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.     

Effect of bile anionic polypeptidic fraction on the fate of cholesterol carried by liposomes in the rat

[14C]Cholesterol associated with liposomes with or without anionic polypeptidic fraction was administered intravenously to the rat. The cholesterol originated from liposomes including anionic polypeptidic fraction is secreted in bile much later, is stored in liver in higher quantity, and is metabolized into bile salts in lesser quantity during the 4 hr of experimentation than the cholesterol issued from liposomes exempt of anionic polypeptidic fraction. From these results it can be postulated that the cholesterol associated with liposomes containing anionic polypeptidic fraction might be directed in a particular liver pathway.     

Intrahepatic distribution of small unilamellar liposomes as a function of liposomal lipid composition

We investigated the intrahepatic distribution of small unilamellar liposomes injected intravenously into rats at a dose of 0.10 mmol of lipid per kg body weight. Sonicated liposomes consisting of cholesterol/sphingomyelin (1:1), (A); cholesterol/egg phosphatidylcholine (1:1), (B); cholesterol/sphingomyelin/phosphatidylserine (5:4:1), (C) or cholesterol/egg-phosphatidylcholine/phosphatidylserine (5:4:1), (D) were labeled by encapsulation of [3H]inulin. The observed differences in rate of blood elimination and hepatic accumulation (A much less than B approximately equal to C less than D) confirmed earlier observations and reflected the rates of uptake of the four liposome formulations by isolated liver macrophages in monolayer culture. Fractionation of the liver into a parenchymal and a non-parenchymal cell fraction revealed that 80-90% of the slowly clearing type-A liposomes were taken up by the parenchymal cells while of the more rapidly eliminated type-B liposomes even more than 95% was associated with the parenchymal cells. Incorporation of phosphatidylserine into the sphingomyelin-based liposomes caused a significant increase in hepatocyte uptake but a much more substantial increase in non-parenchymal cell uptake, resulting in a major shift of the intrahepatic distribution towards the non-parenchymal cell fraction. For the phosphatidylcholine-based liposomes incorporation of phosphatidylserine did not increase the already high uptake by the parenchymal cells while uptake by the non-parenchymal cells was only moderately elevated; this resulted in only a small shift in distribution towards the non-parenchymal cells. The phosphatidylserine-induced increase in liposome uptake by non-parenchymal liver cells was paralleled by an increase in uptake by the spleen. Fractionation of the non-parenchymal liver cells in a Kupffer cell fraction and an endothelial cell fraction showed that even for the slowly eliminated liposomes of type A endothelial cells do not participate to a measurable extent in the elimination process, thus excluding involvement of fluid-phase pinocytosis in the uptake process.     

Inhibition of liver metastases in murine colon adenocarcinoma by liposomes containing human C-reactive protein or crude lymphokine

Previous studies from our laboratory have shown that liposomes containing LYNK or CRP inhibit lung metastases in mice bearing the malignant fibrosarcoma, T241. We have now extended these observations to the murine colon adenocarcinoma (MCA-38), which metastasizes to the liver. MCA-38 tumor cells (1 X 10(6)) were implanted in the wall of the cecum by orthotopic transplantation. Three-hundred twenty-six mice bearing such tumors were divided into four treatment groups as follows: (1) no treatment (2) liposomes containing control medium (3) liposomes containing LYNK, and (4) liposomes containing CRP. Treatment was started from day 2, day 18, or 34 after tumor implantation and it was administered on 3 days per week. Each treatment, given parenterally, consisted of 4 mumol liposomes (PS-PC, 1:1) containing the appropriate agents. Mice receiving liposomes containing LYNK or CRP had significantly fewer and smaller liver metastases (25%-28%), than those in the two control groups (53%-54%). Also, a significantly better survival was noted in the two treated groups than in the two control groups. The most likely mechanism of tumor inhibition appears to be through macrophage activation. In the Winn tumor neutralization test, peritoneal macrophages harvested from normal mice 24 h after a single injection of liposomes (2.5 mumol) containing LYNK or CRP markedly inhibited tumor growth when a mixture of effector-target cells (40 : 1) was injected in the footpad. These studies further confirm the suggested role of CRP as an 'immunomodulator' or 'biological response modifier' in yet another tumor system.     

