Nanoparticles for RNA Interference

Schultes, Stephan (2009): Nanoparticles for RNA Interference: Novel Preclinical Formulations for SIRNA Mediated Gene Therapy. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Efficient and safe protein nanoparticles for the targeted delivery of small molecule, protein and oligonucleotide based drugs will play a key role in the field of science in the upcoming years. Whereas viral and liposomal formulations have been extensively tested throughout the last two decades, their inherent and in the case of viruses sometimes even fatal obstacles not seldom seem impossible to conquer. The time for the development of a new therapeutic option in form of an advanced drug delivery system within pharmaceutical technology, biopharmacy and clinical studies has come. In our eyes gelatin based nanoparticles with polysaccharide and peptide modifications are an optimum to fulfil this need and will therefore be the center of the research presented in this work. Basically, nanoparticles with a size from 150 to 300 nm were prepared by desolvating a clear solution of gelatin through dropwise addition of an organic anti-solvent under heavy stirring. A subsequent destabilization of the water soluble protein chains resulted in round particles with a homogenous size distribution and an even surface. Initially, the polymers used for the formulation of the nanoparticles were characterized by such methods like asymmetric flow field-flow fractionation and nuclear magnetic resonance spectroscopy. Furthermore, established measurement and calculation algorithms were revised into state-of-the-art technology and applied as so called automatic microviscosimetry for in- depth protein analysis. The development of novel nanoparticle formulations based on these polymers was done in a second step using diethyl-amino-ethanol-dextran, polysorbate and polyethylene glycol, as well as methylation and acetylation chemistry. While the modified dextran mainly increased the zeta potential of the nanoparticles, the other modifications were intended to change the pharmacokinetic distribution patterns towards e.g. prolonged circulation times. In novel nanoparticle cytology science the use of a flow chamber device for cell cultivation allowed us to study the interaction patterns of nanoparticles with adherent cells under near to physiological conditions simulating blood vessels, junctions and shear stress. This in-vitro model can be used for online preclinical and high-throughput screenings of new nanoparticle and protein formulations with cell monolayers. The hindrances in traditional static cell culture models were shown to be overcome by comparing several nanoparticle formulations in a static and in a flow model. Proper nanoparticle formulations were tested further in innovative preclinical in-vivo models like the hamster dorsal skin fold chamber and the mouse cremaster model to elucidate their body distribution and targeting properties with a focus on kinetics, blood cell interaction and novel fluorescence detection techniques. In addition, the potential of gelatin nanoparticles as therapeutic options in a model for antigen induced arthritis was demonstrated. Finally, hybrid (sandwich) nanoparticles were formulated by combining gelatin nanoparticle preformulations with the endosomolytic peptide Melittin from bee venom and loading them with small interfering RNA molecules against VEGFR2 and luciferase. The novel hybrid carriers were extensively tested in cell cultures towards their efficiency to induce a protein knock-down based on RNA interference. With these results the door for further, more profound in-vivo studies in the field of oncology might be opened.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Gelatin nanoparticle, drug delivery, RNA interference, siRNA technology, proteomics, oncology therapy, preclinical kinetic modelling, multifunctional nanocarriers
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 10. November 2009
• 1. Bericht­erstatter:in: Coester, Conrad