Novel Method for Obtaining Homogeneous Giant Vesicles from a Monodisperse Water-in-Oil Emulsion Prepared with a Microfluidic Device

Abstract

A novel technique called the "lipid-coated ice droplet hydration method" is presented for the preparation of giant vesicles with a controlled size between 4 and 20 microm and entrapment yields for water-soluble molecules of up to about 30%. The method consists of three main steps. In the first step, a monodisperse water-in-oil emulsion with a predetermined average droplet diameter between 4 and 20 microm is prepared by microchannel emulsification, using sorbitan monooleate (Span 80) and stearylamine as emulsifiers and hexane as oil. In the second step, the water droplets of the emulsion are frozen and separated from the supernatant hexane solution by precipitation, followed by a removal of the supernatant and followed by the replacement of Span 80 by using a hexane solution containing egg yolk phosphatidylcholine, cholesterol, and stearylamine (5:5:1, molar ratio). This procedure is performed at -10 degrees C to keep the water droplets of the emulsion in a frozen state and thereby to avoid extensive water droplet coalescence. In the third step, hexane is evaporated at -4 to -7 degrees C and an external water phase is added to the remaining mixture of lipids and water droplets to form giant vesicles that have an average size in the range of that of the initial emulsion droplets (4-20 microm). The entrapment yield and the lamellarity of the vesicles obtained depend on the lipid/water droplet ratio and on the composition of the external water phase. At high lipid/water droplet ratio, the giant vesicles have a thicker membrane (indicating multilamellarity) and a higher entrapment yield than in the case of a low lipid/water droplet ratio. The highest entrapment yield ( approximately 35%) is obtained if the added external water phase contains preformed unilamellar egg phosphatidylcholine vesicles with an average diameter of 50 nm. The addition of these small vesicles minimizes the water droplet coalescence during the third step of the vesicle preparation, thereby decreasing the extent of release of water-soluble molecules originally present in the water droplets. The GVs prepared can be extruded through polycarbonate membranes to yield large unilamellar vesicles with about 100 nm diameter. This size reduction, however, leads to a decrease in the entrapment yield to about 12% due to solute leakage from the vesicles during the extrusion process.

Giant vesicles: a powerful tool to reconstruct bacterial division assemblies in cell‐like compartments

“…However, the efficiency of macromolecular entrapment is low, and the vesicles produced by this technique are very polydispersed in size (Table 1). Dimitrov, 1986, 1992;Merkle et al, 2008;Pott et al, 2008;Montes et al, 2010;Stachowiak et al, 2012) (Stachowiak et al, 2008;Li et al, 2009) Lipid-coated ice droplets hydration High Controlled monodispersity High Simple equipment (Sugiura et al,2008;Kuroiwa et al, 2012) a. For a more comprehensive view on advantages and disadvantages of these methods the reader is referred to table 3 in Walde et al, 2010. b.…”

“…The production of giant vesicles based on a water-inhexane emulsion formed in a microfluidic device, and stabilized by surfactants, has been developed (Sugiura et al, 2008;Matosevic, 2012). This technique (lipidcoated ice droplet hydration) which produces vesicles of controlled sizes (from 4 μm and 20 μm) consists in four steps: emulsification, surfactant replacement, solvent evaporation, and, finally, hydration.…”

Cross-linking effect of hydrophobic cores on morphology of giant vesicles formed by amphiphilic random block copolymers

“…Such giant vesicles formed by the reconstruction of natural lipids have been widely used for the purpose of elucidating the properties and functions of the biomembranes [32][33][34][35][36][37][38][39]. In recent years, the giant vesicles formed by synthetic polymer surfactants of amphiphilic random block copolymers have been prepared by the photopolymerization-induced self-assembly technique [40][41][42][43][44][45][46].…”

CFD analysis of microchannel emulsification: Droplet generation process and size effect of asymmetric straight flow-through microchannels

“…Advantages of these microfluidic emulsification techniques include the generation of uniform droplets, the precise control of droplet size and droplet shape, and in situ microscopic monitoring that allows fine tuning of the process parameters during droplet generation. In addition, the size uniformity of emulsion droplets is required for making monodisperse dispersions containing microparticles (Sugiura et al 2001a;Xu et al 2005), microcapsules (Nakagawa et al 2004;Utada et al 2005;Seiffert et al 2010), or other new classes of micromaterials (Yi et al 2003;Nie et al 2006;Nishisako and Torii 2007;Sugiura et al 2008). Traditional emulsification devices (e.g., colloid mills, high-pressure valve homogenizers, and ultrasonic homogenizers) use intensive force to repeatedly break up larger droplets into smaller ones (McClements 2004).…”