Neurons are protected from excitotoxic death by p53 antisense oligonucleotides delivered in anionic liposomes.

The potential of anionic liposomes for oligonucleotide delivery was explored because the requirement for a net-positive charge on transfection-competent cationic liposome-DNA complexes is ambiguous. Liposomes composed of phosphatidylglycerol and phosphatidylcholine were monodisperse and encapsulated oligonucleotides with 40-60% efficiency. Ionic strength, bilayer charge density, and oligonucleotide chemistry influenced encapsulation. To demonstrate the biological efficacy of this vector, antisense oligonucleotides to p53 delivered in anionic liposomes were tested in an in vitro model of excitotoxicity. Exposure of hippocampal neurons to glutamate increased p53 protein expression 4-fold and decreased neuronal survival to approximately 35%. Treatment with 1 microm p53 antisense oligonucleotides in anionic liposomes prevented glutamate-induced up-regulation of p53 and increased neuronal survival to approximately 75%. Encapsulated phosphorothioate p53 antisense oligonucleotides were neuroprotective at 5-10-fold lower concentrations than when unencapsulated. Replacing the anionic lipid with phosphatidylserine significantly decreased neuroprotection. p53 antisense oligonucleotides complexed with cationic liposomes were ineffective. Neuroprotection by p53 antisense oligonucleotides in anionic liposomes was comparable with that by glutamate receptor antagonists and a chemical inhibitor of p53. Anionic liposomes were also capable of delivering plasmids and inducing transgene expression in neurons. Anionic liposome-mediated internalization of Cy3-labeled oligonucleotides by neurons and several other cell lines demonstrated the universal applicability of this vector.

Previously reported nerve growth factor levels are underestimated due to an incomplete release from receptors and interaction with standard curve media.

The 1996 research report by Hoener et al. [M.C. Hoener, E. Hewitt, J. M. Conner, J.W. Costello, S. Varon, Nerve growth factor (NGF) content in adult rat brain tissue is several-fold higher than generally reported and is largely associated with sedimentable fractions, Brain Res. 728 (1996) 47-56.] compares levels of nerve growth factor (NGF) found in rat brain by assaying both supernatant and pellet to previously reported data. However, Hoener et al. miscalculated when converting values previously reported in the literature to units of picogram per milliliter. Regardless of this mistake, the method of tissue extraction does affect the extent of release of NGF, which must be maximized in order to accurately determine NGF levels in the central nervous system. We now report that accurate measurement of NGF levels is not only affected by the incomplete release of NGF from receptors, but also the medium in which the standard curve is run. It is the combination of these two variables that has led to the underestimation of NGF levels in previous research.

Interaction between superoxide dismutase and dipalmitoylphosphotidylglycerol bilayers: a fourier transform infrared (FT-IR) spectroscopic study.

PURPOSE: Superoxide dismutase (SOD), an antioxidant enzyme, converts peroxide radicals into hydrogen peroxide. Liposomes have been used as carriers for SOD to enhance its antioxidant effect. Our previous DSC study has suggested that SOD binding to dipalmitoylphosphatidylglycerol (DPPG) may protect lipid membranes against oxygen-mediated injury. We now present FT-IR studies on the effect of DPPG binding on the temperature-induced SOD folding-unfolding process. METHODS: The FT-IR spectra of SOD in D2O or DPPG membranes are measured as temperatures increase from 28 degrees to 121 degrees C at a rate of 0.5 degrees C/min. From the quantitative determination of the changes in the amide I band components of the Fourier self-deconvoluted spectra, the DPPG-induced changes of SOD secondary structure could be detected as a function of temperature. RESULTS: We observe that the relative intensity of the SOD bands from 28 degrees C to 77 degrees C show graduate loss of beta-sheet "distorted" structure, loss of turns, and existence of an intermediate state around 50 degrees C. Beginning at 80 degrees C, changes are obtained in three temperature regions: (i) 80 degrees C, (ii) 92 degrees C, (iii) 109 degrees C. The result suggests that SOD folding/unfolding transition involves mostly the relative changes within the regions of helix-like hydrogen bonding pattern, turn, twisted beta-bend and irregular structures. When SOD is bound to DPPG, the conformational changes shift to lower temperatures, indicating a reduction of SOD thermal stability. In addition, the gel to liquid crystalline phase transition temperature of DPPG increases from 42 degrees C to 43.5 degrees C. CONCLUSIONS: These results suggest that the thermal stability of SOD is reduced by DPPG binding. However, DPPG bilayer is stabilized by the presence of SOD.

