The Complement- but not Mannose Receptor-mediated Phagocytosis is Involved in the Hepatic Uptake of Cetylmannoside-modified Liposomes In Situ

In the elimination of injected liposomes in vivo, it is considered that several serum components play an important role on hepatic uptake of them. This study was conducted to clarify the hepatic uptake mechanism of cetylmannoside (Man)-modified multilamellar vesicles (Man-MLV) using perfused rat liver. In the presence of serum, Man-MLV was taken up by the liver-depending on the serum concentration, and it showed an approximately two-fold higher accumulation than MLV without any surface modifications (PC-MLV). These heptic uptakes of liposomes were obviously inhibited by preheating the serum at 56°C for 30 min or by the treatment with anti-rat C3 antiserum. Further, SDS-PAGE followed by immunoblot analysis showed the deposition of iC3b on the opsonized Man-MLV. These results obtained in the present study suggested that hepatic uptake of Man-MLV was mainly mediated by complement receptor rather than mannose receptor on Kupffer cells-in vivo.     

Enhancing effect of cetylmannoside on targeting of liposomes to Kupffer cells in rats

In order to evaluate whether surface modification of liposomes by cetylmannoside (Man) could be useful for targeting to Kupffer cells, the effect of Man on disposition of liposomes was examined after intravenous administration to rats. In the case of small unilamellar vesicles (SUV), no difference in disposition was observed between control liposomes (PC-SUV) and modified liposomes (Man-SUV). On the other hand, in the case of multilamellar vesicles (MLV), modified liposomes (Man-MLV) were rapidly eliminated from the circulation, and showed higher accumulation (51.4% of dose) in the liver as compared with control liposomes (PC-MLV, 25.7% of dose). In the spleen, splenic clearance of Man-MLV (0.068 ml/min) was comparable to that of PC-MLV (0.068 ml/min), although Man-MLV showed lower accumulation (5.7% of dose) than PC-MLV (14.7% of dose). This lower accumulation in the spleen of Man-MLV might be due to the low blood concentration caused by the high accumulation in the liver. Thus, it is considered that liposomal size is important in revealing the effects of Man, and Man-MLV is able to enhance only the affinity for the liver. The cellular distribution in the liver of Man-MLV 2 h after intravenous administration to rats gave encouraging evidence that Kupffer cells might be involved in the enhanced hepatic uptake of the liposomes. These results suggest the usefulness of Man-MLV for targeting to Kupffer cells. Furthermore, the involvement of plasma protein(s) in the uptake of Man-MLV is suspected.     

Enhancing effect of cholesterol on the elimination of liposomes from circulation is mediated by complement activation

The effects of cholesterol (Chol) content on the biodistribution of liposomes as well as the interaction of liposomes with plasma proteins, primarily complement (C) components, were examined in this study. The elimination of liposomes from blood circulation was enhanced by increasing the Chol content in liposomes. Furthermore, included Chol augmented the rate of liposome degradation as measured by the urinary excretion of ³H-inulin encapsulated in liposomes. We have also examined the effect of liposomal Chol on organ clearance (CL) and renal CL (CL_rel). The values of organ CL and CL_rel reflect the affinity of liposomes for the organ and the degree of liposome degradation in the blood, respectively. Hepatic CL and CL_rel, but not splenic CL, increased with the rise of Chol content in liposomes. The amount of liposome degradation in vitro, which reflects the extent of C activation, was correlated with degradation observed in vivo (CL_rel). However, the amount of plasma proteins bound to the liposomes was inversely proportional to the extent of in vitro liposome degradation. We have investigated the role of C activating factor (CAF) (Funato et al., 1994, Plasma factor triggering alternative complement pathway activation by liposomes, Pharm. Res., 11, 372–376) on Chol-dependent-C activation. Our results showed that binding of CAF to the liposomes is directly proportional to the amount of Chol present in the liposome. Thus, C activation by Chol in liposomes may proceed via a mechanism involving CAF. Taken together, these results suggest that increasing the Chol content of liposomes enhances the binding of CAF to the liposomes, which in turn, mediates Chol dependent-C activation, resulting in the augmentation of both degradation in blood and hepatic uptake of the liposomes.     

