Gene transfer mediated by cationic lipids: lack of a correlation between lipid mixing and transfection

Abstract

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.

Lipoplexes with alkylphospholipid as new helper lipid for efficient in vitro and in vivo gene transfer in tumor therapy

“…On the other hand, the highest transfection with APL-LPs were obtained in vitro with a slightly higher P/N between 1.5 and 2 (L9-LP: 1.69), whereas L9-LP with a P/N ratio of 3.2 was much more effective in vivo. This is consistent with the findings of several other groups [33][34][35][36][37][38] indicating that a slight positive charge (charge ratio between 1 and 2) supports the binding of the complex to the (negatively charged) cell membrane, whereas nucleic acid compaction rather than surface charge was critical for an efficient nuclear trafficking. 39 On the other hand, some reports describe other ratios for more efficient complexes, 10,38,[40][41][42] which indicated that the charge ratio is not the only factor responsible for the transfection efficacy.…”

Lipoplex size is a major determinant of in vitro lipofection efficiency

“…There have been studies which have linked the zeta potential to the transfection efficiency of cationic liposomes, 35 and others where no correlation has been found. 26 Although there may be a correlation, we do not believe that the zeta potential of the lipoplexes plays as great a role in lipofection as does the size of the lipoplex. Our measurements show that, while both the addition of polyanions and serum reduce the zeta potential of MLV lipoplexes at a 2:1 charge ratio to a similar extent, the transfection efficiency increases dramatically with polyanion-induced size increase, but the size and transfection efficiency remain low in the presence of polyanions plus serum (Figures 2a, 3a, 4a and 6a).…”

“…Previous findings have determined that either lipoplex size did not correlate with lipofection 26 or that lipoplexes 27 or liposome-polymer-DNA complexes 37 of smaller size are more efficient than larger sized complexes. In some of these reports, the sizes of complexes studied did not encompass the entire range of sizes necessary to elucidate any correlation.…”

“…Some investigators have argued that size is not associated with activity, 26 or that transfection efficiency is independent of initial vesicle size. 15 Others have determined that either smaller lipoplexes 27 or larger lipoplexes are more efficient, 16,28 and also that larger complexes formed by multilamellar vesicles are more efficient than smaller complexes formed by small unilamellar vesicles without specifying the relative importance of size and liposome type.…”

Improving lipoplex-mediated gene transfer into C6 glioma cells and primary neurons

“…In most cases, these lipids are mixed with an equimolar amount of a ''helper'' lipid. Most frequently, the neutral lipid DOPE is used, since this is known to enhance transfection efficiency (Felgner et al, 1994;Gao and Huang, 1995;Stegmann and Legendre, 1997;Zhou and Huang, 1994), due to its ability to facilitate the formation of cationic liposomes and to its tendency to form non-bilayer phases that may contribute to the destabilization of the endosomal membrane (Farhood et al, 1995;Felgner et al, 1994;Legendre and Szoka, 1992;Zhou and Huang, 1994). On the other hand, cholesterol (Chol) constitutes another promising ''helper'' lipid, since it has been demonstrated that high levels of transfection can be obtained both in vitro and in vivo, upon its association with a cationic lipid (Crook et al, 1998;Templeton et al, 1997).…”