Protein location in liposomes, a drug carrier: a prediction by differential scanning calorimetry.

Location of protein drugs in lipid carriers often determines the stability, loading efficiency, and release rate of these drugs from the carriers following administration. On the basis of conventional differential scanning calorimetry (DSC) measurements, Papahadjopoulos et al. (Biochim. Biphys. Acta 1975, 401, 317-335) proposed that proteins can be classified into three categories depending on their effects on the thermotropic behavior of the lipids, e.g., transition temperature and enthalpy. Interactions are usually electrostatic, hydrophobic, or their combination. The nature of these interactions are reflected by changes in various thermotropic parameters. Our study aims to test the validity of Papahadjopoulos' classification. Hydrophilic ribonuclease A, cytochrome c, and superoxide dismutase (SOD), as well as hydrophobic cyclosporin A, are used as model proteins. Neutral lipids, e.g., dipalmitoylphosphatidylcholine, and/or negatively charged lipids, e.g., dipalmitoylphosphatidylglycerol (DPPG), are used to prepare liposomes. Results from conventional and high-sensitivity DSC are compared. High-sensitivity DSC gives significant, more reproducible results. We find that the classification of Papahadjopoulos et al. needs to be modified. No hydrophilic proteins bind to liposomes exclusively on the surface by electrostatic interactions, and some degree of penetration is observed in most cases. An unexpected binding between SOD and DPPG liposomes is observed. The binding of SOD to negatively charged lipids may account, at least in part, for its ability to protect lipid membranes against oxygen-mediated injury.

Studies on an oral iron chelator: 1,2-dimethyl-3-hydroxy-pyrid-4-one (DMHP). Mechanism of intestinal absorption in rabbits.

Over the last 30 years, desferrioxamine has been the only iron chelator in clinical use. This chelator is expensive and must be given by injection. A new class of chelators, namely 1-alkyl-2-methyl-3-hydroxypyrid-4-ones, have been shown to be orally effective. Using 1,2 dimethyl-3-hydroxy-pyrid-4-one (DMHP), we have carried out a study to clarify the mechanism of intestinal absorption of this new class of drug, using an in-situ system of the intestine from rabbit. The major site of DMHP absorption is in the intestine and is linear with increasing drug concentration. DMHP absorption per unit length of jejunum and ileum is similar; however, due to the larger surface area of jejunum, the absorption by ileum segment is more effective per unit surface. L-Proline, L-tryptophan (amino acids), 2-deoxyglucose, and sodium iodoacetate (metabolic inhibitors) have no effect on DMHP absorption, but L-phenylalanine, an amino acid with a 6-member carbon ring, significantly inhibits the DMHP absorption from the intestinal segment. We conclude that the mechanism of DMHP absorption in the intestine is mainly by simple passive diffusion based on the linear relationship found between drug concentration and absorption. However, the inhibitive effect of L-phenylalanine suggests that the co-existence of a facilitated uptake cannot be ruled out.

Crystal structures of a new oral iron chelator, 1,2-dimethyl-3-hydroxy-4-pyridone, and its solvates with acetic acid and formic acid.