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

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

Targeting lymph nodes with liposomes bearing anti-HLA-DR Fab' fragments.

The ability of liposomes bearing anti-HLA-DR Fab' fragments to target cells expressing the human HLA-DR determinant of the major histocompatibility complex class II (MHC-II) has been evaluated and compared to that of conventional liposomes. Anti-HLA-DR immunoliposomes did not bind to HLA-DR-negative cells. In contrast, a high level of binding was observed following incubation of immunoliposomes with cells bearing important levels of human HLA-DR. The accumulation of conventional and murine anti-HLA-DR immunoliposomes in different tissues has been investigated following a single subcutaneous injection given in the upper back of C3H mice. Anti-HLA-DR immunoliposomes resulted in a much better accumulation in the cervical and brachial lymph nodes when compared to conventional liposomes. The accumulation in the liver was similar for both liposomal preparations, whereas an approximately twofold decrease in accumulation was observed for immunoliposomes in the spleen. Given that HLA-DR surface marker is expressed on monocyte/macrophages and activated CD4+ T lymphocytes, the primary cellular reservoirs of the human immunodeficiency virus (HIV), the use of liposomes bearing surface-attached anti-HLA-DR could constitute a convenient strategy to more efficiently treat this debilitating retroviral disease. Moreover, the reported incorporation of high amounts of host-encoded HLA-DR proteins by HIV particles renders the use of liposomes bearing anti-HLA-DR antibodies even more attractive.     

Alteration of liposome disposition in vivo by bilayer situated carbohydrates

The inclusion of either lactosylcerebroside or dimannosyldiglyceride at a 9% molar ratio in small unilamellar vesicles increased by two-three fold the fraction of the I.V. dose that appeared in mouse liver. For lactosylcerebroside containing liposomes, the half-time for clearance from plasma was 1.2 hours compared to 5.5 hours for liposomes of similar size, charge, and composition but lacking the glycolipid. Uptake of the lactosylcerebroside containing liposomes by the liver could be significantly reduced but not eliminated by the simultaneous injection of asialoorosomucid.     

Studies on membrane proteins involved in ribosome binding on the rough endoplasmic reticulum. Ribophorins have no ribosome-binding activity.

A membrane protein fraction showing affinity for ribosomes was isolated from rat liver microsomes (microsomal fractions) in association with ribosomes by treatment of the microsomes with Emulgen 913 and then solubilized from the ribosomes with sodium deoxycholate. This protein fraction was separated into two fractions, glycoproteins, including ribophorins I and II, and non-glycoproteins, virtually free from ribophorins I and II, on concanavalin A-Sepharose columns. The two fractions were each reconstituted into liposomes to determine their ribosome-binding activities. The specific binding activity of the non-glycoprotein fraction was approx. 2.3-fold higher than that of the glycoprotein fraction. The recovery of ribosome-binding capacity of the two fractions was about 85% of the total binding capacity of the material applied to a concanavalin A-Sepharose column, and about 90% of it was found in the non-glycoprotein fraction. The affinity constants of the ribosomes for the reconstituted liposomes were somewhat higher than those for stripped rough microsomes. The mode of ribosome binding to the reconstituted liposomes was very similar to that to the stripped rough microsomes, in its sensitivity to proteolytic enzymes and its strong inhibition by increasing KCl concentration. These results support the idea that ribosome binding to rat liver microsomes is not directly mediated by ribophorins I and II, but that another unidentified membrane protein(s) plays a role in ribosome binding.     

