Dissertation zur Erlangung des Doktorgrades der Fakultt fr Chemie und Pharmazie der Ludwig-Maximilians-Universitt Mnchen - NANOPARTICLES FOR RNA INTERFERENCE - NOVEL PRECLINICAL FORMULATIONS FOR SIRNA MEDIATED GENE THERAPY

1 1 GENERAL INTRODUCTION 3 1.1 Endothelial related diseases 3 1.2 siRNA and the principle of RNA interference 8 1.3 RNA interference in research and therapy 13 1.4 Viral and non-viral gene delivery 14 1.5 Nanoparticles: definitions, history and current state of knowledge 16 1.6 Gelatin based nanoparticles as colloidal drug delivery vectors 18 1.7 Body distribution of nanoparticles 19 1.8 Nanoparticle modifications 20 1.9 In-vitro and in-vivo models 23 1.10 Barriers to gene transfer 25 1.11 Nanoparticles for siRNA mediated RNA interference 27 1.12 Goal of the thesis 29 2 MATERIALS AND METHODS 31 2.1 Nanoparticle pre-formulation studies 31 2.1.1 Chemicals and reagents 31 2.1.2 Materials 33 2.1.3 Formulation of unmodified gelatin nanoparticles 35 2.1.4 Formulation of cationic gelatin nanoparticles 35 2.1.5 Formulation of diethyl-amino-ethanol-dextran and cholamine modified gelatin nanoparticles 36 2.1.6 Formulation of human serum albumin nanoparticles 38 2.1.7 Formulation of polybutylcyanoacrylate nanoparticles 38 2.1.8 Formulation of chitosan nanoparticles 39 2.1.8.1 Preparation of low molecular weight chitosan 39 2.1.8.2 Synthesis of thiobutylamidine and N-acetylcysteine modified chitosan 39 2.1.8.3 Preparation of unmodified and thiolated chitosan nanoparticles 40 2.2 Post-formulation modification of nanoparticles 40 2.2.1 Methylation and acetylation 40 2.2.1.1 Methylation 40 2.2.1.2 Acetylation 41 2.2.2 Fluorescence labeling 41 2.2.3 Polysorbate and polyethylene glycol modification 44 2.2.4 Polyethylene imine modification 45 2.2.5 Melittin modification 45 2.3 Analytics 48 2.3.1 Chemicals and reagents 48