SIZE-DEPENDENT RELEASE OF CARBOXYFLUORESCEIN FROM CETYLMANNOSIDE-MODIFIED LIPOSOMES IN HUMAN PLASMA

The interaction of liposomes with human plasma was investigated using 6(5)-carboxyfluorescein (CF) as an aqueous phase marker of cetylmannoside-modified multilamellar vesicles (Man-MLVs) of various sizes. The release of CF decreased with increasing liposome concentration. The time courses of the CF release from Man-MLVs were monitored continuously and were analysed kinetically. The curves were characterized by two phases, the first-order release process and the maximum release, which represent the rate and the extent of CF release, respectively. The increase of liposome size increased the rate of release by 42% and the extent of release by 121%, respectively. These effects of liposome size on the release processes were suggested to result from the size-dependent affinities of liposomes to the human complement system. The assay system of liposomally bound fragments of complement component 3 (C3), such as C3b and/or iC3b, was developed by applying a sandwich enzyme-linked immunospecific assay. The percentage of C3 fragments to total proteins bound to liposomes increased with the size of liposomes and there was a good correlation between the extent of CF release and the percentage of C3 fragments bound. These results indicated that Man-MLVs released entrapped CF via activating the human complement system and the affinity of Man-MLV to complement increased with the size of Man-MLVs in human plasma. These in vitro results suggest the role of complement as an opsonin in the disposition of Man-MLVs in humans.     

Antibody Dependent,Complement Mediated Liver Uptake of Liposomes Containing GM1

Purpose. We have previously reported that GM_l exhibits an opposite effect on regulating liposome circulation time in mice and rats (Liu et al. Pharm. Res. Vol. 12:508-512 (1995)). Inclusion of GM₁ into liposomes significantly prolongs liposome circulation time in mice, while it dramatically decreases the blood half life and increases liver uptake of liposomes in rats. The purpose of this study was to elucidate the mechanism that underlies this phenomenon. Methods. Single-pass liver perfusion in vitro and complement mediated liposome lysis assay was used. Results. Serum appeared to play an important role in determining the liver uptake of GM_l liposomes. Specifically, rat serum enhanced the uptake of GM₁ containing liposomes by the perfused liver. Such activity was also found in human and bovine serum, but not in mouse serum. Taking human serum as an example, we demonstrated that such serum activity can be blocked by EDTA and EGTA/Mg^{2 +}. Antibodies against human IgM and the third component of complement system (C3) also inhibited serum activity. Conclusions. The presence of naturally occurring anti-GM₁ antibodies in rats, through the activation of the classic pathway of complement system, is likely the cause of rapid blood clearance of GM₁ liposomes. The third component of complement is likely to serve as the opsonin that is directly involved in mediating liposome clearance.     

In vivo studies on the role of complement in the clearance of liposomes in rats and guinea pigs.

The ability of complement (C) system to remove liposomes from blood circulation was examined in vivo using rat and guinea pig as models. Although the liposomes were not degraded in guinea pig serum in vitro, they were degraded remarkably in guinea pig circulation, as assessed by the urinary excretion of [3H]inulin released from liposomes. The suppression of rat C system to 64% normal C hemolytic activity by treating animals with K76COOH agent resulted in a significant decrease in both the uptake of liposomes by liver and the release of [3H]inulin, providing in vivo evidence for C-mediated clearance of liposomes in rats via uptake by macrophages and degradation in blood circulation, respectively. On the other hand, the K76COOH-induced suppression of C (70% normal hemolytic activity) in guinea pigs slightly increased both the hepatic uptake and the release of [3H]inulin. In addition, the hepatic uptake and in vivo degradation in guinea pigs varied in an opposite manner when the animals were preloaded by empty liposomes or when the liposome size and cholesterol content varied. These results suggest there is a difference between the factors involved in liposome degradation and the factors involved in hepatic uptake and also support the likelihood that there is no C-mediated degradation in guinea pigs.     

