Research Collection Doctoral Thesis Novel Applications of Metallic Nanoparticles for Cancer Treatments Author(s): Herzog, Antoine F. Publication Date: 2018 Permanent Link: https://doi.org/10.3929/ethz-b-000325544 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection . For more information please consult the Terms of use . ETH Library
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Research Collection
Doctoral Thesis
Novel Applications of Metallic Nanoparticles for CancerTreatments
Author(s): Herzog, Antoine F.
Publication Date: 2018
Permanent Link: https://doi.org/10.3929/ethz-b-000325544
Rights / License: In Copyright - Non-Commercial Use Permitted
This page was generated automatically upon download from the ETH Zurich Research Collection. For moreinformation please consult the Terms of use.
ETH Library
DISS. ETH No. 25466
NOVEL APPLICATIONS OF METALLIC
NANOPARTICLES FOR CANCER TREATMENTS
A thesis submitted to attain the degree of
DOCTOR OF SCIENCES of ETH Zurich
(Dr. sc. ETH Zurich)
presented by
ANTOINE FLORENT HERZOG
MSc. Chemistry, ETH Zürich
born on 10.09.1990
citizen of France,
accepted on the recommendation of
Prof. Dr. Wendelin J. Stark, examiner
Prof. Dr. Pablo Rivera-Fuentes, co-examiner
Prof. Dr. Beatrice Beck-Schimmer, co-examiner
2018
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To my family
Be the change that you wish to see in the world.
Mahatma Gandhi
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Acknowledgments
First, I would like to thank Prof. Dr. Wendelin J. Stark for mentoring my PhD thesis as well as
my master thesis and research projects. Wendelin is certainly a unique professor in his way of
thinking, conducting research and his ability to build a great team of people. As a result of his
influence and the ones of my colleagues, the time I spent at the Functional Materials Laboratory
changed me in many aspects with long-lasting effects. As caricatural as at it may sound, my
stay at FML opened my eyes on the endless opportunities life reserves and reinforced my will
not to be a passive witness of my life.
I would like to mention and thank all the group members from my years at the Functional
Materials Laboratory: Julian Koch, Konstantin Schulz-Schönhagen, Lukas Langenegger,
Michele Gregorini, Nadine Lobsiger, Nikita Kobert, Olivier Gröninger, Philipp Antkowiak,
Phillipe Bechtold, Simon Doswald, Urs Lustenberger, Weida Chen, Xavier Kohll, Dr. Carlos
Mora, Dr. Christoph Kellenberger, Dr. Christoph Schumacher, Dr. Corinne Hofer, Dr. Daniela
Paunsecu-Bluhm, Dr. Dirk Mohn, Dr. Elia Schneider, Dr. Gediminas Mikutis, Dr. Jonas Halter,
Dr. Mario Stucki, Dr. Martin Zeltner, Dr. Michael Loepfe, Dr. Michela Puddu, Dr. Nicholas
Cohrs, Dr. Oleksander Stepuk, Dr. Philipp Stoessel, Dr. Samuel Hess and Dr. Vladimir Zlateski.
I express my gratitude to Dr. Elia Schneider for his support regarding the synthesis of caged
gemcitabine and reviewing my manuscript, to Dr. Martin Zeltner for his extensive support for
the projects involving magnetic nanoparticles and to Dr. Carlos Mora for the supervision of one
of my research projects and my master thesis. I would especially like to thank them for the
always uncomplicated and pleasant collaborations. I would also like to thank Prof. Dr. Robert
Grass for always giving great advice whenever I would come with a question.
From the Institute for Chemical- and Bioengineering of ETH Zürich, I would like to thank
Bianca Wittmer, Luca Andres and Urs Krebs. I would like to give special thanks to Elias Halabi
for running HPLC-MS measurements when it was much needed and Andrin Bear for the nice
collaboration. From the University Hospital Zürich, I would like to thank Prof. Dr. Beatrice
Beck-Schimmer and Anja Zabel for the fruitful collaboration on the magnetic nanoparticles
projects.
Finally, I would like to thank my parents, my brother and my girlfriend. They are always here
for me, whenever I need them and I am very grateful for their support throughout all those
years.
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Table of Contents
Acknowledgments 3
Résumé 11
Summary 14
1. Cancer: Origin, Burden and Therapy 18
1.1 Origin of Cancer and its Societal Impact ................................................................. 21
1.1.1 Cancer Biology and Aetiology ..................................................................... 21
1.1.1.1 Hallmarks of Cancer..................................................................................... 21
1.1.1.1.1 Hallmark 1: Sustaining Proliferative Signalling ................................................................... 22
1.1.1.1.2 Hallmark 2: Evading Growth Suppressors ........................................................................... 22
1.1.1.1.3 Hallmark 3: Activating Invasion and Metastasis .................................................................. 22
1.1.1.1.4 Hallmark 4: Enabling Replicative Immortality..................................................................... 23
1.1.1.1.5 Hallmark 5: Inducing Angiogenesis ..................................................................................... 23
1.1.1.1.6 Hallmark 6: Resisting Cell Death ......................................................................................... 23
1.1.1.2 Carcinogenesis ............................................................................................. 23
1.1.1.2.1 Genotoxic Carcinogenesis .................................................................................................... 24
1.1.1.2.1.1 Oxidative Damages ....................................................................................................... 24
1.1.1.2.1.2 Photolesions ................................................................................................................... 24
1.1.1.2.1.3 DNA Alkylation ............................................................................................................ 24
1.1.1.2.2 Non-genotoxic Carcinogenesis ............................................................................................. 25
1.1.1.2.2.1 Hormone-mediated Carcinogenesis ............................................................................... 25
1.1.1.2.2.2 Receptor-mediated Carcinogenesis ............................................................................... 25
1.1.1.2.2.3 Carcinogenesis Mediated by Epigenetic Changes ......................................................... 25
1.1.1.2.3 Viral carcinogenesis .............................................................................................................. 26
1.1.2 Current Cancer Treatments Strategies.......................................................... 26
1.1.2.1 Surgery ......................................................................................................... 26
1.1.2.2 Radiation Therapy ........................................................................................ 27
1.1.2.3 Chemotherapy .............................................................................................. 28
1.1.2.3.1 Alkylating Agents ................................................................................................................. 28
1.1.2.3.2 Antimetabolites ..................................................................................................................... 29
1.1.2.3.2.1 Antifolates ..................................................................................................................... 29
1.1.2.3.2.2 Base Analogues ............................................................................................................. 29
1.1.2.3.3 Anti-microtubules agents ...................................................................................................... 29
1.1.2.3.4 Topoisomerase inhibitors ...................................................................................................... 30
1.1.2.4 Targeted Therapy ......................................................................................... 30
6
1.1.2.4.1 Tyrosine Kinase Inhibitors .................................................................................................... 30
1.1.2.4.2 Angiogenesis Inhibitors ........................................................................................................ 31
1.1.2.4.3 Epidermal Growth Factor Receptor (EGFR) Inhibitors........................................................ 31
1.1.3 Psychological Impact and Financial Burden of Cancer ............................... 31
1.2 Nanomedicine: a Widely Encompassing Concept ................................................... 32
1.2.1 Drug Delivery ............................................................................................... 32
1.2.2 Nanoparticles with a Therapeutic Action ..................................................... 33
1.3 Interactions between Nanoparticles and In Vivo Elements ..................................... 34
1.3.1 Interaction with Blood Components ............................................................ 34
1.3.1.1 Nanoparticle-Protein Interactions ................................................................ 34
1.3.1.2 Nanoparticle-Cell Interactions ..................................................................... 35
1.3.2 Interaction with Organs Influencing Circulation Time ................................ 36
1.3.2.1 Kidneys......................................................................................................... 36
1.3.2.2 Liver ............................................................................................................. 36
1.3.2.3 Spleen ........................................................................................................... 37
1.4 Conclusion ................................................................................................................ 37
2. Hydrogen as a Bio-orthogonal Trigger for Spatiotemporally Controlled Caged
Prodrug Activation 39
2.1 Introduction .............................................................................................................. 40
2.2 Experimental ............................................................................................................ 41
2.2.1 Synthesis of Caged Coumarin (Compound 2a) ............................................ 41
2.2.2 Synthesis of Caged Coumarin (Compound 1a) ............................................ 41
2.2.3 Screening of Catalyst Concentrations .......................................................... 42
2.2.4 Transmission Electron Microscopy Measurements ..................................... 42
2.2.5 Kinetics of Deprotection of Caged Coumarin .............................................. 42
2.2.6 Interval Kinetics of Deprotection of Caged Coumarin ................................ 43
2.2.7 EC50 Experiments ......................................................................................... 43
2.2.8 Extracellular Prodrug Activation in Cancer Cells ........................................ 44
2.2.9 Zeta-Potential Measurement ........................................................................ 44
2.3 Results and Discussion ............................................................................................. 45
2.3.1 EC50 Experiments ......................................................................................... 45
2.3.2 Kinetics of the Uncaging Reaction............................................................... 46
2.3.3 Toxicity of the Nanoparticles ....................................................................... 48
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2.3.4 Extracellular Caged Prodrug Activation ................................................. 49
2.4 Conclusion ................................................................................................................ 50
3. Preliminary Pre-Clinical Trials for the Gemcitabine Caged Prodrug System 52
3.1 Introduction .............................................................................................................. 53
3.2 Experimental ............................................................................................................ 54
3.2.1 Formulations Preparation ............................................................................. 54
3.2.2 Dynamic Light Scattering Measurements .................................................... 54
3.2.3 Nebulization Assays ..................................................................................... 54
3.2.4 Tolerability Profiles of Formulation 2 and of Platinum Nanoparticles
Solutions 55
3.2.4.1 Animals ........................................................................................................ 55
3.2.4.2 Administration Protocol ............................................................................... 55
3.2.4.3 Clinical Follow-up........................................................................................ 56
3.2.4.4 Terminal Procedures .................................................................................... 56
3.2.5 Feasibility Test in Xenograft Mice Model ................................................... 56
3.2.5.1 Animals ........................................................................................................ 56
3.2.5.2 Cells .............................................................................................................. 56
3.2.5.3 Preparation of Cells to Be Implanted ........................................................... 57
3.2.5.4 Implementation of Tumour Cells ................................................................. 57
3.2.5.5 Tumour Measurements and Clinical Follow-up........................................... 57
3.2.5.6 Administration Protocol ............................................................................... 57
3.2.5.7 Terminal Procedures .................................................................................... 58
3.2.5.8 Necropsy and Lung Collection ..................................................................... 58
3.3 Results and Discussion ............................................................................................. 58
3.3.1 Influence of Surfactant on the Average Droplet Size .................................. 58
3.3.2 Freeze-thaw Stability.................................................................................... 59
3.3.3 Nebulization Properties of the Selected Formulations ................................. 60
3.3.4 Tolerability Profile of Formulation 2 and Platinum Nanoparticles Solutions
61
3.3.5 Feasibility Test in Xenograft Mice Model ................................................... 62
3.4 Conclusion ................................................................................................................ 63
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4. Effective Tumour Cells Removal from Patient Blood using Magnetic Nanoparticles.
65
4.1 Introduction .............................................................................................................. 66
4.2 Materials and Methods ............................................................................................. 68
4.2.1 Synthesis of C/Co-PhEtOH .......................................................................... 68
4.2.2 Synthesis of C/Co-PhEtO-Na+ ...................................................................... 69
4.2.3 Synthesis of C/Co@polyglycidin ................................................................. 69
4.2.4 Synthesis of C/Co@polyglycidyl-COOH .................................................... 70
4.2.5 Synthesis of C/Co@polyglycidyl-COO-EpCAM and C/Co@polyglycidin-
COO-IgG 70
4.2.6 Characterization of Antifouling Efficiency .................................................. 71
4.2.7 Characterization of Separation Efficiency ................................................... 72
4.2.8 Cell Line Experiments .................................................................................. 72
4.2.9 Scanning Electron Microscopy of anti-EpCAM Nanoparticles incubated
with HT-29 Cells .............................................................................................................. 72
4.2.10 Influence of Magnetic Nanoparticles Treatment on Lymphocytes
Enumeration in Healthy Subjects Spiked Blood .............................................................. 72
4.2.11 Removal of Circulating Tumour Cells (CTC) from Healthy Subjects Spiked
Blood 73
4.2.12 Removal of Circulating Tumour Cells (CTCs) from Cancer Patients ......... 74
4.3 Results and Discussion ............................................................................................. 74
4.3.1 Technical Aspects of the Newly Designed Nanoparticles ........................... 74
4.3.2 First Tests in Human Blood from Healthy Subjects Spiked with Cancer
Cells 77
4.3.3 Specificity, Safety Aspects and Reproducibility .......................................... 77
4.3.4 First Tests with Blood Samples from Cancer Patients ................................. 80
4.4 Conclusion ................................................................................................................ 80
5. Functionalization Reaction Study and Lymphocytes’ Capture as a Demonstration
of Highly Abundant Cells Removal 82
5.1 Introduction .............................................................................................................. 83
5.2 Materials and Methods ............................................................................................. 85
5.2.1 Synthesis of C/Co-PhEtOH .......................................................................... 85
5.2.2 Synthesis of C/Co-PhEtO-Na+ ...................................................................... 85
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5.2.3 Example of a Standard Synthesis of C/Co@polyglycidin ........................... 86
5.2.4 Synthesis of C/Co@polyglycidyl-COOH .................................................... 86
5.2.5 Synthesis of C/Co@polyglycidyl-COO-Campath and C/Co@polyglycidin-
COO-IgG 86
5.2.6 Removal of Lymphocytes from Healthy Subjects ....................................... 87
5.2.7 Removal of Lymphocytes from Cancer Patients.......................................... 88
5.3 Results and Discussion ............................................................................................. 89
5.3.1 Influence of Various Reaction Parameters on Polymer Length ................... 89
5.3.2 Reproducibility of Polymerization Step ....................................................... 90
5.3.3 B- and T-Lymphocytes Removal from Healthy Subject Blood Samples .... 91
5.3.4 B- and T-Lymphocytes Removal from Cancer Patient Blood Samples ....... 92
5.4 Conclusion ................................................................................................................ 93
6. Conclusion and Outlook 95
A.1 Supplementary Information Chapter 2 ..................................................................... 98
Screening of Hydrogen Concentrations ........................................................................... 98
Screening of Oxygen Concentrations ............................................................................... 99
Toxicity Study of Platinum Nanoparticles and Hydrogen ............................................. 100
Kinetics of Deprotection of Caged Gemcitabine ........................................................... 101
Re-use of Platinum Nanoparticles after Recycling ........................................................ 101
Extracellular Prodrug Activation in Normal Cells ......................................................... 102
Screening of Catalyst Concentrations ............................................................................ 105
Screening of Hydrogen Concentrations ......................................................................... 106
Kinetic of the Uncaging of Caged Gemcitabine ............................................................ 107
Screening of Oxygen Concentrations ............................................................................. 108
Toxicity of Platinum Nanoparticles and Hydrogen ........................................................ 109
Extracellular Prodrug Activation in Normal Cells ......................................................... 110
Reuse of Platinum Nanoparticles after Recycling .......................................................... 111
A.2 Supplementary Information Chapter 3 ................................................................... 112
Macroscopic Observation of the Lungs after Tolerability Profile Experiments ............ 112
A.3 Supplementary Information Chapter 4 ................................................................... 113
Particle Size Distribution................................................................................................ 113
Elemental Analysis ......................................................................................................... 113
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Infrared Spectroscopy..................................................................................................... 113
Rotational Thromboelastometry Measurements ............................................................ 113
Removal Efficiency of pSPM-functinalized Nanoparticles ........................................... 114
Particles Size Distribution .............................................................................................. 114
Materials Reproducibility ............................................................................................... 115
Influence of Magnetic Nanoparticles Treatment on other Blood Cells.......................... 116
Rotational Thromboelastometry Measurements ............................................................ 116
Infrared Spectroscopy..................................................................................................... 117
A.4 Supplementary Information Chapter 5 ................................................................... 119
Elemental Analysis after the Various Synthesis Steps ................................................... 119
Calculations of the Number of Polymer Units (example for standard synthesis) .......... 119
7. References 120
8. Curriculum Vitae 136
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Résumé
Cette thèse décrit deux types de technologies, impliquant des nanoparticules et pouvant trouver
des applications dans le domaine du traitement du cancer. La première technologie est un
nouveau système de pré-médicament qui pourrait éventuellement réduire les effets secondaires
des chimiothérapies du cancer du poumon et/ou en augmenter l’efficacité. La deuxième
technologie implique un changement d’enduit (« coating ») par rapport à des nanoparticules
magnétiques précédemment développées. Les nouvelles nanoparticules permettent une
séparation spécifique et efficace de certaines cellules présentes dans le sang et pourraient être
utilisées pour ôter du sang les cellules tumorales circulantes, à l’origine des métastases, mais
également pour isoler les lymphocytes qui sont impliqués dans de nombreuses maladies comme
les lymphomes ou les maladies auto-immunes.
Le chapitre 1 consiste en une introduction générale aux domaines du cancer et de la
nanomédecine. Premièrement, des faits généraux et des définitions sont décrits. Ceci est suivi
par la présentation des traits caractéristiques (« hallmarks ») du cancer. Ils permettent
notamment de comprendre les différences centrales entre les cellules cancéreuses et normales.
Afin de mieux discerner les origines moléculaires du cancer, une introduction à la biologie de
la carcinogénèse est inclue et une vue d’ensemble des traitements principaux (chirurgie,
radiothérapie, chimiothérapie et thérapie ciblée) du cancer est établie. Aussi, un examen succin
des conséquences sociales et financières du cancer pour les patients et leurs familles est
introduit. Dans la seconde partie, une vue d’ensemble de la nanomédicine est présentée. Cela
inclue la description des systèmes classiques de nanoparticules par l’administration de
médicament (« drug delivery ») ainsi que différentes nanoparticules ayant des effets
thérapeutiques. Cela est complété par quelques éléments clés afin de comprendre les
interactions nanoparticules-protéines, nanoparticules-cellules ainsi que les interactions avec les
organes influençant le temps de circulation (par exemple les reins, le foie et la rate).
Le chapitre 2 présente le concept d'un nouveau système de pré-médicaments en cage. Il utilise
la gemcitabine, une chimiothérapie du cancer du poumon approuvée par la FDA, qui est « mise
en cage » par un groupe protecteur. La réaction de déprotection nécessite l'utilisation de
nanoparticules de platine et est provoquée par l'administration d'hydrogène. Sur une lignée de
cellules de cancer du poumon, il est démontré que le précurseur en cage est beaucoup moins
cytotoxique que son homologue non protégé, que la réaction de déprotection est rapide et peut
être contrôlée en manipulant l'approvisionnement en hydrogène. Dans une autre expérience in
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vitro utilisant une lignée cellulaire de cancer du poumon, il a été démontré que le pré-
médicament, une fois déprotégé, retrouve entièrement la toxicité initiale. Cette expérience est
répétée avec des cellules épithéliales pulmonaires normales où l'on observe que le pré-
médicament en cage n'est pas toxique. De plus, une expérience de recyclage du catalyseur est
effectuée, montrant que l'efficacité catalytique est largement conservée pendant au moins 10
cycles.
Au chapitre 3, des données pré-cliniques préliminaires sont rapportées pour le système de pré-
médicaments en cage. Le chapitre commence par une description du développement d'une
formulation d'émulsion nécessaire à l'administration intratrachéale. À savoir, l'influence de la
concentration en tensioactif sur la taille des gouttelettes et l'influence de diverses températures
de congélation sur la stabilité à la congélation / décongélation est divulguée. Une sélection de
5 formulations est testée pour la formation d'aérosols en utilisant un MicroSprayer. La
meilleure formulation est ensuite administrée une fois par semaine aux souris pendant un mois.
En outre, deux solutions de nanoparticules de platine sont administrées à des souris qui sont
ensuite suivies pendant un mois. Ni l'émulsion, ni les solutions de nanoparticules de platine ne
génèrent de toxicité. Des souris xénogreffées préalablement inoculées avec des cellules
cancéreuses du poumon exprimant la luciférase sont administrées avec l’émulsion (véhicule),
la gemcitabine en cage et la gemcitabine normale. La croissance tumorale semblait être la plus
lente pour la gemcitabine normale, mais la plupart des souris de ce groupe sont décédées de
causes non liées au cancer. Il est supposé que cela est dû à la toxicité de la gemcitabine envers
les cellules normales, mais cela devra être confirmée par les expériences histologiques et
immunohistochimiques qui sont actuellement en cours sur les poumons des souris sacrifiées.
Le chapitre 4 décrit des nanoparticules de cobalt recouvertes de carbone et fonctionnalisées
par une couche de polyglycidol conjuguées à des anticorps anti-EpCAM. Tout d'abord, il est
démontré qu'il existe une relation entre la séparabilité magnétique, les propriétés anti-fouling
et l'efficacité d'élimination des cellules tumorales circulantes (CTC). L'élimination des CTC est
testée avec du sang de personnes en bonne santé dans lequel des cellules cancéreuses sont
ajoutées. La reproductibilité de la synthèse des nanoparticules et de l'efficacité de l'élimination
est rapportée pour trois lots de nanoparticules indépendants. De telles nanoparticules
magnétiques démontrent une efficacité d'élimination élevée associée à une spécificité élevée
puisque les autres cellules sanguines, telles que les lymphocytes, restent intactes pendant la
procédure d'isolement magnétique des CTC. Le chapitre est complété par les données obtenues
sur des échantillons de patients atteints de différents cancers.
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Au chapitre 5, l’étape de polymérisation du processus de production des nanoparticules est
étudiée dans le cadre d’une anticipation de la mise à l’échelle du processus qui sera nécessaire
pour mener des essais sur des animaux. Pour illustrer la possibilité d'utiliser de telles
nanoparticules pour d'autres applications où des quantités plus importantes de cellules doivent
être isolées, un anticorps Campath a été conjugué au polyglycidol. Une procédure rappelant
celle utilisée pour l'élimination des CTC est utilisée pour éliminer les lymphocytes du sang des
patients sains et cancéreux. Cette preuve de concept est une première indication que les
nanoparticules magnétiques fonctionnalisées avec du polyglycidol pourraient être utilisées dans
des maladies impliquant les lymphocytes telles que les lymphomes ou les maladies auto-
immunes. Cependant, pour être plus pertinents sur le plan thérapeutique, des anticorps ciblant
uniquement des sous-types spécifiques de lymphocytes doivent être utilisés.
Le chapitre 6 est une conclusion générale de ce travail et offre une perspective sur les futures
recherches possibles à la fois pour le système de pré-médicaments en cage et les nanoparticules
magnétiques.
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Summary
This thesis describes two types of technologies, involving nanoparticles, that could have
applications in the field of cancer treatments. The first one is a new caged prodrug system that
could eventually result in reduced side effects for lung cancer chemotherapeutics and/or
increased efficacy. The second technology involves a coating change of previously developed
magnetic nanoparticles. The new nanoparticles allow the specific and efficient separation of
cells from whole blood and could find applications in the removal of circulating tumour cells
at the origin of metastasis, but also the isolation of lymphocytes that are involved in numerous
diseases such lymphomas and autoimmune diseases.
In Chapter 1, a general introduction to the field of cancer and nanomedicine. First, general
facts and definitions about cancer are reported. This is followed by presenting the hallmarks of
cancer, which help understand the central differences between cancer and normal cells. To
better discern the molecular origins of cancer, an introduction to the biology of carcinogenesis
is included. An overview of the main cancer treatments (surgery, radiotherapy, chemotherapy
and targeted therapy) is established. Also, a brief examination of the potential social and
financial consequences of cancer for patients and their family is reported. In the second part, an
overview of nanomedicine is presented. It includes a description of the classical types of drug
delivery systems and various nanoparticles having a therapeutic action. This is complemented
by some key elements to understand nanoparticle-protein interactions, nanoparticle-cell
interactions as well as interactions with organs influencing the circulation time (e.g. kidneys,
liver and spleen).
In Chapter 2 the concept of a new caged prodrug system is introduced. It uses gemcitabine, a
FDA-approved lung cancer chemotherapeutic, which is caged with a protecting group. The
uncaging reaction requires the use of platinum nanoparticles and is prompted upon
administration of hydrogen. On a lung cancer cell line, it is shown that the caged prodrug is
much less cytotoxic than its uncaged counterpart and that the uncaging reaction is rapid and can
be controlled by manipulating the hydrogen supply. In a further in vitro experiment using a lung
cancer cell line, it is demonstrated that the prodrug, once uncaged, fully recovers the initial
toxicity. This experiment is repeated with normal lung epithelial cells where it is observed that
the caged prodrug is not toxic. Additionally, a catalyst recycling is done showing that the
catalytic efficiency is largely retained for at least 10 cycles.
