Membrane Permeability of Peptides and Drugs

Abstract

Amphipathic peptides bind to the outer leaflet of membrane bilayers. Do they translocate across the membrane? The answers are directly related to their functions in the case of cell-penetrating peptides and drugs, or to their mechanisms in the case of AMPs. To our knowledge, few permeability experiments have been performed so far, probably because of the difficulty of detecting the peptides and also because a meaningful test must be conducted at biologically relevant concentrations which are usually low. In the case of pore forming peptides, the test must be performed without pore formation. Here we demonstrate a membrane permeation experiment for a peptide drug NYAD-1 (an HIV-1 inhibitor). There are several factors that make this experiment possible. We used a FITC labeled peptide called NYAD-2 for the peptide detection. We discovered that it is possible to produce GUVs in pH 9, although not in pH 7. We found that the intensity of FITC in pH 9 is 1.7 times higher than in pH 7. By using an aspirated GUV, we measured the relative binding coefficients of NYAD-2 to the GUV in pH 7 and pH 9, and found that the former is 2 to 3 times higher than the latter. With all these provisions, we performed the permeability test by transferring an aspirated GUV produced in pH 9 solution containing Texas red dextran (TRD) MW 625 to a solution of pH 7 containing 2 micro-Mole NYAD-2. We found the concentration of NYAD-2 inside the GUV increased with time while the membrane remained intact and there was no leakage of TRD. We are able to extend this method to test the membrane permeability of other peptides and drugs such as Melittin.