The crystal structures of a new oral iron chelator, 1,2-dimethyl-3-hydroxy-4-pyridone (DMHP), and of its 1:1 solvates with formic acid (DMHP,F) and acetic acid (DMHP,A) were determined by single-crystal X-ray diffraction. The data were collected at temperatures of 23 +/- 1 degrees C for DMHP, -64 +/- 1 degrees C for DMHP,F, and -120 +/- 1 degrees C for DMHP,A. The iron chelator DMHP is orthorhombic [Pbca, a = 7.290(5) A, b = 13.046(4) A, c = 13.748(6) A, Z = 8]. The DMHP molecules form centric dimers, each in a 10-membered ring in which the OH group of one molecule is hydrogen-bonded to the CO oxygen of the other [O-H...O; 0.91(4) A, 153(3)degrees, 1.85(4) A]. In each DMHP molecule, the OH group and CO oxygen are insignificantly intramolecularly hydrogen-bonded [O-H...O; 0.91(4) A, 107(3)degrees, 2.33(4) A]. DMHP,F is monoclinic [C2/c, a = 21.825(9) A, b = 3.811(5) A, c = 20.491(6) A, beta = 92.80(3)degrees, Z = 8]. The fundamental intermolecular and insignificant intramolecular hydrogen-bonded dimer structure of DMHP is maintained but is distorted and is supplemented by hydrogen bonds between the CO oxygen of each DMHP molecule and the OH group of one formic acid molecule [O-H...O; 0.99(5) A, 176(3)degrees, 1.53(4) A]. However, the two DMHP and the two formic acid molecules are twisted out of plane like the blades of a four-bladed propeller. DMHP,A is triclinic [P1, a = 8.458(2) A, b = 8.471(2) A, c = 6.986(3) A, alpha = 104.33(2)degrees, beta = 92.57(2)degrees, gamma = 88.78(2)degrees, Z = 2].(ABSTRACT TRUNCATED AT 250 WORDS)

Solid state properties of an oral iron chelator, 1,2-dimethyl-3-hydroxy-4-pyridone, and its acetic acid solvate. I: Physicochemical characterization, intrinsic dissolution rate, and solution thermodynamics.

1,2-Dimethyl-3-hydroxy-4-pyridone (1), a crystalline oral iron chelator, forms an acetic acid solvate (2) on recrystallization from acetic acid and carbon tetrachloride. Compound 2 forms compact prisms, and 1 forms needles from water (mp 274 degrees C). The X-ray powder diffraction patterns of 1 and 2 differ, indicating distinct solid phases. Compound 2 has an extra DSC endotherm at 82 degrees C that is accompanied by a weight loss of 29% in TGA, corresponding to the desolvation of a 1:1 acetic acid solvate. Comparison of the solid-state 13C NMR of 1 and 2 revealed two additional peaks for 2 at 20.3 and 175.6 ppm, characteristic of -CH3 and -COOH, respectively, of acetic acid. The integrated intensities confirmed the 1:1 stoichiometry between 1 and acetic acid. However, 2 underwent desolvation in air at 25 degrees C as suggested by a change in its appearance to opaque crystals and as confirmed by X-ray powder diffraction, DSC, and TGA. Desolvation of 2 at 25 degrees C was a zero-order process with a rate constant of 6.9 mumol.h-1. X-ray powder diffraction showed that crystals or compacted discs of 1 are converted to 2 in contact with glacial acetic acid (A), whereas crystals or discs of 2 are converted to 1 in contact with water. The intrinsic dissolution rate (J) and the apparent solubility (Cs) of compacted discs of 1 and 2 were measured in water at 25 degrees C, and the following relations were determined: J(2)/J(1) = 1.39 and Cs(2)/Cs(1) = 1.70.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of an oral iron chelator: 1,2-dimethyl-3-hydroxy-pyrid-4-one. I. Iron excretion in rats: development of a new rapid microwave method for iron analysis in faeces.

