An ultrastructural study of calcium phosphate formation in multilamellar liposome suspensions

Calcium phosphate precipitation can be induced within liposomes containing buffered inorganic phosphate by the ionophore-mediated loading of calcium ions. Negative staining, positive staining for thin sectioning, and freeze-fracture electron microscopy were used to characterize these synthetic vesicles and to evaluate the liposome-mineral interactions resulting from apatite formation. Suspensions of phosphate (0-50 mM KH2PO4)-encapsulated liposomes were prepared from mixtures of phosphatidylcholine, dicetyl phosphate, and cholesterol in the molar ratios of 7:2:1. Precipitation reactions were initiated by first suspending the liposomes in a buffered solution containing calcium (1.3-2.2 mM Ca(NO3)2) and then adding the cationic ionophore X-537A. All experiments were carried out at 22 degrees C, pH 7.4, and 240 mosm. Transmission electron microscopical analysis showed that the liposome preparation consisted of multilamellar, multicompartmental vesicular structures. The liposomes were typically heterogeneous with respect to both the size and number of phospholipid bilayers surrounding the aqueous cores. In Ca-loaded liposomes, discrete clusters of apatite mineral were present within the lumen, and in close proximity to the inner lipid membranes. These nascent crystallites eventually penetrated the lipid envelope to provide a focus for external precipitation events. Crystalline apatite phases were not observed when the incubation conditions prevented intraliposomal precipitation. The de novo calcification of these liposomes had many features in common with the sequence of mineral deposition occurring in matrix vesicle-mediated calcification. These results reinforce the conclusions of earlier chemical and kinetic studies and further support the use of this system as an experimental model for examining the membrane-mineral interactions associated with tissue mineralization.