Gene transfer mediated by cationic lipids: lack of a correlation between lipid mixing and transfection.
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.
Stegmann
T
T
Department of Biophysical Chemistry, Biozentrum of the University of Basel, Switzerland. stegmann@ipbs.fr
Legendre
J Y
JY
D000643:Ammonium Chloride; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002412:Cations / Q000378:metabolism*; D006224:Cricetinae; D004269:DNA, Bacterial; D004705:Endocytosis; D005229:Fatty Acids, Monounsaturated / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D050356:Lipid Metabolism; D008954:Models, Biological; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D014162:Transfection / Q000502:physiology*
D002412:Cations; D004269:DNA, Bacterial; D005229:Fatty Acids, Monounsaturated; D000644:Quaternary Ammonium Compounds; D000643:Ammonium Chloride; C054005:N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane