Senescence Marker Protein 30 Has a Cardio-Protective Role in Doxorubicin-Induced Cardiac Dysfunction

Background

Senescence marker protein 30 (SMP30), which was originally identified as an aging marker protein, is assumed to act as a novel anti-aging factor in the liver, lungs and brain. We hypothesized that SMP30 has cardio-protective function due to its anti-aging and anti-oxidant effects on doxorubicin (DOX)-induced cardiac dysfunction.

Methods and Results

SMP30 knockout (SMP30 KO) mice, SMP30 transgenic (SMP30 TG) mice with cardiac-specific overexpression of SMP30 gene and wild-type (WT) littermate mice at 12–14 weeks of age were given intra-peritoneal injection of DOX (20 mg/kg) or saline. Five days after DOX injection, echocardiography revealed that left ventricular ejection fraction was more severely reduced in the DOX-treated SMP30 KO mice than in the DOX-treated WT mice, but was preserved in the DOX-treated SMP30 TG mice. Generation of reactive oxygen species and oxidative DNA damage in the myocardium were greater in the DOX-treated SMP30 KO mice than in the DOX-treated WT mice, but much less in the SMP30 TG mice. The numbers of deoxynucleotidyltransferase-mediated dUTP nick end-labeling positive nuclei in the myocardium, apoptotic signaling pathways such as caspase-3 activity, Bax/Bcl-2 ratio and phosphorylation activity of c-Jun N-terminal kinase were increased in SMP30 KO mice and decreased in SMP30 TG mice compared with WT mice after DOX injection.

Conclusions

SMP30 has a cardio-protective role by anti-oxidative and anti-apoptotic effects in DOX-induced cardiotoxicity, and can be a new therapeutic target to prevent DOX-induced heart failure.     

Effects of phosphatidylserine on membrane incorporation and surface protection properties of exchangeable poly(ethylene glycol)-conjugated lipids

Liposomes containing the acidic phospholipid phosphatidylserine (PS) have been shown to avidly interact with proteins involved in blood coagulation and complement activation. Membranes with PS were therefore used to assess the shielding properties of poly(ethylene glycol 2000)-derivatized phosphatidylethanolamine (PE-PEG(2000)) with various acyl chain lengths on membranes containing reactive lipids. The desorption of PE-PEG(2000) from PS containing liposomes was studied using an in vitro assay which involved the transfer of PE-PEG(2000) into multilamellar vesicles, and the reactivity of PS containing liposomes was monitored by quantifying interactions with blood coagulation proteins. The percent inhibition of clotting activity of PS liposomes was dependent on the PE-PEG(2000) content. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG(2000) which transferred out slowly from PS liposomes was able to abolish >80% of clotting activity of PS liposomes at 15 mol%. This level of DSPE-PEG(2000) was also able to extend the mean residence time of PS liposomes from 0.2 h to 14 h. However, PE-PEG(2000) with shorter acyl chains such as 1,2-dimyristyl-sn-glycero-3-phosphoethanolamine-PEG(2000) were rapidly transferred out from PS liposomes, which resulted in a 73% decrease in clotting activity inhibition and 45% of administered intravenously liposomes were removed from the blood within 15 min after injection. Thus, PS facilitates the desorption of PE-PEG(2000) from PS containing liposomes, thereby providing additional control of PEG release rates from membrane surfaces. These results suggest that membrane reactivity can be selectively regulated by surface grafted PEGs coupled to phosphatidylethanolamine of an appropriate acyl chain length.     

Effect of the cholesterol content of small unilamellar liposomes on their stability in vivo and in vitro

Small unilamellar neutral, negatively and positively charged liposomes composed of egg phosphatidylcholine, various amounts of cholesterol and, when appropriate, phosphatidic acid or stearylamine and containing 6-carboxyfluorescein were injected into mice, incubated with mouse whole blood, plasma or serum or stored at 4°C. Liposomal stability, i.e. the extent to which 6-carboxyfluorescein is retained by liposomes, was dependent on their cholesterol content. (1) Cholesterol-rich (egg phosphatidylcholine/cholesterol, 7:7 molar ratio) liposomes, regardless of surface charge, remained stable in the blood of intravenously injected animals for up to at least 400min. In addition, stability of cholesterol-rich liposomes was largely maintained in vitro in the presence of whole blood, plasma or serum for at least 90min. (2) Cholesterol-poor (egg phosphatidylcholine/cholesterol, 7:2 molar ratio) or cholesterol-free (egg phosphatidylcholine) liposomes lost very rapidly (at most within 2min) much of their stability after intravenous injection or upon contact with whole blood, plasma or serum. Whole blood and to some extent plasma were less detrimental to stability than was serum. (3) After intraperitoneal injection, neutral cholesterol-rich liposomes survived in the peritoneal cavity to enter the blood circulation in their intact form. Liposomes injected intramuscularly also entered the circulation, although with somewhat diminished stability. (4) Stability of neutral and negatively charged cholesterol-rich liposomes stored at 4°C was maintained for several days, and by 53 days it had declined only moderately. Stored liposomes retained their unilamellar structure and their ability to remain stable in the blood after intravenous injection. (5) Control of liposomal stability by adjusting their cholesterol content may help in the design of liposomes for effective use in biological systems in vivo and in vitro.     

Augmentation of NK cell activity in tissue specific sites by liposomes incorporating MTP-PE

Liposomes incorporating a variety of immunomodulators have been shown to activate macrophages and monocytes for tumoricidal activity both in vivo and in vitro. We report that in addition to the activation of macrophages, the i.v. injection of liposomes (multilamellar vesicles) that have encapsulated muramyl tripeptide-phosphatidylethanolamine (MTP-PE) could also augment interstitial natural killer (NK) cell activity in the lung and the liver. In contrast, liposomes incorporating MTP-PE were unable to augment NK cell activity in the spleen, peripheral blood, or peritoneal cavity (after i.p. injection). In addition, liposomes did not augment splenic NK cell activity in vitro. This suggests that the augmentation of NK cell activity in the lungs and liver was not due to direct effects of the liposomes but may have been a secondary effect mediated by a monokine. The augmentation of pulmonary NK cell activity was paralleled by the nonspecific immunoprophylaxis of experimental pulmonary metastases. The augmented NK cell activity, as well as the enhanced nonspecific immunoprophylactic activity, was reduced by pretreatment of the mice with anti-asialo GM1 antiserum. Thus, the augmentation of organ-associated NK cell activity by liposomes incorporating MTP/PE plays a major role in the host's increased resistance to the formation of experimental metastases.     

Toxicity and Biodistribution of Liposomes of the Main Phospholipid from the Archaebacterium Thermoplasma Acidophilum in Mice

Abstract

Toxicity and biodistribution of negatively charged liposomes of the main phospholipid (MPL) from the archaebacterium Thermoplasma acidophilum were tested in mice. MPL liposomes with a diameter of 160–220 nm were prepared by extrusion through polycarbonate filters, or by means of a French pressure cell and screened for central nervous system effects after intraperitoneal (i.p.) injection of 4–324 mg of liposomes per kg body weight in NMRI-mice. Besides increased behavioural activity no pharmacological or toxic effects were detected. No alterations were seen in the morphology of the tissues analyzed. Longterm toxicity after life-long oral application of 30 mg MPL per kg body weight per day starting at the age of 10 weeks was tested in immunosuppressed NMRI-mice. Again, there were no toxic effects on survival. Biodistribution of MPL liposomes labeled with ¹¹¹In-diethylenetriaminepentaacetic acid stearylamide was examined 15 min and 2.5 h after intravenous injection into ICR-mice. The liposomes were rapidly cleared from the circulation and the majority accumulated in the liver, followed by the spleen.     

Altered pharmacological properties of liposome-associated human interferon-alpha.

