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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.

The present study explored a novel strategy for attenuation of restenosis after arterial injury by a bisphosphonate encapsulated in polymeric nanoparticles (NP) for transient selective depletion of macrophages. A bisphosphonate (BP), 2-(2-Aminopyrimidino) ethyldiene-1,1-bisphosphonic acid betaine (ISA), was successfully formulated in 400 nm sized polylactide/glycolide-based NP with high yield (69%) and entrapment efficiency (60% w/w). ISA NP, but not blank NP or free ISA, exhibited specific and significant cytotoxic effect on macrophages-like RAW 264 cells, in a dose-dependent manner, with no inhibitory effect on the growth of smooth muscle cells (SMCs). Fluorescent pyrene-labeled NP were shown to be taken up by RAW 264 cells, but not by SMCs. Intravenously (i.v.) administered ISA NP (15 mg/kg, single dose on day-1) resulted in a significant attenuation of neointima to media area ratio (N/M) by 40% and stenosis by 45% 14 days after rat carotid injury, in comparison to animals treated with free ISA, buffer or blank NP. However, the effect was not preserved 30 days post injury, and an insignificant reduction of neointimal formation was observed. Neointimal hyperplasia was also significantly suppressed after subcutaneous (SC) injection of ISA NP (15 mg/kg, single dose on day-1), reducing both N/M and stenosis. Intraperitoneal (i.p.) injection of silica, a known selective toxin for macrophages, (1000 mg/kg), also resulted in a significant inhibition of N/M and stenosis, which further reinforces the cause-effect relationship of macrophage-inactivation and the prevention of neointima formation. Biocompatible and biodegradable NP loaded with ISA characterized by high colloidal stability, reproducible activity, and high drug entrapment warrant further consideration for restenosis therapy, and may be useful in other disease processes involving monocytes/macrophages.