469. Improved Transfection with Mixtures of Cationic Lipids: Application to Human Umbilical Artery Endothelial Cells (HUAEC)

Abstract

Cationic lipids have been widely used for the delivery of plasmid and antisense DNA into eukaryotic cells; however, inefficiency of transfection is a major problem confronting their use in gene therapy. To date, the primary approach to improving the transfection properties of cationic lipids has been the synthesis of new kinds of cationic amphipaths or the inclusion of non-cationic helper lipids. Here it is reported that an alternative approach can be unusually effective, namely, the combination of two cationic lipid derivatives with differing amounts of hydrophobicity. EDLPC (1, 2-dilauroyl-sn-glycero-3-ethylphosphocholine), when combined with EDOPC (1, 2-dioleoyl-sn-glycero-3-ethylphosphocholine), dramatically enhances (up to 30-fold or more) the transfection of human umbilical artery endothelial cells (HUAEC) even though, individually, EDOPC or EDLPC are quite weak transfection reagents for these cells. Similarly, the combinations of EDOPC/di-C14 dimethylammonium, di-C18 dimethylammonium/di-C14 dimethylammonium, EDOPC/DC-cholesterol and EDOPC/DMTAP transfected DNA into HUAEC ~10–100 times more efficiently than either compound separately. A possible explanation of this synergy is that the incorporation of the shorter chain molecule increases the propensity for fusion of the lipoplex lipid with cellular membranes, so that the positive charge becomes neutralized and release of DNA is facilitated. To test this hypothesis, membrane fusion was measured using a FRET assay based on reduction of energy transfer between NBD-PE and Rh-PE in cationic lipids of the lipoplexes upon fusion with egg PC liposomes containing 20% DOPG. The extent of fusion of mixed-lipid lipoplexes was significantly greater than that of pure EDOPC. Also, by fluorescence microscopy of labeled lipid and oligonucleotides, it was seen that EDOPC lipoplexes remained undissociated in the cytoplasm whereas in the case of mixed-lipid lipoplexes, the oligonucleotide accumulated in the nucleus (especially at 2 h) while the lipid remained in the cytoplasm. These results also suggest that inclusion of the medium-chain lipid accelerates mixing of the lipoplex and cellular lipids and release of DNA. Until now, phosphatidylethanolamine and cholesterol have been used as the helper lipids to improve the transfection properties of cationic lipids. These data reveal that a second cationic lipid can dramatically improve the nucleic acid delivery properties of lipoplexes.