STUDY OF IN VITRO STABILITY OF LIPOSOMES AND IN VIVO ANTIBODY RESPONSE TO ANTIGEN ASSOCIATED WITH LIPOSOMES CONTAINING GM1 AFTER ORAL AND SUBCUTANEOUS IMMUNIZATION

ABSTRACT

In order to evaluate the usefulness of liposomes as possible vaccine vehicles (oral and subcutaneous), the stability of liposomes in buffer, plasma and saliva at 25 and 37°C was analyzed via fluorescence and enzymatic methodology. The tested mixtures included [EggPC/Chol] 1 : 1 (mixture I), [EggPC/Chol/SM] 1 : 1 : 1 (mixture II), [EggPC/Chol/SM/GM typeIII] 1 : 1 : 1 : 0.14 (mixture III), [EggPC/Chol/SM/GM1] 1 : 1 : 1 : 0.14 (mixture IV) and [DIAPC/DMPC] 1 : 1 non polymerized (mixture V) and polymerized (mixture VI); all mole ratio. Liposome mixtures I and II were more stable in buffer at 25°C. On the other hand, mixtures III and IV were more stable in plasma at 37°C; mixture VI was more stable in plasma at 37°C than in buffer or saliva. Mixtures IV and V liposomes were both stable in saliva for at least one hour. Blood and feces anti-GM1 response to antigen associated liposomes after subcutaneous and oral administration was also examined. After mixture IV mice immunization, no detectable anti-ganglioside GM1 antibody response was detected. Negative stain transmission electron microscopy, shows that liposomes containing SM, GM1, GM typeIII and DIAPC : DMPC were twice the size of those made with EggPC/Chol. The hydrophobicity factor expressed as A(570/500) was obtained using the probe merocynine 540 (MC540). The order of fluidity increased from: mixture II<mixture I<mixture III<mixture IV<mixtureV<mixture VI. Although the high hydrophobicity factor for polymerizable lipids there are other factors like stability must be taken into account according to the administration via selected. Also, the hydrophilicity of the groups protruding from the membrane interphase into the solution in the case of subcutaneous inoculation is very relevant and for oral administration stability is the property to take into account, as long as they have to last through the different fluids of the gastrointestinal tract. The results obtained suggest that liposomes that showed stability in saliva and plasma at 37°C containing GM1, GM typeIII or DIAPC/DMPC would serve effectively as a delivery vehicle for oral and subcutaneous non-viral vaccines.     

Arsenic trioxide liposomes: encapsulation efficiency and in vitro stability

The use of arsenic-containing compounds in cancer therapy is currently being re-considered, after the recent approval of arsenic trioxide (Trisenox) for the treatment of relapsed promyelocytic leukemia (PML). In an attempt to prepare a carrier system to minimize the toxicity of this drug, the aim of this study is to prepare and characterize liposomes encapsulating arsenic trioxide (ATO). For this, we prepared different types of liposomes entrapping ATO: large multilamellar (MLV), sonicated (SUV) and dried reconstituted vesicles (DRV). The techniques used were: thin film hydration, sonication and the DRV method, respectively. Two lipid compositions were studied for each liposome type, EggPC/Chol (1:1) and DSPC/Chol (1:1). After liposome preparation, drug encapsulation was evaluated by measuring arsenic in liposomes. For this, energy-dispersive X-ray fluorescence spectroscopy or atomic absorption was used. In addition, the retention of the drug in the liposomes was evaluated after incubating the liposomes in buffer at 37 degrees C. The experimental results reveal that encapsulation of ATO in liposomes ranges between 0.003 and 0.506 mol/ mol of lipid, and is highest in the DRV vesicles and lowest in the small unilamellar vesicles, as anticipated. Considering the in vitro stability of ATO-encapsulating liposomes: 1) For the PC/Chol liposomes (DRV and MLV), after 24 hours of incubation, more than 70% (or 90% in some cases) of the initially encapsulated amount of ATO was released. 2) The liposomes composed of DSPC/Chol could retain substantially higher amounts of ATO, especially the DRV liposomes (54% retained after 24 h). 3) In the case of PC/Chol, temperature of incubation has no effect on the ATO release after 24 hours, but affects the rate of ATO release in the MLV liposomes, while for the DSPC/Chol liposomes there is a slight increase (statistically insignificant) of ATO release at higher temperature.     

