Targeting of lactosylceramide-containing liposomes to hepatocytes in vivo.

Incorporation of 8 mol% lactosylceramide in small unilamellar vesicles consisting of cholesterol, dimyristoylphosphatidylcholine and phosphatidylserine in a molar ratio of 5:4:1 and containing [3H]inulin as an aqueous-space marker resulted in a 3-fold decreased half-life of the vesicles in blood and a corresponding increase in liver uptake after intracardial injection into rats. The increase in liver uptake was mostly accounted for by an enhanced uptake in the parenchymal cells, while the uptake by the non-parenchymal cells was only slightly increased. The uptake of both the control and the glycolipid-containing vesicles by the non-parenchymal cell fraction could be attributed completely to the Kupffer cells; no radioactivity was found in the endothelial cells. The effect of lactosylceramide on liver uptake and blood disappearance of the liposomes was effectively counteracted by desialylated fetuin, injected shortly before the liposome dose. This observation supports the notion that a galactose-specific receptor is involved in the liver uptake of lactosylceramide liposomes.

Lactosylceramide-induced stimulation of liposome uptake by Kupffer cells in vivo.

Incorporation of 8 mol percent lactosylceramide into small unilamellar vesicles consisting of cholesterol and sphingomyelin in an equimolar ratio and containing [3H] inulin as a marker resulted in an increase in total liver uptake and a drastic change in intrahepatic distribution of the liposomes after intravenous injection into rats. The control vesicles without glycolipid accumulated predominantly in the hepatocytes, but incorporation of the glycolipid resulted in a larger stimulation of Kupffer-cell uptake (3.2-fold) than of hepatocyte uptake (1.2-fold). Liposome preparations both with and without lactosylceramide in which part of the sphingomyelin was replaced by phosphatidylserine, resulting in a net negative charge of the vesicles, were cleared much more rapidly from the blood and taken up by the liver to higher extents. The negative charge had, however, no influence on the intrahepatic distributions. The fast hepatic uptake of the negatively charged liposomes allowed competition experiments with substrates for the galactose receptors on liver cells. Inhibition of blood clearance and liver uptake of lactosylceramide-containing liposomes by N-acetyl-D-galactosamine indicated the involvement of specific recognition sites for the liposomal galactose residues. This inhibitory effect of N-acetyl-D-galactosamine was shown to be mainly the result of a decreased liposome uptake by the Kupffer cells, compatible with the reported presence of a galactose specific receptor on this cell type (Kolb-Bachofen et al. (1982) Cell 29, 859-866). The difference between the results on sphingomyelin-based liposomes as described in this paper and those on phosphatidylcholine-based liposomes as published previously (Spanjer and Scherphof (1983) Biochim. Biophys. Acta 734, 40-47) are discussed.

The effect of a water-soluble tris-galactoside terminated cholesterol derivative on the in vivo fate of small unilamellar vesicles in rats.

When the water-soluble cholesterol derivative, N-[tris [(beta-D-galactopyranosyloxy)methyl]methyl]-N alpha-[4-(5-cholesten-3 beta-yloxy)succinyl]glycinamide (tris-gal-chol) (Kempen et al. (1984) J. Medicin. Chem. 27, 1306-1312) is added as an aqueous micellar solution to a dispersion of small unilamellar phospholipid vesicles it rapidly associates with the vesicles, without causing significant leakage of liposome contents. Incorporation of 10 mol% tris-gal-chol in the liposomal membrane caused a substantial increase in the rate and extent of rat liver uptake and a shift in intrahepatic distribution of an intravenously administered dose of liposomes. For neutral liposomes composed of equimolar amounts of cholesterol and sphingomyelin incorporation of tris-gal-chol led to a 7-fold increase in total liver uptake, which was mainly accounted for by an increase in uptake by the Kupffer cells (12-fold) and by only a small increase in uptake by the hepatocytes (1.4-fold). The increased liver uptake is blocked by preinjection of N-acetyl-D-galactosamine and not affected by preinjection of N-acetyl-D-glucosamine. This indicates that the increased interaction of liposomes as a result of tris-gal-chol incorporation is mediated by galactose-specific recognition sites on both Kupffer cells and hepatocytes. Targeting of liposomes to the asialoglycoprotein receptor of the hepatocytes is thus frustrated by the highly active galactose-specific receptor on Kupffer cells. Comparable results on lactosylceramide incorporation into liposomes were recently reported by us (Spanjer et al. (1984) Biochim. Biophys. Acta 774, 49-55).

Lipoproteins and liposomes as in vivo cholesterol vehicles in the rat: preferential use of cholesterol carried by small unilamellar liposomes for the formation of muricholic acids.

