Gelatin nanoparticles for targeted oligonucleotide delivery to Kupffer cells [Elektronische Ressource] : analytics, formulation development, practical application / vorgelegt von Jan Carl Zillies

Jan Zillies

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Gelatin Nanoparticles for
Targeted Oligonucleotide Delivery to Kupffer Cells
–
Analytics, Formulation Development, Practical Application





Dissertation



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




vorgelegt von

Jan Carl Zillies
aus Hamburg

2007

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 12. Januar 2007



______________________
Jan Carl Ziles





Dissertation eingereicht am: 12. Januar 2007
1. Gutachter: Prof. Dr. Gerhard Winter
2. Gutachter: Prof. Dr. Wolfgang Frieß
Tag der mündlichen Prüfung: 02. Februar 2007






For my parents


Acknowledgements

The present thesis has been prepared between January 2003 and February 2007 at the
Department of Pharmacy Pharmaceutical Technology and Biopharmaceutics of the
Ludwig-Maximilians-University (LMU) Munich, Germany.
Foremost, I would like to express my honest gratitude to my supervisor Prof. Dr.
Gerhard Winter for giving me the opportunity to be a member of his working group
and to prepare this thesis. I am especially grateful for his guidance that allowed me to
work independently and for the numerous successful scientific discussions driven by
his impressive experience and knowledge. Furthermore, I always enjoyed his
commitment to achieve the great working atmosphere we experienced at his chair.
I would like to thank my tutor Dr. Conrad Coester for his support during my work
and the loyalty to his research team. Special thanks go to his efforts at presenting our
work on international congresses and promoting my research stay at the University
of Colorado.
Many thanks go to Prof. Dr. Angelika Vollmar and her research team at the
Department of Pharmaceutical Biology (LMU Munich) for the great cooperation
within the DFG project. PD Dr. Stefan Zahler is acknowledged for assisting me with
FACS analysis, fluorescence microscopy, and monocyte isolation. My special
appreciation goes to Florian Hoffmann for being more than my cooperation partner.
I am extremely grateful to Prof. Dr. Thomas J. Anchordoquy for giving me the
chance to work in his lab at the School of Pharmacy of the University of Colorado
and for his decisive contribution to the successful development of my research work.
Within his team I especially would like to thank Marion Molina for providing me
with the know-how about freeze-drying, for helping me with the analytics of my
samples, and for joining me for lunch.

Special thanks go to Dr. Torsten Göppert (formerly Department of Pharmaceutical
Technology at the Free University of Berlin) for the pleasant and successful
2D-PAGE cooperation.
I would like to thank Dr. Wolfgang Fraunhofer for telling me the secrets of AF4.
The teams of Postnova Analytics and Wyatt Technology are acknowledged for their
always immediate and uncomplicated help with all kinds of problems concerning
AF4 and light scattering analysis.
I am very thankful to Andrea Hawe, Florian Hoffmann, Rainer Lang, and Klaus
Zwiorek for the quick and helpful proofreading of this thesis, which was essential to
finish my work in time.
I would like to thank all the former and present colleagues of our department for the
great times we had in- and outside the lab. I always enjoyed working with all of you
and it was a pleasure to be member of the team. Especially, I want to highlight Klaus
Zwiorek, who had to cope with my wordy attendance and became a good friend,
after all the years sharing the lab. Special thanks go to Imke Leitner who always
assisted me in supervising the students’ practical course in biopharmaceutics, Alice
Hirschmann for the great supply with lab consumables, Patricia Plath and Sabine
Kersting for providing me with literature, as well as Gabi Hartl for their pleasant help
with the administration.
Finally, I would like to thank my parents, my siblings, and my grandma for being my
family. And, I would like to thank Stefanie!

Table of Contents
TABLE OF CONTENTS
GENERAL INTRODUCTION 1
References 5
CHAPTER I
Asymmetrical Flow Field-Flow Fractionation (AF4) in the Analytical
Description of Gelatin Nanoparticles 7
1.1 Introduction 9
1.2 Materials and Methods 13
Reagents 13
AF4 Running buffers 13
Preparation and surface modification of gelatin nanoparticles 14
PEGylation of gelatin nanoparticles 15
Characterization of nanoparticles 16
Analysis of the gelatin bulk material 16
Quantification of oligonucleotide-loading onto the surface of gelatin
nanoparticles 17
Quantification of gelatin nanoparticle PEGylation 19
Atomic force microscopy analysis of plain and PEGylated gelatin
nanoparticles 20
1.3 Analysis of Gelatin Bulk Material Applied for the Manufacturing of
Gelatin Nanoparticle Drug Delivery Systems Using Asymmetrical Flow
Field-Flow Fractionation (AF4) and Multi-Angle Light Scattering (MALS)
Detection 21
1.3.1 Introduction 21
1.3.2 Results and discussion 25
1.3.3 Summary 28
1.4 Quantifying the Oligonucleotide-Loading of Gelatin Nanoparticle
Drug Delivery Systems Using Asymmetrical Flow Field-Flow Fractionation
(AF4) and UV Detection 29
1.4.1 Introduction 29
1.4.2 Results and discussion 31
I Table of Contents
Characterization of nanoparticles 31
Single-stranded oligonucleotide loading 31
Double-stranded oligonucleotide loading 33
1.4.3 Summary 39
1.5 Quantifying the PEGylation of Gelatin Nanoparticle Drug Delivery
Systems Using Asymmetrical Flow Field-Flow Fractionation (AF4) and
Refractive Index (RI) Detection 41
1.5.1 Introduction 41
1.5.2 Results and discussion 44
Characterization of nanoparticles 44
Quantification of the PEGylation reaction via AF4 45
Visualization of the PEGylation reaction via AFM 51
1.5.3 Summary 52
1.6 Concluding Remarks 53
1.7 References 55
CHAPTER II
Formulation Development of Freeze-Dried Gelatin Nanoparticles 63
2.1 Introduction 65
2.2 Materials and Methods 69
Reagents 69
Preparation and surface modification of gelatin nanoparticles 70
Oligonucleotide-loading of gelatin nanoparticles 70
Freeze-drying of empty and oligonucleotide-loaded gelatin nanoparticles 70
Storage conditions during stability studies 72
Characterization of gelatin nanoparticles 72
Karl-Fischer titration 73
Differential scanning calorimetry (DSC) 73
In vivo hepatic lipopolysaccharide (LPS) (sepsis) rat model 75
2.3 Results and Discussion 77
2.3.1 Applicability of freeze-drying for gelatin nanoparticle suspensions 77
Characterization of nanoparticles 77
Initial freeze-drying experiments 78
II