Permeability and integrity properties of lecithin-sphingomyelin liposomes.

The properties of multibilayered liposomes formed from mixtures of sphingomyelin and phosphatidylcholine in varying mole ratio (all containing one mole dicetylphosphate per 10 moles of phospholipids) have been studied. The principal findings are: (1) Over the range 0 to 1 mole fraction sphingomyelin the liposomes exhibit multibilayer structure as visualized by electron microscopy using negative staining. (2) The two phospholipids differ in their interaction with dicetylphosphate in a bilayer structure. In mixtures of the two the effect of sphingomyelin is dominant. (3) The ability of sphingomyelin to form osmotically active liposomes depends on its fatty acid's composition. (4) Liposomes of all mole fractions of sphingomyelin are osmotically active if the C24: 1 fatty acid content of sphingomyelin exceeds 10% of the total acyl residues. The degree of osmotic activity, however, depends upon the molar ratio between the two phospholipids. The highest initial rate of water permeability was found for lecithin liposomes. The maximal change of volume by osmotic gradients was obtained for liposomes composed of 1:1 lecithin to sphingomyelin (mole ratio). (5) Permeability to glucose increased with increasing lecithin mole fraction. (6) Liposomes composed of 1:1 lecithin to sphingomyelin have the largest aqueous volume per mole of phospholipid as measured by glucose trapping. (7) The osmotic fragility of liposomes made of sphingomyelin is higher than for those made of lecithin but the highest osmotic fragility was obtained for liposomes containing lecithin and sphingomyelin in 1:1 molar ratio. (8) When the temperature is abruptly lowered to about 2 degrees C, lipsomes formed from phosphatidylcholine release about 20% of trapped glucose during a transient increase in permeability. Liposomes containing 0.5 mole fraction sphingomyelin release about 30% of the trapped glucose under these conditions. Liposomes composed of sphingomyelin alone do not exhibit this phenomenon.     

Influence of temperature on complement-dependent immune damage to liposomes

Maximal release of trapped liposomal glucose, in the presence of saturating amounts of liposomal antigen (galactocerebroside), antiserum (anti-galactocerebroside), and complement, was dependent on temperature. At lower temperatures (20--25 degrees C), maximal glucose release was inversely related to liposomal phospholipid fatty acyl chain length (dimyristoyl phosphatidylcholine > dipalmitoyl phosphatidylcholine > distearoyl phosphatidylcholine > sphingomyelin). At higher temperatures (32--35 degrees C) a limiting plateau of glucose release, at approx. 60%, was reached, or approached, by all preparations. Sphingomyelin liposomes still released less glucose than those prepared from other phospholipids, even at 35 degrees C. The titers of antiserum and complement (ABL50/ml and CL50/ml) were dependent on temperature, and differences based on liposomal phospholipid fatty acyl chain length were observed. Analysis of antiserum and complement-dependence on temperature, and on phospholipid type, revealed that although antibody binding to galactocerebroside undoubtedly was subject to steric hindrance due to interference by surrounding phospholipids at 20--25 degrees C, steric hindrance did not play a major role in blocking antibody binding above 32 degrees C.     

Membrane-damaging action of staphylococcal alpha-toxin on phospholipid-cholesterol liposomes

The mechanism of membrane damage by staphylococcal alpha-toxin was studied using carboxyfluorescein (internal marker)-loaded multilamellar liposomes prepared from various phospholipids and cholesterol. Liposomes composed of phosphatidylcholine or sphingomyelin and cholesterol bound alpha-toxin and released carboxyfluorescein in a dose dependent manner, when they were exposed to alpha-toxin of concentrations higher than 1 or 8 micrograms/ml, respectively. In contrast, the other liposomes composed of phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol or phosphatidylinositol plus cholesterol were not susceptible to the toxin even at high concentrations up to 870 micrograms/ml. The insensitive liposomes containing either phosphatidylserine or phosphatidylglycerol were made sensitive to alpha-toxin by inserting phosphatidylcholine into the liposomal membranes. In addition, phosphorylcholine inhibited the toxin-induced marker release from liposomes. These results indicated that the choline-containing phospholipids are required for the interaction between alpha-toxin and liposomal membranes. Susceptibility of liposomes containing phosphatidylcholine or sphingomyelin increased with the increase in cholesterol contents of the liposomes. Based on these results, we propose that the choline-containing phospholipids are possible membrane components or structures responsible for the toxin-membrane interaction, which leads to damage of membranes. Furthermore, cholesterol may facilitate the interaction between alpha-toxin and membrane as a structural component of the membrane.     

