The ability of a molecule to pass through the plasma membrane without the aid of any active cellular mechanisms is central to that molecule's pharmaceutical characteristics. For over a century, the passive transport of small molecules through lipid bilayers has been understood in the context of Overton's rule, which broadly states that more lipophilic molecules cross bilayers more readily. More precisely, the consensus holds that membrane permeability is proportional to the product of the molecular diffusivity D and the oil-water partition coefficient K of the permeating species. So oil-water partition coefficients have been widely used to estimate the membrane permeability.
Standard techniques to observe passive transport processes are flawed and lack reproducibility. Planar lipid bilayers and liposomes are the two most frequently used systems. Planar lipid bilayers are physically unstable and contain residual solvent molecules, which introduce artifacts to the permeation results. Techniques used to test liposome permeation include NMR-based and luminescence methods. The NMR-based technique is conducted in steady-state, and cannot yield dynamic information. This technique also makes use of radioactive chemicals and metals ions, making it relatively complex to implement. Luminescene-based approaches rely on heterogeneous ensembles of liposomes which produce imprecise results.
Another severe limitation of current approaches is the unstirred boundary layer (USL) adjacent to the artificial membrane. The unstirred layer can be though of as a region adjacent to the membrane in which the fluid is static (i.e. there is no convective mixing) and diffusive transport dominates. The USL represents a significant resistance to permeant transport, restricting the permeation coefficients that can be measured. The essence of the USL problem is that while the bulk concentrations on either side of the membrane are easily measured, it is the concentrations directly adjacent to the membrane that determine the membrane flux and that therefore must be known in order to determine the membrane permeability.
We have developed a straightforward solution to the problem of transport artifacts in lipid membrane permeability measurements. Spinning-disk confocal microscopy (SDCM) of giant unilamellar lipid vesicles (GUVs) allows for fluorescent molecules to be tracked as they permeate the lipid bilayer membrane and enter the GUV interior. This approach allows for the temporal development of the concentration field to be directly observed, and further allows us to establish the complete time course of the evolution of the concentration profile. Precise membrane permeability can be determined easily from the transient concentration profile data by fitting the data to a mathematical permeation model.
A series of molecules of increasing hydrophiliciy was constructed by covalently modifying the dye 4-nitrobenzo-2-oxa-1,3-diazole (NBD) with polyethylene glycol (PEG) having 4, 8, or 12 repeating units. Transport of modified NBD molecules was observed by tracking NBD fluorescence as the molecules passed through the GUV membrane. An initial image was captured immediately following addition of GUVs to PEG-n-NBD solution. Subsequent images were taken at regular intervals thereafter. Images were first corrected for illumination heterogeneity by flat-fielding in reference to an image of an empty (no GUVs) field at the same fluorophore concentration. Fluorescence leakage from outside the focal plane was corrected by subtracting internal intensity signal taken from a GUV with the same diameter as the experimental GUV in the presence of the non-penetrating fluorophore fluorescein-dextran. An analytical passive transport model was devised, image intensity data was regressed to the model, and permeability was calculated for each species. Membrane permeability shows that longer chain PEG molecules, which have a smaller octanol-water partition coefficient, permeate more slowly. This trend is consistent with Overton's rule, though it does not seem to fit a simple partition-diffusion model of membrane transport. Finite element modeling (FEM) was used to simulate the experiments. The simulation supported the experimental results well.
Low-molecular-weight carboxylic acids are major end products of metabolism in the human body. They are important in several physiological systems, including the immune system and in blood pressure regulation. Their pharmacological function depends on reaching specific intracellular sites. Some of them have crucial effects on cellular processes such as oxidative apoptosis, phosphorylation, and photosynthesis. The transport of these acids across model cell membranes has consequently been widely studied, but these studies have produced wildly varying values of membrane permeability. We use confocal microscopy to image the transport of carboxylic acids with different lengths of carbon chains into GUVs. Fluorescein-dextran (40kDa), which is a pH sensitive dye, was encapsulated in GUVs to trace the transport of acid. GUVs were immobilized on the surface of a poly(dimethylsiloxane) (PDMS) microchannel by biotin-avidin binding. The PDMS channel was designed with two inlets (allowing for injection of either acid-containing or acid-free buffer) and one outlet. The channel had a width of 1mm, depth of 100Ám, and length of 1cm. This PDMS channel allows the changing of buffer solution to acid solution quickly and uniformly, avoiding nonuniform solution mixing problems which would otherwise introduce artifacts to membrane permeability measurements. Confocal microscopy allows for the interior of GUVs to be trivially distinguished from their exterior. Acid molecules in the exterior space can therefore be optically tracked as they permeate the membrane and enter the GUV interior. By using SDCM, the entire field is captured in a short (< 100 ms) exposure. This allows for the temporal development of the entire concentration field to be tracked at all times with great precision. The results showed that as the chain lengths of acids increase, their permeation through lipid membrane become faster. The permeabilities are consistent with literature octanol-water partition coefficients and demonstrate that Overton's rule applies for this class of molecules.