Development of gene delivery liposome vector for suicide gene therapy

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For injectable sized liposomes with high transfection efficiency of genes, liposomes complexed with plasmid DNA (lipoplexes) and liposomes-entrapping DNA(LED) were studied to optimize the formulae and preparation. For selectivity of gene expression, the thymidine kinase gene controlled by midkine promoter (pMK-tk) was used for herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Liposomes composed of 3([N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), L-dioleoylphosphatidylethanolamine(DOPE) and a biosurfactant such as β-sitosterol β-D-glucoside (Sit-G) or mannosylerythrytol lipid A(MEL)(Sit-G-liposomes or MEL-liposomes, respectively) were prepared by a modified ethanol injection method. Sit-G- and MEL-liposomes produced about 300-nm sized lipoplexes. RA-LED composed of phosphatidylcholine, dioleoyl-3-trimetylammonium propane (DOTAP), DOPE, Sit-G, and retinoic acid(RA), was prepared by a modified dehydration rehydration vesicle method. By optimization of the sucrose : lipid weight ratio, 313-nm sized RA-LED with 66% entrapment efficiency could be formed. Lipoplexes of Sit-G-and MEL-liposomes and RA-LED showed higher transfection efficiency of the luciferase marker gene and thymidine kinase activity in the presence of serum in the cells. The treatment with transfection of pMK-tk by Sit-G-liposome reduced tumor growth at 4-6 days after starting transfection and injection of ganciclovir in a solid tumor model. These liposomes are promising vectors in gene-based therapy.