Giant unilamellar vesicles (GUVs), single walled, closed phospholipid bilayers &gt;1 µm, have gained interest as cellular model for synthetic biology and drug delivery. The assembly of GUVs in salty solutions, &gt;150 mM Na/KCl is challenging due to the increased adhesion potential. Here, we use high resolution confocal microscopy and large dataset analysis to investigate the effect of temperature, macromolecular polymers, and small molecule chemical identity on the molar yield of GUVs in salty solutions. We find that uniquely, low gelling temperature agarose allows for the assembly of free-floating GUVs at molar yields &gt; 10 % in salty solutions. Fructose and macromolecular polymers such as polyvinyl alcohol (PVA) and ultralow gelling temperature agarose produced GUV yields &lt; 7 %. Analysis of surface images reveal that for some of these other compounds, polymer dewetting results in the formation of a large number of lipid-polymer pseudobuds resulting in a reduction of GUV yield. To explain the effects of polymers on the assembly of GUVs, we propose a free energy model of GUV assembly. Experimental data with modulation of the adhesion potential by varying ion concentration and valency supports our model prediction – osmotic pressure from dissolving polymers can reduce the free-energy for bud formation. The results presented herein provide experimental and theoretical framework to guide future studies and provide a quantitative comparison between established and novel methodology.

Membrane interactions and osmotic pressure of agarose enables high yield assembly of cell-like giant phospholipid vesicles in salty solutions

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