Mechanisms of delivery of liposome-encapsulated cytosine arabinoside to CV-1 cells in vitro. Fluorescence-microscopic and cytotoxicity studies

Fluorescence microscopy and assays of the cytotoxicity of liposome-encapsulated cytosine arabinoside (araC) have been used to examine the interactions of CV-1 cells with pH-sensitive liposomes, combining phosphatidylethanolamine (PE) with oleic acid or with double-chain protonatable amphiphiles, and with pH-insensitive liposomes combining phosphatidylcholine (PC) and phosphatidylglycerol (PG). Fluorescence-microscopic observations indicate that double-chain protonatable amphiphiles remain tightly associated with pH-sensitive liposomes during incubations with CV-1 cell monolayers, and that cellular uptake of liposomes is strongly promoted by transferrin coupled to the liposome surface. Liposome-encapsulated araC showed much greater cytotoxicity toward CV-1 cells than did the free drug at equivalent concentrations under the same conditions. The cytotoxicity of encapsulated araC was strongly enhanced by liposome-conjugated transferrin and was maximal using pH-sensitive liposomes combining PE with the double-chain protonatable amphiphile N-(N'-oleoyl-2-aminopalmitoyl)serine. However, the drug was also markedly more cytotoxic when encapsulated in other types of transferrin-conjugated liposomes, including pH-insensitive PC/PG/cholesterol liposomes, than in the free form. The cytotoxicity of liposome-encapsulated araC is significantly attenuated by the nucleoside transport inhibitor nitrobenzothioinosine, and fluorescence microscopy using calcein-containing liposomes provides no evidence for efficient fusion between cellular membranes and any of the types of liposomes examined here. Based on these observations, we suggest that the major mechanism for cytoplasmic delivery of liposome-encapsulated araC is the carrier-mediated transport of drug that has been released from liposomes into the endosomal and/or the lysosomal compartments.     

Interferon production of L929 and HeLa cells enhanced by polyriboinosinic acid-polyribocytidylic acid pH-sensitive liposomes.

The double-stranded RNA polyinosinic acid-polycytidylic acid (PolyIC) is an inducer of interferons alpha and beta (IFN) genes. With L929 and HeLa cells IFN pretreatment (priming) improves the IFN induction by PolyIC by several orders of magnitude. In the absence of the priming we demonstrate that PolyIC encapsulated into pH-sensitive liposomes (and not into pH-insensitive liposomes) enables L929 cells to secrete IFN efficiently and a low toxicity is observed; on primed cells pH-sensitive liposomes containing PolyIC trigger a high toxicity. With HeLa cells, the absence of the priming PolyIC encapsulated into pH-sensitive liposomes induces weak doses of IFN whereas free PolyIC was ineffective. Our experiments established that a pH drop (from 8 to 5.5) provoked a lipid mixing between pH-sensitive liposomes and cell membranes, likely by a fusion mechanism. Entrapment into pH-sensitive liposomes enhances the effect of PolyIC by several orders of magnitude, which might improve its therapeutic ability as an antitumor or anti-HIV agent.     

Interaction of cells from murine organs with liposomes of various composition

The distribution of liposomes prepared from total mouse liver lipids and containing (3H)-labelled platelet activation factor in mouse organs was studied. It was shown that the majority of intraperitoneally injected liposomes prepared from total mouse liver lipids were transported to mouse liver and spleen. The interaction of liposomes with spleen cells in vitro revealed that the affinity of liposomes prepared from total spleen macrophage or total spleen lymphocyte lipids for mouse spleen cells was much higher than that of liposomes prepared from a model lipid mixture. The liposome binding to isolated spleen macrophages or lymphocytes was much higher than the liposome uptake by these cells in the total population of mouse spleen cells.     

