Characterisation of small unilamellar vesicles produced in unsonicated phosphatidic acid and phosphatidylcholine-phosphatidic acid dispersions by pH adjustment

Abstract

As the pH of phosphatidic acid and mixtures of phosphatidylcholine and phosphatidic acid dispersed in water is raised vesicles are formed. These vesicles are stable when the pH is returned to neutrality. Dispersions of phosphatidic acid transiently raised to pH 10–11 have been resolved by Sepharose 4B column chromatography into large structures ( > 100 nm ), which elute at the void volume, and small vesicles ( < 60 nm ), which elute at 0.44–0.48 column volumes (Hauser, H. and Gains, N. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1683–1687). A more detailed characterisation of these vesicles has now been made by including 22Na +, 35SO 4 2− and the fluorochromes, pyranine and harmaline, in the aqueous dispersion before the pH was raised. The encapsulated and unencapsulated ions were then separated, with the vesicles, by Sepharose 4B column chromatography. The material eluting in the void volume consists mainly of unilamellar vesicles with an encapsulated water phase of approx. 15 l per mole of phospholipid. The small vesicles formed from phosphatidic acid encapsulate approx. 0.5 l/mol. Their size is increased by increasing the concentration of sodium chloride in the initial dispersion medium and decreased by both increasing the maximum pH to which the dispersion is raised and increasing the rate of the pH change. Rapidly (in about 1 s) raising the pH to 9 or above results in more than 70% formation of small vesicles. This percentage is decreased in the presence of sodium chloride and increased by both increasing the maximum pH to which the dispersion is raised and increasing the rate of the pH change. The sodium salt of dilauroylphosphatidic acid does not form small vesicles (less than 60 nm) when the pH is rapidly raised to 11, unless the sodium ions have been removed by acid washing. The half-time of efflux of 22Na + and 35SO 4 2− from these vesicles is of the order of days.