IMPROVING THE METHOD TO MAKE SYMMETRIC AND ASYMMETRIC GUVS TO AVOID CHOLESTEROL CRYSTALS

The formation of giant unilamellar vesicles (GUVs), both symmetric and asymmetric, is a widely used tool for studying the physicochemical properties of membranes. However, during the electroformation process—especially in lipid compositions containing cholesterol—the formation of cholesterol crystals can compromise sample quality. These crystals often form on the surface of the slides used, hindering the efficient incorporation of cholesterol into the vesicles and affecting the accuracy of experiments. In this study, we investigate the influence of lipid composition on cholesterol crystallization by varying the concentrations of lipids and cholesterol in different mixtures. As a complementary strategy, we tested a procedure to limit crystal size using a modified 3D printer to selectively scrape the slide surface, creating small “islands” of lipid material. This approach aims to restrict crystal propagation and improve sample homogeneity. We examine cholesterol crystals using optical microscopy, atomic force microscopy, and electron microscopy. Our goal is to optimize the electroformation protocol by reducing or eliminating crystal formation, in order to produce GUVs with higher quality and more reliable cholesterol incorporation, contributing to the development of more robust biological membrane models. The additional steps incorporated into our experimental procedure for the preparation of GUVs — specifically, the utilization of a spin coater and the formation of an island of lipid film — enhance our ability to control the size of GUVs. This approach also facilitates the achievement of a more uniform distribution of dyes across the vesicles, ensuring consistency from one GUV to another. This achievement directly influenced our ability to enhance the method for producing asymmetric bilayers through the hemifusion approach, leading to promising advancements. Hemifusion between a supported lipid bilayer (SLB) and giant unilamellar vesicles (GUVs) allows for the exchange of outer leaflet lipids between the vesicles and the SLB. Consequently, using a procedure that controls the sizes of GUVs improves consistency in the percentage of lipid exchange during the creation of asymmetric GUVs.