Systematic investigation of double emulsion dewetting dynamics for the robust production of giant unilamellar vesicles.

Giant unilamellar vesicles (GUVs) embody biomimetic membranes with compartmentalization and serve as simplified models to better understand complex biochemical and biophysical processes. Recently, double emulsion droplet microfluidics has proven to be a promising platform for their production, offering greater throughput, control, and reproducibility over traditional methods. However, the interplay of parameters that influence the complex multiphase fluid dynamics of the dewetting process has not been thoroughly studied, limiting the democratization of the approach. In this study, we systematically investigate how lipid composition, aqueous phase conditions, droplet confinement, and fluid dynamics promote or impede dewetting. We reveal that successful GUV formation depends on a critical balance between dynamic Marangoni stresses and thermodynamic interfacial forces under confinement. High surfactant concentrations amplify Marangoni flows and necessitate glycerol for vesicle stability. Conversely, reducing surfactant levels minimizes this dynamic barrier to enable rapid on-chip dewetting, yet imposes thermodynamic constraints that are overcome by tuning lipid conditions. Crucially, in the presence of physiological salt, we identify lipid adhesion energy as the governing parameter; increasing membrane packing via saturation successfully overcomes salt-induced inhibition. Our results improve the reliability and accessibility of droplet-microfluidics GUV platforms to catalyze advances in biophysics, synthetic biology, and drug discovery.