Synergistic Effect Between Size and Cholesterol Content in the Enhanced Hepatic Uptake Clearance of Liposomes Through Complement Activation in Rats

Purpose. The effect of liposome size and cholesterol (CH) content on the pharmacokinetics of liposomes was investigated in rats. Methods. The pharmacokinetics of liposomes was examined using 5(6)-carboxyfluorescein (CF) as an aqueous phase marker. The extent of complement activation (ECA) was also measured by the release of CF from liposomes in serum. Results. Both the size and the CH content influenced the mean residence time, total body clearance, and the hepatic uptake clearance (CLh) of liposomes. The increase of the size of liposomes increased the CLh at each CH content. There was no CH dependency of CLh in small liposomes (200 nm in diameter), although the CLh increased with the increase in the CH content in large (800 nm) and medium (400 nm) liposomes. A significant interaction effect was observed between liposome size and the CH content on CLh according to the analysis of variance. The good correlation between CLh and ECA indicated the role of complements as opsonins in enhancing the hepatic uptake of liposomes. The interaction effect between the size and CH content on CLh was explained principally by the product of the size and CH content. Conclusions. A synergistic effect was observed between the size and the CH content on CLh. An underlying hypothesis of the synergistic effect was postulated based on the size dependent recognition of liposomes by complement system.     

The complement system enhances the clearance of phosphatidylserine (PS)-liposomes in rat and guinea pig

In this study, we investigated the contribution of the complement system to the biodistribution of phosphatidylserine (PS)-containing liposomes in rat and guinea pig. It appeared that the inclusion of PS in the liposome formulation accelerates the rate of liposome uptake by liver, resulting in rapid elimination of the liposomes from blood circulation. Pretreatment with K76COOH (K76), an anti-complement agent, decreased the rapid uptake of PS-containing liposomes by guinea pig liver, resulting in increasing blood concentration of the liposomes. Significant complement-dependent liposome destabilization was observed in vitro in both animals, whereas the complement-dependent destabilization in vivo was likely only a part of the process of the clearance of the PS-containing liposomes. This discrepancy suggests that the rate of complement-dependent liposome uptake by liver is much faster than the rate of complement-dependent liposome destabilization in vivo. Pretreatment of K76 dramatically inhibited the binding of C3 fragments, one of dominant opsonins, to PS-containing liposomes in guinea pig under both in vivo and in vitro conditions. This finding suggests that the C3 fragments in the system are responsible for the clearance of the PS-containing liposomes in guinea pig. In rat, in contrast to guinea pig, in vivo binding of C3 fragments was not inhibited by K76-pretreatment, while in vitro binding was inhibited. This discrepancy may be due to different experimental conditions between in vitro and in vivo assay. Nevertheless, based on the observations in this study, the complement components are most likely involved in the clearance of the PS-containing liposomes in rat. Taken together, the activity of PS in enhancing the liposome clearance appears to be mediated by the complement components, presumably C3 fragments, in both guinea pig and rat. This is a first report showing the mechanism on the hepatic uptake of the PS-containing liposomes in guinea pig.     

Monosialoganglioside GMl Shortens the Blood Circulation Time of Liposomes in Rats

Inclusion of monosialoganglioside GM₁ into liposomes significantly enhances the circulation time of liposomes in mice. Conversely, intravenously injected GM₁-containing liposomes were rapidly removed from the blood circulation and accumulated in the liver and spleen in rats. In rats, increasing the GM₁ content in liposomes resulted in more rapid clearance from the blood. Increasing the membrane rigidity of liposomes further increased the liver uptake of GM₁-containing liposomes. The activity of GM₁ in enhancing the liposome uptake by rat liver appears to be mediated by complement components.     

Enhanced Hepatic Uptake of Liposomes Through Complement Activation Depending on the Size of Liposomes

The objective of this study was to differentiate the roles of opsonins and phagocytic cells in the size-dependent hepatic uptake of liposomes in the submicron region. The extent of opsonization decreased with the decrease in size of liposomes (from 800 to 200 nm in diameter) and no enhancement of uptake was observed at 200 nm. There was no effect of liposome size on the uptake of unopsonized liposomes. Serum was pretreated with empty liposomes of each size and its opsonic activity was measured in the perfused liver. The small liposomes could not consume the opsonic activity, while the larger ones did so substantially. These results suggest that opsonins bind to liposomes depending on the size of liposomes and phagocytic cells take up liposomes in proportion to the extent of opsonization. Size-dependent liposome degradation in serum was also found, which was consistent with the size-dependent complement activation, because liposomes with this composition have been shown to be degraded by complement. The mechanism of opsonization was examined by treating serum at 56°C for 30 min or with anti-C3 antiserum. Since both treatments inhibited the opsonic activity, the hepatic uptake of liposomes is considered to occur via complement receptor. In conclusion, the size of liposomes affected complement recognition, and the liposomes were taken up by the liver depending on the extent of opsonization.     

