutic agents across bio-
logical barriers and compartments, to control the release of bioactive agents,
to enhance therapeutic efficacy by selective delivery of drugs to biological
targets and to perform theranostic functions by combining multimodal
imaging and simultaneous diagnosis and therapy into multifunctional
nanoplatforms.5
A handful of nanomaterials and nanoparticles are being studied in clinical
trials or have already been approved by the US Food and Drug Adminis-
tration (FDA) for use in humans and many proof-of-concept studies of
nanoparticles in cell-culture and small-animal models for medical appli-
cations are under way.6 Examples of such nanoparticles and nanomaterials
are provided in Table 1.1.

Table 1.1 Nanoparticles and nanomaterials clinically approved, in clinical trials or

in proof-of-concept research stages.
Nanomaterial/nanoparticle Applications
Metallic
Iron oxide Magnetic resonance imaging/cancer therapy
Gold In vitro diagnostics/cancer therapy
Gold (nanorods, nanoshells, Cancer therapy, diagnosis
nanocages)
Carbon structures
Fullerenes Cancer therapy (photodynamic therapy)
Carbon nanotubes Fluorescence and photoacoustic imaging,
antioxidant
Ceramic nanoparticles
Silica Cancer therapy, diagnosis
Alumina Cancer therapy, diagnosis, computed
tomography
Semiconductor
Quantum dots Fluorescent contrast, in vitro diagnostics
Organic
Protein-based nanoparticles Cancer therapy
DNA-based nanoparticles Cancer therapy
Liposomes Cancer therapy
Polymer nanoparticles Cancer therapy
Polymer–drug conjugates Cancer therapy
Polymeric micelles Cancer therapy
Dendrimers Microbiocides, cancer therapy
Nanogels Gene and drug delivery
Bicelles Topical delivery
Hybrid
Magnetoliposomes Magnetic resonance imaging/cancer therapy
Introductory Aspects of Soft Nanoparticles 5

As can be deduced from Table 1.1, there is a plethora of nanoparticles

suitable for biomedical applications. This large number of nanoparticles is
due to recent developments in synthetic methods, which mostly involve
polymeric formulations, inorganic formulations or a combination of both.
The resulting organic–inorganic hybrid materials inherit properties both of
the polymers and of the metallic compounds.
Magnetoliposomes (liposomes encapsulating iron nanoparticles) are the
most relevant example of hybrid nanoparticles. In this book, however, we
will restrict ourselves to soft nanoparticles. More specifically, our choice is a
compromise between well-known particles (e.g. polymeric micelles) and new
particles whose potential is exciting but not yet widely proved.

1.2 Soft Nanoparticles

Classical micelles were the type of soft nanosystems used in pharmaceutical
applications long before the emergence of nanotechnology. As a result of
micelle formation, an organic compound that would normally be insoluble
in water can be ‘dissolved’ in a surfactant solution because it can move into
the oily interior of the micelle. This phenomenon, known as solubilization,
has been used for solubilize drugs. As a few representative examples,
phenolic compounds are frequently solubilized with soap to form clear
solutions, which are widely used for disinfection. Non-ionic surfactants are
efficient solubilizers of iodine. Such iodine–surfactant systems (referred to
as iodophors) are more stable than iodine–iodide systems. On the other
hand, the low solubility of steroids in water presents a problem in their
formulation for ophthalmic use. The requirement for optical clarity pre-
cludes the use of oily solutions or suspensions. The use of non-ionic sur-
factants permits the production of clear solutions, which are stable to
sterilization. This type of surfactant has also been used to solubilize essen-
tial oils and water-insoluble vitamins.7
Among the first nanotechnology drug-delivery systems were lipid vesicles,
which were described in the mid-1960s and later became known as lipo-
somes.8 At first, they were used to study biological membranes; several
practical applications, most notably in drug delivery, emerged in the 1970s
(in this period, Donald Tomalia invented, named and patented the den-
drimers, although they were not used as drugs until 1980s). Soon, however, it
was observed that liposomes suffered an important drawback when used as
carriers for therapeutically active compounds: they undergo rapid degrad-
ation due to the macrophage phagocyte system (MPS). As with all foreign
colloidal particles, liposomes are quickly recognized as ‘non-self’ and taken
up by the cells of the MPS, chiefly macrophages in the liver and spleen. This
leads to the inability to achieve sustained drug delivery over a prolonged
period of time.9 The incorporation in the bilayer of cholesterol and, mainly,
biocompatible, hydrophilic polymers with a flexible main chain, such as
poly(ethylene glycol) (PEG), led to long-circulating liposomes, also known
as sterically stabilized liposomes or Stealth liposomes.10 Doxil, the first
6 Chapter 1

