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In vitro and in vivo data have indicated that tumor cells actively internalize the low density lipoprotein (LDL) from the circulation. In order to achieve a selective delivery of drugs to tumor cells via the LDL pathway, we have incorporated oleyl derivatives of methotrexate and floxuridine (FdUrd) into LDL particles. Three different incorporation procedures were studied: Method A, the dry film method; Method B, the transfer protein method; and Method C, the delipidation-reconstitution method. In all cases, 3H-labeled drug was incorporated into 125I-labeled LDL to yield double-labeled particles so that the behavior of both drug and carrier could be followed simultaneously. The last method led to the highest drug loading and it was possible to incorporate 50-70 molecules of dioleoyl-FdUrd per LDL particle as compared with about 18 molecules of drug when utilizing the transfer protein procedure. In vitro studies on the interaction of dioleoyl-FdUrd-LDL particles, obtained by the delipidation-reconstitution method, with the hepatocellular carcinoma cell line Hep G2, indicated that these reconstituted particles were equally effective in competing for LDL binding as native LDL. Moreover, drug delivery to Hep G2 cells occurred at the same rate as cellular association of the apolipoprotein B. In vivo studies on the fate of dioleoyl-FdUrd-LDL complexes in rats indicated that the serum decay was increased as compared with native LDL. The half-life of 6-9 min is, however, considerably prolonged as compared to the free drug (t1/2 less than 1 min). It is suggested that the 6-fold increased serum half-life of the drug-LDL complex accompanied by the possibly more specific tumor delivery may lead to an increased therapeutic effect.

The in vivo interaction of low density lipoproteins (LDL) with the liver was investigated by visualizing the endocytic route using light- and immunoelectron microscopic methods in control and 17 alpha-ethinyl estradiol (EE)-treated rats. The fluorescent dye dioctadecyl indocarbocyanine perchlorate allowed the visualization of LDL at the light microscopic level. Cryoimmunocytochemistry using antibodies against apolipoprotein B was applied at the electron microscopic level. In treated, as well as in EE-treated rats, dioctadecyl indocarbocyanine perchlorate-LDL was taken up by Kupffer cells to a substantial extent. Parenchymal cell uptake was strongly increased after EE treatment and at 10 minutes after injection, LDL was found to be attached to the microvilli of the parenchymal cells and some LDL was already localized in multivesicular structures. At later time points, substantial labeling in multivesicular structures, vesicles near bile canaliculi, and also inside bile canaliculi was observed. A low amount of labeling was found in lysosomes. In untreated rats, label was also observed in the aforementioned structures, but at a much lower level. The biliary appearance of LDL was quantified in rats equipped with permanent catheters in the bile duct, duodenum, and heart. After administration of [125I]tyraminecellobiose-LDL in control rats, about 5% of the injected dose was secreted into the bile during the first 3 hours after injection. This value was about 25% for EE-treated rats. The radioactivity secreted into the bile was trichloroacetic acid-precipitable and high molecular weight bands were immunoreactive for apolipoprotein B as revealed by Western blotting. The described events were not observed when methylated [125I]tyraminecellobiose-LDL was administered. It is concluded that, in rat liver, a significant portion of apolipoprotein B derived from LDL is directly transported to the bile. Since this pathway is enhanced in EE-treated rats, it appears to be a route specific for liver parenchymal cells, dependent on uptake via the LDL receptor.

The aim of this study was to set up an in vitro system to study nephrotoxicity of xenobiotics which allows exposure at low concentrations for long periods (1-5 days). A very pure preparation of isolated proximal tubular cells (PTC) from rat kidney (Boogaard et al., Toxicol Appl Pharmacol 101: 135-143, 1989) was brought into primary culture. Cells grew to confluence in 3 days and could be maintained up to 8 days in a modification of Dulbecco's modified Eagle's medium Ham F12 nutrient mixture supplemented with fetal calf serum. Fibroblast growth was completely suppressed by replacement of L-valine by D-valine and of L-arginine by L-ornithine. Polarity was retained: in cells grown on filters organic anions were transported at the basolateral membrane while D-glucose transport was located at the apical membrane. Inhibition of the latter was used to assess the functional integrity of the cells after exposure to nephrotoxins. The newly grown cells expressed gamma-glutamyltranspeptidase activity since incubation with the glutathione-conjugate of 1,1-dichloro-2,2-difluoroethylene (DCDFE) induced cytotoxicity. Both beta-lyase and acylase activities were expressed because the cysteine-S-conjugate and the corresponding mercapturate of DCDFE showed cytotoxicity. Cultured cells showed toxicity on prolonged exposure to very low concentrations of gentamicin, cephaloridine, cisplatin and the cysteine-S-conjugate of chlorotrifluoroethylene. The lowest concentrations at which toxicity can be observed are 1-3 orders of magnitude lower in primary cultures than in freshly isolated PTC in suspension. This indicates that this cell model is suitable to investigate mechanisms of nephrotoxicity in vitro, at prolonged exposure to the low concentrations that are relevant in vivo levels.