Large liposomes containing ganglioside GM1 accumulate effectively in spleen

Large liposomes, with a composition of egg phosphatidylcholine, cholesterol and ganglioside GM1, prepared by an extrusion method, were injected intravenously into mice. After 24 h, up to 50% of injected dose was accumulated in spleen compared with about 15% in spleen for liposomes containing no GM1. The effect of GM1 on spleen accumulation of liposomes was liposome size dependent. Only relatively large liposomes (d greater than 300 nm) showed high accumulation; smaller liposomes were progressively less accumulated. The spleen accumulation increased with increasing injection dose of the liposomes. It was noted that the enhanced uptake by spleen was accompanied by a decrease in the liver uptake, but the total uptake of liposomes by liver and spleen was not dependent on the diameter of liposome or the presence of the ganglioside GM1. Autoradiographs of fixed and sectioned spleen using 125I-labeled tyraminylinulin as a content marker for the liposomes, showed that liposomes localized at the reticular meshwork of the red pulp. These results suggest that larger liposomes containing GM1 are filtered by the spleen during the circulation in blood. The smaller ones with a mean diameter of less than 100 nm are not retained by the filter. The function of GM1 is to prevent liposomes from a rapid uptake by the liver so that liposomes may circulate through the spleen and be filtered. These results, together with the observation that the liposome-entrapped proteins were degraded by the spleen, suggest the potential use of these liposomes for specific drug delivery to the spleen.     

Involvement of Kupffer cells in lipopolysaccharide-induced rapid accumulation of platelets in the liver and the ensuing anaphylaxis-like shock in mice

Intravenous injection of Klebsiella O3 lipopolysaccharide (LPS) into BALB/c mice induces an anaphylaxis-like shock within minutes. Using 5-hydroxytryptamine as a marker for platelets, we previously suggested that a rapid platelet accumulation in the liver and lung precedes the shock, and that a complement-dependent platelet-degradation is involved in the shock. Here, we examined (i) the effect of platelet-depletion (using an anti-platelet monoclonal antibody) on the shock and (ii) the contribution of macrophages to the platelet-accumulation in those organs. LPS-induced platelet-accumulations in the liver and lung were confirmed by immunostaining. In platelet-depleted mice, the shock was largely prevented. The number of F4/80-positive macrophages was much greater in liver than in lung, and the hepatic macrophages were largely lost in mice given clodronate-encapsulated liposomes. In mice treated with such liposomes, both the LPS-induced accumulation of platelets in the liver (but not in the lung) and the shock were largely prevented, and repopulation of hepatic macrophages restored these LPS-induced responses. These results suggest that (i) platelets are indeed involved in the shock, (ii) Kupffer cells mediate the hepatic platelet accumulation, and (iii) preventing this hepatic accumulation can largely prevent rapid shock being induced by LPS (at the dose used here).     

Effects of the monoclonal antibody against porcine 40 kDa adipocyte-specific plasma membrane protein on adipocytes and carcass composition

The effects of the mouse monoclonal antibody against 40 kDa adipocyte-specific plasma membrane protein on porcine adipocytes and carcass composition were investigated in vitro and in vivo. Results revealed that the in vitro complement-mediated cytotoxicity of this monoclonal antibody can lead to adipocyte lysis, remarkable reduction of adipocyte lipid accumulation （P〈0.01）, and significant decrease of well-differentiated fat cells （P〈0.01）. Treatment of adipocytes with this antibody alone in vitro did not induce cell lysis, but could lead to noticeable reduction of well-differentiated cells and lipid accumulation （P〈0.05） at the pre-adipocyte stage. In vivo, pigs injected with 0.5 mg/kg or 1.0 mg/kg of antibody showed smaller adipocyte sizes （P〈0.01） and reduced lipid accumulation of adipocytes （P〈0.01）. Our results also indicated that pigs intraperitoneally or subcutaneously immunized with 0.5 mg/kg of monoclonal antibody at 15 kg or 1.0 mg/kg antibody at 60 kg had a higher lean meat percentage （P〈0.05）, larger loin eye area （P〈0.05）, lower fat meat percentage （P〈0.05）, less backfat thickness （P〈0.05） and smaller leaf fat weight （P〈0.05） than the control pigs, but other carcass traits such as caul fat weight, heart weight, liver weight, spleen weight, kidney weight, lung weight, and dressing percentage were not significantly affected. These results suggested that this monoclonal antibody could be applied to restrain excessive fat deposition in porcine production.     