nanomedicine to secure regulatory approval by the FDA (for the treatment of

AIDS-associated Kaposi’s sarcoma in 1995 and in Europe in 1997 with the
brand name Caelyx), was obtained by encapsulating doxorubicin within
liposomes. At present, liposomes are the most commonly used soft nano-
particles for clinical applications, especially in the treatment of cancer and
systemic fungal infections. The number of liposomal products on the market
or in advanced clinical studies exceeds two dozen. Apart from liposomes,
polymer-based nanoformulations constitute the majority of the nanoparticle
therapeutic agents available for clinical use. Polymer–drug conjugates are
another extensively studied nanoparticle drug-delivery platform currently in
clinical practice. Many polymers have been proposed as drug-delivery car-
riers, but only a few of them with linear architecture have been accepted into
clinical practice. PEG was first introduced into clinical use in the early 1990s.
Today, there are around a dozen examples of PEGylated drugs in clinical
practice. Other macromolecule–drug conjugates have also been developed as
drug carriers, such as Abraxane, a 130 nm albumin-bound paclitaxel that was
approved by the FDA in 2005 as a second-line treatment for patients with
breast cancer.11
Biodegradable polymeric micelles with a size of 10–200 nm have attracted
considerable attention as drug-delivery nanocarriers and have shown re-
markable therapeutic potential. Polymeric micelles are formed by self-
assembly of block copolymers consisting of two or more polymeric chains
with different hydrophobicity. These copolymers spontaneously assemble
into a core–shell micellar structure in an aqueous environment to minimize
the Gibbs energy.12
When short-chain phospholipids were combined with long-chain phos-
pholipids, structures closely related to liposomes and also with micelles were
found: the bicelles. They were used in the 1990s as a model membranes well
suited to magnetic resonance studies of membrane protein structure be-
cause of their ability to orient in a magnetic field, and have been used in
most NMR structural studies of transmembrane proteins. As a biomedical
tool, the use of bicelles has been proposed to favour the penetration of en-
capsulated drugs through the corneum stratus of the skin.13
Dendrimers have emerged as another novel class of drug-delivery soft
nanoparticle platform because of their well-defined architecture and unique
characteristics. The specific molecular structure of dendrimers enables them
to carry various drugs using their multivalent surfaces through covalent
conjugation or electrostatic adsorption. Alternatively, dendrimers can be
loaded with drugs using the cavities in their cores through hydrophobic
interaction, hydrogen bonds or chemical linkages.
As mentioned previously, there are promising candidates for nano-
particles whose clinical applications are still limited. Among them are
polymer-based soft nanoparticles, which are very interesting for use as
nanosized drug carriers. An ideal drug carrier needs to combine both the
targeting property and the stimulus responsiveness to enhance the bio-
availability of the drug together with the reduction of side effects. Therefore,
Introductory Aspects of Soft Nanoparticles 7

the design of stimuli-responsive nanoparticles for drug delivery to release

the drug in a controlled way when arriving at the targeted site is highly
desirable. Among polymer-based soft nanoparticles there are stimulus-
responsive nanoparticles or environment-sensitive nanoparticles having the
ability to change their size or volume when exposed to external changes or
signals. These nanoparticles are also known as micro/nanogels. Stimuli-
responsive soft nanoparticles are classified depending on the stimulus
(physical or chemical) into temperature, electric and magnetic field, light
intensity, pressure, pH, ionic strength, specific (bio)molecules or enzymes
and ultrasound-responsive nanoparticles. The development of soft stimu-
lus-responsive nanoparticles for biomedical applications relies on the
stimulus-sensitive polymer that constitutes their structure. Different
polymer synthesis techniques are used and advanced polymerization pro-
cesses have been developed to produce new stimuli-responsive nano-
particles, which are sensitive to other signals such as microwave, redox and
different chemical substances, in addition to those already mentioned.
Although the synthesis of new stimuli-responsive soft nanoparticles has
attracted considerable interest in recent decades, in particular in the case
of dual/multi-stimulus-responsive types, practical clinical applications re-
main limited except for thermo- and pH-sensitive nanoparticles with high
sensitivities.14
In recent years, several attempts to prepare thermo-responsive hybrid
micro/nanogels with inorganic silica cores have been reported.15–18 These
core–shell hybrid nanogels have interesting additional properties compared
with polymer nanogels, being promising candidates as controlled drug-
delivery vehicles in cancer therapies.18,19 From the synthetic point of view, in
almost all studies the preparation of these nanohybrids consisted of three
steps. First, the silica nanoparticles are prepared. The second step is the
functionalization of silica with a coupling agent, mainly 3-(trimethoxysilyl)
propylmethacrylate (TPM). This coupling agent generates a hydrophobic
surface on the silica particles and in addition can subsequently react by
radical polymerization, allowing chemical coupling between the polymer
network and the inorganic material. The last step consists of the well-known
emulsion polymerization used to synthesize nanogels. Ramos et al.20 re-
ported a facile synthesis of thermo-responsive nanohybrids with a silica core
and a poly(N-vinylcaprolactam) (PVCL)-based thermo-responsive shell. This
was achieved by a batch emulsion polymerization of VCL with TPM and N,N 0 -
methylenebisacrylamide (MBA) as cross-linkers. The hybrid character of
these thermo-responsive nanogels together with the excellent biocompati-
bility conferred by silica and PVCL makes them suitable as carriers for drug
delivery and biosensing.
As Lendlein and co-workers claimed,21 we should conclude that, in order
to bridge the gap between medical-grade material availability and material
demand, there needs to be clear communication between the scientists who
design and produce polymeric materials and those who implement them in
biomedical applications and finally in the clinic.
8 Chapter 1

Gene therapy is another field where soft nanoparticles might play a fun-
damental role in the near future. The concept of gene therapy appeared
formally in the 1970s but the first proof of in vitro gene transfer was not
reported until the 1980s, when white blood cells were extracted from the
body of a girl.22 Certain types of genes were implanted in such cells and then
they were transferred back to the girl’s body.23 An improvement in the im-
mune system of this person was reported. Since then and up to the present,
trials on various diseases have continued.
As genes cannot be directly inserted into a patient’s cells, they need a
shuttle (carrier), named vectors. Usually, these are classified into viral and
non-viral vectors. The first studies started with the use of a harmless virus
altered in the laboratory. The genes were inserted into the virus and then
they were mixed with the cells of the patient. The death of a patient in a
clinical trial24 and a blood disorder in children after another clinical trial
caused a delay in the advancement of these techniques.25 In any case, the
interest in the development of non-viral vectors has grown considerably in
recent times owing to this kind of risk. DNA particles are considered good
non-viral vectors.

