Synthesizing GUV from Wet Lipid Film to Achieve Better Uniformity in Lipid Composition

Abstract

Giant Unilamellar Vesicles (GUVs) are cell-sized model membrane systems that allow direct investigation of membrane-related phenomena using fluorescence microscopy. Understanding the organization and dynamics of lipid bilayers is important for understanding the processes taking place in cell membranes. Previous work showed that micron-scale domains in GUV can be observed in some ternary lipid mixtures composed of a high melting temperature lipid, a low melting temperature lipid and cholesterol. However, it is a well-known problem that GUVs synthesized by the electroformation from dry lipid films have a wide distribution of transition temperature and lipid composition. In this study, we synthesized DOPC/DSPC/cholesterol GUVs and diPhyPC/DPPC/cholesterol GUVs by the standard electroformation method from dry lipid films as well as by a modified method using wet liposomes made from the Rapid Solvent Exchange (RSE) method. We quantified the lipid composition distributions of GUVs synthesized by the two methods by measuring the transition temperature distributions of GUVs using fluorescence microscopy; since a narrower distribution of transition temperature should correspond to a more uniform distribution in GUV lipid composition. Our results show that GUVs synthesized from wet lipid film have a better uniformity in lipid composition, and the standard deviations of transition temperature are about 3 times smaller than that of GUVs prepared from dry lipid films. This improved method not only gives a better control of GUV lipid composition, but also has a potential of synthesizing GUVs from cell membranes containing native proteins without going through a dry film state.