Uptake of phosphatidylserine-containing liposomes by liver sinusoidal endothelial cells in the serum-free perfused rat liver.

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.

The role of beta2-glycoprotein I in liposome-hepatocyte interaction.

Adsorption of serum proteins to the liposomal surface plays a critical role in liposome clearance from the blood. The aim of this study was to investigate the role of liposome-adsorbed serum proteins in the interaction of liposomes with hepatocytes. We analyzed the serum proteins adsorbing to the surface of differently composed small unilamellar liposomes during incubation with human or rat serum, and found that one protein, with a molecular weight of around 55 kDa, adsorbed in a large amount to negatively charged liposomes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). The binding was dependent on the liposomal charge density. The approximately 55-kDa protein was identified as beta2-glycoprotein I (beta2GPI) by Western blotting. Despite the high affinity of beta2GPI for strongly negatively charged liposomes, in vitro uptake and binding experiments with isolated rat hepatocytes, Kupffer cells or liver endothelial cells, and with HepG2 cells showed no enhancing effect of this protein on the association of negatively charged liposomes with any of these cells. On the contrary, an inhibitory effect was observed. We conclude that despite abundant adsorption to negatively charged liposomes, beta2GP1 inhibits, rather than enhances, liposome uptake by liver cells.

A role for scavenger receptor B-I in selective transfer of rhodamine-PE from liposomes to cells.

We investigated the potential role of scavenger receptor B-I (SR-BI) in the selective removal of liposomal markers from blood by hepatocytes. Liposomes were labeled with [(3)H]cholesteryloleyl-ether ([(3)H]COE), 1,2-di[1-(14)C]palmitoyl-phosphatidylcholine ([(14)C]PC), and N-(lissamine rhodamine-B sulfonyl)-phosphatidylethanolamine (N-Rh-PE). The radiolabels were eliminated at identical rates from plasma, while N-Rh-PE was cleared twice as fast. Involvement of SR-BI in the selective removal of N-Rh-PE from liposomes was studied in transfected Chinese hamster ovary cells over-expressing SR-BI. Uptake of N-Rh-PE from liposomes containing phosphatidylserine was higher than [(3)H]COE, and was further enhanced by apolipoprotein A-I, confirming involvement of SR-BI in the selective uptake of liposomal N-Rh-PE by cells.

A potential role of macrophage activation in the treatment of cancer.

One of the functions of macrophages is to provide a defense mechanism against tumor cells. In the last decades the mechanism of tumor cell killing by macrophages have been studied extensively. The tumor cytotoxic function of macrophages requires stimulation either with bacterial cell wall products such as lipopolysaccharide (LPS) or muramyldipeptide (MDP) or with cytokines such as interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Activated macrophages secrete several substances that are directly involved in tumor cell killing i.e. tumor necrosis factor (TNF) and nitric oxide (NO). On the other hand, substances are secreted that are able to stimulate tumor cell growth, depending on the stage and the nature of the tumor. Several clinical trials have been performed aiming at the activation of macrophages or dendritic cells, a subpopulation of the macrophages. In this review we will summarize and discuss experimental studies and clinical trials based on the activation of macrophages.

Pharmacokinetics of differently designed immunoliposome formulations in rats with or without hepatic colon cancer metastases.