1.3 Colloidal Aspects of Soft Nanoparticles

In spite of constituting a motley assortment, nanoparticles share a common
feature: they belong to the colloidal domain and we do not refer to size ex-
clusively. The percentage of atoms at the surface of a material becomes
significant when this material is split into nanosized particles. In fact, many
interesting properties of nanoparticles can be mostly attributed to the large
surface area of the material. The relevance of surface properties is a typical
feature of colloids. Owing to this large surface-to-volume ratio, the Gibbs
energy of a colloidal dispersion is generally higher than the Gibbs energy of
the bulk material. Hence a colloidal dispersion will tend to lower its Gibbs
energy unless there is some substantial energy barrier to prevent it, thus
keeping the system in a metastable state. In any case, the colloidal nature of
nanoparticles should not be neglected. In many cases, they are synthesized,
stored and used in more or less concentrated suspensions.
A long-standing issue in colloid science is the effective force between
colloidal particles,26,27 which is the result of different supramolecular (or
non-covalent) interactions. It should further be stressed that such inter-
actions also affect the supramolecular constituents of soft nanoparticles
(such as polymers, polyelectrolytes or lipids). In fact, the structure and
properties of isolated nanoparticles and their suspensions depend on these
constituents and the interactions between them. Some of these interactions
depend strongly on the specific type of supramolecular entity involved.
However, there are other forces that are rather non-specific, because they
affect all (or almost all) kinds of supramolecular entities. For instance, van
der Waals forces (due to the interaction between either permanent or in-
duced dipoles) are always present (unless the refractive indexes of the
Introductory Aspects of Soft Nanoparticles 9

dispersed and continuous phases are fairly similar). The excluded volume
repulsion between hard entities (due to the impossibility of any overlap) can
also be considered a non-specific interaction.
Polyelectrolytes and, in general, many macromolecules often carry an
electrical charge and therefore attract or repel each other. However, the
treatment of electrostatic forces between charged macromolecules (or
nanoparticles) is not a trivial matter, for several reasons. On the one hand,
the effective electrostatic interaction is screened by ions in aqueous solu-
tions, but the role of ions goes beyond screening. For example, in the case of
multivalent ions, strong ionic correlations can give rise to somewhat coun-
terintuitive phenomena, such as electrostatic attraction between like-
charged particles or (non-specific) charge inversion. On the other hand, the
shape and charge distribution also affect electrostatic ion-mediated forces.
For instance, the attraction between oppositely charged non-spherical pro-
teins with complementary shapes is considerably increased compared with
spherical particles.26 The situation becomes even more complex when highly
specific interactions are considered (such as hydrogen bonding). In some
cases, the role of the solvent is not limited to a mere dielectric screening, as
shown by a couple of examples: (1) water-mediated hydrophobic forces can
be responsible for the collapse of polymer chains in aqueous solutions; and
(2) water molecules play a key role in ion-specific effects (also known as
Hofmeister’s effects). Concerning nanoparticles, we should finally mention
that steric forces between polymer-covered nanoparticles produce an add-
itional repulsive force (which is predominantly entropic in origin), whereas
solutions containing a non-adsorbing polymer can modulate interparticle
forces, producing an attractive depletion force between particles.
In the case of charged soft nanoparticles, the role of electrostatic inter-
actions goes beyond colloidal stabilization. It is well established that such
interactions control processes in different areas of materials science. For
instance, the electrostatic attraction between oppositely charged supramo-
lecular entities is the foundation of assembly techniques (e.g. layer-by-layer
assembly) that allow the synthesis of many nanoparticles.28 However, elec-
trostatic interactions are also essential in the function of biological entities.
As a result of the charges of nucleic acids or proteins, the association of
biomolecules into functional units is strongly dependent on local ionic
concentrations. We should therefore expect that charge distributions will be
important for developing functional soft nanoparticles. For example,
asymmetric ionic profiles and interface charge excesses are crucial in the
organization and function of ionic hydrogels. These interfaces generate a
broad range of responses to external stimuli, including external electric
fields.29

1.4 Measuring the Properties of Soft Nanoparticles

In a general sense, many of the fundamental chemical and physical prop-
erties of nanoparticles (NPs), such as size, shape, surface charge and internal
10 Chapter 1

