Mannosylated liposomes bearing Amphotericin B for effective management of visceral Leishmaniasis

The cationic and mannosylated liposomes were prepared using the cast film method and compared for their antileishmaniasis activity. The surface of the Amphotericin B (Amp B)-bearing cationic multilamellar liposomes was covalently coupled with p-aminophenyl-α-D-mannoside using glutaraldehyde as a coupling agent, which was confirmed by agglutination of the vesicles with concanavalin A. The prepared liposomes were characterized for shape, size, percent drug entrapment, vesicle count, zeta potential, and in vitro drug release. Vesicle sizes of cationic and mannosylated liposomes were found to be 2.32 ± 0.23 and 2.69 ± 0.13 μm, respectively. Zeta potential of cationic liposomes was higher (30.38 ± 0.3 mV), as compared to mannosylated liposomes (17.7 ± 0.8 mV). Percentage drug release from cationic and mannose-coupled liposomes was found to be 45.7% ± 3.1 and 41.9% ± 2.8, respectively, after 24 hours. The in vivo antileishmanial activity was performed on Leishmania donovani-infected golden hamster, and results revealed that Amp B solution was reduced by 42.5 ± 1.8% in the parasite load, whereas the placebo cationic liposomes and drug-containing cationic liposomes showed a reduced parasite load (i.e., 28.1 ± 1.5 and 61.2 ± 3.2%, respectively). The mannose-coupled liposomes showed a maximum reduction in parasite load (i.e., 78.8 ± 3.9%). The biodistribution study clearly showed the higher uptake of mannosylated liposomes in the liver and spleen and hence the active targeting to the reticular endothelial system, which, in turn, would provide a direct attack of the drug to the site where the pathogen resides, rendering the other organs free and safe from the toxic manifestations of the drug.     

Mannosylated liposomes bearing Amphotericin B for effective management of visceral Leishmaniasis

The cationic and mannosylated liposomes were prepared using the cast film method and compared for their antileishmaniasis activity. The surface of the Amphotericin B (Amp B)-bearing cationic multilamellar liposomes was covalently coupled with p-aminophenyl-α-D-mannoside using glutaraldehyde as a coupling agent, which was confirmed by agglutination of the vesicles with concanavalin A. The prepared liposomes were characterized for shape, size, percent drug entrapment, vesicle count, zeta potential, and in vitro drug release. Vesicle sizes of cationic and mannosylated liposomes were found to be 2.32?±?0.23 and 2.69?±?0.13?µm, respectively. Zeta potential of cationic liposomes was higher (30.38?±?0.3 mV), as compared to mannosylated liposomes (17.7?±?0.8 mV). Percentage drug release from cationic and mannose-coupled liposomes was found to be 45.7%?±?3.1 and 41.9%?±?2.8, respectively, after 24 hours. The in vivo antileishmanial activity was performed on Leishmania donovani–infected golden hamster, and results revealed that Amp B solution was reduced by 42.5?±?1.8% in the parasite load, whereas the placebo cationic liposomes and drug-containing cationic liposomes showed a reduced parasite load (i.e., 28.1?±?1.5 and 61.2?±?3.2%, respectively). The mannose-coupled liposomes showed a maximum reduction in parasite load (i.e., 78.8?±?3.9%). The biodistribution study clearly showed the higher uptake of mannosylated liposomes in the liver and spleen and hence the active targeting to the reticular endothelial system, which, in turn, would provide a direct attack of the drug to the site where the pathogen resides, rendering the other organs free and safe from the toxic manifestations of the drug.     

Role of Surface Glycolipids - Natural or Synthetic Origin on the Biodistribution of Liposomes

Abstract

Various sugar residues were incorporated on the surface of liposomes and its effect on the biodistribution and therapeutic importance were discussed here. Galactosylated liposomes were preferentially taken up by liver parenchymal cells whereas mannosylated liposomes were mainly localized in non-parenchyma1 cells. On the other hand, incorporation of dextran on the surface results in increased circulatory half-life of liposomes. The potential application of such liposomes as a carrier of drugs in the diseased condition is also discussed.     

Plasma membrane-mediated leakage of liposomes induced by interaction with murine thymocytic leukemia cells

The interaction of liposomes with BW 5147 murine thymocytic leukemia cells was studied using fluorescent probes (entrapped carboxyfluorescein and fluorescent phosphatidylethanolamine) in conjunction with a Ficoll-Paque discontinous gradient system for rapid separation of liposomes from cells. Reversible liposomal binding to discrete sites on the BW cell surface was found to represent the major form of interaction; uptake of intact liposomal contents by a process such as liposome-BW cell membrane fusion was found to apparently represent a minor pathway of interaction (2%). Liposomal lysis was found to be associated with the process of liposomal binding (perhaps as a result of the binding itself). Lysis was followed by release of the entrapped carboxyfluorescein into the media and its subsequent uptake by the cells. This lysis was shown to be dependent upon discrete membrane-associated sites that have some of the properties of proteins. The results of these studies suggest that liposomal binding to the cells, subsequent lysis of the liposomes and cellular uptake of their contents should be seriously considered in all studies of liposome-cell interactions as an alternate mode of interaction to the four modes (fusion, endocytosis, adsorption and lipid exchange) previously emphasized in the literature.     

