Optimization of the Inverted Emulsion Method for High‐Yield Production of Biomimetic Giant Unilamellar Vesicles

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‐based method is one such technique, which has great potential for rapid production of GUVs with high encapsulation efficiencies for large biomolecules. However, the lack of understanding of various parameters that affect production yields has resulted in sparse adaptation within the membrane and bottom‐up synthetic biology research communities. Here, we optimize various parameters of the inverted emulsion‐based method to maximize the production of GUVs. We demonstrate that the density difference between the emulsion droplets, oil phase, and the outer aqueous phase plays a crucial role in vesicle formation. We also investigated the impact that centrifugation speed/time, lipid concentration, pH, temperature, and emulsion droplet volume has on vesicle yield and size. Compared to conventional electroformation, our preparation method was not found to significantly alter the membrane mechanical properties. Finally, we optimize the parameters to minimize the time from workbench to microscope and in this way 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.

Characterization of lipid composition and diffusivity in OLA generated vesicles

“…Remarkably, we found no significant differences (p < 0.01) in lateral lipid diffusion coefficients between vesicles with or without an octanol pocket. This is an interesting parallel to research by Karamdad et al and Moga et al who compared the bending rigidity of electroformed vesicles to those obtained with a microfluidic technique (using squalene oil) and a phase transfer method, respectively [ 52 , 53 ]. While lipid asymmetry has been shown to alter the bending rigidity of the membrane significantly [ 54 , 55 ], the presence of residual oil in both the microfluidic as well as the phase transfer method do not seem to significantly alter membrane properties [ 52 , 53 ].…”

A Flavone-Based Solvatochromic Probe with A Low Expected Perturbation Impact on the Membrane Physical State

“…One of the most recently developed methods is the “droplet transfer” or “reverse emulsion” [ 26 ], characterized by the stratification of an aqueous phase and a water-in-oil emulsion with the formation of the bilayer at the interface of the two phases. This method permits to easily encapsulate hydrophilic compounds in the aqueous lumen [ 27 , 28 ] and blend hydrophobic species in the membrane, such as fatty acids [ 29 , 30 ] or polymers [ 31 , 32 ]. In this work, the “droplet transfer” method was used for the formation of vesicles of POPC and a ternary mixture of POPC:DPPC:Chol to simulate the two phases Lo and Ld of a membrane.…”

Multivesicular Vesicles: Preparation and Applications

“…Moreover, the presence of small amounts of remaining oil within the vesicle bilayers is possible. Another disadvantage is the need to impose a density gradient (for example using relatively high concentrations of sucrose in the inner phase aqueous medium and glucose in the external aqueous phase) [16,74] . Furthermore, the composition of the amphiphiles in the final vesicle preparation might be different from the expected composition on the basis of the amphiphiles used [74] .…”