We have developed a simple, rapid method for analysing faecal iron using a microwave oven for digestion followed by atomic absorption spectrometry. The chelating effect of 1,2-dimethyl-3-hydroxy-pyrid-4-one (DMHP) has been tested in rats with experimental iron overloading and three routes of DHMP administration, oral, subcutaneous, and intraperitoneal, have been compared. Regardless of the route of administration, we have found that DMHP promotes iron excretion via the urine. We have not observed a difference in the amount of iron excreted in the faeces before and after DMHP administration by any route. The subcutaneous route of administration is the most effective in promoting iron excretion, followed by the intraperitoneal route. Although most convenient for clinical use, oral administration promotes the excretion of only a small fraction of that by the subcutaneous route.

Pharmacokinetics of two alternative dosage forms for cyclosporine: liposomes and intralipid.

Cyclosporine (CsA), commercially available as iv or oral Sandimmune, is a potent immunosuppressant which can induce dose-related nephrotoxocity. In addition, the iv product contains a solubilizing agent, Cremophore EL, which in itself is reported to be nephrotoxic and can induce, in sensitized patients, anaphylactic reactions. Solubilization of CsA with liposomes or lipid emulsions could provide a suitable alternative dosage form for iv administration. With this in mind, male New Zealand white rabbits were given iv CsA (10 mg/kg) in three different dosage forms: (1) CsA:liposomes; (2) CsA:Intralipid (soybean oil and phospholipids); and (3) the commercially available Sandimmune (cyclosporine). The CsA concentration in whole blood samples was analyzed by HPLC. The terminal disposition half-life of CsA (t1/2 beta) ranged from 400 to 475 min and was not statistically different among the three groups. However, the distribution characteristics of CsA changed dramatically depending on the dosage form. The volume of distribution of CsA at steady state (Vdss) in Sandimmune was 2.7 +/- 0.2 L/kg and was significantly lower than that of either Intralipid (10.6 +/- 2.7 L/kg) or liposomes (7.4 +/- 2.3 L/kg). A significantly lower total body clearance (TBC) of CsA also was seen for Sandimmune (12.7 +/- 0.3 mL/min/kg) as compared with that of either Intralipid (24.4 +/- 8.2 mL/min/kg) or liposomes (18.9 +/- 3.9 mL/min/kg). Since CsA is extensively bound to lipoproteins, it is surprising that both test vehicles showed a different distribution pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of cyclosporin A with dipalmitoylphosphatidylcholine.

Cyclosporin A, a hydrophobic cyclic peptide, is a potent immunosuppressant. In an attempt to determine the localization of cyclosporin A in phospholipid membranes, the effect of cyclosporin A on dipalmitoylphosphatidylcholine (DPPC) has been investigated using deuterium nuclear magnetic resonance (2H-NMR) spectroscopy and differential scanning calorimetry (DSC). Cyclosporin A was dispersed within acyl chain per-deuterated DPPC at a concentration of 6 mole percent, hydrated with buffer, and the spectra obtained over a range of temperatures were compared with that of pure DPPC. The changes caused by cyclosporin A were assessed by the first moment (M1) and order parameters calculated from the spectra. The presence of cyclosporin A decreases the magnitude of M1 at temperatures below the gel to liquid-crystalline phase transition temperature but increases M1 at temperatures above the transition. In addition, the change in M1 at the transition temperature was also less abrupt when cyclosporin A was present. For bilayers in the liquid-crystalline state, cyclosporin A causes an increase in the order parameters along the acyl chains which suggests that cyclosporin A is located along the acyl chains of the phospholipid. For DSC, cyclosporin A was dispersed in non-deuterated DPPC at different peptide to phospholipid mole ratios. The endothermic peaks associated with the gel to liquid-crystalline phase transition and pretransition were recorded and compared with similar mole ratios of cholesterol to lipid. At 30 mole percent cyclosporin A, small decreases in the main transition temperature and associated enthalpy were observed, whereas at 30 mole percent cholesterol, the main transition is barely distinguishable from the baseline. The pretransition was not observed with the addition of 11 mole percent of either cyclosporin A or cholesterol. The results of the thermal analysis indicate that although cyclosporin A and cholesterol appear to be both located along the acyl chains of the phospholipids, they have dramatically different interactions with the membrane lipids.