Letters

Abstract

Long-term effects of life-long (>2 year) repeated intravenous injections (up to 17) of high doses of liposomes, lipid A, or liposomes containing lipid A were assessed in BALB/c mice. the liposomes contained dipalmitoylphosphatidylcholine, and cholesterol (1/0.75). When compared with mice injected only with normal saline, there were no statistical differences in life spans observed between the different groups. Animals injected with liposomes or liposomes containing lipid A gradually developed “ruffled” fur, but the animals did not appear sick otherwise, and no differences were observed in the mean weights of the animals in the different groups. All of the animals that were tested in each group, including those injected with normal saline, developed IgG antibodies against one or more of nine lipid antigens. the antibodies were detected by a solid-phase enzyme-linked immunosorbent assay (ELisA) and the antigens consisted of either lipid A or one of eight different phospholipids. After 765 days, when approximately half of the animals had died spontaneously, the surviving animals were sacrificed and subjected to extensive histopathological analysis. In each group (including the normal saline group) certain animals exhibited pathological changes in liver, spleen, or lung that might be expected to occur in highly aged animals. Approximately half of the animals in each group had lymphoproliferative disorders (hyperplasia and/or lymphoma). However, there were no bone marrow abnormalities, and no hepatic or splenic granulomatous reactions were found in any of the animals. From a pathological standpoint the groups were indistinguishable from each other. We conclude that life-long repeated injection of liposomes, lipid A, or liposomes containing lipid A does not alter longevity or cause any overt pathological changes in mice. We also conclude that antibodies to lipid A and a variety of phospholipids occur spontaneously in aged mice that have been injected only with normal saline.     

Interaction of pH-Sensitive Liposomes With Blood Components

Abstract

Avoidance of lysosomal degradation of drugs entrapped in liposomes has been one of the major efforts in liposome research. The achievement of high drug deliver}' efficiency using pH-sensitive liposomes over the pH-insensitive liposomes has greatly influenced our strategies in liposome drug delivery. The success of pH-sensitive liposomes in delivering compounds such as fluorescence dye, anti-cancer reagents, toxins and DNA to target cells with high efficiency in vitro shows a great potential to apply the same strategy to in vivo systems. Using human plasma as a simplified model for blood, we have systematically examined the interaction of pH-sensitive liposomes composed of dioleoylphosphatidyl-ethanolamine (DOPE) and oleic acid (OA) with plasma components. Our results show that the bilayer structure of liposomes in plasma depends on their sizes. Small liposomes (d<200nm) were stabilized by plasma components while the larger ones (d>600nm) were rapidly lysed upon the exposure to plasma. Such differences in their stability in plasma may derive from their differences in lipid packing which determines the surface pressure of the membrane. Using purified serum proteins, we found that albumin such as bovine serum albumin (BSA) lyse liposomes by extracting OA from the bilayer. However, BSA induced lysis could be blocked by lipoproteins including HDL, LDL and VLDL, but not by immunoglobulins. Further studies with purified components of HDL demonstrated that apoAl, not the lipids of the HDL, contains the stabilization activity. The extraction of OA from liposomes and the insertion of plasma components into the bilayer modified the bilayer properties such that plasma stabilized liposomes were no longer pH sensitive. Using dipalmitoylsuccinylglycerol (DPSG), a double-chain pH senser for DOPE liposomes, we could preserve 50% pH sensitivity after plasma treatment. The potential application of such liposomes and other essential properties of pH-sensitive liposomes for drug delivery in vivo are also discussed.     