structure, might have a strong influence on a number of biomedical appli-

cations such as drug delivery, targeting or gene therapy. Concerning the
novel field of applications of NPs in medicine and biology, great emphasis
has been put on the chemistry of new NPs in the past, as some authors have
pointed out.30,31 However, they also suggest the importance of improving
physics-derived solutions (including simulations) for controlling processes
in which NPs are involved. At present, it is clear that the physical properties
mentioned above can have a great impact on the behaviour of NPs inserted
in a biological environment. For that reason, Chapter 2 focuses on key
physical properties of NPs and the methods and techniques used for their
measurement.
Particle size is the first property of a nanoparticle that has to be known.
Several important in vivo and in vitro functions of particles depend on their
size, as can be widely corroborated through this book and further literature.
A typical example can be found in Chapter 6, devoted to DNA particles and
their use in gene therapy. In all the cases studied, the transfection cap-
abilities of different DNA particles are strongly dependent on particles size.
The literature also provides many examples of this kind. Transport and
adhesion of nanoparticles in blood vessels, airways or gastrointestinal tract
is governed by the size of NPs.32,33 In passive and active targeting, as can be
seen in Chapter 5, the size of polymeric micelles makes them suitable as
drug carriers circulating in the bloodstream without losing their properties
and extravasing to specific tissues. Chapter 7 offers another example related
to dendrimers. The high level of control over the dendritic size makes them
ideal systems for enhanced solubility of poorly water-soluble drugs.
The shape of NPs is another valuable property that has to be known.
Particle shapes commonly observed include rods, spheres, filaments, toroids
and globules. Recent studies confirm the important role that this property
plays in biotechnology.34,35 It is worth mentioning some examples. In drug
delivery, phagocytosis by macrophages is strongly dependent on shape.36
The aspect ratio of cylindrical particles influences their degree of internal-
ization, as reported by Gratton et al.37 Geng et al.38 studied the effects of
particle shape (comparing filaments and spheres) on flow and drug delivery.
In comparing spheres ranging in size from 0.1 to 10 mm with an elliptical
disc up to 3 mm, Muro et al.39 concluded that the targeting efficiency of
spheres is less than that of any ellipsoidal disc. Indeed, DeCuzzi et al. also
emphasized the importance of particle geometry in processes such as
endocytosis or intravascular delivery.6,40 Chithrani et al.41 determined the
size and shape dependence of gold nanoparticle uptake into mammalian
cells. In the present book, Chapter 8, devoted to bicelles and their appli-
cation to the skin, provides another illustrative example: by varying the
bicelle lipid composition and/or lipid ratios, the resulting morphologies and
sizes may be varied to obtain more or less superficial effects. In the case of
dendrimers, by varying the composition of the core, the interior branches
and the surface functional groups, it is possible to obtain NPs with different
shapes and sizes that, in consequence, could be good candidates for
Introductory Aspects of Soft Nanoparticles 11

different applications. In DNA particles, when NPs are formed by lipids and
DNA, the possible packing arrangements of the lipid bilayers lead to the
formation of spherical, ellipsoidal or globular vesicles27,42,43 with the cor-
responding influence on the overall transfection efficiency of these systems.
Accordingly, the fundamental techniques used for the measurement of the
size and shape of NPs are presented in Chapter 2. They can be divided into
direct imaging techniques, such as scanning and transmission electron
microscopy (SEM and TEM), and indirect techniques, particularly those
based on the scattering of radiation and/or particles, such as light, neutrons
or X-rays.
Surface charge is another basic physical property of outstanding interest
for NPs. Several examples extracted from this book and from the literature
support this statement. Regarding the magnetoliposome (ML) uptake in
cells, the knowledge of the ML surface charge and the content of anionic-
cationic lipids used are of paramount interest. What is more, the cytotoxicity
of the ML is strongly influenced by the interaction of the cell medium with
the ML surface charge (see Section 3.3.5). In addition, cationic dendrimers
capable of condensing genetic material interact with negatively charged
membranes of cells. Indeed, as can be seen in Section 7.3.3, there is
strong evidence that the charged surface groups in cationic dendrimers
play a key role in the solubilization of protein aggregates, responsible for
Alzheimer’s disease, Huntington’s disease and Creutzfeldt–Jakob disease,
among others.
These examples illustrate the important effects that electrostatic inter-
actions derived from the surface charge of NPs have on their applications. In
our opinion, as Editors, we feel that this aspect has not been soundly ad-
dressed in reviews and books devoted to the application of NPs in bio-
medicine. This is why we decided to include a large section (Sections 2.3 and
2.4) in which the ion cloud surrounding a colloidal particle and the methods
for measuring the surface charge density of NPs are discussed.
In general, the internal and surface structure of NPs has been the focus
of attention of considerable research in recent years, in order to study the
relationship between the morphological characteristics of NPs and their
functional activity in the different processes in which they are involved. Soft
particles, as considered in this book, have non-homogeneous structures. MLs
are pearl-like objects with a shell formed by phospholipids that includes
grains of iron. Micro-nanogels are core–shell structures with the core formed
by a cross-linker and polymer networks and the shell mainly formed by
polymer chains. Polymeric micelles and dendrimers are more or less
branched structures, mainly characterized by holes inside them. Hence in
drug delivery they can bear a therapeutic agent that can be either dispersed
in the polymer matrix or encapsulated by the polymer. Consequently, the
knowledge of the internal and surface structures of the particles is a crucial
issue. DNA particles are structures that can be considered as aggregates
formed by a platform made of a colloidal particle plus nucleic acids (DNA,
RNA), but what about their internal structure? Bicelles, in a rough image,
12 Chapter 1

are considered as disk-shaped aggregates of two kinds of phospholipids.

Finally, core–shell hybrid particles can exhibit a broad variety of structures
where a drug can be encapsulated either in the shell or in the core.
Therefore, for experienced researchers and tyro students, it is of great
interest to establish methods for determining the structure of NPs. Again,
the scattering of light, neutrons or X-rays and also fluorescence and NMR
techniques are valuable tools.

