Peter W. Taylor

In vitro interaction of zinc(II)-phthalocyanine-containing liposomes and plasma lipoproteins

We have studied the interaction of small unilamellar liposomes containing zinc(II)-phthalocyanine (Zn-Pc) with human plasma lipoproteins. High-, low- and very low-density lipoproteins (HDL, LDL and VLDL), were purified from plasma and combined in amounts reflecting their natural abundance in plasma. After short periods of incubation at 37 degrees C, the bulk of Zn-Pc was incorporated into HDL and LDL; very little 14C-labelled palmitoyl oleoyl phosphocholine, the most abundant phospholipid in the formulation, was associated with lipoproteins. When liposomes were incubated in pooled plasma, 73%-85% of Zn-Pc and 27%-34% of radiolabelled phospholipid were recovered with HDL and LDL, indicating a possible role for plasma lipid transfer proteins in the incorporation of phospholipid into lipoproteins. Some Zn-Pc was also found in association with VLDL. The buoyant density of Zn-Pc liposomes increased in a dose-dependent fashion when the particles were incubated with plasma, and it is suggested that this was due, at least in part, to opsonization of liposomes by plasma proteins.

Interaction between zinc(II)-phthalocyanine-containing liposomes and human low density lipoprotein

The interaction of human low density lipoprotein (LDL) and small unilamellar liposomes containing the photosensitiser zinc(II)-phthalocyanine (Zn-Pc) was studied in vitro to determine if Zn-Pc could be directly incorporated into the lipoprotein in the absence of other serum components. Incubation of LDL with increasing concentrations of liposomes resulted in a progressive increase in the net negative charge of LDL as determined by agarose gel electrophoresis and both Zn-Pc and liposomal phospholipid were incorporated into the modified LDL particles. Gel chromatography experiments indicated an increase in the molecular mass of modified LDL and immunoaffinity chromatography provided evidence that apoprotein B epitopes on modified LDL were unable to bind to antibody. The study indicated that the liposomal components could be selectively incorporated into LDL by a process that did not appear to involve either aggregation or fusion of particles.

Uptake of zinc(II)-phthalocyanine by HepG2 cells expressing the low-density lipoprotein receptor: studies with the liposomal formulation CGP55847

Hydrophobic photosensitizers readily intercalate into plasma lipoproteins. Some tumors acquire cholesterol from the circulation as a result of increased low density lipoprotein (LDL) receptor activity. Thus, circulating LDL may function as a vehicle for the delivery of bound Zn-Pc to cells within a tumor. Zn-Pc:LDL complexes, resulting from the interaction of LDL with the liposomal Zn-Pc formulation CGP55847, bind to the LDL receptor expressed on HepG2 cells but with reduced affinity in comparison to LDL. Confocal fluorescence microscopy facilitated the subcellular localization of Zn-Pc in microcolonies of HepG2 cells; the photosensitizer was distributed throughout the cellular membrane systems but was absent from the cell nucleus. Uptake of Zn-Pc in the presence of LDL was twofold greater than in the absence of the lipoprotein. These data suggest that the LDL uptake pathway may contribute to the localization of Zn-Pc in hyperproliferative tissue.

A novel approach to the site specific delivery of potential HMG-CoA reductase inhibitors

Abstract A novel approach to the site specific delivery of potential HMG-CoA reductase inhibitors based on bile acid uptake is described. The synthesis of inhibitors 9 and 16 was achieved from cholic acid methyl ester. Both compounds 9 and 16 are weak inhibitors of HMG-CoA reductase with IC50 values of 39.2 and 12.3μM respectively. Compound 16 is transported non-specifically across an intestinal epithelial monolayer.