PURPOSE: Compare pharmacokinetics of tumor-directed immunoliposomes in healthy and tumor-bearing rats (hepatic colon cancer metastases). METHODS: A tumor cell-specific monoclonal antibody was attached to polyethyleneglycol-stabilized liposomes, either in a random orientation via a lipid anchor (MPB-PEG-liposomes) or uniformly oriented at the distal end of the PEG chains (Hz-PEG-liposomes). Pharmacokinetics and tissue distribution were determined using [3H]cholesteryloleylether or bilayer-anchored 5-fluoro[3H]deoxyuridine-dipalmitate ([3H]FUdR-dP) as a marker. RESULTS: In healthy animals clearance of PEG-(immuno)liposomes was almost log-linear and only slightly affected by antibody attachment; in tumor-bearing animals all liposomes displayed biphasic clearance. In normal and tumor animals blood elimination increased with increasing antibody density; particularly for the Hz-PEG-liposomes, and was accompanied by increased hepatic uptake, probably due to increased numbers of macrophages induced by tumor growth. The presence of antibodies on the liposomes enhanced tumor accumulation: uptake per gram tumor tissue (2-4% of dose) was similar to that of liver. Remarkably, this applied to tumor-specific and irrelevant antibody. Increased immunoliposome uptake by trypsin-treated Kupffer cells implicated involvement of high-affinity Fc-receptors on activated macrophages. CONCLUSIONS: Tumor growth and immunoliposome characteristics (antibody density and orientation) determine immunoliposome pharmacokinetics. Although with a long-circulating immunoliposome formulation, efficiently retaining the prodrug FUdR-dP, we achieved enhanced uptake by hepatic metastases, this was probably not mediated by specific interaction with the tumor cells, but rather by tumor-associated macrophages.

Activation of peritoneal cells upon in vivo transfection with a recombinant alphavirus expressing GM-CSF.

In this study we determined the in vivo localization of recombinant proteins expressed by intraperitoneally (i.p.) injected recombinant Semliki Forest virus (SFV) particles. Subsequently, we investigated the influence of i.p. administered SFV particles encoding recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) on intraperitoneal recruitment and activation of cells. Finally, the therapeutic effect of SFV-GM-CSF treatment on an i.p. growing ovarian tumor was determined. Intraperitoneal injections of recombinant SFV particles encoding the reporter protein luciferase resulted in a high level of luciferase activity in cells of the peritoneal lining and tumor cells in the peritoneal cavity. Low levels of luciferase activity were found in liver, spleen and lungs. Injection of SFV-GM-CSF particles resulted in a slight increase in the number of peritoneal macrophages and in a significant increase in the number of neutrophils. In contrast to multiple i.p. injections with commercially available recombinant GM-CSF, i.p. injected SFV-GM-CSF particles activated the macrophages to tumor cytotoxicity. Although treatment of tumor-bearing mice with SFV-GM-CSF particles did not result in prolonged survival, tumor growth was inhibited for 2 weeks. Our findings indicate that macrophage-activating cytokines expressed by the efficient and safe recombinant SFV system when administered i.p. may provide an immunotherapeutic treatment modality additional to current chemotherapeutic treatment of intraperitoneally growing cancers.

The role of hepatocytes in the clearance of liposomes from the blood circulation.

In this chapter we summarize literature and describe in more detail our own observations over a period of nearly two decennia on the role of hepatocytes in the hepatic clearance of intravenously administered liposomes. Evidence is presented indicating that, although size is an important parameter, it is not decisive in determining access of liposomes to the hepatocytes. Also lipid composition is an important parameter, including charge, rigidity and headgroup composition. The role of the fenestrated sinusoidal endothelial cells in determining liposome accessibility of hepatocytes is discussed as well as the involvement of opsonizing plasma proteins such as apolipoprotein E. Our observations led us to postulate the existence of at least four different mechanisms of interaction of liposomes with hepatocytes, i.e. an endocytic and a non-endocytic one for both neutral and negatively charged vesicles

Uptake and intracellular processing of PEG-liposomes and PEG-immunoliposomes by kupffer cells in vitro 1 *.

