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NanoScience and Technology
Peter Gehr
Reinhard Zellner Editors
Biological
Responses
to Nanoscale
Particles
Molecular and Cellular Aspects and
Methodological Approaches
Series Editors
Phaedon Avouris, IBM Research – Thomas J. Watson Research, Yorktown
Heights, NY, USA
Bharat Bhushan, Mechanical and Aerospace Engineering, The Ohio State
University, Columbus, OH, USA
Dieter Bimberg, Center of NanoPhotonics, Technical University of Berlin,
Berlin, Berlin, Germany
Klaus von Klitzing, Max Planck Institute for Solid State Research, Stuttgart,
Baden-Württemberg, Germany
Cun-Zheng Ning, Electrical, Computer, and Energy Engineering, Arizona State
University, Tempe, AZ, USA
Roland Wiesendanger, Department of Physics, University of Hamburg,
Hamburg, Hamburg, Germany
The series NanoScience and Technology is focused on the fascinating nano-world,
mesoscopic physics, analysis with atomic resolution, nano and quantum-effect
devices, nanomechanics and atomic-scale processes. All the basic aspects and
technology-oriented developments in this emerging discipline are covered by
comprehensive and timely books. The series constitutes a survey of the relevant
special topics, which are presented by leading experts in the field. These books will
appeal to researchers, engineers, and advanced students.
More information about this series at http://www.springer.com/series/3705

Peter Gehr Reinhard Zellner
•
Editors
Biological Responses
to Nanoscale Particles
Molecular and Cellular Aspects
and Methodological Approaches
123
Editors
Peter Gehr Reinhard Zellner
Institute of Anatomy Institute of Physical Chemistry
University of Bern University of Duisburg-Essen
Bern, Switzerland Essen, Germany
ISSN 1434-4904 ISSN 2197-7127 (electronic)

ISBN 978-3-030-12460-1 ISBN 978-3-030-12461-8 (eBook)
https://doi.org/10.1007/978-3-030-12461-8
Library of Congress Control Number: 2018968395
© Springer Nature Switzerland AG 2019

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,
recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission
or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar
methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this
publication does not imply, even in the absence of a specific statement, that such names are exempt from
the relevant protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this
book are believed to be true and accurate at the date of publication. Neither the publisher nor the
authors or the editors give a warranty, expressed or implied, with respect to the material contained
herein or for any errors or omissions that may have been made. The publisher remains neutral with regard
to jurisdictional claims in published maps and institutional affiliations.
This Springer imprint is published by the registered company Springer Nature Switzerland AG
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Foreword
Engineered nanoparticles, as one of the principal components of the twenty-first-century

nanomaterials toolbox, have had a transformative impact on research, research transla-
tion, and development of a wide range of applications in physics, optics, electronics, and
medicine. The research interest in nanoparticles is intense as a result of the emergence and
use of newly acquired size-dependent properties, including quantum confinement in
semiconductor particles, photoactivation in TiO2 and ZnO, surface plasmon resonance in
metals and metal oxides, super-hydrophobicity from surface coatings with organic
molecules, super-paramagnetism of magnetic materials, a dynamic range of surface redox
active properties, oxidative/reductive surface dissolution with the release of reactive ionic
groups, or controllable colloidal behaviors (aggregation, dispersion, diffusion, sorption).
These nanoscale properties can be introduced into new nanoparticles by bottom-up
synthetic techniques, including hydrothermal synthesis, pyrolysis, chemical precipitation,
ion implantation, gas condensation, supramolecular assembly, etc. Moreover, it is also
possible to use top-down methods for the size reduction of macroscale or microscale
particles through techniques such as ball milling. While pristine nanoparticles may be
used as such in the form of powders or suspensions, these materials are often embedded,
conjugated, or incorporated into more complex matrices such as polymers, clothes,
building materials, cosmetics, foods, polymers, electronic circuits, etc.
From the perspective of material properties and functions, nanoparticles can be
thought of as a link between bulk materials and atomic or molecular structures. A key
consideration for the use of nanoparticles in a wide range of industrial and biomedical
applications is the interfacial properties of the particle surface in relation to the
surrounding medium. While the interfacial layer characteristically consists of metallic
surfaces, ions, and organic and inorganic molecules, inorganic particle surfaces are
frequently coated by organic molecules, which serve as capping agents, stabilizers,
surface ligands, or passivating agents. In order to fully understand the use and
interfacial behavior of nanoparticles, it is necessary to characterize newly emerging
materials by an array of physicochemical techniques such as microscopy (e.g., optical
microscopy, transmission electron microscopy, scanning probe microscopy), ele-
mental analysis, spectroscopy (e.g., X-ray, UV-Vis, infrared and nuclear magnetic
resonance spectroscopy, mass spectroscopy), light scattering methods (e.g., laser,
v
vi Foreword
X-ray or neutron scattering), electrophoresis (e.g., zeta potential and charge), the
Brunauer–Emmett–Teller method (to assess surface area), and X-ray diffraction
analysis (to assess crystal structure).
In this challenging book project by Gehr and Zellner, the Biological Responses to
Nanoscale Particles, a number of leading scientists have combined expertise to delineate
the behavior of nanoparticles on a wide range of nano/bio-interfaces, which ranges from
the effects on biomolecules (e.g., the protein corona) to interactions with cells, tissue
barriers, and organs. The authors delineate the importance of a variety of intrinsic
nanomaterial properties (such as chemical composition, size, shape, dimensions,
zeta potential), which are dynamically altered in the presence of biological
media to acquire an additional set of extrinsic material properties
(e.g., a protein corona, colloidal stability, hydrodynamic diameter, charge, dissolution
properties) to shape the outcome at the nano/bio-interface. The various chapters lead us
through the cellular uptake mechanisms for nanoparticles, subcellular localization and
processing, as well as interactions with cell biology and signaling pathways. The spec-
trum of interactions includes the catalysis of biological advantageous outcomes as well as
the generation of adverse cellular effects through the generation of oxidative stress and
genotoxicity. Interestingly, while the generation of danger signals in the immune system
could result in adverse outcomes, it is equally possible to use these danger signals
gainfully to improve vaccination responses, in addition to the use of nanoparticles to
improve antigen delivery. There is also a delineation of the use of nanoscale properties to
study cellular structure and function, including to develop novel advanced cellular
imaging techniques such as optical microscopy, Raman microscopy, optical
near-field microscopy, X-ray microscopy, and spectral microscopy. We also obtain
new insight into the use of nanoparticle physicochemical properties to control fate
and transport in the gastrointestinal tract, with applications in the food industry and
food processing. There is also a description of the nanoparticle interactions with the
skin, including the ability to penetrate into the stratum corneum and the follicular
ducts, but not into the deeper layers. Turning to the use of nanoparticles for thera-
peutic purposes, we learn about the versatility of the design, synthesis, and charac-
terization of polymeric nanocarriers for drug delivery applications, including the use
of nanoscale design features for stimulus–response coupling leading to drug release.
I appreciate the hard work of the editors, who spared no effort in the develop-
ment and successful conclusion of an exciting book project. The readers will
receive an excellent overview of new ideas, findings, and applications of
nanoparticle physicochemical properties in shaping biological outcomes and
possible effects on human health.
Andre Nel
Distinguished Professor of Medicine
and Director of Research, California
NanoSystems Institute (CNSI),
School of Medicine, University of
California, Los Angeles (UCLA)
Preface
The topics of nano-science and nanotechnology and the associated fields of research
applications like nano-biology, nano-toxicology, and nano-medicine have evolved
quickly in recent years. In all these fields, the nature and outcome of the interaction
of nanoparticles with the biological system is of key interest. With all the exciting
applications of nanomaterials in chemistry, physics, electronics, optics, material
science, biology, medicine, and many other fields in mind, one should also carefully
investigate the potential risk, i.e., the adverse health impact of nanomaterials upon
interaction with the organism. There is, therefore, an urgent need for a detailed
understanding of the mechanisms and outcomes of the interaction of nanoparticles
with the biological system including the cellular and even molecular level.
The rapid development of nanotechnology has resulted in increased technical
applications of a variety of nanoparticles, which may be released into the envi-
ronment and to which humans may be exposed accidentally, either at the workplace
or in the ambient environment. A special situation becomes apparent when
nanoparticle is being used in medicine. In this special field of nanotechnology,
called nano-medicine, nanoparticles are designed for diagnostic as well as for
therapeutic applications. Moreover, this field has lately also advanced in a com-
bination of a specific and targeted therapy with specific targeted diagnostic tests,
called theranostics. With these advances, it became imperative to obtain a better
understanding of how and on which sites nanoparticles interact with organs and the
organism. It is expected that with an improved and elementary understanding of this
interaction the risk and the impact of nanomaterials on human health will be better
understood. The present book serves this purpose. It covers our current knowledge
on the interaction of nanoparticles with our organism, nanoparticles which may be
toxic or pathogenic in nature as well as the promising beneficial aspects of
nanoparticles applied in diagnostics or therapeutics or in a combination of the two.
Nanoparticles can enter the body primarily via three different pathways:
inhalation, ingestion, and uptake via the skin. While the intact skin seems to effi-
ciently prevent nanoparticles from entering our organism, the gastrointestinal tract
and probably even more the lungs allow nanoparticles to access the inside of our
organism. After deposition on the inner surface of the gastrointestinal tract and the
vii
viii Preface
lung, they penetrate through liquid layers, cells, and tissues and eventually enter the
capillary network in the sub-epithelial regions. With the blood, these minuscule
particles will translocate into the other organs and may be distributed throughout
the whole organism.
It is our intention to present with this book to the reader the events that
nanoparticles encounter when interacting with our organism, when moving from the
internal surfaces where they had been deposited through tissue into the blood-
stream. In order to understand these pathways, we first need to know the physical,
chemical, and biological properties of individual types of nanomaterials and how
these are manufactured in a reproducible way. In the first part of the book, therefore,
the synthesis and characterization of nanoparticles are presented including their
physical behavior in biologically relevant environments. In the second part, the
mechanisms of interaction with our organism on a cellular and molecular level
including the methodology of investigation are described. In the third part, the
cellular responses and possible health effects are discussed.
We invited experts for each of these topics to cover these sequences of events,
and we were very pleased that many of the scientists we had contacted accepted our
invitation. The different chapters had been thoroughly reviewed by the editors as
well as by external reviewers to ensure the quality of each chapter with the most
updated and comprehensive knowledge.
Finally, we are indebted to the people who were responsible for producing this
book. Foremost we are grateful to the chapter authors for their passionate and very
valuable contributions to this book. We would also like to thank the external
reviewers. Finally, we would like to express our appreciation to the staff of Springer
Nature for their patient and invaluable professional assistance in producing this
book.
Bern, Switzerland Peter Gehr

