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We studied the kinetics of hepatic uptake of liposomes during serum-free recirculating perfusion of rat livers. Liposomes consisted of phosphatidylcholine, cholesterol and phosphatidylserine in a 6:4:0 or a 3:4:3 molar ratio and were radiolabelled with [3H]cholesteryl oleyl ether. The negatively charged liposomes were taken up to a 10-fold higher extent than the neutral ones. Hepatic uptake of fluorescently labelled liposomes was examined by fluorescence microscopy. The neutral liposomes displayed a typical Kupffer cell distribution pattern, in addition to weak diffuse staining of the parenchyma, while the negatively charged liposomes showed a characteristic sinusoidal lining pattern, consistent with an endothelial localization. In addition, scattered Kupffer cell staining was distinguished as well as diffuse parenchymal fluorescence. The mainly endothelial localisation of the negatively charged liposomes was confirmed by determining radioactivity in endothelial and Kupffer cells isolated following a 1-h perfusion. Perfusion in the presence of polyinosinic acid, an inhibitor of scavenger receptor activity, reduced the rate of uptake of the negatively charged liposomes twofold, indicating the involvement of this receptor in the elimination mechanism. These results are compatible with earlier in vitro studies on liposome uptake by isolated endothelial cells and Kupffer cells, which showed that in the absence of serum also endothelial cells in situ are able to take up massive amounts of negatively charged liposomes. The present results emphasize that the high in vitro endothelial cell uptake in the absence of serum from earlier observations was not an artifact induced by the cell isolation procedure.

Objective: To monitor liver regeneration following partial hepatectomy, liver cell proliferation can be measured by assaying in vivo [3H]thymidine incorporation into liver cell DNA. We hypothesized that [3H]thymidine incorporation into whole liver tissue parallels [3H]thymidine incorporation into liver cell DNA, both in high proliferating and low proliferating liver., Study Design: Liver cell proliferation in rats after partial hepatectomy or a sham operation was studied by measuring incorporation of [3H]thymidine into various fractions of liver tissue on days 1, 2, 3, 4 and 10 after surgery., Results: [3H]thymidine incorporation into whole liver tissue and in the protein fraction correlated well with DNA-specific [3H]thymidine incorporation into regenerating (r > .80, P < .0001) and nonregenerating liver (r > .69, P < .005). [3H]thymidine incorporation into DNA was < 5% of the total amount of administered [3H]thymidine in both sham-operated and hepatectomized rats. Significant differences in [3H]thymidine incorporation into partially hepatectomized livers as compared to sham-operated rat livers were found on days 1 and 2 (whole liver tissue and protein fraction) or day 1 (DNA) after surgery., Conclusion: [3H]thymidine incorporation into whole liver tissue is a simple technique that can be used for the study of liver cell proliferation after partial hepatectomy in rats.

Liposomes of 400 nm in diameter can cross the 100-nm fenestrations in the endothelium of the hepatic sinusoid, provided they contain phosphatidylserine (PS) but not phosphatidylglycerol (PG) [Daemen et al. (1997) Hepatology 26, 416]. We present evidence indicating that (i) the PS effect does not involve a pharmacological action of this lipid on the size of the fenestrations, (ii) fluid-type but not solid-type PS liposomes have access to the hepatocytes and (iii) the lack of uptake of PG liposomes by hepatocytes is not due to a lack of affinity of the hepatocytes for PG surfaces. We conclude that the mechanism responsible for the uptake of large PS-containing liposomes by hepatocytes in vivo involves a mechanical deformation of these liposomes during their passage across the endothelial fenestrations.

