Transfer of cholesterol between liposomal membranes.

The transfer of cholesterol between liposomal membranes was examined. On incubation of liposomes compsoed of egg yolk phosphatidylcholine, phosphatidic acid and cholesterol (molar percentage, 65.8 : 1.3 : 32.9 or 65.5 : 6.3 : 31.2), almost complete equilibration of the cholesterol pools was achieved within 6 to 8 h at 37 degrees C. The rate of transfer of cholesterol from the liposomes, in which cholesterol was introduced by 'the exchange reaction', was not significantly different from that from liposomes prepared in the presence of cholesterol, in which the cholesterol was distributed homogenously. These findings indicate that half life for 'flip-flop' of cholesterol molecules in egg yolk phosphatidylcholine liposomes is less than 6 h at 37 degrees C. The transfer of cholesterol between liposomes was strongly dependent on temperature and was affected by the fatty acid composition of the phospholipid, suggesting that the 'fluidity' of the membranes strongly influences the transfer rate. A preferential distribution of cholesterol molecules was observed in heterogeneous liposomes with different classes of phospholipids. The 'affinity order' of cholesterol for phospholipid deduced from the present experiments is as follows: beef brain sphingomyelin greater than dipalmitoylglycerophosphocholine = dimyristoylglycerophosphocholine greater than egg yolk phosphatidylcholine.

Effect of polymyxin B on liposomal membranes derived from Escherichia coli lipids.

The specificity of the action of polymyxin B was studied using liposomes as a model membrane system. Liposomes prepared from total lipids of Gram-negative bacteria Escherichia coli, a mixture of purified E. coli phosphatidylethanolamine and cardiolipin and a mixture of phosphatidylethanolamine and phosphatidylglycerol, were extemely sensitive to polymyxin while those prepared from lipids of Gram-positive bacteria Streptococcus sanguis, lipids of sheep erythrocyte membranes, mixtures of egg lecithin and negatively charged amphiphatic molecules, were less sensitive to the action of the antibiotic. Cholesterol was shown to suppress the polymyxin-induced response in liposomes.

The activity of membranes reconstituted from HVJ envelope proteins and lipids to induce hemolysis and fusion between liposomes and erythrocytes.

A simple method for preparation of lipid-free envelope proteins (HN protein and F protein) of HVJ (Sendai virus) was developed. Reconstituted 'envelopes' were then prepared from envelope proteins and various lipids by the detergent dialysis method, and the activity to induce hemolysis and fusion between liposome and erythrocyte was studied. Lipid-free envelope protein aggregates could induce hemolysis and liposome-erythrocyte fusion. The activity was however greatly augmented by incorporation of envelope proteins into membrane of viral total lipids. Hemolytic and fusogenic activity was somewhat augmented by incorporation of envelope proteins into dipalmitoylphosphatidylcholine/cholesterol (1:1, molar ratio) and dimyristoylphosphatidylcholine/cholesterol (1:1), though the augmentation was lower than that observed with viral total lipids. When 'envelopes' were reconstituted with the proteins and viral total lipids supplemented with phosphatidylethanolamine, two kinds of 'envelopes' were prepared; one was permeable to Dextran (Mr 75000) and hemolytic, and the other was impermeable to Dextran and nonhemolytic. The latter acquired hemolytic activity after subjection to freezing and thawing, and its barrier function was lost concomitantly. The study suggests that envelope proteins (HN protein and F protein) could function without lipids but their activity was greatly influenced by not only the composition of additional lipids but also mode of arrangement of components on the reconstituted membranes.

Selective release of non-electrolytes from liposomes upon perturbation of bilayers by temperature change or polyene antibiotics.

