Factors affecting DNA binding and stability of association to cationic liposomes

► The effect of NaCl concentration on formation and stability of DOTAP/cholesterol-DNA and DOTAP/DOPE-DNA lipoplexes and the effects of two anionic amphiphlies, the monomeric SDS and egg PC/DCP anionic MLV liposome were study by determining DNA distribution between lipoplexes and solution: as lipoplex-bound and unbound-(“free”) DNA as well as degree of DNA condensation. ► The level of lipoplex-associated DNA was determined using two independent methods: ultracentrifugation and agarose gel electrophoresis, which are based on different physical forces. A third method, which employed EtBr as an intercalating dye, was used to study the degree of DNA condensation. These three methods used in this study are good tools for studying interactions of DNA with cationic liposomes of different lipid compositions with salt and biological fluids such as serum. ► The electrical field applied during agarose gel electrophoresis increases the dissociation of the DNA from the DOTAP/cholesterol-DNA lipoplex but not from the DOTAP/DOPE-DNA lipoplex. The results indicate that either the electrostatic association between DOTAP/cholesterol and DNA is weaker as a result of factors such as intimacy of interaction, or level of hydration than that between DOTAP/DOPE and DNA, or that in DOTAP/DOPE-DNA there are forces in addition to electrostatics, which play a role. ► DOTAP/cholesterol-DNA lipoplex, analyzed by agarose gel electrophoresis, was partially destabilized by SDS treatment, and the amount of free DNA detected was higher compared to the amount detected without SDS treatment. SDS, however, caused large changes in DNA condensation state in both DOTAP/DOPE-DNA and DOTAP/cholesterol-DNA lipoplexes. An increase from 7-22% to ∼61% in level of EtBr intensity in DOTAP/DOPE-DNA lipoplex was observed after SDS treatment; and in DOTAP/cholesterol-DNA, EtBr fluorescence intensity increased upon SDS treatment from 6% to 40-50%. ► The effects of both DNA and SDS on the DOTAP/DOPE system are larger than on DOTAP/cholesterol. This may be related to the differences in small counterions released and to SDS interfering with the ion pairing between the quaternary amine of the DOTAP and the phosphate diester negative charge of the DOPE, both effects lacking in DOTAP/cholesterol liposomes.