Effect of liposomal antitumor preparation 5-(5',6'-benzocoumarin-3')-methylaminouracil hydrobromide on cytochrome P-450 in the microsomal liver fraction of tumor bearing rats

Cytochrome P-450 thermal inactivation rate, and content of protein sulfhydryl groups and cytochrome P-450 in the microsomal liver fraction of rats at different stages of Huerin's carcinoma growth were investigated. Liposomal form of BCU administration on the background of preliminary (for 2 hours) administration of phosphatidylcholine liposomes suspension was performed. The low level of cytochrome P-450, protein SH-groups in microsomal liver fraction and increase of the rate of transition of microsomal cytochrome P-450 in P-420 was shown in the dynamics of Huerin's carcinoma growth in an organism. Low microsomal cytochrome P-450 distraction was shown in the rat liver under conditions of antitumor liposomal preparation BCU injection on the 21st day after the transplantation of Huerin's carcinoma. At the same time nonliposomal BCU caused the opposite effect. The preliminary administration of phosphatidylcholine liposomes favours the approach of the investigated parameters to the control values on the terminal stages of tumour growth.     

Interaction of intravenously injected liposomes with mouse liver mitochondria. A fluorescence and electron microscopy study

Megamitochondria, resulting from cuprizone feeding of Swiss ICR mice, were fluorescent in hepatocytes after the intravenous injection to mice of a liposome-encapsulated acridine orange-DNA complex (AO-DNA). Flow cytofluorimetric analysis of isolated megamitochondria showed that the proportion of liposome-encapsulated AO-DNA which localized in megamitochondria increased from 0.02% of the dose injected per liver cell at 3 min after injection to an average of 0.34% at 1 h after injection. Megamitochondria showed negligible fluorescence by fluorescence activated cell sorting (FACS) analysis when free AO-DNA was intravenously injected. Transmission electron micrographs of mouse liver tissue after intravenous injection of liposomes encapsulating iron dextran showed an association of the liposomes with megamitochondria which appeared identical to liposome association with normal mitochondria. These results support and extend our earlier observation that a fraction of the liposomes injected intravenously into mice associate with mitochondria in the liver, and possibly deliver their aqueous contents there.     

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.     

Targeting of lactosylceramide-containing liposomes to hepatocytes in vivo

Incorporation of 8 mol% lactosylceramide in small unilamellar vesicles consisting of cholesterol, dimyristoylphosphatidylcholine and phosphatidylserine in a molar ratio of 5:4:1 and containing [³H]inulin as an aqueous-space marker resulted in a 3-fold decreased half-life of the vesicles in blood and a corresponding increase in liver uptake after intracardial injection into rats. The increase in liver uptake was mostly accounted for by an enhanced uptake in the parenchymal cells, while the uptake by the non-parenchymal cells was only slightly increased. The uptake of both the control and the glycolipid-containing vesicles by the non-parenchymal cell fraction could be attributed completely to the Kupffer cells; no radioactivity was found in the endothelial cells. The effect of lactosylceramide on liver uptake and blood disappearance of the liposomes was effectively counteracted by desialylated fetuin, injected shortly before the liposome dose. This observation supports the notion that a galactose-specific receptor is involved in the liver uptake of lactosylceramide liposomes.     

Amniotic Fluid Stem Cells Inhibit the Progression of Bleomycin-Induced Pulmonary Fibrosis via CCL2 Modulation in Bronchoalveolar Lavage