Grafting of different glycosides on the surface of liposomes and its effect on the tissue distribution of 125I-labelled γ-globulin encapsulated in liposomes

Different glycosides were grafted on the surface of liposomes containing ¹²⁵I-labelled γ-globulin by two ways: (1) by using glycolipid and (2) by covalent coupling of $\text{p-}\text{aminophenyl-}\text{d}\text{-glycosides}$ to phosphatidylethanolamine liposomes using glutaraldehyde. The distribution of ¹²⁵I-labelled γ-globulin was determined in mouse tissues from 5–60 min after a single injection of these liposomes. The liver uptake of encapsulated ¹²⁵I-labelled γ-globulin was highest from liposomes having galactose and mannose on the surface. Competition experiments and cross-inhibition studies indicate that this uptake are mediated by specific recognition of the surface galactose and mannose residues of liposomes by the receptors present on the plasma membrane of liver cells. Stearylamine-containing liposomes were found to be more efficient in mediating the uptake of ¹²⁵I-labelled γ-globulin by the lung, whereas in the case of spleen, phosphatidylethanolamine liposomes were more efficient. The extent of uptake of ¹²⁵I-labelled γ-globulin from all types of liposome decreases as the amount of given liposomes increases. The uptake of ¹²⁵I-labelled γ-globulin from liposomes containing asialogangliosides depends upon the phospholipid/ glycolipid ratio. These experiments clearly demonstrate that enhanced liposome uptake by liver cells could be achieved by grafting galactose and mannose on the liposomal surface.     

Increased circulatory half-life of liposomes after conjunction with dextran

Dextran was covalently coupled to neutral unilamellar liposomes. Dextran conjugated liposomes were cleared from the circulation at a much slower rate than unconjugated liposomes. The uptake of dextran conjugated liposomes by liver and spleen was also decreased. The amount of dextran on the surface of liposomes was found to be a determining factor for their stability in circulation. Dextran conjugated liposomes therefore may be a more effective way of controlled drug release     

Preparation and characterisation of liposomes containing mannosylated phospholipids capable of targetting drugs to macrophages

We have prepared liposomes from mannosylated phosphatidylmyo-inositol, derived from mycobacteria, and cholesterol. The size of the particles so formed could be controlled by membrane filtration. The vesicles encapsulated a significant amount of aqueous phase (about 8 microliter per mg phospholipid). Markers of the liposomal membrane and aqueous phase rapidly associated with mouse peritoneal macrophages and, more slowly, with rat alveolar macrophages. The uptake was saturable at high liposome concentrations, although phagocytosis of latex particles of the same mean diameter was not saturable at these concentrations. An excess of unlabelled liposomes composed of phosphatidylcholine and phosphatidylserine, which were also taken up readily by macrophages, did not inhibit the uptake of mannosylated liposomes. The uptake of fluorescent mannosylated bovine serum albumin was inhibited by these liposomes, suggesting a specific interaction with the macrophage mannose-fucose receptor. We conclude that this type of liposome would be useful for the delivery of immunomodulators to reticuloendothelial cells.     

Covalent attachment of horseradish peroxidase to the outer surface of liposomes

We describe a method by which horseradish peroxidase may be attached covalently to the surface of liposomes under conditions which permit minimal non-covalent association of the enzyme with the lipids. The coupling method adopted does not allow the formation of homopolymers of liposomes or peroxidase. For phosphatidylethanolamine/phosphatidylcholine and stearylamine/phosphatidylcholine vesicles, minimal disruption of vesicular structure is observed, whilst for phosphatidylserine vesicles, the lipid-protein complex appears to form structures much smaller than 25 nm in diameter. Stearylamine/phosphatidylcholine vesicles have been shown to retain entrapped inulin, and activity measurements for the peroxidase suggest that it is located exclusively on the external surface of the liposome membrane. Peroxidase can be localized histochemically which has permitted the morphological study of the coated liposomes and their interactions with cells.     

Distribution of solid liposomes on the surface of an epithelial cell layer

A study was made of the adhesion of liposomes, composed of dipalmitoyl- or di-stearoylphosphatidycholine, on the surface of epithelial cells in culture. Sodium fluorescein was entrapped in liposomes for their visualization by fluorescence microscopy. It is found that sonicated unilamellar liposomes adhere predominantly along the sheet margins. Multilamellar liposomes and lipid-coated carmine particles adhere over the whole cellular surface. However, their adhesion along sheet margins was stronger, as evidenced by a brief trypsin treatment. A prolonged trypsin treatment removed all types of liposomes from the cell surface. After the cells were partly detached from each other, small liposomes readily adhered to the newly accessible cell margins. The existence of special lipid membrane-binding proteins on the cell surface is suggested.     