The possibilities and limitations in using the low-density lipoprotein (LDL) as a carrier for drugs are discussed. LDL, which may be regarded as the natural counterpart of liposomes, possesses a lipid core that may be utilized as a drug reservoir. Unlike most types of liposomes, the endogenous LDL particle is not avidly taken up by the reticuloendothelial system and may persist in the circulation for prolonged times after injection. A well-characterized membrane receptor recognizes LDL, and binding appears to be coupled to uptake and intracellular processing. Since many tumor tissues express a high amount of LDL receptors, there is a rationale for the design of a toxic LDL-cytostatic drug complex. The behavior of LDL in vivo will be discussed and the principles of LDL-mediated targeting to tumor cells will be evaluated. In addition, methods for drug incorporation into LDL will be critically assessed, while evaluation methods will be presented that may set the standards for future research.

The distribution of nicotinamide adenine dinucleotide (NAD) glycohydrolase in rat liver was investigated by subcellular fractionation and by isolation of hepatocytes and sinusoidal cells. The behavior of NAD glycohydrolase in subcellular fractionation was peculiar because, although the enzyme was mainly microsomal, plasma membrane preparations contained distinctly more NAD glycohydrolase than could be accounted for by their content in elements derived from the endoplasmic reticulum or the Golgi complex identified by glucose-6-phosphatase and galactosyltransferase, respectively. When microsomal and plasmalemmal preparations were brought to equilibrium in a linear-density gradient, NAD glycohydrolase differed from these enzymes and behaved like 5'-nucleotidase and alkaline phosphodiesterase I. NAD glycohydrolase was markedly displaced towards higher densities after treatment with digitonin. This behavior in density-gradient centrifugation strongly suggests that NAD glycohydrolase is an exclusive enzyme of the plasma membrane. NAD glycohydrolase differed clearly from other plasmalemmal enzymes when the liver was fractionated into hepatocytes and sinusoidal cells; its specific activity was considerably greater in sinusoidal cell than in hepatocyte preparations. Further subfractionation of sinusoidal cell preparations into endothelial and Kupffer cells by counterflow elutriation showed that NAD glycohydrolase is more active in Kupffer cells. We estimate that the specific activity of NAD glycohydrolase activity is at least 65-fold higher at the periphery of Kupffer cells than at the periphery of hepatocytes. As the enzyme shows not structure-linked latency and is an exclusive constituent of the plasma membranes, we conclude that it is an ectoenzyme that cannot lead to a rapid turnover of the cytosolic pyridine nucleotides.

Small solid liposomes made from distearoylphosphatidyl choline and cholesterol (molar ratio 2:1) showed significant stability in plasma, with a half-life of about 24 hr after intravenous injection in rats. The major cellular uptake of intact liposomes was found in the liver and spleen, peaking after 2-4 hr in the liver and after 24 hr in the spleen. Isolation of parenchymal and non-parenchymal cells from rat livers at various intervals after injection of liposomes showed that both cell types adsorbed liposomal membranes and took up the liposomal contents. Our study has shown that most of the liposomal markers found in the liver shortly (less than 40 min) after administration stemmed from the liposomes adsorbed to extracellular binding sites, and that uptake into the cells took place subsequently. In non-parenchymal cells, uptake was rapid and the intracellular level remained rather constant after 40 min and for up to 4 hr. The uptake of liposomes by parenchymal cells was slower, it showed a lag-phase of approx. 1/2 hr and peaked at 2 hr, whereupon the radioactivity in parenchymal cells dropped. The contents of liposomes behaved in a manner similar to the membranes. It is concluded that, in addition to a rapid uptake of liposomes in non-parenchymal liver cells, there is a significant degree of association with parenchymal cells, provided that the liposomes administered are small (less than 100 nm in diameter) and stable.

