Dynamics of giant vesicle assembly from thin lipid films

Giant unilamellar vesicles (GUVs) are micrometer-scale lipid assemblies that emulate key characteristics of biological cell membranes. GUVs can be obtained when solid-supported thin films of lipids are hydrated in aqueous solutions. However, a comprehensive understanding of their assembly dynamics has been lacking, impeding mechanistic insights. Here, we report the time dependence of the distribution of sizes and molar yield of GUVs obtained through a novel stopped-time technique. We compare three commonly used techniques, PAPYRUS (Paper-Abetted amPhiphile hYdRation in aqUeous Solutions) gentle hydration, and electroformation. We demonstrate that all three techniques show sigmoidal yield curves. Yields increase monotonically before reaching a plateau, with surprisingly high yields 60 seconds after hydration. Gentle hydration shows limited time evolution in contrast to PAPYRUS and electroformation. Exploration of bud dynamics on the surfaces uncovers bud emergence, diameter growth, and merging phenomena. To provide a comprehensive explanation of our observations, we employ the thermodynamic budding and merging model. This work expands our understanding of GUV assembly dynamics and offers fundamental insights into the underlying thermodynamic principles governing this process.