We compare the transfection efficiency of plasmid DNA encoding either luciferase or beta-galactosidase encapsulated in pH-sensitive liposomes or non-pH-sensitive liposomes or DNA complexed with cationic liposomes composed of dioleoyloxypropyl-trimethylammonium:dioleoylphosphatidyl-eth anolamine (1:1, w/w) (Lipofectin) and delivered into various mammalian cell lines. Cationic liposomes mediate the highest transient transfection level in all cell-lines examined. pH-sensitive liposomes, composed of cholestryl hemisuccinate and dioleoylphosphatidylethanolamine at a 2:1 molar ratio, mediate gene transfer with efficiencies that are 1 to 30% of that obtained with cationic liposomes, while non-pH-sensitive liposome compositions do not induce any detectable transfection. Cationic liposomes mediate a more rapid uptake of plasmid DNA, to about an eightfold greater level than that obtained with pH-sensitive liposomes. The higher uptake of DNA mediated by Lipofectin accounts for part of its high transfection efficiency. Treatment of cells with chloroquine, ammonium chloride, or monensin decreases (threefold) transfection using pH-sensitive liposomes and either has no effect on or enhances cationic liposome-mediated transfection. Therefore plasma membrane fusion is not the only mechanism available to cationic liposomes; in certain cell lines DNA delivery via endocytosis is a possible parallel pathway and could augment the superior transfection efficiency observed with cationic liposomes.

Complexes of DNA with cationic lipids are used to transfect eukaryotic cells. The mechanism of transfection is unknown, but it has been suggested that the complexes are taken up into the cell by endocytosis, after which fusion of the cationic lipids with the membranes of intracellular vesicles would allow the DNA to escape into the cytoplasm. Here, we have compared transfection of CHO-K1 cells with lipid mixing measured by fluorescence assays, using liposomes or complexes with plasmid DNA of the cationic lipids 1,2 dioleolyl-3-N, N, N,-trimethylammonium-propane (DOTAP), N-[2,3-(dioleoyloxy)propyl]-N, N, N,-trimethylammonium (DOTMA), or combinations of these lipids with dioleoylphosphatidylethanolamine (DOPE), at various lipid/DNA charge ratios. Mixing of the lipids of the complexes or liposomes with cellular membranes occurred readily at 37 degrees C, and was more efficient with liposomes than with complexes. Lipid mixing was inhibited at low temperatures (0-17 degrees C), by the presence of NH(4)Cl in the medium, and by low extracellular pH, indicating the involvement of the endocytic pathway in entry. In the absence of DOPE, there was no correlation between the efficiency of lipid mixing and the efficiency of transfection. Moreover, although DOPE, which is thought to promote membrane fusion, enhanced transfection, it did not always enhance lipid mixing. Neither the size nor the zeta potential of the complexes were clearly associated with transfection efficiency. Therefore, although fusion between the lipids of the complexes and cellular membranes takes place, a step at a later stage in the transfection process determines the efficiency of transfection.

A DNA transfection protocol has been developed that makes use of the cyclic cationic amphipathic pepffde gramicidin S and dioleoyl phosphatidylethanolamine. The DNA complex is formed by mixing gramicidin S with DNA at a 1:1 charge ratio and then adding phosphatidylethanolamine at a lipid/peptide molar ratio of 5:1. The complex mediates rapid association of DNA with cells and leads to transient expression levels of -galactosidase ranging from 1 to 30% of the transfected cells, with long-term expression being about an order of magnitude lower. The respective roles of peptide and phospholipid are not yet resolved but optimal transfection requires both the cyclic peptide and the hexagonal phasecompetent phospholipid PtdEtn. Transfection in CV-1 cells is not affected by lysomotrophic agents, which suggests that DNA entry into the cell is via the plasma membrane. This technique that is simple, economical, and reproducible mediates transfection levels up to 20-fold higher than cationic liposomes in adherent mammalian cells.Advances in gene therapy depend to a large degree on the development of delivery systems capable of efficiently introducing DNA into the target cell. Although significant progress has been made with retroviruses (1) or adenoviruses (2) for gene delivery, concern about possible recombination with endogenous virus, oncogenic effects, and immunologic hostresponse reactions has encouraged a search for nonviral DNA transfection techniques (3). Nonviral techniques, including cationic or pH-sensitive liposomes (4,5), polylysine conjugates (6, 7), and direct injection of DNA (8) overcome some of the problems of the viral systems. However, there remains a need for improved transfection efficiency in the nonviral systems.Since the efficiency of the viral systems is in large part due to fusogenic amino acid sequences on viral membrane proteins, we examined whether membrane-permeabilizing peptides could be used to create systems that efficiently transfect mammalian cells. The system developed here takes advantage of the DNA binding ability and the membrane destabilization properties of gramicidin S, an amphipathic cyclic decapeptide that bears two positive charges from ornithine residues on one side of the ring and the side chains of the hydrophobic residues arrayed on the opposite face ofthe ring (9). Gramicidin S, a membrane binding peptide (10), strongly interacts with nucleotides (11) and nucleic acids (12) by charge interactions and mediates phase transfer of these molecules into organic solvents. Gramicidin S also can destabilize membranes causing an increase in the permeability of liposomal (13) qTo whom reprint requests should be addressed.-893The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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