Probing the Molecular Mechanism of Passive Transport

The passive transport of small molecules across the plasma membrane is the major pathway by which orally delivered drugs enter circulation and a major route of delivery into target cells. While molecular dynamics simulations suggest that molecules diffusing through lipid bilayers are subject to a complex, dynamic environment, the analytical framework for describing passive transport continues to treat the membrane as a uniform material. The study of passive transport is complicated by the shortcomings of current experimental techniques, in which transport tends to be dominated by diffusion through a stagnant layer adjacent to the membrane.We have developed an approach to probing passive transport that eliminates these artifacts and allows for a study of relationships between molecular structure and transport properties. This approach consists of confocal microscopy of the diffusion of small molecules into giant unilamellar lipid vesicles (GUVs). Experiments and finite element models show that due to small size of GUVs relative to the characteristic diffusive length scale of transported molecules, no significant stagnant layer is established. In addition, confocal imaging allows for observation of the steady-state association of diffusing molecules with the membrane itself, while other technologies only allow for detection of transported molecules.We have concurrently developed a fabrication technology that yields GUVs in which each leaflet of the bilayer has a different lipid composition. This allows a novel investigation of the relevance of the asymmetry of the plasma membrane to passive transport.A series of fluorescent molecules of varying hydrophobicity was synthesized. Time-series images of these molecules crossing GUV membranes were captured, and these images were fit to both an analytical model of membrane permeation and a finite element model of permeation with diffusion. Lipid composition was varied to reproduce the range of compositions observed in human plasma membranes.