Publikationsserver der Universitätsbibliothek Marburg

Titel:Non-Viral Gene Delivery Systems: Studies on HER2-Targeted PEG-PEI Copolymers and Modified Chitosans
Autor:Germershaus, Oliver
Weitere Beteiligte: Kissel, Thomas (Prof. Dr.)
URN: urn:nbn:de:hebis:04-z2008-07787
DDC: Naturwissenschaften
Titel (trans.):Systeme zum nicht-viralen Gentransfer: Studien zu HER2-zielgerichteten PEG-PEI Kopolymeren und modifizierten Chitosanen


Zellkultur, Plasmid, Pharmakokinetik, Polyethyleniminderivate, Gene therapy, Tiermodell, Modified Polymers, Modifizierte Polymere, Polyethylenimin, Pharmacokinetics, Polyethylenimine, Biodistribution, Gentransfer

In this thesis new non-viral gene delivery systems were synthesized and characterized. In the first part different questions concerning in vitro and in vivo gene delivery using HER2-targeted Polyethylenglycol (PEG)-modified Polyethylenimine (PEI) polyplexes were addressed. The second part deals with in vitro gene delivery using chitosan and its trimethyl- and PEG-trimethyl-derivatives. In the first part, different novel targeted gene delivery vectors consisting of PEI, PEG and the HER2 directed monoclonal antibody Trastuzumab are described. Conjugates differed in structure concerning PEG-PEI copolymers and linkers used for synthesis. The resulting PEG-PEI-Trastuzumab conjugates were characterized concerning hydrodynamic diameters and zetapotentials of polyplexes with DNA. All conjugate complexes were well below the size-threshold for endocytosis and the surface of the different conjugates was uncharged, decreasing binding to non-targeted cells. The ability of conjugate complexes to specifically bind to and be taken up into HER2-overexpressing cells was evaluated in vitro. Significant differences in the efficiency of binding and uptake were found depending on polyplex structure. These differences were reflected in transfection efficiency and specificity in HER2 overexpressing cells as well. The best results were obtained when Trastuzumab was coupled to PEI via a short PEG linker. The conjugate polyplexes were also characterized towards in vivo applicability. Blood compatibility was tested by incubation with erythrocytes determining the degree of hemolysis as well as erythrocyte morphology and aggregation. Conjugate polyplexes showed very low levels of hemolysis and erythrocyte morphology was virtually unchanged. Pharmacokinetics of conjugates revealed significant improvements over free plasmid DNA and PEI polyplexes. Furthermore, significantly decreased disposition of polyplexes in spleen and lung was observed in the case of conjugates. A HER2 overexpressing in vivo tumor model was established in SCID mice in an attempt to test conjugates in vivo. In the second part of the thesis, chitosan, trimethyl chitosan or polyethylenglycol-graft-trimethyl chitosan/DNA polyplexes were characterized concerning physicochemical properties such as hydrodynamic diameter, condensation efficiency and DNA release. The cytotoxicity of polymers and copolymers was studied in vitro. Furthermore, uptake- and transfection efficiency of polyplexes were evaluated in vitro. It was found that chitosan on the one hand showed very low cytotoxicity on the other hand, polyplexes tended to form large aggregates under physiological conditions, resulting in only 7% cellular uptake. Quaternization of chitosan improved the solubility, DNA condensation ability, and complex formation at physiological pH. Accordingly, cellular uptake was increased 8.5-fold compared to chitosan polyplexes and transfection efficiency was increased up to 678-fold. PEGylation of trimethyl chitosan not only decreased the cytotoxicity and further increased the solubility but also improved colloidal stability of polyplexes. These improvements resulted in a significant, up to 10-fold increase of transfection efficiency in NIH/3T3, L929 and MeWo cells compared to trimethyl chitosan. Cellular uptake pathways and intracellular processing of chitosan, trimethyl chitosan and polyethylenglycol-graft-trimethyl chitosan/DNA polyplexes were studied in two cell lines. The main uptake pathway in L929 cells was clathrin-mediated endocytosis accompanied by uptake via caveolae in the case of TMC and PTMC polyplexes. Transfection efficiency was strongly influenced by clathrin-mediated endocytosis. In A549 cells, uptake mainly occurred by clathrin-mediated endocytosis which was accompanied by macropinocytosis but transfection mainly depended on the former. However, it was found that besides the respective pathways other steps and barriers downstream polyplex uptake are at least of equal importance for efficient transfection with chitosan based vector systems.

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