15
In Chapter 3, preliminary pre-clinical data are reported for the caged prodrug system. The
chapter starts by a description of the development of an emulsion formulation that is needed for
intratracheal administration. Namely, the influence of the surfactant concentration on the
droplet size and the influence of various freezing temperatures on the freeze/thaw stability is
disclosed. A selection of 5 formulations is tested for aerosol formation by using a
MicroSprayer. The best formulation is weekly administered to mice over the time course of
one month. Also, two platinum nanoparticles solutions are acutely administered to mice which
are then followed for a month. Neither the emulsion formulation, nor the platinum nanoparticles
solutions generate toxicity. Xenograft mice previously inoculated with luciferase-expressing
lung cancer cells are administered with the emulsion formulation (vehicle), the caged
gemcitabine and the uncaged gemcitabine. The tumour growth seemed to be the slowest for the
uncaged gemcitabine but most mice from this group died from non-cancer related causes. It is
hypothesized that this is due to the toxicity of uncaged gemcitabine towards normal cells but it
will have to be confirmed by the histological and immunohistochemical experiments that are
currently being done on the lungs of the sacrificed mice.
In Chapter 4 polyglycidol-functionalized carbon-coated cobalt nanoparticles conjugated with
anti-EpCAM antibodies are described. First, it is shown that there is a relationship between the
magnetic separability, the antifouling properties and the circulating tumour cells (CTCs)
removal efficiency. The CTCs removal is tested by spiking healthy human blood with cancer
cells. The reproducibility of both the nanoparticles synthesis and removal efficiency is reported
for three independent nanoparticles batches. Such magnetic nanoparticles achieved a high
removal efficiency combined with high specificity since other blood cells such as lymphocytes
remain mainly untouched during magnetic CTCs isolation procedure. The chapter is augmented
by first cancer patients’ data for patients with different cancer types.
In Chapter 5 the polymerization step of the production process of the nanoparticles is
investigated in an anticipation of the process scale-up that will be required to conduct animal
trials. To illustrate the feasibility of using such nanoparticles for other applications where larger
quantities of cells need to be isolated, a Campath antibody was conjugated to the polyglycidol
coating. A procedure reminiscent of that used for CTCs removal is employed to eliminate
lymphocytes from the blood of both healthy and cancer patients. This proof-of-concept is a first
hint that the polyglycidol-functionalized magnetic nanoparticles could be used in
lymphocytes-involving diseases such a lymphomas or autoimmune diseases. However, to be
16
more therapeutically relevant, alternative antibodies that target only specific lymphocytes
subtypes should be used.
Finally, Chapter 6 gives a general conclusion of this work and offers an outlook on possible
future investigations for both the caged prodrug system and the magnetic nanoparticles.
17
18
Cancer: Origin, Burden and Therapy
19
According to the World Health Organization, cancer is the second leading cause of death
worldwide with around 8.8 million deaths being directly attributed to cancer in 2015.1 The
number of patients diagnosed with cancer is expected to increase to reach, in 2025, a staggering
worldwide yearly rate of 20 million people newly diagnosed with cancer; most people being
diagnosed in financially rich countries (Figure 1-1).2,3 Already in 2010, it was estimated that
the global economic cost of cancer was around US$ 1.16 trillion (i.e. over 2% of the global
GDP).2,3 Therefore, it is no understatement than saying that cancer is one of humanity’s most
pressing issues.
Figure 0-1. Incidence and mortality of cancer for the various geographical regions. Data are given per
100 000 people. Reproduced from Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C,
Rebelo M, Parkin DM, Forman D, Bray, F. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality
Worldwide: IARC CancerBase No. 11[Internet]. Lyon, France: International Agency for Research on
Cancer; 2013. Available from: http://globocan.iarc.fr, accessed on 19/08/2018.
20
As a consequence of the importance it carries, significant research efforts have been undertaken
to better understand cancer. For instance, it is now known that the incidence of the various types
of cancers strongly depends on environmental and genetic factors, with over 90% of cancer
being attributed to environmental factors such as tobacco and alcohol consumption
(Figure 1-2).4,5 This implies that behavioural changes can strongly reduce the chances of
developing a cancer and also explains the differences in incidence for the various cancer types
at different regional locations.3
To better discern the reasons why cancer arises and how it can be defeated, the first part of this
introductory chapter consists in a brief overview of the biology and aetiology of cancer as well
as the main treatments available. In the second part, the main types of cancer nanomedicines
and their interactions with biological systems will be evoked.
Figure 0-2. The role of genes and environment in the development of cancer. A. The percentage
contribution of genetic and environmental factors to cancer. B. Family risk ratios for selected cancers.
The numbers represent familial risk ratios, defined as the risk to a given type of relative of an affected
individual divided by the population prevalence. C. Percentage contribution of each environmental
factor. Adapted from Pharmaceutical Research, Vol. 25, Anand et al., Cancer is a Preventable Disease
that Requires Major Lifestyle Changes, 2097-2116, Copyright (2008), with permission from Springer.
21
1.1 Origin of Cancer and its Societal Impact
1.1.1 Cancer Biology and Aetiology
As defined by the American National Cancer Institute, cancer is a collection of diseases
involving abnormal and uncontrolled cell growth with the possibility for these cells to invade
surrounding tissues.6 Additionally, cancer is an ever-evolving disease, because cancer cells
have an intrinsic genetic instability due to higher replication errors rates as compared to normal
cells, that can ultimately lead to highly aggressive mutant cells.7 To rationalize and better
apprehend cancer, oncology researchers described specific hallmarks that define cancer. These
hallmarks are key to understand how cancer cells differ from normal cells. Hallmarks are also
useful to explain how many cancer therapies work as well as carcinogenesis. Hanahan and
Weinberg described 6 hallmarks (Figure 1-3) of cancer which are succinctly presented below.8,9
The two seminal papers they wrote cumulate over 50000 citations and de facto constitute the
prevailing literature authority and served as the basis for the next section.
1.1.1.1 Hallmarks of Cancer
Figure 0-3. Scheme summarizing all six hallmarks described by Hanahan and Weinberg. Reprinted
from Cell, Vol. 144, Hanahan and Weinberg, Hallmarks of Cancer: The Next Generation, 646-674,
Copyright (2011), with permission from Elsevier.
22
1.1.1.1.1 Hallmark 1: Sustaining Proliferative Signalling
For normal cells to grow and divide, they need to be stimulated by growth factors. These growth
factors are typically proteins or hormones that act as signalling molecules between cells. Cancer
cells have the capacity to grow without such factors and thus enter the otherwise highly
controlled process of cell division.9 Cancer cells can achieve this via several mechanisms:
- Autocrine signalling, the expression of growth factors by the cell itself,
- Perpetual activation of pathways normally triggered by growth factor binding,
- Destruction of entities responsible of negative feedback controls.
1.1.1.1.2 Hallmark 2: Evading Growth Suppressors
To avoid the growth and division of abnormally functioning cells, tumour suppressor genes can
express growth suppressor proteins that act through several mechanisms to inhibit cell division
or trigger apoptosis. For instance, when cells are in contact with other cells, growth suppressor
proteins repress genes necessary for the cell cycle to continue. This inhibits cell division and
avoids forming cell clusters. Another example of the role of growth suppressor proteins is the
triggering apoptosis in the event of DNA damages that are beyond repair. In the case of cancer
cells, growth suppressors’ expression is altered resulting in either ineffective proteins or in an
insufficient number of proteins.
1.1.1.1.3 Hallmark 3: Activating Invasion and Metastasis
Most normal cells are confined to the tissue from which they originated. The localization of
such cells is maintained by cell-to-cell adhesion molecules (e.g. E-cadherin) and by attachment
to the extracellular matrix (ECM). In the case of cancer cells, some of them acquire traits that
make them able to detach from the tumour, find access to the circulatory system and eventually
later form metastases in other organs. To do so, several mechanisms have been hypothesized
but the main one is epithelial-mesenchymal transition (EMT).10–13 During the EMT, traits such
as the expression of transcription factors repressing the E-cadherin expression or the expression
of matrix-degrading enzymes are acquired thereby “freeing” the cell from its surrounding. Of
note, similar events can be caused by cells from the tumour microenvironment. For instance,
macrophages are able to release matrix-degrading enzymes.14 However, cells in the
mesenchymal state can detach but are not able to invade (i.e. to duplicate). To do so they must
convert back to an epithelial character via the reverse process called mesenchymal-epithelial
transition (MET).9 Only a fraction of cells are able to undergo this transition in the new tumour
23
microenvironment where the tumour is located after extravasation from the circulatory system.
This explains why only a fraction of the disseminated cells can form macrometastases.15
1.1.1.1.4 Hallmark 4: Enabling Replicative Immortality
Normal cells only undergo a limited number of growth and division cycles. One well-known
factor controlling how many cycles a cell can go through is the length of telomeres. Telomeres
are DNA sequences found at the end of the DNA strand and, during each cycle of the DNA
replication, part of the telomere sequence is consumed. Cells stop growing and dividing once
the sequence is too short to protect the DNA strand. Most cancer cells have high expression of
telomerases, the DNA polymerases responsible of incorporating the telomere sequence,
activity, thereby making cells immortal.
1.1.1.1.5 Hallmark 5: Inducing Angiogenesis
Once adult, angiogenesis, the growth of new blood vessels, is stopped with the exception of
processes such as wound healing or the female cycling. Cancer tissues permanently grow and
require increasing supply of oxygen and nutrients. This growing demand can only be met by
producing new vasculature. Cancer cells thus influence angiogenesis by up-regulating
angiogenesis inducers (e.g. vascular endothelial growth factor or VEGF) and down-regulating
angiogenesis inhibitors (e.g. thrombospondin-1 or TSP-1).
1.1.1.1.6 Hallmark 6: Resisting Cell Death
Normal cells use apoptosis as a mean to avoid complications, for instance, after a cell is
damaged or infected. In other words, when a cell is not working normally, apoptosis is
triggered. The induction of apoptosis can be intracellular (intrinsic program) or extracellular
(extrinsic program). Both initiate a cascade of proteolysis by activating proteases called caspase
9 and caspase 8, respectively. These proteolysis cascades ultimately result into the disassembly
of the cell which is then consumed by neighbouring cells or phagocytes. Cancer cells can bypass
apoptosis by modifying abnormality sensors (e.g. p53). The abnormalities not being detected,
the apoptosis mechanisms are not triggered.
1.1.1.2 Carcinogenesis
Normal cells can transform into cancer cells via many mechanisms that can be classified into
two main categories: genotoxic (mutated DNA) or non-genotoxic carcinogenesis (non-mutated
DNA). Both categories involve the abnormal expression of proteins controlled by tumour
suppressor genes and oncogenes. To put it simply, proto-oncogenes are genes that, when not
properly functioning, cause cancer and are then called oncogenes. An example of a proto-
24
oncogene is the myc gene that regulates the transcription of transcription factors that ultimately
induce cell proliferations. Tumour suppressor genes on the other hand are genes that express
proteins that prevent abnormal cell growth. A well-known example is the TP53 gene which
expresses p53, a protein that act as a DNA damage sensor and triggers cell cycle arrest or
apoptosis if necessary.
1.1.1.2.1 Genotoxic Carcinogenesis
In the case of genotoxic carcinogenesis, the abnormal protein expression is due to a DNA
mutation. The origins of such mutations are diverse but can be summarized as follows: DNA is
damaged and if the enzymes or enzyme complexes responsible of DNA repair mechanisms
cannot fix the DNA, the normal DNA polymerases are switched out and translesion (TLS) DNA
polymerases are used instead. Such TLS polymerases have a tolerance for DNA lesions and can
introduce DNA mutations. The mutations can either be single base changes or frame shift-
causing modifications such bases deletions or insertions.
1.1.1.2.1.1 Oxidative Damages
Reactive oxygen species, generated for instance by smoking or a chronic stressed state, can
oxidise the DNA bases at several locations. An example of such a damage is the formation of
8-Oxogunanine (8-oxoG) that leads to a transversion (GC base pair is replaced by AT). Indeed,
8-oxoG can form a base pair with adenine and during replication an adenine is introduced on
the complementary strand in fine resulting into a mutation. However, the base excision repair
(BER) mechanism can repair oxidative damages as well as alkylated bases and AP-sites.
1.1.1.2.1.2 Photolesions
DNA photolesions are a well-known consequence of prolonged sun exposure. For instance,
UV-B (280-315 nm) can generate a dimerization of two thymine nucleobases. The nucleotide
excision repair (NER) mechanism usually repairs such types of helix-distorting DNA lesions
(this also holds true for Pt-induced DNA crosslinks).
1.1.1.2.1.3 DNA Alkylation
DNA alkylation is due to xenobiotics (i.e. a substance normally not found within the organism)
and their potential for DNA damage is diverse among patients due to differences in metabolism.
For instance, aflatoxin, that can be found in contaminated grains or seeds, can be transformed
by cytochrome P450 into an epoxide able to react with DNA to form an adduct that can lead to
the promotion of depurination. Such DNA reactive epoxides can be quenched by glutathione in
a reaction catalysed by glutathione S-transferase (GST). As a consequence of GST
25
polymorphisms, patients are at various cancer risks depending on how effective are their GST
at detoxificating xenobiotics. This type of mechanisms explains how genetic factors can
influence cancer risk.
1.1.1.2.2 Non-genotoxic Carcinogenesis
Non-genotoxic carcinogenesis does not involve DNA mutations directly. Instead, for receptor-
mediated and hormone-mediated carcinogenesis, the mitosis rate is increased thus correlating
with an increased rate of mistake introduction during DNA replication. This can then lead to
mutations. In the case of carcinogenesis originating from epigenetic changes, it is the DNA
transcription that is altered, not the DNA sequence itself.
1.1.1.2.2.1 Hormone-mediated Carcinogenesis
Hormones are a type of signalling molecules that can trigger signal transduction when binding
to receptors on the surface (non-genomic endocrine modulation) or inside cells (genomic
endocrine modulation). A classic example is the oestrogen receptor (ER). Upon genomic
activation, the ER translocates to the nucleus where it activates DNA transcription and leads to
proliferation. Upon non-genomic activation, the ERK and Akt signalling pathways are activated
thereby leading to the prevention of apoptosis. The ER can for instance be stimulated by
xenobiotics such as bisphenol A, phytoestrogens and pesticides (e.g. DDT).
1.1.1.2.2.2 Receptor-mediated Carcinogenesis
Similarly to hormone receptors, some other receptors can trigger signalling pathways that can
lead to tumour initiation or promotion by for instance inhibiting expression of key tumour
suppressor genes such as p53. A typical example is the aryl hydrocarbon receptor (AhR) which
can be activated upon binding of some aromatic hydrocarbons such as dioxin, a commercial
by-product that notably created a food scandal in Belgium in 1999 after being found in livestock
(e.g. chicken, pork).
1.1.1.2.2.3 Carcinogenesis Mediated by Epigenetic Changes
DNA transcription is partially controlled by DNA methylation and histone (i.e. proteins around
which DNA is coiled) modifications. DNA methylation is a natural process used to control
which part of the DNA a specific cell should express. As a result, when DNA is
hypomethylated, its transcription is activated and deactivated when hypermethylated. Histones
regulate chromatin condensation and the extent of condensation is controlled by the histone
acetyl transferase (DNA expansion) and the histone deacetylase (DNA condensation) as well
as by the level of methylation. The DNA methylation can obviously be influenced by some
26
alkylating agents but also indirectly by heavy metals such as Cd, which interfere with DNA
methyltransferases (DNMT), the enzymes that regulate DNA methylation.
1.1.1.2.3 Viral carcinogenesis
12% of human cancer originate from viral infections.16 For instance, human papilloma virus
(HPV), Hepatitis B and Epstein-Barr virus (EBV) are all DNA viruses that, after infecting a
cell, transfer part of their DNA in the host cell DNA thereafter leading to cell proliferation.
Vaccines were developed for viruses such as HPV thereby preventing the apparition of various
cancers (e.g. cervical cancer, vaginal cancer).
1.1.2 Current Cancer Treatments Strategies
1.1.2.1 Surgery
The treatment of cancer originated with surgery. A papyrus dating back from ancient Egypt was
found already describing surgical procedures to remove breast tumours.17 However, for a long
time surgery was synonym of high risks and inconvenience until anaesthesia and the use of
carboxylic soap (antiseptic agent) appeared in 1846 and 1867, respectively. In the 1970s,
techniques such as computed tomography (CT) scanning allowed surgeons to evaluate more
precisely the tumours’ locations. Also, notably with the advent of minimally invasive
techniques such as laparoscopy (see Figure 1-4), mortality during surgical operations has
drastically fallen for many cancers18,19 and modern surgery now also aims at preserving the
form and function of the body parts being operated. Therefore, many techniques such as breast
reconstruction, breast preservation or endoprosthetic reconstruction have been developed.20,21
The positive impact of surgery on the disease course is nowadays further strengthened by
adjuvant therapy. That is, the use of radiotherapy and chemotherapy after surgery. A notable
case is breast-conservation surgery where radiotherapy is applied to maintain local control.22
More recently, neoadjuvant (i.e. the administration of adjuvant therapy before surgery) started
being used to facilitate surgery and/or allow surgery of previously inoperable tumours.23 In the
future, surgery efficiency might be further increased by intraoperative molecular imaging and
the refinement of robot-assisted surgery.24 However, an important drawback of surgery is its
27
potential to release cancer cells in the patient’s systemic circulation thus presumably increasing
the risk of metastatic disease and relapse.25,26
1.1.2.2 Radiation Therapy
In the 1950s, linear accelerators (LINAC) started to be used in medical settings to treat cancer.
Upon exposition to radiations, radicals are formed (e.g. hydroxyl radicals from water and
peroxy radicals from molecular oxygen). Such radicals have the potential to damage DNA and
in the case of peroxy radicals for instance, these damages cannot be repaired and trigger
apoptosis. Nowadays, radiation therapy is frequently used as an adjuvant therapy and proved
to be an efficient tool for local control.27 Because the efficiency is influenced by the selection
and delineation of the target volume, new technologies combining radiotherapy and magnetic
resonance imaging (MRI) are being developed.28 Beyond the classical photon radiotherapy,
methods having the potential to be more specific using protons or heavier atoms have
flourished. Such methods have a reverse depth dose profile (see Figure 1-5) with most energy
deposited in the Bragg peak. By modifying the beam energy, the depth of the Bragg peak is
altered. Proton radiotherapy could therefore be used for cases where vital organs/tissues are
Figure 0-4. Schematic representation of a laparascopic setup. Image reproduced from the “Medical
gallery of Blausen Medical 2014”, WikiJournal of Medicine 1 (2).
28
located near the tumour.27 However, both types of radiotherapies have several drawbacks. First,
there is an heterogeneity in the patients’ responses29 and there can be an increased risk of
developing a secondary cancer30. Also, geographical misses can occur due to many factors such
as weight loss (change in the radiation penetration), changes in tumour volume/location,
breathing cycles or organ filling.27
1.1.2.3 Chemotherapy
Chemotherapy is the use of mainly small cytotoxic molecules to target dividing cells. The
various classes of agents are affecting different processes necessary for cell division. Because
each dose of chemotherapy only kills a fraction of tumour cells, a certain number of treatment
cycles is necessary. Interestingly, already very early in the history of the development of
chemotherapy, it became clear that tumours have a propensity to develop resistance to
chemotherapeutics.31 It was later found that by combining chemotherapies, tumour cells
developed less resistance and this still forms the basis of current strategies in chemotherapy
treatments.31 Chemotherapy is mainly used as adjuvant therapy in combination with surgery for
instance or to treat patients with metastases. Due to its unspecific nature, chemotherapy has
consequent side-effects.
1.1.2.3.1 Alkylating Agents
Alkylating agents are small molecules that alkylate guanine bases of DNA. This alkylation leads
to DNA cross-linking which results into induction of apoptosis. Cancer cells tend to be more
sensitive to such type of agents because they tend to divide more rapidly than normal cells and
Figure 0-5. Depth dose profile of a photon versus proton beam. Reproduced from Nature Reviews
Cancer, Vol. 16, Baumann et al., Radiation oncology in the era of precise medicine, 234-249, Copyright
(2016), with permission from Nature.
29
the DNA error-correcting mechanisms are less efficient than in normal cells. This type of
molecules marked the beginning of the development of chemotherapy. This after the discovery
in the 1940s that mustard gas victims had strong myelosuppression (i.e. a reduction in the
production of blood cells). In 1942, scientists at Yale University administered nitrogen mustard,
a molecule derived from mustard gas, to a non-Hodgkin’s lymphoma patient and observed a
brief remission.32 Molecules such as cyclophosphamide were then developed in order to
improve chemical stability and allow oral administration.31 Of note, a number of alkylating-like
agents such as cisplatin or carboplatin are still being widely used. They do not alkylate DNA
but coordinate to DNA and induce apoptosis.
1.1.2.3.2 Antimetabolites
Antimetabolites constitute a class of chemotherapeutics that work by preventing effective DNA
and RNA synthesis which in turn interferes with cell division and growth. Unlike alkylating
agents, such drugs only intervene in cells being in the S phase of the cell cycle when DNA is
being replicated. There are several subclasses of antimetabolite drugs and the two main ones
will be discussed.
1.1.2.3.2.1 Antifolates
The synthesis of thymidine requires tetrahydrofolic acid which is obtained by reduction of
dihydrofolic acid by dihydrofolate reductase (DHFR), itself synthesized by reduction of folic
acid. Antifolates drugs such as methotrexate block DHFR thereby preventing the production of
thymidines which in turn lack for DNA synthesis.
1.1.2.3.2.2 Base Analogues
Base analogues such as 5-fluorouracil or gemcitabine can work via different mechanisms. One
example of mechanism of action of gemcitabine is via its incorporation into DNA. This creates
an error that cannot be repaired by the base-excision repair mechanism. This irreparable error
leads to inhibition of DNA synthesis ultimately to cell death.33
1.1.2.3.3 Anti-microtubules agents
Microtubules are dynamic tubular structures composed of tubulins. Beyond forming part of the
cytoskeleton, they are involved in the cell division process during which they pull apart the
chromosomes. Anti-microtubules agents, or mitotic inhibitors, disrupt the structure of the
microtubules thereby triggering cell cycle arrest and ultimately apoptosis. There are two main
types of anti-microtubules agents, vinca alkaloids and taxanes. Vinca alkaloids are originally
obtained from a plant, Madagascar periwinkle. Belonging to vinca alkaloids, vincristine and
30
vinblastine work by inhibiting the assembly of tubulin into microtubules. On the other hand,
taxanes such as paclitaxel and docetaxel act by preventing the microtubules disassembly which
is necessary for chromosomes to achieve their metaphase spindle configuration.
1.1.2.3.4 Topoisomerase inhibitors
Topoisomerases I and II are enzymes influencing the DNA topology. The action of these
enzymes is necessary to ensure successful replication and transcription. Two well-known anti-
cancer drugs, irinotecan and etoposide are topoisomerase I and II inhibitors, respectively.
Irinotecan works by intercalating between a DNA cleavage complex and topoisomerase I
thereby inactivating the enzyme. Etoposide forms a complex with topoisomerase II. This
prevents the re-ligation step and the DNA strand remains broken. Such type of errors ultimately
promote apoptosis.
1.1.2.4 Targeted Therapy
Targeted therapy focuses on molecular abnormalities specific to cancer to selectively prevent
the growth of cancer cells. Although it raised large expectations, high failure rates were
observed (drugs working in only 10-20% of patients) for the first generation targeted
therapies.34 This can be explained by a significant intra-tumour and inter-tumour
heterogeneity.35 To address this, came the need to define, via biomarkers, the molecular
subtypes of the various cancers. Another recurrent issue with targeted therapy is the propensity
of tumours to become resistant. This is most likely explained by the fact that a small proportion
of resistant cells are already present in the tumour at the beginning of the treatment and, under
the selective pressure of the therapy, these cells start taking over the other tumour cells.36
Moreover, it has been demonstrated that cells can transiently resist, thereby preventing total
eradication and indicating that a combination of drugs is needed.37
1.1.2.4.1 Tyrosine Kinase Inhibitors
Tyrosine kinases are responsible for the activation of many proteins by phosphorylation.