Cholesterol dynamics in membranes of raft composition: a molecular point of view from 2H and 31P solid-state NMR

Lipidic membrane systems that have been reported to be composed of sphingomyelin (SM)-cholesterol (Chol) microdomains or "rafts" by Dietrich et al. [palmitoyloleoyl-phosphatidylcholine(POPC)/SM/Chol, 1/1/1; Dietrich, C., Bagatolli, L. A., Volovyk, Z. N., Thompson, N. L., Levi, M., Jacobson, K., and Gratton, E. (2001) Biophys. J. 80, 1417-1428] and by Schroeder et al. [SCRL: Liver-PC/Liver-phosphatidylethanolamine/SM/Cerebrosides/Chol, 1/1/1/1/2; Schroeder, R., London, E., and Brown, D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 12130-12134] were investigated under the form of fully hydrated liposomes by the noninvasive solid-state (31)P and (2)H NMR method. Liposomes of binary lipid composition POPC/Chol and SM/Chol were also studied as boundary/control systems. All systems are found to be in the liquid-ordered phase (Lo) at physiological temperatures. Use of deuterium-labeled cholesterol afforded finding both the position of the sterol motional axis and its molecular order parameter. The axis of anisotropic rotation of cholesterol is such that the molecule is, on average, quasiperpendicular to the membrane plane, in all of the four systems investigated. Cholesterol order parameters greater than 0.8 are observed, indicating that the sterol is in a very motionally restricted environment in the temperature range 0-60 degrees C. The binary mixtures present "boundary" situations with the lowest values for POPC/Chol and the highest for SM/Chol. The SCRL raft mixture has the same ordering as the SM/Chol, i.e., the highest order parameter values over the temperature range. It demonstrates that in the SCRL mixture cholesterol dynamics is as in the binary system SM/Chol, therefore, suggesting that it might be depleted from the rest of the membrane to form complexes as if it were alone with SM. On the other hand, the mixture POPC/SM/Chol exhibits an intermediate ordering situation between those of SM/Chol and POPC/Chol. This strongly suggests that cholesterol could be in fast exchange, at the NMR time scale (milli- to microseconds), between two or more membrane regions of different dynamics and questions the statement of "rigid domains" made of SM and cholesterol in the model "raft" system POPC/SM/Chol.     

Tresylated PEG-sterols for coupling of proteins to preformed plain or PEGylated liposomes

A simple and inexpensive method for functionalization of preformed liposomes is presented. Soy sterol-PEG1300 ethers are activated by tresylation at the end of the PEG chain. Coupling of bovine serum albumin as an amino group containing model ligand to the activated lipids can be performed at pH 8.4 with high efficiency. At room temperature, the mixture of sterol-PEG and sterol-PEG-protein inserts rapidly into the outer liposome monolayer with high efficiency (>100 microg protein/mumol total lipid). This method of post-functionalization is shown to be effective with fluid or rigid and plain or pre-PEGylated liposomes (EPC/Chol, 7:3; HSPC/Chol 2:1, and EPC/Chol/MPEG2000-DSPE 2:1:0.16 molar ratios). The release of entrapped calcein upon the insertion of 7.5 mol% of the functionalized sterols is lower than 4%. Incubation of post-functionalized liposomes with serum for 20 h at 37 degrees C shows stable protein attachment at the liposome surface.     

Thermodynamic properties and characterization of proteoliposomes rich in microdomains carrying alkaline phosphatase