A new system for assaying the permeability characteristics of liposomes was established using Amicon cells equipped with a membrane filter (pore size, 0.3 micrometer). In this system, damage of liposomes during the assay procedure was negligible. Changes in permeability to non-electrolytes, such as glucose (Mr 180), sucrose (Mr 342), inulin (Mr 5000) and dextran (Mr 75000), induced by perturbation of the bilayers were examined with this system. The following results were obtained on the barrier properties of multilamellar liposomes modified by various treatments. 1. Amphotericin B and nystatin did not cause any change in permeability to glucose of egg yolk phosphatidylcholine liposomes prepared in physiological saline and containing trace amounts of radioactive markers in their aqueous compartments. Both antibiotics, however, induced nonspecific release of glucose, sucrose, inulin and dextran from liposomes that contained 0.3 M glucose in their aqueous compartments. These antibiotics first seem to form pores through which small ions can permeate; Na+ and Cl- can enter the liposomes through these pores, whereas glucose in the liposomes cannot pass out. As a result, the liposomes become swollen with consequent severe disruption of their membranes. 2. Filipin and digitonin disrupted the membrane structures, resulting in release of large molecules such as dextran even in the absence of an osmotic mechanism. 3. Perturbation of the phase equilibrium by temperature change resulted in formation of 'pores'. The penetration of cations and anions through these 'pores' was apparently much faster than that of glucose, since when liposomes swollen in 0.3 M glucose were incubated in salt solution they were disrupted by an osmotic mechanism releasing not only glucose but also dextran. Most of the 'pores' were not large enough to allow passage of large non-electrolytes, such as inulin and dextran, since no appreciable amounts of these markers were released from liposomes under conditions where there should be no osmotic gradient. 4. At a temperature well above the phase transition temperature, egg yolk phosphatidylcholine liposomes exhibited specific release of glucose. This process did not involve an osmotic gradient, indicating that it was mainly due to diffusion of the solutes through the bilayers.

Studies on the interaction of Sendai virus with liposomal membranes. Sendai virus-induced agglutination of liposomes containing glycophorin.

Liposomes constituted with the major sialoglycoprotein of human erythrocytes, glycophorin, were used as models for studies on cell-virus interactions. Liposomes composed of egg yolk phosphatidylcholine, cholesterol and glycophorin were found to interact with the paramyxovirus HVJ to form aggregates. The aggregation process was temperature dependent: it was maximal at 0 degrees C and decreased with increase of the incubation temperature. The activity of viral neuraminidase is also temperature dependent, and it increases with increase of the incubation temperature; release of N-acetylneuraminic acid was negligible at 0 degrees C. Shift-up of the incubation temperature immediately cancelled HVJ-induced agglutination of liposomes. Viruses attached to liposomes seemed to be released into the supernatant when the 'virus-liposome' complex formed at 0 degrees C was incubated at 37 degrees C, possibly as a result of breakdown of the 'binding' site by neuraminidase. The characteristics of the interaction of HVJ with liposomes containing glycophorin appeared to be phenomenologically similar to those of HVJ-cell interaction.

Haptenic activity of galactosyl ceramide and its topographical distribution on liposomal membranes. I. Effect of cholesterol incorporation.

The relation between the immune-reaction of phosphatidylcholine liposomes containing spin-labeled galactosyl ceramide with or without cholesterol and the topographical distribution of the glycolipid in membranes was studied. In egg yolk phosphatidylcholine liposomes, both immune agglutination and antibody binding occurred, irrespectively of the presence of cholesterol, though the motion of the fatty acyl chain of spin-labeled galactosyl ceramide was restricted by cholesterol. In dipalmitoyl phosphatidylcholine liposomes, unlike in egg yolk phosphatidylcholine liposomes, the immune-reaction depended on the cholesterol content. The electron spin resonance (ESR) spectra of spin-labeled galactosyl ceramide in dipalmitoyl phosphatidylcholine liposomes indicated that cholesterol affected the topographical distribution of spin-labeled galactosyl ceramide in the liposomes. Without cholesterol, most of the spin-labeled galactosyl ceramide was clustered on the dipalmitoyl phosphatidylcholine membrane, but with increase of cholesterol, random distribution of hapten on the membrane increased. The cholesterol-dependent change in the topographical distribution of hapten on the membranes was parallel with that of immune reactivity. 'Aggregates' composed solely of galactosyl ceramide did not show any binding activity with antibody. The findings suggest that the recognition of galactosyl ceramide by antibody depended on the topographical distribution of hapten molecules. Phosphatidylcholine and/or cholesterol may play roles as 'spacers' for the proper distribution of 'active' haptens on the membranes. The optimum density of haptens properly distributed on liposomal membranes is discussed.

