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

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

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.     

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.     

Uptake and processing of immunoglobulin-coated liposomes by subpopulations of rat liver macrophages

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.     

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

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

Differential uptake of liposomes varying in size and lipid composition by parenchymal and kupffer cells of mouse liver

Using liposomes differing in size and lipid composition, we have studied the uptake characteristics of the liver parenchymal and Kupffer cells. Desferal labeled with iron-59 was chosen as a radiomarker for the liposomal content, because Desferal in its free form does not cross cellular membranes. At various time intervals after an intravenous injection of liposomes into mice, the liver was perfused with collagenase, and the cells were separated in a Percoll gradient. It was found that large multilamellar liposomes (diameter of about 0.5 μm) were mainly taken up by the Kupffer cells. For these large liposomes, the rate of uptake by Kupffer cells was rapid, with maximum uptake at around 2 hours after liposome injection. Unexpectedly, small unilamellar liposomes (diameter of about 0.08 μm) were less effectively taken up by Kupffer cells, and the rate of uptake was slow, with a maximum uptake at about 10 hours after liposome injection. In contrast, parenchymal cells were more effective in taking up small liposomes and the uptake of large liposomes was negligible. In addition, liposomes made with a galactolipid as part of the lipid constituents appeared to have higher affinity to parenchymal cells than liposomes made without the galactolipid. These findings should be of importance in designing suitable liposomes for drug targeting.     

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.     

Liposome recognition by resident and newly recruited murine liver macrophages

The evidence in this communication indicate that, unlike resident Kupffer cells, newly recruited liver macrophages (following monocyte migration from the blood to the liver) use complement receptors to recognize and internalize stearylamine-incorporated liposomes. Within two weeks of hepatic residency complement receptors no longer participate in liposome recognition and uptake.     

Interaction of immunoglobulin-coupled liposomes with rat liver macrophages in vitro

The interaction between liposomes coated with covalently linked rabbit immunoglobulin (RbIg-liposomes), and rat liver macrophages (Kupffer cells) in monolayer culture was studied biochemically with radioactive tracers and morphologically by electron microscopy. The attachment of immunoglobulin (Ig) to liposomes caused a five-fold increase in liposome uptake by the Kupffer cells at 37 degrees C, in comparison with uncoated liposomes. The uptake was linear with time for at least 4 h and linear with liposome concentration up to a lipid concentration of 0.2 mM. At 4 degrees C uptake, probably representing cell surface-bound liposomes, was reduced to a level of approx. 20% of the 37 degrees C values. Involvement of the Fc receptor in the uptake process was indicated by the reduction of RbIg-liposome uptake by more than 75% as a result of preincubating the cells with heat-aggregated human or rabbit Ig at concentrations (less than 2 mg/ml) at which bovine serum albumin (BSA) had virtually no effect on uptake. At high concentrations (10-35 mg/ml), however, albumin also reduced liposome uptake significantly (20-30%), which suggests an interaction of the RbIg-liposomes with the Kupffer cells that is partially non-specific. RbIg-liposome uptake was dependent on the amount of RbIg coupled to the liposomes. Maximal uptake values were reached at about 200 micrograms RbIg/mumol liposomal lipid. Electron microscopic observations on cells incubated with horseradish peroxidase-containing RbIg-liposomes demonstrated massive accumulation of peroxidase reaction product in intracellular vacuoles, showing that the uptake observed by label association represents true internalization.     

The effect of reticuloendothelial blockade on the blood clearance and tissue distribution of liposomes

The blood clearance and tissue distribution of liposomes have been studied in mice subjected to reticuloendothelial blockade with dextran sulphate or carbon. The liposomes have been labelled in the lipid membranes with [3H]-cholesterol, [14C]phosphatidylcholine and/or 99mTc and the content with [14C]inulin. Reticuloendothelial blockade has been shown to slow the rate of clearance of neutral, positively and negatively charged liposomes and of both small unilamellar vesicles and large multilamellar vesicles. In normal animals, the liver uptake accounted for only 20-55% of the total injected radioactivity, the amount varying with the charge and size of the liposomes. Following blockade, the liver uptake of charged and neutral multilamellar liposomes was depressed. This was also true for negatively charged small unilamellar vesicles. The degree of depression of hepatic uptake was between 25-50%, which contrasts with the 80-90% reduction in uptake of a wholly phagocytosed particle (sheep red cells). This difference suggests that mechanisms other than Kupffer cell phagocytosis are also responsible for the normal uptake of liposomes into the liver. In the case of neutral and positively charged small unilamellar vesicles, delayed clearance due to blockade was not associated with 'depressed' hepatic uptake. The site of action of blockading agents for these preparations is not clear. With all preparations of liposomes, blockade produced a slight and variable increase in uptake in the lung and spleen. The alteration of distribution of liposomes by reticuloendothelial blockade is therefore not great and the value of the technique in modifying the tissue distribution of substances within liposomes may be limited.     