Human low density lipoprotein (LDL), radiolabeled in the cholesteryl ester moiety, was injected into estrogen-treated and -untreated rats. The hepatic and extrahepatic distribution and biliary secretion of [3H]cholesteryl esters were determined at various times after injection. In order to follow the intrahepatic metabolism of the cholesteryl esters of LDL in vivo, the liver was subfractioned into parenchymal and Kupffer cells by a low temperature cell isolation procedure. In control rats, the LDL cholesteryl esters were mainly taken up by the Kupffer cells. After uptake, the [3H]cholesteryl esters are rapidly hydrolyzed, followed by release of [3H]cholesterol from the cells to other sites in the body. Up to 24 h after injection of LDL, only 9% of the radioactivity appeared in the bile, whereas after 72 h, this value was 30%. Hepatic and especially the parenchymal cell uptake of [3H]cholesteryl esters from LDL was strongly increased upon 17 alpha-ethinylestradiol treatment (3 days, 5 mg/kg). After rapid hydrolysis of the esters, [3H]cholesterol was both secreted into bile (28% of the injected dose in the first 24 h) as well as stored inside the cells as re-esterified cholesterol ester. It is concluded that uptake of human LDL by the liver in untreated rats is not efficiently coupled to biliary secretion of cholesterol (derivatives), which might be due to the anatomical localization of the principal uptake site, the Kupffer cells. In contrast, uptake of LDL cholesterol ester by liver hepatocytes is tightly coupled to bile excretion. The Kupffer cell uptake of LDL might be necessary in order to convert LDL cholesterol (esters) into a less toxic form. This activity can be functional in animals with low receptor activity on hepatocytes, as observed in untreated rats, or after diet-induced down-regulation of hepatocyte LDL receptors in other animals.

The interaction in vivo of 125I-labeled tissue-type plasminogen activator (t-PA) with the rat liver and the various liver cell types was characterized. Intravenously injected 125I-t-PA was rapidly cleared from the plasma (t1/2 = 1 min), and 80% of the injected dose associated with the liver. After uptake, t-PA was rapidly degraded in the lysosomes. The interaction of 125I-t-PA with the liver could be inhibited by preinjection of the rats with ovalbumin or unlabeled t-PA. The intrahepatic recognition site(s) for t-PA were determined by subfractionation of the liver in parenchymal, endothelial, and Kupffer cells. It can be calculated that parenchymal cells are responsible for 54.5% of the interaction of t-PA with the liver, endothelial cells for 39.5%, and Kupffer cells for only 6%. The association of t-PA with parenchymal cells was not mediated by a carbohydrate-specific receptor and could only be inhibited by an excess of unlabeled t-PA, indicating involvement of a specific t-PA recognition site. The association of t-PA with endothelial cells could be inhibited 80% by the mannose-terminated glycoprotein ovalbumin, suggesting that the mannose receptor plays a major role in the recognition of t-PA by endothelial liver cells. An excess of unlabeled t-PA inhibited the association of 125I-t-PA to endothelial liver cells 95%, indicating that an additional specific t-PA recognition site may be responsible for 15% of the high affinity interaction of t-PA with this liver cell type. It is concluded that the uptake of t-PA by the liver is mainly mediated by two recognition systems: a specific t-PA site on parenchymal cells and the mannose receptor on endothelial liver cells. It is suggested that for the development of strategies to prolong the half-life of t-PA in the blood, the presence of both types of recognition systems has to be taken into account.

Administration of phorbol 12-myristate 13-acetate (PMA) to rats in vivo resulted in the induction of ornithine decarboxylase activity in the liver which could be blocked by preinjection of indomethacin, a cyclooxygenase inhibitor. In vitro administration of PMA to primary cultures of rat parenchymal cells did not lead to an induction of ornithine decarboxylase activity. It was investigated to what extent non-parenchymal liver cells could play an intermediary role in the expression of the PMA effect on ornithine decarboxylase activity in parenchymal liver cells. Addition of conditioned medium from PMA-activated Kupffer cells to cultured parenchymal cells led to the induction of ornithine decarboxylase activity in parenchymal cells. This effect was not observed with conditioned medium from untreated Kupffer cells or from Kupffer cells treated with PMA plus indomethacin. Conditioned media from PMA-treated or untreated endothelial liver cells were ineffective in the induction of ornithine decarboxylase activity in parenchymal liver cells. Prostaglandin D2, the main eicosanoid produced by Kupffer cells, was able to stimulate the synthesis of ornithine decarboxylase in parenchymal liver cells (up to 40-fold) in a dose-dependent way. Prostaglandin (PG) D2 appeared to be a more potent inducer of ornithine decarboxylase activity in parenchymal cells than PGE1 and PGE2. It is concluded that intercellular communication inside the liver mediated by prostaglandins derived from activated Kupffer cells may form a mechanism to induce synthesis of specific proteins in parenchymal cells.