Essen, Germany Reinhard Zellner
Contents
Part I Manufacturing and Characterization of Materials

1 Synthesis of Metallic and Metal Oxide Particles . . ............. 3
Kateryna Loza and Matthias Epple
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 Metals (Gold, Silver, Platinum and Copper) . . . . . . . . . . . . . . 5
1.2.1 Gold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.2.2 Silver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.2.3 Platinum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.2.4 Copper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.3 Alloyed Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.4 Nanoscale Oxide Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.4.1 Zinc Oxide Nanoparticles . . . . . . . . . . . . . . . . . . . . 14
1.4.2 Titanium Dioxide Nanoparticles . . . . . . . . . . . . . . . 14
1.4.3 Silica Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . 17
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2 Quantum Dots and Quantum Rods . . . . . . . . . . . . . . . . . . ...... 29
Christin Rengers, Nikolai Gaponik and Alexander Eychmüller
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.2 Quantum-Size-Effect (and Optical Properties) . . . . . . . . . . . . . 31
2.3 Surface Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.3.1 Synthesis Methods . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.4 Nanostructures of Other Geometries . . . . . . . . . . . . . . . . . . . . 45
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3 Polymeric Nanocarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Banu Iyisan and Katharina Landfester
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.1.1 Classification and Types of Polymeric
Nanocarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
ix
x Contents
3.2 Preparation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

3.2.1 Nanocapsules and Nanospheres . . . . . . . . . . . . . . . . 57
3.2.2 Polymersomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.2.3 Dendrimers and Micelles . . . . . . . . . . . . . . . . . . . . 69
3.3 Functional Nanocarriers for Applications in Nanomedicine . . . 69
3.3.1 Stimuli-Responsive Nanocarriers . . . . . . . . . . . . . . . 70
3.3.2 Functionalization of Nanocarriers . . . . . . . . . . . . . . 72
3.4 Characterization of Nanocarriers . . . . . . . . . . . . . . . . . . . . . . 72
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
4 Stability of Nanoparticle Dispersions and Particle
Agglomeration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 85
Kateryna Loza, Matthias Epple and Michael Maskos
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
4.2 Stability of Colloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
4.3 Stabilization Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
4.3.1 Electrostatic Stabilization . . . . . . . . . . . . . . . . . . . . 91
4.3.2 Steric and Electrosteric Stabilization . . . . . . . . . . . . 93
4.4 Possibilities and Limitations of Analytical Methods to
Analyze Nanoparticle Dispersions . . . . . . . . . . . . . . . ...... 94
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 98
5 Nanoparticle Behaviour in Complex Media: Methods for
Characterizing Physicochemical Properties, Evaluating Protein
Corona Formation, and Implications for Biological Studies . . . . .. 101
Wye-Khay Fong, Thomas L. Moore, Sandor Balog,
Dimitri Vanhecke, Laura Rodriguez-Lorenzo,
Barbara Rothen-Rutishauser, Marco Lattuada and Alke Petri-Fink
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
5.2 Biological Fluids: Composition as Colloids . . . . . . . . . . . . . . 104
5.2.1 Cell Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . 105
5.2.2 Model Physiological Fluids . . . . . . . . . . . . . . . . . . . 106
5.2.3 Human Blood and Plasma . . . . . . . . . . . . . . . . . . . . 106
5.3 Fate of NPs in Electrolyte and Protein Crowded
Environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 107
5.4 Interaction with Biomolecules: The Corona of Proteins and
More . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 109
5.4.1 The “Protein Corona” . . . . . . . . . . . . . . . . . . . . . .. 109
5.4.2 Factors Affecting Protein Corona Formation . . . . . .. 109
5.4.3 Beyond Proteins: Other Components of
the Corona . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 111
5.4.4 Biological Effects of the Protein Corona . . . . . . . .. 111
5.4.5 Characterising the Protein Corona . . . . . . . . . . . . .. 112
5.5 Theoretical Considerations with Regards to Colloidal
Stability in Physiological Media . . . . . . . . . . . . . . . . . . . . .. 116
Contents xi
5.5.1 Fundamentals of Nanoparticle Aggregation . . . . . .. 116

5.5.2 Consequences of Nanoparticle Aggregation on Cell
Studies: Dosimetry of Single Particles Versus
Aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 117
5.6 Measuring NP in Complex Cell Culture Media: Different
Methods, Pitfalls and New Developments . . . . . . . . . . . . . .. 118
5.6.1 Scattering and Spectroscopic Methods . . . . . . . . . .. 118
5.6.2 Zeta Potential—Describing the Surface Charge of
NP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 120
5.6.3 Measuring the Effect of Proteins on
Zeta Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 120
5.6.4 Choosing Scattering Methods for Nanoparticles in
Complex Media . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
5.6.5 Characterisation Methods Based on Separation . . . . . 122
5.6.6 Microscopic Methods . . . . . . . . . . . . . . . . . . . . . . . 129
5.6.7 Dynamic Methods . . . . . . . . . . . . . . . . . . . . . . . . . 134
5.7 Conclusions and Future Directions . . . . . . . . . . . . . . . . . . . . . 138
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
Part II Membrane Transfer, Cellular Uptake and Intracellular

Fate: Mechanisms and Detection Methods
6 Nanoparticle-Cell Interactions: Overview of Uptake,
Intracellular Fate and Induction of Cell Responses . . . . . . . ..... 153
Barbara Rothen-Rutishauser, Joël Bourquin and Alke Petri-Fink
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
6.2 Cellular Barriers in Eukaryotic Cells . . . . . . . . . . . . . . . . . . . 154
6.3 Nanoparticle-Cell Interactions . . . . . . . . . . . . . . . . . . . . . . . . 155
6.3.1 Endocytotic Mechanisms . . . . . . . . . . . . . . . . . . . . 156
6.3.2 Intracellular Fate . . . . . . . . . . . . . . . . . . . . . . . . . . 158
6.3.3 Exocytotic Mechanisms . . . . . . . . . . . . . . . . . . . . . 160
6.4 Nanoparticle Induced Cell Responses . . . . . . . . . . . . . . . . . . . 161
6.4.1 Cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
6.4.2 Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
6.4.3 Pro-inflammation Including Inflammasome
Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
6.4.4 Genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
6.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
xii Contents
7 Cellular and Non-cellular Barriers to Particle Transport Across

the Lungs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 171
Nicole Schneider-Daum, Marius Hittinger, Xabier Murgia
and Claus-Michael Lehr
7.1 Introduction: What Distinguishes the Lung from Other
Epithelia? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 171
7.2 Barrier Properties and Transport Mechanisms at the
Pulmonary Epithelia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 173
7.3 Mucus as Non-cellular Barrier to Particle Transport . . . . . . .. 175
7.3.1 Role of Mucus in Health and Disease . . . . . . . . . .. 175
7.3.2 Mucus-Particle Interactions are Primarily
Governed by Particle Size and Surface
Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 177
7.3.3 Considerations for Drug Delivery to the Airways . .. 178
7.4 Surfactant as Outermost Element of the Pulmonary Air-
Liquid Interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 179
7.4.1 Role of Surfactant in Health and Disease . . . . . . . .. 179
7.4.2 Inhaled Particles—Interactions with Surfactant as a
Defense Mechanisms and Transport Facilitator . . . .. 181
7.5 Implications for Particle-Based Pulmonary Drug Delivery . . .. 182
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 184
8 Cellular Uptake Mechanisms and Detection of Nanoparticle
Uptake by Advanced Imaging Methods . . . . . . . . . . . . . . . . . . . .. 191
Kleanthis Fytianos, Fabian Blank and Loretta Müller
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 191
8.2 Non-ligand Dependent Endocytic Uptake . . . . . . . . . . . . . . .. 193
8.3 Receptor-Mediated Cellular Internalization and
Nanoparticles for Targeted Uptake . . . . . . . . . . . . . . . . . . . .. 195
8.3.1 Folate Receptor (FR) Targeting . . . . . . . . . . . . . . .. 196
8.3.2 Transferrin Receptor (TfR) Targeting . . . . . . . . . . .. 196
8.3.3 Epidermal Growth Factor Receptor (EGFR)
Targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 196
8.3.4 Prostate-Specific Membrane Antigen (PSMA)
Targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 197
8.3.5 Integrin Targeting . . . . . . . . . . . . . . . . . . . . . . . . .. 197
8.3.6 Neonatal Fc-Receptor (FcRn) Targeting . . . . . . . . .. 197
8.3.7 Methods of 3D Fluorescence Microscopy and Flow
Cytometry to Determine and Characterize Cellular
Uptake of Nanoparticles . . . . . . . . . . . . . . . . . . . .. 198
8.3.8 Quantum Dots and Inorganic Nanoparticles . . . . . .. 199
8.3.9 Fluorescently Labelled (Nano)Particles . . . . . . . . .. 199
8.3.10 Laser Scanning Confocal Microscopy and Live
Cell Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 200
Contents xiii
8.3.11 Super Resolution Microscopy . . . . . . . . . . . . . . . . . 202

8.3.12 Flow and Imaging Cytometry . . . . . . . . . . . . . . . . . 203
8.4 Conclusions and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
9 Imaging Techniques for Probing Nanoparticles in Cells
and Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 213
Christina Graf and Eckart Rühl
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
9.2 Optical Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
9.3 Fluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
9.4 Single-Photon Confocal Microscopy . . . . . . . . . . . . . . . . . . . 216
9.5 Imaging Flow Cytometry Combined with Confocal
Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
9.6 Multi-photon Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
9.7 Fluorescence Lifetime Imaging Microscopy (FLIM) . . . . . . . . 218
9.8 Super-Resolution Fluorescence Microscopy . . . . . . . . . . . . . . 219
9.9 Photothermal Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
9.10 Photothermal Raster Image Correlation Spectroscopy . . . . . . . 222
9.11 Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
9.12 Scanning Electron Microscopy (SEM) . . . . . . . . . . . . . . . . . . 224
9.13 High-Angle Annular Dark-Field Scanning Transmission
Electron Microscopy (HAADF STEM) . . . . . . . . . . . . . . . .. 226
9.14 Environmental Scanning Electron Microscopy (ESEM) . . . . .. 226
9.15 Liquid Scanning Transmission Electron Microscopy (Liquid
STEM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
9.16 Multimodal Imaging and Correlative Microscopy . . . . . . . . . . 227
9.17 Raman Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
9.18 Atomic-Force Microscopy-Based Techniques . . . . . . . . . . . . . 230
9.19 X-ray Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
9.20 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
Part III Cellular Responses and Health Effects