Human interferon-alpha was associated in different ways with positively (stearylamine) and negatively (phosphatidylserine) charged phosphatidylcholine multilamellar vesicles, depending on the presence or absence of a cholesterol component. Inclusion of cholesterol resulted in interferon that was significantly (P = 0.0001) more deeply internalized within the liposomes, such that detergent disruption was necessary before most of the interferon activity was expressed. Interferon was stably associated with stearylamine-containing liposomes, both with and without a cholesterol component. However, inclusion of cholesterol in the phosphatidylserine-containing liposomes was necessary for stable association of the interferon for more than 2 days at 4 degrees C or for more than 24 h at 37 degrees C. After intramuscular injection into mice, liposome-associated interferon in reverse-phase evaporation vesicles was retained at the local site of injection significantly longer than free interferon. Even 3 days after intramuscular injection, stearylamine-containing liposomes with or without cholesterol resulted in local interferon levels that were comparable to the peak levels obtained 2 to 4 h after free interferon was injected. In contrast, free interferon was not detectable in the local muscles 24 h after injection of 10(4.6) U. Liposomes containing phosphatidylserine and cholesterol resulted in intermediate levels of local interferon retention; without a cholesterol component, phosphatidylserine-containing liposomes resulted in no increased local interferon retention compared with the results when free interferon was injected.     

Effects of Selenium Administration on Erythrocyte and Blood Plasma Glutathione Peroxidase Activity in Goats

The activity of glutathione peroxidase (GSH-Px, E.G. 1.11.1.9.) was determined in heparinized whole blood, blood plasma and washed erythrocytes from goats before and up to 4 weeks after the administration of selenium (0.4 mg/10 kg BW) and vitamin E (20 mg/10 kg BW) or only vit. E (20 mg/10 kg BW). It was found that Se administration caused a significant increase in enzyme activity in whole blood and washed erythrocytes first detected 2 weeks after the intramuscular injection of Se. No changes were observed in plasma from the treated animals. Minor and insignificant changes were seen in the vit. E treated control animals. It is concluded that GSH-Px activity in blood plasma or serum is of no value as a short-term indicator of the selenium status of goats but whole blood is a good indicator of the long-term status.     

Effect of Repeated Intravenous Administration on the Circulation Kinetics of Poly(Ethyleneglycol)-Liposomes in Rats

Abstract

The use of sterically stabilized poly(ethyleneglycol)-coated liposomes (PEG-liposomes) is becoming increasingly important and several preparations based on long-circulating liposomes are already commercially available. From a clinical point of view, it is of importance to study the effect of multiple i.v. administration of PEG-liposomes on their pharmacokinetic behavior. Sterically stabilized liposomes were obtained by incorporation of PEG conjugated to distearoylethanolamine (DSPE) into the liposomal bilayers. Rats received 4 i.v. injections of small (0.12 um) PEG-liposomes at 24 or 48 h dosing intervals. Blood levels of liposomal label were determined at several time-points after injection. Our findings demonstrate that, under the chosen conditions, i.v. injection of PEG-liposomes has no effect on the blood circulation kinetics of subsequent doses of similar liposomes given at 24 or 48 h dosing intervals. These findings suggest that PEG-liposomes are suitable as drug carriers for diagnostic and therapeutic applications that require repeated i.v. injections.     

Prolonged circulation time in vivo of large unilamellar liposomes composed of distearoyl phosphatidylcholine and cholesterol containing amphipathic poly(ethylene glycol).

The effect of poly(ethylene glycol) (PEG) on the circulation time of liposomes in mice was examined by employing amphipathic PEGs (phosphatidylethanolamine (PE) derivatives of PEG) with average molecular weights of 1000, 2000, 5000 and 12,000. The activity of dioleoyl phosphatidylethanolamine-PEG (DOPE-PEG) in prolonging the circulation time of egg phosphatidylcholine/cholesterol large unilamellar liposomes (ePC/CH LUVs) (200 nm) was proportional to the molecular weight of PEG, i.e., 12000 = 5000 greater than 2000 greater than 1000. On the other hand, inclusion of distearoylphosphatidylethanolamine-PEG (DSPE-PEG) or dipalmitoyl-phosphatidylethanolamine-PEG (DPPE-PEG) of low molecular weight such as 1000 and 2000 in distearoylphosphatidylcholine (DSPC)/CH LUVs or dipalmitoyl phosphatidylcholine (DPPC)/CH LUVs effectively increased their blood circulation time. At least 3 mol% of amphipathic PEG in liposomes was required for activity. Addition of CH, which has a bilayer-tightening effect, to DSPC/CH/DSPE-PEG2000 LUVs further increased the blood residence time. A size of less than 300 nm was essential for prolonging the residence time of amphipathic PEG-containing liposomes in blood. DSPC/CH/DSPE-PEG2000 LUVs (1:1:0.13, m/m) containing 6 mol% of PEG and 200 nm in diameter remained in the circulation for over 24 h after injection and may be clinically useful for sustained release of an entrapped drug in the bloodstream and for drug accumulation in solid tumors.     