The influence of retinal on complement-dependent immune damage to liposomes

Retinal was incorporated into liposomes containing dipalmitoyllecithin, cholesterol, dicetyl phosphate and galactocerebroside; the latter substance served as antigen. They were compared to control liposomes, lacking retinal, with regard to glucose release due to complement-dependent immune damage in the presence of anticerebroside serum. The liposomes were indistinguishable from each other in the amount of total glucose trapped, light scattering characteristics and phosphate content. The rate and extent of glucose release in 30 min was inhibited by the incorporation of retinal. In addition, inhibition was directyl related to retinal concentration and was also observed in the presence of a wide range of concentrations of antigen and complement. Damage to liposomes in the presence of either guinea pig or human complement was inhibited by retinal; this was in contrast to the erythrocyte system in which the hemolytic activity of guinea pig complement was inhibited while that of human complement was enhanced by retinal. Addition of retinal to performed liposomes did not influence complement-dependent damage. Inhibition occurred only when retinal was present during the initial formation of the model membranes. Inhibition persisted even after washing the liposomes free of any unincorporated retinal. The data indicate that liposomes may be an excellent model for studying the influences of retinal on complement mechanism in membranes.     

Naturally occurring antibodies to liposomes. II. Specificity and electrophoretic pattern of rabbit antibodies reacting with sphingomyelin-containing liposomes

Sera obtained from rabbits after immunization with a variety of unrelated antigens contain antibodies that induce complement- (C) mediated lysis of sphingomyelin-containing liposomes in the absence of the relevant antigen from the membrane. Absorption or inhibition with dimyristoyl-phosphatidyl choline-containing liposomes were less effective than with sphingomyelin-containing liposomes in decreasing or abolishing C-dependent lysis of target-liposomes. Phosphoryl choline chloride inhibited the C-dependent lysis mediated by these antibodies, but only when used in high molar excess and in the presence of low antibody concentrations. Purified anti-liposome antibodies displayed an isoelectric focusing pattern consistent with a polyclonal response. The findings confirm the antibody nature of the anti-liposome activity of rabbit sera and indicate that their predominant specificity is directed against conformations of the phospholipid molecule in which the polar (phosphoryl choline) group does not have a major contribution.     

Membrane damage of liposomes by the mushroom toxin phallolysin

We have investigated the membrane-damaging effect of phallolysin on liposomes varying in phospholipid composition, net charge and physical constitution. Liposomes were prepared from lipids extracted from bovine or human erythrocyte ghosts. The liposomes composed of bovine lipids (the intact cell showing little sensitivity to phallolysin) were found comparably sensitive to those prepared from lipids of human red cells (these cells being of high sensitivity). In addition, artificial mixtures of lipids were used for the preparation of liposomes, consisting of (a) negatively charged phospholipids such as dicetyl phosphate or phosphatidylserine, (b) cholesterol, and (c) either sphingomyelin (as the major component of erythrocytes from ruminants) or phosphatidylcholine (as the major component of erythrocytes from non-ruminants). Again, we found only little difference in the susceptibilities of sphingomyelin- and phosphatidylcholine-containing liposomes. On the other hand, the susceptibility depended on the presence of phospholipids with negative net charges. Omittance of phosphatidylcholine or dicetyl phosphate, or replacement by the positively charged stearylamine, decreased the susceptibility by a factor of more than 20. Finally, we prepared liposomes from dicetyl phosphate, cholesterol and phosphatidylcholine in two physical states: large unilamellar and smaller multilamellar liposomes. The unilamellar liposomes were about 10-times more sensitive to phallolysin. We conclude: (1) Phallolysin damages phospholipid-membranes in the absence of receptor proteins, but high concentrations of the toxin are required. (2) Membrane damage takes place with liposomes containing phosphatidylcholine as well as those containing sphingomyelin. (3) Phallolysin damages only liposomes containing phospholipids with a negative net charge.     