10 Cellular Defense Mechanisms Following Nanomaterial Exposure:
A Focus on Oxidative Stress and Cytotoxicity . . . . . . . . . . . . . . .. 243
Stephen J. Evans, Gareth J. Jenkins, Shareen H. Doak
and Martin J. D. Clift
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 243
10.2 Paradigms in Particle Toxicology . . . . . . . . . . . . . . . . . . . . .. 244
10.3 Cellular Defense Mechanisms in Mammalian Cells . . . . . . . .. 249
10.4 Oxidative Stress, Antioxidants and Reactive Oxygen
Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 249
xiv Contents
10.5 NMs and Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

10.6 NM Induced Immune Response and Oxidative Stress . . . . . . . 251
10.7 ROS and Cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
10.8 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
11 Nanocarriers and Immune Cells . . . . . . . . . . . . . . . . . . . . . . . .... 255
Lorna Moll and Volker Mailänder
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 255
11.2 Cell Types Comprising the Immune System and Their In
Vivo Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
11.3 Danger-Associated Molecular Patterns . . . . . . . . . . . . . . . . . . 260
11.4 Receptors for DAMPs and Uptake of Nanoobjects . . . . . . . . . 260
11.5 Immune Cells in Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . 262
11.6 Immune Cells In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
11.7 Interaction of Nanocarriers with Immune Cells in Cell
Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 264
11.8 Routes of Applications for Nanoparticles . . . . . . . . . . . . .... 265
11.9 Interaction of Nanocarriers with Immune Cells In Vivo . .... 266
11.10 Uptake and Intracellular Processing of Nanocarriers in
Immune Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 267
11.10.1 Pinocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . .... 267
11.10.2 Phagocytosis and Cross-Presentation After NC
Uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
11.11 Immune Cell Responses to Nanocarrier Interactions . . . . . . . . 270
11.12 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
12 Fate and Translocation of (Nano)Particulate Matter in the
Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 281
Andreas Frey, Katrin Ramaker, Niels Röckendorf,
Barbara Wollenberg, Ingmar Lautenschläger, Gabriella Gébel,
Artur Giemsa, Markus Heine, Denise Bargheer and Peter Nielsen
12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 281
12.2 Architecture of the Gastrointestinal Tract . . . . . . . . . . . . . . .. 283
12.3 Nanoparticulate Matter Confronting the Gastrointestinal
Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 286
12.3.1 Controlled Uptake of Low Molecular Weight
Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 287
12.3.2 Controlled Uptake of Macro- and Supramolecular
Entities and Particulate Matter . . . . . . . . . . . . . . . .. 294
12.3.3 Uncontrolled Influx of Luminal Matter via
Paracellular Leaks . . . . . . . . . . . . . . . . . . . . . . . .. 296
12.3.4 Features Qualifying (Nano)Particulate Matter for
Gastrointestinal Uptake . . . . . . . . . . . . . . . . . . . . .. 298
Contents xv
12.4 Types of Nanoparticles Unintentionally Occuring in the

Alimentary Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 299
12.5 Intentional Administration: Drug Delivery and Contrast
Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 301
12.5.1 Nanoparticulate Drug Delivery Systems . . . . . ..... 302
12.5.2 Particulate Matter for Contrasting the
Gastrointestinal Tract for Medical Imaging . . ..... 303
12.6 Fate of Ingested Particulate Matter: Beeline or Detour . ..... 304
12.6.1 Measuring Gastrointestinal Particle Uptake in
Model Systems . . . . . . . . . . . . . . . . . . . . . . . ..... 305
12.6.2 Deposition, Breakdown and Excretion of
Incorporated Matter . . . . . . . . . . . . . . . . . . . ..... 309
12.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 312
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 313
13 Interactions of Nanoparticles with Skin . . . . . . . . . . . . . . . . . . . .. 329
Fanny Knorr, Alexa Patzelt, Martina Claudia Meinke, Anika Vogt,
Ulrike Blume-Peytavi, Eckart Rühl and Jürgen Lademann
13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 329
13.2 Penetration of NPs into Skin . . . . . . . . . . . . . . . . . . . . . . . .. 331
13.3 Biological Effects and Cellular Uptake of NPs in Skin and
Skin Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 334
13.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 336
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 337
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
Contributors
Sandor Balog BioNanomaterials, Adolphe Merkle Institute, Fribourg, Switzerland

Denise Bargheer Institute of Biochemistry and Molecular Cell Biology,
University Medical Center Eppendorf, Hamburg, Germany
Fabian Blank Respiratory Medicine, Department of BioMedical Research
(DBMR), University of Bern, Bern, Switzerland
Ulrike Blume-Peytavi Department of Dermatology, Venerology and Allergology,
Charité – Universitätsmedizin Berlin, Berlin, Germany
Joël Bourquin BioNanomaterials, Adolphe Merkle Institute, Fribourg,
Switzerland
Martin J. D. Clift In Vitro Toxicology Group, Swansea University Medical
School, Institute of Life Sciences, Swansea, Wales, UK
Shareen H. Doak In Vitro Toxicology Group, Swansea University Medical
Matthias Epple Inorganic Chemistry, University of Duisburg-Essen, Essen,
Germany
Stephen J. Evans In Vitro Toxicology Group, Swansea University Medical
Alexander Eychmüller Physical Chemistry, Technical University of Dresden,
Dresden, Germany
Wye-Khay Fong BioNanomaterials, Adolphe Merkle Institute, Fribourg,
Switzerland
Andreas Frey Division of Mucosal Immunology and Diagnostics, Research
Center Borstel, Borstel, Germany
Kleanthis Fytianos Respiratory Medicine, Department of BioMedical Research
(DBMR), University of Bern, Bern, Switzerland
xvii
xviii Contributors
Nikolai Gaponik Physical Chemistry, Technical University of Dresden, Dresden,

Germany
Gabriella Gébel Institute of Biochemistry and Molecular Cell Biology, University
Medical Center Eppendorf, Hamburg, Germany
Artur Giemsa Institute of Biochemistry and Molecular Cell Biology, University
Christina Graf Department of Chemistry and Biotechnology, University of
Applied Sciences Darmstadt, Darmstadt, Germany
Markus Heine Institute of Biochemistry and Molecular Cell Biology, University
Marius Hittinger Helmholtz Institute for Pharmaceutical Research Saarland
(HIPS), Saarbrücken, Germany;
PharmBioTec GmbH, Saarbrücken, Germany
Banu Iyisan Max Planck Institute for Polymer Research, Mainz, Germany
Gareth J. Jenkins In Vitro Toxicology Group, Swansea University Medical
Fanny Knorr Department of Dermatology, Venerology and Allergology, Charité –
Universitätsmedizin Berlin, Berlin, Germany
Jürgen Lademann Department of Dermatology, Venerology and Allergology,
Charité – Universitätsmedizin Berlin, Berlin, Germany
Katharina Landfester Max Planck Institute for Polymer Research, Mainz,
Germany
Marco Lattuada International Iberian Nanotechnology Laboratory, Water Quality
Group, Braga, Portugal
Ingmar Lautenschläger Clinic for Anesthesiology and Operative Intensive Care
Medicine, University Hospital Schleswig-Holstein, Kiel, Germany
Claus-Michael Lehr Helmholtz Institute for Pharmaceutical Research Saarland
(HIPS), Saarbrücken, Germany;
PharmBioTec GmbH, Saarbrücken, Germany
Kateryna Loza Inorganic Chemistry, University of Duisburg-Essen, Essen,
Germany
Volker Mailänder Department of Dermatology, University Medical Center of the
Johannes Gutenberg-University Mainz, Mainz, Germany;
Max-Planck-Institute for Polymer Research, Mainz, Germany
Michael Maskos Fraunhofer-Institute for Microengineering and Microsystems
(IMM), Mainz, Germany
Contributors xix
Martina Claudia Meinke Department of Dermatology, Venerology and

Allergology, Charité – Universitätsmedizin Berlin, Berlin, Germany
Lorna Moll Department of Dermatology, University Medical Center of the
Johannes Gutenberg-University Mainz, Mainz, Germany;
Max-Planck-Institute for Polymer Research, Mainz, Germany
Thomas L. Moore BioNanomaterials, Adolphe Merkle Institute, Fribourg,
Switzerland
Loretta Müller University Children’s Hospital Basel, Basel, Switzerland;
Department of Pediatrics, Inselspital, Bern University Hospital, Bern, Switzerland
Xabier Murgia Helmholtz Institute for Pharmaceutical Research Saarland (HIPS),
Saarbrücken, Germany
Peter Nielsen Institute of Biochemistry and Molecular Cell Biology, University
Alexa Patzelt Department of Dermatology, Venerology and Allergology, Charité –
Alke Petri-Fink BioNanomaterials, Adolphe Merkle Institute, Fribourg,
Switzerland;
Department of Chemistry, University of Fribourg, Fribourg, Switzerland
Katrin Ramaker Divison of Mucosal Immunology and Diagnostics, Research
Christin Rengers Physical Chemistry, Technical University of Dresden, Dresden,
Germany
Niels Röckendorf Division of Mucosal Immunology and Diagnostics, Research
Laura Rodriguez-Lorenzo BioNanomaterials, Adolphe Merkle Institute,
Fribourg, Switzerland;
International Iberian Nanotechnology Laboratory, Water Quality Group, Braga,
Portugal
Barbara Rothen-Rutishauser BioNanomaterials, Adolphe Merkle Institute,
Fribourg, Switzerland
Eckart Rühl Physical Chemistry, Institute of Chemistry and Biochemistry, Free
University of Berlin, Berlin, Germany
Nicole Schneider-Daum Helmholtz Institute for Pharmaceutical Research
Saarland (HIPS), Saarbrücken, Germany
Dimitri Vanhecke BioNanomaterials, Adolphe Merkle Institute, Fribourg,
Switzerland
xx Contributors
Anika Vogt Department of Dermatology, Venerology and Allergology, Charité –