Specific targeting of drugs to for instance tumors or sites of inflammation may be achieved by means of immunoliposomes carrying site-specific antibodies on their surface. The presence of these antibodies may adversely affect the circulation kinetics of such liposomes as a result of interactions with cells of the mononuclear phagocyte system (MPS), mainly represented by macrophages in liver and spleen. The additional insertion of poly(ethylene glycol) chains on the surface of the immunoliposomes may, however, attenuate this effect. We investigated the influence of surface-coupled rat or rabbit antibodies and of PEG on the uptake of liposomes by rat Kupffer cells in culture with (3)H-cholesteryloleyl ether as a metabolically stable marker. Additionally, we assessed the effects of surface-bound IgG and PEG on the intracellular processing of the liposomes by the Kupffer cells, based on a double-label assay using the (3)H-cholesteryl ether as an absolute measure for liposome uptake and the hydrolysis of the degradable marker cholesteryl-(14)C-oleate as relative measure of degradation. Attachment of both rat and rabbit antibodies to PEG-free liposomes caused a several-fold increase in apparent size. The uptake by Kupffer cells, however, was 3-4 fold higher for the rat than for the rabbit IgG liposomes. The presence of PEG drastically reduced the difference between these liposome types. Uptake of liposomes without antibodies amounted to only about 10% (non-PEGylated) or less (PEGylated) of that of the immunoliposomes. In contrast to the marked effects of IgG and PEG on Kupffer cell uptake, the rate of intracellular processing of the liposomes remained virtually unaffected by the presence of these substances on the liposomal surface. These observations are discussed with respect to the design of optimally formulated liposomal drug preparations, combining maximal therapeutic efficacy with minimal toxicity.

Chemo-immunotherapy of ovarian cancer in a murine tumour model.

BACKGROUND: As a majority of ovarian cancer patients will ultimately develop recurrent disease, there is an urgent need for alternative or additional approaches in the treatment of this cancer. MATERIALS AND METHODS: The antitumour effect of i.p. administered cisplatin, liposomal muramyltripeptide phosphatidylethanulamine (L-MTP-PE) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were investigated using an i.p. growing murine ovarian tumour. Tumour growth was followed by measuring weight and survival of the mice. RESULTS: An i.p. injection of L-MTP-PE in non-tumour bearing mice resulted in an approximately 10-fold increase in the number of peritoneal cells, which were highly cytotoxic. Nonetheless, treatment of mice inoculated with MOT cells with cisplatin, L-MTP-PE and GM-CSF using different treatment schedules did not result in inhibited tumour growth when compared to treatment with cisplatin alone. CONCLUSION: Although L-MTP-PE showed an enormous increase in peritoneal cells with high tumour cytotoxic capacity, the immunotherapeutic treatment with GM-CSF and L-MTP-PE, aimed at the recruitment and activation of the peritoneal cell population, failed to result in a significant prolongation of survival.

Accelerated blood clearance and altered biodistribution of repeated injections of sterically stabilized liposomes.

Sterically stabilized liposomes are considered promising carriers of therapeutic agents because they can facilitate controlled release of the drugs, thereby reducing drug-related toxicity and/or targeted delivery of drugs. Herein, we studied the pharmacokinetics and biodistribution of repeated injections of radiolabeled polyethyleneglycol (PEG) liposomes. Weekly injections of (99m)Tc-PEG liposomes dramatically influenced the circulatory half-life in rats. Biodistribution 4 h after the second dose showed a significantly reduced blood content (from 52.6 +/- 3.7 to 0.6 +/- 0.1% injected dose (ID), P <.01) accompanied by a highly increased uptake in the liver (from 8.1 +/- 0.8 to 46.2 +/- 9.8%ID, P <.01) and in the spleen (from 2.2 +/- 0.2 to 5.3 +/- 0.7%ID, P <.01). At subsequent injections the effect was less pronounced: after the fourth dose, the pharmacokinetics of the radiolabel had almost returned to normal. The same phenomenon was observed in a rhesus monkey, but not in mice. The enhanced blood clearance of the PEG liposomes also was observed in rats after transfusion of serum from rats that had received PEG liposomes 1 week earlier, indicating that the enhanced blood clearance was caused by a soluble serum factor. This serum factor was a heat-labile molecule that coeluted on a size exclusion column with a 150-kDa protein. In summary, i.v. administration of sterically stabilized PEG liposomes significantly altered the pharmacokinetic behavior of subsequently injected PEG liposomes in a time- and frequency-dependent manner. The observed phenomenon may have important implications for the repeated administration of sterically stabilized liposomes for targeted drug delivery.

Uptake of long-circulating immunoliposomes, directed against colon adenocarcinoma cells, by liver metastases of colon cancer.

