Membrane electropermeabilization effects of frequency and membrane surface order on liposome leakage

Liposomes containing fluorescence marker were exposed to an alternating electric field of 80 V peak to peak square electric waves at different frequencies 0.01, 1, and 100 kHz to perturb the liposome permeation. The efflux of fluorescence dye after application of the electric field was measured by recording the fluorescence emission due to the complex formation reaction between the fluorescence dye and calcium ions in the bulk medium solution. Two independent sets of experiments were conducted: 1) calcium ions were present during electropulsation; and 2) they were added after electric field application. Two parameters, fluorescence emission intensity and increment of temperature of the solution in the chamber, were studied. The effect of membrane surface order on the fluorescence dye leakage from the liposomes was studied by addition of urea at threshold concentration before the liposomes sealed. The data demonstrate the existence of frequency dependency window at 1 kHz. Furthermore, the data were interpreted according to the theory of interactions of electromagnetic fields with highly polarized and deformed materials such as liposome particles. The urea caused an enhancement of the fluorescence dye leakage at frequency of 100 kHz. This effect could be explained as a decrease of the membrane binding rigidity due to the disordering effect of urea on the membrane lipid surface. Our conclusion is that the frequency and the membrane surface order are additional parameters that influence the processes of membrane electropermeabilization.