Liposomes as carriers for paramagnetic gadolinium chelates as organ specific contrast agents for magnetic resonance imaging (mri)

Abstract

Small unilamellar liposomes were used as carriers for chelates of gadolinium as organ specific magnetic resonance imaging (MRI) contrast agents. The pharmacokinetic and imaging properties of the lipophilic liposome membrane associated chelate diethylenetriaminepentaacetate-stearylamide (DTPA-SA) were investigated. Gadolinium-DTPA-SA liposomes accumulated in the liver of rats at a peak concentration of 60% of the injected dose 4 hours after application. The elimination half-life from the liver was 61 h. Tl-weighted MR images of this liposomal Gd-chelate in rats and dogs gave a strong signal enhancement of the abdominal organs, liver and spleen. High blood concentrations of the Gd-DTPASA liposomes, reaching 60% of the injected dose after 30 min., decreasing to 40% after 2 hours, suggest their potential as a contrast agent for the blood pool. The gadolinium chelate benzoyloxypropionictetraacetate (Gd-BOPTA) was entrapped in liposomes of different lipid composition. Pharmacokinetic studies of liposome preparations containing a poly(ethylene)glycol (PEG) modified lipid showed that high levels of 80 - 60 % of the injected dose remained in the blood, 15 to 60 minutes after application. Peak blood concentrations of liposomes without PEG reached only 30%, with a correspondingly higher uptake in the liver and the spleen. Thus, both the lipophilic chelate Gd-DTPA-SA, as well as Gd-BOPTA entrapped within the aqueous volume of liposomes possess not only a potential as a liver and spleen specific contrast agent, but also for the imaging of the vascular system.     

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.     

Targeting of anti-Thy 1.1 monoclonal antibody conjugated liposomes in Thy 1.1 mice after intraveneous 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.     

亚油酸铂靶向脂质体抗肿瘤特性的研究

用超声波制备了内部包裹亚油酸铂,表面有抗人乳腺癌单克隆抗体ＭｃＡｂＧｐ－１Ｄ８的亚油酸铂靶向脂质体和亚油酸铂非靶向脂质体,研究了这些脂质体绎腹部注射到荷瘤裸鼠之后的组织分布和抑瘤效果,实验结果表明,靶向脂质体亚油酸铂在肿瘤组织的含量明显高于游离亚油酸铂组;在肾,肝,肺,脾,心脏等器官中,前者的含量比后者有所降低,分别在接种癌细胞后６天,１２天和２４天,按６ｍｇ／ｋｇ的剂量分别注射ＰＢＳ,游离亚油酸     

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.     

Proton-induced fusion of oleic acid-phosphatidylethanolamine liposomes

Liposomes composed of oleic acid and phosphatidylethanolamine (3:7 mole ratio) aggregate, become destabilized, and fuse below pH 6.5 in 150 mM NaCl. Fusion is monitored by (i) the intermixing of internal aqueous contents of liposomes, utilizing the quenching of aminonaphthalene-3,6,8-trisulfonic acid (ANTS) by N,N'-p-xylylenebis(pyridinium bromide) (DPX) encapsulated in two separate populations of vesicles, (ii) a resonance energy transfer assay for the dilution of fluorescent phospholipids from labeled to unlabeled liposomes, (iii) irreversible changes in turbidity, and (iv) quick-freezing freeze-fracture electron microscopy. Destabilization is followed by the fluorescence increase caused by the leakage of coencapsulated ANTS/DPX or of calcein. Ca2+ and Mg2+ also induce fusion of these vesicles at 3 and 4 mM, respectively. The threshold for fusion is at a higher pH in the presence of low (subfusogenic) concentrations of these divalent cations. Vesicles composed of phosphatidylserine/phosphatidylethanolamine or of oleic acid/phosphatidylcholine (3:7 mole ratio) do not aggregate, destabilize, or fuse in the pH range 7-4, indicating that phosphatidylserine and phosphatidylcholine cannot be substituted for oleic acid and phosphatidylethanolamine, respectively, for proton-induced membrane fusion. Freeze-fracture replicas of oleic acid/phosphatidylethanolamine liposomes frozen within 1 s of stimulation with pH 5.3 display larger vesicles and vesicles undergoing fusion, with membrane ridges and areas of bilayer continuity between them. The construction of pH-sensitive liposomes is useful as a model for studying the molecular requirements for proton-induced membrane fusion in biological systems and for the cytoplasmic delivery of macromolecules.     

