Formation of asymmetric phospholipid membranes via spontaneous transfer of fluorescent lipid analogues between vesicle populations

Abstract

A method is presented for generating artificial lipid vesicles bearing an asymmetric distribution of either of the fluorescent lipid analogues 1-acyl-2-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]caproyl]phosphatidylcholine or 1-acyl-2-[12[(7-nitro-2,1,3-benzoxadiazol-4-yl]amino]dodecanoyl]-phosphatidylcholine, in which the fluorescent lipid is located predominantly in either the outer or inner leaflet of the vesicle bilayer. The procedure is based on the observation that these lipid analogues undergo rapid spontaneous transfer (exchange) between vesicle populations [Nichols, J. W., & Pagano, R. E. (1981) Biochemistry 20, 2783-2789]. When an excess of nonfluorescent acceptor vesicles is mixed with small unilamellar vesicles containing 5 mol % fluorescent lipid, approximately 50% of the fluorescent lipid is transferred to the acceptor vesicles, whereas if fluorescent multilamellar vesicles are used, only approximately 10% of the analogues is available for transfer. These fractions of fluorescent lipid available for intervesicular transfer correspond closely to the amount of phospholipid residing in the outermost leaflet of the donor vesicles, suggesting that only fluorescent lipids present in the outer surface of the vesicles can spontaneously transfer between vesicles populations. Evidence demonstrating that the movement of the fluorescent lipid between vesicle population is the result of a net transfer process rather than lipid exchange is also presented. A novel assay based on resonance energy transfer is described for determining the size of the exchangeable fluorescent lipid pool, a measure of the degree of asymmetry of these preparations. Finally, for demonstration of the usefulness of asymmetric vesicles in distinguishing various pathways of vesicle-cell association, preliminary results are presented on their interactions with Chinese hamster fibroblasts in vitro.