Phase separation on giant liposomes prepared by gel-assisted swelling.

Abstract

Giant unilamellar vesicles (GUVs) are often used as models to study lipid membrane structure and function. The most common method for GUV formation is electroformation, though this technique has its own inherent drawbacks such as low yield in ionic solutions or in the presence of charged lipids. In recent years, the method of gel-assisted swelling has been explored as an alternative for electroformation and has been reported to overcome some of the limitations of this technique. This study aims to assess the ability of gel-assisted swelling to form GUVs of ternary lipid compositions that are known to undergo phase separation. To this end, we utilize different ratios of a 3-component mixture of DOPC/DPPC/Cholesterol to form vesicles capable of separating into phases of liquid-ordered (Lo) and liquid-disordered (Ld). This lipid composition was chosen as previous studies have shown that at room temperature, many of its possible compositions exhibit distinct phase separation. We apply confocal fluorescence microscopy to evaluate the resultant GUVs, in terms of yield, size, and phase behavior. We compare the results of this method to that of electroformation with a focus on GUV size and quantity, as well as formation of lipid domains, their number and area fraction on GUVs. Ultimately, the results show that agarose gel-assisted swelling can parallel electroformation in forming phase-separating GUVs over a series of ternary lipid compositions.