Distribution of phospholipids and triglycerides in multivesicular lipid particles

Abstract

The distribution of phospholipid and triglyceride molecules in the membranes forming the nonconcentric vesicular network within a multivesicular lipid particle (MLP) and its precursors was investigated. MLP formulations afford controlled release of encapsulated pharmaceuticals over a time period ranging from a few days to a few weeks. The formation of MLP requires the use of a double emulsification process and a neutral lipid such as a triglyceride. MLP formulations with the antineoplastic agent cytarabine encapsulated in the aqueous compartments were prepared that further contained [13C]carbonyl-enriched triolein, and quantitative 13C nuclear magnetic resonance (NMR) experiments were performed to quantitate membrane-soluble and bulk triglyceride. In the NMR spectra of MLP, components attributable to triolein residing in a location similar to that of bilayer phospholipid were not observed. Spectral components attributable to triolein in a liquid-like phase were observed, and the intensity of these components accounted for the triolein present in the sample. Laser scanning confocal microscopy with two phospholipid fluorescent probes, Rhodamine-DHPE and NBD-PG, and a triglyceride fluorescent probe, Bodipy-Triglyceride, was used to visualize the distribution of the lipid components in the emulsion precursors of MLP and in MLP themselves with cytarabine, amikacin, morphine, or sucrose encapsulated within the aqueous phases. The confocal micrographs revealed a uniform distribution of the two phospholipid probes in the plane of the membrane, whereas the triglyceride probe accumulated at discrete locations. The NMR and microscopy results are consistent with a structural model for MLP in which triolein acts as a hydrophobic space filler at bilayer intersection points and stabilizes these junctions, and is also present as oil droplets dispersed in the encapsulated aqueous compartments. The experimental approach presented here could provide a rational approach to the development of MLP formulations with variable rates of sustained release, modulated by changes in the distribution of various phospholipids and triglycerides.