The interaction with liver cells of liposomes containing different mol fractions of phosphatidylserine was investigated in vivo and in vitro. Increasing the amount of liposomal phosphatidylserine from 10 to 30 mol% leads to a faster blood disappearance of the liposomes. Within the liver, which is mainly responsible for this elimination, these liposomes are only taken up by the hepatocytes and Kupffer cells. By contrast, sinusoidal endothelial cells, in vitro, do bind and internalize liposomes containing >/=30% phosphatidylserine at least as actively as Kupffer cells. The uptake by endothelial and Kupffer cells is inhibited by poly(inosinic acid) and other anionic macromolecules, suggesting the involvement of scavenger receptors. The lack of liposome uptake by endothelial cells under in vivo conditions can be attributed to plasma effects since addition of various sera caused severe reduction of in vitro uptake of liposomes. In vivo the phosphatidylserine head groups may be masked by plasma proteins adsorbed to the liposomal surface, thus preventing recognition by receptors, which are intrinsically able to recognize phosphatidylserine., (Copyright 1999 Academic Press.)

Human serum albumin (HSA) derivatized with cis-aconitic anhydride was covalently coupled to liposomes with a size of approximately 100 nm [polyaconitylated HSA (Aco-HSA) liposomes]. Within 30 min after injection into a rat, Aco-HSA liposomes were completely cleared from the blood and almost exclusively taken up by the liver, whereas in control liposomes 80% was still present in the blood at that time. Endothelial cells were shown to account for almost two-thirds of the hepatic uptake of the Aco-HSA liposomes, the remainder being recovered mainly in the liver macrophages (Kupffer cells). With fluorescently labeled liposomes it was shown that the Aco-HSA liposomes target a vast majority (>85%) of the cells in the endothelial cell population. Control liposomes were not taken up to a significant extent by the endothelial cells. Uptake of Aco-HSA liposomes by both endothelial and Kupffer cells was inhibited by preinjection with polyinosinic acid, indicating the involvement of scavenger receptors in the uptake process. The uptake of Aco-HSA liposomes by liver endothelial cells was dependent on liposome size; with increasing liposome diameter endothelial cell uptake decreased in favor of Kupffer cell uptake. We have demonstrated that massive in vivo targeting of liposomes to a defined cell population other than macrophages is possible. Aco-HSA liposomes thus may represent an attractive drug carrier system for treatment of various liver or liver endothelium-associated disorders.

Liposomes with diameters of 200 to 400 nm containing phosphatidylserine (PS) or phosphatidylglycerol (PG) were injected intravenously into rats. Two hours after injection, 75% of the injected dose of PS liposomes was found in the liver and only 10% found in the spleen, while 35% of the PG liposomes was found in the liver and as much as 40% was found in the spleen. Cell-isolation experiments revealed the following remarkable difference in the intrahepatic distribution between the two liposome formulations: the PS liposomes distributed in about equal amounts to Kupffer cells and hepatocytes, despite their size (200-400 nm) exceeding that of the endothelial fenestrae (average 150 nm), whereas the PG liposomes were only taken up by the Kupffer cells and not at all by the hepatocytes. Double-label studies, using liposomes in which the lipid-moiety was radio labeled with [3H]cholesteryloleylether ([3H]CE) and the water phase with [14C]sucrose, showed that the liposomes were taken up as intact particles. These observations were confirmed through electron microscopy by determining the in situ localization of liposome-encapsulated colloidal gold particles in thin sections of liver and spleen. The differences in organ distribution are ascribed to differences in opsonization patterns of the two liposomal surfaces. For the difference in intrahepatic distribution, we offer the following two explanations: the exploitation of the blood cell-mediated forced sieving concept and the indication of a PS-specific pharmacological effect on the dimensions of the fenestrations.