The treatment of intravenously implanted Lewis lung carcinoma with two sustained release forms of 1-beta-D-arabinofuranosylcytosine.

Liposomes have been used in recent years as carriers for drugs and molecules of biological importance. In cancer chemotherapy, however, the advantages of liposome encapsulation of antitumor drugs remain uncertain, with the possible exception of the usefulness of encapsulated 1-beta-D-arabinofuranosyl-cytosine (ara-C), an antitumor drug of a very short half-life. Liposome-encapsulated ara-C has been shown by others to enhance significantly the survival time of mice bearing leukemia, and the enhancement may be attributable to the role of liposomes as a slow release system for ara-C. We now further explore the advantages of two sustained release systems for ara-C, namely the liposome-encapsulated ara-C and 1-beta-D-arabinofuranosylcytosine-5'-diphosphate-L-1,2-dipalmitin (ara-CDP-L-dipalmitin, a prodrug of ara-C). Intravenously implanted Lewis lung carcinoma is used as a solid tumor model. The therapeutic effectiveness of the two slow release forms of ara-C given by either i.v. or i.p. injections is examined. Viable tumor cells (1.0 X 10(5) cells/mouse) were inoculated i.v. and treatment was initiated 24 hr later using three schedules of multiple treatments for liposomal ara-C and single or multiple injections of ara-CDP-L-dipalmitin. Liposomal ara-C given by the i.p. route consistently increased the number of cures (greater than 120 days survival). For example, when nine small doses (10 mg/kg) were given on consecutive days by i.p. injections, 50% of mice given liposomal ara-C were cured, compared with 10% cures in the group given ara-C liposomes by i.v. and no cures in mice receiving free ara-C given according to the same schedules. On the other hand, ara-CDP-L-dipalmitin given at a single dose is more effective than an equal dose divided in five injections. However, no cures have been obtained by treatments with ara-CDP-L-dipalmitin. These results have further demonstrated the advantage of liposomes as carriers for antitumor drugs of short half-life.

The effects of charge and size on the interaction of unilamellar liposomes with macrophages.

The effect of the positive surface charge of unilamellar liposomes on the kinetics of their interaction with rat peritoneal macrophages was investigated using three sizes of liposomes: small unilamellar vesicles (approx. 25 nm diameter), prepared by sonication, and large unilamellar vesicles (100 nm and 160 nm diameter), prepared by the Lipoprep dialysis method. Charge was varied by changing the proportion of stearylamine added to the liposomal lipids (egg phosphatidylcholine and cholesterol, molar ratio 10:2.5). Increasing the stearylamine content of large unilamellar vesicles over a range of 0-25 mol% enhanced the initial rate of vesicle-cell interaction from 0.1 to 1.4 microgram lipid/min per 10(6) cells, and the maximal association from 5 to 110 micrograms lipid/10(6) cells. Cell viability was greater than 90% for cells incubated with large liposomes containing up to 15 mol% stearylamine but decreased to less than 50% at stearylamine proportions greater than 20 mol%. Similar results were obtained with small unilamellar vesicles except that the initial rate of interaction and the maximal association were less sensitive to stearylamine content. The initial rate of interaction, with increasing stearylamine up to 25 mol%, ranged from 0.5 to 0.7 microgram lipid/min per 10(6) cells, and the maximal association ranged from 20 to 70 micrograms lipid/10(6) cells. A comparison of the number and entrapped aqueous volume of small and large vesicles containing 15 mol% stearylamine revealed that although the number of large vesicles associated was 100-fold less than the number of small vesicles, the total entrapped aqueous volume introduced into the cells by large vesicles was 10-fold greater. When cytochalasin B, a known inhibitor of phagocytosis, was present in the medium, the cellular association of C8-LUV was reduced approx. 25% but association of SUV increased approx. 10-30%. Modification of small unilamellar vesicles with an amino mannosyl derivative of cholesterol did not increase their cellular interaction over that of the corresponding stearylamine liposomes, indicating that cell binding induced by this glycolipid may be due to the positive charge of the amine group on the sugar moiety. The results demonstrate that the degree of liposome-cell interaction with macrophages can be improved by increasing the degree of positive surface charge using stearylamine. Additionally, the delivery of aqueous drugs to cells can be further improved using large unilamellar vesicles because of their greater internal volume. This sensitivity of macrophages to vesicle charge and size can be used either to increase or reduce liposome uptake significantly by this cell type