The complement system in liposome clearance: Can complement deposition be inhibited?

The activation of complement results in the opsonization of particles for removal by the reticuloendothelial system. Experimental evidence suggests that complement-mediated clearance of liposomal systems may significantly contribute to liposome biodistribution. Because of the multiplicity of complement activation mechanisms and the large number of proteins in the pathway, there are multiple opportunities to reduce or eliminate the opsonic effects of complement activation. This review addresses the state of our understanding of the interaction of liposomes with complement proteins and suggests some approaches to minimize complement activation.     

Recognition by Macrophages and Liver Cells of Opsonized Phospholipid Vesicles and Phospholipid Headgroups

The interaction of liposomes with blood proteins is believed to play a critical role in the clearance pharmacokinetics and tissue distribution of intravenously injected liposomes. In this article we have focused our discussion on the interaction of liposomes with key blood proteins, which include immunoglobulins, complement proteins, apolipoproteins, fetuin, von Willebrand factor, and thrombospondin, and their role in liposome recognition by professional phagocytes and nonmacrophage hepatic cells. Alternatively, macrophages as well as hepatocytes and liver endothelial cells may phagocytose/endocytose liposomes via direct recognition of phospholipid headgroups. A number of plasma membrane receptors such as lectin receptors, CD14, various classes of scavenger receptors (e.g., classes A, B, and D), Fc-gammaRI and FcgammaRII-B2 may participate in phospholipid recognition. These concepts are also discussed.     

Biodistribution of liposomes and C3 fragments associated with liposomes: evaluation of their relationship

The biodistribution of liposomes with two different kind phospholipids (hydrogenated egg phosphatidylcholine and egg phosphatidylcholine) plus cholesterol (CHOL) were investigated after intravenous administration to rats. Elimination of liposomes from blood circulation was affected by the lipid composition. It appeared that the inclusion of CHOL in liposomes accelerates the rate of liposome uptake by liver, resulting in rapid elimination of liposomes. The amount of C3 fragments bound to liposomes was quantitatively determined to assess the contribution of the complement system to liposome accumulation into organs and liposome destabilization in vivo and in vitro. The amount of bound C3 fragments was directly proportional to CHOL content, and the amount was also proportional to the CLh, CLs as well as CLrel. This relationship suggests that the complement system is responsible for the elimination of liposomes from blood circulation, presumably as a consequence of opsonization by C3 fragments and assembly of membrane attack complex (MAC) onto liposomes. In addition, substitution of cholesteryl methyl ether into the liposome formulation for CHOL significantly diminished not only the binding of C3 fragments but also the CLh, CLs and CLrel, resulting in increased mean resident time (MRT) of the liposomes. This result suggests that the hydroxyl-group on CHOL is a binding site for C3 fragments on the liposomes and that CHOL in a liposome formulation promotes the accumulation of liposomes into the liver and spleen, probably due to their uptake by phagocytic cells, and impairs the stability of the liposomes in blood circulation, via a mechanism involving the complement system.     

Thermosensitive Sterically Stabilized Liposomes: Formulation and in Vitro Studies on Mechanism of Doxorubicin Release by Bovine Serum and Human Plasma