Four subpopulations of hepatic macrophages, differing in size, were isolated from rat liver. The secretion of nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), prostaglandin E (PGE) and interleukin-1 (IL-1) by freshly isolated as well as cultured cells was studied after in vitro stimulation with the immunomodulator muramyl dipeptide (MDP) in free or liposome-encapsulated form. Freshly isolated liver macrophages could be induced to secrete significant levels of NO, TNF-alpha, PGE and IL-1. The extent of secretion, however, varied substantially between macrophages of different size. The highest levels of secretion of TNF-alpha, PGE and IL-1 were observed in the fraction containing the large-size macrophages, while progressively lower levels of secretion were observed with decreasing size. In contrast, the highest levels of NO secretion were observed by small macrophages and steadily decreased with increasing size. Hepatic macrophages of different size displayed differences in secretory potential during in vitro culture. The ability of small liver macrophages to secrete NO, TNF-alpha, or PGE, following activation with MDP, gradually increased with time in culture. In contrast, large liver macrophages gradually lost their secretory ability after 1-2 days and the intermediate-size cells after 2-3 days in culture. This functional heterogeneity in secretory properties among rat liver macrophages of different size is discussed with reference to their potential role and significance in host defense against metastatic tumor growth in the liver.

Subfractions of the hepatic macrophage population, differing in cell size, were isolated from normal rats and rats treated with liposomal muramyl dipeptide (lipMDP) and analyzed histochemically and by ultrastructural peroxidase cytochemistry. The majority of cells in all subfractions of control rats displayed the ultrastructural endogenous peroxidase pattern of resident liver macrophages and showed positive staining with the general macrophage markers nonspecific esterase (NSE) and monoclonal antibody ED1. Heterogeneity in intensity of NSE and ED1 staining was observed among macrophages of different size. Generally, the intensity of NSE and ED1 staining decreased with decreasing cell size. After injection of lipMDP, we observed the appearance of a discrete subpopulation of cells in the liver in addition to the resident macrophages. These cells, containing a nucleus with a characteristic shape, were predominantly recovered in the small-sized fractions and were characterized by an immature ultrastructural macrophage morphology (no or only a few lysosomes and phagosomes) and a lack of ED1 reactivity, NSE, and endogenous peroxidase. We suggest an important role for these cells in lipMDP induced antitumor capacity of the liver.

Intraportal inoculation of CC531 adenocarcinoma cells into syngeneic rats causes an increase of liver macrophage cell number but not of major histocompatibility complex class II antigen expression. On day 1 after inoculation of 10(5) CC531 cells, a fixed number of isolated liver macrophages lysed significantly more target cells in vitro than did control cells. This effect was still present after 4 weeks. A 10-fold higher initial tumor dose significantly suppressed the macrophage response during the first 2 weeks. In contrast to tumoricidal activity induced by lipopolysaccharide in vitro, the tumoricidal response following in vivo challenge with tumor cells appeared not closely related to the production of reactive nitrogen intermediates, as in the latter case it was not abrogated in the presence of nitric oxide synthase inhibitor. Furthermore, the liver macrophage population appeared not fully activated after tumor inoculation as lipopolysaccharide further increased tumoricidal activity in vitro. The observed numerical and functional response of liver macrophages to intraportally inoculated tumor cells points at an important role of these cells in aspecific immune reactivity aimed at the reduction of local tumor growth. Results suggest that mechanistic differences exist between macrophage tumoricidal activity induced by tumor cells as compared with lipopolysaccharide.

We examined the effects of several hemopoietic growth factors on proliferation of rat liver macrophages in vitro. The proliferative response of liver macrophages to hemopoietic growth factors was assayed on the basis of [methyl-3H]thymidine uptake. Macrophage colony-stimulating factor and recombinant murine granulocyte-macrophage colony-stimulating factor stimulated [methyl-3H]thymidine incorporation in a concentration-dependent manner. With granulocyte-macrophage colony-stimulating factor, maximum incorporation was observed at 50 U/ml, whereas with macrophage colony-stimulating factor no incorporation plateau was observed up to 50% L929-conditioned medium. Incubation of liver macrophages with various concentrations of recombinant human interleukin-2, recombinant murine interleukin-3 and recombinant human interleukin-6 or culture medium alone did not result in significant incorporation of [methyl-3H]thymidine. When liver macrophages were fractionated according to cell size, highest incorporation was observed in the large macrophages. Proliferating cells in cultures of all subfractions were microscopically identified as typical macrophages by the use of macrophage-specific monoclonal antibodies. After 6 days in culture, these macrophages had functional properties similar to those of resident liver macrophages with respect to phagocytosis and in vitro activation with immunomodulators to tumorcytotoxicity and secretion of nitric oxide and tumor necrosis factor-alpha. These results suggest that macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor play important roles among the regulatory factors that support local proliferation of rat liver macrophages.

