High Transfection Activity Research Articles

The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer

In a reporter gene assay, cationic liposomes containing the cationic lipid 3β-( N-( N′, N′-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol) and a neutral phospholipid dioleoylphosphatidylethanolamine (DOPE) showed high transfection activity. DNA/liposome complex which contained low amount of liposomes could bind to the cell surface but failed to transfect the cells. We have designed a two-step protocol to examine this phenomenon in more detail. A431 human cells were incubated on ice (pulse) with DNA complexed to a low level of cationic liposomes. The cells were washed and incubated at 37° C (chase) with or without free cationic liposomes of various composition (helper liposomes). Only liposomes enriched with DOPE showed helper activity; liposomes containing dioleoylphosphatidylcholine (DOPC), a structural analog of DOPE, had no helper activity. The delivery was inhibited by the lysosomotropic agent chloroquine and was optimal if the helper liposome chase was initiated immediately after the pulse. An endocytosis model of DNA delivery by cationic liposomes is proposed in which the principal function of the chase liposomes is to destabilize the endosome membrane and allow the release of DNA into the cytosol. This model is consistent with the known activity of DOPE to assume non-bilayer structures, hence destabilizing the endosome membrane.

Targeted delivery of DNA by liposomes and polymers

Cationic quaternary ammonium detergents stabilized the lamellar phase of dioleoylphosphatidylethanolamine (DOPE) to form liposomes. Such cationic liposomes interacted with and delivered DNA to tissue culture cells with high efficiency. Positively charged polymers conjugated with lipids were also active in DNA delivery. The transfection activity of the lipopoly- l-lysine under optimal conditions was approximately 3-fold higher than that of Lipofectin, a commercially available liposome reagent. Moreover, the conjugate was much more resistant to serum neutralizing effect. Specific delivery of DNA to target cells was achieved by using the anionic pH-sensitive immunoliposomes. It was found that both pH-sensitivity and incorporation of antibody on liposome surface were important for the high transfection activity. Both the short- and long-term transformation efficiency had been determined and shown to be more superior to those of traditional calcium phosphate precipitation method. Successful delivery and expression of the exogenous genes mediated by pH-sensitive immunoliposomes had been demonstrated in both tissue cultured cells and in a mouse model.