Calcium phosphate precipitation in aqueous suspensions of phosphatidylserine-containing anionic liposomes

Summary

Liposomes prepared from 6.3∶1.8∶0.9∶ 1.0 molar mixtures of phosphatidylcholine, dicetyl phosphate, cholesterol, and phosphatidylserine, respectively, (PS(+) liposomes) were compared with similarly prepared liposomes without the phosphatidylserine (PS(−) liposomes) for their effect on calcium phosphate precipitate formation in aqueous solutions at pH 7.4 and 22°C. The liposomes, encapsulated with 50 mM phosphate (P_I), were suspended in buffered 2.2 mM CaCl₂, 0 or 1.5 mM KH₂PO₄ solutions and made permeable to Ca²⁺ fluxes with the ionophore, X-537A. External solution Ca²⁺ losses were found to be small in both PS(+) and PS(−) liposome suspensions when no ionophore was added. Even with 1.5 mM P_I in the external solution, these losses did not exceed 0.2 mM. However, inoculating both liposome preparations with X-537A resulted in rapid, appreciable losses in solution Ca²⁺. Previous studies showed that in PS(−) liposomes, these latter losses were due to calcium phosphate precipitation, with the precipitate confined to the interior of the liposomes when no external P_I was present, but extending to outside the liposomes when the suspending medium was rendered metastable. In the present study, Ca²⁺ losses resulting from intraliposomally confined precipitation were found to be marginally greater in PS(+) liposomes due primarily to a larger volume of entrapped P_I available for reaction in these liposomes. However, with the addition of P_I to the external solution, the reverse was observed, i.e., considerably less Ca²⁺ was lost in PS(+) than in PS(−) suspensions, a result of markedly less X-537A-induced precipitate forming outside PS(+) liposomes. The most probable explanation for this latter decrease was a PS-induced adherence of the outer liposome membranes to the surfaces of developing crystals, restricting the avialability of these surfaces as sites for further growth.