Radiolabeled ([3H]cholesteryloleyl ether) immunoliposomes directed against rat colon adenocarcinoma CC531 cells were prepared by random coupling of a tumor cell-specific antibody, CC52, via a thio ether bond. In vitro binding experiments demonstrated a saturable and specific interaction of CC52-immunoliposomes, which could be inhibited by free non-coupled CC52 but not by irrelevant antibodies. The in vivo targeting potential of CC52-immunoliposomes, which were pegylated to achieve prolonged circulation times, was tested in an established rat liver CC531 metastasis model. Twenty-four hours after injection of the liposomes, 25% of the CC52-immunoliposomes were still present in the blood, which was comparable with the control liposomes (either with or without antibody). Liposomes were mainly taken up from the blood by the liver and the spleen, although hepatic uptake of the immunoliposomes was higher and splenic uptake was lower as compared to liposomes without antibody. Within the metastatic tumor nodules in the liver, uptake of both the CC52-immunoliposomes and non-specific immunoliposomes was significantly higher than that of control liposomes without antibody. Visualization of fluorescently or gold labeled CC52-immunoliposomes revealed that, although targeting to liver metastases was achieved, the immunoliposomes were mostly not associated with tumor cells but rather localized in tumor associated cells, probably macrophages.

Antiproliferative effect of immunoliposomes containing 5-fluorodeoxyuridine-dipalmitate on colon cancer cells.

We have investigated the antiproliferative action towards CC531 colon adenocarcinoma cells of target cell-specific immunoliposomes containing the amphiphilic dipalmitoyl derivative of 5-fluorodeoxyuridine (FUdR-dP). FUdR-dP incorporated in immunoliposomes caused a 13-fold stronger inhibition of CC531 cell growth in vitro, during a 72-h treatment, than FUdR-dP in liposomes without antibody, demonstrating that the prodrug is efficiently hydrolysed to yield the active drug, FUdR, intracellularly. The intracellular release of active FUdR was confirmed by determining the fate of 3H-labelled immunoliposomal FUdR-dP. Treatments shorter than 72 h with FUdR-dP in immunoliposomes resulted in anti-tumour activities comparable to, or even higher than, that of free FUdR. The shorter treatments reflect more closely the in vivo situation and illustrate the potential advantage of the use of immunoliposomes over non-targeted liposomal FUdR-dP or free FUdR. Association of tumour cell-specific immunoliposomes with CC531 cells was up to tenfold higher than that of liposomes without antibody or with irrelevant IgG coupled, demonstrating a specific interaction between liposomes and target cells which causes an efficient intracellular delivery of the drug. Since biochemical evidence indicates a lack of internalization or degradation of the liposomes as such, we postulate that entry of the drug most likely involves the direct transfer of the prodrug from the immunoliposome to the cell membrane during its antigen-specific interaction with the cells, followed by hydrolysis of FUdR-dP leading to relatively high intracellular FUdR-levels. In conclusion, we describe a targeted liposomal formulation for the anticancer drug FUdR, which is able to deliver the active drug to colon carcinoma cells with high efficiency, without the need for the cells to internalize the liposomes as such.

Selective transfer of a lipophilic prodrug of 5-fluorodeoxyuridine from immunoliposomes to colon cancer cells.