Purpose. To formulate thermosensitive sterically stabilized liposomes and to study the effects of plasma and serum components in vitro. Methods. The rate of release of encapsulated doxorubicin (Dox) from liposomes of various compositions was followed by fluorometric assay at 37°, 42° and 45°C, in buffer and also in both calf serum and human plasma up to 50% by volume. Results. The optimal composition for the maximal differential release of doxorubicin between 37°C and 42°C in human plasma was a mixture of dipalmitoylphosphatidylcholine/hydrogenated soy phosphatidylcholine/cholesterol and distearoylphosphatidylethanolamine derivatized with polyethylene glycol at a molar ratio of 100:50:30:6. In experiments designed to study the mechanism causing increased permeability of liposomes in bovine serum, we found two different distinct release patterns: a slow linear rise of rate of Dox release for fluid liposomes and fast exponential rise reaching plateau within 5 minutes for solid phase (rigid) liposomes. This release of Dox from rigid but not fluid liposomes was inhibited by pre-heating serum at 55°C for 30 minutes or by addition of EDTA (but not EGTA) or antiserum to the C3 component of complement. Conclusions. A formulation of sterically stabilized liposomes with the proper thermal sensitivity in human plasma has been obtained. In addition, the results suggest that complement may play an important role in the interaction of rigid but not fluid liposomes with bovine serum. Human plasma did not show this effect.     

Delivery of Plasmid DNA into Mammalian Cell Lines Using pH-Sensitive Liposomes: Comparison with Cationic Liposomes

We compare the transfection efficiency of plasmid DNA encoding either luciferase or (-galactosidase encapsulated in pH-sensitive liposomes or non-pH-sensitive liposomes or DNA complexed with cationic liposomes composed of dioleoyloxypropyl-trimethylammonium:dioleoylphosphatidyl-ethanolamine (1:1, w/w) (Lipofectin) and delivered into various mammalian cell lines. Cationic liposomes mediate the highest transient transfection level in all cell-lines examined. pH-sensitive liposomes, composed of cholestryl hemisuccinate and dioleoylphosphatidylethanolamine at a 2:1 molar ratio, mediate gene transfer with efficiencies that are 1 to 30% of that obtained with cationic liposomes, while non-pH-sensitive liposome compositions do not induce any detectable transfection. Cationic liposomes mediate a more rapid uptake of plasmid DNA, to about an eightfold greater level than that obtained with pH-sensitive liposomes. The higher uptake of DNA mediated by Lipofectin accounts for part of its high transfection efficiency. Treatment of cells with chloroquine, ammonium chloride, or monensin decreases (threefold) transfection using pH-sensitive liposomes and either has no effect on or enhances cationic liposome-mediated transfection. Therefore plasma membrane fusion is not the only mechanism available to cationic liposomes; in certain cell lines DNA delivery via endocytosis is a possible parallel pathway and could augment the superior transfection efficiency observed with cationic liposomes.     

Influence of Spacer Length on Interaction of Mannosylated Liposomes with Human Phagocytic Cells

Purpose. To improve target specificity and uptake of liposomes by macrophages, one can improve high-affinity receptor binding to mannose determinants with their 175-kDa mannose receptor (MR), which is mainly influenced by the length and flexibility of the spacer between the carbohydrate head group and liposome surface. Liposomes containing alkylmannosides with hydrophilic spacers 0 to 8 ethyleneoxy units (EO) long (Man0...Man8) were used to investigate systematically the effects of spacer length on liposome-cell interactions. Methods. Concanavalin A (ConA)-induced liposome aggregation was studied by turbidity measurement and cell uptake using PMA-induced HL-60 cells or native human macrophages by determining 6-CF after cell lysis or NBD-fluorescence with flow cytometry. Detection of MR in native cell populations was carried out by an antibody assay using flow cytometry; MR-representing cells were selected analytically. Results. Liposomes containing mannosides with more than one EO spacer length were specifically aggregated by ConA, indicating accessibility of the carbohydrate ligands of these derivatives. Increase in EO spacer units of incorporated mannosides (two or more EO) led to suppression of cellular uptake of mannosylated liposomes by phago- cytes lacking MR (HL60, U937). The extent of suppression increased with spacer length. Liposome uptake by native macrophages expressing MR was, on the contrary, improved, particularly by Man6 and Man8. Conclusions. Uptake of liposomes modified with Man6 or Man8 by native cells was enhanced but did not reach an optimum. Thus, Man6, Man8, and mannosides with even longer spacer arms are of potential use in receptor-mediated targeting.     