Permeability properties of liposomes prepared from dipalmitoyllecithin, dimyristoyllecithin, egg lecithin, rat liver lecithin and beef brain sphingomyelin

Liposomes have been prepared from dipalmitoyllecithin, dimyristoyllecithin, egg lecithin, rat liver lecithin and beef brain sphingomyelin.Permeability properties of liposomes thus prepared were studied toward glucose. The glucose permeability of liposomes with saturated lecithins (dipalmitoyllecithin and dimyristoyllecithin) and sphingomyelin appears to be more strongly temperature dependent than that of liposomes with lecithin containing unsaturated fatty acyl chains (egg and rat liver lecithins). The permeability of glucose through vesicles of dipalmitoyllecithin or dimyristoyllecithin was enhanced drastically at their transition temperatures, while the incorporation of about 25 mole% of egg lecithin into liposomes of saturated lecithins suppressed the enhanced permeation rates of glucose above the transition temperatures.The incorporation of small amounts of cholesterol enhanced the temperature-dependent permeability of glucose through the bilayer of saturated lecithins or sphingomyelin. This tendency was best shown in the case of dipalmitoyl-lecithin, in which 20 mole% of cholesterol had the most stimulating effect on the temperature-dependent permeability. The introduction of more than 33 mole% of cholesterol showed, however, reduced effects on the temperature-dependent permeability through liposomes with saturated lecithins or sphingomyelin. It was also shown that cholesterol had a much larger effect on the regulation of the temperature-dependent permeability of liposomes prepared with saturated lecithins or sphingomyelin than on that of liposomes prepared with phospholipids containing unsaturated fatty acids.     

Release of macromolecular markers (enzymes) from liposomes treated with antibody and complement: An attempt at correlation with electron microscopic observations

Antibody-complement dependent damage to liposomal model membranes has been previously investigated by measuring the release of low molecular weight markers such as glucose. To determine whether larger solutes are also released under these conditions, experiments have been performed using immunologically sensitive liposomes that contained not only trapped glucose, but also enzymes (hexokinase, glucose-6-phosphate dehydrogenase, β-galactosidase) as macromolecular markers. The largest of these enzymes (β-galactosidase) has dimensions which closely approximate the diameter of the lesions detected by negative staining in natural membranes after immune lysis. Liposomes prepared with lecithin, and either actively sensitized with globoside or passively sensitized with alkali-treated lipopolysaccharide, released the enzymes in parallel with glucose upon incubation with the appropriate antiserum and native guinea pig serum as source of complement. Immune damage to sphingomyelin liposomes was characterized by a significantly lower loss of the enzymes in comparison to the percentage of glucose released; a comparable response was manifested by liposomes prepared from sheep erythrocyte lipids. Electron microscopic examination of negatively stained lecithin liposomes, which had released the macromolecular markers, failed to reveal the characteristics lesions; these findings are consistent with evidence obtained by other laboratories suggesting that the lesions may not correspond to functional holes. Lesions were, however, consistently observed in liposome preparations that had been treated with the polyene antibiotics, filipin; this antibiotic causes appreciable loss of both glucose and enzymes from either lecithin or sphingomyelin liposomes.     

Cholesterol-dependent tetanolysin damage to liposomes.

Tetanolysin caused membrane damage, resulting in release of trapped glucose from liposomes containing cholesterol. Maximum glucose release occurred from liposomes that contained 50 mol% cholesterol. At higher or lower levels of cholesterol, glucose release was reduced and glucose release did not occur at all below 40 mol% cholesterol. The apparent activity of tetanolysin was not influenced by temperature (24 degrees C compared to 32 degrees C) or by liposomal phospholipid fatty acyl chain length. We conclude that tetanolysin caused cholesterol-dependent lysin-mediated damage to liposomes, possibly by means of a pore consisting of a complex of toxin and cholesterol.     

Physicochemical consequences of opsonization: perturbation of liposomal membranes by Salmonella typhimurium 395 MS opsonized with IgG antibodies.

When phagocytosis-resistant Salmonella typhimurium 395 MS bacteria sensitized with anti-MS IgG antibodies were incubated with liposomes composed of phosphatidylcholine, cholesterol, and dicetylphosphate, a previously sequestered liposomal marker (4-methylumbelliferylphosphate) was released. Unsensitized or F(ab')2-sensitized bacteria had no such effect. This perturbation was neither dependent upon negatively charged dicetylphosphate nor upon cholesterol. It was further evident that palmitoyl-poly(ethyleneglycol) caused release of the trapped marker in a similar way as sensitized bacteria. These findings demonstrate a similarity between sensitized bacteria and a hydrophobic probe and lend support to the hypothesis that the perturbation was brought about by hydrophobic interaction. The observations indicate that liposomes, like phagocytes, possess "receptor sites" for the activated part of IgG and raise the possibility that phagocytic effectors can operate in a relatively nonspecific manner.     