Hepatic cholesterol metabolism was studied in rats with a permanent biliary drainage. Three cholesterol vehicles were used to discriminate between metabolic pathways of cholesterol in the liver. [3H]Cholesterol was administered intravenously associated with rat serum lipoproteins, multilamellar (MLV) or small unilamellar (SUV) liposomes. The liposomes were made from cholesterol, sphingomyelin and phosphatidylserine in a 5:4:1 molar ratio. Initial blood elimination differed markedly for the three vehicles: 15 min after injection the 3H radioactivity content of blood for MLV, SUV and lipoprotein was 3, 50 and 54% of the injected dose, respectively. After about 30 min, MLV-cholesterol label started to reappear in the blood, probably after processing of the vehicle by the Kupffer cells. For all vehicles about 80% of the cholesterol label had been excreted in bile after 120 h, predominantly as bile acids. Initial biliary excretion was highest for lipoproteins (5.7% at 1 h), followed by MLV and SUV (1.3 and 1.2%, respectively). No differences in the radioactivity of excreted bile acids were detectable between the three vehicles at 12 h after injection. However, at 1 h the radioactivity in the muricholic acid fraction was markedly increased, as compared to the other bile acids after injection of SUV-cholesterol, but not after injection of MLV- or lipoprotein-cholesterol. Also, the glycine/taurine conjugation ratio of bile acids was increased for SUV-cholesterol at 1 h as compared to that for the other two vehicles. Since SUV appear to donate their cholesterol to a pool which preferentially supplies cholesterol for muricholic acid synthesis, we conclude that more than one cholesterol pool exists in the hepatocytes from which cholesterol can be recruited for bile acid synthesis. Zonal heterogeneity might be responsible for the observed differences.

Intrahepatic distribution of small unilamellar liposomes as a function of liposomal lipid composition.

We investigated the intrahepatic distribution of small unilamellar liposomes injected intravenously into rats at a dose of 0.10 mmol of lipid per kg body weight. Sonicated liposomes consisting of cholesterol/sphingomyelin (1:1), (A); cholesterol/egg phosphatidylcholine (1:1), (B); cholesterol/sphingomyelin/phosphatidylserine (5:4:1), (C) or cholesterol/egg-phosphatidylcholine/phosphatidylserine (5:4:1), (D) were labeled by encapsulation of [3H]inulin. The observed differences in rate of blood elimination and hepatic accumulation (A much less than B approximately equal to C less than D) confirmed earlier observations and reflected the rates of uptake of the four liposome formulations by isolated liver macrophages in monolayer culture. Fractionation of the liver into a parenchymal and a non-parenchymal cell fraction revealed that 80-90% of the slowly clearing type-A liposomes were taken up by the parenchymal cells while of the more rapidly eliminated type-B liposomes even more than 95% was associated with the parenchymal cells. Incorporation of phosphatidylserine into the sphingomyelin-based liposomes caused a significant increase in hepatocyte uptake but a much more substantial increase in non-parenchymal cell uptake, resulting in a major shift of the intrahepatic distribution towards the non-parenchymal cell fraction. For the phosphatidylcholine-based liposomes incorporation of phosphatidylserine did not increase the already high uptake by the parenchymal cells while uptake by the non-parenchymal cells was only moderately elevated; this resulted in only a small shift in distribution towards the non-parenchymal cells. The phosphatidylserine-induced increase in liposome uptake by non-parenchymal liver cells was paralleled by an increase in uptake by the spleen. Fractionation of the non-parenchymal liver cells in a Kupffer cell fraction and an endothelial cell fraction showed that even for the slowly eliminated liposomes of type A endothelial cells do not participate to a measurable extent in the elimination process, thus excluding involvement of fluid-phase pinocytosis in the uptake process.

A water-soluble cholesteryl-containing trisgalactoside: synthesis, properties, and use in directing lipid-containing particles to the liver.

The synthesis of a trisgalactoside-terminated cholesterol derivative is described. Tris(galactosyloxymethyl)-aminomethane is coupled to cholesterol by using glycyl and succinyl as intermediate hydrophilic spacer moieties. The resulting cholesteryl ester dissolves easily in water, forming monodisperse micelles. When added to dispersions of liposomes or plasma lipoproteins in water, the substance becomes incorporated rapidly into these structures, causing an increase of their buoyant density. Liposomes or low-density lipoproteins, preloaded with the substance, are rapidly cleared from the circulation and taken up by the liver after intravenous injection in rats. This uptake is inhibited by N-acetylgalactosamine but not by N-acetylglucosamine, indicating the specificity of this process.

Liposomes in chemo- and immunotherapy of cancer.

In this paper, we report on the in vivo behavior of liposomes as a function of their size and composition. It is emphasized that by varying these parameters we can influence not only the rate of blood elimination but also the intrahepatic destination of the liposomes. Thus, we show that small liposomes with diameters well below 100 nm can reach and be internalized by the parenchymal cells of the liver, i.e. the hepatocytes. The rate and the extent at which this occurs depends on the liposomal composition. With respect to the application of liposomes as a drug carrier system in anticancer therapy, we put emphasis on the liver macrophage, i.e. the Kupffer cell, as a target cell. Large liposomes with diameters well over 100 nm exclusively are taken up by these cells as far as hepatic uptake is concerned. By encapsulation within liposomes, a drug may be delivered specifically to these macrophages; this will prevent its rapid excretion from the body and/or undesired accumulation in other cell types. Two examples of the way in which this condition may be exploited are presented. First, we demonstrate the formation of intracellular depots in the macrophages of the cytostatic drug 5-fluorodeoxyuridine (FUdR), thus preventing the rapid metabolism of the drug by the hepatocytes and allowing its sustained release from the macrophages and subsequent uptake by adjacent metastatic tumor cells.(ABSTRACT TRUNCATED AT 250 WORDS)

In situ study of haemopoiesis in human fetal liver.

