(694d) Engineering Asymmetric Lipid Bilayers in Giant Vesicles

Synthetic lipid bilayer vesicles have been essential tools for investigating the biophysical properties of the cell membrane. These structures, however, differ from the actual cell membrane in that both leaflets (or monolayers) of the lipid bilayer have the same lipid composition. In eukaryotic cells, the inner leaflet is concentrated in charged lipids while the outer leaflet is concentrated in glycolipids and sphingolipids. This asymmetry has profound implications for interactions between the cell and its environment and for cell signaling processes.

We have developed a microfluidic layer-by-layer fabrication technique that allows for the controlled assembly of giant vesicles (diameter 10-50 micrometers) with asymmetric bilayer membranes. This technique is based on a droplet flow configuration in which microdroplets of water are formed in an oil flow stream in the presence of lipid surfactant, forming the inner leaflet. The outer leaflet is then added off-device by transfer of the droplets from oil to water.

Membrane asymmetry is confirmed by differential fluorescence quenching experiments. The response of fluorescently labeled lipid headgroups to a charged (i.e. bilayer impermeable) chemical photoquencher is observed as a function of which leaflet the lipids are localized to. This simple technique also allows for the measurement of the rate of lipid flip-flop diffusion from one leaflet to the other. Asymmetry is further confirmed by binding of labeled annexin V to membranes formed with an asymmetric composition of the negatively charged lipid phosphatidylserine.

These asymmetric giant vesicles are accessible to optical microscopy, calorimetry, and micropipette aspiration. These experimental techniques allow for the detailed study of the effects of membrane asymmetry on the properties of biomembranes, including passive transport of small molecules, structure formation in lipid mixtures, and membrane bending mechanics.