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Characterization of peptide dendrimers for DNA delivery in living cells

Spassova, Christina

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The hope of a successful treatment of genetically based diseases like cancer increased with the development of gene therapy in the early 1990’s. The major challenge thereof is the safe and efficient transfer of nucleic acids into eukaryotic cells. As viral vectors (genetically modified viruses) are toxic and immunogenic, non-viral gene delivery systems (synthetic polymers) lacking these disadvantages continue to gain a high profile. To achieve an efficient and safe delivery of nucleic acids into living cells, low generation asymmetric peptide dendrimers comprised of the basic amino acids lysine and arginine were synthesized using the well established solid phase peptide synthesis approach. The mechanisms of peptide dendrimer/DNA complex(dendriplex) formation as well as the complex stoichiometry were inspected by electrophoretic mobility shift assay and a PicoGreen exclusion assay. The dendriplex formation is of electrostatic nature and strongly depends on the charge ratio as well as on the number of dendrimer head group charges. The peptide dendrimers are able to form stable complexes with DNA of various sizes at a charge ratio of two and higher. For effective tracing of the pathway through the cell membrane and quantitative evaluation of cellular uptake the peptide dendrimer was site-specifically labelled with a fluorophore. The optimal transfection charge ratio and the capacity of the peptide dendrimers as a DNA delivery system were examined in two different cell lines (NIH3T3 EpoR and HeLa) by confocal laser scanning microscopy. Efficient transfection of plasmid DNA as well as of single and double stranded oligonucleotides of various lengths with minimal cell toxicity was achieved for both cell lines at a charge ratio of five. The intracellular distribution of the dendriplexes was limited to the cell cytoplasm. The cellular internalization of dendriplexes was found to occur by energy dependent endocytosis including both clathrin-dependent and clathrin-independent uptake pathways. Furthermore, the actin cytoskeleton and the lipid rafts/caveolae are not involved in the dendriplexe uptake mechanism. Due to their low pH (between 5.5-6.5), fluorescent dendriplexes are mainly directed to lysosomes and partially to mitochondria. These studies has demonstrated that low fluorescent labelled generation asymmetric peptide based dendrimers can transfect different mammalian cells in vitro. To possess a potential as a non-viral gene delivery vectors for gene therapy a significant improvement of their transfection efficiency is needed.

Item Type: Dissertation
Supervisor: Herten, PD Dr. Dirk-Peter
Place of Publication: Heidelberg
Date of thesis defense: 1 February 2013
Date Deposited: 04 Mar 2013 14:24
Date: 1 January 2014
Faculties / Institutes: Fakultät für Chemie und Geowissenschaften > Institute of Physical Chemistry
Subjects: 540 Chemistry and allied sciences
Uncontrolled Keywords: dendrimers, gene delivery,
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