Tissue-nonspecific alkaline phosphatase (TNAP) is associated to the plasma membrane via a GPI-anchor and plays a key role in the biomineralization process. In plasma membranes, most GPI-anchored proteins are associated with "lipid rafts", ordered microdomains enriched in sphingolipids, glycosphingolipids and cholesterol. In order to better understand the role of lipids present in rafts and their interactions with GPI-anchored proteins, the insertion of TNAP into different lipid raft models was studied using dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), sphingomyelin (SM) and ganglioside (GM1). Thus, the membrane models studied were binary systems (9:1 molar ratio) containing DPPC:Chol, DPPC:SM and DPPC:GM1, ternary systems (8:1:1 molar ratio) containing DPPC:Chol:SM, DPPC:Chol:GM1 and DPPC:SM:GM1 and finally, a quaternary system (7:1:1:1 molar ratio) containing DPPC:Chol:SM:GM1. Calorimetry analysis of the liposomes and proteoliposomes indicate that lateral phase segregation could be noted only in the presence of cholesterol, with the formation of cholesterol-rich microdomains centered above Tc=41.5°C. The presence of GM1 and SM into DPPC-liposomes influenced mainly ΔH and Δt(1/2) values. The gradual increase in the complexity of the systems decreased the activity of the enzyme incorporated. The presence of the enzyme also fluidifies the systems, as seen by the intense reduction in ?H values, but do not alter Tc values significantly. Therefore, the study of different microdomains and its biophysical characterization may contribute to the knowledge of the interactions between the lipids present in MVs and its interactions with TNAP.     

Arsenic Trioxide Liposomes: Encapsulation Efficiency and In Vitro Stability

The use of arsenic‐containing compounds in cancer therapy is currently being re‐considered, after the recent approval of arsenic trioxide (Trisenox®) for the treatment of relapsed promyelocytic leukemia (PML). In an attempt to prepare a carrier system to minimize the toxicity of this drug, the aim of this study is to prepare and characterize liposomes encapsulating arsenic trioxide (ATO). For this, we prepared different types of liposomes entrapping ATO: large multilamellar (MLV), sonicated (SUV) and dried reconstituted vesicles (DRV). The techniques used were: thin film hydration, sonication and the DRV method, respectively. Two lipid compositions were studied for each liposome type, EggPC/Chol (1:1) and DSPC/Chol (1:1). After liposome preparation, drug encapsulation was evaluated by measuring arsenic in liposomes. For this, energy‐dispersive X‐ray fluorescence spectroscopy or atomic absorption was used. In addition, the retention of the drug in the liposomes was evaluated after incubating the liposomes in buffer at 37°C. The experimental results reveal that encapsulation of ATO in liposomes ranges between 0.003 and 0.506 mol/ mol of lipid, and is highest in the DRV vesicles and lowest in the small unilamellar vesicles, as anticipated. Considering the in vitro stability of ATO‐encapsulating liposomes: 1) For the PC/Chol liposomes (DRV and MLV), after 24 hours of incubation, more than 70% (or 90% in some cases) of the initially encapsulated amount of ATO was released. 2) The liposomes composed of DSPC/Chol could retain substantially higher amounts of ATO, especially the DRV liposomes (54% retained after 24 h). 3) In the case of PC/Chol, temperature of incubation has no effect on the ATO release after 24 hours, but affects the rate of ATO release in the MLV liposomes, while for the DSPC/Chol liposomes there is a slight increase (statistically insignificant) of ATO release at higher temperature.     

Biodistribution and immunotargetability of ganglioside-stabilized dioleoylphosphatidylethanolamine liposomes.

The biodistribution and immunotargetability of liposomes composed primarily of dioleoylphosphatidylethanolamine (DOPE) or dioleoylphosphatidylcholine (DOPC) in mice injected via the tail vein were examined and compared. The ganglioside GM1 (7 mol%) prolonged the circulation of DOPC but not DOPE liposomes. Gangliosides GD1a and GT1b (7 mol%) also increased the amount of DOPC liposomes remaining in circulation, and to a similar extent as GM1, at 15 min post injection. However, these liposomes were cleared from the circulation by 2.5 h. Monoclonal antibody 34A, which specifically binds to a surface glycoprotein (gp 112) of the pulmonary endothelial cell surface, was coupled with N-glutarylphosphatidylethanolamine and incorporated into liposomes by a dialysis procedure. These 34A-immunoliposomes, composed of DOPE and GM1 (7 mol%), but not the antibody-free liposomes, accumulated efficiently (approximately 24% of the injected dose) in the lungs. Inclusion of cholesterol (31 mol%) enhanced the lung accumulation of both DOPE/GM1 immunoliposomes and DOPC/GM1 immunoliposomes to 33% and 51% of the injected dose, respectively. The transient increase in DOPC liposome circulation provided by GD1a and GT1b was sufficient to enhance DOPC immunoliposome binding, where 44% and 43% of the injected dose of DOPC/Chol/GD1a and DOPC/Chol/GT1b immunoliposomes accumulated in lung at 15 min after injection, respectively. In general, cholesterol-containing DOPC liposomes were more targetable than DOPE liposomes, and the degree to which these liposomes avoid RES uptake influences their targetability. The results presented here are relevant to the design of targetable drug delivery vehicles.     