Tyrosine kinase inhibitors (TKIs) can work either by competing with adenosine triphosphate,
the substrate or both or act in an allosteric manner.38 Unlike classical chemotherapeutics, TKIs
do not kill cells but rather interfere in signalling pathways that are key to cancer growth. They
can, for instance, interfere with kinases that are constitutively activated in cancer cells which
are otherwise regulated in normal cells.39
31
1.1.2.4.2 Angiogenesis Inhibitors
Tumours require the formation of new blood vessels (angiogenesis) to sustain their growth and
cannot grow to a size larger than 1-2 mm3 without angiogenesis.40 Therefore, by preventing
angiogenesis, drugs have the potential to contain tumour growth. Angiogenesis inhibitors use
various mechanisms but many target the vascular endothelial growth factor (VEGF); a key
signalling protein for angiogenesis.41
1.1.2.4.3 Epidermal Growth Factor Receptor (EGFR) Inhibitors
The epidermal growth factor receptor (EGFR) is an important transmembrane protein that is
activated by extracellular ligands. Upon activation of EGFR, a cascade of signals is triggered
in the cell leading to the activation of pathways resulting in DNA synthesis and cell
proliferation.42 In cancer cells, EGFR tend to be overexpressed or dysregulated and drugs have
been developed that are either directed to the EGFR extracellular domain or to its tyrosine
kinase domain.43
1.1.3 Psychological Impact and Financial Burden of Cancer
Beyond the physical impairments that can result from cancer treatment, the psychological and
social outcomes of cancer are central aspects that are often overlooked.44 For instance, cancer
is frequently associated with anxiety and depression that can be caused by the treatment (e.g.
altered body image), the release of chemicals (e.g. for pancreatic and lung cancers) or the
prolonged presence in a hospital setting.45 Moreover, patients report a decline in cognitive
functions such as memory and attention/concentration impairments as well as a reduced
processing speed.46 Reminiscences of such cognitive impairments can sometimes be observed
even years after treatment arrest.47 Also, due to the shock of cancer diagnosis and the resulting
mental stress, some patients contract post-traumatic stress disorders.48 A non-negligible fraction
of cancer patients report sexual dysfunction during or following treatment, a factor known to
potentially highly affect quality of life.49 Less expectedly, some patients declare having post-
traumatic positive outcomes and notably “improved relationships”, “better outlook on life” and
enhanced coping skills.44
Financial toxicity is described as an additional side-effect of cancer.50 This is especially in
countries like the U.S. where a significant fraction of the treatments costs are covered by the
patients or the patients’ families. This can lead to deleterious coping strategies such as the use
of family savings or patients that do not take all medications and skip medical appointments.51
32
1.2 Nanomedicine: a Widely Encompassing Concept
Nanomedicine is a hard to define word given the fact that it englobes a wide variety of
technologies having not much more in common than a given size range. Indeed, cancer
nanomedicines englobe liposomes and polymeric micelles, belonging to the subfield of drug
delivery, but also, nanoparticles used for radiotherapy, chemodynamic therapy or magnetic
hyperthermia.
1.2.1 Drug Delivery
The field of nanomedicine is very largely dominated by drug delivery technologies. As early as
1995, a liposomal formulation was approved by the FDA for doxorubicin.52 The rationale
behind using nanoparticulate formulations is to improve the pharmacodynamics and
biodistribution.53 However, until recently no marketed liposomal therapeutic agent increased
the overall survival when compared to the non-liposomal parent drug.54 In 2017, a first example
of a liposome formulation improving overall survival was demonstrated for Vyxeos, liposome-
encapsulated daunorubicin and cytarabine to treat acute myeloid leukemia.55 Another important
category of nanoparticles used for drug delivery purposes are polymeric micelles. A wide
variety of polymers have been used by researchers. For instance poly(ethylene glycol),
polypeptides (e.g. poly(L-lysine) or poly(L-glutamic acid)), polyethylenimine or
poly(lactic-co-glycolic acid) all have been exploited in polymeric micelles.56–58
To improve the specificity of the drug delivery systems, stimuli-responsive nanocarriers have
been developed.59 Such nanocarriers can take advantage of the fact that the chemical
environment of tumours is different than in normal tissues and/or use an external stimulus to
trigger the release of the active entity. For instance, systems targeting the decreased pH and
more reducing properties of the TME were developed.60,61 Also, technologies based on external
stimuli such as heat, light or a magnetic field have flourished.59,62 Additionally, antibodies and
protein fragments have been conjugated on the surface of various drug delivery systems to
improve the cellular uptake selectivity (i.e. targeted delivery). However, clinical translation
turned out to be arduous. Indeed, large discrepancies between rodent and human models
(accumulation being much lower in humans than in rodents) were observed.63 Moreover, an
33
improved internalization does not necessarily translate into improved clinical efficiency since
factors, other than just accumulation, might be limiting to the drug action.63 Despite the many
limitations, numerous nanoparticulate drug delivery systems are nowadays marketed or in late
clinical trials.53
1.2.2 Nanoparticles with a Therapeutic Action
Although more confidential than drug delivery, the subfield of nanomedicine using
nanoparticles to generate or improve a therapeutic action has witnessed a plethora of research
directions. For example, nanoparticles, especially gold nanoparticles, were used for their
radiosensitising potential. Such nanoparticles, via the emission of secondary electrons, can
increase free radicals production, thereby improving the efficiency of radiotherapy treatments.64
Nanoparticles used for magnetic hyperthermia constitute another broad field of research with
already at least one marketed product.53 Magnetic hyperthermia relies on the fact that magnetic
nanoparticles, such as iron oxide, when placed in a magnetic field dissipate heat and locally
increase the temperature. This may enhance the results of radio- and chemotherapy.65 More
recently, nanoparticles used for chemodynamic therapy emerged.66 Iron-based nanoparticles
were used to generate radicals via H2O2-dependent Fenton reaction, thereby exploiting the fact
that tumour cells are overproducing H2O2.67 Even though there is a wealthy literature of
nanomedicines having a therapeutic action, they had a marginal impact in the clinic as
compared to their drug delivery counterparts.
It is worth noting that the field of nanomedicine is ever-evolving as new knowledge is acquired
and an interesting perspective is the exploitation of synergistic interactions between
nanoparticles and biological entities. For instance, it has been observed that nanoparticles below
around 50 nm diameter tend to accumulate in the lymph nodes, a secondary immune system
organ where antigen-presenting cells, such as dendritic cells, present antigens to T-cells.68 To
take advantage of this, researchers developed nanoparticles that can deliver tumour antigens
directly to the dendritic cells which can in turn activate other leukocytes.69 Such type of
technologies might provide sturdy applications of nanoparticles by avoiding having to
outcompete the immune system which, as a result of evolution, has set up a profusion of
strategies to target nano- and microscale exogenous entities.
34
1.3 Interactions between Nanoparticles and In Vivo Elements
1.3.1 Interaction with Blood Components
1.3.1.1 Nanoparticle-Protein Interactions
Proteins account for over 90% of the dry weight of human blood plasma and when entering
blood circulation nanoparticles are rapidly covered by proteins.70 This process, often named
biofouling, is largely described in the literature since over a decade.71,72 Akin to any adsorption
process, biofouling is determined by the frequency of protein collision as well as the adsorption
and desorption rates. Key equations describing this phenomenon are reported in the review from
Lane et al.70 A visual representation summarizing the principles of biofouling can be found on
Figure 1-6. Of note, the equilibrium dissociation constant (Kd) is defined as the ratio of the
desorption rate over the adsorption rate and the latter is also proportional to the frequency of
collision. Therefore, it can be predicted that proteins displaying a strong interaction and/or a
high frequency of collision will have a low Kd. That is, a low probability of desorbing from the
Figure 0-6. (a) Schematic illustration of the interparticle distance, d, defined as the distance from the
nanoparticle surface to the protein surface. (b) Plot of a typical potential energy curve between a
nanoparticle and a protein molecule. (c) Potential energy plot of a particle with a deep primary energy
well for adsorption and a small barrier for protein adsorption, leading to surface fouling that is
thermodynamically stable. (d) Potential energy plot of a nanoparticle with a small primary well and a
high-energy barrier for protein adsorption, leading to surface fouling that is thermodynamically
unstable. Reprinted from Annu. Rev. Phys. Chem., Vol. 66, Lane et al., Physical Chemistry of
Nanomedicine: Understanding the Complex Behaviors of Nanoparticles in Vivo, 521-547, Copyright
(2015), with permission from Annual Reviews.
35
surface. This first layer defines the so-called hard corona (i.e. slowly exchanging proteins) in
opposition to the soft corona formed by proteins loosely bound to the hard corona.73,74 Since
the corona is what cells actually “see”, it is known that its composition influences the biological
course of the nanoparticles.75 In other terms, nanoparticles with different surface properties will
have different protein coronas and ultimately different interactions with the various types of
blood cells. For instance, if there are many opsonins in the corona of nanoparticles, they tend
to be rapidly eliminated by the immune system.70,75
1.3.1.2 Nanoparticle-Cell Interactions
Due to the lower concentration and larger size of cells, cell-nanoparticle interactions are
happening at slower timescales as compared to protein-nanoparticle interactions. Beyond the
classical types of intermolecular interactions responsible of the affinity between proteins and
nanoparticles, there are some cell-specific aspects to consider. First, in some cases the
nanoparticle-cell interaction will lead to cellular uptake; a feature typically wanted for drug
delivery applications. In order to be internalized, there are some active processes for which the
affinity of the nanoparticle for the cell receptors has to be high enough to overcome the
energetic cost of wrapping the cellular membrane around the nanoparticle (Figure 1-7).70
Alternatively, internalization can occur via diffusion through the lipid bilayer for nanoparticles
that are small enough and exhibit rather lipophilic properties. Interestingly, it is not fully known
whether, even in the case of targeted nanoparticles, the interaction between the cell and the
nanoparticles indeed occurs via the targeting protein present on the surface of the nanoparticles
are rather via the proteins present in the corona.70 Moreover, successful internalization does not
necessarily translate into effective therapeutic action. In fact it was found that many
nanoparticles are trapped into endocytic vesicles.76 This potentially explains why, despite
reaching more efficiently the required cells, most targeted drug delivery system did not result
in an improved overall survival when compared to their standard treatment counterpart.63
36
1.3.2 Interaction with Organs Influencing Circulation Time
1.3.2.1 Kidneys
Blood is filtered by the kidneys through the glomerulus which is composed of three consecutive
elements with different filtration capacities. The first one, the endothelium, has fenestrae of
around 60 nm while the last element only lets entities with a small (<5-6 nm) hydrodynamic
diameters pass through.75,77 The entities that are not retained during glomerular filtration are
directly being reabsorbed into the blood circulation.
1.3.2.2 Liver
The liver receives blood from the gut (via the portal vein) and the heart (via the hepatic artery).
It has important immunological functions since this is where Kupffer cells, a type of
Figure 0-7. (a) Multivalent interactions, by forming multiple ligand-receptor bond pairs as opposed
to a single bond, significantly increase the affinity of nanoparticles to the cell membrane. (b)
Nanoparticles with multiple affinity ligands increase the flux of receptors toward the particle, leading
to more binding events to gain the energy required to wrap the membrane around the particle. (c) A
free nanoparticle with affinity ligands (1) can contact the cell surface at which it binds multiple cell
surface receptors to create energetically favorable conditions for membrane wrapping and
endocytosis (2 and 3). Reprinted from Annu. Rev. Phys. Chem., Vol. 66, Lane et al., Physical
Chemistry of Nanomedicine: Understanding the Complex Behaviors of Nanoparticles in Vivo, 521-
547, Copyright (2015), with permission from Annual Reviews.
37
macrophage accounting for 80-90% of the total body macrophages, reside.75 Once internalized,
the nanoparticles are phagocytosed by the Kupffer cells. Additionally, the liver produces bile
and biliary excretion is the only way to excrete non-biodegradable nanoparticles which are
larger than 5-6 nm (smaller particles can be excreted by the kidneys).75
1.3.2.3 Spleen
The spleen is a lymphatic organ having roles in lymphocytes maturation as well as pathogens
filtration.75 The efferent lymph vessels connect the spleen to the lymphatic circulation. This has
direct consequences for nanoparticles that need to be administered more than once. Indeed, in
the case of prolonged retention in the spleen, B-cells trigger antibodies secretion which may
result in the opsonization of the nanoparticles at the second injection, ultimately leading to their
faster clearance.75 This resistance mechanism is called accelerated blood clearance (ABC) and
has been observed for different types of nanoparticles.78
1.4 Conclusion
In this first chapter we touched various aspects of cancers. In addition to contextualizing the
disease by describing how it affects patients and their families, we introduced key components
of the biology and aetiology of cancer. This helped us to understand the risks of contracting
cancer and how those risks can be alleviated. It also provided us with the possibility to better
capture how current treatments function. A brief overview of nanomedicines and of the
interactions between nanoparticles and various biological components was presented.
The next four chapters are dedicated to two technologies related to the field of nanomedicine;
albeit in a less classical way. Chapters 2 and 3 report a caged prodrugs system relying on
catalytic nanoparticles to convert a prodrug into its drug counterpart with the former focusing
on in vitro data and the latter on in vivo. Chapters 4 and 5 describe magnetic nanoparticles to
physically remove cells from the blood. In chapter 4, the nanoparticles are applied to remove
circulating tumour cells. Chapter 5 focuses on first aspects towards scaling up the production
in view of future pre-clinical trials and discloses the application of the nanoparticles to remove
lymphocytes as further illustration of the therapeutic potential of such magnetic particles.
38
39
Hydrogen as a Bio-orthogonal Trigger for
Spatiotemporally Controlled Caged Prodrug Activation
Manuscript published in part as:
A. F. Herzog, E. M. Schneider, W. J. Stark,
Helvetica Chimica Acta, 2018, 10.1002/hlca.201800134
Author contributions: concept/design by AFH and WJS. Experiments planned/conducted by
AFH with support of EMS. Data Analysis/interpretation by AFH and WJS. Drafting article:
AFH. Critical revision of article by AFH, EMS and WJS. Approval of article by AFH, EMS
and WJS.
40
2.1 Introduction
In the last decades, extensive efforts have been made to improve the prognosis of lung
cancer. Indeed, lung cancer remains a staggering cause of human deaths and accounted for
around 1.6 millions of deaths in 2012 alone, thereby making it the leading cause of cancer
deaths worldwide.79 Stage IV patients, as well as patients having undergone surgery, are being
given chemotherapy; most of which are untargeted and have significant negative side effects.80–
82
To reduce the side effects of cancer treatments the field of caged prodrugs has emerged.83,84
The so-called cage is a chemical protecting group and the resulting caged prodrug is a less
potent version of its uncaged analogue. Unlike classical prodrugs, which are converted to the
drug under physiological conditions, caged prodrugs require the use of an exogenous catalyst
to remove the protecting group. Seminal works were based on ruthenium and palladium metals
to catalytically cleave allyl carbamates.85,86 Recently, gold was successfully utilized in vivo to
carry out similar allyl carbamate cleavages.87,88 Most of these bio-orthogonal bond cleavage
reactions rely on heterogeneous catalysis, thus possibly conferring spatial control. Within recent
months, ruthenium complexes have been utilized as caging agents rather than as catalysts. The
uncaging of the reported complexes was achieved upon light exposure.89,90 This strategy gives
a temporal control over the uncaging; however, it is limited by the shallow depth of light tissue
penetration.91 Consequently, applications further than cutaneous or peri-cutaneous applications
remain out of reach.
To combine the advantages of both research directions (heterogeneous catalytic uncaging
and light-triggered metal complexes decomposition) and to avoid the shortcomings of the latter,
we developed a catalytic uncaging prompted upon activation via an exogenous bio-orthogonal
trigger. Herein, we disclose an example of a temporally- and spatially-controlled
caging/uncaging bio-orthogonal system. The method uses platinum, a metallic catalyst that has
not been exploited so far in the domain of caged prodrugs. The protecting group p-
nitrobenzyloxycarbonyl (pNZ) is used as the cage while hydrogen serves as the trigger for the
uncaging (Figure 2-1A).
For this in vitro proof-of-concept, we focused upon one specific possible clinical application -
the treatment of lung cancer. Hence, the A549 cell line (human lung carcinoma) and
gemcitabine were selected. The latter being an antimetabolite which, in combination with
41
carboplatin, form the gemcarbo regimen, a clinically used treatment for non-small cell lung
cancer.81 With the objective of a later in vivo application, hydrogen-containing gas mixtures
inspired by those encountered in deep diving breathing gas (e.g. hydrox composition: 96%
hydrogen and 4% oxygen) were employed.92 The nanoparticles used could be applied via an
aerosol (inhalation), thereby making the administration of the catalyst minimally invasive.93
This is in contrast to the injection required for most of the aforementioned systems. Moreover,
it has been reported that particles remained confined to the lungs of mice administered with
ultrafine particles aerosols.94–96 This should thus confer a spatial control to our system.
The absence of significant cytotoxicity of the materials and of hydrogen are here reported
for conditions relevant to the in vitro proof-of-concept of the working principle of our system.
2.2 Experimental
2.2.1 Synthesis of Caged Coumarin (Compound 2a)
7-amino-4-methylcoumarin (200 mg, 1.14 mmol) was dissolved in 5 mL of dry DCM under N2
atmosphere. Pyridine (100 µL, 1.24 mmol) was added and the solution was cooled to 0°C.
Subsequently, 4-Nitrobenzyl chloroformate (365 mg, 1.7 mmol) was added and the reaction
stirred overnight. 20 mL of 0.5 M HCl was added which lead to the precipitation of the product.
The precipitate was filtered off and washed using Et2O. After drying, 212 mg of product was
obtained (53% yield).
1H NMR (200 MHz, DMSO-d6) δ 10.45 (s, 2H), 8.50 (s, 1H), 8.36 – 8.22 (m, 4H), 7.73 (d, J
= 8.6 Hz, 6H), 7.57 (d, J = 2.0 Hz, 2H), 7.44 (dd, J = 8.7, 2.1 Hz, 2H), 6.26 (d, J = 1.4 Hz,
2H), 5.36 (s, 4H), 2.40 (d, J = 1.2 Hz, 6H). 13C NMR (50 MHz, DMSO-d6) δ 153.64, 124.12.
HRMS (m/z) [M + H]+ calculated for C18H14N2O6, 355.0930; found: 355.0923.
2.2.2 Synthesis of Caged Coumarin (Compound 1a)
Gemcitabine hydrochloride (299 mg, 1 mmol) was dissolved in dry DMA (4 mL) with NaHCO3
(252 mg, 3 mmol) under N2 atmosphere. The mixture was then cooled to 4°C in an ice bath. 4-
Nitrobenzyl chloroformate (324 mg, 1.5 mmol) was subsequently added to the mixture. The
mixture was stirred overnight at room temperature. The solution was filtered and the filtrate
was concentrated in vacuo. The filtrate was added in its liquid form to a silica column (66 g).
The column was run with 500 mL of a mixture of hexane/ethyl acetate (20:80), followed by
500 mL of ethyl acetate. After drying, 93 mg of product was obtained (21% yield).
42
1H NMR (200 MHz, DMSO-d6) δ 11.15 (s, 1H), 8.26 (dd, J = 8.1, 4.2 Hz, 3H), 7.69 (d, J =
8.5 Hz, 2H), 7.09 (d, J = 7.6 Hz, 1H), 6.32 (d, J = 6.4 Hz, 1H), 6.17 (t, J = 7.5 Hz, 1H), 5.33
(d, J = 10.7 Hz, 3H), 4.19 (tt, J = 13.0, 7.4 Hz, 1H), 3.98 – 3.72 (m, 2H), 3.75 – 3.54 (m, 1H),
2.09 (s, 1H). 13C NMR (50 MHz, DMSO-d6) δ 147.59, 144.18, 128.73, 124.07, 59.25.
HRMS (m/z) [M + Na]+ calculated for C17H16F2N4O8, 465.0834; found: 465.0827.
2.2.3 Screening of Catalyst Concentrations
In a 96-well plate, wells were filled with 90 L of fresh F-12K medium containing caged 7-
amino-4-methylcoumarin (110 M) and DMSO (2% v/v). Positive controls wells were filled
with 90 L of fresh F-12K medium containing caged 7-amino-4-methylcoumarin (110 µM) and
DMSO (2% v/v). To treated wells, 10 L of a platinum solution (125 ppm, 250 ppm or 500
ppm; 3 nm diameter; Sigma-Aldrich) was added and 10 L of Milli-Q water was added to
control wells. The plate was placed in a custom-designed gas chamber (see Scheme A1-1). The
chamber was placed in the incubator, connected to a gas bottle outside the incubator and to an
outlet. The gas chamber was flushed for 0.5 min with hydrogen gas at 0.07 bar and closed. The
plate was incubated at 37°C for 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 80, 100,120 min in the gas
chamber. After each time point, the plate was taken out of the gas chamber and fluorescence
emission was measured using a microplate reader (Tecan, Infinite 200 PRO; ex: 340 nm; em:
460 nm). Values were normalized by the mean value of the positive controls wells.
2.2.4 Transmission Electron Microscopy Measurements
An aqueous solution of platinum nanoparticles (3 nm diameter, Sigma-Aldrich) was prepared
at a concentration of 5 ppm. 1.5 µL of the solution was deposited on carbon-coated copper
grids. After overnight drying, the samples were measured on a Tecnai F30 (FEI, 300 kV).
2.2.5 Kinetics of Deprotection of Caged Coumarin
In a 96-well plate, wells were filled with 90 L of fresh MEM or F-12K medium containing
caged 7-amino-4-methylcoumarin (110 M) and DMSO (2% v/v). All cell culture media were
supplemented with 10% Foetal Bovine Serum. To treated wells, 10 L of a platinum
nanoparticles solution (1000 ppm, 3 nm diameter, Sigma-Aldrich) was added and 10 L of
Milli-Q water was added to control wells. The plate was placed in a custom-designed gas
chamber (see Scheme A1-1). The chamber was placed in the incubator, connected to a gas
bottle outside the incubator and to an outlet. The gas chamber was flushed for 0.5 min with
43
hydrogen gas at 0.07 bar and closed. The plate was incubated at 37°C for 0, 5, 10, 15, 20, 25,
30, 100 min in the gas chamber. After each time point, the plate was taken out of the gas
chamber and fluorescence emission was measured using a microplate reader (Tecan, Infinite
200 PRO; ex: 340 nm; em: 460 nm). Values were normalized by the mean value of the positive
controls wells.
2.2.6 Interval Kinetics of Deprotection of Caged Coumarin
In a 96-well plate, wells were filled with 100 L of fresh MEM medium containing caged 7-
amino-4-methylcoumarin (110 M) and DMSO (2% v/v). All cell culture media were
supplemented with 10% Foetal Bovine Serum. To treated wells, 10 L of a platinum
nanoparticles solution (250 ppm or 500 ppm, 3 nm diameter, Sigma-Aldrich) was added and 10
L of Milli-Q water was added to control wells. The plate was placed in a custom-designed gas
chamber (see Scheme A1-1). The chamber was placed in the incubator, connected to a gas
bottle outside the incubator and to an outlet. The gas chamber was flushed for 0.5 min with
hydrogen gas at 0.07 bar and closed. The plate was incubated at 37°C for 0.5 min in the gas
chamber. The plate was taken out of the gas chamber and left open for 2 min in order to
equilibrate with the ambient atmosphere of the room. Fluorescence emission was measured
using a microplate reader (Tecan, Infinite 200 PRO; ex: 340 nm; em: 460 nm). The plate was
placed back in the incubator for 2 min before measuring fluorescence again. Four such cycles
were made, followed by two cycles with 1 min incubation in the gas chamber and 2 min of
incubation directly in the incubator. Two last cycles were done with 4 min incubation with
hydrogen and 2 and 4 min, respectively, of incubation directly in the incubator. Values were
normalized by the mean value of the positive controls wells.
2.2.7 EC50 Experiments
A549 cells were seeded in 96-well plates at a concentration of 2,000 cells/well and incubated
overnight at 37°C before treatment. The supernatant of each well was then replaced with 100
L of fresh F-12K medium containing protected gemcitabine (concentration ranging from 104
M to 10-4 M) and DMSO (2% v/v), control wells were replaced with 100 L of fresh F-12K
medium and DMSO (2% v/v). All cell culture media were supplemented with 10% Foetal
Bovine Serum. Five biological replicates for each condition were included in the plate. Control
and treated plates were incubated at 37°C for 72 h before PrestoBlue™ cell viability reagent
(10% v/v) was added to each well and the plate incubated at 37°C for 2 h. Fluorescence emission
44
was measured using a microplate reader (Tecan, Infinite 200 PRO; ex: 560 nm; em: 590 nm).
Values were normalized by the mean value of the positive controls wells.
2.2.8 Extracellular Prodrug Activation in Cancer Cells
A549 cells were seeded in 96-well plates at a concentration of 4,000 cells/well and incubated
overnight at 37°C before treatment. The supernatant of each well was then replaced with 100
L of fresh F-12K medium containing protected gemcitabine or gemcitabine (0.05 M) and
DMSO (2% v/v), control wells were replaced with 100 L of fresh F-12K medium and DMSO
(2% v/v). All cell culture media were supplemented with 10% Foetal Bovine Serum. Plates
were incubated at 37°C for 1 h before 10 L of a platinum nanoparticles solution (100 ppm, 3
nm diameter, Sigma-Aldrich) was added to each well and 10 L of Milli-Q water was added to
control wells. Six biological replicates for each condition were included in the plates. Plates
were incubated at 37°C for 1 h before placing them in a custom-designed gas chamber (see
Scheme A1-1). Control plates were left untouched in the incubator. The chamber was placed in
the incubator, connected to a gas bottle outside the incubator and to an outlet. The gas chamber
was flushed for 1 min with hydrogen gas at 0.07 bar and closed. Plates were incubated at 37°C
for 2 h in the gas chamber before placing them back in the incubator with the control plates.
