The involvement of parenchymal,Kupffer and endothelial liver cells in the hepatic uptake of intravenously injected liposomes effects of lanthanum and gadolinium salts

¹²⁵I-labeled albumin or poly(vinyl pyrrolidone) encapsulated in intermediate size multilamellar or unilamellar liposomes with 30–40% of cholesterol were injected intravenously into rats. In other experiments liposomes containing phosphatidyl[Me-¹⁴C]choline were injected. 1 h after injection parenchymal or non-parenchymal cells were isolated. Non-parenchymal cells were separated by elutriation centrifugation into a Kupffer cell fraction and an endothelial cell fraction. From the measurements of radioactivities in the various cell fractions it was concluded that the liposomes are almost exclusively taken up by the Kupffer cells. Endothelial cells did not contribute at all and hepatocytes only to a very low extent to total hepatic uptake of the ¹²⁵I-labels. Of the ¹⁴C-label, which orginates from the phosphatidylcholine moiety of the liposomes, much larger proportions were recovered in the hepatocytes. A time-dependence study suggested that besides the involvement of phosphatidylcholine exchange between liposomes and high density lipoprotein, a process of intercellular transfer of lipid label from Kupffer cells to the hepatocytes may be involved in this phenomenon. Lanthanum or gadolinium salts, which effectively block Kupffer cell activity, failed to accomplish an increase in the fraction of liposomal material recovered in the parenchymal cells. This is compatible with the notion that liposomes of the type used in these experiments have no, or at most very limited, access to the liver parenchyma following their intravenous administration to rats.     

The reaction of bisulfite with liposomal membranes

Bisulfite has been shown to induce leakage of encapsulated substances from liposomal vesicles. The bisulfite induced leakage of either DNP-tyrosine, potassium ferricyanide, or [³H]glycine was observed to be greater with lipsomes composed of phospholipids containing unsaturated fatty acids. The leakage of encapsulated substances from liposomes was found to be concentration dependent when incubated for a constant time interval and time dependent when incubated at a constant bisulfite concentration. In addition, bisulfite caused the leakage of approximately 5 times more [³H]glycine from unilamellar liposomes than from multilamellar liposomes. These findings are consistent with the interaction of bisulfite with liposomal membranes via reaction with sites of unsaturation.     

The effect of reticuloendothelial blockade on the blood clearance and tissue distribution of liposomes

The blood clearance and tissue distribution of liposomes have been studied in mice subjected to reticuloendothelial blockade with dextran sulphate or carbon. The liposomes have been labelled in the lipid membranes with [3H]-cholesterol, [14C]phosphatidylcholine and/or 99mTc and the content with [14C]inulin. Reticuloendothelial blockade has been shown to slow the rate of clearance of neutral, positively and negatively charged liposomes and of both small unilamellar vesicles and large multilamellar vesicles. In normal animals, the liver uptake accounted for only 20-55% of the total injected radioactivity, the amount varying with the charge and size of the liposomes. Following blockade, the liver uptake of charged and neutral multilamellar liposomes was depressed. This was also true for negatively charged small unilamellar vesicles. The degree of depression of hepatic uptake was between 25-50%, which contrasts with the 80-90% reduction in uptake of a wholly phagocytosed particle (sheep red cells). This difference suggests that mechanisms other than Kupffer cell phagocytosis are also responsible for the normal uptake of liposomes into the liver. In the case of neutral and positively charged small unilamellar vesicles, delayed clearance due to blockade was not associated with 'depressed' hepatic uptake. The site of action of blockading agents for these preparations is not clear. With all preparations of liposomes, blockade produced a slight and variable increase in uptake in the lung and spleen. The alteration of distribution of liposomes by reticuloendothelial blockade is therefore not great and the value of the technique in modifying the tissue distribution of substances within liposomes may be limited.     