In this study, we investigated different functional characteristics of the rat liver macrophage population after a single i.v. injection of liposome-encapsulated muramyl dipeptide (MDP) or its lipophilic derivative muramyl tripeptide-phosphatidylethanolamine (MTP-PE). The in situ induced tumoricidal activity of the liver macrophage population was determined in vitro against C26 colon adenocarcinoma cells. For investigating which cells are responsible for the observed cytotoxic effects, subfractions of the liver macrophage population, differing in cell size, were isolated at different intervals after injection of the liposomal muramyl peptides. From these subfractions, the number of cells, degree of cytotoxicity, and the response to an additional activation with free MDP in vitro were determined. Maximal induction of tumoricidal activity of the liver macrophage population was reached between 12 and 24 hours after injection of liposomal MDP, while no significant differences between the subfractions were observed. Heterogeneity of tumor cytolytic capacity was observed in subfractions of macrophages isolated at 2 and 48 hours after injection. At these time points, highest cytolytic activity was observed for the small to intermediate-size macrophages. No significant cytotoxicity was detectable in any subfraction 72 hours after injection of liposomal MDP. An identical pattern of macrophage tumoricidal activity was observed after injection of liposomal MTP-PE, although slightly lower cytotoxicity levels were found. When isolated during the first 12 hours after injection of liposomal MDP, the macrophage population was unable to respond to a subsequent in vitro exposure to MDP, with respect to tumor cytotoxicity. Twenty-four and 48 hours after injection, the smallest cells could be slightly reactivated, whereas the larger cells still remained unresponsive.(ABSTRACT TRUNCATED AT 250 WORDS)

While investigating the effects of 5-fluorouracil (5FU) and 5-fluoro-2'-deoxyuridine (FUdR) on the tumoricidal state of rat liver macrophages activated in vitro by means of liposome-encapsulated muramyl dipeptide (MDP), we observed that 5FU in combination with macrophages produced substantially higher extents of cytolytic activity on tumor cells than 5FU alone. In contrast, FUdR failed to produce this effect; rather, at relatively low FUdR concentrations, lytic activity in the presence of macrophages was even significantly diminished as compared with FUdR in the absence of macrophages. Both 5FU and FUdR were able to enhance the cytolytic activity of macrophages activated by liposome-encapsulated MDP. This finding indicates that, rather than inhibiting the activation of macrophages by liposomal MDP, 5FU can act as a stimulator of macrophage activation by itself. This is further supported by the observations that (i) in combination with 5FU, the secretion of TNF induced by liposomal MDP was synergistically enhanced and (ii) that a second treatment of macrophages with the drug, 24 h after the first, fails to produce increased macrophage cytotoxicity. Our results also show that neither 5FU nor FUdR are likely to unfavorably influence the induction of cytotoxic activity of the macrophages. Rather, combinations of 5FU or FUdR and liposomal MDP may result in an additive or synergistic tumoricidal effect.