Control and treated plates were incubated at 37°C for 72 h before PrestoBlue™ cell viability
reagent (10% v/v) was added to each well and the plate incubated at 37°C for 2 h. Fluorescence
emission was measured using a microplate reader (Tecan, Infinite 200 PRO; ex: 560 nm; em:
590 nm). Values were normalized by the mean value of the positive controls wells.
2.2.9 Zeta-Potential Measurement
100 µL of a platinum nanoparticles solution (1000 ppm, 3 nm diameter, Sigma-Aldrich) was
added to a 1.5 mL Eppendorf tube containing 1000 µL of 0.9% saline solution. The Eppendorf
tube was centrifuged for 1.5h at 22130g (Eppendorf, Centrifuge 5424 R). The supernatant was
discarded and replaced by 1000 µL saline solution. The nanoparticles were redispersed with an
ultrasonic horn (Hielsher, UP50H; 30 kHz) before being transferred to a Disposable Capillary
Cell (DTS1070, Malvern) and measured using a Zetasizer Nano (Malvern).
45
2.3 Results and Discussion
2.3.1 EC50 Experiments
Inspired by research in the field of solid-phase peptide synthesis, the caged prodrug was
generated by introducing a p-nitrobenzyloxycarbonyl protecting group via chloroformate
chemistry (Figure 2-1A and Supporting Information).97–99 Both molecules were dissolved in
DMSO and then diluted in F-12K medium, which was used as the cell culture medium for the
cancer cells. The dose-response behaviour of both the uncaged and caged gemcitabine were
investigated using the A549 cell line. After 72h of incubation at 37°C, viability was evaluated
Figure 0-1 A) Caging and uncaging reactions for gemcitabine and 7-amino-4-methylcoumarin (model
compound). In their uncaged forms, gemcitabine and 7-amino-4-methylcoumarin are active and
fluorescent, respectively. Once caged the former is inactive and the latter is not fluorescent. Upon
uncaging with platinum nanoparticles and hydrogen, they return to their initial state. B) Transmission
electron micrographs of the platinum nanoparticles. The red symbols delimit the distance between two
crystalline layers. C) Photographs of reaction mixtures under a 360 nm UV-lamp. The cuvettes 1-4
correspond to the four possible reaction conditions (+/- hydrogen and +/- Pt nanoparticles). When both
hydrogen and Pt nanoparticles are present, the fluorescence is restored to a level similar to that of the
control with 7-amino-4-methylcoumarin.
46
using a PrestoBlue™ assay and a plate reader (Figure 2-2). As anticipated, gemcitabine and its
caged counterpart cause notably different responses. The caged prodrug is much less cytotoxic
than gemcitabine with an EC50 difference spanning over several orders of magnitude.
Gemcitabine is fully active in a sub-micromolar range while such a level of cytotoxicity cannot
be reached, even at over millimolar concentrations, for the caged molecule.
2.3.2 Kinetics of the Uncaging Reaction
Having confirmed the first hypothesis of our system, the catalysis of the drug activation
was studied. This was achieved with a model reaction using the fluorophore 7-amino-4-
methylcoumarin as a drug surrogate (Figure 2-1A). The fluorescence of the caged coumarin is
quenched upon protection of the amino group. The fluorescence is recovered upon uncaging
(Figure 2-1C). For our application, small nanoparticles exhibiting a high surface-to-volume
ratio and a potential ability to cross the cellular membrane are needed. Therefore, small (3 nm
diameter) platinum nanoparticles were used as catalysts. To fully characterize the reaction,
catalyst concentrations ranging from 12.5 to 50 ppm were screened (Figure A1-1) and gas
mixtures containing various hydrogen levels were investigated (Figure A1-2). Kinetic
Figure 0-2. Dose-response curve of gemcitabine (1a) and caged gemcitabine (1b). A A549 human lung
adenocarcinoma cell line was used. Solutions were prepared in Ham’s F-12K (Kaighn’s) Medium (F-
12K). After 72h of incubation at 37°C, the viability was evaluated using the PrestoBlue™ assay and a
plate reader (n = 5; 𝜆ex/em = 560/590 nm).
47
measurements were done in two frequently encountered cell culture media; namely, F-12K and
MEM. A custom-designed gas chamber accommodating 96-well plates was built (Scheme A1-
1); this allowed the concomitant use, within a standard incubator, of both non-standard gas
mixtures (containing from 3.2% to 100% of hydrogen) and of standard incubator atmosphere
(air supplemented with 5% CO2). The reactions were conducted without stirring or bubbling
and the hydrogen supply was thus diffusion-controlled. The rapid reaction rates found (τ1/2 <
10 min, Figure 2-3) make this reaction propitious for our application. To ensure that the kinetic
of the uncaging of caged gemcitabine remains akin to that of the caged fluorophore, further
kinetic experiments were carried out with caged gemcitabine. Caged prodrug to drug
conversion was followed by HPLC-MS and a comparable reaction half-life was observed
(Figure A1-3).
To investigate the hypothesis of using hydrogen as an on/off switch, an interval kinetics
experiment was designed (Figure 2-4). The model reaction with 7-amino-4-methylcoumarin
was used and after each hydrogen interval, the plate was left open at ambient conditions to let
it equilibrate before measuring fluorescence intensity. This alternative regimen is reminiscent
to that of a breathing cycle with normal air, and one cycle with a gas mixture containing
hydrogen. Intervals of 30 seconds incubation are enough to trigger the uncaging and thus
Figure 0-3. Kinetics of the uncaging of caged prodrug surrogate 2b. Solutions of 99 µM of caged
prodrug surrogate 2b were prepared in F-12K and Minimum Essential Medium Eagle (MEM) cell
media. A concentration of platinum nanoparticles of 100 ppm and pure hydrogen were employed.
48
support the idea of breathing two alternating gas mixtures. The reaction is virtually stopped
during the hydrogen-free intervals.
2.3.3 Toxicity of the Nanoparticles
It is well known that the tumour microenvironment is hypoxic and patients median percentage
of oxygen in lung tumours have been reported between 1.9 and 2.2% while normal tissues had
an average of 5.6%.100 The influence of the oxygen concentration on the uncaging reaction was
studied (Figure A1-4). The presence of oxygen decreases both the reaction yield and kinetics.
Such a phenomenon could thus be at the origin of a partial selectivity between cancer and
normal tissues because of the lower oxygen tension in cancer tissues. The toxicity of the
platinum nanoparticles and hydrogen were investigated (Figure A1-5). As expected, the cells
treated with hydrogen display a biologically insignificant decrease in viability. Cells were
treated with three different platinum nanoparticles concentrations. Interestingly, cells treated
with the two lowest platinum concentrations exhibit a small increase in viability. This supports
the idea of a beneficial reducing environment in the presence of Pt nanoparticles.94,101–104
Additionally, the zeta-potential of the Pt nanoparticles measured in physiological saline
solution was -0.08±0.27 mV. According to the results of Cho et al. such nanoparticles should
Figure 0-4. Interval kinetics of the uncaging of caged prodrug surrogate 2b. A solution of 99 µM of
caged prodrug surrogate 2b was prepared in F-12K medium. For both experiments a plate reader was
used (n = 5; 𝜆ex/em = 340/460 nm).
49
thus be non-hemolytic and have a low acute lung inflammogenic potential.105 It also
corroborates the results from Onizawa et al. who found no pathological changes in mice that
were intranasally administered with platinum nanoparticles.94 Having proved that hydrogen
supply can act as an on/off switch and that neither hydrogen, nor platinum are toxic in vitro at
the required concentrations, we combined all these elements into a single experiment.
2.3.4 Extracellular Caged Prodrug Activation
To prove that the activity is fully restored after the uncaging, an experiment was designed
where cells were administered with caged gemcitabine and were treated with a 2x2 matrix of
conditions: presence/absence of hydrogen and presence/absence of a catalyst. Positive controls
were performed with Milli-Q water and negative controls with uncaged gemcitabine. Two
plates were made and incubated: one within the hydrogen-containing gas chamber and the other
freely standing in the incubator. After 2h, the plate was taken out and both plates were incubated
for 72h before a PrestoBlue™ assay was used to evaluate the viability (Figure 2-5). The addition
of caged gemcitabine slightly lowers the viability compared to the positive control with water.
In a similar experiment with non-cancerous lung cells (Figure A1-6), this was not observed.
The difference could potentially be explained by a constitutive reduction of the nitro moiety of
the protecting group by cancer cells.106,107 Interestingly, the cells treated with caged gemcitabine
where platinum and hydrogen were applied exhibited a statistically significant lower viability
compared to the gemcitabine control (p-value < 0.01, assumption of homoscedascity, two-tailed
unpaired t-test). This may be caused by an improved cellular uptake of the caged gemcitabine,
due to the increased lipophilicity compared to its uncaged counterpart, followed by an
intracellular uncaging. It has been shown on mice that using elevated gemcitabine
concentrations lead to a more marked tumour growth inhibition; however, this was at the
expense of considerable side effects.108 We believe that through use of our system, this could
be alleviated. Indeed, the observation that at equimolar concentrations the drug activity is fully
recovered upon uncaging, suggests that higher drug doses could be used while keeping side
effects to a level similar to that encountered today.
50
2.4 Conclusion
In summary, we have shown in vitro the advantageous application of hydrogen gas for
selective uncaging of a drug thus conveying spatiotemporal control. The fast kinetics enable
the fascinating use of hydrogen as an on/off switch locally triggering the bio-orthogonal bond
cleavage reaction. The catalyst can be administered in a minimally invasive manner
(inhalation). The toxicity of here used hydrogen and platinum particles concentrations under
realistic conditions has been reported as non-inhibiting such applications. Overall, the system
described expands upon the growing repertoire of bond cleavage reactions in medicinal
chemistry. These early results only constitute a preliminary step and need to be followed by
studies in higher level models such as in xenografted mice. However, if similar results are
obtained in vivo, we believe this system could provide a strategy to reduce side effects,
particularly for lung cancer chemotherapeutics.
Figure 0-5. Extracellular caged prodrug activation. A A549 human lung adenocarcinoma cell line was
used. Solutions of gemcitabine (1a) and caged gemcitabine (1b) were prepared at a concentration of
0.05 µM in the F-12K cell medium. A concentration of platinum nanoparticles of 10 ppm was used. A
custom-designed gas chamber was used for incubation with hydrogen (2h). After 72h of incubation in
normal cell culture atmosphere, the viability was evaluated using the PrestoBlue™ assay and a plate
reader (n = 6; 𝜆ex/em = 560/590 nm). ٭٭٭ ,٭٭ ,٭ = p < 10-7 (assumption of homoscedascity, two-tailed
unpaired t-test).
51
52
Preliminary Pre-Clinical Trials for the Gemcitabine
Caged Prodrug System
Manuscript in preparation:
A. F. Herzog, E. M. Schneider, W. J. Stark,
Author contributions: concept/design by AFH and WJS. Experiments planned/conducted by
AFH with support of EMS. Data Analysis/interpretation by AFH and WJS. Drafting article:
AFH. Critical revision of article by AFH and WJS. Approval of article by AFH, EMS and WJS.
53
3.1 Introduction
Clinical translation is a long and risky process with new drug approval taking an average
of 12 years with an average success rate from pre-clinical to phase III trials as low as 11%.109
In the US, the regulatory process begins with an Investigational New Drug (IND) application.
For the IND approval, it is necessary to document information about the drug production but,
most importantly, data from animal studies are required describing the drug’s safety and
efficacy.110
The vast majority of pre-clinical studies (over 80% of the studies published in 2016 in
the 8 most important journals) use cell-line derived models and xenograft mice in particular.111
In such models, the mice used are immune-compromised and human cancer cells are introduced
directly in the target organ or inoculated systemically. Although standard, xenograft models
have serious disadvantages such as the lack of a competent immune system, a much faster
tumour growth and an altered tumour microenvironment as compared to naturally developing
tumours.111 These limitations most likely reduce the clinical translation predictive power and
in many cases reproduction of key experiments in more complex models such as
environmentally-induced and genetically engineered mouse models is recommended.111
However, xenograft models can provide useful information regarding the toxicity of the various
components of the caged prodrug system and a lung cancer xenograft model was selected where
bioluminescent A549 cells were injected in the caudal vein of immuno-deficient mice.112 As a
result, such a model displays a cancer cell biology that closely resemble the one of the in vitro
experiments. Therefore, this has the potential to allow us to develop the treatment methodology
(e.g. how hydrogen is supplied) in a model for which it is known that the uncaged drug is active.
In other words, this reduces the number of unknowns and facilitates the development process.
Gagnadoux et al. administered aqueous aerozolized gemcitabine solutions to a similar
mice model.108 Their work served as a basis when planning the in vivo experiments reported
here. However, due to the insufficient water solubility of the caged prodrug, a formulation had
to be developed. In early tests, a proportion of ethanol and DMSO was added to ensure
solubility of the caged prodrug. However, if acute administration did not engender
complications, during chronic administration a large fraction of mice experienced strong lung
inflammation leading to death in some cases. To overcome this issue, it was decided to develop
emulsion formulations. In an attempt to facilitate future clinical translation, only materials
approved by the FDA for intratracheal or inhalation were used.113
54
Preliminary formulation tests, using peanut oil, TWEEN® 80, propylene glycol and a
saline solution are here described. Additionally, toxicity profiles of the vehicle and platinum
nanoparticles solutions are disclosed. Finally, first data are reported for an experiment where
the lung cancer xenograft mice were administered with both caged gemcitabine and
gemcitabine.
3.2 Experimental
3.2.1 Formulations Preparation
The desired amounts of peanut oil and TWEEN® 80 were weighted and vortexed for 10 seconds
(Vortex-Genie 2, Scientific Industries). When applicable, the drug/prodrug was added to the
peanut oil/TWEEN® 80 mixture. The solution was vortexed for 1 minute, placed in an
ultrasonic bath (Sonorex Digitec DT 103 H, Bandelin) and vortexed for an additional minute.
A saline solution (0.9% NaCl) or a saline solution and propylene glycol were added. The
resulting solution was vortexed for 10 seconds before using an ultrasonic horn (UP50H,
Hielscher) for 30 seconds or until formation of a deep white emulsion.
3.2.2 Dynamic Light Scattering Measurements
Solutions were prepared by dissolving 10 µL of 990 µL of milli-Q water and vortexing 10
seconds (Vortex-Genie 2, Scientific Industries). The solutions were transferred to disposable
cuvettes (ZEN0040, Malvern) before the average size of the nanoparticles was determined by
dynamic light scattering (Zetasizer Nano ZS, Malvern).
3.2.3 Nebulization Assays
The following formulations of vehicle were nebulized using a IA-1C MicroSprayer® (Penn-
Century, USA) connected to a FMJ-250 high-pressure syringe (Penn-Century, USA). Right
before nebulisation assays, the formulations were vortexed for 30 seconds, sonicated for 1
minute and vortexed for additional 30 seconds to obtain stable emulsions.
Formulation 1: 0.3% TWEEN® 80, 20% peanut oil, 25% propylene glycol, rest 0.9% NaCl
Formulation 2: 0.3% TWEEN® 80, 20% peanut oil, rest 0.9% NaCl
Formulation 3: 1% TWEEN® 80, 20% peanut oil, 25% propylene glycol, rest 0.9% NaCl
Formulation 4: 1% TWEEN® 80, 20% peanut oil, rest 0.9% NaCl
Formulation 5: 0.3% TWEEN® 80, 20% peanut oil, 5% propylene glycol, rest 0.9% NaCl
55
3.2.4 Tolerability Profiles of Formulation 2 and of Platinum Nanoparticles Solutions
3.2.4.1 Animals
Experiments were carried out using 9 female NOD/SCID mice (Janvier Labs, France), 9-10
weeks old on the day of the administration. Mice were housed in social groups of 3-6
individuals in polysulfone individually ventilated cages (IVC) (floor area = 501 cm²). An
external ventilation unit supplied the cages individually with fresh HEPA (High Efficiency-
Particulate Air)-filtered air (75 air volume replacements/hour). A positive pressure ventilation
was maintained in each cage. Cages were equipped to provide food and sterile water ad libitum.
Irradiated food (RM1 from SDS Dietex) and irradiated poplar wood bedding (MP 16/4 from
Anibed) were used. Cages and water bottles were cleaned and decontaminated using hydrogen
peroxide once a week. Ventilated cages were maintained in a specific pathogen-free (SPF)
animal facility under controlled temperature (22±2°C), hygrometry (55±10%), and photoperiod
(12h light/12h dark, light on at 8:00 am) conditions. Mice were numbered by marking their tail
with indelible markers. Mice were allowed to acclimate to their new environment for at least 5
days prior to the first day of the experiments.
3.2.4.2 Administration Protocol
On the first dosing day (D0), mice were randomly allocated to 3 groups (with 3 mice per group)
and administered as follows:
- Formulation 2 was administered once weekly aerosol for 4 weeks on D0, D7, D14 and
D21.
- Platinum nanoparticles solution (10 ppm, sterile-filtered) was administered as a single
aerosol administration on D0.
- Platinum nanoparticles solution (100 ppm, sterile-filtered) was administered as a single
aerosol administration on D0.
Aerosol administration were performed using a IA-1C MicrosSprayer® (Penn-Century, USA)
connected to a FMJ-250 high-pressure syringe (Penn-Century, USA) with an administration
volume of 25 μL/mouse, under isoflurane anaesthesia. Briefly, the mice were anaesthetised with
isoflurane and anaesthesia was maintained all along the procedure via a mask on the nose. Each
mouse was placed on its back on a platform hanging by its incisors to clearly visualize the
background of the throat. The MicroSprayer® was introduced into the animal’s trachea using a
laryngoscope to slowly inject 25 µL of the respective solutions. The mice were observed until
they recovered and displayed normal activity.
56
3.2.4.3 Clinical Follow-up
From the first day of administration and until the sacrifice of the animals (on day 28), mice
were clinically observed daily for their general health status and weighed twice a week, and
then daily if substantial body weight loss was observed.
3.2.4.4 Terminal Procedures
Animals were sacrificed by sodium pentobarbital overdose, given by intraperitoneal (i.p.)
injection 28 days after the first administration (D28). An autopsy was performed with a detailed
macroscopic observation of the lung.
3.2.5 Feasibility Test in Xenograft Mice Model
3.2.5.1 Animals
Experiments were carried out using 21 female NOD/SCID mice (Janvier Labs, France), 7-8
weeks old on the day of the bioluminescent A549 cell implantation. From the 21 mice, 18 mice
were randomized on the first dosing day. Mice were housed in social groups of 3-6 individuals
in polysulfone individually ventilated cages (IVC) (floor area = 501 cm²). An external
ventilation unit supplied the cages individually with fresh HEPA (High Efficiency-Particulate
Air)-filtered air (75 air volume replacements/hour). A positive pressure ventilation was
maintained in each cage. Cages were equipped to provide food and sterile water ad libitum.
Irradiated food (RM1 from SDS Dietex) and irradiated poplar wood bedding (MP 16/4 from
Anibed) were used. Cages and water bottles were cleaned and decontaminated using hydrogen
peroxide once a week. Ventilated cages were maintained in a specific pathogen-free (SPF)
animal facility under controlled temperature (22±2°C), hygrometry (55±10%), and photoperiod
(12h light/12h dark, light on at 8:00 am) conditions. Mice were numbered by marking their tail
with indelible markers. Mice were allowed to acclimate to their new environment for at least 5
days prior to the first day of the experiments.
3.2.5.2 Cells
Human NSCLC A549 (ATCC® CCL-185™) cells were genetically modified to express
luciferase. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM High Glucose
+ GlutaMax™-I; ref 61965-026; Gibco, Life Technologies) supplemented with 10% foetal
bovine serum (FBS; ref 10270-098; Gibco, Life Technologies) and 1% cell culture
contamination preventive (ZellShield; ref 13-0150; Minerva-Biolabs). Cells were maintained
in an incubator with 5% CO2 at 37°C. Cells were routinely passaged two to three times per
week using 0.05% Trypsin 0.53 mM EDTA (ref 25300-056; Gibco).
57
3.2.5.3 Preparation of Cells to Be Implanted
On the day of cell inoculation, cells were harvested by trypsinizing with 0.25% Trypsin-EDTA.
Cells were then washed with Dulbecco’s phosphate-buffered saline (D-PBS, ref. 14190-094,
Gibco, Life Technologies), centrifuged at 150 x g for 5 minutes at 4°C and suspended in serum-
free culture media at a concentration of 1x104 cells/µL, allowing for an inoculation of 1x106
cells/100 μL per mouse. Cells were kept on ice until the intravenous injection in mice.
3.2.5.4 Implementation of Tumour Cells
Each mouse received an intravenous injection of a suspension of 1x106 tumour cells in the
caudal vein, with an administration volume of 100 μL per mouse, on day 0 (D0). Cells were
carefully resuspended just prior to injection.
3.2.5.5 Tumour Measurements and Clinical Follow-up
After A549 cell injection and until the sacrifice of the animals, mice were clinically observed
daily for their general health status and weighed twice a week, and then daily if substantial body
weight loss was observed. Mice were monitored for 8 weeks after A549 cell injection. Tumour
growth was assessed on days 2, 10, 23, 37 and 52 (D2, D10, D23, D37 and D52) after cell
inoculation by non-invasive in vivo bioluminescence imaging. Each mouse was injected
intraperitoneally (i.p.) with 150 mg/kg D-luciferin (XenoLight D-Luciferin Potassium Salt, Ref
122799, Perkin Elmer) and anesthetized with isoflurane. Mice were positioned in the imaging
chamber (PhotonImager, Biospace Lab) and anaesthesia was maintained during the acquisition
via a mask on the nose. Analysis of bioluminescence data was performed using M3Vision
Software (Biospace Lab).
3.2.5.6 Administration Protocol
According to the bioluminescence imaging data obtained on D2 and D10, 15 out of the 21
implanted mice were randomly allocated to 3 groups (with 5 mice per group), in such a manner
that the tumours bioluminescence were homogeneous between the different groups. Mice not
allocated to a treatment group were sacrificed. Treatment was initiated 14 days after cell
implantation (D14) to let the tumour grow. Each mouse received once weekly aerosol
administration of protected gemcitabine, non-protected gemcitabine or vehicle for 4 weeks, i.e.
on D14, D21, D28 and D35, as follows:
1. Vehicle (0 mg/kg, aerosol)
2. Protected gemcitabine (5.9 mg/kg, aerosol)
3. (Non-protected) Gemcitabine (4 mg/kg, aerosol)
58
Aerosol administrations were performed using a IA-1C MicroSprayer® (Penn-Century, USA)
connected to a FMJ-250 high-pressure syringe (Penn-Century, USA) with an administration
volume of 25 μL/mouse, under isoflurane anaesthesia. Briefly, the mice were anaesthetized
with isoflurane and anaesthesia was maintained all along the procedure via a mask on the nose.
Each mouse was placed on its back on a platform hanging by its incisors to clearly visualize the
background of the throat. The MicroSprayer® was introduced into the animal’s trachea using a
laryngoscope to slowly inject 25 µL of the respective solutions. The mice were observed until
they recovered and displayed normal activity.
3.2.5.7 Terminal Procedures
Animals were sacrificed by sodium pentobarbital overdose, given by intraperitoneal (i.p.)
injection 28 days after the first administration (D28).
3.2.5.8 Necropsy and Lung Collection
A necropsy of mice, even those found dead, was performed. The lungs were carefully observed
and the presence of tumour nodules was evaluated. After the sacrifice of each mouse, the lungs
were fixed by intratracheal perfusion of 4% formaldehyde solution, collected, and a picture was
taken. Lungs were placed in 4% formaldehyde solution for a minimum of 24 hours. All samples
were kept at room temperature.
3.3 Results and Discussion
3.3.1 Influence of Surfactant on the Average Droplet Size
After screening various surfactants and co-solvents, several stable formulations were prepared
with peanut oil, TWEEN® 80 as the surfactant, propylene glycol as the co-solvent and normal
saline solution (0.9 w/v% of NaCl). To test the influence of surfactant concentration on the
droplet size and stability, formulations composed of 0 to 5 wt. % of TWEEN® 80 were prepared
in 20 wt. % peanut oil, 25 wt. % propylene glycol and a corresponding saline solution
concentration of 50 to 55 wt. %. Formulations having less than 0.3 wt. % of TWEEN® 80 were
only stable for few minutes and were thus discarded from further tests. Above the 0.3 wt. %
threshold level, the average droplet size, as measured by dynamic light scattering (DLS), was
not significantly influenced by the surfactant concentration and was comprised between 560
nm and 840 nm (Figure 3-1). The small average droplet size ensures that the formulations are
compatible with the MicroSprayer® aerolizer utilized for the mice study. Of note, formulations
with higher TWEEN® 80 concentrations were stable at room temperature for weeks while
59
formulations containing 0.3 wt. % were stable for few hours only. However, few hours stability
is long enough to carry out the mice administration procedure with a comfortable margin.