Effect of glycolipids contained in liposomes on the incorporation of beta-galactosidase into specific organs

A series of glycolipids were examined to find a system capable of targeting liposomes into specific organs of rats. Sulfatide was found to be the best among the components of liposomes examined for delivering the entrapped enzyme, beta-galactosidase from Aspergillus oryzae, into the brain and liver; gangliosides, for the spleen; and sphingomyelin, for the lung. To introduce the enzyme into the liver, galactocerebroside was far better than glucocerebroside. These data suggest that the sugar residues of glycolipids function to target the liposomes into specific organs.     

Surface modified liposomes by mannosylated conjugates anchored via the adamantyl moiety in the lipid bilayer

The aim of the present study was to encapsulate mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides, namely [(2R)-N-(adamant-1-yl)-3-(α,β-d-mannopyranosyloxy)-2-methylpropanamide and (2R)-N-[3-(α-d-mannopyranosyloxy)-2-methylpropanoyl]-d,l-(adamant-2-yl)glycyl-l-alanyl-d-isoglutamine] in liposomes. The characterization of liposomes, size and surface morphology was performed using dynamic light scattering (DLS) and atomic force microscopy (AFM). The results have revealed that the encapsulation of examined compounds changes the size and surface of liposomes. After the concanavalin A (ConA) was added to the liposome preparation, increase in liposome size and their aggregation has been observed. The enlargement of liposomes was ascribed to the specific binding of the ConA to the mannose present on the surface of the prepared liposomes. Thus, it has been shown that the adamantyl moiety from mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides can be used as an anchor in the lipid bilayer for carbohydrate moiety exposed on the liposome surface.     

Targeting of plant glycoside-bearing liposomes to specific cellular and subcellular sites

The possibility of using liposomes as an effective drug delivery system has been studied by incorporation of two plant glycosides of varying terminal sugar residues onto the surface of liposomes and examination of their distribution in different tissues. The two glycosides, corchorusin D and asiaticoside having glucose and rhamnose respectively at the terminal ends wee selected for the purpose. The hepatic uptake of liposomes made from egg lecithin, cholesterol and dicetyl phosphate and either of the two glycosides was compared. The hepatic uptake of asiaticoside bearing liposomes was reduced, whereas that of corchorusin D bearing liposomes was enhanced and was specific for glucose. Liver perfusion followed by cell separation showed that the uptake is mostly into the non-parenchymal cells of liver. The distribution of corchorusin D bearing liposomes was maximal in the lysosomal fraction of the non-parenchymal cells. Ways of using corchorusin D bearing liposomes as delivery systems for drugs or enzymes to lysosomes have been sought.     

Enzyme therapy VI: Comparative in vivo fates and effects on lysosomal integrity of enzyme entrapped in negatively and positively charged liposomes

Entrapment of enzyme in liposomes, biodegradable lipid vesicles, offers an intriguing strategy for the intracellular delivery of these macromolecules to the lysosomal apparatus for enzyme replacement endeavors in selected lysosomal storage diseases. Therefore, the in vivo tissue and subcellular fate and effect on the subcellular distribution of endogenous lysosomal hydrolases was determined following intravenous administration of β-glucuronidase entrapped in positively and negatively charged liposomes into C3H/HeJ β-glucuronidase-deficient mice. Enzyme entrapped in negatively charged liposomes was rapidly cleared from the circulation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 4}\text{min}$); maximal tissue recovery, 75% of dose, was detected in the liver at 1 h, was maintained for 48 h and then gradually declined to non-detectable levels by 8 days. A similar circulatory clearance and reciprocal hepatic uptake was observed for positively charged liposomes; however, the β-glucuronidase was retained in murine liver for 11 days. Significant activity, 15% of dose, was found in the kidneys up to 1 and 4 days post-injection of positively and negatively charged liposomes, respectively. No activity was recovered in neural or other visceral tissues except in spleen and lungs (?5% of dose). Exogenous β-glucuronidase activity administered in negatively charged liposomes was primarily localized in the lysosomally-enriched hepatic subcellular fraction, compared to the predominantly soluble localization of exogenous activity entrapped in positively charged liposomes. Administration of negatively charged liposomes caused no detectable change in the subcellular localization of several endogenous lysosomal hydrolase activities compared to their distribution in untreated mice. In contrast, a marked but temporary translocation of these hydrolase activities into the soluble fraction was observed following the administration of positively charged liposomes, identifying possible deleterious effects on cellular physiology.     