Liposomes containing chelating agents. Cellular penetration and a possible mechanism of metal removal

Electron microscope studies were done on mouse liver, from 5 min to 8 wk after an intravenous injection of liposomes containing ethylenediaminetetraacetic acid (EDTA). Livers of mice receiving an injection of liposomes containing KCL instead of EDTA or an injection of a solution of EDTA were also examined. Liposomes were shown to be phagocytized by hepatocytes as well as by Kupffer cells within minutes after the injection. Initially, there was a close contact between the liposomal membrane and the cellular membrane, followed by an invagination of the latter and the formation of a distinct vesicle surrounding a single liposome or a cluster of several liposomes. No fusion between the liposomal membrane and the cell membrane was observed. Between 15 min and 6 h after liposome injection, the Kupffer cells were found to have an increased number of lysosomes and autophagic vacuoles. Within the latter, morphologically intact liposomes or remnants of liposomes could be seen. At 12 h after injection, a striking increase in macrophages was observed in the liver sinusoids of EDTA-liposome-injected mice, but not in those of KCl- liposome-injected mice. Within the macrophages, remnants of liposomes occasionally could be observed. However, the origin and the physiological role of these cells are unknown. In the hepatocytes, morphological changes were first observed 24 h after injection; there were large numbers of autophagic vacuoles, and some cells showed extensive areas of focal cytoplasmic degeneration. The morphology of the liver cells returned to normal about 7 days after injection. No morphological changes were observed in livers of mice receiving EDTA solution without liposomes. A possible mechanism by which the liposome- encapsulated chelating agents can successfully remove intracellular toxic metals is discussed. The use of liposomes as carriers seems to be a useful tool for intracellular delivery of chelating agents or drugs in general.     

The influence of repeated injections on pharmacokinetics and biodistribution of different types of sterically stabilized immunoliposomes

Sterically stabilized immunoliposomes (IL) with diameters of about 135 nm carrying mouse IgG, either coupled directly to the liposome surface, or linked to the terminal ends of grafted poly(ethylene glycol) (PEG) chains by a recently described conjugation procedure (Cyanur-PEG-PE), were intravenously injected into rats and the elimination kinetics and biodistribution were determined and compared with control liposomes. The amounts of conjugated antibodies were about 30 μg/μmol total lipid for all IL. In naive rats, plain pegylated liposomes displayed the longest blood circulation time, whereas the terminal-coupled IL exhibited the fastest elimination. Liposomes containing the underivatized anchor molecules circulate nearly as long as plain pegylated liposomes, indicating that the fast elimination of the IL can be attributed to the presence of antibodies.A second injection of identical liposomes 14 days after the first injection had a considerable influence on the pharmacokinetic parameters of the liposomes. The circulation time of plain pegylated liposomes drastically dropped by half and their uptake by the liver increased concomitantly, indicating that the PEG, upon repeated injection, ceases to function as an efficient barrier reducing opsonization and/or immune reactions. The circulation time of conventional IL was moderately reduced upon a second injection, whereas that of the terminally coupled IL was nearly unaffected. These differences among the IL demonstrate that the pharmacokinetic behavior of IL is strongly dependent on the antibody conjugation site on the liposome. The observed effects of repeated injections were similar for liposomes of 90-nm diameter. The phenomena described may have important implications for the repeated application of IL as drug carriers.     