Resorcinolic lipids improve the properties of sphingomyelin–cholesterol liposomes

Alkylresorcinolic lipids isolated from cereal grains and their semi-synthetic myristoyl-sulphonyl derivatives (MSAR) were used to modify small long-circulating sphingomyelin–cholesterol liposomes. Those SM:Chol vesicles modified with 10–30 mol% resorcinolic lipids had stable size and low membrane permeability in vitro at 4 °C and 37 °C. Liposomes containing 30 mol% MSAR showed very fast solute release in the presence of human plasma at 37 °C, which was drastically diminished in heat-inactivated plasma. In vivo studies showed that unmodified SM:Chol liposomes and those modified with alkylresorcinols were eliminated from the circulation more slowly than liposomes with the highest concentration of MSAR in membrane and were located mostly in the liver and spleen.     

Phase behavior of fluorocarbon and hydrocarbon double-chain hydroxylated and galactosylated amphiphiles and bolaamphiphiles. Long-term shelf-stability of their liposomes.

This paper describes the morphological characterization, by freeze-fracture electron microscopy, and the thermotropic phase behavior, by differential scanning calorimetry and/or X-ray scattering, of aqueous dispersions of various hydroxylated and galactosylated double-chain amphiphiles and bolaamphiphiles, several of them containing one or two hydrophobic fluorocarbon chains. Colloidal systems are observed in water with the hydroxylated hydrocarbon or fluorocarbon bolaamphiphiles only when they are dispersed with a co-amphiphile such as rac-1,2-dimyristoylphosphatidylcholine (DMPC) or rac-1,2-distearoylphosphatidylcholine (DSPC). Liposomes are formed providing the relative content of bolaamphiphiles does not exceed 20% mol. Most of these liposomes can be thermally sterilized and stored at room temperature for several months without any significant modification of their size and size distribution. The hydrocarbon galactosylated bolaamphiphile HO[C24][C12]Gal forms in water a lamellar phase (the gel to liquid-crystal phase transition is complete at 45 degrees C) and a Im3m cubic phase above 47 degrees C. The fluorocarbon HO[C24][F6C5]Gal analog displays a more complex and metastable phase behavior. The fluorinated non-bolaform galactosylated [F8C7][C16]AEGal and SerGal amphiphiles form lamellar phases in water. Low amounts (10% molar ratio) of the HO[C24][F6C5]Gal or HO[C24][C12]Gal bolaamphiphiles or of the single-headed [F8C7][C16]AEGal improve substantially the shelf-stability of reference phospholipon/cholesterol 2/1 liposomes. These liposomes when co-formulated with a single-headed amphiphile from the SerGal series are by far less stable.     

Tresylated PEG-sterols for coupling of proteins to preformed plain or PEGylated liposomes

A simple and inexpensive method for functionalization of preformed liposomes is presented. Soy sterol-PEG₁₃₀₀ ethers are activated by tresylation at the end of the PEG chain. Coupling of bovine serum albumin as an amino group containing model ligand to the activated lipids can be performed at pH 8.4 with high efficiency. At room temperature, the mixture of sterol-PEG and sterol-PEG-protein inserts rapidly into the outer liposome monolayer with high efficiency (>100 μg protein/μmol total lipid). This method of post-functionalization is shown to be effective with fluid or rigid and plain or pre-PEGylated liposomes (EPC/Chol, 7:3; HSPC/Chol 2:1, and EPC/Chol/MPEG₂₀₀₀-DSPE 2:1:0.16 molar ratios). The release of entrapped calcein upon the insertion of 7.5 mol% of the functionalized sterols is lower than 4%. Incubation of post-functionalized liposomes with serum for 20 h at 37 °C shows stable protein attachment at the liposome surface.     

Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains

A mixture of sphingomyelin (SM) and cholesterol (Chol) exhibits a characteristic lipid raft domain of the cell membranes that provides a platform to which various signal molecules as well as virus and bacterial proteins are recruited. Several proteins capable of specifically binding either SM or Chol have been reported. However, proteins that selectively bind to SM/Chol mixtures are less well characterized. In our screening for proteins specifically binding to SM/Chol liposomes, we identified a novel ortholog of Pleurotus ostreatus, pleurotolysin (Ply)A, from the extract of edible mushroom Pleurotus eryngii, named PlyA2. Enhanced green fluorescent protein (EGFP)-conjugated PlyA2 bound to SM/Chol but not to phosphatidylcholine/Chol liposomes. Cell surface labeling of PlyA2-EGFP was abolished after sphingomyelinase as well as methyl-β-cyclodextrin treatment, removing SM and Chol, respectively, indicating that PlyA2-EGFP specifically binds cell surface SM/Chol rafts. Tryptophan to alanine point mutation of PlyA2 revealed the importance of C-terminal tryptophan residues for SM/Chol binding. Our results indicate that PlyA2-EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes.     

Use of liposomes as carriers for radiation synovectomy

Using [^99mTc]pertechnetate as an aqueous space marker, the permeability of liposomes composed of seven different mixtures of distearoylphosphatidylcholine (DSPC) and sphingomyelin (SM) was determined. Liposomes containing 20–33% SM were the least permeable in the presence of rheumatoid synovial fluid. Following injection of ^99mTc-containing liposomes into the knee joints of rabbits, retention of ^99mTc in the knee was more than 200 times greater than following injection of nonencapsulated [^99mTc]pertechnetate. The knee clearance biologic half time of ^99mTe with DSPC/SM (4:1) liposomes was 64 h. Most of the activity that had leaked from the knee was not found in extra-articular tissues, suggesting rapid excretion. When DSPC/SM (4:1) liposomes were labeled with ¹¹¹In(oxine), a knee clearance biologic half time of greater than 1200 h was observed.     

Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes

Incorporation of dioleoyl N-(monomethoxy polyethyleneglycol succinyl)phosphatidylethanolamine (PEG-PE) into large unilamellar liposomes composed of egg phosphatidylcholine:cholesterol (1:1) does not significantly increase the content leakage when the liposomes are exposed to 90% human serum at 37 degrees C, yet the liposomes show a significant increase in the blood circulation half-life (t1/2 = 5 h) as compared to those without PEG-PE(t1/2 less than 30 min). The PEG-PE's activity to prolong the circulation time of liposomes is greater than that of the ganglioside GM1, a well-described glycolipid with this activity. Another amphipathic PEG derivative, PEG stearate, also prolongs the liposome circulation time, although its activity is less than that of GM1. Amphipathic PEGs may be useful for the sustained release and the targeted drug delivery by liposomes.     

Formation of transition metal-doxorubicin complexes inside liposomes

Doxorubicin complexation with the transition metal manganese (Mn(2+)) has been characterized, differentiating between the formation of a doxorubicin-metal complex and doxorubicin fibrous-bundle aggregates typically generated following ion gradient-based loading procedures that rely on liposome encapsulated citrate or sulfate salts. The physical and chemical characteristics of the encapsulated drug were assessed using cryo-electron microscopy, circular dichroism (CD) and absorbance spectrophotometric analysis. In addition, in vitro and in vivo drug loading and release characteristics of the liposomal formulations were investigated. Finally, the internal pH after drug loading was measured with the aim of linking formation of the Mn(2+) complex to the presence or absence of a transmembrane pH gradient. Doxorubicin was encapsulated into either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/cholesterol (Chol) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/Chol liposomes, where the entrapped salts were citrate, MnSO(4) or MnCl(2). In response to a pH gradient or a Mn(2+) ion gradient, doxorubicin accumulated inside to achieve a drug-to-lipid ratio of approximately 0.2:1 (wt/wt). Absorbance and CD spectra of doxorubicin in the presence of Mn(2+) suggested that there are two distinct structures captured within the liposomes. In the absence of added ionophore A23187, drug loading is initiated on the basis of an established pH gradient; however, efficient drug uptake is not dependent on maintenance of the pH gradient. Drug release from DMPC/Chol is comparable regardless of whether doxorubicin is entrapped as a citrate-based aggregate or a Mn(2+) complex. However, in vivo drug release from DSPC/Chol liposomes indicate less than 5% or greater than 50% drug loss over a 24-h time course when the drug was encapsulated as an aggregate or a Mn(2+) complex, respectively. These studies define a method for entrapping drugs possessing coordination sites capable of complexing transition metals and suggest that drug release is dependent on lipid composition, internal pH, as well as the nature of the crystalline precipitate, which forms following encapsulation.     

