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.     

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.     

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

Multilamellar 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 G_M1, monosialoganglioside G_M2, monosialoganglioside G_M3, disialoganglioside G_D1a, or sialic acid. The liposomes containing brain phospholipids were mixtures of either sphingomyelin and cholesterol or a brain total phospholipid extract and cholesterol. Distribution of ¹⁴C-labeled EDTA were determined in mouse tissues from 15 min to 6 h or 12 h after a single injection of liposome prepartion. Liver uptake of 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 the 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 G_D1a. 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 G_D1a. 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.     

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.     

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.     

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.     

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.     

Expression of hepatitis B virus surface antigen in adult rat liver. Co-introduction of DNA and nuclear protein by a simplified liposome method.

We established a simple and efficient method for gene transfer in vitro (to cultured cells) and in vivo (to an adult organ) using liposomes. Plasmid DNA and proteins were efficiently co-encapsulated in liposomes by agitation and sonication, and were co-introduced into cells by hemagglutinating virus of Japan (HVJ)-mediated membrane fusion. Introduction of the Escherichia coli beta-galactosidase gene with non-histone chromosomal protein high mobility group 1 (HMG1) into LLCMK2 cells resulted in about 3 times higher beta-galactosidase activity than that on introduction of the gene alone. Two days after injection of HVJ-liposomes containing the beta-galactosidase gene and HMG1 under the perisplanchnic membrane of adult rat liver, hepatic cells near the injection site were found by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining to have beta-galactosidase activity. After similar injection of HVJ-liposomes containing the hepatitis B virus surface antigen (HBsAg) gene and HMG1, HBsAg was detected in the serum for 9 days with a maximum of 25-45 ng/ml on day 2 after the injection.     

Purification and characterization of the L-fucose transporter

L-Fucose is a monosaccharide present in low levels in the serum. It is, however, a common structural component of glycoproteins. L-Fucose is accumulated in eukaryotic cells by a specific, facilitative diffusion transport system which has been designated the fucose transporter. In this study, purification of the transporter from mouse brain was performed by detergent extraction followed by ion-exchange and reactive dye ligand column chromatography. Purification was followed using a transport assay into reconstituted liposomes. A 111-fold purification with 5% yield was achieved from the crude homogenate. The apparent molecular weight of the protein was 57 kDa. Transport was found to be saturable. The K(m) and V(max) values are estimated at 3 microM and 275 pmol/min/mg, respectively. The tissue distribution of fucose transport was examined in liver, kidney, heart, lung, spleen, brain, muscle, adipose, ovary, pancreas, and thymus. Some fucose transport was found in all tissues examined. Very low levels were observed in the liver relative to all other tissues examined. The only monosaccharide which could inhibit the uptake of L-[5,6-(3)H]fucose was fucose itself.     

Effect of long-term Intralipid administration in mice

Intralipid was administered intravenously to mice at a level of 2 g kg-1 day-1 for 23 days. No alterations in phagocytic index, liver or spleen size were observed in the chronically injected mice as compared with control mice that received saline injections. Tissue distribution of 0.45 micron multilamellar liposomes of egg phosphatidylcholine:cholesterol (2:1) was similar in mice that had been chronically injected with Intralipid to that in control mice. Mice chronically given the same total amount of phospholipid in the form of 0.2 micron liposomes of phosphatidylcholine:cholesterol (2:1) rather than as a lipid-triglyceride emulsion showed altered tissue distribution of entrapped label with decreased liver uptake and increased splenic uptake, which is indicative of reticuloendothelial blockade. Tissue distribution of [14C]dipalmitoylphosphatidylcholine Intralipid was compared with that of [14C]dipalmitoylphosphatidylcholine 0.2 micron MLV of phosphatidylcholine:cholesterol (2:1). Intralipid was taken up 2- to 3-fold less by liver and 5- to 10-fold less by spleen than liposomes. Blood levels of Intralipid were higher than those of liposomes. [14C]dipalmitoylphosphatidylcholine Intralipid was eliminated from the body at a faster rate than [14C]dipalmitoylphosphatidylcholine liposomes. The lack of reticuloendothelial blockade caused by Intralipid as compared with liposomes appears to be related to its diminished uptake into reticuloendothelial tissues. This diminished uptake may be related to differences in apolipoprotein uptake of Intralipid, which is primarily in the form of a phospholipid monolayer, and liposomes, which have their phospholipid organized into a bilayer.     