Peroxidation of liposomes in the presence of human erythrocytes and induction of membrane damage of erythrocytes by peroxidized liposomes.

Hemolysis (Kobayashi, T., Takahashi, K., Yamada, A., Nojima, S. and Inoue, K. (1983) J. Biochem. 93, 675-680) and shedding of acetylcholinesterase-enriched membrane vesicles (diameter 150-200 nm) were observed when human erythrocytes were incubated with liposomes of phosphatidylcholine which contained polyunsaturated fatty acyl chains. These events occurring on erythrocyte membrane were inhibited by radical scavengers or incorporation of alpha-tocopherol into liposomes, suggesting that lipid peroxidation is involved in the process leading to membrane vesiculation and hemolysis. The idea was supported by findings that generation of chemiluminescence, formation of thiobarbituric acid reactive substance, accumulation of conjugated diene compounds in liposomes and decrease of polyunsaturated fatty acids in liposomes occurred concomitantly during incubation. Hemolysis was also suppressed by the addition of extra liposomes, insensitive to peroxidation, or of serum albumin even after the completion of peroxidation of liposomes. These results suggest that peroxidized lipids, responsible for vesiculation and hemolysis, may be formed first in liposomes and then gradually transferred to erythrocyte membranes. The accumulation of these lipids peroxides may eventually cause membrane vesiculation followed by hemolysis.

Liposomal membranes. XI. A suggestion to structural characteristics of acido-thermophilic bacterial membranes.

To understand the role of omega-cyclohexyl fatty acid residue of lipids in acido-thermophilic bacterial membranes, three unusual phosphatidylcholines, 1, 2-di-11-cyclohexylundecanoyl-L-alpha-phosphatidylcholine (11CYPC), 1,2-di-13-cyclohexyltridecanoyl-L-alpha-phosphatidylcholine (13CYPc), and 1-13-cyclohexyltridecanoyl-2-11-cyclohexylundecanoyl-L-alpha- phosphatidylcholine (1-13CY-2-11CYPC) were prepared and the steady-state fluorescence anisotropy of 1, 6-diphenylhexatriene (DPH) in the hydrophobic domain of these liposomal bilayers was determined. Compared with the case of dipalmitoyl (DPPC) or dimyristoyl phosphatidylcholine (DMPC), introducing the omega-cyclohexyl moiety onto lecithins makes the bilayers fluid below the phase transition temperature, while immobilizes them above the phase transition temperatures. The properties of the unusual phosphatidylcholine liposomes suggested by the steady-state fluorescence anisotropy investigation were in good agreement with those obtained from the thermotropic and permeability investigations. Results obtained are discussed from the view point of the role and function of lipid membranes of acido-thermophilic bacteria which contain unusual fatty acids.

Electron microscopic study on the interaction of Sendai virus with liposomes containing glycophorin.

The interaction of liposomes containing glycophorin, a major sialoglycoprotein of human erythrocytes, with Sendai virus was studied by freeze-fractures and negative staining electron-microscopy. Viral envelopes were absorbed on liposomal membranes at 0 degrees C. When the temperature was shifted up to 37 degrees C, the viral envelopes fused with the liposomal membranes (envelope fusion). Particles representing viral membrane components formed clusters on liposomal membranes after incubation for more than 1 h at 37 degrees C.

ESR study on synthetic glyceroglycolipid liposomal membranes.

