Recombinant human tumor necrosis factor-α covalently conjugated to long-circulating liposomes

Abstract

Recombinant tumor necrosis factor-α (rHuTNF) was covalently conjugated to a phospholipid, N-glutaryl phosphatidylethanolamine (NGPE). The resultant rHuTNF-NGPE conjugates were incorporated into liposomes composed of phosphatidylcholine (PC) and cholesterol (Chol) with or without polyethyleneglycol conjugated to phosphatidylethanolamine (PEG3000-PE). Efficient incorporation (35–50%) of rHuTNF-NGPE conjugates into liposomes was obtained for both PC/Chol and PC/Chol/PEG3000-PE liposomes. An in vitro cytotoxicity assay showed that rHuTNF-NGPE conjugates incorporated into liposomes exhibit a reduced biological activity as compared to the free rHuTNF. Biodistribution studies using 125I-labeled rHuTNF showed a significant increase in the circulation time of rHuTNF by incorporation into PC/Chol/PEG3000-PE liposomes, but not conventional PC/Chol liposomes. However, studies using a radioactive lipid as a liposome marker showed that incorporation of rHuTNF-NGPE conjugates resulted in increased clearance from the blood and accumulation in the spleen and liver of both liposomal formulations. The liposome clearance from the blood depends on the protein/lipid ratio of liposomes. The higher the protein/lipid ratio, the higher the liposome clearance from the blood and accumulation in the spleen and liver, suggesting that accumulation of rHuTNF-bound liposomes in the spleen and liver involves interactions with TNF-receptors in these organs.