THE ROLE OF HELPER LIPIDS IN CATIONIC LIPOSOME-MEDIATED GENE-TRANSFER

In the procedure for cationic liposome-mediated transfection, the cati onic lipid is usually mixed with a ''helper lipid'' to increase its tr ansfection potency. The importance of helper lipids, including dioleoy lphosphatidylcholine (DOPC) and phosphatidylethanolamine (dioleoyl PE) , DO was examined. Freeze-fracture electron microscopy of DNA:cationic complexes containing the pSV-beta-GAL plasmid DNA, the cationic lipid dioleoyl trimethylammonium propane, and these helper lipids showed th at the most efficient mixtures were aggregates of ensheathed DNA and f used liposomes. PE-containing complexes aggregated rapidly when added to culture media containing polyanions, whereas PC-containing complexe s did not. However, more granules of PC-containing complexes were form ed on cell surfaces after the complexes were added to Chinese hamster ovary (CHO) cells in transfection media. Pronase treatment inhibited t ransfection, whereas dilute poly-L-lysine enhanced transfection, indic ating that the attachment of DNA:liposome complexes to cell surfaces w as mediated by electrostatic interaction. Fluorescence spectroscopy st udies confirmed that more PC-containing complexes than PE-containing c omplexes were associated with CHO cells, and that more PC-containing c omplexes were located in a low pH environment (likely to be within end osomes) with time. Cytochalasin-B had a stronger inhibitory effect on PC-containing liposome-mediated than on PE-containing liposome-mediate d transfection. Confocal microscopic recording of the fluorescently la bel lipid and DNA uptake process indicated that many granules of DNA:c ationic liposome complexes were internalized as a whole, whereas some DNA aggregates were left out on the cell surfaces after liposomes of t he complexes fused with the plasma membranes. For CHO cells, endocytos is seems to be the main uptake pathway of DNA:cationic liposome comple xes. More PC-containing granules than PE-containing granules were form ed on cell surfaces by cytoskeleton-directed membrane motion, after th eir respective DNA:liposome complexes attached to cell surfaces by ele ctrostatic means. Formation of granules on the cell surface facilitate d and/or triggered endocytosis. Fusion between cationic liposomes and the cell membrane played a secondary role in determining transfection efficiency.