Phospholipid-nucleoside conjugates. 5. The interaction of selected 1-beta-D-arabinofuranosylcytosine-5'-diphosphate-L-1,2-diacylglycerols with serum lipoproteins.

The phospholipid-nucleoside conjugates 1-beta-D-arabinofuranosylcytosine-5'-diphosphate-L-1,2-dipalmitin (1), -distearin (2), and -diolein (3) have been shown to interact rapidly with canine high density lipoprotein and with both high density and low density lipoproteins isolated from human serum. The extent of interaction with the high density lipoproteins appears to be dependent upon the characteristic gel-liquid crystalline phase transition of the conjugate's phospholipid. Since the phospholipid-nucleoside conjugates under study represent sustained release forms of the antileukemic agent 1-beta-D-arabinofuranosylcytosine, the therapeutic efficacy of these conjugates should now be considered in light of these interactions.

Enhanced iron removal from liver parenchymal cells in experimental iron overload: liposome encapsulation of HBED and phenobarbital administration.

The effectiveness of N,N'-bis[2-hydroxybenzyl]-ethylene-diamine-N,N'-diacetic acid (HBED) in removing radioiron introduced into the parenchymal cells of mouse liver as 59Fe-ferritin has been investigated. The effectiveness of HBED, an iron chelator of low water solubility, has also been compared with that of desferrioxamine (DF), an iron chelator of high water solubility and currently in clinical use for treatment of transfusional iron overload. Using the 59Fe excretion as the measure of effectiveness of chelation therapy and a standardized single chelator dose of 25 mg/kg, we have found that: (1) a saline suspension of HBED, prepared by sonication and given intraperitoneally to mice, promotes a small but significant increase in excretion of radioiron compared to the untreated controls, whereas DF, in its free form, is ineffective; (2) HBED encapsulated in lipid bilayers of liposomes and given intravenously is superior to nonencapsulated HBED; (3) DF encapsulated in small unilamellar liposomes is ineffective in removing iron given in the form of ferritin; (4) administration of phenobarbital in drinking water, at a concentration of 1 g/liter, induces a 30%-55% increase of iron excretion from untreated control mice and also from mice given HBED either in liposome-encapsulated or nonencapsulated form. We have demonstrated that HBED is superior to DF for removal of storage iron from liver parenchymal cells and that liposomes are useful carriers for iron chelators of low water solubility.

Improvement of iron removal from the reticuloendothelial system by liposome encapsulation of N,N'-bis[2-hydroxybenzyl]-ethylenediamine-N,N'-diacetic acid (HBED). Comparison with desferrioxamine.