Encapsulation of immunoadjuvant [24C]peptidoglycan monomer into liposomes: Effect on metabolism and immune response in mice

¹⁴C-labeled peptidoglycan monomer was encapsulated into negatively charged, multilamellar liposomes composed of egg phosphatidylcholine, cholesterol and dicetylphosphate. Excretion and tissue distribution of the label in mice were studied after intravenous injections. Encapsulation of peptidoglycan monomer into liposomes as compared to free peptidoglycan monomer, resulted in increased retention of the label, particulary in the liver and to a lesser extent in spleen. The excretion was drastically reduced and delayed even after 4 days when cholesterol-rich (phosphatidylcholine/cholesterol, 7:5 molar ratio) liposomes were used for encapsulation of peptidoglycan monomer. Peptidoglycan monomer and liposomes, when tested separately, stimulate the immune response to sheep erythrocytes in mice. However, there was no significant additive or synergistic effect when peptidoglycan monomer was encapsulated into liposomes.     

Uptake and biodistribution of free and liposomally incorporated lipopolysaccharide of aeromonas salmonicida administered via different routes to rainbow trout: (Oncorhynchus mykiss)

Abstract

The effects on uptake and biodistribution of radiolabelled lipopolysaccharide (LPS) due to changing routes of administration, encapsulation of LPS within liposomes and altering liposomal surface charge were examined in rainbow trout (Oncorhynchus mykiss). ³H-labelled LPS, positively- and negatively-charged (¹⁴C-labelled) liposomes or ¹⁴C-labelled liposomes containing ³H-LPS were administered to trout via intravenous, intraperitoneal, intramuscular, or oral routes. Twenty-four hours following administration, relative uptake of LPS and multilamellar vesicles (MLV) based on detection of ³H and ^1AC, respectively, was determined in samples taken from the kidney, spleen, liver, plasma, blood cells and skeletal muscle. In general, regardless of the route of administration, ³H-LPS, ^1AC-MLV and liposomally encapsulated LPS were recovered primarily in the kidney and spleen. Intravenous administration resulted in the greatest uptake of radiolabel by the kidney and spleen, followed by the intraperitoneal and intramuscular routes. Although oral administration yielded the lowest overall uptake of labelled material, detection of ³H and ¹⁴C in the liver was enhanced when compared with the other routes. Negatively-charged MLV were delivered more efficiently to the kidney and spleen than positively-charged MLV; but negatively- and positively-charged MLV containing LPS demonstrated the opposite relationship between charge and distribution among the kidney and spleen. These results suggest that liposomal encapsulation (particularly within positively-charged MLV) enhances delivery of LPS to the primary hemopoietic organs in rainbow trout.     

Binding of insulin to the external surface of liposomes: Effect of surface curvature,temperature, and lipid composition

The binding of insulin to the external surface of phosphatidylcholine liposomes as a function of the temperature, the surface curvature, and the composition of lipids was studied. The amount of the saturated binding of insulin to liposomes was assessed by gel-filtration chromatography. The binding of insulin to small unilamellar vesicles was highly dependent upon the temperature, favoring low temperatures. As the temperature increased, there was a distinct temperature range where the binding of insulin to small unilamellar vesicles decreased. The temperature ranges for dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) small unilamellar vesicles were found to be 10–20°C and 21–37°C, respectively. These temperature ranges were quite different from the reported ranges of the gel → liquid crystalline phase transition temperatures ($\text{T}{}_{\mathrm{<mtext>c</mtext>}}$) for DMPC or DPPC small unilamellar vesicles. In contrast to other proteins, the amount of insulin bound to DMPC and DPPC small unilamellar vesicles was negligible at or above the upper limit of the above temperature ranges, and increased steadily to 6–7 μmol of insulin per mmol of phospholipid as the temperature decreased to or below the lower limit of these temperature ranges. On the other hand, the binding of insulin to the large multilamellar liposomes cannot be detected at all temperatures tested. The affinity of insulin to neutral phosphatidylcholine small unilamellar vesicles appeared to be related to the surface curvature of the liposomes, favoring the liposomes with a high surface curvature. Furthermore, the amount of insulin bound to small unilamellar vesicles decreased as the content of the cholesterol increased. The presence of 10% molar fraction of phosphatidic acid did not appear to affect the binding of insulin to small unilamellar vesicles. However, the presence of 5% molar fraction of stearylamine in DPPC small unilamellar vesicles increased the amount of bound insulin as well as the extent of aggregation of liposomes. The results of the present study suggest that the interstitial regions of the acyl chains of phospholipids between the faceted planes of small unilamellar vesicles below $\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ may be responsible for the hydrophobic interaction of insulin and small unilamellar vesicles. The tight binding of insulin to certain small unilamellar liposomes could lead to an overestimation of the true amount of insulin encapsulated in liposomes, if care is not taken to eliminate the bound insulin during the procedure of encapsulating insulin in liposomes.     