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.

Escherichia coli endotoxin (lipopolysaccharide) was shown to increase glycogenolysis in the perfused liver 2-3-fold. In isolated parenchymal liver cells, however, endotoxin did not influence glycogenolysis, whereas stimulation by endotoxin of glycogenolysis in the perfused liver could be blocked by aspirin. This suggests that the effect of endotoxin on liver glycogenolysis is mediated by eicosanoids. The amount of prostaglandin D2 (which is the major prostanoid formed by Kupffer cells) in the liver perfusates was increased 5-fold upon endotoxin addition, with a time course which preceded the increase in glucose output. It is concluded that endotoxin stimulates glycogenolysis in the liver by stimulating prostaglandin D2 release from Kupffer cells, with a subsequent activation of glycogenolysis in parenchymal liver cells. This mechanism of intercellular communication may be designed to provide the carbohydrate source of energy necessary for the effective destruction of invaded microorganisms, by phagocytic cells, including the Kupffer cells.

Isolation and separation of rat liver cells into endothelial, Kupffer, and parenchymal cell fractions were performed at different times after injection of human 125I-acetyl low density lipoproteins (LDL). In order to minimize degradation and redistribution of the injected lipoprotein during cell isolation, a low temperature (8 degrees C) procedure was applied. Ten min after injection, isolated endothelial cells contained 5 times more acetyl-LDL apoprotein per mg of cell protein than the Kupffer cells and 31 times more than the hepatocytes. A similar relative importance of the different cell types in the uptake of acetyl-LDL was observed 30 min after injection. For studies on the in vitro interaction of endothelial and Kupffer cells with acetyl-LDL, the cells were isolated with a collagenase perfusion at 37 degrees C. Pure endothelial (greater than 95%) and purified Kupffer cells (greater than 70%) were obtained by a two-step elutriation method. It is demonstrated that the rat liver endothelial cell possesses a high affinity receptor specific for the acetyl-LDL because a 35-fold excess of unlabeled acetyl-LDL inhibits association of the labeled compound for 70%, whereas unlabeled native human LDL is ineffective. Binding to the acetyl-LDL receptor is coupled to rapid uptake and degradation of the apolipoprotein. Addition of the lysosomotropic agents chloroquine (50 microM) or NH4Cl (10 mM) resulted in more than 90% inhibition of the high affinity degradation, indicating that this occurs in the lysosomes. With the purified Kupffer cell fraction, the cell association and degradation of acetyl-LDL was at least 4 times less per mg of cell protein than with the pure endothelial cells. Although cells isolated with the cold pronase technique are also still able to bind and degrade acetyl-LDL, it appeared that 40-60% of the receptors are destroyed or inactivated during the isolation procedure. It is concluded that the rat liver endothelial cell is the main cell type responsible for acetyl-LDL uptake.

Morphological characteristics of the interaction of low density lipoproteins (LDL) and acetylated low density lipoproteins (AcLDL) with rat liver cells are described. These liver cell types are mainly responsible for the catabolism of these lipoproteins in vivo. Isolated rat liver Kupffer, endothelial, and parenchymal cells were incubated with LDL or AcLDL conjugated to 20 nm colloidal gold. LDL was mainly internalized by Kupffer cells, whereas AcLDL was predominantly found in endothelial cells. Kupffer and endothelial cells displayed different morphological characteristics in the processing of these lipoproteins. Kupffer cells bound LDL at uncoated regions of the plasma membrane often at the base of pseudopodia, and internalized the particles via small smooth vesicles. These uptake characteristics differ from the classical LDL uptake pathway, as described for other cell types, and may be related to the unique recognition properties of the receptor of Kupffer cells as observed in biochemical studies. Liver endothelial cells bound AcLDL in coated pits, followed by rapid uptake. Uptake proceeded through small coated vesicles, and after 5 min of incubation large (600-1200 nm) electron-lucent vacuoles (endosomes) with AcLDL-gold particles arranged along the membrane region were present. The endosomes were often associated closely with the cell membrane which might enable direct recycling of AcLDL receptors. These observations might explain the high efficiency of these cells in the processing of modified LDL in vivo.