Short-term handling of the slimming agent oleoyl-estrone in liposomes (Merlin-2) by the rat

Female adult rats were injected in the jugular vein with oleoyl-³H-estrone incorporated into liposomes. The label rapidly disappeared from the blood, being taken up by the tissues, mainly liver, spleen and lung, which filtered most of the label. However, many other tissues, such as the heart, brown adipose tissue, adrenals and visceral fat incorporated significant amounts of oleoyl-estrone. The analysis of the form in which the label remained 10 min after the injection showed that it was hydrolysed in a large proportion even in liver and lungs. However, in most tissues (brain, brown and white - periovaric - adipose tissues and ovaries), intact oleoyl-estrone accounted for less than one quarter of all tissue label, and less than 10% in the case of subcutaneous adipose tissue and uterus. This rapid destruction of oleoyl-estrone is in agreement with the active role of this compound in the control of body weight.     

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.     

Effects of alkyl glycosides incorporated into liposomes prepared from synthetic amphiphiles on their tissue distribution in Ehrlich solid tumor-bearing mice.

A study of the effects of alkyl glycosides incorporated into synthetic liposomes with respect to their stability, their in vivo distribution in Ehrlich solid tumor-bearing mice and their in vitro interaction with liver cells was undertaken. The synthetic liposomes were prepared from N,N-didodecyl-N alpha-[6-(trimethylammonio)hexanoyl]-L-alaninamide bromide (N+C5Ala2C12) and labeled with 99mTc. n-Dodecyl glucoside (DG) and n-dodecyl sucrose (DS) were used as alkyl glycosides. The stability was hardly changed by incorporation of alkyl glycosides into the liposomes in saline and serum. The uptake of DG- and DS-modified N+C5Ala2C12 liposomes decreased in liver and spleen compared with that of unmodified N+C5Ala2C12 liposomes, resulting in an increase in blood and other tissues such as tumor, duodenum and kidney, where the DS-modified N+C5Ala2C12 liposomes had a marked tendency. It was observed with electron micrographs that the size of N+C5Ala2C12 liposomes became small by incorporation of alkyl glycoside. The smaller N+C5Ala2C12 liposomes were found to result in the lower uptake in liver. The interaction of the liposomes with liver cells in vitro indicated that both DG- and DS-modified liposomes had a low affinity for liver cells compared with the unmodified liposomes and the extent of interaction of the DS-modified liposomes was weaker than that of the DG-modified liposomes.     

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.     

Hepatic glycosphingolipid abnormalities in a patient with GM1-gangliosidosis

Glycosphingolipids from the liver, kidney, and spleen of a patient with type 1 II3-N-acetylneuraminosylgangliotetraosylceramide (GM1)-gangliosidosis were quantitatively analyzed. It was noted that large amounts of unusual glycosphingolipids other than GM1 ganglioside or gangliotetrasylceramide accumulated in the liver of the patient. Particularly, the prominent accumulation of III3-alpha-fucosylneolactotetraosylceramide, galactosylceramide I3-sulfate and cholesterol sulfate was observed in addition to a small but significant increase of galabiosylceramide and neolacto-or lactotetraosylceramide. None of these lipids except cholesterol sulfate can be detected in normal liver. None of the lipids accumulated in the liver can be the direct substrates for acid beta-galactosidase which is deficient in the patient. Thus, it was suggested that secondary effects due to the defect in acid beta-galactosidase might cause the abnormal accumulation of various lipids in the liver.     