Chlorpromazine interaction with glycerophospholipid liposomes studied by magic angle spinning solid state (13)C-NMR and differential scanning calorimetry

Phosphatidylserine (PS) extracted from pig brain and synthetic dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were used to make DPPC/DMPC and DPPC/PS large unilamellar liposomes with a diameter of approximately 1 microm. Chlorpromazine-HCl (CPZ), an amphipathic cationic psychotropic drug of the phenothiazine group, is known to partition into lipid bilayer membranes of liposomes with partition coefficients depending on the acyl chain length and to alter the bilayer structure in a manner depending on the phospholipid headgroups. The effects of adding CPZ to these membranes were studied by differential scanning calorimetry and proton cross polarization solid state magic angle spinning (13)C-nuclear magnetic resonance spectroscopy (CP-MAS-(13)C-NMR). CP-MAS-(13)C-NMR spectra of the DPPC (60%)/DMPC (40%) and the DPPC (54%)/DMPC (36%)/CPZ (10%) liposomes, show that CPZ has low or no interaction with the phospholipids of this neutral and densely packed bilayer. Conversely, the DPPC (54%)/PS (36%)/CPZ (10%) bilayer at 25 degrees C demonstrates interaction of CPZ with the phospholipid headgroups (PS). This CPZ interaction causes about 30% of the acyl chains to enter the gauche conformation with low or no CPZ interdigitation among the acyl chains at this temperature (25 degrees C). The DPPC (54%)/PS (36%)/CPZ (10%) bilayer at a sample temperature of 37 degrees C (T(C)=31.2 degrees C), shows CPZ interdigitation among the phospholipids as deduced from the finding that approximately 30% of the phospholipid acyl chains carbon resonances shift low-field by 5-15 ppm.     

Temperature and pressure effects on structural and conformational properties of POPC/SM/cholesterol model raft mixtures--a FT-IR, SAXS, DSC, PPC and Laurdan fluorescence spectroscopy study

We report on the effects of temperature and pressure on the structure, conformation and phase behavior of aqueous dispersions of the model lipid "raft" mixture palmitoyloleoylphosphatidylcholine (POPC)/bovine brain sphingomyelin (SM)/cholesterol (Chol) (1:1:1). We investigated interchain interactions, hydrogen bonding, conformational and structural properties as well as phase transformations of this system using Fourier transform-infrared (FT-IR) spectroscopy, small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), and Laurdan fluorescence spectroscopy. The IR spectral parameters in combination with the scattering patterns from the SAXS measurements were used to detect structural and conformational transformations upon changes of pressure up to 7-9 kbar and temperature in the range from 1 to about 80 degrees C. The generalized polarization function (GP) values, obtained from the Laurdan fluorescence spectroscopy studies also reveal temperature and pressure dependent phase changes. DSC and PPC were used to detect thermodynamic properties accompanying the temperature-dependent phase changes. In combination with literature fluorescence spectroscopy and microscopy data, a tentative p,T stability diagram of the mixture has been established. The data reveal a broad liquid-order/solid-ordered (lo+so) two-phase coexistence region below 8+/-2 degrees C at ambient pressure. With increasing temperature, a lo+ld+so three-phase region is formed, which extends up to approximately 27 degrees C, where a liquid-ordered/liquid-disordered (lo+ld) immiscibility region is formed. Finally, above 48+/-2 degrees C, the POPC/SM/Chol (1:1:1) mixture becomes completely fluid-like (liquid-disordered, ld). With increasing pressure, all phase transition lines shift to higher temperatures. Notably, the lo+ld (+so) phase coexistence region, mimicking raft-like lateral phase separation in natural membranes, extends over a rather wide temperature range of about 40 degrees C, and a pressure range, which extends up to about 2 kbar for T=37 degrees C. Interestingly, in this pressure range, ceasing of membrane protein function in natural membrane environments has been observed for a variety of systems.     

