Analysis of the response of vesicles to the incorporation of transportan into POPC membrane

J.Majhenc1, G.Gomišček1, U.Langel3, S.Svetina1, M.Zorko2 and B.Žekš1

1 Institute of Biophysics, Faculty of Medicine, Univ. of Ljubljana, Slovenia; 2 Institute of Biochemistry, Faculty of Medicine, Univ. of Ljubljana, Slovenia; 3 Dept. Neurochemistry Neurotoxicology, Stockholm University, Sweden

Several cell-penetrating peptides (CPP) that can be used for drug delivery have recently been developed. One of them is transportan (TP), consisting of the active part of neuropeptide galanin attached to mastoparan, the component of wasp venom. TP can efficiently enter cells even at very low concentrations (nM range) and accumulates mainly in cellular membranous structures. Several models of the internalization mechanism of CPP were proposed (reverse micelles, pore formation), but the mechanism of membrane translocation has not been established. We studied the effect of TP on POPC membranes at relatively high concentrations by monitoring the changes of vesicle shapes and the transport of small (sugars) and large (dextrans) molecules across the membrane of giant vesicles. The permeability of the membrane was determined through the time course of halo effect or vesicle internal fluorescence. There was no effect well below the critical micelle concentration (CMC, 20 µM). Near CMC, the decrease in the intensity of the fluorescence and brightness of halo was observed. At intermediate TP concentrations (~30 µM) vesicles destructions were observed. At higher concentrations (above 50 µM) vesicles survives, but the internal fluorescence and halo fade in few seconds. The increase of the membrane surface was also observed. The behavior of vesicles (relaxation by the consequent transition tension pores, destruction and final survival) was modeled by assumption of forming of TP induced pores, where the size and the number of pores were TP concentration dependent. The model predicts, that destruction of a vesicle is possible only if the difference in the solute dimension and the concentration are large enough. The size of TP induced pores should allow large permeability, but the diameter of the pore must still be comparable to the diameter of solute molecules. For the used system (0.2 M sucrose/glucose solution) the diameter of pores between 1.5 nm and 5 nm was determined.

Email: janja.majhenc@biofiz.mf.uni-lj.si

Address: Janja Majhenc, Inštitut za biofiziko, Lipičeva 2, 1000 Ljubljana