Ketorolac tromethamine liposomes: encapsulation and release studies

Liposomes loaded with ketorolac tromethamine salt were prepared by using a thin layer evaporation method. The physical properties of liposomes were studied by using atomic force microscopy (AFM) and transmission electron microscopy (TEM). The relationship between lipid composition, encapsulation efficiency, vesicle size, and the release of ketorolac tromethamine-loaded liposomes was studied. The drug content was found to be dependent on the lipidic composition used in the preparations and, in particular, vesicles containing both cationic lipids (dimethyldioctadecylammonium bromide and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride), and phosphatidylcholine had a higher entrapped efficiency than liposomes with phosphatidylcholine alone or in the presence of cholesterol. Finally, the cationic liposomes appear to be useful as carriers for ketorolac tromethamine to control its in vitro release.     

Enzyme replacement via liposomes variations in lipid composition determine liposomal integrity in biological fluids

Liposomes survive exposure to biological fluids poorly, extruding trapped enzymes, drugs, or solutes upon interaction with serum or plasma constituents. We have quantified the disruptive effects of human serum on liposomes and have studied whether various modifications in their phospholipid composition might produce liposomes with an increased carrier potential for applications in vivo. Multilamellar liposomes (phosphatidylcholine 70:dicetyl phosphate 20: cholesterol 10) were prepared with ³H-labeled phosphatidylcholine as the lipid phase marker and [¹⁴C]inulin and horseradish peroxidase as aqueous phase markers. Gel exclusion chromatography showed that 32 ± 3% of [¹⁴C]inulin and 27 ± 7% of horseradish peroxidase were lost after 1 h incubation with 10% (v/v) human serum. Loss of aqueous solutes was reduced to 20 ± 5%/h and 17 ± 2%/h, respectively, after treatment with decomplemented serum (56°C, 30 min). Loss induced by serum was concentration and time dependent: to 57 ± 2% at 1 h and 67 ± 14% at 24 h, with 50% serum; plasma was slightly less perturbing whereas human serum albumin was not at all disruptive. By incorporating sphingomyelin (35 mol%) into multilamellar liposomes, the leakage of [¹⁴C]-inulin in the presence of 10% serum was reduced to 12 ± 4%/h; increasing the molar percentage of cholesterol to 35% also stabilized the lipid bilayers, reducing leakage to 20 ± 7%/h. Both small and large unilamellar vesicles could not be stabilized against serum-mediated leakage by the incorporation of sphingomyelin. The data suggest that cholesterol and sphingomyelin enhance liposomal integrity in the presence of serum or plasma and promise to yield enhanced survival of drug-laden lipid vesicles in biological fluids in vivo.     

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.     

Antilipose antisera activity against negatively charged phosphate amphiphils.

Complement-immune lysis of liposomes loaded with water-soluble spin label 2,2,6,6-tetramethyl-piperidinoxy-choline chloride (TEMPO-choline) was used to measure specificity of rabbit antisera generated by complete adjuvant with liposomes consisting of sphingomyelin; cholesterol; dicetylphosphate; 5-N-thyroxine-2,4-dinitrophenyl -phosphatidylethanolamine (T₄-Dnp-PE) in the ratio 2.0:1.5:0.2:15. Antisera so generated induced complement lysis of the liposomes containing negatively charged amphiphils. No immune lysis of positively charged liposome was observed. This antiliposome specificity is retained in partially purified antibodies.     

Cholesterol-dependent tetanolysin damage to liposomes

Tetanolysin caused membrane damage, resulting in release of trapped glucose from liposomes containing cholesterol. Maximum glucose release occurred from liposomes that contained 50 mol% cholesterol. At higher or lower levels of cholesterol, glucose release was reduced and glucose release did not occur at all below 40 mol% cholesterol. The apparent activity of tetanolysin was not influenced by temperature (24°C compared to 32°C) or by liposomal phospholipid fatty acyl chain length. We conclude that tetanolysin caused cholesterol-dependent lysin-mediated damage to liposomes, possibly by means of a pore consisting of a complex of toxin and cholesterol.     