The potential for amniotic fluid stem cell (AFSC) treatment to inhibit the progression of fibrotic lung injury has not been described. We have previously demonstrated that AFSC can attenuate both acute and chronic-fibrotic kidney injury through modification of the cytokine environment. Fibrotic lung injury, such as in Idiopathic Pulmonary Fibrosis (IPF), is mediated through pro-fibrotic and pro-inflammatory cytokine activity. Thus, we hypothesized that AFSC treatment might inhibit the progression of bleomycin-induced pulmonary fibrosis through cytokine modulation. In particular, we aimed to investigate the effect of AFSC treatment on the modulation of the pro-fibrotic cytokine CCL2, which is increased in human IPF patients and is correlated with poor prognoses, advanced disease states and worse fibrotic outcomes. The impacts of intravenous murine AFSC given at acute (day 0) or chronic (day 14) intervention time-points after bleomycin injury were analyzed at either day 3 or day 28 post-injury. Murine AFSC treatment at either day 0 or day 14 post-bleomycin injury significantly inhibited collagen deposition and preserved pulmonary function. CCL2 expression increased in bleomycin-injured bronchoalveolar lavage (BAL), but significantly decreased following AFSC treatment at either day 0 or at day 14. AFSC were observed to localize within fibrotic lesions in the lung, showing preferential targeting of AFSC to the area of fibrosis. We also observed that MMP-2 was transiently increased in BAL following AFSC treatment. Increased MMP-2 activity was further associated with cleavage of CCL2, rendering it a putative antagonist for CCL2/CCR2 signaling, which we surmise is a potential mechanism for CCL2 reduction in BAL following AFSC treatment. Based on this data, we concluded that AFSC have the potential to inhibit the development or progression of fibrosis in a bleomycin injury model during both acute and chronic remodeling events.     

Studies on lysophospholipases. VI. The action of two purified lysophospholipases from beef liver on membrane-bound lysophosphatidylcholine.

The action of two lysophospholipases purified from beef liver on lysophosphatidylcholine in microsomal membranes has been studied. Enzyme I, which has been shown to be localized in the soluble fraction of the beef liver cell, has a higher specific activity on microsomal lysophosphatidylcholine than Enzyme II, which originates from the microsomal cell fraction. This trend is also observed with phosphatidylcholine liposomes and single bilayer vesicles in which lysophosphatidylcholine has been incorporated. At low mol fractions of lysophosphatidylcholine in liposomes, the maximum enzymatic rate is proportional to this mol fraction. Similar results are obtained with mixed micelles of lysophosphatidylcholine and Triton X-100. The results are explained in terms of a model in which the two-dimensional substrate density in the membrane surface controls the rate of enzyme action.     

Liver microsomal membrane fluidity and lipid characteristics in vitamin A-deficient rats.

Rat liver microsomes (microsomal fraction) were isolated from vitamin A-deficient and -sufficient rats and analysed for membrane lipid characteristics. Membrane fluidity was found to be significantly decreased in microsomes from the vitamin A-deficient rats, but not in liposomes prepared from lipid extracts. Microsomes from vitamin A-deficient animals showed a significant decrease in C18:2, omega 6 and an increase in C22:5, omega 6 fatty acids.     

The use of phospholipid vesicles for in vitro studies on cholesteryl ester hydrolysis.

Radiolabeled cholesteryl oleate was incorporated into vesicles prepared from egg yolk lecithin and utilized as a substrate for studies of sterol ester hydrolases present in rat liver homogenates. The cholesteryl oleate was shown to be associated with vesicles (unilamellar liposomes) using Sepharose 4B chromatography. With this substrate, two different cholesteryl ester hydrolytic enzymes were demonstrated in subcellular fractions from the liver homogenates. In the lysosome-rich fraction an acid hydrolase was present, while in the cytosol fraction (150,000 g supernatant), hydrolytic activity was shown to occur with an optimum pH between 8 and 8.5. The substrate was characterized by Sepharose chromatography both before and after incubation with the liver fraction and was not dramatically altered even by rigorous incubation conditions. The lysosomal enzyme preparation was capable of hydrolyzing almost all the cholesteryl oleate in the vesicles. Hydrolysis of the phospholipid was proportionately much less than that of the cholesteryl oleate. Comparisons were performed between the vesicle preparation and an alternate substrate preparation involving the direct addition of cholesteryl oleate in acetone solution. The vesicles appeared to be a better substrate for the lysosomal enzyme whereas the activity in the cytosol fraction did not distinguish between the two substrate preparations. Unsonicated suspensions of cholesteryl oleate and lecithin did not serve as suitable substrates for the enzymes. These studies demonstrate the applicability of cholesteryl ester-containing vesicles as a useful substrate for studying cholesteryl ester hydrolysis in vitro.     