Barbara Wollenberg Department of Ear, Nose and Throat, University Hospital
Schleswig-Holstein, Lübeck, Germany
Part I
Manufacturing and Characterization of
Materials
Chapter 1
Synthesis of Metallic and Metal Oxide
Particles
Kateryna Loza and Matthias Epple
Abstract The diversity of applications in catalysis, energy storage and medical

diagnostics utilizes unique and fascinating properties of metal and metal oxide
nanostructures. Confined to the nanometer scale, materials may display properties
that are different from the equivalent bulk compounds. To meet the requirements
for various applications, numerous production techniques were developed to control
particle size, morphology, aggregation state, crystal structure, surface charge and
composition. This chapter presents an overview of the preparation of metallic and
metal oxide nanoparticles by bottom-up and top-down approaches. We describe basic
synthetic routes for prominent cases of metals (gold, silver, platinum and copper)
and metal oxides (zinc oxide, titania, and silica).
1.1 Introduction
Metal nanostructures attract particular interest because of their unique and fascinating
properties compared to their bulk counterparts. The variety of applications comprises
biological sensing [1, 2], imaging [3–9], medical diagnostics [10–12], cancer therapy
[13, 14], catalysis [15, 16], and energy storage [17, 18]. The observed new chemical,
optical, and thermal properties of metallic nanoparticles occur when the size is con-
fined to the nanometer length scale [19]. Numerous techniques were developed to
produce metal nanoparticles to meet the requirements for various applications. In gen-
eral, there are two strategies to manufacture materials on the nanoscale: “Top-down”
and “bottom-up” (Fig. 1.1) [20, 21]. The first method is based on breaking down
a system (i.e., the bulk material) into smaller units. Common “top-down” techniques
are lithography, milling, ultrasound treatment, and laser ablation. These processes
K. Loza (B) · M. Epple

Inorganic Chemistry, University of Duisburg-Essen, Universitätsstrasse 2,
45141, Essen, Germany
e-mail: Kateryna.Loza@uni-due.de
M. Epple
e-mail: Matthias.epple@uni-due.de
© Springer Nature Switzerland AG 2019 3

P. Gehr and R. Zellner (eds.), Biological Responses to Nanoscale Particles,
NanoScience and Technology, https://doi.org/10.1007/978-3-030-12461-8_1
4 K. Loza and M. Epple
Fig. 1.1 Schematic illustration of synthetic methods for metal nanoparticles. (Adapted with per-
mission from New J. Chem., 1998, 1179–1201. Copyright 1969 The Royal Society of Chemistry)
[22]
are comparatively simple and usually lead to ligand-free (“naked”) nanoparticles.

However, there is a limited control over the manufacturing process, e.g., an exact
size or shape adjustment of resulting particles. The “bottom-up” method relies on
material synthesis from atomic or molecular species via a suitable chemical reaction,
allowing the particles to grow from smaller units. This approach uses the chemical
properties of single molecules or atoms to cause self-organization into the desired
particle shape.
The “bottom-up” approach is commonly associated with wet-chemical methods,
because colloidal metallic particles are commonly produced by chemical reduction
of metal salts dissolved in a suitable solvent in the presence of surfactants or ligands
that cover the surface [23]. A wide range of reducing agents have been used in the
colloid-chemical synthesis of metal nanoparticles [24]. For example, H2 , hydrazine,
hydroxylamine, hydrides (e.g. NaBH4 or B2 H6 ), ascorbic acid or ascorbate, citric acid
or citrate, reducing polymers (e.g., PVA) and solvents (like alcohols, diols, aldehydes,
and DMF) have been used to prepare metal nanoparticles [25–29]. Reduction can
take place at room temperature or at elevated temperatures, depending on the relative
reduction potentials of the precursor and the reducing agents [30–33].
In the following, we discuss the cases of gold, silver, copper, and platinum as
representative examples, and also the preparation of alloyed nanoparticles by various
synthetic methods. The described methods can typically be transposed to other kinds
of nanoparticles, typically of noble metals.
1 Synthesis of Metallic and Metal Oxide Particles 5
1.2 Metals (Gold, Silver, Platinum and Copper)
1.2.1 Gold
The first systematic synthesis of Au colloids was reported 160 years ago by Michael
Faraday using phosphorus to reduce AuCl4 − ions [34]. In the 1950s, an easier
approach was established and standardized by Turkevich [35]. He used the mildly
reducing agent trisodium citrate, added to a boiling aqueous solution of HAuCl4 , to
obtain monodisperse gold nanoparticles in the size range from 10 to 40 nm. Due to
its simplicity, this synthesic method was adapted in many variations [36, 37]. For
example, switching to a mixture of reducing agents (e.g. citrate and tannine) allows
to clearly shorten the reaction time and to enhance the stability of the formed colloid
[38, 39].
The reduction of tetrachloroauric acid in an aqueous medium is a versatile syn-
thetic route and possible with many different reducing agents like sodium borohy-
dride (NaBH4 ), ascorbic acid, and hydroquinone [40–42]. The use of NaBH4 as
reducing agent results in a fast reduction and a gold particle size of 1–5 nm [29,
43, 44]. In general, the choice of the reducing agent has a strong influence on the
resulting particle size, since with increasing reduction potential the number of the
formed nuclei increases and the growth of particles is limited. On the nanometer
scale, metals tend to nucleate and grow into multiply twinned particles with their
surfaces defined by the lowest-energy facets [45]. Anisotropic gold nanoparticles
(rod-, rectangle-, hexagon-, cube-, triangle- and star-like shapes) with less stable
facets were kinetically achieved by adding chemical capping reagents, i.e. agents
that selectively block certain crystal faces, to the reaction mixture [25, 46, 47].
The previously described methods are based on a synthesis from atomic or molec-
ular species by chemical reaction, so called “bottom-up” approach. In liquid media,
dispersed metallic nanoparticles can be generated by the pulsed laser ablation process,
a “top down” technique [48]. This method provides ligand-free nanoparticles [49].
The size of obtained particles can be varied to some extent by the laser parameters
and by subsequent laser fragmentation steps [50, 51]. Furthermore, an in situ conju-
gation of nanoparticles with biomolecules by laser ablation in an aqueous medium
is a highly promising one-step method for the production of functional nanoparticles
[52].
The polydispersity of nanoparticles is a key concern in nanoscience research.
Even though reasonably monodisperse nanoparticles can be produced, usually not
all nanoparticles are fully identical (see Fig. 1.2 for an example). This fact leads to
the ultimate aim for a synthesis of atomically precise nanoparticles [53]. In the case
of gold, this was accomplished for ultrasmall gold nanoparticles (containing 10–300
atoms, often called nanoclusters) [54, 55]. Several groups established synthetic routes
to produce a gold core in the size range of 1–3 nm. Such ultrasmall nanoparticles are
typically formed by metal salt reduction in the presence of phosphanes (PR3 ) [56,
57] or thiols (HS–R) [44]. Exerting a strict control over the size of a cluster strongly
affects the activity and the selectivity in a catalytic process [58]. Furthermore, a
Fig. 1.2 Transmission electron micrographs of PVP-stabilized gold nanoparticles, prepared by

the standard citrate method after Turkevich. (Reproduced from the dissertation of D. Mahl, 2011,
University of Duisburg-Essen) [60]
supracolloidal self-assembly of atomically precise nanoparticles is a promising plat-

form for novel 2D and 3D materials with additional plasmonic functionalities, novel
mechanical properties, and inherent flexibility [59].
1.2.2 Silver
Colloidal silver is known since about 120 years [61]. The manufacturing of silver
nanoparticles can be done by physical processes such as ultrasonication, chemical
vapor deposition, or pulsed laser ablation in liquids [62–64]. However, wet-chemical
“bottom-up” syntheses offer more possibilities for the variation of particle size, mor-
phology and functionalization. The most commonly used precursor for preparing
silver nanoparticles in wet-chemical reductions is silver nitrate (AgNO3 ) because of
its high solubility in many polar solvents and dispersability in less polar solvents,
sometimes after adding surfactants and/or using ultrasonication. The reducing agents
used in the synthesis of nanoparticles from silver(I) ions are comparable to those used
for gold nanoparticle preparation. Already in 1889, M. C. Lea published the syn-
thesis of citrate-stabilized silver nanoparticles [65]. In general, one-pot methods for
the reduction of silver nitrate have evolved, where different reducing agents such
as sodium citrate [66], glucose [67], ascorbate [68], sodium borohydride [69, 70],
polyols [71, 72], and ammonium formiate were used [73]. Typically, the reactions
are performed at elevated temperatures by conventional heating in an oil bath. Alter-
natively, microwave-assisted syntheses can increase the reaction rates and yields as
well as selectivity and reproducibility [30].
The particle properties depend not only on their size but also on their morphol-
ogy. As a result, a shape-controlled synthesis of silver nanoparticles is of special
Fig. 1.3 Transmission electron micrographs of different kinds of PVP-stabilized silver nanoparti-
cles, prepared by glucose reduction (a) [67], a microwave-assisted reduction (b) [30], a modified
polyol synthesis (c) [79], and a microwave-assisted modified polyol process (d) [80]. (Adapted
with permission from Cryst. Growth Des. 16, 7, 3677–3687. Copyright 2016 American Chemical
Society) [81]
interest. Xia et al. and others described the structural evolution of silver nanoseeds
to nanoparticles with defined shapes like platelets [74], cubes [75], rods [76], rings
[77], and bipyramids [78] (Fig. 1.3).
It is critical to understand not only the growth mechanism of nanostructures, but
the process of seed formation, because the number of twin planes in the initial stage
is the key factor for determining the shape of the final product (single-crystal seeds
form cubes, multiply-twinned decahedral seeds form wires) [82].
Fig. 1.4 Transmission electron micrographs of platinum nanoparticles, prepared by solution-phase

synthesis using metal carbonyls as reducing agents. This synthetic method produces highly monodis-
perse Pt octahedral, icosahedra, cubes, truncated cubes, cuboctahedra, spheres, tetrapods, star-
shaped octapods, multipods, and hyper-branched structures. (Reproduced with permission from
ACS Nano 7, 1, 645–653. Copyright 2012 American Chemical Society) [89]
1.2.3 Platinum
Platinum nanostructures are of particular interest for many industrial applications

