Tissue distribution of EDTA encapsulated within liposomes containing glycolipids or brain phospholipids.

Multilameller liposomes were prepared with various asialoglycolipids, gangliosides, sialic acid, or brain phospholipids in the liposome membrane and with ethylenediaminetetraacetic acid (EDTA) encapsulated in the aqueous compartments. The liposomes containing glycolipids or sialic acid were prepared from a mixture of phosphatidylcholine, cholesterol, and one of the following test substances: galactocerebroside, glucocerebroside, galactocerebroside sulfate, mixed gangliosides, monosialoganglioside GM1, monosialoganglioside GM2, monosialoganglioside GM3, disialoganglioside GD1a, or sialic acid. The liposomes containing brain phospholipids were mixtures of either sphingomyelin and cholesterol or a brain total phospholipid extract and cholesterol. Distributions of 14C-labeled EDTA were determined in mouse tissues from 15 min to 6 h or 12 h after a single injection of liposome preparation. Liver uptake up encapsulated EDTA was lowest from all liposome preparations containing sialic acid or sialogangliosides, regardless of the amount of sialic acid moiety present or the identity of the particular ganglioside; highest uptake of encapsulated EDTA by liver was from liposomes containing galactocerebroside or brain phospholipids. Lungs and brain took up the largest amounts of EDTA from liposomes containing sphingomyelin and lesser amounts from liposomes containing GD1a. Use of mouse brain phospholipid extract to prepare liposomes did not increase uptake of encapsulated EDTA by the brain. EDTA in liposomes containing monosialogangliosides, brain phospholipids, galactocerebroside, or sialic acid was taken up well by spleen and marrow. Highest thymus uptake of encapsulated EDTA was from liposomes containing GD1a. These results demonstrate that inclusion of sialogangliosides in liposome membranes decreases uptake of liposomes by liver, thus making direction of encapsulated drugs to other organs more feasible. Liposomes containing glycolipids also have potential uses as probes of cell surface receptors.     

Tissue distribution of EDTA encapsulated within liposomes of varying surface properties

Liposomes containing ethylenediaminetetraacetic acid (EDTA) were prepared with different surface properties by varying the liposomal lipid constituents. Positively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and stearylamine. Negatively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and phosphatidylserine. Neutral liposomes were prepared with phosphatidylcholine alone, dipalmitoyl phosphatidylcholine alone, or with a mixture of phosphatidylcholine and cholesterol. Distributions of ¹⁴C-labeled EDTA were determined in mouse tissues from 5 min to 24 h after a single intravenous injection of liposome preparation. Differences in tissue distribution were produced by the different liposomal lipid compositions. Uptake of EDTA by spleen and marrow was highest from negatively charged liposomes. Uptake of EDTA by lungs was highest from positively charged liposomes; lungs and brain retained relatively high levels of EDTA from these liposomes between 1 and 6 h after injection. Liver uptake of EDTA from positively or negatively charged liposomes was similar; the highest EDTA uptake by liver was from the neutral liposomes composed of a mixture of phosphatidylcholine and cholesterol. Liposomes composed of dipalmitoyl phosphatidylcholine produced the lowest liposomal EDTA uptake observed in liver and marrow but modrate uptake by lungs. Tissue uptake and retention of EDTA from all of the liposome preparations were greater than those of non-encapsulated EDTA. The results presented demonstrate that the tissue distribution of a molecule can be modified by encapsulation of that substance into liposomes of different surface properties. Selective delivery of liposome-encapsulated drugs to specific tissues could be effectively used in chemotherapy and membrane biochemistry.     

Modified in vivo behavior of liposomes containing synthetic glycolipids

Liposomes with synthetic saccharide determinants were prepared from synthetic cholesterol conjugates of D-mannose and 6-amino-6-deoxy-D-mannose and labeled with [⁵¹Cr]chromate. The kinetics and tissue distribution of label in mice were determined after footpad and subcutaneous injection. Liposomes bearing either of these saccharide determinants greatly increased retention of label at the injection sites compared to control liposomes, which contain no glycolipid, and to free [⁵¹Cr]chromate. Draining lymph nodes contained small fractions of the injected radioactivity but in some cases this retention was saccharide-dependent and highly concentrated. These results show that incorporation of synthetic glycolipids can substantially alter the in vivo lifetime and distribution of liposomes outside the bloodstream. Such surface-modified liposomes may be useful for sustained release or selective delivery of therapeutic or diagnostic agents.     