1.5 Computer Simulations and Soft Nanoparticles

Computer simulations of nanoparticles are one of the greatest challenges
that soft matter science faces today owing to the time and length scales
involved in these systems. For the same reason, the level of detail employed
in computer simulations varies greatly. Two widely used representations of
reality in simulations are atomistic models and coarse-grained (CG) models.
The number of atomistic simulations, in which macromolecules and the
surrounding solvent are explicitly modelled in full detail, has grown recently
together with the size of computationally tractable systems. However, many
questions regarding soft matter involve length and time scales that signifi-
cantly exceed those that can be treated in atomistic simulations (not only
nowadays but also for many years to come). Hence simulating the solvent
explicitly is prohibitively expensive and avoided in many GC simulations. In
addition, a single bead can even represent a small group of atoms in this
kind of simulation (see Figure 1.2). In this way, the computational time is
reduced by orders of magnitude (compared with atomistic simulations) and
a meaningful exploration of the system is feasible. It should be stressed,
however, that there is also a wide variety of different approaches in coarse-
graining of soft materials, including integrated multiscale modelling, in
which several models at different scales are interconnected.44
In any case, computer simulations are currently a powerful tool for
achieving a better understanding of diverse aspects of soft nanoparticles,
and some illustrative examples related to nanoparticles studied in this book
are presented. Both atomistic and GC models have been employed in the
simulation of lipid bilayers.45 Their conclusions can be extremely useful for
the comprehension of liposomes, MLs and bicelles. In fact, bicelles have
been explicitly simulated.46 For instance, the effect of the system com-
position on the edge structure, the edge composition and the line tension
has been analysed.47 A considerable excess of the short-chain lipids at
the bilayer edge and a reduction in line tension were observed in bilayers
predominantly formed of the long-chain component. This enrichment of
short-chain lipids at the bicelle edge is corroborated by experiments. How-
ever, there is an excess of long-chain lipids near the edge and a slight de-
crease in line tension (compared with a bilayer of pure short-chain lipids) in
bilayers predominantly composed of short-chain lipids. Bicelles have also
been used as starting structures in GC simulations of the formation of
vesicles.48
Another random document with
no related content on Scribd:
cubría de los pies á la cabeza, y
en la mano derecha una lanza
pintada de colores. Amainaron
entrambos las velas, y á fuerza de
remos arremetieron el uno contra
el otro con furia muy grande.
Movióse grande alarido de las
Nymphas y salvajes, y de los que
con sus voces los favorescían.
Los remeros emplearon allí todas
sus fuerzas, procurando los unos
y los otros llevar mayor ímpetu y
hacer más poderoso encuentro. Y
viniéndose á encontrar los
salvajes con las lanzas en los
escudos, era cosa de gran deleite
lo que les acaescía. Porque no
tenían tantas fuerzas ni destreza,
que con la furia con que los
barcos corrían y con los golpes de
las lanzas quedassen en pie, sino
que unas veces caían dentro de
los bajeles y otras en el río. Con
esto allí se movía la risa, el
regocijo y la música, que nunca
cessaba. Los justadores la vez
que caían en el agua iban
nadando, y siendo de las
Nymphas de su parcialidad
recogidos, volvían otra vez á
justar, y cayendo de nuevo,
multiplicaron el regocijo. Al fin el
barco de carmesí vino con tanta
furia y su justador tuvo tanta
destreza, que quedó en pie,
derribando en el río á su
contrario. A lo cual las Nymphas
de su escuadrón levantaron tal
vocería y dispararon tan extraña
música, que las adversarias
quedaron algo corridas, y
señaladamente un salvaje
robusto y soberbio, que afrentado
y muy feroz dijo: ¿Es possible que
en nuestra compañía haya
hombre de tan poca habilidad y
fuerza que no pueda resistir á
golpes tan ligeros? Quitadme,
Nymphas, esta cadena, y sirva en
mi lugar por remero quien ha sido
tan flojo justador, veréis cómo os
dejaré á vosotras vencedoras y á
las contrarias muy corridas. Dicho
esto, librado por una hermosa
Nympha de la cadena, con un
bravo denuedo tomó la lanza y el
escudo, y púsose en pie sobre la
proa. A la hora los salvajes con
valerosos ánimos comenzaron á
remar, y las Nymphas á mover
grande vocería. El contrario barco
vino con el mesmo ímpetu, pero
su salvaje no hubo menester
emplear la lanza para quedar
vencedor, porque el justador, que
tanto había braveado, antes que
se encontrassen, con la furia que
su barco llevaba, no pudo ni supo
tenerse en pie, sino que con su
lanza y escudo cayó en el agua,
dando claro ejemplo de que los
más soberbios y presumptuosos
caen en mayores faltas. Las
Nympas le recogieron, que iba
nadando, aunque no lo merescía.
Pero los cinco barcos de morado
que aparte estaban, viendo su
compañero vencido, á manera de
afrentados todos arremetieron.
Los otros cinco de carmesí
hicieron lo mesmo, y comenzaron
las Nymphas á tirar
muchedumbre de pelotas de cera
blanca y colorada, huecas y
llenas de aguas olorosas,
levantando tal grita y peleando
con tal orden y concierto, que
figuraron allí una reñida batalla,
como si verdaderamente lo fuera.
Al fin de la cual los barcos de la
devisa morada mostraron quedar
rendidos, y las contrarias
Nymphas saltaron en ellos á
manera de vencedoras, y luego
con la mesma música vinieron á
la ribera, y desembarcaron las
vencedoras y vencidas con los
captivos salvajes, haciendo de su
beldad muy alegre muestra.
Passado esto, Felicia se volvió á
la fuente donde antes estaba, y
Eugerio y la otra compañía,
siguiéndola, hicieron lo mesmo. Al
tiempo que vinieron á ella,
hallaron un pastor que en tanto
que había durado la justa había
entrado en la huerta y se había
sentado junto al agua.
Parescióles á todos muy gracioso,
y especialmente á Felicia, que ya
le conoscía, y ansí le dijo: A mejor
tiempo no pudieras venir, Turiano,
para remedio de tu pena y para
augmento desta alegría. En lo
que toca á tu dolor, después se
tratará, mas para lo demás
conviene que publiques cuanto
aproveche tu cantar. Ya veo que
tienes el rabel fuera del zurrón,
paresciendo querer complacer á
esta hermosa compañía; canta
algo de tu Elvinia, que dello
quedarás bien satisfecho.
Espantado quedó el pastor que
Felicia le nombrasse á él y á su
zagala, y que á su pena alivio
prometiesse; pero pensando
pagarle más tales ofrescimientos
con hacer su mandado que con
gratificarlos de palabras, estando
todos assentados y atentos, se
puso á tañer su rabel y á cantar lo
siguiente:

