Imaging Molecular Transport Across Lipid Bilayers

Abstract

The passive transport of small molecules across the plasma membrane is a key physiological process. Literature measurements of membrane permeability to small molecules have varied widely. We used confocal microscopy to image the transport of molecules into a giant unilamellar lipid vesicle (GUV). Fluorescent dyes were used to trace the transport of molecules. The GUV was immobilized on the surface of a microfluidic channel by biotin-avidin binding. This microchannel allows the rapid and uniform exchange of the solution surrounding the GUV. Using a spinning-disk confocal microscope, the entire concentration field is captured in a short exposure. We used this system to study the passive transport of carboxylic acids, which have many properties common to small-molecule drugs. The transport of these acids across cell membranes has been widely studied, but there is much variation in the reported permeabilities. By using pH-sensitive fluorescein-dextran to track the acids permeating through the GUV membrane, our results showed that more lipophilic acids cross the bilayer more quickly. A finite difference model was developed to simulate the experimental process and derive precise permeability values. The permeabilities change with the same trend as oil-water partition coefficients, demonstrating that Overton's rule applies to this class of molecules. We used the imaging technique described above to study the transport of protons across compositionally asymmetric lipid bilayers. Synthetic asymmetric GUVs were prepared via a microfluidic multiphase droplet flow technology to mimic membrane charge asymmetry. Negatively charged phosphatidylserine was added to an asolectin GUV on either the internal or external leaflet. The permeation rates of protons into and out of these GUVs were measured. The proton distribution across the asymmetric GUV membrane at equilibrium was also studied. This research can reveal how asymmetric cell membrane composition affects small molecule transport behavior in physiological processes.