Antifibrotic effect of a proline analogue delivered in liposomes to cells in culture.

Proline analogues inhibit procollagen triple helix formation and are antifibrotic in vivo. Efficacy of the proline analoguecis-4-hydroxy-L-proline (cHyp) on vascular collagen accumulation is improved by in vivo delivery in liposomes. This effect may be due to local release of drug from liposomes taken up by vascular endothelium. To test this postulate, we used a co-culture system to assess the antifibrotic effect of cHyp in liposomes taken up by endothelium (upper well) by measuring inhibition of growth of smooth muscle cells and fibroblasts (lower well). We also studied whether release of cHyp was prolonged in poly(ethyleneglycol) (PEG)-conjugated liposomes compared to liposomes not conjugated with PEG (control liposomes). In fibroblasts, free (unencapsulated) cHyp (1 mg/ml) added to the upper well inhibited growth for 3 days; an equivalent dose of cHyp in control liposomes inhibited growth for 4 days. cHyp in PEG-liposomes produced greater growth inhibition than cHyp in control liposomes. cHyp in liposomes did not inhibit growth of smooth muscle cells more than free cHyp. Washing free cHyp from endothelium after 1 day incubation restored growth of smooth muscle cells whereas washing liposomes containing cHyp failed to restore cell growth. These results suggest that liposomes enhance drug efficacy of cHyp by prolonging the release of drug from endothelium.