Interactions of liposomes with the reticuloendothelial system: II. Nonspecific and receptor-mediated uptake of liposomes by mouse peritoneal macrophages

Liposomes are taken up as intact vesicles by mouse peritoneal macrophages in a process which is temperature sensitive and is affected by inhibitors of glycolytic metabolism and of microfilament activity. Macrophages take up negatively charged vesicles more readily than positively charged vesicles (2-fold) or neutral vesicles (4-fold). Macrophages take up similar amounts of multilamellar liposomes, reversed phase liposomes and small unilamellar liposomes in terms of lipid, however this corresponds to vastly different numbers of particles and amounts of trapped volume. Coating the liposomes with macromolecular ligands capable of interacting with macrophage surface receptors can markedly promote liposome uptake. Thus, formation of an IgG-antigen complex on the liposome surface results in a 10²-fold enhancement of liposome uptake, while coating the vesicles with fibronectin results in a 10-fold augmentation of uptake. Uptake via IgG-mediated and fibronectin-mediated processes seem to be independent since excess unlabelled, IgG-coated liposomes will inhibit the uptake of radioactively-labelled IgG-coated liposomes much more effectively than the uptake of radioactively-labelled fibronectin-coated liposomes. Cell-bound liposomes can readily be visualized on and inside of the macrophages using fluorescence microscopy techniques.     

In vitro drug delivery mediated by ecto-NAD+-glycohydrolase ligand-targeted liposomes

We have studied the growth-inhibitory potency of methotrexate and methotrexate-γ-aspartate encapsulated in liposomes conjugated to ligands of ecto-NAD⁺-glycohydrolase (Salord, J. et al., Biochim. Biophys. Acta 886 (1986) 64–75). The ability of targeted liposomes to enhance growth inhibition, which amounted to a 4-fold reduction of the drug concentration required to inhibit cell growth by 50% as compared to nontargeted liposomes, was observed only with cells expressing this ecto-enzyme activity, i.e., Swiss 3T3 fibroblasts and RAJI, a Burkitt-type lymphoma cell line. Delivery of the encapsulated drugs was inhibited by NH₄Cl and varied with the endocytic capacity of the cells. Only small unilamellar vesicles affected the growth of the lymphoma cells, whereas the fibroblasts were more sensitive to large unilamellar vesicles. With vesicles of appropriate size, there was a good correlation between the specific binding of the targeted liposomes to cells and drug delivery. Our results suggest that ecto-NAD⁺-glycohydrolase can provide a recognition site on target cells and mediate the internalization of targeted liposomes by a mechanism most probably related to adsorptive endocytosis.     

Phagocytosis of liposomes by human alveolar macrophages

The ability of cells recovered from lung lavages to phagocytose liposomes has been investigated.Inulin (¹⁴C-labelled), entrapped in multilamellar immunoglobulin-G coated liposomes with ³H-labelled cholesterol as the lipid phase marker, was fed to the recovered cells. Fifteen patients with diffuse interstitial pulmonary disease (DIPD), prior to steroid treatment, and eight normal controls were lavaged for the study. Uptake was found for both groups and it was concluded that the liposomes enter the cells predominantly via endocytosis. Follow-up lavage, three months after the initial lavage, was repeated on three patients receiving 60 mg prednisone per day. A decrease in the uptake of liposomes was observed after steroid treatment.     

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.     

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.     

Liposomal Hexadecylphosphocholine: Characterization and Effects on Adherent Tumor Cells

Abstract

We describe the preparation of small unilamellar and multilamellar vesicles from hexadecylphosphocholine, cholesterol and 1,2-dipalmitoyl-sn-glycero-phosphoglycerol in the molar ratio 4/5/1. Particle size and chemical stability of two types of liposomes, small unilamellar vesicles and lyophilized, freshly resuspended multilamellar vesicles were proved to be stable for at least 12 months. Compared to hexadecylphosphocholine in free form, liposomal hexadecylphosphocholine showed remarkably reduced hemolysis which did not change during storage. Fluorescence microscopy showed the uptake of propidium iodide containing hexadecylphosphocholine liposomes by KB and MDA-MB 231 tumor cells. Free propidium iodide was not incorporated into these cells. Although cytotoxicity seemed to be reduced in liposomal preparations, hexadecylphosphocholine liposomes still affected cultured tumor cells to a great extent. In relatively low concentrations they induced shape alteration, smoothing of the cell surface and blebbing.     