Liposomes in immunology: Evidence that their adjuvant effect results from surface exposition of the antigens

The immune responses against human serum albumin (HSA) and bovine gamma globulin (BGG) were studied in rabbits after intravenous injections of various preparations of these antigens. Antigens were injected free in saline, coated on “empty” liposomes or both coated on liposomes, and entrapped in their inner compartments. The earlier established adjuvant effect of the liposomes was confirmed for both antigens. Although the amount of antigen entrapped in the liposomes was much higher than the amount coated on their outer surfaces, liposomes containing the antigen both in their inner compartments and on their outer surface showed no stronger adjuvant effect than “empty” liposomes coated with the antigen only. The results support the hypothesis that the adjuvant effect of liposomes is mediated by antigens exposed on the outer surfaces of the liposomes. Suggestions are made for the use of liposomes as a practical immunoadjuvant with definite advantages over many other adjuvants.     

Biologically active, recombinant DNA in clathrin-coated vesicles isolated from rat livers after in vivo injection of liposome-encapsulated DNA

DNA entrapped in liposomes containing lactosylceramide in the bilayers is found to be associated with clathrin-coated vesicles isolated from the rat livers after intravenous injection of these liposomes. The presence of the exogenous DNA in the coated vesicles was detected by Southern blotting. The amount of DNA present in the coated vesicles does not appear to vary up to 4 h after injection of the liposomes into the animals. The recognition of the lactosyl group present in the liposome by the galactose receptor present on the surface of the different liver cells may lead to their internalization in a way analogous to receptor-mediated endocytosis of various macromolecules. DNA present in the lumen of the coated vesicles is found to be biologically active as evidenced by its replication in bacterial cells and mouse fibroblasts.     

Mannose receptor dependent uptake of a ricin A chain--antibody conjugate by rat liver non-parenchymal cells

Mannose receptor mediated uptake by the reticuloendothelial system has been suggested as an explanation for the rapid removal of ricin A chain antibody conjugates from the circulation after their administration. We have measured, in the rat, hepatic uptake of a ricin A chain antibody conjugate in vivo and its susceptibility to inhibition by a mannosylated protein and have measured uptake of the conjugate in vitro by rat parenchymal and non-parenchymal liver cells. The results indicate that rapid hepatic uptake of conjugate does occur in vivo; cultured non-parenchymal cells accumulate the conjugate to a much greater degree than cultured parenchymal cells and that mannose receptors appear to be involved in the process.     

Receptor-mediated uptake of a mannose-6-phosphate bearing glycoprotein by isolated chicken osteoclasts

We have recently shown that degradation of bone collagen by osteoclasts occurs via proteolytic enzyme activity that depends on an acidic milieu. Since bone resorption occurs in an extracellular, acidic compartment located at the cell-matrix attachment site, the osteoclast must deliver the acid collagenolytic enzymes to the cell surface. These observations raise the possibility that the mannose-6-phosphate (M-6-P) receptor, known to sort acidic proteases in other cells, is involved in trafficking lysosomal enzymes to the plasmalemma of bone resorbing cells. To this end we studied receptor-mediated uptake, distribution and release, by isolated chicken osteoclasts, of 125I-hexosaminidase, a M-6-P bearing enzyme. We found that at 4 degrees C, the bone-resorbing polykaryons bind approximately 10,000 molecules of radioligand/cell with a Kd of 0.7 nM, which is endocytosed by osteoclasts at 37 degrees C by a calcium-independent process. Furthermore, 125I-hexosaminidase uptake is unaffected by mannosylated albumin, documenting specificity of the receptor-mediated event. Release of endocytosed enzyme from the cell is also much more rapid than its degradation, attesting to a pathway of uptake and secretion. By autoradiography, the M-6-P bearing ligand is concentrated at the site of osteoclast-bone attachment. Thus, osteoclasts also have the capacity to deliver M-6-P bearing degradative enzymes to their surface at the site of matrix degradation.     

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.     

Increased affinity of liposomes derived from total spleen and liver lipids to spleen cells

The interaction of liposomes derived from total lipids of mouse spleen and liver with mouse spleen cells was studied. It was shown that the binding of these liposomes is much higher than the binding of liposomes obtained from a model lipid mixture--phosphatidylcholine--phosphatidylethanolamine--cholesterol (2:1:1). Adherent and nonadherent spleen cells were found to have affinity for liposomes derived from total lipids of spleen or liver. Removal of gangliosides and protein contaminants from the liposomes derived from total spleen lipids caused an increased binding of liposomes to spleen cells. Multilamellar liposomes bound more effectively to ultrasonicated vesicles having a homologous lipid composition than the liposomes with a different lipid composition. The increased affinity of liposomes derived from total lipids of spleen or liver for spleen cells may account for the identical fluidity of the lipid bilayer of liposomes and plasma membranes of spleen cells.     

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.