Rimas provenzales.
Cuando con mil colores
devisado
viene el verano en el ameno
suelo,
el campo hermoso está,
sereno el cielo,
rico el pastor y próspero el
ganado.
Philomena por árboles floridos
da sus gemidos:
hay fuentes bellas,
 y en torno dellas,
cantos suaves
de Nymphas y aves.
Mas si Elvinia de allí sus
ojos parte,
habrá contino hibierno en
toda parte.

Cuando el helado Cierzo de

hermosura
despoja hierbas, árboles y
flores,
el canto dejan ya los
ruiseñores
y queda el yermo campo sin
verdura.
Mil horas son más largas que
los días
las noches frías,
espessa niebla
con la tiniebla
escura y triste
el aire viste.
Mas salga Elvinia al campo,
y por doquiera
renovará la alegre
primavera.

Si alguna vez envía el cielo

airado
el temeroso rayo ó bravo
trueno,
está el pastor de todo
amparo ajeno,
triste, medroso, atónito y
turbado.
Y si granizo ó dura piedra
arroja,
la fruta y hoja
gasta y destruye,
el pastor huye
á passo largo,
triste y amargo.
Mas salga Elvinia al campo,
y su belleza
desterrará el recelo y la
tristeza.

Y si acaso tañendo estó ó

cantando
á sombra de olmos ó altos
valladares,
y están con dulce acento á
mis cantares
la mirla y la calandria
replicando;
Cuando suave expira el fresco
viento,
cuando el contento
más soberano
me tiene ufano,
libre de miedo,
lozano y ledo:
si assoma Elvinia airada,
assí me espanto,
que el rayo ardiente no me
atierra tanto.

Si Delia en perseguir silvestres

fieras,
con muy castos cuidados
ocupada
va de su hermosa escuadra
acompañada,
 buscando sotos, campos y
riberas;
Napeas y Hamadriadas
hermosas
con frescas rosas
le van delante,
está triunfante
con lo que tiene;
pero si viene
al bosque donde caza
Elvinia mía,
parecerá menor su lozanía.

Y cuando aquellos miembros

delicados
se lavan en la fuente
esclarescida,
si allí Cyntia estuviera, de
corrida
los ojos abajara
avergonzados.
Porque en la agua de aquella
transparente
y clara fuente
el mármol fino
y peregrino
con beldad rara
se figurara,
y al atrevido Acteon, si la
viera,
no en ciervo, pero en
mármol convertiera.

Canción, quiero mil veces

replicarte
en toda parte,
por ver si el canto
 amansa un tanto
mi clara estrella,
tan cruda y bella.
Dichoso yo si tal ventura
hubiesse
que Elvinia se ablandasse ó
yo muriesse.

No se puede encarescer lo que

les agradó la voz y gracia del
zagal, porque él cantó de manera,
y era tan hermoso, que paresció
ser Apolo, que otra vez había
venido á ser pastor, porque otro
ninguno juzgaron suficiente á
tanta belleza y habilidad.
Montano, maravillado desto, le
dijo: Grande obligación tiene,
zagal, la pastora Elvinia, de quien
tan subtilmente has cantado, no
sólo por lo que gana en ser
querida de tan gracioso pastor
como tú eres, pero en ser sus
bellezas y habilidades con tan
delicadas comparaciones en tus
versos encarescidas. Pero siendo
ella amada de ti, se ha de
imaginar que ha de tener última y
extremada perfección, y una de
las cosas que más para ello la
ayudarán, será la destreza y
ejercicio de la caza, en la cual con
Diana la igualaste, porque es una
de las cosas que más belleza y
gracia añaden á las Nymphas y
pastoras. Un zagal conoscí yo en
mi aldea, y aun Ismenia y
Selvagio también le conoscen,
que, enamorado de una pastora
nombrada Argía, de ninguna
gentileza suya más captivo
estaba que de una singular
destreza que tenía en tirar un
arco, con que las fieras y aves
con agudas y ciertas flechas
enclavaba. Por lo cual el pastor,
nombrado Olympio, cantaba
algunas veces un soneto sobre la
destreza, la hermosura y crueldad
de aquella zagala, formando entre
ella y la Diosa Diana y Cupido un
desafío de tirar arco, cosa harto
graciosa y delicada, y por
contentarme mucho le tomé de
cabeza. A esto salio Clenarda
diciendo: Razón será, pues, que
tengamos parte de esse contento
con oirle. A lo menos á mí no me
puede ser cosa más agradable
que oirtele cantar, siquiera por la
devoción que tengo al ejercicio de
tirar arco. Pláceme, dijo
Montano, si con ello no he de ser
enojoso. No puede, dijo
Polydoro, causar enojo lo que
con tan gran contento será
escuchado. Tocando entonces
Montano un rabel, cantó el
soneto de Olympio, que decía:

Soneto.
 Probaron en el campo su
destreza
Diana, Amor y la pastora
mía,
flechas tirando á un árbol,
que tenía
pintado un corazón en la
corteza.
Allí apostó Diana su belleza,
su arco Amor, su libertad
Argía,
la cual mostró en tirar más
gallardía,
mejor tino, denuedo y
gentileza.
Y ansí ganó á Diana la
hermosura,
las armas á Cupido, y ha
quedado
tan bella y tan cruel desta
victoria,
Que á mis cansados ojos su
figura,
y el arco fiero al corazon
cuitado
quitó la libertad, la vida y
gloria.