Intrahepatic Distribution of Long-Circulating Liposomes Containing Polyethylene Glycol) Distearoyl Phosphatidylethanolamine

Abstract

We investigated the intrahepatic distribution in rats of liposomes of 85 or 130 nm diameter, which were sterically stabilized with a polyethylene glycol) derivative of phosphatidylethanolamine (PEG-PE) so as to increase their circulation time in blood. Various times after intravenous injection of radiolabeled ([³H-]cholesterylether) liposomes, parenchymal and non-parenchymal cells of the liver were isolated and their radioactivity content was determined. Control liposomes of 85 nm without PEG-PE distributed in an approximately 80:20 ratio to hepatocytes (H) and macrophages (M), respectively; the 130-nm control liposomes showed a 50:50 H/M distribution. Incorporation of PEG-PE reduced the rate of total liver uptake about 4-fold for liposomes of either size and shifted the H/M ratio to 60:40 for the smaller vesicles and to 40:60 for the larger ones. For both liposome sizes, PEG-PE apparently causes a shift in intrahepatic distribution in favor of the macrophages. It is concluded that PEG-PE has a stronger inhibitory effect on liposome uptake by hepatocytes than on uptake by macrophages. Attempts to shift liposome uptake more in favor of hepatocytes, by incorporation of lactosylceramide, failed. This compound, although causing an increase in hepatic uptake, particularly for the 130-nm liposomes, shifted the H/M ratio further towards the macrophages. We conclude that the galactose moiety of the glycolipid is sufficiently exposed on the surface of (PEG-PE)-containing liposomes to allow interaction with the galactose-binding lectin at the surface of the liver macrophage and that the extent of exposure is dependent on vesicle size.     

LIPOSOME RECOGNITION BY RESIDENT AND NEWLY RECRUITED MURINE LIVER MACROPHAGES

ABSTRACT

The evidence in this communication indicate that, unlike resident Kupffer cells, newly recruited liver macrophages (following monocyte migration from the blood to the liver) use complement receptors to recognize and internalize stearylamine-incorporated liposomes. Within two weeks of hepatic residency complement receptors no longer participate in liposome recognition and uptake.     

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.     

Receptor-mediated uptake of asialoganglioside liposomes: sub-cellular distribution of the liposomal marker in isolated liver cell types

The use of asialo GM1-containing small unilamellar liposome preparations in vivo caused a 2.8-fold increase in the uptake by the liver as compared with the control (neutral) preparations (without asialo GM1). The uptake of negatively charged dicetylphosphate and dipalmitoyl phosphatidic acid-containing small unilamellar liposomes was found to be 1.6-and 1.8-fold respectively higher than that of the neutral preparations. In studies with isolated liver cell types, inhibition of the galactosylated liposome uptake by asialofetuin indicated a possible involvement of hepatic galactose receptors in the recognition of asialo GM1 liposomes by the hepatic parenchymal cells, which in turn were found to be mainly responsible for the enhanced incorporation of these liposomes in the liver. Sub-cellular distribution studies with isolated liver cell types indicated lysosomal localization of the liposomes both in parenchymal and nonparenchymal cells, and it has been proposed that the asialo GM1 liposomes are cointernalized with asialofetuin through a common lysosomal route of ligand internalization.     

