Spontaneous formation of giant unilamellar liposomes in gentle hydration process, as well as the adhesion energy between liposomal membranes, has been found to be dependent on the concentration of divalent alkali cations, Ca2+ or Mg2+, in the medium. With electrically neutral phosphatidlycholine (PC), Ca2+ or Mg26+ at 1- 30 mM greatly promoted liposome formation compared to low yields in nonecletrolyte or potassium chloride solutions. When negatively charged phosphatidylglycerol (PG) was mixed at 10%, the yield was high noneletrolytes but liposomes did not form at 3-10 mM CaCl2. In the adhesion test with micropipette manipulation, liposoma membranes adhered to each other only in a certain range of CaCl2 concentrations, which agreed with range where liposome did not form. The adhesion range shifted to higher Ca2+ concentrations as the amount of PG was increased. These results indicate that the divalent cations bind to and add positive charges to the lipids, and that membranes are separated and stabilized in the form of unilamellar liposomes when bet charges on the membranes produce large enough electrostatic repulsion. Under the assumption that the maximum of adhesion energy within an adhesive range corresponds to exact charge neutralization by added Ca2+ were estimated at 7.3 M-1, respectively, in good agreement with literature values.
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