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     

Differential uptake of liposomes varying in size and lipid composition by parenchymal and kupffer cells of mouse liver

Using liposomes differing in size and lipid composition, we have studied the uptake characteristics of the liver parenchymal and Kupffer cells. Desferal labeled with iron-59 was chosen as a radiomarker for the liposomal content, because Desferal in its free form does not cross cellular membranes. At various time intervals after an intravenous injection of liposomes into mice, the liver was perfused with collagenase, and the cells were separated in a Percoll gradient. It was found that large multilamellar liposomes (diameter of about 0.5 μm) were mainly taken up by the Kupffer cells. For these large liposomes, the rate of uptake by Kupffer cells was rapid, with maximum uptake at around 2 hours after liposome injection. Unexpectedly, small unilamellar liposomes (diameter of about 0.08 μm) were less effectively taken up by Kupffer cells, and the rate of uptake was slow, with a maximum uptake at about 10 hours after liposome injection. In contrast, parenchymal cells were more effective in taking up small liposomes and the uptake of large liposomes was negligible. In addition, liposomes made with a galactolipid as part of the lipid constituents appeared to have higher affinity to parenchymal cells than liposomes made without the galactolipid. These findings should be of importance in designing suitable liposomes for drug targeting.     

Distribution of liposome-entrapped cations in tumor-bearing mice.

The effect of entrapment of ⁸⁶Rb⁺ and ²²Na⁺ in multilamellar, negatively charged phospholipid liposomes on their clearance from the bloodstream and uptake into a variety of tissues in tumor-bearing mice was studied. Although differences were seen between the distribution of free and entrapped ions, these were smaller than might be expected from the uptake of liposomes containing labelled phospholipid. One problem detected was that addition of mouse serum caused a large increase in the efflux of ²²Na⁺ from liposomes, suggesting that a large amount of the injected, trapped ions may have been free in the bloodstream within 1–2 hours. It is concluded that if liposomes are to be fully effective in increasing the uptake of entrapped substances into tissues, the type of liposome used as well as the nature of the entrapped substance are important variables.     

Inhibitory effect of cholesterol on the uptake of liposomes by liver and spleen

The effect of cholesterol content of small unilamellar (SUV) and reverse phase (REV) liposomes on blood clearance and tissue distribution has been studied. [¹⁴C]Inulin has been used as an aqueous marker of liposomes to represent the uptake of intact liposomes in tissues. The blood clearance of the intravenously-injected SUV and REV liposomes depends on the cholesterol content of liposomes. The cholesterol-free (0 mol%) liposomes are cleared more readily from the circulation than the cholesterol-poor liposomes (20 mol%) and the cholesterol-poor are cleared more rapidly than the cholesterol-rich (46.6 mol%) liposomes. This clearance pattern of liposomes from the circulation is not attributed to the change of size of liposomes due to the increase in cholesterol content of liposomes. However, poor stability of cholesterol-free or cholesterol-poor liposomes in the circulation is partly responsible, but the predominant factor responsible for the observed blood clearance pattern is the inhibitory effect of cholesterol on the uptake of liposomes by reticuloendothelial-rich tissues liver and spleen. Uptake of liposomes by these organs is decreased with increasing cholesterol content of vesicles. It is suggested that to produce liposome preparations with a long circulating half life in vivo it is necessary to inhibit their uptake by liver and spleen.     

