Engineering Lipid Self-Assembly to Build Biomimetic Cell Membranes

The lipid bilayer of the eukaryotic plasma membrane is compositionally asymmetric. That is, the outer monolayer of lipids, facing extracellular space, contains different lipids at different concentrations than the inner monolayer, facing the cytoplasm. While the outer monolayer is relatively concentrated in sphingolipids and cholesterol and net neutral, the inner monolayer is concentrated in phosphatidylserine and carries a net negative charge. The cell actively maintains this asymmetry, expending metabolic energy to prevent the membrane from equilibrating to a symmetric bilayer.

While lipid bilayer asymmetry has been long known to be a feature of the cell membrane, there is little agreement on what physiological purpose this asymmetry serves. This is due in part to the difficulty in studying asymmetric bilayers: the tools to build minimal model systems that can accurately mimic cell membrane composition have been lacking.

Here, we present a new technology for fabricating giant unilamellar lipid vesicles (GUVs) with compositional asymmetry. Rather than relying on the standard spontaneous formation methods typically used to fabricate GUVs, we use a microfluidic droplet flow to construct GUVs in a layer-by-layer fashion. In the first step, droplets of aqueous buffer are formed in an oil stream. This stream contains dissolved lipids, which self-assemble at the oil-water interface. These monolayer-coated droplets are then dispensed into another oil phase, which contains another set of dissolved lipids. This second oil phase floats atop more aqueous buffer, and when the droplets pass through this second water-oil interface, a second lipid monolayer is assembled, forming a complete lipid bilayer.

We have demonstrated the facile formation of GUVs using this method and have demonstrated lipid asymmetry in these GUVs using both labeled and natural lipids.

These spherical, cell-sized biomimetic membranes are useful for a range of fundamental membrane biophysical studies, including studies of membrane transport, mechanics, and lipid raft dynamics. They are also a potential foundation for synthetic artificial cells.