Using Agarose-Assisted Formation as an Alternative to Electroformation for Producing Phase Separating Vesicles



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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, gel-assisted swelling has been explored as an alternative to 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 five 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 agarose-assisted swelling to that of electroformation with a focus on GUV size and quantity, as well as the number of lipid domains formed, the lipid domain area fraction, and the lipid domain perimeter on GUVs. Here, we show that the phase behavior of giant liposomes formed from the gentle hydration of hybrid films of agarose and lipids is comparable to the phase behavior of giant liposomes formed from electroformation. This work shows that agarose-assisted swelling is a simple, rapid, reproducible, and affordable approach to generate phase-separating giant liposomes of ternary lipid compositions.



Phase-separating giant liposomes, Agarose-assisted swelling, Electroformation, Giant unilamellar vesicles