Antineoplastic activity of N-maleamide homocysteine thiolactone amide encapsulated within liposomes

The antineoplastic activity of N-maleamide homocysteine thiolactone amide (MHTA) encapsulated within liposomes was studied in mice with transplanted tumors. Tumor weight was decreased by 4-5 biweekly intraperitoneal injections of MHTA in liposomes in DBA/2N females with MTG mammary adenocarcinoma (35% of control value, P less than 0.005) and in C57B1/6N males with MUO4 rhabdomyosarcoma (11% of control value, P less than 0.0000001). Tumor incidence was reduced from 84 to 63% (P less than 0.05) and from 100 to 32% (P less than 0.001) in the two systems, respectively. When the compound was administered in dimethyl sulfoxide to A/HeJ females with A10 mammary adenocarcinoma by daily intraperitoneal injection, tumor weight was reduced to 70% of control value (P less than 0.05), and there was no decrease in tumor incidence (100%). No toxicity was observed at the therapeutic dose utilized, 10 mg/kg/day. N-Maleamide homocysteine thiolactone amide is a derivative of the normal biochemical constituents, maleic acid and homocysteine thiolactone. The results show that the N-substituted maleamide derivative of homocysteine thiolactone decreases the growth of murine tumors of two different histological types, when administered encapsulated within liposomes.     

Tissue distribution of 99mic-labeled liposomes prepared from synthetic amphiphiles containing amino acid residues

Abstract

The tissue distribution of ^99mTc-labeled liposomes prepared from synthetic amphiphiles containing amino acid residues was investigated for application to radiopharmaceuticals. The amphiphiles used were N,N-didodecyl-N α-[6-(trimethylammoniohexanoyl]-L-ala-ninamide bromide (N+C₅Ala2C₁₂), N,N-didodecyl-N_α-{6-[dimethyl(2-carboxyethyl)ammonio]hexanoyl}-L-alaninamide bromide (CAC₂N⁺C₅Ala2C₁₂) and S-{l-carboxy-2-([2,3-bis (he xadecyloxy)propoxy]carbony1)ethyl}homocy ste ine. These liposomes were stable in saline and 50% serum at 37° for at least 24h in comparison with the liposomes prepared from phosphatidylcholine and cholesterol (1:1). Most of the radioactivity of N+C₅Ala2C₁₂ and CAC₂N+C₅Ala2C₁₂ liposomes was firmly bound to Ehrlich ascites tumor cells in vitro. But the accumulation of three liposomes into the tumor of Ehrlich solid tumor-bearing mice after intravenous injection was low and most of the liposomes was taken up highly in liver and spleen which belong to the reticuloendothelial system (RES). Some approaches were made to reduce the RES uptake of N+C5Ala2C12 liposomes as follows: (1) the pretreatment of dextran sulfate depressed the uptake of the liposomes in the liver accompanied by increasing uptake in tumor and other tissues except stomach, (2) the modification of the liposomes with n-dodecyl glucoside or n-dodecyl sucrose depressed the uptake in liver and spleen, resulting in an increase in blood and other tissues such as tumor, duodenum and kidney, (3) the modification of the liposomes with ganglioside GM3 or GM1 reduced the uptake in liver and spleen, but increased scarcely the uptake in blood and tumor because of the rapid excretion into urine, (4) the intraperitoneal injection reduced the uptake of the liposomes in liver and increased significantly the accumulation in pancreas.     

Effects of calcium-induced aggregation on the physical stability of liposomes containing plant glycolipids

Membranes containing either negatively charged lipids or glycolipids can be aggregated by millimolar concentrations of Ca(2+). In the case of membranes made from the negatively charged phospholipid phosphatidylserine, aggregation leads to vesicle fusion and leakage. However, some glycolipid-containing biological membranes such as plant chloroplast thylakoid membranes naturally occur in an aggregated state. In the present contribution, the effect of Ca(2+)-induced aggregation on membrane stability during freezing and in highly concentrated salt solutions (NaCl+/-CaCl(2)) has been determined in membranes containing different fractions of uncharged galactolipids, or a negatively charged sulfoglucolipid, or the negatively charged phospholipid phosphatidylglycerol (PG), in membranes made from the uncharged phospholipid phosphatidylcholine (PC). In the case of the glycolipids, aggregation did not lead to fusion or leakage even under stress conditions, while it did lead to fusion and leakage in PG-containing liposomes. Liposomes made from a mixture of glycolipids and PG that approximates the lipid composition of thylakoids were very unstable, both during freezing and at high solute concentrations and leakage and fusion were increased in the presence of Ca(2+). Collectively, the data indicate that the effects of Ca(2+)-induced aggregation of liposomes on membrane stability depend critically on the type of lipid involved in aggregation. While liposomes aggregated through glycolipids are highly stable, those aggregated through negatively charged lipids are severely destabilized.     

