Electroformation of Giant Unilamellar Vesicles from Damp Films in Conditions Involving High Cholesterol Contents,...

Giant unilamellar vesicles (GUVs) are frequently used as membrane models in studies of membrane properties. They are most often produced using the electroformation method. However, there are a number of parameters that can influence the success of the procedure. Some of the most common conditions that have been shown to have a negative effect on GUV electroformation are the presence of high cholesterol (Chol) concentrations, the use of mixtures containing charged lipids, and the solutions with an elevated ionic strength. High Chol concentrations are problematic for the traditional electroformation protocol as it involves the formation of a dry lipid film by complete evaporation of the organic solvent from the lipid mixture. During drying, anhydrous Chol crystals form. They are not involved in the formation of the lipid bilayer, resulting in a lower Chol concentration in the vesicle bilayer compared to the original lipid mixture. Motivated primarily by the issue of artifactual Chol demixing, we have modified the electroformation protocol by incorporating the techniques of rapid solvent exchange (RSE), ultrasonication, plasma cleaning, and spin-coating for reproducible production of GUVs from damp lipid films. Aside from decreasing Chol demixing, we have shown that the method can also be used to produce GUVs from lipid mixtures with charged lipids and in ionic solutions used as internal solutions. A high yield of GUVs was obtained for Chol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) samples with mixing ratios ranging from 0 to 2.5. We also succeeded in preparing GUVs from mixtures containing up to 60 mol% of the charged lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and in NaCl solutions with low ionic strength (<25 mM).