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thelial cells. Liver endothelial cells have numerous pores (feLiposomes with diameters of 200 to 400 nm containing nestrae), with an average diameter of 150 nm. 1 Larger bloodphosphatidylserine (PS) or phosphatidylglycerol (PG) were borne particles are therefore supposed to be prevented from injected intravenously into rats. Two hours after injection, contact with the hepatocytes situated across the endothelial 75% of the injected dose of PS liposomes was found in the lining. liver and only 10% found in the spleen, while 35% of the PG In a previous study 2 we found that the hepatic uptake of liposomes was found in the liver and as much as 40% was 200-nm liposomes (ie, phosphatidylcholine (PC), cholesterol found in the spleen. Cell-isolation experiments revealed the (chol), and phosphatidylserine (PS) in a 4:5:1 molar ratio) following remarkable difference in the intrahepatic distribuwas virtually unaffected in rats that were depleted of their tion between the two liposome formulations: the PS liposomesKupffer cells by pretreatment with liposomal doxorubicin. distributed in about equal amounts to Kupffer cells and hepaCell-isolation experiments showed that the liposomes were tocytes, despite their size (200-400 nm) exceeding that of mainly found to be associated with the hepatocyte populathe endothelial fenestrae (average 150 nm), whereas the PG tion. Transmission electron microscopy revealed that the enliposomes were only taken up by the Kupffer cells and not dothelial lining was damaged due to treatment with doxoruat all by the hepatocytes. Double-label studies, using lipobicin, which, thus, could explain the uptake of liposomes by somes in which the lipid-moiety was radio labeled with hepatocytes. Surprisingly, however, we also found that al- 423.) AnimalsHepatic uptake of conventional multilamellar liposomes Specific pathogen-free female Wistar or male Wag/Rij rats (Harlarger than 150 nm in diameter has so far been attributed to lan CPB, Zeist, The Netherlands), weighing between 200 and 250 phagocytosis by Kupffer cells. Kupffer cells are situated in g, were used in all experiments. The animals received care in accorthe liver sinusoids, which are lined by a single layer of endo-dance with the institution's guidelines.

Human serum albumin (HSA) derivatized with cis-aconitic anhydride (Aco-HSA) that was earlier shown to inhibit replication of human immunodeficiency virus type 1 (HIV-1), was covalently coupled to conventional liposomes, consisting of phosphatidylcholine, cholesterol and maleimido-4-(p-phenylbutyryl)phosphatidylethanolamine, using the heterobifunctional reagent N-succinimidyl-S-acetylthioacetate (SATA). The amount of HSA that could be coupled to the liposomes depended on derivatization of the HSA and ranged from 64.2 +/- microgram HSA/micromol total lipid for native HSA to 29.5 +/- 2.7 microgram HSA/micromol total lipid for HSA in which 53 of the epsilon amino groups of lysine were derivatized with cis-aconitic anhydride (Aco53-HSA). Incorporation of 3.8 mol% of total lipid of a poly(ethylene glycol) derivative of phosphatidylethanolamine (PEG-PE) in the liposomes resulted in a lower coupling efficiency of Aco-HSA. The elimination and distribution of the liposomal conjugates in rats in vivo was largely dependent on the modification of the HSA coupled to the liposomes. With native HSA-liposomes, more than 70% of the conjugate was still found in the blood plasma 30 min after i.v. injection in rats, while at this time Aco-HSA-liposomes were completely cleared from the circulation. The rapid clearance of conventional Aco-HSA-liposomes was due to a rapid uptake into the liver and could be considerably decreased by incorporating PEG-PE in the liposomal bilayer. After 3 h 60% of Aco-HSA-PEG-liposome conjugates were found in the blood. In an in vitro anti-HIV-1 assay, the 50% inhibitory concentrations (IC50) for Aco39-HSA-liposomes and Aco53-HSA-liposomes expressed as protein weight, were 2.87 microgram/ml and 0.154 microgram/ml, respectively. When PEG-PE was incorporated, the Aco53-HSA-liposomes retained anti HIV-1 activity (IC50:3.13 microgram/ml). The possibility to modulate the residence time in the bloodstream of Aco-HSA-liposomes and the potent anti-HIV-1 activity of these conjugates, may allow the development of an intrinsically active drug carrier system. By incorporating anti HIV-1 drugs such as AZT into such liposomes a drug delivery system can be designed that might act simultaneously on the virus/cell binding by virtue of the coupled Aco-HSA and on the RNA/DNA transcription of the HIV-1 replication cycle through the nucleoside analogue.

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