Studies on the uptake of low molecular weight monomeric tris-galactosyl conjugates by the rat liver

We have attempted to direct low molecular weight compounds to the liver via the internalizing asialoglycoprotein receptor on parenchymal cells by conjugation to a monomeric triantennary galactosyl cluster. Acetate and a hypolipidaemic ansamycin were derivatized and the biodistribution of the conjugates was determined 250 sec and 30 min after administration to Wistar rats. The ansamycin conjugate (CGH46) was rapidly cleared from the circulation by the liver; after 250 sec, 64% of the radiolabelled dose was found in the liver compared to 18% in the blood. However, the distribution of the conjugate did not differ significantly from that of unconjugated ansamycin (CGH45). Tris-galactosyl acetate showed no capacity to localize in the liver, with only 2% recovered from that organ 250 sec after administration compared to 38% in the blood and 13-18% in the kidneys, skin and muscle. Extraction efficiency of CGH46 by isolated perfused rat livers was almost 20% of the administered dose and this value was not significantly changed by co-administration of specific inhibitors of the uptake process. It is concluded that derivatization of low molecular weight molecules with monomeric triantennary galactosyl residues is unlikely to increase their affinity for the liver.

Engineering proteins that bind to cell surface carbohydrates.

Carbohydrate residues covalently linked to plasma membrane proteins and lipids often provide specific markers at the cell surface. Traditionally such carbohydrate structures have been identified using antibodies and lectins. However problems of affinity and lack of specificity have restricted their usefulness. Protein engineering offers a way round these difficulties. In the case of some specialised cell surface carbohydrate structures, such as polysialic acid, enzymes may be useful analytical tools. Endosialidases specific for polysialic acid have recently been cloned and sequenced.

The Mode of C5b-9 Attack on Susceptible Gram Negative Bacteria

The lysis of erythrocytes by colloid osmotic deregulation upon assembly of heteropolymeric C5b-9 channels on the target membrane became the subject of intense investigation following the realization that the haemolysis assay provided a clear opportunity to unravel the complexities of the complement system (Mayer, 1984). In contrast, relatively little effort has been spent on the elucidation of the mechanism of lysis of nucleated cells and of killing of Gram-negative bacteria by complement, processes that do not follow non-cooperative or one-hit kinetics and are not due exclusively to colloid osmotic deregulation (Born and Bhakdi, 1986; Kim et al., 1987).

Localization of zinc(II)-phthalocyanine within implanted tumors after intravenous administration of a liposomal formulation

The liposomal zinc(II) phthalocyanine (Zn-Pc) formulation CGP55847 was administered intravenously (0.5 mg Zn-Pc/kg) to C57/BL6 mice bearing subcutaneously implanted B16 melanomas or to Swiss mice bearing intramuscularly implanted Ehrlich carcinomas. Tumors were removed 3 or 24 h after dosing, and the Zn-Pc content and intratumoral distribution determined by extraction and quantitative fluorescence microscopy. Localization of the photosensitizer within the tumor mass occurred more rapidly in the highly vascularized Ehrlich carcinoma compared to the less highly vascularized B16 melanoma. Zn-Pc was evident in and around blood vessels 3 but not 24 h after dosing. More Zn-Pc was found in necrotic areas compared to viable tumor tissue; little or no Zn-Pc was detected in the muscle tissue invaded by the Ehrlich carcinoma. At the cellular level, Zn-Pc was associated with membranes and the cytosol but not the nucleus.

Tissue distribution of liposomal zinc(II)-phthalocyanine in normolipaemic and hyperlipaemic rats following intravenous administration

Liposomal formulations of the photosensitizer zinc(II)-phthalocyanine (Zn-Pc) readily interact with plasma lipoproteins in vitro and in vivo, leading to a redistribution of the photosensitizer amongst the major lipoprotein classes. As lipoprotein binding may facilitate the uptake of lipophilic photosensitizers by proliferating tissues, we have examined the fate of intravenously administered liposomal Zn-Pc in normolipaemic rats and rats rendered hyperlipaemic through dietary intervention. Differences were found in the plasma decay profiles of Zn-Pc and liposomal phospholipid between the two groups of animals.