We previously reported that glyceroglycolipid liposomes without cholesterol activated mouse peritoneal macrophages in vivo and in vitro, whereas glyceroglycolipid liposomes containing equimolar cholesterol did not. In order to characterize the properties of the glyceroglycolipid membranes, ESR spectroscopic studies were carried out with an acyl spin-labeled galactosyl ceramide (SL-GC) or a headgroup spin-labeled phospholipid (SL-6-DPPA) in 1,2-dipalmitoyl[beta-cellobiosyl-(1'---3)]glycerol (Cel-DAG) liposomal membranes. The ESR spectrum of the SL-GC in the Cel-DAG liposomes at 37 degrees C was a single broad line, indicating that the SL-GC molecules were excluded almost completely from Cel-DAG domains and formed clusters in the membranes. The spectrum of SL-6-DPPA in the Cel-DAG liposomes at 37 degrees C showed broad resonance lines with the central peak being the highest, while that at 60 degrees gave narrow lines with the low-field peak being the highest. This observation and rotational correlation time analysis showed that the molecular motions of spin-label moiety of the SL-6-DPPA were extremely restricted at 37 degrees C but not above Tc. These results suggest that below Tc the Cel-DAG molecules are packed tightly and restricted in motion in the membrane. Incorporation of cholesterol into the Cel-DAG liposomal membranes gave (1) the spectra of the SL-GC triplet, and (2) the spectra of the SL-6-DPPA narrow resonance with the low-field peak being the highest. These results suggest that cholesterol disturbs the rigid-packed structure of the Cel-DAG membrane and increases the molecular motions of the Cel-DAG. The DSC analysis of Cel-DAG with and without cholesterol agreed well to the results of the ESR technique. Thus we assume that peritoneal macrophages recognize the rigid-packed carbohydrate residues which are restricted in motion on the Cel-DAG membranes.

HVJ-mediated fusion between erythrocyte membranes and liposomes containing glycophorin.

The HVJ-mediated interaction of liposomes containing glycophorin with human erythrocytes was studied. A ternary complex of erythrocyte-HVJ-liposome was formed on incubation at 4 degrees C. Spin-labeled phosphatidylcholine and spin-labeled galactosylceramide, both of which were incorporated into liposomes, were effectively transferred into erythrocytes upon shifting-up of the incubation temperature to 37 degrees C. This finding indicates that fusion between glycophorin liposomes and erythrocytes occurs in the presence of HVJ. Fusion could be also inferred from the transfer of radio-labeled lipids embedded in the liposomal membranes to erythrocyte membranes as well as uptake of solutes entrapped in the aqueous space of the liposomes by erythrocytes. The total amount of liposomes fused with erythrocytes depended on the glycophorin content of liposomes and the amount of HVJ added. No transfer was observed when trypsinized HVJ was used in place of HVJ, suggesting that the fusion is F protein dependent.

Reactivity of human C-reactive protein with positively charged liposomes.

Interaction of CRP with liposomes was studied by agglutination, complement consumption, and the binding assay. Liposomes composed of phosphatidylcholine and stearylamine were agglutinated with CRP. The interaction of CRP and liposomes was further demonstrated by the consumption of hemolytic complement activity. Stearylamine could be substituted by cetyltrimethylammonium bromide, indicating that positively charged molecules were required for the interaction. Agglutination of liposomes caused by CRP was dependent on the fatty acid composition of phosphatidylcholine, cholesterol content and temperature. Differential scanning calorimetry measurement revealed that both the agglutination of liposomes and the consumption of complement caused by CRP required the fluid state of membranes. Particle electrophoresis measurement showed that stearylamine in fluid state membranes was more dispersed than in rigid state membranes indicating that lateral distribution of stearylamine affected the reactivity of liposomes. The mode of complement activation by the interaction of CRP with positively charged liposomes was similar to that observed in the system of protamine and heparin.

Immune reactions of liposomes containing cardiolipin and their relation to membrane fluidity.

The relation between the immune reactions of phosphatidylcholine liposomes containing cardiolipin with or without cholesterol and the physical state of the liposomes was studied. In egg yolk phosphatidylcholine liposomes, both immune agglutination and complementmediated immune damage occurred irrespective of the presence of cholesterol. Immune agglutination also occurred at all temperatures tested (0--40 degrees C). On the other hand, in dipalmitoylphosphatidylcholine liposomes, both reactions depended on the cholesterol content, but immune agglutination occurred even in the absence of cholesterol above the phase transition temperature at which liposomal membranes became fluid. The finding of antibodies to liposomes was found to be independent of the physical state of the liposomal membranes. The requirement of the fluid state of liposomal membranes for immune reactions is discussed.

