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Abstract

In the field of bottom-up synthetic biology, lipid vesicles provide an important role in the construction of artificial cells. Giant unilamellar vesicles (GUVs), due to their membrane’s similarity to natural biomembranes, have been widely used as cellular mimics. So far, several methods exist for the production of GUVs with the possibility to encapsulate biological macromolecules. The inverted emulsion method has great potential for rapid production with high encapsulation of biomolecules. However, the lack of understanding of the parameters that affect production has resulted in sparse adaptation within the membrane and bottom-up synthetic biology research communities. Here, we optimize the parameters of the inverted emulsion method to maximize the yield of GUVs. We show that the density difference between the emulsion droplets, oil phase and the outer aqueous phase plays a crucial role in vesicle formation. We investigated the impact that centrifugation speed/time, lipid concentration, pH, temperature, and emulsion volume has on yield and size. Compared to electroformation, our method was not found to alter the membrane mechanics. Finally, we optimize the parameters to minimize the time from workbench to microscope to open up the possibility of time-sensitive experiments. In conclusion, our findings will promote the usage of the inverted emulsion method for basic membrane biophysics studies as well as the development of GUVs for use as future artificial cells.