Repeated injections of PEG-PE liposomes generate anti-PEG antibodies

Liposomes containing the polyethylene glycol (PEG) derivative of phosphatidyl ethanolamine (PE) have recently been found to be promising drug carriers, as they facilitate controlled and target-oriented release of therapeutics. They also reduce the side effects of many drugs. Here, we present the results of a study on antiliposomal properties of rabbit sera obtained after weekly injections of small liposomes containing 20% PEG-PE. The effect was analysed as the level of induced carboxyfluorescein release from these liposomes in vitro. The incubation of liposomes with rabbit serum taken after the injections induced the release of carboxyfluorescein at a higher level than was seen for incubation with untreated animal's serum. The strongest effect was observed for serum obtained after the second injection, i.e. during the second week of the study. The effect was much smaller after the serum samples were preheated at 56 degrees C. The binding of serum proteins by PEGylated liposomes was analysed via gel filtration and via the immunoblot technique using goat anti-rabbit IgG; this revealed that the serum protein which bound to the liposomes in vitro had a molecular weight of 55 kD and reacted with the anti-IgG antibody. Competition with PEG or lipids indicate that this IgG has an anti-PEG activity. We therefore assume that these antibodies are responsible for the activation of complement and leakage induction of PEG-liposomes. Such antibodies could be responsible for increased phagocytosis by RES macrophages (in particular liver macrophages) and decreased circulation time.     

The influence of repeated injections on pharmacokinetics and biodistribution of different types of sterically stabilized immunoliposomes

Sterically stabilized immunoliposomes (IL) with diameters of about 135 nm carrying mouse IgG, either coupled directly to the liposome surface, or linked to the terminal ends of grafted poly(ethylene glycol) (PEG) chains by a recently described conjugation procedure (Cyanur-PEG-PE), were intravenously injected into rats and the elimination kinetics and biodistribution were determined and compared with control liposomes. The amounts of conjugated antibodies were about 30 microg/micromol total lipid for all IL. In naive rats, plain pegylated liposomes displayed the longest blood circulation time, whereas the terminal-coupled IL exhibited the fastest elimination. Liposomes containing the underivatized anchor molecules circulate nearly as long as plain pegylated liposomes, indicating that the fast elimination of the IL can be attributed to the presence of antibodies.A second injection of identical liposomes 14 days after the first injection had a considerable influence on the pharmacokinetic parameters of the liposomes. The circulation time of plain pegylated liposomes drastically dropped by half and their uptake by the liver increased concomitantly, indicating that the PEG, upon repeated injection, ceases to function as an efficient barrier reducing opsonization and/or immune reactions. The circulation time of conventional IL was moderately reduced upon a second injection, whereas that of the terminally coupled IL was nearly unaffected. These differences among the IL demonstrate that the pharmacokinetic behavior of IL is strongly dependent on the antibody conjugation site on the liposome. The observed effects of repeated injections were similar for liposomes of 90-nm diameter. The phenomena described may have important implications for the repeated application of IL as drug carriers.     

Lysosomal localization of β-fructofuranosidase-containing liposomes injected into rats. Some implications in the treatment of genetic disorders

Yeast beta-fructofuranosidase (invertase) or (131)I-labelled albumin were entrapped into liposomes composed of phosphatidylcholine, cholesterol and phosphatidic acid. Of the beta-fructofuranosidase activity in the liposomal preparations 96-100% was latent. The following observations were made in experiments with rats injected with protein-containing liposomes. 1. After injection of beta-fructofuranosidase-containing liposomes (220 units or 1.5mg of beta-fructofuranosidase and 17.5mg of lipid), beta-fructofuranosidase activity in blood retained its latency but the activity declined to 50% of the injected dose in 1h. Within 6h much of this activity was recovered in the liver and spleen (respectively 45% and 10% of that injected). For up to 21h after injection, the mitochondrial-lysosomal fraction was the principal location of the hepatic beta-fructofuranosidase activity. 2. Lysosomal localization of liposomal protein was supported by the observed increase in the trichloroacetic acid-soluble radioactivity during incubation of the lysosome-rich fraction of the liver of rats injected with liposomes containing (131)I-labelled albumin. 3. Association of liposomal protein with lysosomes was demonstrated on subfractionation of the mitochondrial-lysosomal fraction of the liver of rats injected with beta-fructofuranosidase-containing liposomes in a Ficoll-mannitol gradient. beta-Fructofuranosidase, lysosomal and mitochondrial enzyme marker activities were found to exhibit similar distribution patterns along the gradient. However, in similar experiments with rats previously injected with Triton WR-1339 or dextran (known to alter the specific gravity of lysosomes), only beta-fructofuranosidase and lysosomal marker moved along the gradient, in strikingly similar patterns. 4. The lysosomal localization of injected liposome-entrapped material can probably be utilized in the treatment of certain disorders in man.     

