Gelatin nanoparticles as delivery system for nucleotide-based drugs [Elektronische Ressource] / vorgelegt von Klaus Zwiorek

Klaus Zwiorek

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• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 32
• Poids de l'ouvrage: 7 Mo

Extrait

Dissertation

zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München




Gelatin Nanoparticles as Delivery System
for Nucleotide-Based Drugs










vorgelegt von
Klaus Zwiorek
aus München


2006
published by Verlag Dr. Hut, Munich, www.dr.hut-verlag.de, ISBN: 3-89963-451-9 Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung
vom 29. Januar 1998 von Herrn Prof. Dr. Gerhard Winter betreut.


Ehrenwörtliche Versicherung

Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.


München, am 03. August 2006







Klaus Zwiorek




Dissertation eingereicht am 03.08.2006
1. Gutachter: Prof. Dr. Gerhard Winter
2. Gutachter: Prof. Dr. Wolfgang Frieß
Mündliche Prüfung am 24.10.2006
published by Verlag Dr. Hut, Munich, www.dr.hut-verlag.de, ISBN: 3-89963-451-9







Meinen Eltern
in Liebe und Dankbarkeit
gewidmet

published by Verlag Dr. Hut, Munich, www.dr.hut-verlag.de, ISBN: 3-89963-451-9 Table of contents
Table of contents


Preface
General introduction, aims and organization of this thesis

1. General introduction 1
2. Aims of this thesis 2
3. Organization of this thesis 2
4. References 4


Chapter I
Optimized preparation of gelatin nanoparticles

1. Introduction 5
2. Materials and Methods 12
2.1 Reagents 12
2.2 Preparation of gelatin nanoparticles by two-step desolvation 12
2.3 Particle size determination 13
2.3.1 Dynamic light scattering (DLS) 13
2.3.2 Asymmetrical flow field-flow fractionation (AF4) in combination with multi-angle
light scattering (MALS) 15
2.3.3 Scanning electron microscopy (SEM) 16
2.4 Molecular weight analysis of gelatin 16
2.4.1 Size exclusion HPLC (SE-HPLC) analysis of various gelatin samples 16
2.4.2 AF4 analysis of gelatin 17
2.5 Zetapotential ( ζ potential) determination of gelatin 17

I
published by Verlag Dr. Hut, Munich, www.dr.hut-verlag.de, ISBN: 3-89963-451-9 Table of contents
3. Results and Discussion 18
3.1 Comparison of different analytical tools for gelatin nanoparticle size
determination 18
3.2 Molecular weight characterization of gelatin 22
3.3 Optimization of the two-step desolvation technique 27
3.3.1 Temperature at the first and second desolvation step 27
3.3.2 Amount of sediment after the first desolvation step 28
3.3.3 pH value before the second desolvation step 30
3.3.4 Acetone addition-rate during the second desolvation step 32
3.3.5 Amount of the applied crosslinking reagent 33
3.3.6 Evaluation of the conditions for tailor-made nanoparticles 34
3.4 Gelatin nanoparticles by one-step desolvation 35
4. Conclusion 40
5. References 41


Chapter II
Cationic gelatin nanoparticles as non-viral gene delivery system

1. Introduction 45
2. Materials and Methods 49
2.1 Reagents 49
2.2 Preparation of cationized gelatin nanoparticles 49
2.3 Cell culture 51
2.4 Plasmid DNA (pDNA) and oligonucleotide (ODN) loading of gelatin
nanoparticles 52
2.4.1 Experiments to determine the maximum pDNA or ODN payload on surface-
modified nanoparticles 52
2.4.2 pDNA loading of cholamine-modified nanoparticles for subsequent in vitro
application 52
II
published by Verlag Dr. Hut, Munich, www.dr.hut-verlag.de, ISBN: 3-89963-451-9 Table of contents
2.5 Preparation and pDNA loading of cationic liposomes 52
2.6 Preparation of PEI polyplexes 53
2.7 Transfection 53
2.8 Luciferase assay 53
2.9 Cell viability 54
2.10 Hemolysis assay 54
3. Results and Discussion 55
3.1 Characterization of the surface-modified nanoparticles 55
3.2 Nucleotide loading efficiency of surface-modified nanoparticles 58
3.3 Optimized pDNA transfection conditions with cholamine-modified
gelatin nanoparticles 61
3.4 Cytotoxicity of cationized gelatin nanoparticles 65
4. Conclusion 68
5. References 69


Chapter III
In vitro delivery of immunogenic CpG oligonucleotides

1. Introduction 73
1.1 The immune system and the role of toll-like receptors 73
1.2 History and mechanism of action of CpG ODNs 75
1.3 Synthetic CpG ODN classes for humans and their effects on the
immune system 77
1.4 Fields of applications for synthetic CpG ODNs 79
1.5 Clinical trials with CpG ODNs 82
1.6 CpG ODN transport by delivery systems and aim of the study 82