Interaction of phospholipid vesicles with rat hepatocytes in primary monolayer culture

We studied the interaction of positively and negatively charged unilamellar and multilamellar phospholipid vesicles (liposomes) with rat-liver parenchymal cells in primary monolayer culture. Radioactive liposomal phosphatidylcholine was taken up more rapidly and to a larger extent from unilamellar than from multilamellar vesicles. No significant difference in uptake characteristics was observed between vesicles of different charge. The presence of serum greatly reduced uptake of liposomal phosphatidylcholine of both unilamellar and multilamellar vesicles. This serum effect was independent of surface charge of the vesicles. When cells were allowed to take up radioactive liposomal phospholipid and then incubated further in absence of vesicles, part of the radioactivity associated with the cells was released into the medium, most of it as water soluble degradation products. When cells were preincubated with vesicles containing horseradish peroxidase and then, after removal of the vesicles, further incubated, peroxidase activity could be demonstrated in the culture medium, part of it only after addition of Triton X-100. These observations were taken to indicate that part of the phospholipid taken up the cells represented vesicles binding to the cell surface rather than having been internalized. Vesicle-entrapped [125I]albumin was taken up by the cells and rapidly hydrolyzed as indicated by the appearance of radioactivity soluble in trichloroacetic acid within minutes after starting the incubation. No uptake of free albumin could be demonstrated. The kinetics of albumin uptake and release of trichloroacetic acid-soluble radioactivity from the cells suggest that, initially, liposomes are internalized predominantly by endocytosis, while during prolonged incubation fusion of the liposomal membrane with the plasma membrane gradually contributes more substantially to the overall uptake process. The significance of these findings is emphasized with special reference to the use of liposomes as intravenous carriers of enzymes or drugs.     

Liposome-Encapsulated Bacteriophages for Enhanced Oral Phage Therapy against Salmonella spp.

Bacteriophages UAB_Phi20, UAB_Phi78, and UAB_Phi87 were encapsulated in liposomes, and their efficacy in reducing Salmonella in poultry was then studied. The encapsulated phages had a mean diameter of 309 to 326 nm and a positive charge between +31.6 and +35.1 mV (pH 6.1). In simulated gastric fluid (pH 2.8), the titer of nonencapsulated phages decreased by 5.7 to 7.8 log units, whereas encapsulated phages were significantly more stable, with losses of 3.7 to 5.4 log units. The liposome coating also improved the retention of bacteriophages in the chicken intestinal tract. When cocktails of the encapsulated and nonencapsulated phages were administered to broilers, after 72 h the encapsulated phages were detected in 38.1% of the animals, whereas the nonencapsulated phages were present in only 9.5%. The difference was significant. In addition, in an in vitro experiment, the cecal contents of broilers promoted the release of the phages from the liposomes. In broilers experimentally infected with Salmonella, the daily administration of the two cocktails for 6 days postinfection conferred similar levels of protection against Salmonella colonization. However, once treatment was stopped, protection by the nonencapsulated phages disappeared, whereas that provided by the encapsulated phages persisted for at least 1 week, showing the enhanced efficacy of the encapsulated phages in protecting poultry against Salmonella over time. The methodology described here allows the liposome encapsulation of phages of different morphologies. The preparations can be stored for at least 3 months at 4°C and could be added to the drinking water and feed of animals.     

Comparison of large unilamellar vesicles prepared by a petroleum ether vaporization method with multilamellar vesicles: ESR,diffusion and entrapment analyses

Large unilamellar vesicles, prepared by a petroleum ether vaporization method, were compared to multilamellar vesicles with respect to a number of physical and functional properties. Rotational correlation time approximations, derived from ESR spectra of both hydrophilic (3-doxyl cholestane) and hydrophobic (3-doxyl androstanol) steroid spin probes, indicated similar molecular packing of lipids in bilayers of multilamellar and large unilamellar liposomes. Light scattering measurements demonstrated a reduction in apparent absorbance of large unilamellar vesicles, suggesting loss of multilamellar structure which was confirmed by electron microscopy. Furthermore, large unilamellar vesicles exhibited enhanced passive diffusion rates of small solutes, releasing a greater percentage of their contents within 90 min than multilamellar vesicles, and reflecting the less restricted diffusion of a unilamellar system. The volume trapping capacity of large unilamellar vesicles far exceeded that of multilamellar liposomes, except in the presence of a trapped protein, soy bean trypsin inhibitor, which reduced the volume of the aqueous compartments of large unilamellar vesicles. Finally, measurement of vesicle diameters from electron micrographs of large unilamellar vesicles showed a vesicle size distribution predominantly in the range of 0.1–0.4 μm with a mean diameter of 0.21 μm.