Inner leaflet cationic lipid increases nucleic acid loading independently of outer leaflet lipid charge in asymmetric liposomes

Use of cationic lipid vesicles (liposomes) can yield large amounts of nucleic acid entrapped inside the vesicles and/or bound to the external surface of the vesicles. To show a method to prepare asymmetric lipid vesicles (liposomes) with high amounts of entrapped nucleic acid is possible, symmetric and asymmetric lipid vesicles composed of mixtures of neutral (zwitterionic), anionic, and/or cationic phospholipids were formed in the presence of oligo DNA. For symmetric large unilamellar vesicles nucleic acid association with vesicles was roughly 100 times greater for vesicles with a net cationic charge than for vesicles having a net neutral or anionic net charge. A high degree of association between nucleic acid and lipid was also achieved using asymmetric large unilamellar vesicles with a net cationic charge in their inner leaflet, even when they had an anionic charge in their outer leaflet. In contrast, asymmetric vesicles in which only the outer leaflet had a net cationic charge had only low amounts of vesicle-associated nucleic acid, similar in amount to the amount of nucleic acid associated with asymmetric vesicles with an outer leaflet having a net anionic charge. These results indicate that in asymmetric vesicles with cationic lipid enriched inner leaflets nucleic acid is largely entrapped inside the vesicle lumen rather than bound to their external surface, and that asymmetric vesicles can be used to trap high amounts of nucleic acid even when using a lipid composition in the outer leaflet of a lipid vesicle that does not associate with nucleic acids. Such asymmetrically charged vesicles should have applications in studies of membrane protein-nucleic acid interactions as well as in studies of how membrane charge asymmetry can influence membrane protein structure, orientation, and function.