Saposins (Sphingolipid Activator Proteins) in the Twitcher Mutant Mouse

The twitcher mutant mouse, the animal model of Krabbe disease (human globoid cell leukodystrophy), is characterized by apparent deficiency of galactosylceramide beta-galactosidase activity. Saposin A and C, the heat-stable small sphingolipid activator glycoproteins, stimulate the activity of galactosylceramide beta-galactosidase as well as glucosylceramide beta-glucoside. The role of these saposins in the twitcher mutation was investigated. Boiled supernatant fractions, which contained saposins, were prepared from homogenates of twitcher brain, liver, kidney, and spleen. These preparations showed an almost identical effect on the activity of purified glucosylceramide beta-glucosidase (measured by hydrolysis of 4-methylumbelliferyl-beta-glucoside) with similar preparations from control tissues. The effect on the activity of galactosylceramide beta-galactosidase as well as 4-methylumbelliferyl-beta-glucoside beta-glucosidase in the twitcher brain and liver homogenates by authentic saposin A and C was similar to that in control tissues. These results suggest that the twitcher mutation does not affect the concentrations of saposin A or C or their interaction with galactosylceramide beta-galactosidase.     

The use of liposomal form of phenylimide of cis-aconitic acid in herpes therapy

The results of study of the antiviral activity and pharmacokinetics of phenylimide of cis-aconitic acid (PCAA) is presented. The 20% increase of the antiviral activity of PCAA incorporated into liposomes in comparison with the antiviral activity of the pure substance was shown. Liposomes with PCAA were tropic to lymphocytes and macrophages with maximum fluorescence being observed in the spleen, while empty liposomes were accumulated mainly in the liver. After the treatment with liposomal PCAA the symptoms of herpetic meningoencephalitis became less severe with 100% survival of the experimental animals. In the control group of rabbits 50% of the animals died, and in the surviving animals blindness or paralysis developed.     

Organ-specific expression of the lacZ gene controlled by the opsin promoter after intravenous gene administration in adult mice

BACKGROUND: The tissue-specific expression of an exogenous gene, under the influence of a tissue-specific promoter, has been examined in the past with pro-nuclear injections of the transgene and the development of transgenic mouse models. 'Adult transgenics' is possible with the acute expression of an exogenous gene that is administered to adult animals, providing the transgene can be effectively delivered to distant sites following an intravenous administration. METHODS: The organ specificity of exogenous gene expression in adult mice was examined with a bacterial beta-galactosidase (LacZ) expression plasmid under the influence of the bovine rhodopsin gene promoter. The 8-kb plasmid DNA was delivered to organs following an intravenous administration with the pegylated immunoliposome (PIL) non-viral gene transfer technology. The PIL carrying the gene was targeted to organs with the rat 8D3 monoclonal antibody (MAb) to the mouse transferrin receptor (TfR). RESULTS: The rhodopsin/beta-galactosidase gene was expressed widely in both the eye and the brain of adult mice, but was not expressed in peripheral tissues, including liver, spleen, lung, or heart. Ocular expression included the retinal-pigmented epithelium, the iris, and ciliary body, and brain expression was observed in neuronal structures throughout the cerebrum and cerebellum. CONCLUSIONS: The expression of trans-genes in adult animals is possible with the PIL non-viral gene transfer method. The opsin promoter enables tissue-specific gene expression in the eye, as well as the brain of adult mice, whereas gene expression in peripheral tissues, such as liver or spleen, is not observed.     

Uptake of liposome-entrapped mannitol by diaphragm.

Tissue uptake of liposome-entrapped radioactive mannitol was examined in rats and mice after both intravenous and intraperitoneal injection. In accord with results from other laboratories, liver and spleen effectively accumulated liposomes. Diaphragm also took up significant amounts of label. In nearly all cases the radioactive content of perfused tissues was less than tissues which were not perfused but this was statistically significant in only a few comparisons.