Circular dichroism study of membrane dynamics focused on effect of monosialogangliosides

Monosialogangliosides (GM) purified from bovine brain were incorporated into circular dichroism (CD)-active liposomes and the effects of GM on the membrane dynamics were studied by CD spectroscopy. In the presence of 7 mol% of GM, the phase transition temperature (Tc) of the membrane increased by ca. 10 degrees C compared with the membrane without GM and characteristic CD spectra were observed for CD-active liposomes incorporating GM at low temperature. Asialogangliosides had no effect on the CD spectra or Tc. We have also studied the role of GM in reducing leakage of [3H]sucrose from liposomes composed of egg phosphatidylcholine, dipalmitoyl phosphatidic acid, cholesterol and alpha-tocopherol with a molar ratio of 4 : 1 : 5 : 0.1 in the presence of human plasma at 25 degrees C. The half-life of [3H]-sucrose leakage was 173 h for liposomes incorporating 7 mol% of GM. On the other hand, the half-lives for liposomes incorporating 7 mol% of asialogangliosides and liposomes without glycolipids were 45 and 42 h, respectively. These results indicate that sialic acid on the membrane surface contributes to the increase of Tc, to the change of the aggregation state of phospholipids and to the stabilization of liposomes in plasma.     

Solubilization of Multilamellar Liposomes in the Presence of Non-ionized Drug

The solubilization of multilamellar liposomes by metoprolol tartrate (MPL) has been studied as a function of pH, [MPL], [dimyristoylphosphatidylcholine (DMPC)], temperature and lipid composition. The solubilization of liposomes at 37° C by 7.3 mM MPL occurred at different rates at different pH values. MPL completely solubilized by 7.2 mM DMPC liposomes after about 17 hat pH 12, but only a partial solubilization occurred at pH 10 and 11. Between pH 7 and 9 no change in turbidity was observed after 1 week. Addition of cholesterol (CHOL) to DMPC (2:1 mol) had very little effect on solubilization after 24 h, however with DMPC:CHOL (5:1 mol) the decrease in turbidity was observed after 24 h, even though solubilization was much less compared with that of DMPC alone. The rate of solubilizaiton was decreased when dipalmitoylphosphatidylcholine liposomes were employed. Addition of dicetylphosphate (DCP) to DMPC liposomes reduced the rate of solubilization significantly. The solubilization of liposomes by 7.3 mM MPL as a function of [DMPC], indicated that the lower the liposome concentration the greater the effect on solubilization. It is concluded that MPL in the non-ionized form has a solubilizing effect on liposomes, and addition of CHOL or DCP to DMPC has a stabilizing effect against solubilization.     

Bending elasticities of model membranes: influences of temperature and sterol content

Giant liposomes obtained by electroformation and observed by phase-contrast video microscopy show spontaneous deformations originating from Brownian motion that are characterized, in the case of quasispherical vesicles, by two parameters only, the membrane tension sigma and the bending elasticity k(c). For liposomes containing dimyristoyl phosphatidylcholine (DMPC) or a 10 mol% cholesterol/DMPC mixture, the mechanical property of the membrane, k(c), is shown to be temperature dependent on approaching the main (thermotropic) phase transition temperature T(m). In the case of DMPC/cholesterol bilayers, we also obtained evidence for a relation between the bending elasticity and the corresponding temperature/cholesterol molecular ratio phase diagram. Comparison of DMPC/cholesterol with DMPC/cholesterol sulfate bilayers at 30 degrees C containing 30% sterol ratio shows that k(c) is independent of the surface charge density of the bilayer. Finally, bending elasticities of red blood cell (RBC) total lipid extracts lead to a very low k(c) at 37 degrees C if we refer to DMPC/cholesterol bilayers. At 25 degrees C, the very low bending elasticity of a cholesterol-free RBC lipid extract seems to be related to a phase coexistence, as it can be observed by solid-state (31)P-NMR. At the same temperature, the cholesterol-containing RBC lipid extract membrane shows an increase in the bending constant comparable to the one observed for a high cholesterol ratio in DMPC membranes.