Abstract Giant unilamellar vesicles serve as membrane models and primitive mockups of natural cells. With respect to the latter use, amphiphilic polymers can be used to replace phospholipids in order to introduce certain favorable properties, ultimately allowing for the creation of truly synthetic cells. These new properties also enable the employment of new preparation procedures that are incompatible with the natural amphiphiles. Whereas the growth of lipid compartments to micrometer dimensions has been well established, growth of their synthetic analogs remains underexplored. Here, the influence of experimental parameters like salt type/concentration and magnitude of agitation on the fusion of nanometer‐sized vesicles made of poly(dimethylsiloxane)‐poly(ethylene oxide) graft copolymer (PDMS‐ g ‐PEO) is investigated in detail. To this end, dynamic light scattering, microscopy, and membrane mixing assays are employed, and the process at different time and length scales is analyzed. This optimized method is used as an easy tool to obtain giant vesicles, equipped with membrane and cytosolic biomachinery, in the presence of salts at physiological concentrations.
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