Preparation of Alendronate Liposomes for Enhanced Stability and Bioactivity: In Vitro and In Vivo Characterization

Abstract

Abstract. Liposomes containing bisphosphonates have been shown to deplete circulating monocytes and reduce experimental restenosis. However, acceptable shelf life was not achieved, and the disruption extent and rate of the vesicles in the circulation has not been examined. Designing an optimal liposomal formulation in general, and for an anti-inflammatory effect in particular, requires careful consideration of the factors that contribute to their in vitro stability and integrity in the blood after injection. An improved liposomal alendronate formulation was prepared by a modified thin lipid film hydration technique followed by extrusion, resulting in relatively smaller size vesicles, narrow size distribution, and low drug to lipid ratio in comparison to the reverse phase evaporation method. In order to rule out premature leakage of the drug, the integrity of the vesicles was examined by means of size-exclusion chromatography in vitro and in vivo, with subsequent analysis of size, drug (fractions of encapsulated and free) and lipid concentrations. Vesicles were found to be stable in serum, with 15±3% leakage of the drug after 10 min in rabbit's circulation, and intact liposomes were detected in the circulation 24 h following administration. It is concluded that the new formulation results in increased stability (2.5 years) as determined by the insignificant changes in vesicle size, drug leakage, lipid and drug stability, in vitro bioactivity (macrophages inhibition), as well as in vivo in depleting circulating monocytes and inhibition of restenosis in rabbits. Our in vitro stability results regarding dilution in serum paralleled in vivo data. Thus, in vitro assessment may provide a valuable tool in assessing in vivo integrity of liposomal formulations.

Liposomal Simvastatin Attenuates Neointimal Hyperplasia in Rats

“…After internalization, as have been demonstrated for this type of formulation (14,42,43), lysosomal action disrupts the fatty bilayer of the liposome and simvastatin is released into the cell, causing cell death. Circulating blood monocytes fully recovered 7 days after injection.…”

“…Circulating blood monocytes fully recovered 7 days after injection. The effects of liposomal simvastatin are limited to cells with phagocytic capacity with no effect on other cells, such as SMC or endothelial cells, as has been demonstrated in animals treated with liposomal BPs (12)(13)(14)(15)(16)42,46). Simvastatin leaking from inactivated monocytes and the delivery system would accumulate mainly in the liver.…”

“…The preferable physicochemical properties of monocytetargeted liposomes are those of the so called 'conventional' liposomes, neither hydrophilic nor neutral membrane, and not of ultra-small size (36,(40)(41)(42). A charged membrane, particularly negative, enhances the internalization of liposomes into phagocytic cells through adsorptive endocytosis (36,50), in part following opsonization (39,51).…”

“…A preferential uptake of intact 'conventional' liposomes containing serotonin by circulating monocytes has been demonstrated recently (44). We hypothesized that encapsulation of statins in a suitable liposomal formulation, similarly to liposomal BPs (14)(15)(16)42,(44)(45)(46), will divert the statins from their main target (hepatocytes) to circulating monocytes resulting in systemic inactivation of monocytes and consequently, attenuation of neointimal formation. To the best of our knowledge, there are no reports in the literature on targeting circulating monocytes by a liposomal delivery system of statins for preventing restenosis.…”

“…In contrast to the biodistribution of drugs following parenteral administration of a solution, 'conventional' liposomes (charged and not of ultra-small size) accumulate in the mononuclear phagocytic system (MPS, formerly known as the reticuloendothelial system; [36][37][38][39][40][41][42][43]. A preferential uptake of intact 'conventional' liposomes containing serotonin by circulating monocytes has been demonstrated recently (44).…”