due to their extraordinary catalytic properties in various industrial syntheses like
petrochemistry or energy conversion [83–86]. Conventional techniques to prepare
platinum nanoparticles are based on wet-chemical methods [87–89]. Typically, the
reaction involves the reduction of a Pt(II) precursor (like K2 PtCl4 or Pt(acac)2 ) or a
Pt(IV) precursor (like K2 PtCl6 ) in the presence of a stabilizing polymer by reducing
agents such as hydrogen [90], carbon monoxide [91], sodium borohydride [92],
lithium borohydride [93], and ethylene glycol [94]. The resulting nanoparticles may
be considered as monodisperse in size, but they are often irregular in shape and
lack well-defined facets [95]. Further modifications may include sonication during
the reaction [96] or microwave-assisted heating [94]. Because the reactivity and the
selectivity of Pt nanoparticles are highly dependent on the exposed facets [97], the
synthesis of uniformly shaped particles is decisive for high catalytic performance
[98]. Their morphological evolution is often controlled by the reduction kinetics
of the platinum precursor [95], the reaction temperature [98], or the use of shape-
directing reagents [99, 100] (Fig. 1.4).
The previously syntheses were based on the “bottom-up” approach. However,
chemical synthesis methods often lead to impurities of the nanoparticle colloids
caused by additives and precursor reaction products [101]. In contrast, Barcikowski
et al. demonstrated the preparation of ligand-free platinum nanoparticles by laser
ablation in liquids (“top-down” technique) for the surface modification of electrodes

for neural stimulation [102].
1.2.4 Copper
Since the ninth century, copper nanoparticles are known as coloring agents in
Mesopotamia [103]. Nowadays the application range comprises biomedicine [104,
105], sensors [106], conductive inks [107], and organic catalysis [108–110]. Being
inexpensive and rather abundant in nature, copper is utilized in large scale for the
fabrication of plasmonic solar cells [111]. Recently established methods for copper
nanoparticle synthesis include laser ablation [112], thermal decomposition [113],
the polyol process [114, 115], and other chemical reduction methods [116]. Typi-
cal precursors for copper nanoparticle wet-chemical syntheses are CuSO4 , copper
acetylacetonate (Cu(acac)2 ), CuCl2 , and Cu(NO3 )2 [117]. Reducing agents comprise
ascorbic acid [118], sodium borohydride [119], and hypophosphite [120]. It should
be mentioned that the preparation of Cu nanoparticles is challenging due to its high
sensitivity to air because copper is easily oxidized to copper oxides, being less noble
than silver, gold, or platinum metals [121]. The oxidation of copper nanoparticles
can be avoided if the synthesis is conducted in non-aqueous media (sometimes under
inert gas) and in the presence of CO or H2 . Previously described synthetic routes
result in spherical multi-twinned nanoparticles in the size range between 10 and
70 nm. As shown in Fig. 1.5, by variation of the ratio of copper acetylacetonate to
oleylamine, different particle size distributions can be achieved. If a hydrothermal
treatment is applied, anisotropic copper particles such as nanowires or nanorods can
be produced [110].
1.3 Alloyed Nanoparticles
The properties of metallic systems can be significantly extended by mixing ele-

ments to generate intermetallic compounds and alloys. Due to synergetic effects, an
enhancement in desired properties is possible. The diversity of compositions, struc-
tural organizations, and tunable properties of metallic alloys makes them suitable
for a wide range of applications in electronics, engineering, biomedicine, and het-
erogeneous catalysis [123–126]. For example, alloyed silver and gold nanoparticles
utilize the physicochemical properties of both metals, e.g., the optical properties of
gold and the toxicity towards bacteria or cells of silver [127, 128]. Surface structure,
composition, and segregation properties [129] of nanoalloys are of great importance
for the chemical reactivity and the selectivity in catalysis [130, 131]. If confined to
the nanometer scale, they may display properties that are different from the equiv-
alent bulk compounds. For example, iron and silver are immiscible in the bulk, but
can be mixed in nanoparticles [132] (Fig. 1.6).
Fig. 1.5 Transmission electron micrographs and UV/VIS spectra of copper nanoparticles, prepared
with different ratios of copper acetylacetonate and oleylamine. Scale bars in a and b are 50 nm,
those in c, d, f, and h are 20 nm, and that in i is 2 nm. (Reproduced with permission from J. Phys.
Chem. C, 2010, 114 (37), pp 15612–15616. Copyright 2010 American Chemical Society) [122]
Due to the heterogeneity of different properties of individual components (e.g.,