Fué muy agradable á todos este

soneto, y más la suavidad con
que por Montano fué cantado.
Después de consideradas en
particular todas sus partes, y
passadas algunas pláticas sobre
la materia dél, Felicia, viendo que
la noche se acercaba,
paresciéndole que para aquel día
sus huéspedes quedaban asaz
regocijados, haciendo señal de
querer hablar, hizo que la gente,
dejado el bullicio y fiesta, con
ánimo atento se sossegasse, y
estando todos en reposado
silencio, con su acostumbrada
gravedad habló ansí:
Por muy averiguado tengo,
caballeros y damas, pastores y
pastoras de gran merescimiento,
que después que á mi casa
venísteis, no podréis de mis
favores ni de los servicios de mis
Nymphas en ninguna manera
quejaros. Pero fué tanto el deseo
que tuve de complaceros y el
contento que recibo en que
semejantes personas le tengan
por mi causa, que me paresce
que, aunque más hiciera, no
igualara de gran parte lo mucho
que merescéis. Solos quedan
entre vosotros descontentos
Narcisso con la aspereza de
Melisea y Turiano con la de
Elvinia. A los cuales por agora les
bastará consolarse con la
esperanza; pues mi palabra, que
no suele mentir, por la forma que
más les conviene, presta y
cumplida salud ciertamente les
promete. A Eugerio veo alegre
con el hijo, hijas y yerno, y tiene
razón de estallo, despues que á
causa dellos se ha visto en tantos
peligros y ha sufrido tan fatigosas
penas y cuidados.
Acabadas las razones de Felicia,
el viejo Eugerio quedó espantado
de tal sabiduría, y los demás
satisfechos de tan saludable
reprensión, sacando della
provechoso fruto para vivir de allí
adelante muy recatados. Y
levantándose todos de entorno la
fuente, siguiendo á la sabia,
salieron del jardín, yendo al
palacio á retirarse en sus
aposentos, aparejando los ánimos
á las fiestas del venidero día. Las
cuales y lo que de Narcisso,
Turiano, Tauriso y Berardo
acontesció, juntamente con la
historia de Danteo y Duardo,
portugueses, que aquí por
algunos respetos no se escribe, y
otras cosas de gusto y de
provecho, están tratadas en la
otra parte deste libro, que antes
de muchos días, placiendo á
Dios, será impressa.

FIN DE LA DIANA ENAMORADA

DE GASPAR GIL POLO
 EL PASTOR DE FILIDA

COMPUESTO POR

LUIS GÁLVEZ DE
MONTALVO
GENTIL-HOMBRE CORTESANO

CARTA DEDICATORIA DEL

AUTOR AL MUY ILUSTRE
SEÑOR DON ENRIQUE DE
MENDOZA Y ARAGÓN

Considerando que desde el

tiempo que U. S. se criaba en
casa de sus excelentíssimos
abuelos, aquel gran Duque del
Infantado, tan digno deste
nombre, y aquella gran señora,
digna hija del Infante Fortuna,
siempre U. S. fué amador de la
virtud; y siempre, desde aquella
edad tierna, ha ido
resplandeciendo en su pecho la
gloriosa llama de su sangre, hasta
ser el mayor testimonio della, de
dó nace ser U. S. entre los suyos
el más virtuoso de los ricos y el
más rico de los virtuosos, con
aquel don del cielo que por mayor
premio el mundo puede dar:
amado de grandes y menores, y
de todos conocidas las
excelencias con que fué criado,
sin que rabia de tiempo ni rigor de
envidia lo puedan negar ni
deshacer. Entre los venturosos
que á U. S. conocen y tratan, he
sido yo uno, y estimo que de los
más, porque deseando servir á U.
S. se cumplió mi deseo, y assi
dejé mi casa y otras muy
señaladas, dó fui rogado que
viviesse, y vine á ésta, donde
holgaré de morir, y donde mi
mayor trabajo es estar ocioso,
contento y honrado, como criado
de U. S. Y assi, á ratos
entretenido en mi antiguo ejercicio
de la divina alteza de la Poesía,
donde son tantos los llamados y
tan pocos los escogidos, he
compuesto El Pastor de Filida,
libro humilde y pequeño,
digníssimo de su nombre, de
aquel favor con que U. S. suele
amparar á los necessitados dél,
en lo cual fiado se le ofrezco, rudo
y mal ataviado, como viene de las
Selvas, para que U. S. le
despierte y componga de su
mano, que cuanto es soberbio en
pensamientos, es humilde en
voluntad; y sabrá conocer la
merced que se le hiciere, sin
miedo de que nadie le ose enojar;
y yo que le envío, me atreveré á
trocar su zampoña en trompeta
heroica, que cante el bien que el
mundo de U. S. tiene y espera:
cuya muy ilustre persona y estado
nuestro Señor guarde y
acreciente, como todo el mundo
desea. De Madrid, y Febrero 20
de 1582.
Las muy ilustres manos de U. S.
besa su criado
Gálvez de Montalvo.