A monoclonal antibody against the rat colon carcinoma CC531 was covalently coupled to liposomes containing a dipalmitoylated derivative of the anticancer drug FUdR as a prodrug in their bilayers. We investigated the in vitro interaction of these liposomes with CC531 target cells and the mechanism by which they deliver the active drug FUdR intracellularly to the cells by monitoring the fate of the liposomal bilayer markers cholesterol-[(14)C]oleate and [(3)H]cholesteryloleylether as well as the (3)H-labeled prodrug and colloidal gold as an encapsulated liposome marker. After binding of the immunoliposomes to the cell surface, only limited amounts were internalized as demonstrated by a low level of hydrolysis of liposomal cholesterol ester and by morphological studies employing colloidal gold-labeled immunoliposomes. By contrast, already within 24 h immunoliposome-incorporated FUdR-dP was hydrolyzed virtually completely to the parent drug FUdR intracellularly. This process was inhibited by a variety of endocytosis inhibitors, indicating that the prodrug enters and is processed by the cells by a mechanism involving an endocytic process, resulting in intracellular FUdR concentrations up to 3000-fold higher than those in the medium. Immunoliposomes containing poly(ethyleneglycol) (PEG) chains on their surface, with the antibody coupled either directly to the bilayer or at the distal end of the PEG chains were able to deliver the prodrug into the tumor cells at the same rate as immunoliposomes without PEG. Based on these observations, we tentatively conclude that during the interaction of the immunoliposomes with the tumor cells the lipophilic prodrug FUdR-dP is selectively transferred to the cell surface and subsequently internalized by constitutive endocytic or pinocytic invaginations of the plasma membrane, thus ultimately delivering the prodrug to a lysosomal compartment where hydrolysis and release of parent drug takes place. This concept allows for an efficient delivery of a liposome-associated drug without the need for the liposome as such to be internalized by the cells.

Effect of intraperitoneally administered recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) on the cytotoxic potential of murine peritoneal cells.

We studied the effect of recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) on the cytotoxic potential of murine peritoneal cells. Mice received rmGM-CSF intraperitoneally using different dosages and injection schemes. At different time points after the last injection, mice were sacrificed, peritoneal cells isolated and their tumour cytotoxicity was determined by a cytotoxicity assay using syngeneic [methyl-3H]thymidine-labelled colon carcinoma cells. Also, the cytotoxic response to a subsequent in vitro stimulation with lipopolysaccharide was determined. Upon daily injection of 6000-54,000 U rmGM-CSF over a 6-day period, the number of peritoneal cells increased over ten fold with the highest rmGM-CSF dose. Increases in cell numbers was mainly due to increases in macrophage numbers. Upon injection of three doses of 3000 U rmGM-CSF per day for 3 consecutive days, the number of macrophages remained elevated for minimally 6 days. Although the peritoneal cells from rmGM-CSF-treated mice were not activated to a tumoricidal state, they could be activated to high levels of cytotoxicity with an additional in vitro stimulation of lipopolysaccharide. Resident cells isolated from control mice could be activated only to low levels of tumour cytotoxicity with lipopolysaccharide. Tumour cytotoxicity strongly correlated with nitric oxide secretion. When inhibiting nitric oxide synthase, tumour cell lysis decreased. Thus, the expanded peritoneal cell population induced by multiple injections of rmGM-CSF has a strong tumour cytotoxic potential and might provide a favourable condition for immunotherapeutic treatment of peritoneal neoplasms.

On the mechanism of hepatic transendothelial passage of large liposomes.

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.

Uptake of liposomes containing phosphatidylserine by liver cells in vivo and by sinusoidal liver cells in primary culture: in vivo-in vitro differences.

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.

[3H]thymidine incorporation into whole liver as an alternative to [3H]thymidine incorporation into DNA as a parameter of cell proliferation in regenerating liver tissue in rats.

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.

Serum obtained from rats after partial hepatectomy enhances growth of cultured colon carcinoma cells.

Tumour-bearing rats were randomized to a 70% partial hepatectomy or a sham operation. At days 1, 3 or 14, portal and systemic serum was obtained and colon carcinoma cells were cultured in the presence of 5, 10, 20 or 50% serum. Proliferation and epidermal growth factor receptor (EGFr) expression was measured in tumour cells. Proliferation was 25-40% higher in tumour cells cultured with portal serum after hepatectomy than after sham operation when using serum obtained at day 3, but not days 1 and 14 after operation. In cultures with serum obtained at day 14 after operation CC 531 cells showed a 30% higher proliferation rate with systemic hepatectomy serum than CC 531 cells with sham systemic serum. These effects were not mediated by a change in EGFr mRNA and protein levels as the used colon carcinoma cells did not reveal EGFr activity by any of the three detection methods used.

Massive targeting of liposomes, surface-modified with anionized albumins, to hepatic endothelial cells.

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.

Different intrahepatic distribution of phosphatidylglycerol and phosphatidylserine liposomes in the rat.

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.