III
published by Verlag Dr. Hut, Munich, www.dr.hut-verlag.de, ISBN: 3-89963-451-9 Table of contents
2. Materials and Methods 84
2.1 Materials 84
2.1.1 Reagents 84
2.1.2 Fluorescent dyes and monoclonal antibodies 85
2.1.3 Applied ODNs 85
2.1.4 Cell culture reagents 86
2.2 Preparation of cationized gelatin nanoparticles 86
2.3 Preparation of fluorescent gelatin nanoparticles 86
2.3.1 Covalent linkage of aminoreactive fluorescent dyes 86
2.3.2 Incorporation of dextran-linked fluorescent dyes 87
2.4 Preparation of fluorescent HSA nanoparticles 87
2.5 Preparation of fluorescent PLGA nanoparticles 87
2.6 Characterization of the nanoparticles 87
2.7 ODN loading of cationized gelatin nanoparticles 88
2.8 Experiments with murine myeloid dendritic cells (MDCs) 88
2.8.1 Culture of MDCs 88
2.8.2 FACS analysis of nanoparticle uptake by MDCs 88
2.8.3 Visualization of nanoparticle uptake by MDCs via CLSM 89
2.8.4 Quantification of cytokine secretion 89
2.8.5 Special conditions for kinetic experiments 89
2.9 Experiments with primary human blood cells 90
2.9.1 Isolation of PBMCs 90
2.9.2 Isolation of PDCs 90
2.9.3 Isolation of B cells 91
2.9.4 Cell cultures 91
2.9.5 Cytokine quantification by ELISA 91
2.9.6 CLSM experiments 91
3. Results and Discussion 93
3.1 Comparative uptake study of various biodegradable colloidal carrier
systems into murine myeloid dendritic cells (MDCs) 93
3.1.1 Nanoparticle characterization via DLS 93
3.1.2 FACS analysis of the cellular uptake 93
IV
published by Verlag Dr. Hut, Munich, www.dr.hut-verlag.de, ISBN: 3-89963-451-9 Table of contents
3.1.3 Visualization of the cellular uptake by CLSM 95
3.1.4 Discussion and Summary 95
3.2 Delivery of CpG ODN-loaded nanoparticles (CpG-GNPs) into murine
MDCs 96
3.2.1 Characterization of the nanoparticles 96
3.2.2 FACS analysis of CpG-GNP uptake by MDCs 98
3.2.3 Visualization of CpG-GNP uptake by CLSM 99
3.2.4 Phenotypic maturation of MDCs following treatment with CpG-GNPs 100
3.2.5 Cytokine secretion following treatment of MDCs with CpG-GNPs 101
3.2.6 Assessment of CpG-GNP uptake and immune-stimulation kinetics 105
3.2.7 Discussion and summary 109
3.3 Transfer from murine setup to primary human cells 113
3.3.1 Activation of CpG ODN sensitive human cell-lines after CpG-GNP treatment 113
3.3.1 Morphological investigations of CpG-A ODN 2216 116
3.3.2 Effects of CpG ODN-loading on particle mean size and homogeneity 117
3.3.3 Is enhanced cellular uptake the key for the enhanced activation of the
immune system? 119
3.3.4 Discussion and summary 121
4. Conclusion 124
5. References 126


Chapter IV
In vivo characterization of CpG ODN-loaded gelatin
nanoparticles – Application as efficient and safe vaccine adjuvant

1. Introduction 135
1.1 Novel vaccine adjuvants 135
1.2 Aim of the study and potential safety hazards 139
2. Materials and Methods 141
2.1 Reagents 141
V
published by Verlag Dr. Hut, Munich, www.dr.hut-verlag.de, ISBN: 3-89963-451-9 Table of contents
2.2 Preparation of gelatin nanoparticles 141
2.2.1 Cationized gelatin nanoparticles 141
2.2.2 Gelatin nanoparticles with encapsulated OVA 141
2.3 CpG ODN or OVA loading of cationized gelatin nanoparticles 142
2.4 Mice 142
2.5 Immunostimulation in vivo and immunization 142
2.6 Quantification of cytokines in serum 143
2.7 OVA-specific immune response 143
2.8 Biodistribution studies via CLSM 144
2.9 FACS analysis of lymph node tissue 144
2.10 Gelatin-specific immune response 144
3. Results and Discussion 145
3.1 Systemic immunogenic activity of CpG-GNPs in vivo 145
3.1.1 Intravenous injection 145
3.1.2 Subcutaneous 147
3.1.3 In vivo localization of fluorescence labeled CpG-GNPs 148
3.1.4 Summary 151
3.2 CpG-GNPs as immunization adjuvant for chicken egg ovalbumin
(OVA) as model protein vaccine 152
3.2.1 Orientation study to evaluate the applicability of CpG-GNPs as adjuvant for OVA 152
3.2.2 Rational design of an applicable OVA delivery by gelatin nanoparticles 153
3.2.3 Comparison of the adjuvant potential of CpG-GNPs vs. soluble CpG ODN 156
3.2.4 Summary 157
3.3 Do gelatin nanoparticles induce anti-gelatin IgGs in mice? 158
4. Conclusion 160
5. Ref