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

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

Effect of Macrophage Elimination Using Liposome-Encapsulated Dichloromethylene Diphosphonate on Tissue Distribution of Liposomes

Abstract

Effect of macrophage elimination using liposomal dichloromethylene diphosphonate (C1₂MDP)1 on tissue distribution of different types of liposomes was examined in mice. Intravenously administration into mice with CI2MDP encapsulated in liposomes composed of phosphatidylcholine, cholesterol and phosphatidylserine exhibits a temporary blockade of liver and spleen function for liposome uptake. At a low dose of 90 (ig/mouse, the liposome uptake by the liver was significantly decreased. Such decrease was accompanied by an increase in liposome accumulation in either spleen or blood depending on liposome composition and size. Direct correlation between the administration dose of liposomal CI2MDP and the liposome circulation time in blood was also obtained even for liposomes with an average diameter of more than 500 nm. These results indicate that temporary elimination of macrophages of the liver and spleen using liposomal CI2MDP may prove to be useful to enhance the drug delivery efficiency of liposomes.     

Release of Active Drug from Intracellular Liposomal Prodrug Depots in Macrophages Controlled by Liposome Composition

Abstract

Several drugs have limited potency due to their rapid elimination or inactivation. The anticancer drug 5-fluoro-deoxyuridine (FUdR), which is frequently used in therapeutic treatment of liver metastases from colon tumors, is an example of such drugs. It is rapidly eliminated from circulation and metabolized, mainly by the hepatocytes in the liver. Over the past few years we have investigated the possibility to keep the drug away from the hepatocytes and to save it from rapid inactivation by encapsulating it in liposomes. In this way the liposomal drug is expected to accumulate in the macrophages of the liver (Kupffer cells), which form a major target site for intravenously administered liposomes. There, as the liposomal structure is gradually degraded by lysosomal enzymes, the drug will be released, initially within the lysosomal compartment, while subsequently it will leak out of the lysosomes and eventually out of the cells so as to become available for uptake by intrahepatically situated tumor cells. In this contribution we describe this system for the prodrug dipalmitoyl-FUdR, incorporated in the liposomal bilayer, requiring an additional step for the drug to become available, i.e. the enzymatic deacylation of the prodrug. It is demonstrated that the rate of intralysosomal degradation of liposomes in Kupffer cells varies substantially with liposomal lipid composition and that the rate of release of active drug from the Kupffer cells parallels the rate of liposome degradation. In addition, it is demonstrated that in this way the antitumor activity of the FUdR can be enhanced by more than two orders of magnitude and that the degree of antitumor activity reflects, to a limited extent, the rate at which the liposomes are degraded.     

UPTAKE AND INTRACELLULAR PROCESSING OF PEG-LIPOSOMES AND PEG-IMMUNOLIPOSOMES BY KUPFFER CELLS IN VITRO*

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 ³H-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 ³H-cholesteryl ether as an absolute measure for liposome uptake and the hydrolysis of the degradable marker cholesteryl-¹⁴C-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.     

The role of β2-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 ∼55-kDa protein was identified as β₂-glycoprotein I (β₂GPI) by Western blotting. Despite the high affinity of β₂GPI 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, β₂GP1 inhibits, rather than enhances, liposome uptake by liver cells.     

Targeting of anti-Thy 1.1 monoclonal antibody conjugated liposomes in Thy 1.1 mice after intravenous administration

125I-labeled liposomes, conjugated to an anti-Thy 1.1 monoclonal antibody (MRCOX7), demonstrated up to 7.4-fold greater lymph node uptake than liposomes conjugated to non-specific monoclonal antibody (R-10) after intravenous injection into Thy 1.1 (AKR-J) mice. Uptake of anti-Thy 1.1-conjugated liposomes by the lymph nodes of AKR-J mice was 3-times greater than their uptake by lymph nodes of Thy 1.2 (AKR-Cu) mice. Lymph node localization of anti-Thy 1.1-liposomes was equal to that of control monoclonal antibody-liposomes in Thy 1.2 mice. Conjugation to either monoclonal antibody substantially increased liposome clearance by the liver, while decreasing liposome uptake in a number of organs outside the reticuloendothelial system. Changes in liposome size and phospholipid composition did not significantly alter these results. Administration of a large predose of unconjugated liposomes prior to injection of MRCOX7-conjugated liposomes increased blood levels and reduced liver uptake of the monoclonal antibody-liposome conjugates, but did not further enhance lymph node uptake. This study demonstrates that targeting of liposomes by conjugation to the appropriate monoclonal antibody, can significantly increase their uptake in lymph nodes which contain high levels of cells expressing the target antigen. However, conjugation to monoclonal antibody also increases clearance of liposomes by the liver. To increase the uptake of monoclonal antibody-conjugated liposomes in target tissue, substantial reduction of their clearance by the reticuloendothelial system will be required.