(425j) High Throughput Fabrication of Synthetic Asymmetric Bacterial Membranes

Giant unilamellar vesicles (GUVs) are of great interest as versatile model systems to emulate the function and fundamental properties of the plasma membrane in living cells. The double-membrane cellular envelope in Gram-negative bacteria enables them to endure harsh environments and represents a barrier to many clinically available antibiotics. The outer membrane (OM), which is exposed to the environment, is believed to be the first point of contact involved in fundamental bacterial processes such as signaling, pathogenesis, and motility. In this work, we report on a microfluidic technique for synthesizing monodispersed asymmetric GUVs possessing the Gram-negative bacterial OM lipid composition. As in the cytoplasmic membrane, the OM in Gram-negative bacteria has a phospholipid-rich inner leaflet; however, the outer leaflet is predominantly composed of lipopolysaccharide (LPS).

Our continuous microfluidic fabrication technique generates 50 to 100 µm diameter water-in-oil-in-water double emulsions at high-throughput. The water-oil and oil-water interfaces facilitate the self-assembly of phospholipid and LPS molecules to create the inner- and outer-leaflet of lipid bilayer, respectively. Bilayer membrane asymmetry is evaluated using a fluorescence quenching assay. Our approach addresses many of the deficiencies found in existing biotechnological techniques for building vesicles possessing strong membrane asymmetry. The effect of cholesterol and divalent metal ions (i.e. Mg²⁺) are investigated on the membrane stability. The GUVs built using this strategy are collected off-chip and transferred to an aqueous solution to be studied as bacterial model membranes in biological processes.