3.3.2 Freeze-thaw Stability
For the mice study it is necessary to store the drug-containing formulations over long period of
time (at least several weeks). To reduce the risk of degradation, it was decided to freeze the
formulations. It is well known that freeze-thaw stability represents a significant hurdle for
oil-in-water emulsions.114 Because the crystal size influences the freeze-thaw stability, samples
were placed in either a -20°C or -80°C freezer. As can be seen on Figure 3-2, the emulsions
remained relatively stable after one freezing/thawing cycle. However, after two cycles
significant phase separation could be observed. Therefore, it was decided to prepare aliquots
for the various administration steps of the in vivo study and to apply ultrasonication briefly
before administration.
Figure 0-1. Influence of surfactant concentration on the average droplet size. The average droplet size
was measured by dynamic light scattering (DLS) for 4 solutions containing 0.3 to 5 wt. % of
TWEEN® 80 in 20 wt. % peanut oil, 25 wt. % propylene glycol and a corresponding saline solution
concentration of 50 to 54.7 wt. %.
60
3.3.3 Nebulization Properties of the Selected Formulations
A pre-selection of five formulations were passed through an aerolizer. Formulations were pre-
selected based on the following criteria: a low TWEEN® 80 content, a high peanut oil content
and varying degrees of propylene glycol content. The maximal peanut oil and propylene glycol
concentrations values were taken from the FDA database for inactive ingredients.113 It was
found that none of the formulations generated an aerosol but rather a thin jet instead. The
formulation giving best results was formulation 2 and was therefore selected for the mice
experiments. Remarkably, the formulation selected was the one containing the lowest
TWEEN® 80 concentration and no propylene glycol.
Figure 0-2. Influence of freezing temperature on the freeze-thaw stability. Two freezing/thawing cycles
were made at both -20°C and -80°C. After thawing, a short vortexing step was made before the average
droplet size was measured with a dynamic light scattering device.
Table 0-1. Table indicating the compositions of the various formulations and the macroscopic
observations after passing through a MicroSprayer® aerolizer.
61
3.3.4 Tolerability Profile of Formulation 2 and Platinum Nanoparticles Solutions
Tolerability profiles were determined to ensure that no toxicity originates from neither the
vehicle (formulation 2) itself, nor from platinum nanoparticles. Three groups of 3 mice were
made. Mice from group 1 were chronically administered (once a week over one month) with
formulation 2. Mice from group 2 and 3 were acutely (on D0) administered with platinum
aqueous dispersions of 10 and 100 ppm, respectively. Mice from all 3 groups were regularly
clinically followed and were weighted twice a week during one month. The mice were then
sacrificed and their lungs macroscopically observed (Figure A2-1). No toxicity signs could be
observed for the lungs. Furthermore, the bodyweight curves (Figure 3-3) indicated normal
growth. In conclusion, neither formulation 2, nor the platinum nanoparticles solutions should
be toxic at the here used concentrations over a one-month period.
Figure 0-3. Tolerability profiles of formulation 2 and platinum nanoparticles solutions. Mice from
group 1 were administered weekly over one month with formulation 2. Mice from group 2 and 3 were
administered once on D0 and were clinically followed during one month.
62
3.3.5 Feasibility Test in Xenograft Mice Model
Before testing the uncaging reaction in vivo, it was decided to first study the caged prodrug in
vivo toxicity and study its influence on the growth of tumours as compared to both the vehicle
and gemcitabine. This was done by weekly administering the various formulations to mice
previously inoculated with luciferase-expressing A549 cells. Bioluminescence was recorded
biweekly, mice weight was measured twice a week and mice were clinically observed daily
(Figure 3-4).
Due to the restricted number of animals of the current study, the statistical power is not
significant enough to draw definitive conclusions. However, several comments can already be
made from those preliminary data. First, caged gemcitabine did not, at least at a macroscopic
level, trigger any toxicity. This confirms what was observed in vitro with normal lung epithelial
cells. Second, the gemcitabine-containing formulation triggered significant toxicity as is
exemplified by the fact that many mice from this group prematurely died despite the slower
tumour growth. At the time of writing, histological and immunohistochemical studies are being
undertook on the lungs obtained after sacrificing the animals at the end of the study. The
information that will be gathered might provide more insights into the causes of death for mice
belonging to the gemcitabine group and indicate whether caged gemcitabine actually generated
some toxicity that was not detectable at the macroscopic level. If not, this would permit to either
go to higher drug concentration and/or have more frequent drug administrations. Another aspect
that was assessed during these experiments was whether the reducing environment, generally
present in tumours, was sufficient to trigger the uncaging reaction. This was not the case but it
has to be noted that tumours in such models are much less hypoxic that their human or
environmentally induced and genetically engineered mice models counterparts.63,111
Further studies are required to determine whether and with which conditions can the
prodrug be activated in vivo. Also, more therapeutically realistic, but costlier, models could be
envisaged to more precisely determine the therapeutic potential of this caged prodrug system.
63
3.4 Conclusion
In this preliminary pre-clinical study, a formulation suitable for intratracheal lung
administration of caged gemcitabine was developed. Moreover, it could be shown that neither
the vehicle, the caged prodrug nor the platinum nanoparticles introduce toxicity. The
gemcitabine-containing formulation lead to significant toxicity and an important fraction of the
mice prematurely died from non-cancer related causes. The reducing environment of the tumour
was not sufficient to uncage the prodrug and new experiments are required to establish an in
vivo uncaging protocol. Overall, the results were encouraging but request further experiments.
For instance, once a prodrug uncaging protocol is found, it would have to be determined
whether the uncaging indeed translates into an elevated cytotoxicity and whether a higher drug
concentration and/or a more frequent administration could be used while keeping side-effect at
a level similar to that of gemcitabine. Also, more complex and more expensive mouse models
mimicking more closely human cancer could be used in the perspective of phase I clinical trials.
Figure 0-4. A) Growth of tumours as measured by the tumour bioluminescence signal. Of note, the
dashed lines are only indicative since the average does not correspond to the same number of animals
as in the beginning of the experiments. At t = 2 d, there were 6 animals in the vehicle and protected
gemcitabine groups and 5 animals in the gemcitabine group (one animal was removed from the analysis
because at t= 10 d, it displayed a bioluminescence signal two orders of magnitude higher than the other
animals within the same group). B) Kaplan-Meier curves for the time period during which
bioluminescence signals were regularly acquired.
64
65
Effective Tumour Cells Removal from Patient Blood
using Magnetic Nanoparticles.
Manuscript submitted:
A. F. Herzog, M. Zeltner, A. Zabel, N. Schläpfer, A. Siebenhüner, W. J. Stark,
B. Beck-Schimmer
Author contributions: concept/design by AFH, MZ, NS, WJS and BBS. Experiments
planned/conducted by AFH, MZ and AJ. Data Analysis/interpretation by AFH, MZ, AZ, NS,
WJS and BBS. Drafting article by AFH, MZ, AZ and BBS. Critical revision of article by AFH,
MZ, AZ, NS, AS, WJS and BBS. Approval of article by AFH, MZ, AZ, NS, AS, WJS and BBS.
66
4.1 Introduction
Metastatic disease is responsible for over 90% of cancer deaths.115–118 Cells that detach
from the primary tumour and join the vascular system are also known as circulating tumour
cells (CTCs). They are thought to be involved in metastasis formation, and a direct correlation
between the CTCs number and patient prognosis has been found.119–122 Apart from the
continous release of CTCs from the primary tumour, a large increase in CTCs may be recorded
during and after cancer surgery,123–127 which further puts cancer patients at risk for recurrent
relapses as well as a decreased overall survival.25,26,128 Therefore, perioperative removal of
CTCs could potentially provide a way to improve long-term prognosis. However, targeted
elimination of blood cells is a challenging task, particularly with focus on CTCs, which often
present in low concentrations (down to 1 CTCs per 107 leukocytes or per 1010 erythrocyte).129
Here, we show a way to magnetically remove CTCs from blood. It relies on highly magnetic
and antifouling carbon-coated cobalt nanoparticles functionalised with polyglycidol and
epithelial-selective anti-epithelial cell adhesion molecule (EpCAM) antibodies. By using these
magnetic particles, our approach allows for the reproducible and specific removal of CTCs-
spiked blood. Moreover, first translation into a clinical scenario was possible, eliminating CTCs
from blood obtained from cancer patients. When evaluating safety aspects, such as possible
interference of the magnetic particles with the blood coagulation system, no major deviations
were observed. Our system is flexible in that the nanoparticles can be functionalized with other
antibodies, thereby potentially allowing further applications, as well as the possibility to adapt
to the discoveries in the field of CTCs biomarkers. We anticipate the development of such a
platform to translate into the feasibility of filtering the entire blood of patients, thus
hypothetically increasing patients’ prognosis, especially for the ones undergoing tumour
resection.
Although the significance of circulating tumour cells (CTCs) has been discussed for a long
time, their detection in the peripheral blood of cancer patients has received increasing interest
in the last decade. Reviews summarizing prognostic studies in breast, prostate, colon and lung
cancer highlighted the importance of CTCs as prognostic factors for tumour progression and
overall survival.118,130 The following examples further support these findings: For
adenocarcinoma of the breast, several trials suggested that patients tested “positive” for CTCs
had a shorter progression-free and overall survival.119,131 Also, in patients suffering from
urothelial cancer the presence of CTCs in the blood indicated poor outcome.132 Similarly, for
67
the entity of gastric and gastro-oesophageal junction adenocarcinoma, CTCs were associated
with worse progression-free survival as well as impaired overall survival.133
Circulating tumour cells are cancer cells, which detach from the primary tumour and find
access to the blood. This way, they are carried around the body to other locations and, as they
leave the blood system and find access to the extravascular space, possibly lead to cancer
metastasis.134 Various technologies were developed for CTCs detection and isolation. Namely,
immunocytological-based approaches (e.g. immunocytochemistry, CellSearch system, flow
cytometry), molecular RNA-based technologies (e.g. qPCR, FISH) as well as functional assays
were reported.130,135 Among those technologies, the CellSearch system is the only detection
system to be approved by the FDA for tumour entities such as breast, prostate and colorectal
cancer, all of them of epithelial origin and therefore defined as carcinomas. Consequently,
CellSearch serves as the gold standard for CTCs enumeration. Akin to many of the methods
available for the detection of CTCs, the technology is taking advantage from the fact that CTCs
of epithelial origin express epithelial cell adhesion molecule (EpCAM) as a cell surface marker.
Many different EpCAM antibodies directed against this epitope are available, which allow a
specific interaction with it. This way CTCs of epithelial origin can be identified. This implies
that to detect CTCs from other tumour entities such as sarcoma or melanoma other markers are
needed for CTCs enrichment.136 Of note is that most of the malignancies are solid tumours.137–
139
While the diagnostic field of CTCs detection in the blood has been extensively
explored140–142, there is still no technique available to therapeutically remove CTCs in vivo from
a complex biological fluid, such as the blood, in a specific and efficient way without interacting
with the single blood components. The objective of the present work is to provide means and
methods to enable the efficient removal of CTCs from peripheral blood. This was done using
highly magnetic carbon-coated cobalt (C/Co) nanoparticles functionalized with polyglycidol
and conjugated with anti-EpCAM antibodies (Figure 4-1 and Figure 4-2a).
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4.2 Materials and Methods
4.2.1 Synthesis of C/Co-PhEtOH
Following previously described synthesis143, 10 g carbon-coated cobalt nanoparticles (C/Co)
were added to 400 mL of distilled water. The nanoparticles were dispersed by using an
ultrasonication bath (10 min, Sonorex Digitec DT 103 H, Bandelin). In a round-bottom flask,
4-aminophenethyl alcohol (1.2 g, 8.76 mmol) was mixed with 30 mL of distilled water and
dissolved by addition of 10 mL hydrochloric acid (HCl conc./37% fuming). The solution of 4-
aminophenyl alcohol was added to the dispersed particles before being placed for an additional
5 min in an ultrasonication bath. The nanoparticle dispersion was then placed in an ice bath
before a sodium nitrite solution (1.2 g, 17.4 mmol in 10 mL of distilled water) was added
dropwise. Instantaneous evolution of nitrogen gas (N2) could be observed. The reaction vessel
Figure 0-1. Overview of working principles. Human blood is sampled from a cancer patient (step 1) and a
dispersion of anti-EpCAM-functionalized nanoparticles is added (step 2). After gentle mixing, the nanoparticles
bind to CTCs and are then recovered by magnetic separation (step 3). In practice, this is done within a few minutes
by passing the sample through a magnetic beads-containing column. The filtrate is then subject to CTCs
enumeration (step 4). In later clinical applications, the purified blood could be reinjected to the patient after
extracorporeal purification.
69
was then placed in an ultrasonication bath for 2 h. The nanoparticles were thoroughly washed
with distilled water (3 x 100 mL), EtOH (3 x 100 mL) and acetone (3 x 100 mL). At every step,
the particles were dispersed in an ultrasonication bath for 3 min and separated by application
of a permanent magnet (magnetic decantation). After washing, the particles were dried
overnight in a vacuum oven at 50°C.
4.2.2 Synthesis of C/Co-PhEtO-Na+
Following previously described syntheses144,145, 10 g of C/Co-PhEtOH were added to 20 mL of
a sodium methoxide solution (2 M in dry methanol). After being dispersed with an ultrasonic
bath for 2 min, the solution was stirred overnight at 65 °C. The particles were washed with dry
methanol (8 x 10 mL). At every step, the particles were dispersed in an ultrasonication bath for
3 min and separated by application of a permanent magnet (magnetic decantation). After
washing, the particles were dried overnight in a vacuum oven at 50 °C.
Elemental microanalysis:
[C] =5.13 %, [H] = 0.24%, [N] = 0.1%, [S] =0%
4.2.3 Synthesis of C/Co@polyglycidin
Following previously described synthesis146, 500 mg C/Co-PhEtO-Na+ were added to 20 mL of
dry toluene. After being dispersed with an ultrasonic bath for 2 h, the reaction mixture was
degassed by bubbling nitrogen through for 30 min. After installation of a reflux condenser, the
reaction mixture was heated up to 140 °C under inert conditions. Once the reaction mixture
reached 140 °C, 10 mL (+/-)-glycidol (+/- -oxiran-2-ylmethanol) was slowly added with a
syringe pump (1.3 mL/h). The reaction mixture was then stirred for 16 h before being down to
room temperature. The nanoparticles were thoroughly washed with toluene (100 mL), MeOH
(100 mL) and distilled water (100 mL). At every step, the particles were dispersed in an
ultrasonication bath for 3 min and separated by application of a permanent magnet (magnetic
decantation). The washing process with water was repeated until no foam generation could be
observed anymore. After washing, the particles were dried overnight in a vacuum oven at 50
°C.
Elemental microanalysis:
[C] = 10.3 %, [H] = 1.2 %, [N] = 0.07 %, [S] = 0 %
ΔC = 5.17 % = 4.31 mmol/g , ΔH = 1.08 % = 9.6 mmol/g, ΔN =-0.03 % = -0.02 mmol/g, ΔS
=0 %
70
Calculated amount of polyglycidol: 1.44 mmol/g nanoparticles.
Calculated average chain length: 15 units per starter.
Infrared Spectroscopy:
Peak list: 2873 cm-1, 2356 cm-1, 1469 cm-1, 1328 cm-1, 1068 cm-1, 923 cm-1, 862 cm-1
4.2.4 Synthesis of C/Co@polyglycidyl-COOH
Following previously described synthesis147, 300 mg C/Co@polyglycidin were added to 15 mL
of dry dimethylformamide. After being dispersed with an ultrasonic bath for 3 min, succinic
anhydride (150 mg, 1.3 mmol) was added. The solution was placed for 10 min in an
ultrasonication bath before N,N-dimethylpyridin-4-amine (DMAP, 180 mg, 1.5 mmol) and
triethylamine (1.5 mL, 10.8 mmol) were added. The reaction mixture was degassed by bubbling
nitrogen through for 30 min. The reaction mixture was heated up to 70 °C and stirred overnight
under inert conditions. The nanoparticles were thoroughly washed with distilled water (3 x 100
mL). At every step, the particles were dispersed in an ultrasonication bath for 3 min and
separated by application of a permanent magnet (magnetic decantation).
Elemental Analysis:
[C] = 14.3 %, [H] = 1.4 %, [N] = 0.26 %, [S] = 0 %;
ΔC = 4 % = 3.3 mmol/g, ΔH = 0.2 % = 2 mmol/g, ΔN =0.19 % = 0.1 mmol/g, ΔS =0 %
Infrared Spectroscopy:
Peak list: 2939 cm-1, 2873 cm-1, 2675 cm-1, 2360 cm-1, 2356 cm-1, 1725 cm-1, 1560 cm-1,
1406 cm-1, 1244 cm-1, 1168 cm-1, 1068 cm-1, 838cm-1
4.2.5 Synthesis of C/Co@polyglycidyl-COO-EpCAM and C/Co@polyglycidin-
COO-IgG
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide
(sulfo-NHS) were left at room temperature until equilibration. Each compound was dissolved
in activation buffer (OceanNanotech) at concentrations of 4 mg/mL and 2 mg/mL, respectively.
The solutions were homogenised by vortexing for 10 sec. C/Co@polyglycidyl-COOH
nanoparticles solutions (5 mg/mL in activation buffer) were prepared. After being dispersed
71
with an ultrasonic bath for 2 min, 200 µL of the solution were added to 1.5 mL Eppendorf tubes
containing 100 µL of activation buffer. The EDC and sulfo-NHS solutions were mixed in a 1:1
volume ratio and 10 µL were added to each nanoparticles-containing Eppendorf tube. To ensure
homogeneous dispersion, the solutions were vortexed for 10 sec, followed by 20 sec in an
ultrasonication bath. The activation step was done by shaking the Eppendorf tubes at 1200 rpm
for 10 min at 25°C in a thermomixer (ThermoMixer Comfort, Eppendorf). 100 µL of an
antibody solution (anti-EpCAM or IgG isotype control; 1 mg/mL) were added to the Eppendorf
tubes. To ensure homogeneous dispersion, the solutions were vortexed for 10 sec followed by
20 sec in an ultrasonication bath. The Eppendorf tubes were then shaken at 1200 rpm for 4 h at
25°C in a thermomixer. To quench the reaction, 10 µL of quenching buffer (OceanNanotech)
were added to each Eppendorf tube. To ensure homogeneous dispersion, the solutions were
vortexed for 10 sec followed by 20 sec in an ultrasonication bath. The quenching reaction was
done by shaking the Eppendorf tubes at 1200 rpm for 30 min at 25 °C in the thermomixer. The
particles were washed by placing the reaction vessel in a pre-cooled (4 °C) SuperMag separator
(OceanNanotech). To ensure complete separation, the magnet was placed in a fridge for 1.5 h
before the supernatant was discarded and replaced with 420 µL of fresh cold PBS (pH 7.4, Life
Technologies). The nanoparticles were dispersed by vortexing for 10 sec followed by 20 sec in
an ultrasonication bath. The Eppendorf tubes were placed back in the fridge and the washing
procedure was repeated 3 times. Once the washing process completed, the solutions were
aliquoted (30 µL aliquots) and stored at -20 °C until use.
4.2.6 Characterization of Antifouling Efficiency
Various nanoparticles batches were added to PBS (concentration of 2 mg/mL). Dispersion was
achieved by using an ultrasonic horn (3x30 sec on ice, UP50H, Hielscher).
Tetramethylrhodamine-conjugated bovine serum albumin (rhodamine-BSA) was dissolved in
PBS (concentration of 0.4 mg/mL), and the rhodamine-BSA solution was then diluted 3 times
to reach a concentration range where linear behaviour is observed for the antifouling tests. For
each nanoparticle batch 500 µL of the rhodamine-BSA and 500 µL of a nanoparticle solution
were added to an Eppendorf tubes. Particles having no polymer coating were used as the
negative control while milli-Q water was used as the positive control. To ensure homogeneous
dispersion, the solutions were vortexed for 10 sec followed by 30 sec in an ultrasonication bath.
To ensure optimal contact between the proteins and the nanoparticles surface, the samples were
shaken for 90 min at 1000 rpm at 25 °C (Thermomixer Comfort, Eppendorf). The particles were
washed by placing the Eppendorf tubes for 1 h in a SuperMag separator. Supernatant aliquots
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(5x100 µL) of each sample were made and transferred to a 96-well plate before fluorescence
was measured (ex: 540 nm, em: 620 nm; Spark 10M; Tecan).
4.2.7 Characterization of Separation Efficiency
Solutions of nanoparticles were prepared at a concentration of 2 mg/mL in PBS. Four mL of
the solutions were transferred to 5 mL glass vials. The vials were placed for 10 min in an
ultrasonic bath. The separation was started by placing one glass vial on each side of a permanent
magnet (1.3T, Webcraft AG). The separation was recorded using two CMOS sensors (sensor
1: 12 MP, 1.25 µm, f/2.2; sensor 2: 12 MP, 1.0 µm, f/2.6; Xiaomi A1, Xiaomi Inc.). The
quantification was done using an image processing program (ImageJ, NIH).
4.2.8 Cell Line Experiments
The EpCAM-expressing human colon cancer cell line HT-29 (HTB38™, ATCC) was cultured
in RPMI 1640 medium, supplemented with glutamax (Gibco), 10% foetal bovine serum (FBS,
Gibco) and a mixture of penicillin/streptomycin (final concentration 100 U/ml/ 100 µg/ml,
Gibco). The cells were incubated at 37°C in 5% CO2. Once cells reached confluence, they were
detached with accutase (Gibco) and resuspended in blood at a concentration of 5x105 cells/mL.
Cell membrane of the HT-29 cells was stained with the PKH26GL labelling kit (Sigma-Aldrich)
before the tumour cells were added to PBS or whole blood. Passages between 5 and 40 were
chosen for the experiments.
4.2.9 Scanning Electron Microscopy of anti-EpCAM Nanoparticles incubated with
HT-29 Cells
HT-29 cells were cultured for 48h in 12-well plates where carbon-coated sapphire disks were
deposited. 200 L of an anti-EpCAM nanoparticles solution (2.38 mg/mL in PBS) were added
to each well. The sapphire disks were then freeze substituted (-90°C to room temperature) in
dry ethanol containing 1% uranyl acetate. Room temperature samples were washed 3 times with
dry ethanol and critical point dried with liquid CO2 (Autosamdri 931, Tousimis). The samples
were coated with 7 nm of Au (CCU-010, Safematic) and imaged by SEM (Leo 1530, Zeiss).
4.2.10 Influence of Magnetic Nanoparticles Treatment on Lymphocytes Enumeration in
Healthy Subjects Spiked Blood
After informed patient consent (ethics approval: KEK-ZH-Nr. 2012-0274), up to 20 mL blood
samples were drawn from healthy subjects into heparin tubes (BD Biosciences). Eppendorf
73
tubes containing either an anti-EpCAM nanoparticles solution (2.38 mg/mL in PBS) or an IgG-
isotype control nanoparticles solution (2.38 mg/mL in PBS) were sonicated (5x1 min with 1
min breaks in between) in an ultrasonication bath (Sonorex Digital 10P, Bandelin) on ice-cold
water. Once sonicated, the 25 L of the nanoparticles were added to 475 L of blood. In
addition to the IgG-isotype control, a control was made with 25 L PBS. The samples were
incubated for 2 min on an orbital shaker (16 mot/min, WS 10, Edmund Bühler) before being
passed through a column magnet system (MS columns, MACS, Miltenyi Biotec). Each column
was washed twice with 500 µL of PBS. Lymphocytes were labelled by transferring a third of
the filtrates to Eppendorf tubes and incubating with 1 L of Human CD19 APC-conjugated
(SJ25-C1, ThermoFisher) and 1 L of Human CD3d Alexa Fluor 48-conjugated (7D6,
ThermoFisher) for 30 min at 4°C. Samples were then prepared for flow cytometry analysis.
Red blood cells were lysed by adding 10 mL of a red blood cells lysis buffer (Biolegend, USA).
The samples were centrifuged for 10 min at 400 g (centrifuge 5810R, Eppendorf). The
supernatant was discarded and the pellet resuspended with 200 µL of PBS. The cells were fixed
with 200 µL of a 4% formalin solution. 25 µL of counting beads solution (concentration of
5.2x104 counting beads per 50 µL, CountBright Absolute, Life Technologies) were added to
each sample before analysing them using a flow cytometer (BD Canto II, BD Biociences).
Acquisition was carried out with the BD FACSDiva Software (BD Biosciences). Forward and
side scatter area as well as signal height were recorded. Lymphocytes were detected using either
side scatter area or forward scatter area vs either APC-A or Alexa Fluor 488-A. FACS data
were processed using FlowJo V10.0.8.