Transfer of steroids and alpha-tocopherol between liposomal membranes.

The transfer of various steroids and alpha-tocopherol between liposomes was examined. The transfer rate of cholest-4-en-3-one and epicholesterol between egg yolk phosphatidylcholine liposomes was much higher than that of cholesterol. Rapid transfer was also noted for coprostanol and 5-cholesten-3 beta-ol-7-one, while the transfer of cholestanol, which has a 3 beta-hydroxyl residue and a planar nucleus structure, was similar to that of cholesterol. These results suggest that the mode of interaction between steroid and phospholipid may be an important factor controlling the rate of transfer. It was found that alpha-tocopherol could also be transferred between liposomes. The transfer rate of alpha-tocopherol was dependent on the temperature and the phospholipid composition.

Properties of liposomal membranes containing lysolecithin.

Liposomes have been prepared with lysolecithin (1-acyl-sn-3-glycerylphosphorylcholine), egg lecithin (3-sn-phosphatidylcholine), dicetyl phosphate, and cholesterol. The ability to function as a barrier to the diffusion of glucose marker and the sensitivities of the liposomes to hypotonic treatment and other reagents which modified the permeability were examined. Generally, lysolecithin incorporation decreased the effectiveness of the membranes as a barrier to glucose and made the membranes more "osmotically fragile." Cholesterol incorporation counteracted the effect of incorporated lysolecithin. The more cholesterol incorporated into liposomes, the more lysolecthin could be incorporated into the membrane without loss of function as a barrier. With more than 50 mole% of colesterol, lysolecithin alone could form membranes which were practically impermeable to glucose. The hemolytic activity of lysolecithin was affected by mixing with various lecithins or cholesterol. Liposomes containing lysolecithin, which have the ability to trap glucose marker, showed poor hemolytic activity, while lipid micelles with lysolecithin (which could trap little glucose) showed almost the same hemolytic activity as lysolecithin itself. There seems to be a close correlation between hemolytic activity and barrier function of lipid micelles.

Effect of lipid composition of liposomes on their sensitivity to peroxidation.

The effect of lipid composition of liposomes on peroxidation induced by ferrous ion and ascorbate was examined. Temperature affects the sensitivity of liposomes; the peroxidation rate was increased with increase of the incubation temperature. With liposomes consisting of 1-palmitoyl-2-arachidonyl phosphatidylcholine (substrate) and a peroxidation-insensitive lipid, 1-palmitoyl-2-oleoyl phosphatidylcholine, peroxidation was dependent on the density of the substrate. No appreciable peroxidation was observed with liposomes containing less than 10 mol% of the substrate at 37 degrees C. When 1 mol substrate was mixed with 9 mol dimyristoyl phosphatidylcholine, peroxidation occurred below 10 degrees C, but not above 20 degrees C. Above 20 degrees C, the substrates should be located homogeneously on the membranes, whereas they should be clustered below 10 degrees C, since the gel-liquid crystalline phase transition temperature of matrix membrane of dimyristoylphosphatidylcholine was 17-21 degrees C. Peroxidation of liposomes consisting of 1-palmitoyl-2-arachidonyl phosphatidylcholine was also suppressed by cholesterol. These findings indicate that the lateral distribution as well as the density of the substrate on membranes affects the sensitivity of the substrate to peroxidation. It was also found that alpha-tocopherol is preferentially located in the 1-palmitoyl-2-arachidonyl phosphatidylcholine-rich regions of membranes consisting of mixed phospholipids, and efficiently suppresses peroxidation of liposomal lipids.

Physical properties and barrier functions of synthetic glyceroglycolipids.

