Effect of membrane cholesterol on calcium phosphate formation in aqueous suspensions of anionic liposomes

Summary

The present study examined the effect of membrane cholesterol on liposome-mediated calcium phosphate precipitation in metastable aqueous solutions (2.25 mM Ca²⁺ and 1.5 mM inorganic phosphate) at 22°C, pH 7.4 and 240 mOsm. The liposomes were prepared from 7:2:X molar mixtures of phosphatidylcholine, dicetylphosphate, and cholesterol (x=0, 1, 5, or 9) and contained either 0 or 50 mM encapsulated phosphate. The membranes were made permeable to Ca²⁺ by addition of the cationophore, X-537A. Changes in external Ca²⁺ concentration were used as the principal monitor of the course of precipitation. Without encapsulated phosphate, 7:2:X liposomes (with or without ionophore) induced no precipitation. With 50 mM encapsulated phosphate and in the presence of ionophore, precipitation significantly depended on the cholesterol level in the membrane. At 0 and 10 mole% cholesterol, precipitate developed rapidly both within and outside the liposomes. At 35 and 50 mole% cholesterol, no observable intraliposomal precipitation occurred, and extraliposomal precipitation started only after an induction period of 24 hours. Delayed extraliposomal precipitation also took place in PO₄-containing liposomes without added ionophore. In this latter case, however, cholesterol was essential for this precipitation to occur with the optimum level being around 10 mole%. Suppression of ionophore-mediated intraliposomal precipitation at higher cholesterol levels could be related to the inflexible cholesterol molecules making the membrane more rigid, thereby restricting Ca-ionophore transport. This restriction could be reversed with ethanol. Delayed extraliposomal precipitation in the absence of added ionophore (or at higher cholesterol levels in its presence) could be explained by seeding from low, unobserved levels of intraliposomal precipitate formed during slow, unfacilitate Ca²⁺ leakage into the liposomal interior.