We have investigated the processing of the non-exchangeable fluorescent phospholipid analogue phosphatidyl(N-sulphorhodamine B sulphonyl)ethanolamine (N-Rh-PE) by rat liver cells. In the hepatocyte couplet system, N-Rh-PE was incorporated into the plasma membrane at 2 degrees C and readily internalized upon warming to 37 degrees C. Fluorescence was initially found to be concentrated in vesicles clustered throughout the cell, but subsequently it started to accumulate in pericanalicular vesicles, tentatively identified as lysosomes, and in the bile canalicular lumen. Analysis of cells and media by t.l.c. revealed the slow formation of at least two metabolites. After intravenous injection into bile-fistula rats of [9,10-3H-oleoyl]N-Rh-PE incorporated in small unilamellar liposomes, the initial rates of elimination from plasma of 3H and rhodamine label were virtually identical. However, biliary secretion of the 3H label (5.5% of dose at 2 h) was much slower than that of the rhodamine label (49.3% at 2 h). The rhodamine label in bile was chloroform-soluble, but not identical to the native molecule, and was resistant to phospholipase A2 and alkaline hydrolysis. To gain insight in the mechanism of the rapid bile secretion of this metabolite, we compared the processing of N-Rh-PE, its deacylated form [glycerophospho(N-sulphorhodamine B sulphonyl)ethanolamine; Gly-N-Rh] and the rhodamine label itself (sulphorhodamine B sulphonyl chloride; SRho). Intravenous injection of chloroform-soluble N-Rh-PE and of methanol/water-soluble Gly-N-Rh complexed with albumin both resulted in rapid bile secretion of chloroform-soluble fluorescent compounds (60.2% and 86.3% respectively at 2 h), which showed behaviour identical to that of the metabolite of liposomal N-Rh-PE on t.l.c. Methanol/water-soluble SRho was also rapidly secreted into bile (89.5% at 2 h) without being metabolized. Bile secretion of the chloroform-soluble metabolite of N-Rh-PE and of SRho was markedly impaired (-31% and -52% respectively) in GY Wistar rats, which express a genetic defect in the hepatobiliary transport of organic anions. Our data show that the rat hepatocyte is capable of modifying the structure of N-Rh-PE, a process which proceeds considerably faster in vivo than in vitro. The chloroform-soluble metabolite is subsequently rapidly removed via the bile. The canalicular organic anion transporting system, which is deficient in GY rats, appears to be involved in the excretion of this apolar product of hepatic metabolism.

The liver macrophage population was fractionated according to cell size into three subpopulations by means of elutriation centrifugation. The total liver macrophage population and the three subpopulations were cultured and exposed to the immunomodulators muramyl dipeptide (MDP), in a free or liposome-encapsulated form, and/or lipopolysaccharide (LPS). The tumor cytotoxic activity thus induced in the populations, the preservation of this activity and the response to a second stimulus were studied. The in vitro induced cytolytic activity was determined by a radioactivity release assay, using C26 colon adenocarcinoma cells, labeled with [methyl-3H]thymidine, as target cells. MDP or LPS readily activated the total macrophage population in maintenance culture to a tumor cytotoxic state during the first 2 days after isolation. Four days after isolation, the activation induced with both MDP and LPS was strongly reduced. The small to intermediate-size macrophages could be activated to tumor cytotoxic activity with MDP for up to 3 days and with LPS for up to 4 days in culture. The large-size macrophages could only be activated up to day 2 in culture with MDP or LPS or both. The combination of MDP and LPS, however, induced all cell populations in a synergistic way to become cytolytic for up to 4 days in culture. With free MDP as an activator, the activated state decayed within 1 day to almost zero levels, but less rapidly in the small cells than in the large cells. With liposome-encapsulated MDP, the activated state was preserved considerably longer, except in the largest cells.(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate the contribution of plasma-derived phosphatidylcholine (PC) to bile PC, the hepatic processing and biliary secretion of liposome-associated PC was studied in rats. For this purpose, small unilamellar vesicles (SUV), containing trace amounts of [2-palmitoyl-9,10-3H]dipalmitoylphosphatidylcholine ([palmitoyl-3H]DPPC), [choline-14C]-dipalmitoylphosphatidylcholine ([choline-14C]DPPC), di[14C]palmitoylphosphatidylcholine ([14C]DPPC) or di[1-14C]-oleoylphosphatidylcholine ([14C]DOPC), were administered intravenously to unanaesthetized rats, equipped with permanent catheters in heart and bile duct. Biliary secretion of the 14C-head-group label of DPPC was very slow (0.3% of injected dose in 4 h), whereas the [3H]palmitoyl label was secreted at a much higher rate (16% in 4 h), but only after substantial catabolism of the acyl chain. To study the latter process in more detail, we compared hepatic metabolism and biliary secretion of [1-14C]acyl-labelled DPPC and DOPC. In rats with an 8-day bile drainage, degradation products of the oleoyl chain were utilized for synthesis of bile acids, which were subsequently secreted into the bile (2% in 6 h). A much smaller fraction (0.6% in 6 h) was secreted as PC and lyso-PC. When bile drainage was started immediately after SUV injection, i.e. a situation with a low hepatic bile acid synthesis rate and a high phospholipid secretion, the secretion of [14C]DOPC-derived radioactivity in the form of bile acids was decreased (0.2% in 6 h), and that as (lyso-)PC increased (1.5% in 6 h). Biliary secretion of DPPC palmitoyl chains in bile-diverted rats was much less than that of the oleoyl chains, and occurred predominantly as PC and lyso-PC (0.6%, compared with 0.4% as bile acids in 6 h). Breath analyses demonstrated that a considerable fraction of both acyl chains was oxidized to CO2 and expired: 25.1% of the administered label for oleoyl chains and 13.4% for palmitoyl chains respectively in a 4 h period. The results of this study indicate that liposomal PC is only minimally secreted into bile via a direct pathway; the bulk is extensively degraded in the liver. Resulting products are partly secreted into bile, as bile acid or as resynthesized PC. There appears to be a quantitative difference in the metabolism of oleoyl and palmitoyl acyl chains.