Improvement of in vivo stability of phosphodiester oligonucleotide using anionic liposomes in mice

Antisense phosphodiester oligonucleotides (ODN) are unstable in biological fluids due to nuclease-mediated degradation and therefore cannot be used in most antisense therapeutic applications. We describe here an in vitro and in vivo stabilization of a 15 mer phosphodiester sequence using anionic liposomes. Two formulations have been studied: DOPC/OA/CHOL and DOPE/OA/CHOL (pH-sensitive liposomes). Our in vitro findings reveal the same stabilization effect in mouse plasma for both anionic liposomes. In vivo investigation showed a great protective effect for both formulations after intravenous administration to mice. By contrast with in vitro results, a higher protection of ODN was observed with DOPC/OA/CHOL liposomes compared to the DOPE/OA/CHOL formulation. The latter was degraded in blood (75% of the injected dose at 5 min) probably due to interactions with blood components, and the remaining (25% at 5 min) was distributed mostly to the liver and spleen. DOPC liposomes were remarkably stable in blood and were distributed more slowly to all studied organs (liver, spleen, kidneys and lungs). Intact ODN was still observed in some organs (liver, spleen, lungs), but not in blood, 24 hours after DOPC liposome administration. These results suggest that this antisense strategy using carrier systems may be applicable to the treatment of diseases involving the reticuloendothelial system.     

Interaction between oleic acid-containing pH-sensitive and plain liposomes. Fluorescent spectroscopy studies.

The energy transfer method has been applied to study the interaction between pH-sensitive liposomes (phosphatidyl ethanolamine/oleic acid/cholesterol, 4:2:4 molar ratio) and plain liposomes (phosphatidyl choline/phosphatidyl ethanolamine/cholesterol, 4:2:3 molar ratio). It was shown that a slow fusion process occurs between two types of liposomes. Also, the transfer of oleic acid from pH-sensitive liposomes to plain liposomes takes place. This transfer results in the increased permeability of both pH-sensitive and plain liposomes, facilitating the release of liposome-entrapped fluorescent dye. The data obtained were used for a possible explanation of the mechanism of intracytoplasmic drug delivery by pH-sensitive oleic acid-containing liposomes.     

Production of a monoclonal antibody directed against rat liver glutathione-insulin transhydrogenase

A hybridoma cell line secreting monoclonal antibody specific for glutathione-insulin transhydrogenase has been produced by fusing mouse myeloma cells with spleen cells from mice immunized to purified rat liver glutathione-insulin transhydrogenase. The secreted antibody isotypes were found to be: Ig gamma 1 heavy chains and kappa light chains. This monoclonal antibody has been used to screen glutathione-insulin transhydrogenase in various rat tissue extracts (liver, fat, heart, testis, spleen, lung and kidney) following separation on NaDodSO4/urea polyacrylamide disc-gel electrophoresis and electrophoretic transfer to nitrocellulose. Screening with the monoclonal antibody showed the presence of one immunoreactive protein band equal in molecular weight to that of purified rat liver GIT (Mr 53,000) in extracts of all tissues studied and a second immunoreactive protein band of lower molecular weight (Mr 49,000) in spleen and lung tissue extracts. Separation of these two proteins by HPLC using a TSK-DEAE column demonstrated that both proteins exhibit insulin degrading activity. These data indicate that GIT may occur in multiple forms in some tissues.     

Application of liposomes to generation of monoclonal antibody to glycosphingolipid: production of monoclonal antibody to GgOse4Cer