Incorporation of mitochondrial membrane proteins into liposomes containing acidic phospholipids.

Cytochrome oxidase, QH2-cytochrome c reductase, and the oligomycin-sensitive adenosine triphosphatase were incorporated into liposomes by a new procedure which yielded unidirectional orientation of the proteins. Cytochrome oxidase was reconstituted in the mitochrondrial orientation and the adenosine triphosphatase in the submitochondrial orientation. Reconstitutions were achieved by incubating the proteins at room temperature with liposomes which contained phosphatidylcholine, phosphatidylethanolamine, and an acidic phospholipid (cardiolipin, phosphatidylinositol, or phosphatidylserine). The incorporation occurred without added detergent or sonication. This incorporation procedure may serve as a model for the insertion of proteins in vivo.     

Tissue distribution and cellular distribution of liposomes encapsulating muramyltripeptide phosphatidyl ethanolamide

In previous studies it was shown that administration of liposome-encapsulated MTPPE (LE-MTPPE) led to resistance againstKlebsiella pneumoniae infection. To get more insight in the cell types that are involved in this by LE-MTPPE induced antibacterial resistance, the tissue distribution of liposomes encapsulating MTPPE and the distribution over the cells in the main target organs were investigated. After intravenous injection of the liposomes in mice a substantial amount was recovered from liver and spleen and a smaller amount from the lung. In the liver 83% of the liposomes was taken up by the macrophages. In the spleen also most liposomes were taken up by macrophages of the red and white pulp as well as by dendrocytes. The liver and spleen were also the organs in which, after intravenous inoculation,K. pneumoniae was trapped. It was observed that cells containing LE-MTPPE often had not taken up bacteria. Most bacteria, about 73%, were found in cells not containing liposomes. The capacity of the liposome-containing cells to take up bacteria did not change with time. This suggests that the by LE-MTPPE immunostimulating effect is due to the production of cytokines by the cells that take up LE-MTPPE. These cytokines might stimulate other cells to the killing of bacteria.     

The dispersion of cholesterol with phospholipids and glycolipids

Of the polar lipids studied (phospholipids and glycolipids), only phosphatidylcholine and sphingomyelin can disperse in water with up to 2 mol cholesterol/mol polar lipid. However, mixtures of phosphatidylethanolamine with small amounts of phosphatidylcholine and mixed lipids from mitochondria and myelin will also form sterol-rich dispersions. Steroids in which the 3β-OH group is replaced by an oxo function do not form such steroid-rich dispersions. Electron microscopy and optical rotatory dispersion (ORD) show that sterols disperse with cerebrosides and gangliosides to form cylindrical structures with the regions around C atoms 3 and 7 of the sterol in less polar environments than those they occupy in phospholipid liposomes.

It is proposed that choline-containing phospholipids facilitate entry of sterol molecules into the outer leaflet of cell surface membranes but that the phospholipid composition itself will not give rise to an asymmetric distribution of sterol in membranes with a high cholesterol content.     

Molecular volumes of phospholipids and glycolipids in membranes

Data on the molecular volumes of phospholipids and glycolipids in membranes are collected together in order to determine the contributions from the component groups, for as wide a range of lipids as possible, including sphingolipids. Wherever possible, the volumes of the methylene groups in the lipid chains are established from the dependence on chain length at fixed temperature in a given phase. In this way, it is also possible to determine the constant contribution from cis double bonds in the chains of monoenoic unsaturated phosphatidylcholines, and the volume of the branched methyl groups in isoacyl phosphatidylcholines. Issues concerning separation of contributions from the polar head groups from those of the chain terminal methyl groups are discussed. Molecular volumes of lipids in crystals are also analysed to provide information on head-group packing that can be compared with the situation in membranes, and used to set limits on the relative contributions from polar groups and terminal methyl groups. Comparisons are made with volumetric analyses based on diffraction studies of bilayers of single lipids. The parameters derived can be used to estimate molecular volumes of lipids for which dilatometric or densitometric data are lacking. Lipid volumes are determining parameters for lipid dynamics, membrane partitioning and permeation of solutes, and are essential quantities for the structural analysis of lipid membranes.     

Oral application of insulin encapsulated liposomes

125Iodine labelled insulin encapsulated liposomes were introduced orally to rats. After different intervals of feeding, blood from the portal vein and heart was collected. The plasma was passed through a Sepharose 2B column to establish the presence of liposomes or intact hormone. Liposomal and hormonal fractions from the column loaded with plasma from the portal vain contained radioactivity. It was found that approx. 60% of the loaded radioactivity was associated with the liposomes whereas 20% of the loaded count was found with the hormonal fractions. It is noted that undegraded protein is not detected in portal plasma from rats fed with free insulin. When plasma from the heart of rats fed with liposomal insulin was passed through Sepharose 2B column, neither liposomes nor insulin was detected.     