Reversible depression of the reticuloendothelial system by liposomes

The effect of various doses of different types (reverse phase evaporation vesicles and small unilamellar vesicles) of intravenously injected liposomes on reticuloendothelial activity, as measured by the blood clearance rate of intravenously injected carbon, was investigated. Also the effect of pretreatment with reverse phase evaporation vesicles on blood clearance and tissue distribution of a second dose of similar vesicles was determined. For all concentrations used reverse phase evaporation vesicles caused reduction in reticuloendothelial activity at least up to 4 h after injection. 24 h after administration the rate of carbon clearance returned to the control level. On the contrary small unilamellar vesicles did not block reticuloendothelial activity. Pretreatment with reverse phase evaporation vesicles (250 μmol/kg) caused an increased blood level and a decreased hepatic uptake of a second dose of the vesicles, injected 1 h after the first dose. This seems to be due to a depression of reticuloendothelial activity and not to a depletion of opsonins. Pretreatment with small unilamellar vesicles (250 μmol/kg) had no significant influence on the tissue distribution of a second dose of vesicles. Our results clearly indicate that reverse phase evaporation vesicles cause a reversible depression of reticuloendothelial activity and this depression seems to be induced by a saturation of reticuloendothelial cells with liposomes.     

Systemic activation of macrophages by liposome-entrapped muramyl tripeptide in mice pretreated with the chemotherapeutic agent adriamycin

Summary The purpose of these studies was to determine whether macrophages of mice pretreated with the chemotherapeutic agent adriamycin (ADR) could be systemically activated by IV injection of liposomes containing muramyl tripeptide phosphatidylethanolamine (MTP-PE), a lipophilic derivative of muramyl dipeptide. Lower than normal levels of alveolar macrophages or peritoneal exudate macrophages were found in mice following IV injection of ADR. This decrease was dose-dependent and, in mice given <10 mg ADR/kg, it was transient (14 days). Peritoneal macrophages surviving the administration of 15 mg ADR/kg were tumoricidal.At various times after single or repeated administration of ADR, mice were given IV or IP injections of liposomes containing MTP-PE. One day thereafter, the cytotoxic activity of the in situ-activated macrophages (alveolar or peritoneal exudate) was assessed in culture against syngeneic melanoma cells. Our data demonstrate that under defined conditions the systemic administration of ADR does not interfere with the in situ activation of tumoricidal properties of murine macrophages after IV injection of liposomes containing a macrophage-activating agent.     

Metabolism of liposomes prepared from a labelled ether analog of 1,2-dioleoyl-sn-glycero-3-phosphocholine in the rat