EL AUTOR AL LIBRO
Pastor de mis
pensamientos,
guardador de mis cuidados,
si quieres trocar los prados
por soberbios aposentos,
seráte fuerza volar
sin alas con que subir,
y habréme de lastimar,
de mí, por verte partir;
de ti, por verte quedar.
Dejarás la gravedad;
no me parezcas en esto;
también será deshonesto
que pierdas mi autoridad.
Si te vieres en aprieto,
mostraréte á ser bastante
para quedar sin defeto,
sei con el necio arrogante
y humilde con el discreto.
Cuando entre damas te
vieres,
honestas, sabias, hermosas,
encubrirás cuantas cosas
contra su opinion tuvieres;
mas si te catan los senos
y en sus orejas dissuenas,
diles, con ojos serenos,
que si todas fueran buenas
las buenas valdrían menos.
No llevas capas, ni ornatos
de Parnassos, ni Helicones,
que por mis pobres rincones
apenas tenías zapatos.
Y si los Faunos acaso
por los montes te encontraren,
passa quedo, habla passo;
que donde ellos agradaren
harán de ti poco caso.
No te quiero yo obligar
á hablar de mí por tassa;
que lo que passa ó no passa,
ya sé que lo has de contar;
y si causares porfía
con lo que te enseño yo,
bajarás la fantasía,
y di que el que te enseñó
quizá menos lo entendía.
Si te aprobaren los más,
no te mueva hichazón,
que la perfeta eleción
en los menos la verás;
pero si los pocos ves
contar tus hechos por vanos,
no pretendas tu interés,
ni te cures de las manos,
que más te valdrán los pies.
Para derramar tus obras,
no tomes larga carrera:
si agradas, vas tras do quiera,
si enfadas, do quiera sobras.
Donde tus prendas están
no temas los enemigos,
y si te ves en afán
acógete á mis amigos,
que éstos no te faltarán.
No quiero negarte aquí,
que otro gallo me cantara
si á mí se me aconsejara
lo que te aconsejo á ti;
lo que sé te significo,
haz lo que será cordura,
no puedo dejarte rico;
mas si tuvieres ventura,
podrás valer por tu pico.
Bien conviene que
recuerden
los Hados á te ayudar,
si te tienes de ganar
por lo que tantos se pierden,
podría ser que muriesses
como han hecho más de dos;
ó tantos siglos viviesses,
que hoy pidiesses por Dios,
y tú mañana lo diesses.
Si se rompiere la hebra
de mi nombre y de tu vida,
la hechura irá perdida,
como vidrio que se quiebra.
Y pues de vivir honrado
te partes tan sospechoso,
no debes juzgar tu estado
por larga vida dichoso,
ni por corta desdichado.
Mas ¡ay! que me llevas
cuanto
me tenía enriquecido,
 que como lo he padecido
por fuerza lo estimo en tanto,
y otras prendas que no
cuento,
que parece poco seso
mezclarlas en este intento;
mas van para contrapeso,
porque no te lleve el viento.
Ora cantes, ora llores,
ora provoques á risa,
siempre será tu devisa:
la causa de mis dolores.
Este es el blasón que quiero,
y dél quiero que presumas;
y en lo demás te requiero,
que te faltarán las plumas
si te picas de altanero.

CENSURA
Por comissión de los Señores del
Consejo de su Majestad, he visto
este libro, cuyo título es El
Pastor de Filida, compuesto por
Luis Gálvez de Montalvo, en
prosa y verso castellano; y
habiéndole passado con atención,
me parece no sólo digno de salir
á luz, en conformidad de la
pretensión de su autor, más aun
que me parece, por su pureza,
propiedad, facilidad y dulzura, por
la novedad de las invenciones,
por la orden y disposición con que
las trata, ser estimado por uno de
los más aceptos que hasta ahora
en este género han salido á juicio
del mundo; y aunque la materia,
siendo pastoril y amorosa, parece
que de suyo requiere humildad y
llaneza, no le ha costado tan poco
guardar el decoro que en ella se
pide, que no haya hecho por igual
el estilo y acomodarle al propósito
que se sigue, guardando las
partes á él necessarias, todo lo
que, con mucho estudio, de un
aventajado ingenio se puede
esperar: y assí, libre de pasión,
me parece que se le debe
conceder la licencia que pide. En
Madrid á dos de Junio de 1581.
Pedro Laínez.

PRIMERA PARTE
DEL PASTOR DE FILIDA

Cuando de más apuestos y

lucidos pastores florecía el Tajo,
morada antigua de las sagradas
Musas, vino á su celebrada ribera
el caudaloso Mendino, nieto del
gran rabadán Mendiano, con cuya
llegada el claro río ensoberbeció
sus corrientes: los altos montes
de luz y gloria se vistieron; el fértil
campo renovó su casi perdida
hermosura, pues los pastores dél,
incitados de aquella sobrenatural
virtud, de manera siguieron sus
pisadas que, envidioso Ebro,
confuso Tormes, Pisuerga y
Guadalquivir admirados,
inclinaron sus cabezas, y las
hinchadas urnas manaron con un
silencio admirable: sólo el felice
Tajo resonaba, y lo mejor de su
son era Mendino, cuya ausencia
sintió de suerte Henares, su
nativo río, que con sus ojos
acrecentó tributo á las arenas de
oro. Bien le fué menester al
gallardo pastor, para no sentir la
ausencia de su caríssimo
hermano, hallar en esta ribera al
gentil Castalio, su primo, al
caudaloso Cardenio, al galán
Coridón, con otros muchos
valerosos pastores y rabadanes,
deudos y amigos de los suyos,
con quien passaba dulce y
agradable vida Mendino, en quien
todos hallaban tan cumplida
satisfación, que como olvidados
de sus propias cabañas, sitios y
albergues, los de Mendino
estaban siempre acompañados
de la mayor nobleza de la