Repeated injections of PEG-PE liposomes generate anti-PEG antibodies

Liposomes containing the polyethylene glycol (PEG) derivative of phosphatidyl ethanolamine (PE) have recently been found to be promising drug carriers, as they facilitate controlled and target-oriented release of therapeutics. They also reduce the side effects of many drugs. Here, we present the results of a study on antiliposomal properties of rabbit sera obtained after weekly injections of small liposomes containing 20% PEG-PE. The effect was analysed as the level of induced carboxyfluorescein release from these liposomes in vitro. The incubation of liposomes with rabbit serum taken after the injections induced the release of carboxyfluorescein at a higher level than was seen for incubation with untreated animal's serum. The strongest effect was observed for serum obtained after the second injection, i.e. during the second week of the study. The effect was much smaller after the serum samples were preheated at 56 degrees C. The binding of serum proteins by PEGylated liposomes was analysed via gel filtration and via the immunoblot technique using goat anti-rabbit IgG; this revealed that the serum protein which bound to the liposomes in vitro had a molecular weight of 55 kD and reacted with the anti-IgG antibody. Competition with PEG or lipids indicate that this IgG has an anti-PEG activity. We therefore assume that these antibodies are responsible for the activation of complement and leakage induction of PEG-liposomes. Such antibodies could be responsible for increased phagocytosis by RES macrophages (in particular liver macrophages) and decreased circulation time.     

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.     

Enzyme replacement via liposomes. Variations in lipid compositions determine liposomal integrity in biological fluids.

Liposomes survive exposure to biological fluids poorly, extruding trapped enzymes, drugs, or solutes upon interaction with serum or plasma constituents. We have quantified the disruptive effects of human serum on liposomes and have studied whether various modifications in their phospholipid composition might produce liposomes with an increased carrier potential for application in vivo. Multilamellar liposomes (phosphatidycholine 70:dicetyl phosphate 20:cholesterol 10) were prepared with 3H-labeled phosphatidylcholine as the lipid phase marker and [14C]inulin and horseradish peroxidase as aqueous phase markers. Gel exclusion chromatography showed that 32 +/- 3% of [14C]inulin and 27 +/- 7% of horseradish peroxidase were lost after 1 h incubation with 10% (v/v) human serum. Loss of aqueous solutes was reduced to 20 +/- 5%/h and 17 +/- 2%/h, respectively, after treatment with decomplemented serum (56 degrees C, 30 min). Loss induced by serum was concentration and time dependent: to 57 +/- 2% at 1 h and 67 +/- 14% at 24 h, with 50% serum; plasma was slightly less perturbing whereas human serum albumin was not at all disruptive. By incorporating sphingomyelin (35 mol%) into multilamellar liposomes, the leakage of [14c]-inulin in the presence of 10% serum was reduced to 12 +/- 4%/h; increasing the molar percentage of cholesterol to 35% also stabilized the lipid bilayers, reducing leakage to 20 +/- 7%/h. Both small and large unilamellar vesicles could not be stablilized against serum-mediated leakage by the incorporation of sphingomyelin. The data suggest that cholesterol and sphingomyelin enhance liposomal integrity in the presence of serum or plasma and promise to yield enhanced survival of drug-laden lipid vesicles in biological fluids in vivo.     

Raman spectroscopic investigation of the interaction of gramicidin A with dipalmitoyl phosphatidylcholine liposomes.

The interaction of gramicidin A with dipalmitoyl phosphatidylcholine liposomes is investigated by Laser-Raman spectroscopy. As revealed by the methylene C-H stretching mode the phase transition of the hydrocarbon chains near 40 degree C is eliminated in the presence of gramicidin A. Liposomes prepared from a mixture of lecithin and cholesterol seem to be unaffected by gramicidin A and show only the normal broadened phase transition.     

Differential scanning calorimetry characterization of oxidized egg phosphatidylcholine liposomes

Differential scanning calorimetry has been used to characterize liposomes made from mixtures of unoxidized and singlet oxygen oxidized egg phosphatidylcholine. Cooling scans reveal that trapped water decreases when the oxidized phosphatidylcholine content is increased in the liposomes. Liposomes made from mixtures containing more than 50% by weight of oxidized phosphatidylcholine do not show trapped water.