An iron chelator of low water solubility, HBED, has been encapsulated in the lipid bilayers of unilamellar and multilamellar liposomes. The effectiveness of liposome-encapsulated HBED for removing excess iron burden from the RE system of the mouse liver (i.e., Kupffer cells) has been compared to that of the most commonly used iron chelator, DF, a water-soluble drug. We report the following: (1) At a single dose of 25 mg/kg, HBED in liposomes is more effective in removing excess iron than free nonencapsulated HBED. (2) HBED is a more potent iron chelator than DF; after a single dose of 25 mg/kg, about 25% of the originally injected iron is excreted within 7 days from mice given HBED either in small unilamellar or in large multilamellar liposomes, whereas about 18% is excreted from mice given the same dose of liposome-encapsulated DF. (3) Although the iron burden is introduced into the Kupffer cells, liposome-encapsulated HBED promotes iron excretion mainly via the bile and feces, whereas liposome-encapsulated DF promotes iron excretion through the kidney. (4) Cell fractionation studies show that encapsulation of HBED in the lipid bilayers of liposomes does not alter the uptake pattern of liposomes by the Kupffer and parenchymal cells of the liver; in other words, Kupffer cells are more effective in taking up large-sized multilamellar liposomes while parenchymal cells take up small-sized unilamellar liposomes more effectively. (5) Electron microscopic studies demonstrate that the liver biliary canaliculi are enlarged and filled with vesicular materials in mice given liposome-encapsulated HBED and that this condition does not occur in control mice or mice given liposome-encapsulated DF. Our results have thus demonstrated that liposomes could be very useful as injection vehicles for metal chelators that are not readily soluble in water. HBED is also demonstrated to be far superior to DF, the iron chelator of choice for therapy of transfusional iron overload.

Liposome uptake into human colon adenocarcinoma cells in monolayer, spinner, and trypsinized cultures.

The purpose of this study was to begin investigating the nature of liposome interactions with colon tumor cells. Thus, experiments were performed to study the uptake and incorporation of multilamellar and of reverse-phase evaporation liposomes of neutral charge into monolayers, suspended spinner cultures, and trypsinized cells of a human colon adenocarcinoma cell line, LS174T. The results showed that the same tumor cells cultured under each condition exhibited a distinct pattern of vesicle uptake as determined at 0, 15, 30, 60, and 120 min. In monolayer cultures of LS174T cells, the uptake of liposomes bearing [3H]actinomycin D in the lipid bilayers was linear throughout the incubation period. In contrast, in trypsinized and spinner suspension cultures, uptake of liposomes was biphasic. There was a proportional uptake of both liposome (labeled with [3H]phosphatidylcholine or [14C]cholesterol) and of actinomycin D (trace labeled with 3H) into the cells under all culture conditions, indicating quantitative delivery of the drug with the intact lipid vesicle. Although the amount of actinomycin D presented to tumor cells by the two liposomes was equivalent, reverse-phase evaporation liposomes were more effective than multilamellar vesicles in inhibiting uridine uptake. In the presence of excess liposomes (10 times the uptake studies), saturation of the tumor cell surface occurred by 120 min. However, the liposomes remained accessible to enzymatic removal for 60 min. Liposome-saturated tumor cells remained refractory to further binding of liposomes for at least 2 hr. The results thus revealed that differences in cell uptake were due to the state of the target cells and not the liposome types, or their differential leakage of labels.

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 micron) 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 micron) 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.

In vitro uptake and therapeutic application of liposome-encapsulated methotrexate in mouse hepatoma 129.

The biological activities of liposome-encapsulated and non-encapsulated methotrexate (MTX) were compared in vitro and in vivo using mouse Hepatoma 129 ascites tumor cells. Under in vitro conditions, cells accumulated up to 29% of [3H]-MTX when the drug was incorporated in the lipid bilayers of positively charged, unilamellar liposomes. There was no significant uptake of non-encapsulated MTX under the same conditions. A single intraperitoneal injection of liposome-encapsulated MTX (3 mg MTX/kg) increased the mean survival time of tumor-bearing mice to 42.5 +/- 11.2 days, compared to 23.5 +/- 2.2 days for untreated controls. Non-encapsulated MTX had no significant effect on survival time. Thus the in vivo treatment studies appear to agree with the in vitro uptake measurements. Addition of galactolipids to the lipid bilayers of liposomes did not increase in vitro uptake of encapsulated MTX and gave no additional improvement in therapeutic effectiveness. Encapsulation of MTX in liposomes might thus be used to increase uptake of the drug in cells which may be deficient in MTX transport.