Four glyceroglycolipids containing dipalmitylglycerol were synthesized. The thermotropic behavior and barrier function of aqueous suspensions of these glyceroglycolipids were studied. The gel to liquid crystalline phase transition temperatures of these glycolipids were higher than that of dipalmitoylphosphatidylcholine. The interaction between headgroups in glyceroglycolipids might be stronger than that in dipalmitoylphosphatidylcholine. Diglycosyl dipalmitylglycerols could form liposomes and function as a barrier against water-soluble small molecules (glucose, UmP, and H+) when assayed below their phase transition temperatures, like dipalmitoylphosphatidylcholine liposomes. The fundamental properties of glyceroglycolipid membranes so far examined were rather similar to those of phospholipid membranes, with the exception that the permeability rate of water through glycolipid membranes was higher than that through phospholipid membranes. This property might cause the apparent sensitivity difference between liposomes prepared from glycolipids and those from phospholipids to amphotericin B.

Intermolecular interaction between glycolipids and glycophorin on liposomal membranes.

The haptenic activity of Forssman glycolipid was affected by glycophorin, and the interaction of glycophorin with lectins was also affected by the glycolipids, when both glycophorin and Forssman glycolipid or globoside were incorporated into the same phosphatidylcholine liposomes. Glycophorin incorporated into phospholipid membranes could interact with lectins, whereas that in glyceroglycolipid membranes could not. These findings suggest that glycophorin and glycolipids may interact, affecting the conformation of their sugar residues, probably through a carbohydrate-carbohydrate interaction. Such an interaction may cause suppression or stimulation of the reactivity of glycoconjugates with lectins or antibodies. Lactosylceramide showed only a slight effect on the interaction between glycophorin and lectins. We suggest that the structure of the sugar residues as well as the lateral distribution of molecules was important for the intermolecular interaction.

The interactions of lysophosphatidylcholine with protein-containing liposomes.

The effect of exogenous lysophosphatidylcholine on the membranes of liposomes containing protein has been studied. Lysophosphatidylcholine severely damaged liposomes prepared in the presence of membrane proteins such as glycophorin and "band 3" protein of human erythrocytes. Some basic proteins such as cytochrome c, lysozyme and polylysine also could sensitize liposomes to lysophosphatidylcholine. As described in previous papers (Inoue, K., et al. (1974) Biochim. Biophys. Acta 363, 361-372; Utsumi, H., et al. (1978) Biochemistry 17, 1990-1996), large multilamellar liposomes without protein were affected by lysophosphatidylcholine only under certain conditions where a phase boundary could exist. Sonicated liposomes without protein were almost completely insensitive to lysophosphatidylcholine. Liposomes prepared by the cholate dialysis method were also insensitive to lysophosphatidylcholine, irrespective of the incubation temperature. It is known that natural membranes such as membranes of erythrocytes and of other mammalian cells are rather sensitive to lysophosphatidylcholine. The difference observed between natural membrane and liposomal membrane seems to be removed by the introduction of proteins into lipid bilayers. The mode of interaction of lysophosphatidylcholine with membranes of liposomes containing protein is discussed.

Effect of albumin and methylated albumin on the glucose permeability of lipid membranes.

The diffusion of glucose from phospholipid membranes has been measured in the presence of serum albumin or methylated serum albumin. At neutral pH, serum albumin enhanced the rate at which glucose diffused from liposomes containing more than a certain amount of lysolecithin. Net charge of the membrane is not important for the reaction, since positively charged membranes containing stearylamine showed almost the same reactivity as negatively charged liposomes containing dicetyl phosphate. Carboxylmethylated albumin showed enhancement of the diffusion rate of glucose from negatively but not from positively charged liposomes. The amount of methylated albumin required to affect liposomes was much smaller than the amount of albumin required to damage liposomes containing lysolecithin. Cholesterol incorporation suppressed the sensitivity of liposomes to both proteins, albumin and methylated albumin. The effect of temperature and fatty acid composition of phospholipids on the sensitivity of liposomes to proteins suggests the importance of the fluidity of the membrane, especially in the case of methylated albumin.