To synthesize the ether analog of 1,2-diacyl-sn-glycero-3-phosphocholine (PC), 1-O-cis-9'- octadecenyl -2-O-cis-9'-[9',10'(n)-3H] ocatadecenyl -sn-glycero-3- phosphocholine, we have adapted available methodology and have obtained a product of high specific activity and purity. The labelled dioleyl ether phosphatidylcholine ( DOEPC ) was used to prepare 250-350 A unilamellar liposomes, which contained also PC and free cholesterol. Following intravenous injection into rats, labelled PC was cleared from the plasma at a faster rate than DOEPC . The uptake of both labelled compounds by the liver increased up to 3 h, at which time there was about 40% of injected PC and 60% of DOEPC . The PC disappeared more rapidly than the DOEPC , so that 17 and 48% of injected label were present in the liver 24 h after injection of PC and DOEPC , respectively. Ten days after injection of DOEPC , about 10% of the label was still present in the liver. During the first 5 days after injection of DOEPC , 10% of radioactivity was found in the gastrointestinal tract and about 20% in the carcass; no increase in carcass radioactivity occurred during the loss of label from the liver. 24 and 48 h after injection of DOEPC , 40% of liver radioactivity was present in a neutral lipid, which on TLC comigrated with triacylglycerol. Since after alkaline hydrolysis this compound comigrated with diacylglycerol, it appears that the ether bond of DOEPC was not hydrolyzed, but after removal of phosphocholine, presumably by phospholipase C, the diether glycerol was reacylated . In experiments in vitro, the rate of exchange of labelled PC with red blood cell phospholipids exceeded that of DOEPC . Incubation of cultured hepatocytes with liposomes containing PC and/or DOEPC resulted in uptake of both phospholipids and metabolism of DOEPC to neutral lipids. The present findings indicate that DOEPC undergoes slow metabolism and can be eliminated from the body. These properties could prove advantageous for the use of DOEPC as a carrier of drugs and possibly as a carrier of free cholesterol in reverse cholesterol transport.     

Localization of retinal dehydrogenase type 1 in the stomach and intestine

Rats were injected with liposomes containing iodixanol (CTP10 Injection; 100 mg iodine per kg body weight) followed by a second injection of 125I-tyramine-cellobiose-albumin microspheres. The amounts of phagocytosed and degraded labelled albumin in liver were measured. A reduced uptake and degradation of albumin microspheres was observed when the labelled microspheres were injected 2 h or 24 h after the liposomes compared with that obtained in control animals receiving saline. No effect on the uptake and degradation of labelled microspheres was observed when the time lag between the injection of liposomes and labelled microspheres was 1 week. The data show that the uptake and degradation of 125I-tyramine-cellobiose-albumin microspheres can be used as indicators of Kupffer cell phagocytotic function following drug uptake by these cells.     

Immunization against experimental rabbit cysticercosis using liposome-associated antigen preparations

Rabbits were vaccinated once, by subcutaneous and intradermal injection, with sonicates of oncospheres (TpO) or conditioned media from in vitro maintained mature metacestodes (TpMcES) of Taenia pisiformis. Extracts were either incorporated into or mixed with unilamellar liposomes (reverse phase evaporative vesicles) or emulsified in Freund's Incomplete Adjuvant (FIA). Control groups received liposomes or FIA without antigen, or antigen preparation without adjuvant. Rabbits were challenged orally two weeks after vaccination with approximately 1500 eggs of T. pisiformis and necropsied eight weeks after challenge. A mean of 155 cysts was recovered from seven control rabbits. A 67% reduction in peritoneal cyst numbers was obtained in TpO-IFA vaccinated rabbits compared to 75% for the TpO-liposome entrapped group. The highest level of protection (86%) was obtained when TpO was mixed with but not entrapped in liposomes. Only 32% and 39% reduction in peritoneal cyst numbers was obtained after immunizing with the TpMcES preparation in liposomes or IFA respectively, however greater than 85% of peritoneal metacestodes were dead (necrotic or calcified) and suggests a different immune response than occurs after vaccination with oncosphere extracts. Specific anti-oncospheral or anti-metacestode ES antibody (IgG) responses at two weeks post vaccination were similar in rabbits immunized with liposome or IFA associated extracts.     

Suppression of liver uptake of orally fed liposomes by injection (I.P.) of dextran sulfate 500

¹²⁵Iodine labelled human immunoglobulin-G encapsulated liposomes were administered orally to rats. Distribution of radioactivity was checked in various tissues and in portal blood. The effect of dextran sulfate (DS 500,000 m. wt., liver blockade agent) injection (i.p.) on the liver uptake of liposomes and on the amount of liposomes appearing in the portal blood from the gastrointestinal tract have been studied. An increased amount of radioactivity was observed in the portal blood and the amount of radioactivity in the liver decreased appreciably after injection of dextran sulfate. In both the cases the action of dextran sulfate started 2 hours after injection and reached maximum at 12 hour, falling slightly at 24 hour.