The anatomy of haemopoietic cells in human fetal liver was examined using immunohistological techniques on frozen sections of 31 fetuses (10-28 weeks gestational age). The immunohistological findings were consistent with reported cell suspension data. With regard to the location of haemopoietic activity no particular relationship existed between the various haemopoietic cell lineages. A large number of proliferating cells was present; only a few of these were reactive with haemopoietic progenitor cell monoclonal antibodies (MoAb) CD34. A population of haemopoietic cells expressed CD43 antigen (MoAb MT1) alone or together with anti-vimentin MoAb reactivity; this population needs further delineation. Erythropoiesis and myelopoiesis occurred in clusters around sinusoids and portal triad vessels respectively. Lack of MoAb reacting exclusively with early developmental stages of erythropoiesis and myelopoiesis precluded dissection of these lineages. Lymphopoiesis occurred in a loosely scattered pattern without any sign of focal development. Pre-B and B-cell numbers increased with gestational age. Cells expressing markers of more mature B cells (surface IgD, CD35, and CD21) were rare. Also, few cells reacted with mature T-cell markers, but CD7+ cells were obviously present. This expression of CD7 on haemopoietic fetal liver cells suggests that T-cell precursors develop in fetal liver as well as B cells.

Hepatic disposition of cationic drugs bound to asialoorosomucoid: lack of coendocytosis and evidence for intrahepatic dissociation.

A combination of protein binding, liver clearance, subcellular distribution and cell separation experiments was employed to investigate the influence of binding of cationic drugs to asialoorosomucoid (ASOR) on their hepatic uptake and intrahepatic distribution. Two quaternary ammonium drugs, d-tubocurarine and N-methyldeptropine, were selected because of their marked differences in hepatic processing and binding to ASOR. In spite of an increase in protein binding of 560% for d-tubocurarine and 380% for N-methyldeptropine, perfusate clearance of both drugs in isolated perfused rat livers was not influenced by addition of 75 mg of ASOR. Absence of coendocytosis was indicated by subcellular distribution studies revealing no extra enrichment of quaternary ammonium drugs in lysosomal fractions compared with control studies. Isolation of parenchymal and sinusoidal liver cells demonstrated d-tubocurarine to be present solely in hepatocytes; binding to ASOR did not affect the relative distribution in the various cell types. It is concluded that binding of cationic drugs to ASOR does not result in endocytosis of a drug-protein-receptor complex by the liver. This result rather suggests that dissociation of the organic cations from the asialoglycoprotein occurs within the liver before endocytosis of the glycoprotein.

The effect of a water-soluble tris-galactoside-terminated cholesterol derivative on the fate of low density lipoproteins and liposomes.

A triantennary galactose-terminated cholesterol derivative, N-(tris(beta-D-galactopyranosyloxymethyl) methyl)-N alpha-(4-(5-cholesten-3 beta-yloxy)succinyl)glycinamide (Tris-Gal-Chol), which dissolves easily in water, was added to human low density lipoproteins (LDL) in varying quantities. Upon addition to LDL, Tris-Gal-Chol was immediately incorporated, and after intravenous injection into rats, the iodine-labeled apolipoprotein B radioactivity was readily associated with the liver. The incorporation of 5 or 13 micrograms of Tris-Gal-Chol into LDL (20 micrograms of protein) stimulates the parenchymal cell association of LDL 6- and 10-fold, respectively, at 10 min after injection. For non-parenchymal cells, the cell association is 60- and 70-fold stimulated, respectively. It can be calculated that non-parenchymal cells (mainly Kupffer cells) are for 80-90% responsible for the increased, galactose-mediated, interaction of Tris-Gal-Chol LDL with the liver. The increased interaction of LDL with the cells upon Tris-Gal-Chol incorporation is followed by degradation of the apolipoprotein B in the lysosomes. Incorporation of Tris-Gal-Chol into unilamellar liposomes (10 mol %) leads to an increased cell association of the enclosed [3H]inulin to parenchymal cells (1.4-fold) and non-parenchymal cells (11.8-fold). It is concluded that Tris-Gal-Chol incorporation into LDL leads to a markedly increased catabolism of LDL by the liver which might be used for lowering serum LDL levels. The possibility of increasing the interaction of LDL or liposomes with specific liver cell types by Tris-Gal-Chol might also have an application for targeting drugs or other compounds of interest to these cells.