Oxidation of glycolipids in liposomes by galactose oxidase

Small unilamellar phosphatidylcholine vesicles containing globo-series glycolipids were labeled by the galactose oxidase/NaB[3H]4 procedure. The major glycolipid of human red cells, globoside, was the best substrate for galactose oxidase both in vesicles and in tetrahydrofuran-containing buffer. The oxidation rates of membrane-bound ceramide trihexoside and Forssman glycolipid were one-fourth and one-tenth, respectively, of the oxidation rate of globoside. Membrane-bound ceramide dihexoside was not a substrate for galactose oxidase, although it was readily oxidized in tetrahydrofuran-containing buffer. Soluble sialoglycoproteins and membrane-incorporated glycophorin A stimulated the oxidation of globoside-containing vesicles, whereas membrane-bound GD1a ganglioside had no effect on globoside oxidation.     

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.     

Interactions of plant lectins with glycolipids in liposomes

A panel of five plant lectins with different binding specificities was used to determine if plant lectins could bind specifically to membrane-associated glycolipids. $\text{Ricinis communis}$ and wheat germ agglutinins both bound specifically to mixed brain gangliosides and globoside I from human erythrocytes. Wheat germ agglutinin also bound to ganglioside G_M1 and human erythrocyte ceramide trihexoside, but not to ceramide dihexoside, mono-, or digalactosyl diglycerides. Concanavalin A bound to liposomes with or without glycolipid substituents, and this binding was partially inhibited by α-methyl mannoside. This study indicates that lectins can specifically recognize and bind to certain glycolipids in membranes.     

Transverse and lateral distribution of phospholipids and glycolipids in the membrane of the bacterium Micrococcus luteus

The photodimerization of anthracene was used to investigate the transverse and lateral distribution of lipids in the membrane of the Gram-positive bacterium Micrococcus luteus. 9-(2-Anthryl)nonanoic acid (9-AN) is incorporated at a high rate into various membrane lipids of M. luteus. On irradiation of intact bacteria at 360 nm, anthracene-labeled lipids form stable photodimers which can be extracted and separated by thin-layer chromatography. We present here the results of a study on the distribution of two major lipids, phosphatidylglycerol (PG) and dimannosyldiacylglycerol (DMDG), within each leaflet of the membrane lipid bilayer. After metabolic incorporation of a tritiated derivative of 9-AN in M. luteus, the radioactivity associated with the photodimers issued from PG and DMDG was counted. In the bacterial membrane, the ratio of PG-DMDG heterodimer with respect to PG-PG and DMDG-DMDG homodimers is around half of what should be obtained for a homogeneous mixture of the two lipids. In order to find out whether this was due to an asymmetric distribution of the two lipids between the two membrane leaflets or a heterogeneous distribution of the two lipids within the same membrane leaflet, the transverse distribution of PG and DMDG was also investigated. This was carried out by following the kinetics of oxidation of the two lipids by periodic acid in the membrane of M. luteus protoplasts. PG predominated slightly in the outer layer (60%), while DMDG was found to be symmetrically distributed between the two leaflets. By itself, this lipid asymmetry cannot account for the lipid distribution determined from the photodimerization experiments. This indicates that PG and DMDG are not homogeneously distributed in the plane of the bacterial membrane.     

Induction of immunity against experimental tuberculosis with mycobacterial mannophosphoinositides encapsulated in liposomes containing lipid A

Abstract Mycobacterial mannophosphoinositides (PIMs) encapsulated in liposomes made of egg phosphatidylcholine (EPC) and cholesterol (CH) (2:1.5 molar ratio) were able to induce humoral and delayed type hypersensitivity (DTH, foot-pad swelling reaction) responses in mice without the help of any carrier protein. Animals immunized with this glycophospholipid antigen demonstrated enhanced percent survival on intravenous challenge with virulent M. tuberculosis . On fractionation of PIMs, pentamannophosphoinositide (PIM₅) was found to induce higher antibody and DTH reaction and proved to be more immunoprotective than other fractions. Inclusion of lipid A as immunomodulator in liposomes containing PIMs or PIM₅ resulted in a significantly increased immune response. Further, mice immunized with PIMs or PIM₅ in lipid A-containing liposomes exhibited decreased mortality on challenge with M. tuberculosis H₃₇Rv, which was comparable to BCG vaccinated animals.