LAUSR

All biological cell membranes maintain an electric transmembrane potential of around 100 mV, due in part to an asymmetric distribution of charged phospholipids across the membrane. This asymmetry is crucial to cell health and physiological processes such as intracell signaling, receptor-mediated endocytosis, and membrane protein function. Experimental artificial membrane systems incorporate essential cell membrane structures, such as the phospholipid bilayer, in a controllable manner where specific properties and processes can be isolated and examined. Here, we describe a new approach to fabricate and characterize planar, free-standing, asymmetric membranes and use it to examine the effect of headgroup charge on membrane stiffness. The approach relies on a thin film balance used to form a freestanding membrane by adsorbing aqueous phase lipid vesicles to an oil-water interface and subsequently thinning the oil to form a bilayer. We validate this lipid-in-aqueous approach by analyzing the thickness and compressibility of symmetric membranes with varying zwitterionic DOPC and anionic DOPG content as compared to previous lipid-in-oil methods. We find that as the concentration of DOPG increases, membranes become thicker and stiffer. Asymmetric membranes are fabricated by controlling the lipid vesicle composition in the aqueous reservoirs on either side of the oil. Membrane compositional asymmetry is qualitatively demonstrated using a fluorescence quenching assay and quantitatively characterized through voltage-dependent capacitance measurements. Stable asymmetric membranes with DOPC on one side and DOPC/DOPG mixtures on the other were created with transmembrane potentials ranging from 15 to 80 mV. Introducing membrane charge asymmetry decreases both the thickness and stiffness in comparison to symmetric membranes with the same overall phospholipid composition. These initial successes demonstrate a viable pathway to quantitatively characterize asymmetric bilayers that can be extended to accommodate more complex membranes and membrane processes in the future.