Structural and fusogenic properties of cationic liposomes in the presence of plasmid DNA

The structural and fusogenic properties of large unilamellar vesicles (LUVs) composed of the cationic lipid N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and 1,2-dioleoyl-3-phosphatidylethanolamine (DOPE) have been examined in the presence of pCMV5 plasmid and correlated with transfection potency, It is shown, employing lipid mixing fusion assays, that pCMV5 plasmid strongly promotes fusion between DOTMA/DOPE (1:1) LUVs and DOTMA/1,2-dioleoyl-3-phosphatidylcholine (DOTMA/DOPC) (1:1) LUVs such that at a cationic lipid-to-DNA charge ratio of 3.0, similar to 80% fusion is observed, The anions citrate and chloride can also trigger fusion, but at much higher concentrations, Freeze-fracture electron microscopy studies demonstrate the tendency of cationic vesicles to form clusters at low pCMV5 content, whereas macroscopic fused aggregates can be observed at higher plasmid levels, P-31 NMR studies of the fused DNA-DOTMA/DOPE (1:1) complexes obtained at high plasmid levels (charge ratio 1.0) reveal narrow ''isotropic'' P-31 NMR resonances, whereas the corresponding DOPC containing systems exhibit much broader ''bilayer'' P-31 NMR spectra, In agreement with previous studies, the transfection potency of the DOPE-containing systems is dramatically higher than for the DOPC-containing complexes, indicating a correlation between transfection potential and the motional properties of endogenous lipids. Interestingly, it was found that the complexes could be separated by centrifugation into a pellet fraction, which exhibits superior transfection potencies, and a supernatant fraction, Again, the pellet fraction in the DOPE-containing system exhibits a significantly narrower P-31 NMR resonance than the corresponding DOPC-containing system, It is suggested that the P-31 NMR characteristics of complexes exhibiting higher transfection potencies are consistent with the presence of nonbilayer lipid structures, which may play a direct role in the fusion or membrane destabilization events vital to transfection.