These investigations are focused on the design and formulation of novel nonviral lipopolyamine vectors capable of efficiently and safely delivering DNA to the nucleus, and siRNA to the cytoplasm, in two tissue cultured (primary and cancer) cell lines. The thesis starts with a focussed literature review on the non-viral gene therapy (NVGT) vectors currently used in the formulation of DNA and siRNA. The first experimental part tests the ability of our novel lipospermines in NVGT, this includes structure-activity relationship (SAR) studies changing the: position, length, saturation or symmetry of the fatty chains of N4,N9-diacyl, N1,N12-diacyl and N4,N9-dialkyl spermines. The ability of these lipospermines in DNA condensation is investigated using ethidium bromide fluorescence-quenching, and gel electrophoresis (including gel shift and DNase protection) assays followed by nanoparticle characterization techniques (particle size and zeta potential). Transfection efficiency of pEGFP (using FACS) and cytotoxicity (using MTT) were studied in both cancer and primary cell lines and compared with Lipogen™ (N4,N9-dioleoyl spermine). Some of these novel lipospermines are shown to be as good as, but not better than N4,N9-dioleoyl spermine as efficient DNA transfecting agents. N4,N9-Dioleoyl spermine is the best transfecting agent from the all tested novel lipospermines displaying the lowest N/P ratio, highest transfection efficiency and the lowest cytotoxicity on both tested cell lines. We extended this SAR study to examine the same lipospermines in siRNA delivery. The ability of these compounds to bind siRNA was studied using the RiboGreen intercalation assay followed by similar nanoparticle characterization techniques. Transfection efficiency for delivery of Label IT® RNAi Delivery Control (using FACS) and cytotoxicity (MTT) were also studied in both cancer and primary cell lines, and compared with a market leader siRNA transfecting agent Trans-IT™. Twelve of these non-viral vectors, led by N4,N9-dieicosenoyl spermine and N4,N9-dierucoyl spermine, showed both transfection efficiency and cell viability over 75%.
|Date of Award||1 Sept 2008|
|Supervisor||Ian Blagbrough (Supervisor)|
- gene delivery