Conditioned media of isolated Kupffer and endothelial liver cells were added to incubations of parenchymal liver cells, in order to test whether secretory products of Kupffer and endothelial liver cells could influence parenchymal liver cell metabolism. With Kupffer cell medium an average stimulation of glucose production by parenchymal liver cells of 140% was obtained, while endothelial liver cell medium stimulated with an average of 127%. The separation of the secretory products of Kupffer and endothelial liver cells in a low and a high molecular weight fraction indicated that the active factor(s) had a low molecular weight. Media, obtained from aspirin-pretreated Kupffer and endothelial liver cells, had no effect on the glucose production by parenchymal liver cells. Because aspirin blocks prostaglandin synthesis, it was tested if prostaglandins could be responsible for the effect of media on parenchymal liver cells. It was found that prostaglandin (PG) E1, E2, and D2 all stimulated the glucose production by parenchymal liver cells, PGD2 being the most potent. Kupffer and endothelial liver cell media as well as prostaglandins E1, E2, and D2 stimulated the activity of phosphorylase, the regulatory enzyme in glycogenolysis. The data indicate that prostaglandins, present in media from Kupffer and endothelial liver cells, may stimulate glycogenolysis in parenchymal liver cells. This implies that products of Kupffer and endothelial liver cells may play a role in the regulation of glucose homeostasis by the liver.

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 apolipoprotein E-free high density lipoproteins (HDL) in varying quantities. Incorporation of 5 or 13 micrograms of Tris-Gal-Chol into HDL (20 micrograms of protein) stimulates the liver association of the HDL apoprotein radioactivity 24- and 55-fold, respectively, at 10 min after intravenous injection into rats. The increased interaction of Tris-Gal-Chol HDL with the liver is blocked by preinjection of asialofetuin or N-acetylgalactosamine but not influenced by N-acetylglucosamine. The parenchymal liver cell uptake of HDL is stimulated 42- or 105-fold, respectively, by incorporation of 5 or 13 micrograms of Tris-Gal-Chol into HDL (20 micrograms of protein), while the association with nonparenchymal cells is stimulated only 1.7- or 5-fold. It can be calculated that 98.0% of the Tris-Gal-Chol HDL is associated with parenchymal cells. In contrast, incorporation of 13 micrograms of Tris-Gal-Chol into LDL (20 micrograms of protein) leads to a selective association of LDL with nonparenchymal cells (92.3% of the total liver uptake). It is concluded that Tris-Gal-Chol incorporation into HDL leads to a specific interaction of HDL with the asialoglycoprotein (galactose) receptor on parenchymal cells whereas Tris-Gal-Chol incorporation into LDL leads mainly to an interaction with a galactose receptor from Kupffer cells. Probably this highly selective cellular targeting of LDL and HDL by Tris-Gal-Chol is caused by the difference in size between these lipoproteins. The increased interaction of HDL with the parenchymal cells upon Tris-Gal-Chol incorporation is followed by degradation of the apolipoprotein in the lysosomes. It is concluded that Tris-Gal-Chol incorporation into LDL or HDL leads to a markedly increased catabolism of LDL by way of the Kupffer cells and HDL by parenchymal cells which might be used for lowering serum cholesterol levels. The use of Tris-Gal-Chol might also find application for targeting drugs or other compounds of interest to either Kupffer or parenchymal liver cells.

In this study, binding and degradation of tissue-type plasminogen activator (t-PA) by the human hepatoma cell line Hep G2 was investigated. Binding at 4 degrees C was time-dependent and reached a maximum after ca. 2 hours. Scatchard analysis of saturation experiments showed about 170,000 high affinity binding sites for t-PA per cell with an apparent Kd of 90 nM. These binding sites were calcium-dependent. Part of the binding to the hepatoma cells was non-saturable, owing to a large amount of low affinity binding sites which are at least partially located on the extracellular matrix of the cells. Competition with mannose- and galactose-terminated glycoproteins had no effect on total binding of 125I-t-PA. Degradation products of 125I-t-PA were found in the supernatant after a short lag phase and then increased linearly for at least 5 hours at 37 degrees C. Degradation could be inhibited by chloroquine, NH4Cl and NaN3. We conclude that the human hepatoma cell line Hep G2 has a specific binding mechanism for t-PA which is not mediated by known carbohydrate receptor systems. Binding is followed by cellular uptake and degradation in the lysosomes.