4.2.11 Removal of Circulating Tumour Cells (CTC) from Healthy Subjects Spiked Blood
After informed patient consent (ethics approval: KEK-ZH-Nr. 2012-0274), up to 20 mL blood
samples were drawn from healthy subjects into heparin tubes (BD Biosciences) and spiked with
labelled HT-29 cells (see above) to reach a concentration of 5x105 cells/mL. Eppendorf tubes
containing either an anti-EpCAM nanoparticles solution (2.38 mg/mL in PBS) or an IgG-
isotype control nanoparticles solution (2.38 mg/mL in PBS) were sonicated (5x1 min with 1
min breaks in between) in an ultrasonication bath (Sonorex Digital 10P, Bandelin) on ice-cold
water. Once sonicated, the 25 L of the nanoparticles were added to 475 L of cancer cells-
containing blood. In addition to the IgG-isotype control, a control was made with 25 L PBS.
The samples were incubated for 2 min on an orbital shaker (16 mot/min, WS 10, Edmund
Bühler) before being passed through a column magnet system (MS columns, MACS, Miltenyi
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Biotec). Each column was washed twice with 500 µL of PBS. A third of the filtrates were
transferred to Eppendorf tubes. Samples were then prepared for flow cytometry analysis. Red
blood cells were lysed by adding 10 mL of red blood cells lysis buffer lysis buffer (Biolegend,
USA). The samples were centrifuged for 10 min at 400 g (centrifuge 5810R, Eppendorf). The
supernatant was discarded and the pellet resuspended with 200 µL of PBS. The cells were fixed
with 200 µL of a 4% formalin solution. 25 µL of counting beads solution (concentration of
5.2x104 counting beads per 50 µL, CountBright Absolute, Life Technologies) were added to
each sample before analysing them using a flow cytometer (BD Canto II, BD Biociences).
Acquisition was carried out with the BD FACSDiva Software (BD Biosciences). Forward and
side scatter area as well as signal height were recorded. CTCs were detected using forward
scatter area vs the PE-. FACS data were processed using FlowJo V10.0.8.
4.2.12 Removal of Circulating Tumour Cells (CTCs) from Cancer Patients
After informed patient consent (ethics approval: KEK-ZH-Nr. 2016-01140), up to 25 mL blood
samples were drawn from cancer patients into either CellSave (Menarini-Silicon Biosystems),
heparin or EDTA tubes. After confirming the presence of circulating tumour cells with the
CellSearch system (Menarini-Silicon Biosystems), 7.5 mL CTCs-containing blood samples
were treated with either 375 µL of an anti-EpCAM nanoparticles solution (2.38 mg/mL in PBS)
or with 375 µL of PBS. Before being added to the blood samples, the nanoparticles solutions
were sonicated (5x1 min with 1 min breaks in between) in an ultrasonication bath (Sonorex
Digital 10P, Bandelin) on ice-cold water. The samples were incubated for 5 min on an orbital
shaker (16 mot/min, WS 10, Edmund Bühler) and passed through a column magnet system (LS
columns, MACS, Miltenyi Biotec). The filtrate was analysed with the CellSearch system
(Menarini-Silicon Biosystems) and evaluated by trained experts with CellSpotter.
4.3 Results and Discussion
4.3.1 Technical Aspects of the Newly Designed Nanoparticles
As a prerequisite for a successful translation into a clinical scenario, efficiency is highly
desirable, which allows blood purification of the patient’s blood within a short period of time.
This can be reached by using particles that are rapidly separated from the blood, allowing high
filtration rates. Magnetic separability is influenced by the metallic core’s magnetic properties
and, to a notable extent, by the agglomeration properties. In order to obtain efficient
separability, a paradigm shift was necessary for the design of the here described nanoparticles.
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Indeed, generally the focus lies on engineering nanoparticles as stable as possible. This is
because, for most applications, agglomeration is an undesirable outcome. As an example of
this, recently, C/Co nanoparticles were developed for later drug delivery applications.148 The
nanoparticles were functionalised with poly(3-sulfopropyl methacrylate potassium salt)
(pSPM), which affords ultra-stable particles. However, in our preliminary tests, the high
stability lead to a curtailed separability and poor performances for filtration applications (Figure
S1, green line). To overcome this problem, polyglycidol was selected as an alternative polymer
to pSPM because electrosteric repulsions are much lower than for pSPM, yet retain interesting
antifouling properties. Beyond the adequate separability properties, polyglycidol has further
advantages over pSPM. It is biologically inert and is approved by the FDA.149–151 The
preparation of the nanoparticles involves established syntheses that provide for a robust
manufacture (Figure 4-2a and Table A3-1). Additionally, the assessment of the degree of
Figure 0-2. Nanoparticles synthesis and characterisation. a, Overview of the synthesis of antibody-
functionalized C/Co nanoparticles. b, Infrared spectra of the C/Co nanoparticles coated with polyglycidol
and identical particles modified with a carboxylic group. The introduction of the carboxylic can be
monitored by the introduction of a peak around 1735 cm-1. c, Transmission electron micrographs of the
anti-EpCAM-functionalised C/Co nanoparticles. d, Transmission electron micrographs of the HT-29
cancer cells after incubation with anti-EpCAM-functionalised C/Co nanoparticles. The blue area delimits
a HT-29 cell, the red area nanoparticles bound on the cell surface.
76
polymerisation as well as the number of carboxyl moieties incorporated can easily be
determined. The former is obtained by elemental microanalysis while the introduction of the
carboxyl group gives a clear infrared (IR) peak (Table A3-1, Figure 4-2b and Figures A3-4 to
A3-6). The resulting nanoparticles were further characterised by transmission electron
microscopy (Figure 4-2c). In an additional characterisation experiment where the nanoparticles
were incubated with cancer cells, nanoparticles aggregates could be observed on the surface of
the cells (Figure 4-2d). Assuming that colloidal stability is directly influenced by the extent of
polymer coating, three nanoparticles batches, with varied polymer lengths, were manufactured.
The batches had coating with average polymer lengths of 0 (no polymer spacer), 16 and 48
polymer units, respectively. An experiment was designed to obtain qualitative information
about the relative magnetic separability of the various batches. The nanoparticles were
dispersed in phosphate-buffered saline (PBS) and collected using a permanent magnet. The
separation was recorded using a camera and quantified using an image processing software. As
hypothesised, the larger the polymer chain, the slower the separation (Figure 4-3a, Figure A3-
1).
Removal of CTCs implies using nanoparticles that do not display extensive unspecific
protein adsorption (i.e. designing nanoparticles with good antifouling properties). As a
consequence, a protocol using rhodamine-labelled albumin, the most abundant plasma
Figure 0-3. Polymer coating optimisation. a, Qualitative information about the polymer length influence on
the magnetic separability. Nanoparticles solutions were placed next to a permanent magnet. The separation
was recorded using a camera and quantification was done using an image processing software. b, Polymer
length influence on the protein adsorption. Rhodamine-modified bovine serum albumin (BSA) was incubated
in PBS with the nanoparticles. After magnetic separation, the fluorescence of the supernatant was measured
with a plate reader (n = 5 technical replicates; 𝜆ex/em = 540/620 nm). c, Polymer length influence on the CTC
removal. Healthy subjects’ blood was spiked with HT-29 cells. Anti-EpCAM-functionalised C/Co nanoparticles
were added to samples which were passed through a magnetic column system. The enumeration of the HT-29
cells in the filtrate was assessed by flow cytometry (n = 3 biological replicates).
77
protein152, was established. After incubation, the nanoparticles were collected and the
fluorescence of the supernatant measured. The protein adsorption was back-calculated using a
standard curve. As expected, antifouling properties were directly influenced by the degree of
polymerisation. Particles functionalised with no spacer had a 6.8-fold higher bovine serum
albumin (BSA) adsorption on their surface as compared to particles having 48 polyglycidol
units (Figure 4-3b). Particles with an intermediate polymer chain length had an adsorption level
comprised between the two aforementioned extremes.
4.3.2 First Tests in Human Blood from Healthy Subjects Spiked with Cancer Cells
After the magnetic separability and antifouling tests, CTCs removal efficiency of the three types
of nanoparticles was assessed in blood samples from healthy subjects. Nanoparticles, coated
with anti-EpCAM antibodies, were supplemented to fresh human blood previously spiked with
HT-29 cells, a human colorectal adenocarcinoma cell line. After mixing, the samples were
passed through magnetic microbeads-containing columns for positive selection. Erythrocytes
present in the filtrate were then lysed and CTC enumeration was obtained by flow cytometry.
Interestingly, the removal efficiency followed a divergent pattern as compared to the antifouling
properties, and an optimum was found for particles functionalised with an average of 16 units
per polyglycidol chain (Figure 4-3c). This could indicate that two competing factors are at play.
On one hand, the removal efficiency increases with improved antifouling properties (over 7-
fold higher efficiency for particles with 16 units as compared to particles with no polymer
coating). On the other hand, particles with superior antifouling properties dreadfully performed
(over 28-fold higher efficiency for particles with 16 units as compared to particles with 48
units). This suggests that, in our case, separability plays a more substantial role for cell removal
efficiency over antifouling properties. A plausible explanation is that, for more stable particles,
the forces obtained by placing the separation columns in a permanent magnet are not enough to
overcome the electrosteric repulsion forces and the forces resulting from the flow through the
magnetic column. Hence, the antifouling properties might only be necessary to ensure that
nanoparticles are moderately covered by other blood components and thereby playing only a
secondary role.
4.3.3 Specificity, Safety Aspects and Reproducibility
When eliminating CTCs from blood, the removal is expected be specific through the EpCAM
epitope-antibody interaction. That is, the CTCs removal should not originate from unspecific
binding of the tumour cells on the nanoparticles’ surface. To study this, nanoparticles were
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conjugated with either the IgG isotype as control or the anti-EpCAM antibodies. Both groups
of nanoparticles were subject to the protocol for CTCs removal from healthy subjects’ blood
spiked with cancer cells (HT-29 cells). When using IgG isotype nanoparticles between 83%
and 91.4% of the spiked cells remained in the blood samples, hence demonstrating the
specificity of the nanoparticles (Figure 4-4a and Figure 4-4b).
Another aspect of specificity concerns elimination of CTCs without interacting with other blood
cells. Therefore, experiments were performed to test the hypothesis that EpCAM-functionalised
nanoparticles do not eliminate important immunological blood components such as B- and T-
lymphocytes. Blood was exposed to anti-EpCAM and purified using the magnetic column
system. Lymphocytes counts were assessed in the filtrate via flow cytometry. The level of B-
and T-lymphocytes after treatment with anti-EpCAM nanoparticles did not differ significantly
from control blood that had run over the column (Figure 4-4c and Figure A3-3).
To explore the possible interaction of the nanoparticles with the coagulation system, rotational
thromboelastometry (ROTEM®) measurements were performed. ROTEM® is a clinically
well-established viscoelastic method to test haemostasis in whole blood, which allows not only
the definition of diagnostic, but also therapeutic processes as a reliable point-of-care tool. It
provides detailed information about clot formation such as clotting time (CT; start of reaction
until clots starts forming) or maximum clot firmness (MCF), reflecting the strength of the clot.
For the anti-EpCAM nanoparticles, all ROTEM® data were found to be within the reference
values not only for the EXTEM with activation of the clotting cascade via tissue factor, but also
for the INTEM with contact activation (Table A3-2)153 ROTEM®
The safest way to avoid in vivo complications, is to prevent nanoparticles from entering into
the patient’s bloodstream. This can be obtained by efficient magnetic separation followed by
monitoring before blood reinjection into the patient’s circulatory system. To achieve this, we
recently co-developed a method to detect nanoparticles in blood down to sub-picomolar
concentrations.154 The method can be setup in line with our previously developed
extracorporeal continuous blood purification device.155
Reproducibility being one of the key requirements of the FDA’s manufacturing process
validation, and indeed for the clinical application itself as well, several batches of the optimised
nanoparticles were synthesised. The synthesis was particularly robust; as exemplified by the
limited batch to batch variation in the extent of carboxyl moiety introduction, less than 2.5%,
as measured by elemental microanalysis (Table A3-1). The batches were later functionalised
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with either anti-EpCAM or IgG isotype control antibodies and CTCs removal from spiked
blood experiments were repeated. All batches yielded similar results, with over 98% CTCs
removal, thereby confirming the biological reproducibility beyond the chemical reproducibility
(Figure 4-4b).
Figure 0-4. CTCs removal from spiked blood and from cancer patient blood samples. a, Flow
cytometry data performed on filtrates obtained after CTCs removal procedure in healthy subject’s
spiked blood. The gate indicates the region where the spiked HT-29 cells appear. b, Specific
elimination of CTCs with particles functionalised with anti-EpCAM. Also, this graph displays batch
to batch reproducibility of CTC removal. The CTC removal procedure was reproduced three times
with each of the three optimised anti-EpCAM nanoparticles batches. c, Table showing B- and T-
lymphocyte count after treatment with anti-EpCAM coated magnetic nanoparticles. d, CTCs
removal from cancer patient blood samples. Blood samples were taken from cancer patients with
metastatic disease. CTCs enumeration was carried out on a CellSearch device for samples with
and without treatment with nanoparticles.
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4.3.4 First Tests with Blood Samples from Cancer Patients
In a subsequent step, the optimised nanoparticles were used to remove CTC from blood samples
obtained from cancer patients. This included blood from patients suffering from prostate
(patient no. 1, 2 and 3), pancreatic (patient no. 4 and 6) and colon cancer (patient no. 5). The
protocol consisted in a direct 15 times scale up of the spiked blood experiments (7.5 mL vs. 0.5
mL). All cancer patients had suffered from metastatic disease, and blood was drawn before they
received standard of care chemotherapy. In blood of two cancer patients with colon cancer,
results were not conclusive. Of the remaining six patients, the average removal efficiency was
over 68% (Figure 4-4d). These preliminary results obtained are encouraging, especially given
the fact that patients had very different initial CTCs counts and their CTCs most likely exhibit
various EpCAM-expressing profiles.
4.4 Conclusion
In summary, carbon-coated cobalt (C/Co) nanoparticles were covalently functionalised with a
polyglycidol coating and anti-EpCAM antibodies. Such nanoparticles exhibited a high
magnetisation and the antifouling properties necessary to provide the desired ability to remove
CTCs from peripheral blood with a remarkable efficiency, both from blood of healthy subjects
spiked with tumour cells as well as from cancer patients. Of highlight is extraction of CTCs of
different tumour entities as well as elimination of tumour cells of various concentrations.
Additionally, the nanoparticles exhibited adequate specificity towards EpCAM-expressing
cancer cells without elimination of other blood cells such as lymphocytes. Moreover, the
coagulation system, a major component of the blood, does not seem to be impaired, neither
towards thrombosis or thrombolysis. Finally, the material could be manufactured reproducibly
using robust syntheses procedures. These preliminary findings constitute a first step towards
whole blood filtration for CTCs removal. If confirmed by further preclinical and clinical data,
this might provide a new tool in the field of cancer surgery.
81
82
Functionalization Reaction Study and Lymphocytes’
Capture as a Demonstration of Highly Abundant Cells
Removal
Manuscript in preparation:
A. F. Herzog, M. Zeltner, A. Zabel, N. Schläpfer, A. Siebenhüner, W. J. Stark,
B. Beck-Schimmer
Author contributions: concept/design by AFH, MZ, NS, WJS and BBS. Experiments
planned/conducted by AFH, MZ and AJ. Data Analysis/interpretation by AFH, MZ, AZ, NS,
WJS and BBS. Drafting article by AFH, MZ, AZ and BBS. Critical revision of article by AFH,
MZ, AZ, NS, AS, WJS and BBS. Approval of article by AFH, MZ, AZ, NS, AS, WJS and BBS.
83
5.1 Introduction
Extracorporeal blood purification using magnetic nanoparticles falls into the class III of
the FDA’s medical devices classification.156 Such devices must undergo the most stringent
process of medical devices approval. The development costs are typically comprised between
$10 and $20 million dollars and take an average of 3 to 7 years until reaching the market.156
Similarly to new drugs, for a class III medical device to be approved, it is required to
demonstrate its safety and efficacy.156
In the perspective of pre-clinical and clinical trials, consequent quantities of the carbon-
coated cobalt nanoparticles, described in Chapter 4, are needed and production at the upper
gram scale is required. The current limiting step for scaling up is the polyglycidol
polymerization. Therefore, the influence of various reaction parameters was studied in order to
ease the scale up process.
In the previous chapter, the use of such magnetic nanoparticles to remove rare cells was
reported. To demonstrate that such nanoparticles can further be used to remove highly abundant
cells, the polyglycidol-functionalized magnetic nanoparticles were conjugated with Campath,
a monoclonal antibody binding to the CD52 protein present on the surface of mature
lymphocytes.157 Normal lymphocytes counts are in the range of several millions cells per
millilitre blood.158 This is at least 4 to 6 orders of magnitude more than circulating tumour cells.
Moreover, abnormal lymphocyte counts or abnormal lymphocytes behaviour are encountered
in numerous diseases. Therefore, the use of lymphocyte-removing nanoparticles could find
applications for diseases such as the ones exemplified below.
Autoimmune diseases are a broad class of conditions where the immune system
recognizes self-antigens as foreign and mounts an immune response against self-entities.
Autoimmune diseases include heavy conditions such as multiple sclerosis or rheumatoid
arthritis. There are no cure for autoimmune diseases and, up to now, treatments where mainly
aiming at improving symptoms. For instance, the burden of several autoimmune diseases could
be alleviated by administering patients with rituximab; an antibody reducing the number of B-
cells which are notably responsible of presenting antigens to T-cells.159,160 However, anti-
rituximab immune responses can decrease the effectiveness of the drug.159,161 This serious issue
could be bypassed by achieving B-cell depletion via magnetic nanoparticles-based
extracorporeal blood purification with which, due to the shorter exposition time, the likeliness
of mounting an adaptive immune response would be diminished.
84
Allergies, also called type I hypersensitivities, form a class of conditions where the
immune system mounts an inappropriate immune response when presented to an actually
inoffensive antigen. Removal of especially T-lymphocytes could weaken the symptoms
observed during chronic allergic inflammation (i.e. when the allergen exposure is continuous)
or mitigate the extent of late-phase allergy, which occurs few hours after early-phase reaction
(i.e. acute reaction).162
Lymphomas constitute a group of cancers involving lymphocytes. There are over 30
types of lymphomas and currently, to establish a diagnostic, a lymph node biopsy is required to
observe the cells under a microscope.163 By using magnetic nanoparticles to isolate
lymphocytes circulating in the blood, it would provide a mean to obtain a liquid biopsy that
would be much less invasive and would benefit both the surgeon and the patient.
This chapter thus contains two parts. First, in view of the future need to scale up the
nanoparticles’ production process, the influence of various reaction variables for the
polymerization step are reported as well as its reproducibility. Also, this chapter constitutes an
illustration of the possibility to use polyglycidol-functionalized carbon-coated cobalt
nanoparticles to remove the highly abundant B- and T-lymphocytes from the blood of healthy
and cancer patients.
Figure 0-1. Schematic overview of the extracorporeal lymphocytes’ removal and examples of diseases
for which polyglycidol-functionalized C/Co nanoparticles could find applications.
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5.2 Materials and Methods
5.2.1 Synthesis of C/Co-PhEtOH
To a 500 mL Schott® flask containing 200 mL of distilled water, 10 g carbon-coated cobalt
nanoparticles (C/Co) were added. Nanoparticles were dispersed by placing the Schott® flask
for 10 min in an ultrasonication bath (Sonorex Digitec DT 103 H, Bandelin). Concomitantly,
in 20 mL of distilled water, 2-(4-aminophenyl)ethanol (1.5 g, 10.95 mmol) was added. A
hydrochloric acid solution (5 mL, HCl conc. / 37% fuming) was used to dissolve 4-aminophenyl
alcohol before addition to the nanoparticles solution. A sodium nitrite (NaNO2, 1.5 g, 21.8
mmol) was prepared in 5 mL of distilled water. Before the dropwise addition of the sodium
nitrite solution, the nanoparticles solution was cooled down with an ice bath. The nanoparticles
solution was left reacting for 2 h by placing back the Schott® flask in the ultrasonication bath.
Once the reaction over, the nanoparticles were washed several times by magnetic decantation.
The general washing procedure was: nanoparticles dispersion by placing the container for 3
min in the ultrasonication bath and subsequent application of a magnetic field with a permanent
magnet. After discarding the supernatant, fresh solvent was added and the washing procedure
repeated. The following solvents were used: distilled water (3 x 100 mL), EtOH (3 x 100 mL)
and acetone (3 x 100 mL). The particles were then dried overnight in a vacuum oven at 50°C.
5.2.2 Synthesis of C/Co-PhEtO-Na+
To a 50 mL two-neck round-bottom flask, 10 g of C/Co-PhEtOH nanoparticles and a magnetic
stirrer were added. A reflux condenser was installed and the installation was placed under inert
atmosphere. A commercial sodium methoxide solution (20 mL, 2 M in dry methanol, Fluka)
was added. Nanoparticles were dispersed by placing for 30 s an ultrasonic horn (UP50H,
Hielscher) in the over-pressurized installation. The solution was left reacting by stirring
overnight at 65°C. The general washing procedure was: nanoparticles dispersion by placing the
ultrasonic horn for 30 s in the solution and subsequent application of a magnetic field with a
permanent magnet. After discarding the supernatant with a syringe, fresh solvent was added
and the washing procedure repeated. The following solvents were used: dry methanol (8 x 100
mL). Five needles were placed in the septum be the contained was placed overnight in a vacuum
oven at 50°C to dry the nanoparticles.
86
5.2.3 Example of a Standard Synthesis of C/Co@polyglycidin
To a 100 mL three-neck round-bottom flask, 500 mg C/Co-PhEtO-Na+ were added to 20 mL of
dry toluene. A reflux condenser and a high shear mixer (1050 FME-P Geradschleifer, Kress)
were installed while the last opening was closed with a septum for injection. The particles were
dispersed by placing the high shear mixer on maximal speed (25000 rpm). The reaction mixture
was degassed by bubbling nitrogen through for 30 minutes and temperature was then increased
to 140°C under inert conditions. Once the reaction mixture reached 140°C, the shear speed was
reduced to 16500 rpm and 10 mL (+/-)-glycidol (+/- -Oxiran-2-ylmethanol) was slowly added
with a syringe pump (1.3 mL/h, Model 100, KD Scientific). From the time of injection, the
reaction mixture was stirred for 16 h before being cooled down to room temperature. The
nanoparticles were washed 7 times with toluene distilled water (100 mL). At every step, the
particles were dispersed in an ultrasonic bath (Sonorex Digitec DT 103 H, Bandelin) for 3 min
and separated by application of a permanent magnet (magnetic decantation). After washing, the
particles were dried overnight in a vacuum oven at 50°C.
5.2.4 Synthesis of C/Co@polyglycidyl-COOH
300 mg C/Co@polyglycidin were added to 15 mL of dry dimethylformamide. After being
dispersed with an ultrasonic bath (3 min, Sonorex Digitec DT 103 H, Bandelin), succinic
anhydride (150 mg, 1.3 mmol) was added. The solution was placed for 10 min in an
ultrasonication bath before N,N-Dimethylpyridin-4-amine (180 mg, 1.5 mmol) and
triethylamine (1.5 mL, 10.8 mmol) were added. The reaction mixture was degassed by bubbling
nitrogen through for 30 minutes. The reaction mixture was heated up to 70°C and stirred
overnight under inert conditions. The nanoparticles were thoroughly washed with distilled
water (3 x 100 mL). At every step, the particles were dispersed in an ultrasonication bath for 3
min and separated by application of a permanent magnet (magnetic decantation).
5.2.5 Synthesis of C/Co@polyglycidyl-COO-Campath and C/Co@polyglycidin-COO-
IgG
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide
(sulfo-NHS) were left at room temperature until equilibration. Each compound was dissolved
in activation buffer (OceanNanotech) at concentrations of 4 mg/mL and 2 mg/mL, respectively.
