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Masters Degrees (Genetics)

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Browsing Masters Degrees (Genetics) by Author "Arbuthnot, Patrick Brian."

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    Novel cationic lipoplexes : characterization in cell culture in vitro and in vivo.
    (2010) Sewbalas, Alisha.; Ariatti, Mario.; Singh, Moganavelli.; Arbuthnot, Patrick Brian.
    Amongst the more promising non-viral DNA vehicles are liposomes, with those derived from cationic lipids showing significant potential, despite moderate transfection levels in vivo. This study has investigated the effect of liposome-anchored ionophore crown ethers on lipoplex-mediated gene transfer in vitro and in vivo. Several liposomes were constructed to include the cytofectin 3β[N(N’,N’-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T), the co-lipid dioleoylphosphatidylethanolamine (DOPE), and 5% (mole/mole) of the cholesteryl crown ethers RUI-128 (aza-18-crown-6) or RUI-129 (aza-15-crown-5). Liposome size and lamellarity were established by transmission electron microscopy. All liposome preparations were shown to bind, condense and protect DNA avidly in the respective band shift, ethidium displacement and nuclease protection assays. Lipoplex targeting to hepatocytes may be achieved via the asialoglycoprotein receptor (ASGP-R), which is abundantly expressed on the human hepatoblastoma cell line HepG2. Therefore six additional liposomes were formulated to include 5% (mole/mole) of the cholesteryl galactosyl RUI-90 (Gal) and cholesteryl glucosyl RUI-92 (Glu) ligands. Their hepatotropic gene delivery was examined in the HepG2 cell line using the pCMV-luc plasmid. Transfection studies in the human embryonic kidney cell line HEK293 (ASGP-R-negative) revealed an increase in transgene activity in lipoplexes displaying the RUI-129 cholesteryl derivative. No ionophore-mediated enhancement of transfection activity was observed in HepG2 cells although Chol-T:DOPE, Chol-T:DOPE:RUI-128 and Chol-T:DOPE:RUI-129 liposomes achieved very high transfection levels, exceeding those of their hepatocyte targeted counterparts. Liposome-anchored crown ethers have been shown to potentiate in vitro transfection activity of lipoplexes in the HEK293 cell line. The novel cholesteryl glycosyl derivatives were, however, unable to enhance the targeted entry of lipoplexes into HepG2 cells. The three most effective preparations from in vitro studies were taken forward for in vivo assessment in NMRI mice at the University of the Witwatersrand Molecular Medicine and Haematology unit. Three groups of mice were employed for the evaluation of Chol-T:DOPE, Chol-T:DOPE:RUI-129 and Chol-T:DOPE:RUI-129-Gal lipoplexes with the Psi-CHECK plasmid. Mice treated with hydrodynamic injection and untreated animals made up two control groups. Luciferase activity was determined on examination of the harvested liver homogenates. All liposomes showed modest, but significant transfection activity (p<0.05) and were well tolerated. The assemblies examined therefore warrant further development.