crystal system, redox potential, crystal symmetry, or surface charge), the successful
mixture of these materials into a finite nanoparticle is challenging [123, 133]. In
general, the methods for preparation of nanoalloys are the same as for single metal
nanoparticles. Ligand-free manufacturing methods of nanoparticles are based on
laser ablation of solids in liquid environment [134, 135], pulsed arc discharge, and
sputtering techniques [123]. These approaches start with single, bimetallic or ternary
targets or mixed metallic powders. Figure 1.7 shows a typical setup and the obtained
alloyed Ag/Au nanoparticles by laser ablation in liquids.
Bimetallic colloids can be generated by chemical reduction of a suitable mixture of
salts (metal precursor) in the solution, using appropriate reducing agent. To avoid the
formation of core-shell structures due to the difference in redox potentials, different
ligands can be used [137]. Another variation is based on the reduction of double metal
complexes [22]. Instead of chemical reduction, an electrochemical process can be
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I like, and I had made him come to the station because I was afraid
of meeting Rahas alone. And I told him to take tickets for him and
me, and we went back by the next train to the station where Rahas
got out. The porter said two gentlemen had got out and gone across
the fields; and I knew who the other one was, and I screamed, and
told William my husband had come back. But he said it was a fancy.
We walked across the wet fields in the dark, and I was trembling so
that I could scarcely stumble along, and William carried a lantern,
and said I had better go back, for we were on a wild-goose chase.
And when we came down to the wood, my foot slipped, and I fell on
to the grass, and as he stooped to pick me up, William saw marks on
the ground, as if something had been pulled along over it. He went a
little way slowly until I heard him give a cry, and I ran to him, and—
and we found you.”
She could not say more, her voice was suffocated, her lips were
shaking. But the whitewashed walls of the room in which he was
lying, the hayrick he could see through the window, told George that
it was to a farmhouse he had been brought; and there they spent two
days, until he was well enough to get up and go with Nouna back to
her friends in Plymouth. Then began for them both in the pretty
southern town a new and sweeter honeymoon, marred only for each
by a secret fear for the other. In the first days of their re-union
happiness gave their wasted frames a new vitality which made each
feel on the high road to health, but which made to each only the
more evident the pale face and heavy breathing of the other.
They were sitting together in the sunlight one May afternoon, the
window wide open, the breeze coming in straight from the sea,
drinking in the joy of each other’s presence as they were never tired
of doing, when George passed his hand slowly down his wife’s
cheeks, and shivered.
“Are you cold?” she asked anxiously, nestling up to him and
putting her little arms round him as if to protect him from the spring
air.
“No,” he said in a troubled voice, “I’m all right. But I’m afraid this
place doesn’t suit you, Nouna; you’re getting so thin and white. You
are paler than when I came back.”
Nouna’s face changed; after a moment’s pause she sprang up
with her old vivacity, and running to a looking-glass, gazed at her
own reflection for some minutes, and then crept back to her
husband’s side with a bright light in her eyes. As he looked at her
inquiringly, she drew up the sleeve of his coat as far as she could,
very gently, and then baring her own arm also, laid it beside his, and
glanced up into his face with an odd, tender, yearning expression
which, after a moment’s wonderment, opened his own dull eyes. For
a few seconds neither spoke again. Then he snatched her into his
arms and their eyes held each other’s for some minutes in an
ecstasy of relief and gratitude. George had loved his wife better than
his career, better than his own happiness. Nouna, since the fall of
her first idol—her mother—had turned all her devotion to the
husband who had cherished her so tenderly. Both, therefore,
dreaded life without the other a thousand times worse than death,
and when it dawned upon them that they were not to be parted
again, there was no further sorrow possible for them in this world.
“George,” said Nouna at last, in a broken whisper, “if you had
never met me you would have been much happier, for you would
have married that good Ella and have got on in the world and
become a great man.”
“Yes,” said he at once.
“Well, aren’t you sorry?”
“No.”
“Why?”
It was not easy to explain. The sailor, sinking with his ship at
twenty-five, does not in his last moments wish that he had been a
grocer, though if he had he might have gone on contentedly selling
tea and candles for half a century. George, struck down by
misfortune in the prime of his youth, had tasted some of life’s
supremest joys, and the rolling years could give him no delight such
as he had felt in running the whole gamut of an absorbing passion.
He hesitated before he answered her.
“If I had not married you,” he said at last, “you would never have
been poor, you would have had as many lovely dresses and
diamonds as you wanted, and nobody would ever have teased you
to tell the truth, or to do anything you didn’t want to do. And yet you
are not sorry you married me. What’s the reason?”
She curled herself about him. “I don’t know,” she said shyly.
“You’ve made me feel things I didn’t—feel—before.”
“Well, Nouna, and you’ve done the same to me. Are you
satisfied?”
“Yes,” she said.
“Then so am I.”
And in this state of placid but languid contentment these two
shipwrecked creatures drifted on day by day, tired out by the buffets
of fortune, and making no effort to escape from the black archer who
seemed to have marked them down. The young come to this stage
more easily than the middle-aged; when their strong passions and
eager desires burn low, quenched suddenly by ill-health or desperate
misfortune, all the busy wheels of the world seem to stand still with
them, and they cry, when they feel that the pulse of life beats weakly:
“This is the end!” While older sufferers, who have shaken Time by
the hand, and know his ways, and have learnt to bear his penalties
patiently, see only the daily work interrupted against their will, waiting
to be taken up again when the storm is over.
There came to Plymouth, when Lauriston and his wife had been a
week there together, a friend who saw something of this, and set her
wits to work, after her custom, to put right what she saw was wrong.
Ella Millard had brought her whole family to the town on the plea that
a fortnight of the Devonshire air would improve her sisters’
complexions, and arm them for the triumphs of the coming season.
Having gained over her mother, from whom she inherited her own
strong will, the rest yielded like lambs, and within a week of her
resolution to come they were all installed in a house at the upper end
of Lockyer Street, near the Hoe. By Sir Henry and his two eldest
daughters, who all enjoyed a serene animal health, and to whose
lymphatic temperament trials of the nervous system were
meaningless words, the wan faces and languid movements of the
Lauristons were looked upon as altogether fatal signs. But the more
discriminating Ella would not give up hope so easily. It seemed to her
contrary to common sense, and to the lofty qualities she attributed to
him that the man who had been her ideal should allow himself to be
snuffed out of life so easily. Afraid to depend entirely upon her own
judgment in such an important matter, she refrained from setting her
scathing little tongue to jibe at him for the inertness of his mind until
she had found some person of authority to pronounce upon the
health of his body. But George had never before in his life been in
need of a doctor, and scouted the idea of seeing one now; while
Nouna, on whose behalf Ella then pleaded, shrank sensitively from
the ordeal of meeting a stranger, and only consented at last to see
the physician whom George had called in to dress her arm on the
memorable evening of his first visit to Mary Street. The very next day
Dr. Bannerman arrived, and had an interview with both his patients.
The entrance of the tall, slightly stooping figure, the sight of his dark,
penetrating face, lean, lined, and impressive as that of a magician,
raised a flush of excitement to Nouna’s face, and brought back to her
husband’s mind a vivid recollection of the prophecy uttered by the
doctor on that May evening. If the sharp eyed man of science knew
all the circumstances that had chequered Lauriston’s life since he
disregarded that warning, he would indeed think that his sinister
prophecy had been amply fulfilled.
The interview was a short one. The doctor affected to have no
recollection of either of his patients until George followed him out of
the room, and stood face to face with him on the landing.
“You remember me, doctor, I suppose,” he said in a rather
shamefaced way.
“Perfectly.”
“The first time you met me you were kind enough to read me a
sermon. You might read me one to more purpose now.”
“More purpose! No. You can read your own sermon now, and I
come to my proper function, that of curing the results of the acts my
warning could not save you from.”
“If you knew the whole story, doctor, you would hardly blame me.”
“I don’t blame you. How can I blame conduct which brings me a
patient? If all men were wise, we poor medicine men might go sweep
crossings.”
“But, doctor, if I had been a wiser man I should have been a worse
one.”
“Not necessarily. And it shows no more virtue than wisdom to
throw up the sponge when you are beaten by Fortune at the first
round.”
George reddened. “First and last round too, isn’t it, doctor? Come,
tell me honestly how long you give me to live.”
Dr. Bannerman looked at him steadily.
“If you remain mooning about here, hovering along like a moth in
the sunshine, brooding over things which are past and beyond
remedy, I give you a year. If you buckle to, make yourself new
interests in life, start on a new career, and get new air into your lungs
and new thoughts into your brain, I give you any time from ten years
to five-and-twenty.”
George instinctively drew himself up into a more martial attitude.
“And my wife?” he asked with fresh interest and eagerness.
“I give her as long as she has a strong heart and a brave arm to
take care of her.”
The young man turned his eyes away with a new light burning in
them. At last he said with a tremor in his voice:
“You would not deceive us about this, of course, just to keep us
lingering on a little longer?”
“Not a bit of it. You are both suffering from severe shock to the
nervous system, and because each of you thought you were going to
lose the other, neither has had the energy or the desire to pull round.
You besides have a weak lung, and I tell you frankly you would not
make her majesty such a smart young officer again. But a man of
your intelligence must have other resources.”
George saw by the foregoing speech that very little of his history
during the past year was unknown to the doctor. On the whole, this
knowledge made him feel easier.
“I think I could write,” said he reflectively. “I have already given
myself some sort of training for it, and if only all my ideas did not
seem to be locked up somewhere out of my reach, I think I could
express them at least intelligibly.”
“Good,” said Dr. Bannerman. “Then all we have to do is to find the
key. I think I know a friend of yours whom we can consult about that.
You shall hear the result of our conference very shortly. In the
meanwhile, keep up your spirits and keep out of draughts, and
English literature may yet thank your wife for taking you out of the
army.”
George shook his hand warmly, and the doctor left the house. Half
way down Lockyer Street he met Ella Millard, who was burning with
impatience to know the result of the interview. As he came up she