The solutions were homogenized by vortexing for 10 s. C/Co@polyglycidyl-COOH
nanoparticles solutions (5 mg/mL in activation buffer) were prepared. After being dispersed
with an ultrasonic bath (2 min, Sonorex Digitec DT 103 H, Bandelin), 200 µL the solution were
87
added to 1.5 mL Eppendorf tubes containing 100 µL of activation buffer. The EDC and sulfo-
NHS solutions were mixed in a 1:1 volume ratio and 10 µL were added to each nanoparticles-
containing Eppendorf tube. To ensure homogeneous dispersion, the solutions were vortexed for
10 seconds followed by 20 seconds in an ultrasonication bath (Sonorex Digitec DT 103 H,
Bandelin). The activation step was done by shaking the Eppendorf tubes at 1200 rpm for 10
min at 25°C in a ThermoMixer (ThermoMixer Comfort, Eppendorf). 100 µL, 200 µL or 300
µL of an antibody solution (Campath or IgG isotype control; 30 mg/mL and 1 mg/mL,
respectively) was added to the Eppendorf tubes. To ensure homogeneous dispersion, the
solutions were vortexed for 10 seconds followed by 20 seconds in an ultrasonication bath
(Sonorex Digitec DT 103 H, Bandelin). The Eppendorf tubes were then shaken at 1200 rpm for
4 h at 25°C in a ThermoMixer (ThermoMixer Comfort, Eppendorf). To quench the reaction, 10
µL of quenching buffer (OceanNanotech) were added to each Eppendorf tube. To ensure
homogeneous dispersion, the solutions were vortexed for 10 seconds followed by 20 seconds
in an ultrasonication bath (Sonorex Digitec DT 103 H, Bandelin). The quenching reaction was
done by shaking the Eppendorf tubes at 1200 rpm for 30 min at 25°C in a ThermoMixer
(ThermoMixer Comfort, Eppendorf). The particles were washed by placing the reaction vessel
in a pre-cooled (4°C) SuperMag separator (OceanNanotech).To ensure complete separation,
the magnet was placed in a fridge for 1.5 h before the supernatant was discarded and replaced
with 420 µL of fresh cold PBS (pH 7.4, Life Technologies). The nanoparticles were dispersed
by vortexing for 10 seconds followed by 20 seconds in an ultrasonication bath (Sonorex Digitec
DT 103 H, Bandelin). The Eppendorf tubes were placed back in the fridge and the washing
procedure was repeated 3 times. Once the washing process completed, the solutions were
aliquoted (30 µL aliquots) and stored at -20°C until use.
5.2.6 Removal of Lymphocytes from Healthy Subjects
After informed patient consent (ethics approval: KEK-ZH-Nr. 2012-0274), up to 20 mL blood
samples were drawn from healthy subjects into heparin tubes (BD Biosciences). Eppendorf
tubes containing either a Campath nanoparticles solution (2.38 mg/mL in PBS) or an IgG-
isotype control nanoparticles solution (2.38 mg/mL in PBS) were sonicated (5x1 min with 1
min breaks in between) in an ultrasonication bath (Sonorex Digital 10P, Bandelin) on ice-cold
water. Once sonicated, the 25 L of the nanoparticles were added to 250 L of blood. In
addition to the IgG-isotype control, a control was made with 25 L PBS. The samples were
incubated for 2 min on an orbital shaker (16 mot/min, WS 10, Edmund Bühler) before being
passed through a column magnet system (MS columns, MACS, Miltenyi Biotec). Each column
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was washed twice with 500 µL and 750 µL of PBS. Lymphocytes were labelled by transferring
a third of the filtrates to Eppendorf tubes and incubating with 1 L of Human CD19 APC-
conjugated (SJ25-C1, ThermoFisher; final concentration: 0.2 mg/mL) and 1 L of Human
CD3d Alexa Fluor 48-conjugated (AB, ThermoFisher; final concentration: 0.2 mg/mL) for 30
min at 4°C. Samples were then prepared for flow cytometry analysis. Red blood cells were
lysed by adding 10 mL of a red blood cells lysis buffer (Biolegend, USA). The samples were
centrifuged for 10 min at 400 g (centrifuge 5810R, Eppendorf). The supernatant was discarded
and the pellet resuspended with 200 µL of PBS. The cells were fixed with 200 µL of a 4%
formalin solution. 25 µL of counting beads solution (concentration of 5.2x105 counting beads
per 50 µL, CountBright Absolute, Life Technologies) were added to each sample before
analysing them using a flow cytometer (BD Canto II, BD Biociences). Acquisition was carried
out with the BD FACSDiva Software (BD Biosciences). Forward and side scatter area as well
as signal height were recorded. Lymphocytes were detected using either side scatter area or
forward scatter area vs either APC-A or Alexa Fluor 488-A. FACS data were processed using
FlowJo V10.0.8.
5.2.7 Removal of Lymphocytes from Cancer Patients
After informed patient consent (ethics approval: KEK-ZH-Nr. 2016-01140), up to 25 mL blood
samples were drawn from cancer patients into heparin tubes (BD Biosciences). Eppendorf tubes
containing either a Campath nanoparticles solution (2.38 mg/mL in PBS) or an IgG-isotype
control nanoparticles solution (2.38 mg/mL in PBS) were sonicated (5x1 min with 1 min breaks
in between) in an ultrasonication bath (Sonorex Digital 10P, Bandelin) on ice-cold water. Once
sonicated, the 25 L of the nanoparticles were added to 250 L of blood. In addition to the IgG-
isotype control, a control was made with 25 L PBS. The samples were incubated for 2 min on
an orbital shaker (16 mot/min, WS 10, Edmund Bühler) before being passed through a column
magnet system (MS columns, MACS, Miltenyi Biotec). Each column was washed twice with
500 µL and 750 µL of PBS. Lymphocytes were labelled by transferring a third of the filtrates
to Eppendorf tubes and incubating with 1 L of Human CD19 APC-conjugated (SJ25-C1,
ThermoFisher; final concentration: 0.2 mg/mL) and 1 L of Human CD3d Alexa Fluor 48-
conjugated (AB, ThermoFisher; final concentration: 0.2 mg/mL) for 30 min at 4°C. Samples
were then prepared for flow cytometry analysis. Red blood cells were lysed by adding 10 mL
of a red blood cells lysis buffer (Biolegend, USA). The samples were centrifuged for 10 min at
400 g (centrifuge 5810R, Eppendorf). The supernatant was discarded and the pellet resuspended
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with 200 µL of PBS. The cells were fixed with 200 µL of a 4% formalin solution. 25 µL of
counting beads solution (concentration of 5.2x105 counting beads per 50 µL, CountBright
Absolute, Life Technologies) were added to each sample before analysing them using a flow
cytometer (BD Canto II, BD Biociences). Acquisition was carried out with the BD FACSDiva
Software (BD Biosciences). Forward and side scatter area as well as signal height were
recorded. Lymphocytes were detected using either side scatter area or forward scatter area vs
either APC-A or Alexa Fluor 488-A. FACS data were processed using FlowJo V10.0.8.
5.3 Results and Discussion
5.3.1 Influence of Various Reaction Parameters on Polymer Length
It has been reported that one of the limiting factors to achieve successful clinical translation of
nanomedicines, was the limited reproducibility and/or scalability.53 Empirically, it has been
found that, for such polyglycidol-functionalized carbon-coated cobalt nanoparticles, the
polymerization is the most delicate step. Therefore, the influence of three key reaction
parameters of the polymerization reaction have been tested by reproducing the procedure and
changing one variable at a time in ranges near the values obtained for the batch displaying
optimal properties in Chapter 4. The first parameter to be changed was the speed of mixing. To
obtain results that are more easily transferrable to industrial processes, the magnetic stirrer used
in chapter 4 was replaced by a high shear mixer. This provides a more controlled mixing as
compared to what is achieved with a traditional chemistry setup (i.e. magnetic stirrer combined
with a magnetic plate) but it also facilitates the scale-up process. It was found that increasing
the shear speed only resulted in minor increase of the number of polymer units introduced
(Figure 5-2A). Second, the excess of glycidol value was modified while keeping the rate of
addition constant. As can be seen on Figure 5-2B, going to larger volumes did not result in
much change with respect to the number of polymer units. Lastly, the reaction temperature was
modified. Temperature appeared to be a key factor in controlling the extent of polymer
functionalization (Figure 5-2C). Moreover, for the values reported here, a linear correlation was
found between the reaction temperature and the number of polymer units. This could therefore
provide an easy mean to control the amount of polyglycidol introduced by simply altering the
temperature.
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5.3.2 Reproducibility of Polymerization Step
Materials reproducibility is one of the key requirements from the U.S. FDA. Polyglycidol
functionalization was thus repeated three times with identical reaction conditions and the
batches characterized by elemental analysis. As can be seen on Figure 5-3A, the carbon content
was comprised within a narrow range for all three batches thus indicating the introduction of
similar amounts of polymer as calculated (Appendix A4 and Figure 5-3D). On Figure 5-3C, a
decrease in nitrogen content is observed for the third batch. However, since the nitrogen content
is very low, the differences measured are not significant and are more likely to be due to a
residual error of the instrument or the introduction of air in the sample. Moreover, the
polymerization step does not introduce any nitrogen and the starting material was the same for
all three batches. Of note, one of the limitations from elemental analysis is that it does not
provide any information about the structure of the polymer layer introduced which, in the case
of polyglycidol, can differ greatly (i.e. ratio of linear vs branched polymer). However, in their
Figure 0-2. Influence of various reaction parameters on the number of polymer units introduced. A)
Influence of the speed of mixing. B) Influence of the glycidol volume excess. C) Influence of temperature.
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draft report of the guidance for industry, the U.S. FDA does not require the description of the
polymer structure but rather the “coating properties”.164
5.3.3 B- and T-Lymphocytes Removal from Healthy Subject Blood Samples
As a demonstration of the possibility of using polyglycidol-functionalized carbon-coated cobalt
nanoparticles for lymphocytes removal, the nanoparticles were functionalized with Campath
(also called Alemtuzumab and Lemtrada). This antibody is used in the clinic for the treatment
of multiple sclerosis.165 Three batches were synthesized with varying excesses of antibody
solutions volumes (100 µL, 200 µL and 300 µL, respectively) to determine whether this would
influence the conjugation and, ultimately, the removal efficiency. To test the latter, blood from
a healthy subject was sampled and magnetic nanoparticles were added before passing the
resulting samples through a magnetic column system. The B- and T-lymphocytes counts were
then assessed by flow cytometry. Additionally, the lymphocytes removal experiments were
repeated at different time points, always with blood from the same subject, in order to get
Figure 0-3. Elemental analysis of three batches produced with the same reaction conditions. A) Carbon
content B) Hydrogen content C) Nitrogen content D) Carboxyl functionalities content calculated from
the carbon content (see Appendix A4 for a description of such calculations).
92
insights about the storability of such nanoparticles. As can be seen on Figure 5-4, all batches
reduced the number of both B- and T-lymphocytes although Batch 3 turned out to be less
efficient than the two first ones having a small excess. Moreover, the nanoparticles retained
their efficiency even after at least 2 months of storage at 5°C. This is an important feature in
the view of an eventual clinical translation. However, after 6 months of storage, the efficiency
was drastically reduced. The variability between the results obtained after 1 and 2 months of
storage could presumably be due to the fact that the CD52 expression might slightly differ at
various time points even for a single subject. Of note, the removal efficiency was considerable
for both B- and T-lymphocytes although there is an order of magnitude difference between the
two types of cells.
5.3.4 B- and T-Lymphocytes Removal from Cancer Patient Blood Samples
To explore the robustness of the lymphocytes removal procedure, blood samples from cancer
patients were used to repeat the experiments done with healthy subjects samples. Although this
is not therapeutically relevant for the cancers involved to remove a large fraction of the
lymphocytes, it provides insights on the efficiency of the nanoparticles for widely differing
lymphocytes levels and in a disease setting. Indeed, cancer patients exhibit lymphocytes counts
that can be several times lower than healthy subjects. Batch 2 from the experiments with healthy
subject blood was used and yielded efficient lymphocytes removal from the blood samples of
cancer patients (Figure 5-5). However, if the results are encouraging and show that the
Figure 0-4. Removal of B- and T-lymphocytes after treatment of blood samples from healthy subjects.
Campath-functionalized C/Co nanoparticles were added to samples which were passed through a
magnetic column system before measuring lymphocytes counts by flow cytometry. Batches with slight
synthesis changes were produced and stored at 5°C.
93
nanoparticles are efficient in several contexts, the study would require to be extended to more
relevant types of cancers such as lymphomas. Indeed, the isolation of lymphocytes in the case
of lymphoma could provide a mean of obtaining a liquid biopsy and this could be an alternative
to lymph node biopsy which is the current diagnosis standard.
5.4 Conclusion
In conclusion, we provided insights into the polymerization step, a central aspect for the
successful scale up of the production of polyglycidol-functionalized carbon-coated cobalt
nanoparticles. We also reported data about the reproducibility of the synthesis. In addition, we
disclosed the successful use of Campath-conjugated nanoparticles to remove lymphocytes from
the blood of patients. In this proof-of-principle, the focus was not on specifically removing
either B- or T-lymphocytes, as would require some of the mentioned therapeutic applications,
but rather to show that such nanoparticles are not only suitable to isolate rare cells but abundant
cells as well. However, already the Campath-nanoparticles could find applications in field of
diagnosis where they could allow surgeons to obtain a liquid biopsy, necessary for the diagnosis
of lymphoma, in a less-invasive manner as what is currently done. Finally, by conjugating the
nanoparticles with antibodies that are specific to a given type of lymphocytes, therapeutically
relevant applications could be developed. For instance, by magnetically removing B-cells only,
one could potentially circumvent the resistance mechanism encountered for B-cell depleting
drugs in the treatment of autoimmune diseases.
Figure 0-5. Removal of B- and T-lymphocytes after treatment of blood samples from cancer patients.
Campath-functionalized C/Co nanoparticles were added to samples which were passed through a
magnetic column system before measuring lymphocytes counts by flow cytometry. Patients had widely
varying lymphocytes counts notably with three patients having very low B-lymphocytes counts.
94
95
Conclusion and Outlook
This thesis covered two concepts of cancer treatments using nanoparticles. First,
platinum nanoparticles were used as part of a caged prodrug system with applications for lung
cancer. The caged prodrug is a less potent form of a chemotherapeutic that is deactivated by
introducing a chemical protecting that can later be removed in situ thanks to the catalytic
properties of nanoparticles. This system could potentially lead to either a reduction in side-
effects or allow the use of higher drug concentrations and/or more frequent drug administration.
A second concept was presented where polyglycidol-functionalized magnetic nanoparticles
were conjugated with antibodies to remove cells from the blood in a targeted manner. By
removing circulating tumour cells, this could translate into a tool having the potential to reduce
the appearance of metastases, the main cause of cancer deaths. Also, such nanoparticles were
used to remove lymphocytes and could therefore find use in applications such as lymphoma
diagnosis by providing a way to obtain a liquid biopsy and, additionally, in the treatment of
autoimmune diseases thereby avoiding resistance mechanisms of current B-cell depletion
treatments.
In Chapter 2, we could show that the introduction of a protecting group, a “cage”, on
gemcitabine resulted in a caged prodrug that was in vitro much less cytotoxic as compared to
the uncaged counterpart. The system was further composed of platinum nanoparticles and
hydrogen in order to uncage the prodrug in an efficient and timely manner. It could be shown
that the prodrug could recover desired cytoxicity.
Preliminary pre-clinical data for the caged prodrug system were then presented. A
formulation for intratracheal administration was developed and administered to mice which did
not display any subsequent toxicity. Also, at least at a macroscopic level, aqueous platinum
nanoparticles solutions did not trigger toxicity in the mice models. Formulations containing
caged gemcitabine, gemcitabine or the vehicle itself were administered to mice previously
inoculated with bioluminescent A549 cells. In this experiment it was observed that caged
gemcitabine was not toxic to the mice and that it did not provide a significant advantage with
respect to tumour growth as compared to the vehicle. Gemcitabine on the other hand triggered
significant complications and mice prematurely died from non-cancer related causes.
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Polyglycidol-functionalized carbon-coated cobalt nanoparticles were used as another
nanomedicine application. First, they were functionalized with an anti-EpCAM antibody to
target circulating tumour cells present in the blood. We could establish a relationship between
the antifouling and separability properties of the nanoparticles and their efficiency to remove
CTCs. Experiments with spiked blood from healthy subjects demonstrated the high efficiency,
selectivity and reproducibility of the CTCs removal procedure. The work was extended by
showing that the nanoparticles do not generate blood coagulation and applying the procedure
to blood samples originating from cancer patients.
In Chapter 5, the polymerization step of the nanoparticles’ functionalization was
studied. This provided key insights that will be necessary for the scale up of the nanoparticles
production in prevision of pre-clinical animal studies. Furthermore, it was shown that the
synthesis is reproducible, thereby fulfilling one of the central requirements of the U.S. FDA for
regulatory approval. The chapter was augmented by demonstrating the use of such
nanoparticles for the removal of lymphocytes from the blood. An interesting perspective given
the vast field of applications ranging from lymphoma diagnosis to autoimmune treatments.
Both technologies require some further development before entering clinical studies.
For the caged prodrug system, a protocol for the in vivo uncaging reaction has to be developed
and thereafter similar mice experiments have to be repeated to determine the feasibility of such
a system to reduce the side-effects of lung chemotherapy and/or improve its efficacy by
increasing the dosage and frequency of chemotherapy administrations. The technology could
be extended to other free amino-containing chemotherapeutics and eventually other triggering
systems could be developed. Regarding the magnetic nanoparticles for specific cell isolation
from blood, the next step will be the scale up of the material production in order to conduct
animal safety tests. There are also some new development ideas such as evolving antibodies
that are specific to the antigen receptors that recognize self-antigens on the surface of immune
cells. This would allow one to selectively remove the pathogenic cells while not reducing the
immune protection of the patients. Additionally, nanoparticles could be developed for which
the captured cells could be released upon the action of a biorthogonal trigger. This could find
applications and facilitating a new trend in research consisting in training immune cells for a
specific target in vitro before reintroducing them in the patient’s circulatory system.
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Appendix
98
A.1 Supplementary Information Chapter 2
Additional Materials and Methods
Screening of Hydrogen Concentrations
Experiment with 3.2% hydrogen
In a 96-well plate, wells were filled with 90 L of fresh F-12K medium containing caged 7-
amino-4-methylcoumarin (110 M) and DMSO (2% v/v). Positive controls wells were filled
with 90 L of fresh F-12K medium containing caged 7-amino-4-methylcoumarin (110 µM) and
DMSO (2% v/v). To treated wells, 10 L of a platinum nanoparticles solution (1000 ppm, 3
nm diameter, Sigma-Aldrich) was added and 10 L of Milli-Q water was added to control wells.
The plate was placed in a custom-designed gas chamber (see Scheme A1-1). The chamber was
placed in the incubator, connected to a gas bottle outside the incubator and to an outlet. The gas
chamber was flushed for 0.5 min with hydrogen gas at 0.07 bar and closed. The gas mixture is
further composed of 5% of CO2 and 91.8% of nitrogen. The plate was incubated at 37°C for 0,
0.25, 1, 2, 3 hours in the gas chamber. After each time point, the plate was taken out of the gas
chamber and fluorescence emission was measured using a microplate reader (Tecan, Infinite
200 PRO; ex: 340 nm; em: 460 nm). Values were normalized by the mean value of the positive
controls wells.
Experiment with 20% hydrogen
In a 96-well plate, wells were filled with 90 L of fresh F-12K medium containing caged 7-
amino-4-methylcoumarin (110 M) and DMSO (2% v/v). Positive controls wells were filled
with 90 L of fresh F-12K medium containing caged 7-amino-4-methylcoumarin (110 µM) and
DMSO (2% v/v). To treated wells, 10 L of a platinum nanoparticles solution (1000 ppm, 3
nm diameter, Sigma-Aldrich) was added and 10 L of Milli-Q water was added to control wells.
The plate was placed in a custom-designed gas chamber (see Scheme A1-1). The chamber was
placed in the incubator, connected to a gas bottle outside the incubator and to an outlet. The gas
chamber was flushed for 0.5 min with hydrogen gas at 0.07 bar and closed. The gas mixture is
further composed of 5% of CO2 and 75% of nitrogen. The plate was incubated at 37°C for 0, 5,
20, 35, 50, 65, 100, 130, 160, 190 min in the gas chamber. After each time point, the plate was
taken out of the gas chamber and fluorescence emission was measured using a microplate reader
(Tecan, Infinite 200 PRO; ex: 340 nm; em: 460 nm). Values were normalized by the mean value
of the positive controls wells.
99
Experiment with 100% hydrogen
In a 96-well plate, wells were filled with 90 L of fresh F-12K medium containing caged 7-
amino-4-methylcoumarin (110 M) and DMSO (2% v/v). Positive controls wells were filled
with 90 L of fresh F-12K medium containing caged 7-amino-4-methylcoumarin (110 µM) and
DMSO (2% v/v). To treated wells, 10 L of a platinum nanoparticles solution (1000 ppm, 3
nm diameter, Sigma-Aldrich) was added and 10 L of Milli-Q water was added to control wells.
The plate was placed in a custom-designed gas chamber (see Scheme A1-1). The chamber was
placed in the incubator, connected to a gas bottle outside the incubator and to an outlet. The gas
chamber was flushed for 0.5 min with hydrogen gas at 0.07 bar and closed. The plate was
incubated at 37°C for 0, 5, 10, 15, 20, 25, 30, 150 min in the gas chamber. After each time
point, the plate was taken out of the gas chamber and fluorescence emission was measured
using a microplate reader (Tecan, Infinite 200 PRO; ex: 340 nm; em: 460 nm). Values were
normalized by the mean value of the positive controls wells.
Screening of Oxygen Concentrations
Experiment with 0% oxygen
In a 96-well plate, wells were filled with 90 L of fresh F-12K medium containing caged 7-
amino-4-methylcoumarin (110 M) and DMSO (2% v/v). Positive controls wells were filled
with 90 L of fresh F-12K medium containing caged 7-amino-4-methylcoumarin (110 µM) and
DMSO (2% v/v). To treated wells, 10 L of a platinum nanoparticles solution (1000 ppm, 3
nm diameter, Sigma-Aldrich) was added and 10 L of Milli-Q water was added to control wells.
The plate was placed in a custom-designed gas chamber (see Scheme A1-1). The chamber was
placed in the incubator, connected to a gas bottle outside the incubator and to an outlet. The gas
chamber was flushed for 0.5 min with the gas mixture at 0.07 bar and closed. The gas mixture
is further composed of 90% of hydrogen and 10% of nitrogen. The plate was incubated at 37°C
for 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 90 minutes in the gas chamber. After each
time point, the plate was taken out of the gas chamber and fluorescence emission was measured
using a microplate reader (Tecan, Infinite 200 PRO; ex: 340 nm; em: 460 nm). Values were
normalized by the mean value of the positive controls wells.
Experiment with 1% oxygen
100
In a 96-well plate, wells were filled with 90 L of fresh F-12K medium containing caged 7-
amino-4-methylcoumarin (110 M) and DMSO (2% v/v). Positive controls wells were filled
with 90 L of fresh F-12K medium containing caged 7-amino-4-methylcoumarin (110 µM) and
DMSO (2% v/v). To treated wells, 10 L of a platinum nanoparticles solution (1000 ppm, 3
nm diameter, Sigma-Aldrich) was added and 10 L of Milli-Q water was added to control wells.
The plate was placed in a custom-designed gas chamber (see Scheme A1-1). The chamber was
placed in the incubator, connected to a gas bottle outside the incubator and to an outlet. The gas
chamber was flushed for 0.5 min with the gas mixture at 0.07 bar and closed. The gas mixture
is further composed of 90% of hydrogen and 9% of nitrogen. The plate was incubated at 37°C
for 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 90 minutes in the gas chamber. After each
time point, the plate was taken out of the gas chamber and fluorescence emission was measured
using a microplate reader (Tecan, Infinite 200 PRO; ex: 340 nm; em: 460 nm). Values were
normalized by the mean value of the positive controls wells.
Experiment with 2.1% oxygen
In a 96-well plate, wells were filled with 90 L of fresh F-12K medium containing caged 7-
amino-4-methylcoumarin (110 M) and DMSO (2% v/v). Positive controls wells were filled
with 90 L of fresh F-12K medium containing caged 7-amino-4-methylcoumarin (110 µM) and
DMSO (2% v/v). To treated wells, 10 L of a platinum nanoparticles solution (1000 ppm, 3
nm diameter, Sigma-Aldrich) was added and 10 L of Milli-Q water was added to control wells.
The plate was placed in a custom-designed gas chamber (see Scheme A1-1). The chamber was
placed in the incubator, connected to a gas bottle outside the incubator and to an outlet. The gas
chamber was flushed for 0.5 min with the gas mixture at 0.07 bar and closed. The gas mixture
is further composed of 90% of hydrogen and 7.9% of nitrogen. The plate was incubated at 37°C
for 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 90 minutes in the gas chamber. After each
time point, the plate was taken out of the gas chamber and fluorescence emission was measured
using a microplate reader (Tecan, Infinite 200 PRO; ex: 340 nm; em: 460 nm). Values were
normalized by the mean value of the positive controls wells.
Toxicity Study of Platinum Nanoparticles and Hydrogen
A549 cells were seeded in 96-well plates at a concentration of 4,000 cells/well and incubated
overnight at 37°C before treatment. The supernatant of each well was then replaced with 100
L of fresh F-12K medium and DMSO (2% v/v). All cell culture media were supplemented
101
with 10% Foetal Bovine Serum. Plates were incubated at 37°C for 1 h before 10 L of a
platinum nanoparticles solution (1, 5 or 10 ppm; 3 nm diameter; Sigma-Aldrich) was added to
each well and 10 L of Milli-Q water was added to control wells. Six biological replicates for
each condition were included in the plates. Plates were incubated at 37°C for 1 h before placing
them in a custom-designed gas chamber (see Scheme A1-1). A control plate was left untouched
in the incubator. The chamber was placed in the incubator, connected to a gas bottle outside the
incubator and to an outlet. The gas chamber was flushed for 1 min with hydrogen gas at 0.07
bar and closed. Plates were incubated at 37°C for 2 h in the gas chamber before placing them
back in the incubator with the control plate. Control and treated plates were incubated at 37°C
for 72 h before PrestoBlue cell viability reagent (10% v/v) was added to each well and the plate
incubated at 37°C for 2 h. Fluorescence emission was measured using a microplate reader
(Tecan, Infinite 200 PRO; ex: 540 nm; em: 590 nm). Values were normalized by the mean value
of the positive controls wells.