Receptor versus non-receptor mediated clearance of liposomes

Numerous studies have appeared over the years dealing with liposome-cell interaction mechanisms, most of them performed under in vitro conditions with isolated cell populations or cell lines. It is remarkable that, nonetheless, there hardly seem to exist established and generally accepted views on how precisely liposomes interact with cells and by what parameters this is influenced. In this article we will summarize and discuss the most relevant studies (in our opinion) on this matter in relation to in vivo conditions and with special attention to the relation between scavenger, complement and PS receptors.Researchers in the field have long been aware of the interaction of liposomes with blood proteins and their potential involvement in the process of liposome elimination from the blood circulation. A few of these 'opsonizing' proteins have been identified, but it is not clear to what extent each of them determines the fate of the liposome in the blood stream and how liposomal parameters such as size, charge and rigidity play a role in this process. We will include in this article our own recent observations on a thus far largely ignored class of such liposomal 'opsonins', the apolipoproteins. This class of plasma proteins, which physiologically are instrumental in hepatic lipoprotein clearance and processing, has been shown to contribute specifically to hepatocyte-mediated uptake of liposomes.Separately, as opposed to the fate of plain liposomes, we briefly touch on the clearance of surface-modified liposomes, which are designed to actively target specific cells or tissues. Plasma proteins are not usually supposed to play a significant role in the clearance of such liposomes. We will summarize these studies and address in this connection the question of how plasma proteins may interfere with such active targeting attempts.     

Factors in cobra venoms affecting the complement system

Three constituents of cobra venoms are known to interact with complement: the “cobra venom factor” (CVF), a high-molecular-weight factor (H-CoF), and the “cobra inhibitor” (CI). CVF and CI act by forming complexes with certain complement components. In doing so, CVF replaces an endogenous component, C3b, and hence activates the alternative pathway and leads to C3 (and C5) consumption. In contrast, CI competes with essential complex formations of endogenous components and thus inhibits various reactions. Both, CVF and CI, are useful tools to study the biochemistry of complement and its pathophysiological involvements. The effect of H-CoF on complement has not yet been studied in detail.     

应用补体活化体外模型评价姜黄素脂质体

目的建立补体活化体外模型,评价姜黄素脂质体补体激活能力。方法使用混合的人血清作为标准反应血清,用人IgG和zymosan A激活标准反应血清中的补体成分后得到标准阳性对照血清,在标准反应血清中加入姜黄素脂质体进行补体激活反应,并检测SC5b-9含量。结果姜黄素脂质体组SC5b-9浓度与NC组相比,增加了约330%。结论使用补体活化体外模型可以评价姜黄素脂质体补体激活能力和比较不同批次姜黄素脂质体补体激活能力的差异。     

Pharmacokinetics, excretion, and mass balance of 14C after administration of 14C-cholesterol-labeled AmBisome to healthy volunteers

Amphotericin B (AmB) in small unilamellar liposomes (AmBisome) provides higher plasma concentrations and greater safety than the conventional deoxycholate formulation. The authors compared the disposition of the liposome's drug and cholesterol components by measuring AmB and radioactivity in plasma, urine, and feces for 1 week after a single 2-hour infusion of 14C-cholesterol-labeled AmBisome (2 mg/kg, 1 microgCi/kg) in healthy adults (4 males, 1 female). The plasma profile of 14C-cholesterol differed from that of AmB, lacking an initial rapid disappearance phase, having a lower total clearance, and having a volume of distribution (0.13 L/kg) close to that of the plasma compartment. The biphasic disappearance and long plasma half-life (147 h) of 14C-cholesterol were similar to those of other low-clearance liposomes. This and the low clearance of 14C-cholesterol from the plasma compartment suggest that it served as a liposome marker. The plasma drug-lipid ratio fell during the study, showing that AmB was cleared from plasma more rapidly than cholesterol or liposomes and suggesting that the composition of the liposomes changed over time. 14C-radioactivity was recovered mainly in the feces (9.5% of dose), consistent with the catabolism of cholesterol to bile salts. Combined fecal and renal clearances were < 18% of total clearance, suggesting that most of the liposomal drug and lipid remained in the body 1 week after dosing. Thus, AmBisome remains in the circulation for an extended period of time while releasing AmB, resulting in its markedly altered pharmacokinetic and safety profiles.