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Klöckner, Julia (2006): Novel biodegradable gene carriers based on oligomerized polyamines. Dissertation, LMU München: Faculty of Chemistry and Pharmacy



Gene therapy is a very promising approach to treat or to prevent diseases. However, progress in this field is hindered by lack of suitable vectors. Current research focuses on the development of novel nonviral biodegradable gene carriers with improved gene transfer activity and low toxicity. In the course of this thesis, a library of degradable DNA compacting domains based on oligomerized polyamines was synthesized and analyzed. Degradation of the originated polymers was either based on site-specific reductive cleavage of disulfide bonds or on time-dependent ester/amide hydrolysis. DNA binding activity, polyplex stability, transfection efficiency, toxicity, and hemocompatibility studies were performed in order to identify promising candidates. Some of the novel gene carriers, especially the degradable oligoethylenimine (OEI) derivatives were successfully applied for in vitro transfection and could easily compete with the current ‘golden standard’ linear polyethylenimine with an average Mw of 22 kDa (PEI22lin). Furthermore, screening results revealed critical structure activity relationships which were very helpful for improving the polymer design. According to transfection and biocompatibility results, efficiency and toxicity correlated to some degree. Polymers with an overall high charge density and a high molecular weight like OEI-HD-1 provided polyplex stability and formed small uniform particles. On the other hand these polymers tended to induce erythrocyte aggregation and exhibited a pronounced cytotoxicity when applied at high concentrations. Polycation with a lower molecular weight (~ 10 kDa) like e.g. OEI-IP-1 were essentially nontoxic, but had to be applied at high concentrations in order to achieve efficient gene transfer. Intrinsic membrane activity of certain polymers could damage cellular membranes but may also trigger endosomal release and therefore boost transfection activity. Crosslinking of OEI 800 with 1,6-hexanedioldiacrylate resulted in highly efficient degradable polycations. Different reaction temperatures during OEI-HD-1 synthesis had a strong impact on molecular weight and the ester/amide ratio. Despite structural differences, both OEI-HD-1 (synthesized at 60°C) and lt-OEI-HD-1 (synthesized at 20°C) possessed equal gene transfer activity as the ‘golden standard’ PEI22lin when applied at their optimal polymer/DNA-ratio (w/w). It was important to note that lt-OEI-HD-1, the LMW-derivative which is predominantly based on ester linkages, was significantly less toxic than its HMW amide-linked counterpart. OEI-HD displayed a very promising basis for the development of further powerful gene carriers. A two-step synthesis protocol was established in order to generate OEI-HD cores bearing excessive linker which could be subsequently modified with various functionalities like spermine. OEI-HD-Sper pseudo-dendrimers were characterized by a pronounced intrinsic membrane activity and possess high transfection efficiency. Since current nonviral vectors are still very inefficient as compared to their viral competitors, natural viruses present an ideal example educating us how to further optimize polycationic gene carriers in terms of specific cell-targeting and improved endosomal release. Modification of polyplexes towards a “smart” virus-like system was achieved in the following way. Degradable DNA compacting domains (OEI-HD-1) were utilized for complex formation. Furthermore, epidermal growth factor (EGF) was incorporated as targeting ligand into OEI-HD-1 polyplexes and thus allowed cell-specific cellular uptake via the EGF receptor (EGFR). Gene transfer potential of EGFR-targeted degradable polyplexes was further improved by applying technologies which promoted the endosomal release of endocytosed particles. Photochemical intracellular release (PCI) is based on accumulation of amphiphilic photosensitizers (PS) in endosomal membranes. Illumination of PS pre-treated transfected cells results in activation of the PS and subsequent light-induced rupture of endocytic vesicles. Combination of biological (EGFR) and physical (PCI) targeting greatly enhanced reporter gene delivery mediated by OEI-HD-1 polyplexes. Finally, the incorporation of membrane active melittin derivatives into EGF/OEI-HD-1 polyplexes was the first example of a biodegradable synthetic virus for gene delivery.