Tetrodotoxin-sensitive protein in the extracts from excitable tissues

The sodium permeability of liposomes preincubated with the soluble fraction of brain and heart muscle homogenates was increased veratrine. The veratrine increment was decreased by tetrodotoxin. The effect was specific for the extracts from excitable tissues. Bovine serum and soluble fraction of liver homogenate induced neither veratrine- nor tetrodotoxin-sensitivity of the liposomes. Treatment of the excitable tissue extracts by pronase and heat denaturation caused their complete inactivation. Tetrodotoxin-sensitive factor could be fractionated by ammonium sulfate precipitation and by DEAE-Servacel chromatography. On a column of Sephadex G-200 it was eluted with the void volume. It is suggested that the tetrodotoxin-sensitive factor is a protein which could be a soluble precursor of the voltage-dependent sodium channels.     

Involvement of the membrane fluidity of lactosylceramide-targeted liposomes in their intrahepatic uptake

Incorporation of N-lignoceroyldihydrolactocerebroside (lactosylceramide) enhanced liver uptake of small unilamellar liposomes consisting of dipalmitoylphosphatidylcholine, cholesterol and dicetyl phosphate (molar ratio, 4:5:1). The increase in liver uptake was mostly accounted for by an enhanced uptake into the parenchymal cells. The enhancing effects of lactosylceramide on uptake of the liposomes into liver in vivo and into isolated parenchymal cells in vitro were greater with dipalmitoylphosphatidylcholine-based liposomes than with dimyristoylphosphatidylcholine-based ones. In contrast, addition of lactosylceramide had no significant effect on egg phosphatidylcholine vesicle uptake. The stimulated uptake of lactosylceramide liposomes by parenchymal cells was counteracted by added asialofetuin. These observations suggest that transfer of the targeted liposomes via a galactose-specific receptor into parenchymal cells may be controlled by the membrane fluidity of the liposomes.     

Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions

Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.     

Differentiation in hepatic and splenic phagocytic activity during reticuloendothelial blockade with cholesterol-free and cholesterol-rich liposomes

We have shown earlier that liver and spleen reticuloendothelial cells have low affinity to phagocyte liposomes containing cholesterol. In the present study, we predosed mice with cholesterol-rich (identical to = 46.6 mol% cholesterol content) and cholesterol-free (identical to 0 mol%) liposomes to saturate the reticuloendothelial cells and examined the tissue distribution of the second dose of the test liposomes containing an aqueous marker, 125I-labelled poly(vinylpyrrolidone). The result shows that both preparations of the predosed liposomes caused suppression in hepatic uptake and delay in the blood clearance of the test liposomes, but the cholesterol-free liposomes were more effective in producing these effects than the cholesterol-rich liposomes. The suppression in hepatic phagocytic function, in accordance with the 'spillover' phenomenon [16, 17], caused an enhancement in spleen and lung uptake. The increase in lung uptake was proportionally related to the degree of suppression in the hepatic uptake, but the results of the splenic uptake showed some discrepancy. The predosed cholesterol-free liposomes which caused the maximum spillover of the test liposomes from the liver did not achieve maximum enhancement in the splenic uptake. Instead, the maximum enhancement was recorded with the predosed cholesterol-rich liposomes. This discrepancy in splenic uptake suggests that the predosed liposomes caused saturation of not only liver also the spleen reticuloendothelial system. However, instead of suppression in the splenic uptake due to the saturation, enhancement in uptake of the test liposomes was observed. We suggest the cause of this apparent increase the splenic phagocytic activity may be due to stimulation, by some unknown mechanism of splenic macrophages endothelial cells and/or lymphocytes, to phagocyte the excess of the test liposomes spillover from the liver with impaired phagocytic function.