We synthesized the 3',5'-O-dipalmitoyl derivative of 5-fluoro-6-[3H]-2'-deoxyuridine and incorporated it into the bilayers of multilamellar liposomes (400 nm diameter) of various lipid compositions. The prodrug-containing liposomes were incubated with rat liver macrophages (Kupffer cells) in monolayer culture and with lysosomal fractions from whole rat liver homogenates. The release of water-soluble radioactive degradation products from the cells was measured and we found the rate of release strongly dependent on the lipid composition of the liposomes. After 4 hours of incubation the release of radioactivity was 9-fold higher from egg-phosphatidylcholine/phosphatidylserine/cholesterol liposomes than from distearoylphosphatidylcholine/dipalmitoylphosphatidylglycerol/cholest ero l or dioctadecyl-sn-glycero-phosphorylcholine/dipalmitoylphosphatidylg lycerol/ cholesterol liposomes. A somewhat less pronounced difference in rate of prodrug degradation was found when the liposomes were incubated with lysosomal fractions. The water-soluble products that were formed showed anti-tumor activity against C26-adenocarcinoma tumor cells in vitro. Preliminary evidence suggests this activity to be caused by 5-fluoro-2'-deoxyuridine. We conclude that incubation of liposomes of varied composition containing diacylated 5-fluoro-2' deoxyuridine derivatives with Kupffer cells in culture, results in the formation of an intracellular prodrug depot in these cells from which compounds with anti-tumor activity are released with controllable rates.

We compared the metabolic fate of [3H]cholesteryl[14C]oleate, [3H]cholesteryl hexadecylether, 125I-labeled bovine serum albumin and [3H]inulin as constituents of large immunoglobulin-coupled unilamellar lipid vesicles following their internalization by rat liver macrophages (Kupffer cells) in monolayer culture. Under serum-free conditions, the cholesteryl oleate that is taken up is hydrolyzed, for the greater part, within 2 h. This occurs in the lysosomal compartment as judged by the inhibitory effect of the lysosomotropic agents monensin and chloroquin. After hydrolysis, the cholesterol moiety is accommodated in the cellular pool of free cholesterol and the oleate is reutilized for the synthesis mainly of phospholipids and, to a lesser extent of triacylglycerols. During incubation in plasma, however, substantial proportions of both the cholesterol and the oleate are shed from the cells, predominantly in the unesterified form. When the liposomes are labeled with the cholesteryl ester analog [3H]cholesteryl hexadecylether only a very small fraction of the label is released from the cells, even in the presence of plasma. Similar to the label remaining associated with the cells, the released label is identified in that case as unchanged cholesteryl ether. The liposomal aqueous phase marker 125I-labeled bovine serum albumin is also readily degraded intralysosomally and the radioactive label is rapidly released from the cells in a trichloroacetic acid-soluble form. Also, as much as 20% of the aqueous phase marker [3H]inulin that becomes cell-associated during a 2-h incubation with inulin-containing liposomes, is released from the cells during a subsequent 4-h incubation period in medium or rat plasma. The usefulness of the various liposomal labels as parameters of liposome uptake and intracellular processing is discussed.

