(732f) Continuous Microfluidic Fabrication of Synthetic Asymmetric Vesicles As Biological Membrane Model Systems

Engineered synthetic vesicles (i.e. asymmetric liposomes) support comprehensive in vitro studies on membrane organization and associated biological processes (e.g. signaling, adhesion, protein transport, endocytosis, etc.). Membrane vesicles are spherical structures consisting of a single lipid bilayer enclosing an aqueous lumen. In nature, biological membranes possess many different lipid compositions with some degree of lipid asymmetry across the bilayer. A robust strategy for preparing customizable asymmetric vesicles will have a significant impact on basic and applied research in membrane biology because they more accurately represent the complexity of natural membranes. Here, we report a novel microfluidic-based fabrication process to build synthetic asymmetric vesicles while simultaneously controlling membrane unilamellarity, monodispersity, vesicle size, and luminal content.

Our microfluidic device consists of a triangular post region and two flow-focusing regions that can generate synthetic vesicles at high-throughput. The vesicle fabrication process has four steps: (1) generate highly monodispersed water-in-oil (w/o) emulsions in an oil/inner-leaflet-lipid solution that serve as precursors to form asymmetric vesicles based on the spontaneous assembly of lipid molecules; (2) replace the oil/inner-leaflet-lipid solution that surrounds the w/o emulsions with an oil/outer-leaflet-lipid solution inside the triangular post region; (3) form water-in-oil-in-water (w/o/w) double emulsions; and (4) extract excess oil/outer-leaflet-lipid solution from the double emulsions. Bilayer membrane asymmetry and unilamellarity are assessed by conducting a fluorescence quenching assay and an α-hemolysin (α-HL) protein insertion assay, respectively. Our approach addresses many of the deficiencies found in current technologies for building vesicles. This method enables us to observe the vesicle formation process on-chip (i.e. due to the transparency of the polydimethylsiloxane microfluidic device) and conduct off-chip experiments immediately after vesicle formation (i.e. since the last step for forming the vesicles occurs off-chip). Over 80% of the vesicles remained stable for at least 6 weeks and the membrane asymmetry was maintained for over 30 hours. The asymmetric vesicles built using this strategy are used to investigate fundamental properties of membranes, lipid-protein interactions, and can be used as drug delivery vehicles.