hastily dismissed a fair-haired young fellow of three or four and
twenty, who trotted meekly off at once towards the Hoe. She was too
deeply interested in what the doctor had to tell to utter more than the
word “Well!” in a tremulous voice. She thought, however, by the
expression of his face that his news could not be very bad.
“Well!” he repeated after her.
“Is it well?” said she impatiently.
The doctor smiled. “I think so.”
Her face softened. “I thought it could not be the worst; it would
have been too dreadful—and too foolish,” she added sharply.
“That is just what I told him. Oh! I was very hard with him; I thought
he wanted it. He has had an awful time of it lately, and the poor boy
hardly knows even yet whether he is on his head or his heels. But it
is quite time now that he made an effort to pull himself together. I
gave him a good talking to, I can tell you.”
Her look seemed to implore mercy, but she said nothing. He
continued: “They ought to go away. He thinks he could write, and I
should encourage him to try.”
“And—his wife?” she asked, with a scarcely perceptible diminution
of interest.
“There is nothing organically wrong with her at all. She will be
herself again before him, and then help his recovery.”
“Help him! Do you think so?” asked Ella doubtfully.
“Yes.”
“I thought you told me, that when you first saw her she produced
on you a very different impression.”
“So she did. But then—she was a very different woman.”
Ella’s mouth twitched rather scornfully. She thought that the weird
prettiness of Nouna’s little wasted face had bewitched even this
middle-aged doctor.
“She is scarcely even yet an ideal companion for a man of
intellect,” she said with a slight touch of her worst, most priggish
manner.
“H’m, I don’t know,” said the doctor. “Your man of intellect is
generally a man of something else besides; and the housekeeper-
wife and the blue-stocking wife both frequently leave as much to be
desired as—well, say, the flower-wife, if once the flower learns to
turn to the sun, as, I think, little Mrs. Lauriston has done.”
“She is fond of him,” agreed Ella rather grudgingly.
“And what more does he ask of her?”
“Nothing more now; but will it be always so?”
“Who can tell? But love on both sides is a good matrimonial
foundation. Have they any money?”
“Enough to live upon as quietly as they are doing now.”
“Ah! but they want something more than that. He ought to move
about, to travel, and she ought to be tempted back to interest in life
with some of the pretty things she is so fond of. Haven’t they any
relations who could manage that?”
Ella’s face brightened with a little smile as she nodded assent. “I
think the relations can be found,” she said.
Apparently the doctor thought he had put the suggestion into good
hands, for he looked at her very good-humouredly as he held out his
hand and bade her good-bye.
“The gentleman who was dismissed for me will be wishing me all
the nauseous draughts I ever prescribed,” said he drily.
Ella grew superbly disdainful.
“Oh no,” she cried with haughty emphasis. “He is only a silly young
fellow who was a fellow-officer of Mr. Lauriston’s, and who is so fond
of him that he has come down here on purpose to see him, although
he puts off doing so from day to day for fear of waking in him
recollections which might distress him.”
The doctor was more than satisfied with this elaborate
explanation.
“I dare say he manages to fill up his time agreeably enough—in
this pleasant neighbourhood,” said he gravely.
And he raised his hat and left her before she had time to utter
another protest.
Now, quite unintentionally, Dr. Bannerman had done a very ill turn
to a most harmless and kindly fellow-mortal. Clarence Massey, the
humble companion whom he displaced at Ella’s side, having been
attracted to Ella by the devotion with which she had worked for his
friend George Lauriston, had raised up an altar to her in his most
affectionate and warm heart, on which, figuratively speaking, he
burned incense all day long. Whenever and wherever she would let
him, he followed like a dog, bearing her snappish fits with beautiful
meekness, accepting any remarks she liked to throw to him, as
precious pearls to be treasured in his memory; gentle, loyal, and
devoted always. Ella, who had begun by laughing at him, had been
thawed by his distracted anxiety and misery over George Lauriston’s
misfortunes, until from tolerating she had begun to like him. And
now, just as she was getting so amiable to him that he had begun to
entertain hopes which he had the sense and modesty to think
extravagant, this light suggestion on the part of a stranger chilled her
into anger at the thought that any one should think her capable of a
serious thought for so unintellectual a person as Clarence Massey.
She had promised, on Doctor Bannerman’s approach, to rejoin
Clarence on the Hoe; but it was with the step of an offended
empress that the plain little girl met this well-provided young fellow,
on whom a dozen mammas of marriageable daughters now fixed
longing eyes.
“Well, what does he say?” asked Clarence, afraid from the
expression of her face that the report was bad.
She told him briefly and coldly the substance of the doctor’s
opinion, but without any hint of his last suggestion except the vague
information that the pair had better go abroad. Then she walked
briskly on in the direction of the Fort, and to Clarence’s meek request
for permission to accompany her, she gave the most brusque, most
chilling answer that he could “do as he pleased.” Of course he
pleased to go, and when they got on to the narrow footpath which is
only wide enough for one, he followed with tears in his eyes at the
change in her, wondering what in the world had happened to make
her so unkind to him. Meanwhile, however, an idea had come into
her busy little head which helped the effect of the spring air in
restoring her to good humour; so that when she stopped to look
reflectively out to sea and caught sight of his disconsolate face, she
smiled at him with mingled mercy and majesty and asked him why
he looked so miserable.
“I’m not miserable now,” said he, brightening up at once. “It was
only that I was afraid you didn’t want me.”
Ella grew prim again.
“It is very kind of you to come,” said she.
“Ella, don’t say that. How can you say that, when you know very
well how happy it makes me to be with you!”
“Happy! How absurd! I wish, Clarence, you wouldn’t say such
ridiculous things.”
“But, Ella, why is it ridiculous? It’s true, you know it’s true. You
know very well I would follow you to the end of the world if you’d let
me, that I’d do anything you wanted me to, that I’m never so happy
as when I’m with you. Well, why is it ridiculous to say what is true
and what you know?”
“But I don’t want to know it,” said Ella sharply. “If I had thought you
would ever talk to me in such a silly way I would never have let you
come out with me. When I’m thinking about serious things, too!”
“Can’t you see that this is serious to me?”
“It’s only all the more ridiculous. You must either promise never to
talk such nonsense to me again, or you must give up the walks.”
“Very well, then, I must give up the walks,” said Clarence
resignedly, “for I can’t make the promise.”
And he walked away over the rough grass, and began to look out
to sea on his own account. Ella, in spite of the “serious things” which
had occupied her thoughts, was forced to turn her attention to this
importunate and foolish person close at hand, and she did so with a
much graver countenance than was her wont in matters relating to
him. The fact was that this unexpected threat of withdrawing his
despised attentions woke her suddenly to the fact that she should
miss them. Ella discovered all at once that she was not so insensible
as she had imagined to the ordinary feminine pleasure in the
possession of a devoted slave. Even a Clarence who occasionally
talked nonsense would be better than no Clarence at all. Some
expression of these conclusions found its way to her face, for the
crestfallen swain was emboldened by her glance to draw near her
again. She said no kind word however, and he was afraid that further
pleading at the moment might be injudicious, so they stood very
quietly side by side until Ella broke out vehemently:
“I wish I had twenty thousand pounds!”
The wish and the fiery manner in which it was uttered took
Clarence so completely by surprise that instead of assuring her that
she had only to say the word, and he would lay that sum at her feet,
as perhaps she had expected of the impulsive little Irishman, he only
said simply:
“What for?”
“To throw into the sea,” was her surprising answer.
He laughed, supposing that this was a faint sort of joke.
“I mean it,” she added gravely. “I can get five thousand pounds of
my fortune from papa, but I want twenty thousand more.”
“But what a strange use for it; you are not in earnest about that!”
“Oh, yes, but I am.”
“Well, if anybody were to offer you twenty thousand pounds, what
would you say to—him?”
“I should say, Thank you.”
“Prettily?”
Ella paused. He was bending his head to look into her eyes, and
putting into that word a great deal of impertinent meaning. Then she
flashed up into his face a grand glance full of magnificent
haughtiness.
“Of course, because I am not handsome, you think I ought to jump
at you!”
“Oh, no, I don’t. But whether you jump at me or away from me, you
shall have the twenty thousand pounds.”
“What, without knowing what I am going to do with it?”
“You said you wanted it to throw into the sea.”
“Oh, yes, yes, so I do. But supposing I were to throw it to another
man—a merman, for example?”
Clarence winced. “Whatever you do is right, Ella,” he said, at last.
“You can throw it to whoever or whatever you like.”
“When can I have it?”
“I shall have to go up to town. I can raise it by next week.”
Ella put her hand on his arm impulsively.
“You’re a good fellow,” she said, in a very sweet voice.
And Clarence, who had never had such a mark of her favour
before, felt all on fire, and wished he dared to hold her fingers where
they had so unexpectedly placed themselves. But the overwhelming
reverence he felt for this small girl taught him discretion, and you
might have thought, by the stiffness with which he held himself under
her touch, that a wasp had settled upon him, and that he was afraid
to move for fear of provoking it to sting. But they walked back
together to the Hoe in a very amicable manner, Clarence feeling that
luck had helped him to make a splendid move, and Ella wondering
whether by the acceptance of twenty thousand pounds from a man
she could be considered in any way to have compromised herself.
CHAPTER XXXII.
Three weeks passed very quietly for George Lauriston and his
wife without any markedly apparent result of the doctor’s visit, except
that George, trying to shake off the lethargy into which he had sunk
since his imprisonment, had put himself into harness for a new battle
with fortune by writing articles on the condition of the army for a local
paper. He also took a journey to London to fulfil his long-promised
revenge upon Rahas, and would probably have got himself into fresh
trouble by using other than legal means of chastisement upon the
Arabian, if that ingenious gentleman had not just got into a little
difficulty with the excise officers over a large consignment of choice
tobacco which was more than suspected of having paid no duty, and
some silver goods not up to standard, the hall-mark on which had
been forged, which forced him to leave the land of his adoption for
shores where genius is more respected.
Both George and Nouna for a long time refrained from mentioning
her mother’s name, and it was with some emotion that they both
recognised her handwriting one day outside a letter directed to the
husband, the postmark of which was Bath. George took it away to
read, and Nouna made no remark, but when he came back to her,
holding it open in his hand, he found that she was trembling with
intense excitement. She took it from him with a passionately anxious
glance, but gathered comfort from his gravely smiling face.
Nouna then read these words:
“My dear Mr. Lauriston,