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.

We investigated the in vitro activation of rat liver macrophages to a tumoricidal state with free and liposome-encapsulated immunomodulators. The cytolytic activity of liver macrophages was determined by a radioactivity release assay using murine B16 melanoma cells, labeled with [methyl-3H]thymidine. Exposure of the liver macrophages to concentrations of 50 micrograms of free, nonencapsulated, muramyl dipeptide (MDP) per ml resulted in maximal levels of tumor cell lysis of approximately 20%. Encapsulation of the MDP within liposomes (multilamellar vesicles, 0.3 to 0.5 micron in diameter, consisting of egg phosphatidylcholine, cholesterol, and dicetylphosphate, 4:5:1) not only caused a 500-fold reduction in the amount of MDP required to obtain the same levels of cytolysis but also increased the maximally obtainable level of cytolysis more than 2-fold. A synergistic effect of lipopolysaccharide and free or encapsulated MDP on cytolytic activity was observed when the macrophages were exposed to a combination of the two agents simultaneously. Besides causing tumor cell lysis, activated macrophages were also able to suppress tumor cell proliferation by 80 to 90% as determined by a [methyl-3H]thymidine incorporation assay. With a fixed amount of MDP, encapsulated in different amounts of liposomal lipid, the extent of macrophage activation was found to increase with a larger amount of encapsulating lipid. This increase in macrophage activation may be the result of a sustained intracellular release of encapsulated MDP from the liposomes. Liposome structure and composition will thus be important parameters in the in vivo application of liposomes as carriers of immunoactive substances.

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 vivo uptake and processing by liver macrophages (Kupffer cells) of liposomes, covalently coated with rabbit immunoglobulin (Ig liposomes) was studied following intravenous injection in rats. Rabbit Ig liposomes were labeled with trace amounts of cholesteryl[14C]oleate and [3H]cholesteryl hexadecyl ether. 1 h after injection of the liposomes, the non-parenchymal cells were isolated and subjected to centrifugal elutriation with stepwise-increasing flow rates; thus, five sub-fractions of Kupffer cells were obtained ranging in size from 9 to 14 micron in diameter. The cells were assayed for peroxidase activity and protein content. Rabbit Ig liposomes were taken up preferentially by Kupffer cells with diameters larger than 11 micron, which constitute less than 25% of the total Kupffer cell population. The intralysosomal degradation of the ingested liposomes was monitored by measuring the 3H/14C ratio of the cells. Due to the rapid release from the cells of the [14C]oleate formed from the cholesteryl[14C]oleate and the virtually complete retention of the non-metabolizable [3H]cholesteryl hexadecyl ether the 3H/14C ratio of the cells increases with proceeding hydrolysis of the liposomes. Thus, we were able to show that, in vivo, the Kupffer cells of the larger size classes, are not only more active in liposome uptake, but are also substantially more active in liposome degradation than smaller cells. The maintenance of the observed heterogeneity of rat liver Kupffer cells, with respect to liposome uptake under in vitro culture conditions, was examined. Subfractions were maintained in monolayer culture for 2 days and incubated with rabbit Ig liposomes. Binding and uptake of liposomes by the cells was monitored by measuring cell-associated radioactivity at 4 degrees C and 37 degrees C, respectively. In contrast to our in vivo results, we observed maximal in vitro liposome binding and uptake in those subfractions containing small cells (10-11 micron diameter), while the fractions containing cells larger than 12 micron, which were more active in vivo, were substantially less active than the smaller cells. The maximum we observed was even more pronounced when the liposome concentration was increased. We conclude that liver macrophage subfractions that barely participate in liposome uptake from the bloodstream in vivo, possess the potential to develop the capacity in vitro to phagocytose rabbit Ig-coated liposomes to extents equal to or even higher than the cells belonging to those subfractions containing the phagocytically most active cells under in vivo conditions.

