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.

BACKGROUND AND PURPOSESystemic glucocorticoid therapy may effectively attenuate lung inflammation but also induce severe side-effects. Delivery of glucocorticoids by liposomes could therefore be beneficial. We investigated if liposome-encapsulated dexamethasone inhibited ventilator-induced lung inflammation. Furthermore, we evaluated whether targeting of cellular Fcg-receptors (FcgRs) by conjugating immunoglobulin G (IgG) to liposomes, would improve the efficacy of dexamethasone-liposomes in attenuating granulocyte infiltration, one of the hallmarks of lung inflammation. EXPERIMENTAL APPROACHMice were anaesthetized, tracheotomized and mechanically ventilated for 5 h with either 'low' tidal volumes~7.5 mL·kg -1 (LVT) or 'high' tidal volumes~15 mL·kg -1 (HVT). At initiation of ventilation, we intravenously administered dexamethasone encapsulated in liposomes (Dex-liposomes), dexamethasone encapsulated in IgG-modified liposomes (IgG-Dex-liposomes) or free dexamethasone. Non-ventilated mice served as controls. KEY RESULTSDex-liposomes attenuated granulocyte infiltration and IL-6 mRNA expression after LVT-ventilation, but not after HVT-ventilation. Dex-liposomes also down-regulated mRNA expression of IL-1b and KC, but not of CCL2 (MCP-1) in lungs of LVT and HVT-ventilated mice. Importantly, IgG-Dex-liposomes inhibited granulocyte influx caused by either LVT or HVTventilation. IgG-Dex-liposomes diminished IL-1b and KC mRNA expression in both ventilation groups, and IL-6 and CCL2 mRNA expression in the LVT-ventilated group. Free dexamethasone prevented granulocyte influx and inflammatory mediator expression induced by LVT or HVT-ventilation. CONCLUSIONS AND IMPLICATIONSFcgR-targeted IgG-Dex-liposomes are pharmacologically more effective than Dex-liposomes particularly in inhibiting pulmonary granulocyte infiltration. IgG-Dex-liposomes inhibited most parameters of ventilator-induced lung inflammation as effectively as free dexamethasone, with the advantage that liposome-encapsulated dexamethasone will be released locally in the lung thereby preventing systemic side-effects. AbbreviationsALI, acute lung

We prepared immunoliposomes by covalent coupling of a randomly thiolated monoclonal antibody against the rat colon adenocarcinoma cell line CC53 1 to MPB-PE on the outer surface of conventional as well as PEGylated liposomes of about 100-nm diameter. We attempted to target these immunoliposomes in vivo to CC531 cells growing metastatically in the liver of syngeneic rats. Only when the immunoliposomes contained PEG-DSPE, did we observe, both with fluorescent and radioactive labels, accumulation of label in many, but not all, metastatic nodules. The fluorescent label concentrated in scattered areas within the nodules. By means of transmission electronmicroscopy, using colloidal gold particles as an encapsulated morphological marker, we established that the large majority of the tumor-associated gold particles located in areas not containing tumor cells. Most of the gold was detected in cells with a macrophage morphology. We tentatively ascribe this to either tumor morphology or to the coupling procedure we applied for the preparation of the immunoliposomes, or both. The random thiolation step of the antibody molecule conceivably allows for the exposure of the Fc portion of (part of) the antibody molecules so as to permit interaction with Fc receptors on the macrophages. Experiments with immunoliposomes prepared either by coupling of the antibody specifically via its Fc portion or by using F(ab')? fragments are in progress. The crucial condition of liposomal longevity as in the above experiments, where PEG-ylation of the immunoliposomes was necessary in order to achieve accumulation in the tumor area, by no means represents a general requirement for successful liposome targeting. We have shown that for efficient liposome targeting to a cell population which is readily accessible from the 419 Copyright 0 1997 by Marcel Dekker, Inc. Journal of Liposome Research Downloaded from informahealthcare.com by Nanyang Technological University on 08/23/15 For personal use only. 420 SCHERPHOF, KAMPS, AND KONINGcirculation, and has a high affinity for the liposomes, i.c. the hepatic sinusoidal endothelial cells, the presence of PEG chains may even be counter-productive.Journal of Liposome Research Downloaded from informahealthcare.com by Nanyang Technological University on 08/23/15For personal use only.Journal of Liposome Research Downloaded from informahealthcare.com by Nanyang Technological University on 08/23/15For personal use only.

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