Description
Bottom-up synthetic biology aims to construct synthetic cells that recapitulate, understand, and repurpose biological behaviors. Phospholipid giant vesicles (GVs) represent an excellent model for a simplified cell membrane and have been extensively explored in the field. To produce meaningfully biomimetic compartments, GVs need to be cell-sized, unilamellar, and need to encapsulate molecules of interest in high yield. Double emulsion templated techniques excel at this, guaranteeing vesicle unilamellarity, control over size, and near 100% encapsulation efficiency, representing the state-of-the-art for the formation of GV-based synthetic cells. The transition from thin-shell double emulsion to a phospholipid bilayer involves de-wetting of the oil carrier from the membrane, which is governed by the spreading coefficient of the oil over the two aqueous phases. This balance of surface tensions needs to favor contacts between the inner and outer aqueous phases, mediated by a phospholipid bilayer, compared to oil-water interfaces stabilized by a lipid monolayer. This constrains the composition of the outer aqueous phase to include additional surfactants that lower the interfacial tension between it and the oil carrier. The spreading coefficient assigns a thermodynamically favored endpoint to the system. However, thermodynamics alone do not account for the kinetics of oil removal. De-wetting initiation is a stochastic process that requires adhesion of the two lipids monolayers to begin [1]. The monolayer contact area expands, driven by the balance of interfacial tensions, until a droplet-associated GV is formed, without full detachment of the oil droplet from the underlying aqueous compartment. Our results show these droplet-associated GVs are stable in suspension for several days and full de-wetting can be achieved by mechanical means (e.g. narrow channels in microfluidic devices, centrifugation) or osmotic deflation of the vesicle [2,3]. To date the inconsistency of full de-wetting is a major bottleneck in the wide adoption of microfluidic techniques to assemble synthetic cells.
We aim to address this issue systematically, by characterizing oil de-wetting dynamics and putting them in relation with the oil content of the double emulsion, membrane composition, and surfactant presence in the outer aqueous phase. Lipid composition in particular is critically underexplored and represents a promising avenue for limiting the use of additional surfactants. These results further our understanding of the kinetic barriers to oil removal, ultimately enabling more robust microfluidic assembly of synthetic cells.