Kinetics of Deprotection of Caged Gemcitabine
In a 96-well plate, wells were filled with 100 L of fresh F-12K medium containing caged
gemcitabine (0.1 M) and DMSO (2% v/v). All cell culture media were supplemented with
10% Foetal Bovine Serum. To treated wells, 10 L of a platinum concentration (200 ppm, 3
nm diameter, Sigma-Aldrich) was added and 10 L of Milli-Q water was added to control wells.
The plate was placed in a custom-designed gas chamber (see Scheme A1-1). The chamber was
placed in the incubator, connected to a gas bottle outside the incubator and to an outlet. The gas
chamber was flushed for 1 min with hydrogen gas at 0.07 bar and closed. The plate was
incubated at 37°C for 0, 15, 30, 120 min in the gas chamber before placing them back in the
incubator with the control plates. After each iteration, the plate was taken out of the gas chamber
and the content of 6 wells were pooled together in an Eppendorf tube. The plate was placed
back in the gas chamber and back in the incubator, flushed for 1 min with hydrogen gas at 0.07
bar and closed until the next sampling. The Eppendorf tubes collected were centrifuged at
21130 g for 1 h and the supernatant was transferred to MS sample vials before measurement
with an HPLC-MS (HPLC: Agilent 1260 Infinity, MS: Agilent 6130).
Re-use of Platinum Nanoparticles after Recycling
In a 24-well plate, wells were filled with 1.5 mL of fresh F-12K medium containing caged 7-
amino-4-methylcoumarin (100 µM) and DMSO (2% v/v). Positive controls wells were filled
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with 1.5 mL of fresh F-12K medium containing caged 7-amino-4-methylcoumarin (100 µM)
and DMSO (2% v/v). All cell culture media were supplemented with 10% Foetal Bovine Serum.
To treated wells, 150 µL of a platinum nanoparticles solution (1000 ppm, 3 nm diameter,
Sigma-Aldrich) was added and 150 µL of Milli-Q water was added to negative control wells.
The plate was placed in a custom-designed gas chamber (see Scheme A1-1). The chamber was
placed in the incubator, connected to a gas bottle outside the incubator and to an outlet. The gas
chamber was flushed for 0.5 min with hydrogen gas at 0.07 bar and closed. The plate was
incubated at 37°C for 40 min in the gas chamber. Fluorescence emission was measured using a
microplate reader (Tecan, Spark 10M; ex: 340 nm; em: 460 nm). Values were normalized by
the mean value of the positive controls wells. The wells content was transferred to 1.5 mL
Eppendorf tubes and were centrifuged for 1.5h at 22130g (Eppendorf, Centrifuge 5424 R). The
supernatant was discarded and replaced by 1.5 mL of fresh solution. The nanoparticles were
redispersed with an ultrasonic horn (Hielsher, UP50H; 30 kHz). The content of the Eppendorf
tubes was transferred to the 24-well plate before it was placed back in the incubator and another
cycle was done. Ten such cycles were made.
Extracellular Prodrug Activation in Normal Cells
The surface of 96-well plates was modified by adding to each well 50 L of a 0.02 mg/mL
poly-L-lysine solution. The plates were incubated overnight at 37°C and were washed twice
with 60 L of Milli-Q water. Human Pulmonary Alveolar Epithelial (HPAEpiC) cells were
seeded in 96-well plates at a concentration of 4,000 cells/well and incubated overnight at 37°C
before treatment. The supernatant of each well was then replaced with 100 L of fresh
HPAEpiC medium containing protected gemcitabine or gemcitabine (0.05 M) and DMSO
(2% v/v), control wells were replaced with 100 L of fresh HPAEpiC medium and DMSO (2%
v/v). All cell culture media were supplemented with 10% Foetal Bovine Serum. Plates were
incubated at 37°C for 1 h before 10 L of a platinum nanoparticles solution (100 ppm, 3 nm
diameter, Sigma-Aldrich) was added to each well and 10 L of Milli-Q water was added to
control wells. Five biological replicates for each condition were included in the plates. Plates
were incubated at 37°C for 1 h before placing them in a custom-designed gas chamber (see
Scheme A1-1). Control plates were left untouched in the incubator. The chamber was placed in
the incubator, connected to a gas bottle outside the incubator and to an outlet. The gas chamber
was flushed for 1 min with hydrogen gas at 0.07 bar and closed. Plates were incubated at 37°C
for 2 h in the gas chamber before placing them back in the incubator with the control plates.
103
Control and treated plates were incubated at 37°C for 48 h before PrestoBlue cell viability
reagent (10% v/v) was added to each well and the plate incubated at 37°C for 2 h. Fluorescence
emission was measured using a microplate reader (Tecan, Spark 10M; ex: 560 nm; em: 590
nm). Values were normalized by the mean value of the positive controls wells.
104
Scheme A1-1. Plan of the custom-designed box. The box is made out of aluminium. It is sealed thanks to a toric
joint and screws that maintain the cover tight. One can place a typical well plate inside.
105
Additional Results and Discussion
Screening of Catalyst Concentrations
To determine the catalyst concentration required for the uncaging reaction to take place, a
screening of several platinum concentrations was done (Figure A1-1). Above a concentration
of 25 ppm, the uncaging is particularly fast with the reaction reaching completion within about
20 minutes. At a concentration of 12.5 ppm, the maximum is reached after two hours.
Additionally, such a reaction rate is obtained without stirring, nor bubbling and is thus
particularly fast for a reaction that is only diffusion-controlled. In order to reduce the risk of
platinum-induced toxicity, the concentration for cell experiments was reduced to 10 ppm. It is
noteworthy that hydrogen without platinum (0 ppm Pt curve on Figure A1-1) does not lead to
any conversion. This feature translates into a background that is particularly low.
Figure A1-1. Kinetics of deprotection of prodrug surrogate 2a for various catalyst concentrations. A
solution of 99 µM of caged coumarin was prepared in Ham’s F-12K (Kaighn’s) Medium (F-12K). Pt
nanoparticles concentrations of 0, 12.5, 25, and 50 ppm and pure hydrogen were used. Fluorescence
was measure with a plate reader (n = 5; 𝜆ex/em = 340/460 nm).
106
Screening of Hydrogen Concentrations
To study the influence of the hydrogen concentration present in the gas mixture, a screening of
3 hydrogen concentrations was done (3.2%, 20% and 100%, Figure A1-2). The kinetics is
extensively affected by the hydrogen level and the reaction half-life is shifted from about 10
minutes for 100% hydrogen to about an hour for 20% hydrogen while for 3.2% hydrogen,
completion was not reached within the timeframe of the experiment. Among the three options,
we opted for the fast kinetics and carried out an interval kinetics experiment (Figure 2-4) that
mimics alternating breathing regimen in order to cope with the absence of oxygen in the gas
mixture.
Figure A1-2. Kinetics of deprotection of prodrug surrogate 2a for various hydrogen concentrations. A
solution of 99 µM of caged coumarin was prepared in Ham’s F-12K (Kaighn’s) Medium (F-12K)
supplemented with 2% DMSO. Hydrogen concentrations of 3.2, 20 and 100% and a Pt nanoparticles
concentration of 100 ppm were used. Fluorescence was measured with a plate reader (n = 5;
𝜆ex/em = 340/460 nm).
107
Kinetic of the Uncaging of Caged Gemcitabine
The kinetics of the uncaging reaction was done with caged gemcitabine (Figure A1-3). The
nanoparticles were removed by ultracentrifugation before measuring the supernatant via HPLC-
MS. The reaction turned out to be as fast as with the caged fluorophore. Namely, a half-life of
about 10 minutes was found for the uncaging reaction of caged gemcitabine.
Figure A1-3. Kinetics of deprotection of caged gemcitabine. A solution of 0.1 µM of caged coumarin
was prepared in Ham’s F-12K (Kaighn’s) Medium (F-12K) supplemented with 2% DMSO and 10%
FBS. Pt nanoparticles solutions with concentrations of 20 ppm and pure hydrogen were used. The
reaction was followed by HPLC-MS.
108
Screening of Oxygen Concentrations
To determine the influence of oxygen on the uncaging reaction to take place, a screening of
oxygen concentrations was done (Figure A1-4). A reduction in kinetics can be observed when
going from 0 to 1% of oxygen. When increasing the oxygen fraction to 2.1%, the extent of
reaction is reduced. As mentioned in the manuscript, it has been reported that the median
percentage of oxygen in lung tumours were 1.9 and 2.2% of oxygen while normal tissues had
an average of 5.6%. The exact values cannot be reproduced as such due to gas supplier’s internal
regulations implying that no gas mixtures can be manufactured with oxygen concentration
higher than 2.1% if the hydrogen concentration is above 5%. However, the trends are expected
to be similar with higher oxygen concentrations. Namely, the higher the oxygen concentration,
the slower and less extensive the uncaging reaction.
Figure A1-4. Kinetics of deprotection of prodrug surrogate 2a for various oxygen concentrations. A
solution of 99 µM of caged coumarin was prepared in Ham’s F-12K (Kaighn’s) Medium (F-12K)
supplemented with 2% DMSO. Oxygen concentrations of 0, 1 and 2.1% and a Pt nanoparticles
concentration of 100 ppm were used. A hydrogen concentration of 90% was used. Fluorescence was
measured with a plate reader (n = 5; 𝜆ex/em = 340/460 nm).
109
Toxicity of Platinum Nanoparticles and Hydrogen
The toxicity of hydrogen and of platinum was studied (Figure A1-5). The addition of platinum
did not hinder the growth of the cells and even seemed to slightly increase it for the two lowest
concentrations. The impact of hydrogen administration even over an extended period of time
(2h) did not seem to impair cellular growth. When both were combined, the viability remained
close to the positive control (no Pt, no H2).
Figure A1-5. Influence of platinum and of hydrogen on A549 cell viability. The F-12K cell medium was
supplemented with 2% DMSO and 10% FBS. A custom-designed gas chamber was used for incubation
with hydrogen (2h). After 72h of incubation in normal cell culture atmosphere, the viability was evaluated
using the PrestoBlue™ assay and a plate reader (n = 6; 𝜆ex/em = 560/590 nm).
110
Extracellular Prodrug Activation in Normal Cells
The extracellular prodrug activation experiment was repeated in normal cells (Figure A1-6).
Gemcitabine being an untargeted chemotherapy, the results obtained are similar to the ones
with cancer cells. As is usually the case in the field of caged prodrugs, the selectivity originates
from the spatial restriction of the catalyst. However, the system here introduced is sensitive to
the oxygen level (Figure A1-4) and partial selectivity might thus arise from the different oxygen
tensions that are found in normal and cancer tissues.
Figure A1-6. Extracellular caged prodrug activation. Human pulmonary alveolar epithelial
(HPAEpiC) cells were used. Solutions of gemcitabine (1a) and caged gemcitabine (1b) were prepared
at a concentration of 0.05 µM in the HPAEpiC cell medium. A concentration of platinum nanoparticles
of 10 ppm was used. A custom-designed gas chamber was used for incubation with hydrogen (2h). After
48h of incubation in normal cell culture atmosphere, the viability was evaluated using the PrestoBlue™
assay and a plate reader (n = 5; 𝜆ex/em = 560/590 nm).
111
Reuse of Platinum Nanoparticles after Recycling
The platinum nanoparticles were used for the uncaging of caged coumarin. The reaction was
done in F-12K medium with 100% hydrogen. After each reaction cycle, the nanoparticles were
recycled by ultracentrifugation. The activity is slowly decreasing over time (Figure A1-7).
Figure A1-7. Reuse of platinum nanoparticles for the deprotection of prodrug surrogate 2a. A solution
of 99 µM of caged coumarin was prepared in Ham’s F-12K (Kaighn’s) Medium (F-12K) supplemented
with 2% DMSO. A hydrogen concentration of 100% and a Pt nanoparticles concentration of 100 ppm
were used. Fluorescence was measured with a plate reader (n = 6; 𝜆ex/em = 340/460 nm).
112
A.2 Supplementary Information Chapter 3
Macroscopic Observation of the Lungs after Tolerability Profile Experiments
Figure A2-1. Macroscopic observations of the lung after the tolerability profile experiments. Group 1
mice were administered the vehicle. Group 2 mice were administered a 10 ppm aqueous platinum
nanoparticles solution. Group 3 mice were administered a 100 ppm aqueous platinum nanoparticles
solution.
113
A.3 Supplementary Information Chapter 4
Additional Materials and Methods
Particle Size Distribution
An aqueous solution of carbon-coated cobalt nanoparticles (28 nm diameter, NanoAmor) was
prepared at a concentration of 5 ppm in Milli-Q water. 1.5 µL of the solution was deposited on
carbon-coated copper grids. After overnight drying, the samples were measured on a Tecnai
F30 (FEI, 300 kV). The quantification was done for 428 nanoparticles using an image
processing program (ImageJ, NIH).
Elemental Analysis
Carbon, hydrogen and nitrogen content of the samples were measured by vario MICRO cube
(Elementar). The micro analyzer was calibrated with sulfanilamide. 2 mg of sample were
oxidized/pyrolized over a period of 70 seconds (standard program: 2mg70s). All samples were
double determined.
Infrared Spectroscopy
The nanoparticles were analyzed by FTIR spectroscopy (5% in KBr using a Tensor 27
Spectrometer, Bruker Optics equipped with a diffuse reflectance accessory, DiffusIR™, Pike
Technologies, 200 scans). The measurements were usually done against precursor material (5%
in KBr, as well).
Rotational Thromboelastometry Measurements
Blood from healthy subjects was supplemented with a nanoparticles solution to a final
concentration of 2.38 mg/mL, which had been previously sonicated (5x1 min with 1 min breaks
in between) in an ultrasonication bath (Sonorex Digital 10P, Bandelin) on ice-cold water. A
control group was treated adding only the carrier solution phosphate-buffered saline (PBS). The
samples were incubated for 2 min on an orbital shaker (16 mot/min, WS 10, Edmund Bühler)
before nanoparticles were removed with the help of an external magnet. The blood was then
used to measure EXTEM and INTEM on a rotational thromboelastometry device (ROTEM
sigma, Tem Innovations).
114
Additional Results and Discussion
Removal Efficiency of pSPM-functinalized Nanoparticles
In early attempts, the previous generation of pSPM particles was used. These were poorly
functioning for CTC removal application. It was empirically determined that this was due to
their low separability within our setup, which was confirmed in the magnetic separability
experiment
Particles Size Distribution
To determine the particle size distribution, a transmission electronic microscope and an image
processing software (ImageJ) were used. From 428 nanoparticles, an average diameter size of
37.2 ± 27.5 nm was determined.
Figure A3-1. Nanoparticle separability. This graph includes the data from the previous generation
pSPM nanoparticles.
115
Materials Reproducibility
To demonstrate that the overall synthesis protocol is robust, three nanoparticles batches were
produced starting from naked carbon-coated cobalt nanoparticles. All batches exhibited similar
compositions after a 4-step synthesis thereby illustrating the robustness of the procedure.
Figure A3-2. Particle size distribution. A transmission electronic microscope was used to take several
micrographs. The images were analysed using ImageJ and particle size distribution was determined
using 428 nanoparticles.
Table A3-1. Nanoparticles manufacture reproducibility. The compositions of the three manufactured batches were
determined by elemental analysis. Based o the compositions, the amount of carboxyl functionalities could be
determined.
116
Influence of Magnetic Nanoparticles Treatment on other Blood Cells
To assess the effect of magnetic nanoparticles treatment on other blood cells, filtrates from three
blood experiments using anti-EpCAM functionalised nanoparticles were subject to flow
cytometry analysis. In an overview, no major differences were observed for the different cell
types such as granulocytes, monocytes and lymphocytes.
Rotational Thromboelastometry Measurements
To assess the effect of magnetic nanoparticles treatment blood coagulation, rotational
thromboelastometry measurements were performed with blood from healthy subjects
supplemented with anti-EpCAM nanoparticles (concentration identical to the one used in CTC
removal experiments). A control was made with PBS only. Results represent one of several
experiments from different subjects, which are found within the normal range.
Figure A3-3. Flow cytometry results after treatment of blood with anti-EpCAM-functionalised nanoparticles
focusing on granulocytes, monocytes and lymphocytes.
117
EXTEM INTEM
CT CFT MCF CT CFT MCF
(s)
______________
(s)
______________
(mm)
______________
(s)
______________
(s)
______________
(mm)
______________
Control 69 95 61 177 72 60
anti-EpCAM
particles
74 116 55 207 71 76
Normal range [1] 38-79 34-159 50-72 100-240 30-110 50-72
Table A3-2. Rotational thrombolelastometry measurement in blood from a healthy subject. The
measurements were made in blood supplemented with either anti-EpCAM nanoparticles or PBS
(negative control). Clotting time (CT), clot formation time (CFT) and maximum clot firmness (MCF) for
the EXTEM channel (activation via tissue factor) and for the INTEM channel (contact activation) are
displayed. Reference: [1] Lang T, Bauters A, Braun SL, Pötzsch B, von Pape KW, Kolde HJ, Lakner M.
Multi-centre investigation on reference ranges for ROTEM thromboelastometry. Blood Coagul
Fibrinolysis. 2005 Jun;16(4):301-10.
Infrared Spectroscopy
Figure A3-4. Infrared Spectrum from anti-EpCAM nanoparticles Batch 1.
118
Figure A3-5. Infrared Spectrum from anti-EpCAM nanoparticles Batch 2.
Figure A3-6. Infrared Spectrum from anti-EpCAM nanoparticles Batch 3.
119
A.4 Supplementary Information Chapter 5
Elemental Analysis after the Various Synthesis Steps
Nanoparticles Type Variable
changed
Amount C
(%)
Amount N
(%)
Amount H
(%)
Amount S
(%)
C/Co - 4.11 0.09 0.04 0
C/Co-PhEtO-Na+ - 5.33 0.23 0.11 0
C/Co@polyglycidin Standard 9.55 1.54 0.21 0
C/Co@polyglycidin 5 mL 8.37 0.95 0.09 0
C/Co@polyglycidin 15 ml 10.04 1.29 0.09 0
C/Co@polyglycidin 90°C 6.48 0.69 0.23 0
C/Co@polyglycidin 190°C 12.5 1.77 0.26 0
C/Co@polyglycidin 7700 rpm 8.75 0.98 0.11 0
C/Co@polyglycidin 25000 rpm 10.65 1.56 0.23 0
Calculations of the Number of Polymer Units (example for standard synthesis)
Determination of the molar amount of carbon introduced per theoretical gram of particle:
(𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐶𝑎𝑓𝑡𝑒𝑟 𝑝𝑜𝑙𝑦𝑚𝑒𝑟𝑖𝑧𝑎𝑡𝑖𝑜𝑛 − 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐶𝑏𝑒𝑓𝑜𝑟𝑒 𝑝𝑜𝑙𝑦𝑚𝑒𝑟𝑖𝑧𝑎𝑡𝑖𝑜𝑛)
𝐴𝑡𝑜𝑚𝑖𝑐 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑐𝑎𝑟𝑏𝑜𝑛
=(0.0955𝑔 − 0.0518𝑔)
12 𝑔/𝑚𝑜𝑙= 3.6 𝑚𝑚𝑜𝑙 𝐶/𝑔
Determination of the amount of polymer repeating units per theoretical gram of particle:
𝑚𝑜𝑙𝑎𝑟 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐶 𝑖𝑛𝑡𝑟𝑜𝑑𝑢𝑐𝑒𝑑
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐶 𝑎𝑡𝑜𝑚𝑠 𝑐𝑜𝑛𝑡𝑎𝑖𝑛𝑒𝑑 𝑖𝑛 𝑜𝑛𝑒 𝑟𝑒𝑝𝑒𝑎𝑡𝑖𝑛𝑔 𝑢𝑛𝑖𝑡=
3.6 𝑚𝑚𝑜𝑙 𝐶/𝑔
3
= 1.2 𝑚𝑚𝑜𝑙 𝑜𝑓 𝑟𝑒𝑝𝑒𝑎𝑡𝑖𝑛𝑔 𝑢𝑛𝑖𝑡𝑠/𝑔
120
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Curriculum Vitae
Antoine Florent Herzog
Functional Materials Laboratory
Institute for Chemical and Bioengineering Private Address:
Department of Chemistry and Applied Biosciences Hofwiesenstrasse 378
ETH Zürich, HCI E103 8050 Zürich
8093 Zurich Switzerland
Switzerland
Phone: +41 44 633 37 70
Email: [email protected]
Born: September 10th, 1990 in Mulhouse, France
Citizen of France
Languages: French (native), English (fluent), German (intermediate)
137
Education
07/2015 – current PhD studies at the Department of Chemistry and Applied Biosciences,
Institute of Chemical- and Bioengineering, Functional Materials
Laboratory, ETH Zurich, Zurich, Switzerland
Advisor: Prof. Dr. Wendelin J. Stark
Title: Novel Applications of Metallic Nanoparticles for Cancer
Treatments
09/2012 – 04/2015 MSc studies in Chemistry, Department of Chemistry and Applied
Biosciences, ETH Zurich, Zurich, Switzerland.
Master thesis: In Vitro Intracellular Metabolite Reduction with an
Exogenous Platinum Nanocatalyst under Hydrogen-Containing
Atmosphere, Prof. Dr. Wendelin J. Stark, Institute for Chemical and
Bioengineering, ETH Zurich.
Research project: Feasibility Study of Living Surface Having the Ability
to Inducibly Express Emerald Green Fluorescent Protein,
Prof. Dr. Wendelin J. Stark, Institute for Chemical and Bioengineering,
ETH Zurich.
09/2009 - 07/2012 BSc studies in Chemistry, Chemistry and Chemical Engineering
Section, EPFL, Lausanne, Switzerland.
09/2011 – 06/2012 Erasmus exchange studies at Imperial College London, London, United
Kingdom.
Research project: On the Stereoselectivity of Photocycloadditions
between Benzene and Ethylene Derivatives, Prof. Dr. Michael
Bearpark, Department of Chemistry, Imperial College London.
09/2005 – 06/2009 High-school, Baccalauréat Scientifique, Section Sport-Étude (judo)
Lycée Louis Pasteur, Strasbourg, France.
Core subjects: Chemistry and Physics
Until 09/2005 Collège Pierre Pflimlin, Section Sport-Étude (judo), Brunstatt, France.
138
Refereed Journal Articles
3. A. F. Herzog, E. M. Schneider, W. J. Stark, Hydrogen as a bio-orthogonal trigger for
spatiotemporally controlled caged prodrug activation, Helvetica Chimica Acta, 2018,
10.1002/hlca.201800134
2. C. J. Hofer, R. N. Grass, E. M. Schneider, L. Hendriks, A. F. Herzog, M. Zeltner, D.
Günther, W. J. Stark, Water dispersible surface-functionalized platinum/carbon
nanorattles for size-selective catalysis, Chemical Science, 2018, 9, 362.
1. C. A. Mora, A. F. Herzog, R. A. Raso, W. J. Stark, Programmable living material
containing reporter micro-organisms permits quantitative detection of oligosaccharides,
Biomaterials, 2015, 61, 1-9.
Patents
2. A. F. Herzog, W. J. Stark, M. Zeltner, B. Beck-Schimmer, A. Zabel, Bioconjugates of
antibodies and functionalized magnetic nanoparticles, 2018, EP18159763.
1. A. F. Herzog, W. J. Stark, Combination medicament comprising a prodrug and
inhalable catalyst, 2017, EP17176199.
Honours and Awards
09/2011 – 06/2012 Erasmus Scholarship.
Conference Presentations and Proceedings
1. A. F. Herzog, E. M. Schneider, W. J. Stark, In vitro prodrug activation over platinum
nanoparticles for side effect reduction in lung cancer (poster), MRS Fall Meeting 2017,
Boston MA, USA, November 26 – December 1, 2017.
Student supervision
2. Stéphanie Nguengoue (Research Project, 09/2016 – 12/2016): Proof-of-principle of
intracellular polymer/oligomer synthesis for cytotoxicity application.
1. Riccardo Tarchini (Research Project, 02/2016 – 04/2016): Development of an
odour-adsorbing insole for shoes.
Professional experience
139
05/2013 - 11/2013 Internship, Organic Electronics Group, BASF, Basel, Switzerland.
07/2012 - 08/2012 Summer Internship, Cheminformatics Group, Novartis Institute for
Biomedical Research, Basel, Switzerland.
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