“I am writing to make a request which I pray you will
generously grant. I know there are differences between us
which would make another meeting undesirable and perhaps
painful to both, I would not suggest that we should see each
other again: but I implore you to let me see my daughter just
once more. Six months ago I could have claimed this as a
right, or I would have contrived it by a trick. But I have learnt
to respect you, and I only ask. I am a different woman, I have
grown old, I am changed, you would not mind her coming now
—I swear it. Lord F. has been very generous, and I want
nothing but just one more look at my daughter. Let her come
and see the Condesa di Valdestillas, that is the name I bear
here, and shall bear to the end of my life. A foreign title covers
whatever of eccentricity is left in
“Yours very sincerely,

“Lakshmi di Valdestillas.”
Nouna was crying quietly as she finished. She clung to her

husband’s arm.
“Must I go?” she whispered.
“Oh, yes,” said George promptly. “She has always loved you,
Nouna; I will write to tell her you are coming.”
“Oh, George, George,” panted the little creature in the same low
voice, “I feel so wicked for not wanting to go! But all my heart has
turned to you now, and I can’t get the old feeling back.”
He clasped his hands round her shoulders.
“But you will, Nounday, you will have just the feeling that is right
when you see her all by herself, lonely, waiting for you whom she
has always loved better than anything in the world.”
All the sting had now gone out of his feelings towards the creature
who, with all her odd mixture of coarseness and refinement,
corruption and generosity, had lived to see the very virtues she had
fostered in her child turn against her in the loneliness of her
premature age. For George had learnt from Lord Florencecourt, who
ran down to Plymouth two or three times to see him and Nouna, to
whom he was beginning to be reconciled, that Chloris White had
indeed retired from her old life, broken up and suddenly middle-
aged, and had fixed her retreat in the pretty old city of Bath, where
she lived safe from recognition in a colony of what the Colonel
irreverently called “old tabbies,” feeling neither contrition for the past
nor discontent with the present, and passing her time, with a serenity
born of dulled faculties and worn-out energies, in petty charities and
petty scandal.
Two days after the receipt of the letter George arrived with Nouna
in Bath, left her at the door of her mother’s residence, a small, well-
kept house in a quiet street, and walked up and down outside until
she should rejoin him. When she reappeared at the door she was
very serious, and she beckoned him to come up the steps to her.
“Mamma wishes—to say—good-bye to you,” she said in a
tremulous voice.
Standing aside she let George see a bent figure, dressed in black,
with greyish hair, and a wan dark face, who raised her great black
burning eyes, but not with the old boldness, to his face. He took his
hat off, and held out his hand. The lean little dark fingers she put into
his were shaking.
“Good-bye, Mr. Lauriston. I shall not see you again. It has made
me happy to see you. Remember when you think of me that I had no
chance—from the beginning. But I kept my child pure, and so God
sent you to her. I dare not bless you, but I thank you; if I were better I
would pray for you. Good-night. Good-bye.”
The long evening shadows were creeping over the quiet streets,
as George and his wife, walking slowly away, caught the final
glimpse of a pale, drawn face, and great eyes like flaming fires,
straining in the gloom for a last look at them. Nouna was very quiet,
but she was much happier than she had been in coming.
“George,” she said in a low voice, “I can think about her and love
her now just as I used to do. When may I see her again? She would
not tell me.”
And George could not tell her either, though he gave her a ready
assurance that she should come whenever she was summoned; for
he had a shrewd suspicion that, in spite of Lord Florencecourt’s
belief that she was happy and contented, the restless spirit of the
reputed Countess was untamed still, and chafed in secret under the
new bonds of broken health, changed habits, and disappointed
ambition. Two days later this suspicion was confirmed, when he
received the tidings, conveyed to him only, of the sudden end of the
Condesa di Valdestillas, who had been found dead in her bed from
an overdose of a sleeping-draught. But as she left a sealed letter for
George with instructions to keep the news of her death from her
daughter until Nouna was stronger, full of passionate thanks to him,
and equally passionate regrets that she might not leave what she
possessed to her child, he was not deceived, though he was the only
person who ever knew the secret.
Poor Sundran, who was with her mistress to the last, implored
George, who went at once to Bath on learning the tidings, to let her
come back to her darling Missee Nouna. And as he was sure
enough now of his influence over his wife no longer to dread that of
the black woman, he promised that, at no distant time, she should
return to her service.
On hearing that the “Condesa di Valdestillas” was dead, Lord
Florencecourt, finally relieved from his fears, openly acknowledged
Nouna as his daughter “by a former wife,” as indeed poor Chloris,
thinking over the position of affairs on the eve of her first and last
attempt at reparation, had foreseen that he would do, and settled a
handsome allowance upon her. He came down to Plymouth in the
last week of May to make this determination known to his son-in-law.
He was accompanied by his niece Ella, who was in a state of strong
but subdued excitement, but who gave no reason which her uncle
could consider adequate for her entreaty that she might thus leave
London for a few days in the height of the gaieties of the season.
On their arrival in Plymouth, Ella chose to remain alone at the
hotel while the Colonel went to call upon the Lauristons. He thought
this decision very extraordinary; but on his return a light came to him;
for in the sitting-room, standing close by his niece’s side, and
bending over her to speak with a passionate earnestness which
seemed to infect the usually self-contained girl, was Clarence
Massey. They both started guiltily on Lord Florencecourt’s entrance,
and Clarence shook with nervousness as he greeted him. Ella
rushed at her uncle, and asked about the health of the invalids with
great vivacity and interest.
“What were you talking about when I came in?” asked the Colonel
bluntly, when he had informed her that George and Nouna were
neither better nor worse than they had been three weeks ago.
“We were talking about them—about the Lauristons,” answered
Ella.
And Clarence echoed her words. The Colonel looked from the one
to the other incredulously. His niece seized both his hands
impulsively, with a light-hearted laugh.
“We must tell you—it’s a great secret, but it’s coming out now, and
you shall be the first to hear it,” said she.
Then she made him sit down, and told him, rather breathlessly, a
long story, to which Clarence played Chorus, and to which the
Colonel listened with amazement, admiration, and something like
consternation too.
“And who’s to pay for it all?” he asked at last in bewilderment.
“Oh, we’ve arranged all that,” said Ella airily.
Again Clarence echoed, “We’ve arranged all that.”
And this astonishing unanimity naturally led Lord Florencecourt to
a conclusion the expression of which would have filled Ella with the
loftiest indignation. In the meantime, having been informed of the
plot, he was pressed into the service of the conspirators, and that
evening, when it had grown dark, they all three went to the house
where the Lauristons were staying, and the Colonel entered, leaving
the two young people to walk up and down outside in a state of
breathless expectancy.
“Break it gently!” was Ella’s last injunction as he left them.
Lord Florencecourt found his way up stairs to his son-in-law’s
sitting-room in a state of great nervousness. He found George and
Nouna, pale, thin, and languid as ever, the former sitting at the table,
writing, while his tiny wife, curled up on the sofa with a large ball of
wool, some long wooden pins, and a small, misshapen piece of work
which was the result of many evenings’ labour, flattered herself that
she was knitting. They were both surprised by this second visit from
the Colonel, and by the fact that now he had come he seemed to
have nothing to say.
“What are you doing?” he asked Nouna at last.
“I’m making George a comforter,” she answered proudly. “I can’t
be idle while my husband’s at work.”
“Well, it keeps you quiet at any rate,” he observed injudiciously, a
glance at the comforter having convinced him that if ever it should be
finished and worn it would belie its name. The Colonel fidgeted for a
few moments, and the young people began to assume an attitude of
expectancy, perceiving that something was to come of this unusual
restlessness. “I suppose you wouldn’t like to leave Plymouth—to go
anywhere—to—India, for instance,” he blurted out at last.
Nouna sprang up with a cry, a great light in her eyes. George’s
face flushed; he crossed the room and came to support his wife, who
was tottering.
“Why does he say it? why does he say it? It can’t be true, oh, it
can’t be true!” sobbed she, burying her face in his breast.
“What does it mean, Colonel? Are you serious?” asked George in
a hoarse voice.
He hated England just now, sore and beaten down as he still felt,
but he had felt that to run away from it was cowardly, even if he had
been able to afford it. This suggestion of change for himself and joy
for Nouna therefore came upon his heart like a ray of bright light in
the dead grey level of their languid lives.
“Make all your preparations to-night,” said the Colonel, “for you will
have to start to-morrow.”
And, as if afraid of committing himself by any explanation, he left
the room, and darted out of the house like a lad before they had time
to stop him. In the street Ella and Clarence met him, full of
excitement.
“Well?” said they at the same time, both quivering with excitement.
“It’s all right. I told them—just enough and no more. I said it rather
suddenly perhaps, but I was afraid they’d ask questions. They’re to
be ready to start to-morrow, I said. You couldn’t have managed
better yourself, Ella. They were delighted, absolutely delighted.”
The Colonel was right. To these two beings, whose hearts and
minds were still scarcely as convalescent as their bodies after the
trials of the preceding few months, the suggestion of this great
change came as the grant of a new bright life to them. Nouna, in
particular, was half crazy with delight, and seemed to recover in a
moment all her lost vivacity, as she babbled of palms and sunshine,
palaces with stately domes and graceful minarets, of elephants with
rich trappings, birds with bright plumage, and dark depths of jungle
where the tiger was known to lurk, and where every step was
hedged with fascinating peril. That night she scarcely slept, and next
morning, when Lord Florencecourt again made his appearance,
accompanied this time by Ella, he was quite bewildered by the
change in his daughter’s looks. Ella herself, although very quiet, was
almost as much excited, as she asked whether they were ready.
George, with dull masculine pertinacity, worried everybody by asking
for details of the journey for which they had so hastily prepared; but
at last perceiving, by the evasive answers he got, that some surprise
was intended, he was in the end content to hold his tongue, and to
wait patiently till the proper time should bring enlightenment.
Arrangements had been made, they were told, for the transport of
their luggage, and they had nothing to do but to start in the company
of Ella and the Colonel. They set out on foot, which was one
astonishing thing, and they were taken in the direction of the Hoe,
which was another. It was a beautiful, bright May morning. From the
seat by the camera obscura they all stood for a moment, looking
down at the water, when suddenly Nouna burst forth into a cry of
admiration at the sight of a beautiful yacht which was anchored half-
way between the shore and Drake’s Island.
“When did it come?” she cried with much interest. “It wasn’t here
yesterday. What a beautiful little thing!”
“Little thing!” cried Lord Florencecourt, with untimely impetuosity.
“Why it’s 150 tons; big enough to go round the world in!”
Then an awkward silence fell upon everybody, for, vulgarly
speaking, the cat was out of the bag. And the conversation was kept
up with difficulty until, descending the cliff, they all came to the little
landing-pier, where a small boat was waiting with Clarence Massey
standing up in it, waving his hat frantically and beaming with
unspeakable enthusiasm. Neither George nor Nouna asked any
questions now; and they all got into the little boat in a state of
surprising silence, and were rowed straight out towards the beautiful
yacht without anybody’s remarking upon the strangeness of the
circumstance. But as they drew near her, Nouna caught sight of the
name, painted in bright gold letters on the stern—“Scheherazade.”
She touched her husband’s arm, and made him read it too. Before
he could speak, they were close under the yacht, and Lord
Florencecourt was leading the way on board. Nouna climbed up next
like a cat, and the rest followed quickly.
Then Ella took the young wife by the hand, and, leaving the three
men on the deck, led her on a tour of inspection. The yacht was a
tiny floating palace, fitted up by the dainty taste of one woman to suit
the luxurious fancy of another. The rooms were hung with rich
tapestry, and with delicate China silks embroidered in gold and pale
colours. The woodwork was painted with birds and flowers on a
background of faint grey landscape. The bed-room was fitted up with
satin-wood, and hung with rose-coloured silk; while in order that
George might have a corner better suited to masculine taste in this
dainty little craft, a very small room, dark with old oak and
serviceable leather, had been appointed for him as a study. Every
corner of the yacht held something beautiful and curious: skins of
white bears, mounted in maroon velvet; carvings in ivory, securely
fixed on dark brackets that showed off their lacelike outlines;
treasures in bronze, in delicate porcelain, in exquisitely tinted glass
from Salviati’s, met the eyes at every turn. The whole furnishing and
fitting of the little vessel, down to the choice of silver-gilt teaspoons
from Delhi and a lamp which was said to have been dug up at
Pompeii, had clearly been a labour of love.
Nouna was overwhelmed; she walked along with her hand in
Ella’s, scarcely uttering a sound, until at last she heard the words
whispered in her ear: “This is a present for you—all for you, with my
love. You are to make good use of it, and be very happy in it. No”—
she stopped Nouna, who was breaking into tears, and incoherent,
passionate thanks—“you may thank me when you and your husband
both come sailing back strong and rosy and well.”
Nouna smiled at her with glistening eyes as she put her little
hands round the girl’s shoulders.
“I can’t thank you, I can scarcely try. You were born to be a good
fairy to everybody. Kiss me, kiss me hard, and give me some of your
own sweetness that I may be a better wife.”
When they came on deck again they were both very quiet; and
George, who had in the meantime learnt that this fairy yacht was a
present to his wife, and also that, in common with the fairy presents
of tradition, for a whole year at least it would entail no expense upon
its owner, could do nothing but shake Ella’s hand warmly and
murmur some incoherent words.
All the visitors on board now felt that their task was done. The
luggage was on board, the steam was up, the hands were ready to
hoist the anchors; and both George and Nouna showed signs of
having suffered as much excitement as their still weak frames could
bear. Lord Florencecourt, Ella and Clarence took their leave quickly,
descended from the yacht into the little boat, and rowed away in the
sunshine, while the young husband and wife waved them good-bye.
“Where are we going to, George?” asked Nouna, when the little
boat had reached the pier, and the passengers were landed.
“Just where you like. You are its mistress, you know.”
She drew a long breath of pleasure.
“Tell the captain to go, as quickly as possible, to some place—
nearer than India—where there are palms and blue skies, and bright
birds.”
George obeyed, and, coming back, told her that they were going
first to Malta. She was satisfied, considering that Valetta was a pretty
name, and remembering she had heard the air was good for people
with weak lungs.
“Yes, yes, let us go to Malta, George, and there you will get well,”
said she.
And she drew him towards a pretty little pavilion which had been
erected on the deck. The hanging curtains were crimson and gold,
and could be looped back to command a view of the sea in any
direction.
“Why didn’t Ella take me in there?” she said.
“Perhaps it contains some great treasure which she kept as a
bonne bouche at the last,” suggested he, smiling.
Already she had an inkling of the truth, and when she tore back
the nearest curtain and found, kneeling on the ground on a leopard’s
skin among white silken cushions which were to support her young
mistress’s head, the old servant Sundran trembling with joy, she
gave way, and fell sobbing into the Indian woman’s arms.
“Oh, George, George,” she whispered passionately, springing up
again to her husband’s side, “Ella must have an angel from heaven
hovering about her to whisper to her just what will make people
happiest! Aren’t you afraid of waking up and finding it isn’t real?”
“No, Nounday,” said he, tenderly, but with a thoughtful face; “I’d
rather think that we have been in a dreary, feverish sleep, and that
we are sent away to wake us up to life again!”

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Biological Responses to Nanoscale

Particles Molecular and Cellular

Sarcopenia-Molecular, Cellular, and Nutritional Aspects

Cellular and Molecular Basis of Mitochondrial

Clinical, Biological and Molecular Aspects of COVID-19

Computational Methods for Nanoscale Applications

Technoscientific Research Methodological And Ethical

Cellular and Molecular Immunology 9th Edition Abul K.

Methodological Approaches 1st Edition Ruth Evans

Molecular and Cellular Biology of Viruses 1st Edition

More information about this series at http://www.springer.com/series/3705

ISSN 1434-4904 ISSN 2197-7127 (electronic)

Library of Congress Control Number: 2018968395

© Springer Nature Switzerland AG 2019

Engineered nanoparticles, as one of the principal components of the twenty-ﬁrst-century

Bern, Switzerland Peter Gehr

Part I Manufacturing and Characterization of Materials

3.2 Preparation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

5.5.1 Fundamentals of Nanoparticle Aggregation . . . . . .. 116

Part II Membrane Transfer, Cellular Uptake and Intracellular

7 Cellular and Non-cellular Barriers to Particle Transport Across

8.3.11 Super Resolution Microscopy . . . . . . . . . . . . . . . . . 202

Part III Cellular Responses and Health Effects

10.5 NMs and Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

12.4 Types of Nanoparticles Unintentionally Occuring in the

Sandor Balog BioNanomaterials, Adolphe Merkle Institute, Fribourg, Switzerland

Nikolai Gaponik Physical Chemistry, Technical University of Dresden, Dresden,

Martina Claudia Meinke Department of Dermatology, Venerology and

Anika Vogt Department of Dermatology, Venerology and Allergology, Charité –

Kateryna Loza and Matthias Epple

Abstract The diversity of applications in catalysis, energy storage and medical

K. Loza (B) · M. Epple

© Springer Nature Switzerland AG 2019 3

are comparatively simple and usually lead to ligand-free (“naked”) nanoparticles.

1.2 Metals (Gold, Silver, Platinum and Copper)

Fig. 1.2 Transmission electron micrographs of PVP-stabilized gold nanoparticles, prepared by

supracolloidal self-assembly of atomically precise nanoparticles is a promising plat-

Fig. 1.4 Transmission electron micrographs of platinum nanoparticles, prepared by solution-phase

Platinum nanostructures are of particular interest for many industrial applications

ablation in liquids (“top-down” technique) for the surface modification of electrodes

1.3 Alloyed Nanoparticles

The properties of metallic systems can be significantly extended by mixing ele-

Due to the heterogeneity of different properties of individual components (e.g.,

“My dear Mr. Lauriston,

“Yours very sincerely,

Nouna was crying quietly as she finished. She clung to her

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