Mechanisms and Factors\nInfluencing the Production\nof Uniform-Sized Giant Unilamellar Vesicles by Discrete Lipid Film\nArrays

In this paper, we innovatively proposed a highly uniform\nvesicle\npreparation scheme based on the intervesicle mechanical self-constraint\neffect of vesicle crowding. By adjusting the spacing of discrete microwell\nstructures, we observed that during the self-assembly of phospholipid\nmolecules in microwells to form giant unilamellar vesicles (GUVs),\nthe scale swelling of the vesicles during the continuous growth process\nwould lead to the crowding of vesicles in adjacent microwells, thus\ninducing the formation of intervesicle mechanical self-constraint\neffect. The results of the experiment showed that this paper obtained\nthe optimized discretized microwell structure (micropillar side: 30\nμm; pitch: 0 μm), and the corresponding lipid mass was\nmeasured and determined, yielding homogeneous giant GUVs of 37.9 ±\n2.0 μm. In this paper, homogenized GUVs (∼40 μm)\nwith different cholesterol concentrations (10, 20, and 30%) were obtained\nby this method, and the above vesicles were subjected to controlled\nelectroporation experiment under external electric fields of 23, 31,\nand 41 kV/cm, respectively. It showed that the mechanical self-constraint\neffect of vesicle crowding induced by patterned microstructures during\nthe self-assembly of phospholipid molecules significantly enhances\nthe size homogeneity of GUVs, which would be helpful for the wide\napplications of GUVs in other areas such as cell-like models and controlled\nrelease of drugs.