Liposomes were applied to the immunization with GgOse4Cer and screening for production of monoclonal antibody to GgOse4Cer. Four-week-old and 22-week-old Balb/c mice were immunized with GgOse4Cer and Salmonella minnesota R595 lipopolysaccharides incorporated liposomes which were composed of dipalmitoyl-phosphatidylcholine and cholesterol. Since antibody response to GgOse4Cer was higher in 22-week-old than 4-week-old Balb/c mice after immunization, 22-week-old Balb/c mice were used for the immunization prior to generation of the monoclonal antibodies to GgOse4Cer. The screening of monoclonal antibodies was performed by complement-dependent liposome immune lysis assay using GgOse4Cer-containing liposomes. Six kinds of monoclonal antibodies, AG-1, -2, -3, -4, -5, and -6, of the IgM class were established. The specificities of the monoclonal antibodies obtained were defined by complement-dependent liposome immune lysis assay using various glycosphingolipids incorporated in liposomes and by thin-layer chromatography (TLC) with immunostaining. All of the monoclonal antibodies reacted only with GgOse4Cer in the liposome immune lysis assay. In addition, the monoclonal antibodies reacted only with GgOse4Cer in the TLC immunostaining. However, none of the monoclonal antibodies obtained was capable of removing natural killer activity from C3H/He mice spleen cell suspensions in vitro. Liposomes may be useful in the procedures of immunization and screening for generation of antiserum and monoclonal antibody to GSLs.     

Disposition and hepatoprotection by phosphatidyl choline liposomes in mouse liver

Small unilamellar liposomes with an average diameter of 80 nm were prepared from phosphatidyl choline of various sources using the dialysis method with cholate as a detergent. When 14C-labeled soybean liposomes were intravenously injected into male NMRI mice, up to 10% of the total label was found in the liver lipid. The uptake was dose-dependent and reached an apparent saturation 4 h after injection. The liver maintained a constant radioactivity corresponding to 1.9 +/- 0.13 mg phospholipid/g liver until ten hours after injection of 850 mg labeled phosphatidyl choline/kg body wt. Little radioactivity was taken up by the spleen. Analogous doses of liposomes prepared from egg yolk phosphatidyl choline led to a radioactivity corresponding to 1.3 +/- 0.4 mg lipid/g liver 4 h after injection. Liposomes with a similar size were prepared from hydrated, i.e., saturated phosphatidyl choline. After intravenous administration of these liposomes, an amount of 5.3 +/- 0.5 mg labeled lipid was found per g liver after 4 h. In contrast to unsaturated liposomes, 5.8 +/- 0.8 mg lipid per gram spleen was trapped by the spleen. The pharmacodynamic effect of these different liposomes was studied in benzo[a]pyrene-pretreated mice intoxicated with 400 mg/kg paracetamol. Animals which received paracetamol exhibited serum alanine aminotransferase activities of 4220 +/- 1140 units/l after 4 h and exhaled 120 +/- 19 nmol ethane kg-1 h-1. When pretreated with 850 mg soybean phosphatidyl choline/kg body wt. (i.v.) 2 h prior to paracetamol, the increase in serum transaminase activity was reduced to 117 +/- 104 units/l and ethane exhalation amounted to 18 +/- 8 nmol kg-1 h-1. In contrast, similar pretreatment with egg yolk phosphatidyl choline or hydrated phosphatidyl choline failed to protect against paracetamol-induced hepatotoxicity. The different pharmacodynamic effects of the two phosphatidyl cholines of plant or animal origin cannot be explained on the basis of their different pharmacokinetics. In the case of soybean phosphatidyl choline liposomes, the amount of radioactive lipid found in the liver correlated with the hepatoprotective potency.     

Highly efficient immunoliposomes prepared with a method which is compatible with various lipid compositions

Monoclonal antibody was conjugated to N-glutaryl-phosphatidylethanolamine in the presence of octylglucoside by using N-hydroxysulfosuccinimide as a carboxyl-activation reagent. The conjugated antibody was then incorporated into liposomes by a simple dialysis method. The method is mild and is compatible with various lipid compositions of the liposomes. We have prepared immunoliposomes containing a lung endothelium-specific monoclonal antibody and showed excellent target binding (approximately 75% injected dose) of the immunoliposomes in mouse. Immunoliposomes can be prepared to contain other acidic lipids such as phosphatidylserine and various amounts of cholesterol. The presence of 20% or more cholesterol in liposomes resulted in high level of target binding. We have used in these experiments a new radioactive lipid-phase marker, 111In-DTPA-SA, which was very stable in vivo. The halflife of clearance in mouse exceeded 3 weeks.