We investigated the effect of cholesterol on the uptake and intracellular degradation of liposomes by rat liver and spleen macrophages. Multilamellar vesicles (MLV) consisting of distearoylphosphatidylcholine/phosphatidylserine (molar ratio 9:1) or distearoylphosphatidylcholine/cholesterol/phosphatidylserine (molar ratio 4:5:1) were labeled with [3H]cholesteryl hexadecyl ether and/or cholesteryl [14C]oleate. After i.v. injection the cholesterol-containing liposomes were eliminated less rapidly from the bloodstream and taken up to a lesser extent by the liver (macrophages) than the cholesterol-free liposomes. Assessment of the 3H/14C ratios in liver and spleen cells revealed that the cholesterol-containing liposomes are substantially more resistant towards intracellular degradation than the cholesterol-free liposomes. These results could be confirmed by measuring the release of 111In from liposomes after uptake by liver and spleen by means of gamma-ray perturbed angular correlation spectroscopy. Experiments with cultured Kupffer cells in monolayer also revealed that incorporation of cholesterol results in a decrease of the uptake and an increase of the intracellular stability of cholesteryl [14C]oleate-labeled liposomes. Finally, incubation of both types of liposomes with lysosomal fractions prepared from rat liver demonstrated a difference in susceptibility to lysosomal degradation: the cholesterol-free vesicles were much more sensitive to lysosomal esterase than the cholesterol-containing liposomes. These results may be relevant to the application of liposomes as a drug carrier system to liver and spleen (macrophages).

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.

Intravenously administered gadolinium chloride caused only a slight decrease in the rate of elimination of small unilamellar liposomes from the blood and had no influence on the total hepatic uptake of these vesicles, but did alter their intrahepatic distribution substantially. Uptake by the non-parenchymal cells was substantially decreased, whereas uptake by the parenchymal cells showed a concomitant increase. Our earlier observations (Roerdink et al. (1981) Biochim. Biophys. Acta 677, 79-89) on the effect of lanthanides on the in vivo distribution of multilamellar liposomes have been extended, in that we demonstrate, in addition to the drop in elimination rate from the blood and in the over-all hepatic uptake, a shift of liposome distribution within the Kupffer cell population. While the larger Kupffer cells, which normally take up a major fraction of an injected liposome dose, were strongly inhibited in liposome uptake, the more numerous small macrophages showed a 3-4-fold increase in uptake.

Highly purified mitochondria from rat liver contain a phospholipase A that catalyzes removal of 2-fatty acids, with a pH optimum above pH 8.0. Lysosomal preparations appeared to have two phospholipases A associated with them, one with a pH optimum at about pH 4.0, the second between pH 6.0 and 7.0. Mitochondrial phospholipase A hydrolyzed exogenous phospholipid as fast as or faster than endogenous phospholipid. The difference in specific radioactivity of (14)C-ethanolamine-labeled endogenous mitochondrial phospholipid before and after incubation indicates that a fraction of mitochondrial phosphatidyl ethanolamine is hydrolyzed more rapidly than the mitochondrial phospholipids as a whole. Acyl bond hydrolysis of exogenous and endogenous phospholipid by mitochondria was stimulated by free fatty acid, Ca(++), or in certain cases, monoacyl phospholipids or by treatments that disrupt the mitochondrial membrane. Of various fatty acids tested, lauric, myristic, oleic, and linoleic were most effective. ADP and ATP inhibited mitochondrial phospholipase, probably because they compete for Ca(++). Mg(++) also behaved as a competitive inhibitor; the effect was overcome by relatively little Ca(++).