Effects of diclofenac on EPC liposome membrane properties

In this work the interaction of a non-steroidal anti-inflammatory drug (NSAID), diclofenac, with egg yolk phosphatidylcoline (EPC) liposomes, used as cell-membrane models, was quantified by determination of the partition coefficient. The liposome/aqueous phase partition coefficient was determined by derivative spectrophotometry, fluorescence quenching, and measurement of zeta-potential. Theoretical models based on simple partition of the diclofenac between two different media, were used to fit the experimental data, enabling the determination of K_p. The three techniques used yielded similar results. The effects of the interaction on the membranes characteristics were further evaluated, either by studying membrane potential changes or by effects on membrane fluidity. The liposome membrane potential and the size and size-homogeneity of liposomes were measured by light scattering. The effects of diclofenac on the internal viscosity or fluidity of the membrane were determined by use of spectroscopic probes—a series of n-(9-anthroyloxy) fatty acids in which the carboxyl terminal group is located at the interfacial region of the membrane and the fluorescent anthracene group is attached at different positions along the fatty acid chain. The location of the diclofenac on the membrane was also evaluated, by fluorescence quenching using the same series of fluorescent probes. Because the fluorescent anthracene group is attached at different positions along the fatty acid chain, it is possible to label at a graded series of depths in the bilayer. The interactions between the drug and the probe are a means of predicting the location of the drug on the membrane.     

Photodamage in a Mitochondrial Membrane Model Modulated by the Topology of Cationic and Anionic Meso‐Tetrakis Porphyrin Free Bases

The photodynamic effects of the cationic TMPyP (meso‐tetrakis [N‐methyl‐4‐pyridyl]porphyrin) and the anionic TPPS4 (meso‐tetrakis[4‐sulfonatophenyl]porphyrin) against PC/CL phosphatidylcholine/cardiolipin (85/15%) membranes were probed to address the influence of phorphyrin binding on lipid damage. Electronic absorption spectroscopy and zeta potential measurements demonstrated that only TMPyP binds to PC/CL large unilamellar vesicles (LUVs). The photodamage after irradiation with visible light was analyzed by dosages of lipid peroxides (LOOH) and thiobarbituric reactive substance and by a contrast phase image of the giant unilamellar vesicles (GUVs). Damage to LUVs and GUVs promoted by TMPyP and TPPS4 were qualitatively and quantitatively different. The cationic porphyrin promoted damage more extensive and faster. The increase in LOOH was higher in the presence of D₂O, and was impaired by sodium azide and sorbic acid. The effect of D₂O was higher for TPPS4 as the photosensitizer. The use of DCFH demonstrated that liposomes prevent the photobleaching of TMPyP. The results are consistent with a more stable TMPyP that generates long‐lived singlet oxygen preferentially partitioned in the bilayer. Conversely, TPPS4 generates singlet oxygen in the bulk whose lifetime is increased in D₂O. Therefore, the affinity of the porphyrin to the membrane modulates the rate, type and degree of lipid damage.     

Uptake of transition metal ions using liposomes containing dicetylphosphate as a ligand.

The uptake of Cu2+ was investigated using various types of liposomes composed of phosphatidylcholine (PC), cholesterol (Chol) and dicethylphosphate (DCP). DCP played a role as a ligand for Cu2+. Multilamellar vesicles (MLVs) were more effective for the uptake of Cu2+ compared to unilamellar vesicles prepared by the extrusion technique. The uptake efficiency of MLVs for Cu2+ was dependent on the molar ratio of DCP in MLVs. The uptake percent of Cu2+ was 92% using MLVs having a PC:DCP:Chol molar ratio of 4:3:3; 95% of the total vesicle Cu2+ was bound to DCP of the outer membrane surface of the MLVs, and the remaining 5% of the total Cu2+ was distributed into the interior side of the MLVs. MLVs having a PC:DCP:Chol molar ratio of 4:3:3 were also effective as separation media for Mn2+, Co2+, Ni2+ and Zn2+. The uptake efficiency of the MLVs for the transition-metal ions increased in the order Co2+ < Zn2+ < Ni2+ < Mn2+ < Cu2+.     

INTERACTION BETWEEN UPOSOMES AND MONOLAYERS IN THE PRESENCE OF CALCIUM

Abstract

The interaction between phospholipid vesicles (phosphatidylcholine : phosphatide acid, 90:10 w/w) and phosphatidylcholine : cholesterol (70:30, molar ratio) monolayers at air/water interfscks has been studied at. several concentrations of calcium cation ( Ca²⁺). The liposome vesicles were SUVs and MLVS.

The vesicles interact with the monolayers, rapidly causing a large increase in surface pressure. Limiting values of surface pressure, 2.07-6.99 mN.m-1 for SUVs, and 7.01-11.11 mN.m^?1 for MLVs, were reached in less than 40?min.

Calcium ion concentration affects the liposome size in MLVs, producing an increase of gyration radius. The SUVs are little influenced. The change in size can be due to a variation of liposome composition induced by calcium: cholesterol molecules can migrate from monolayer to liposomes and the redistribution of exchanged lipids in the outer bilayer can also explain the size variation.     

Solubilization of egg phosphatidylcholine liposomes by sodium taurocholate: partition behavior and morphology

Solubilization process of egg yolk phosphatidylcholine (EggPC) liposomes by sodium taurocholate (TC) was performed using membrane dialysis technique. Turbidity, apparent particle size, Cl⁻ permeability, membrane fluidity measured by electron spin resonance spectroscopy (ESR), and structural changes observed by freeze-fracture electron microscopy were investigated. The concentrations of TC were analyzed by high-performance liquid chromatography to determine the incorporation of TC in membrane phase and apparent partition coefficient. When TC concentration in water phase (D _w) was lower than 1.41 mM, or effective ratio of TC to EggPC (R _e) was less then 0.14, the partition coefficient was independent of the TC concentration and was 90 M⁻¹, where the membrane permeability of Cl⁻ was rather small. Upon increasing the concentrations in the range of 1.41 mM≤D _w<3.10 mM (0.14≤R _e<0.21), the apparent partition coefficient decreased and membrane permeability markedly increased. The coexistence of small vesicles and bilayer fragments were observed in this region. At 3.10 mM≤D _w<3.53 mM (0.21≤R _e<0.59), the steep decrease in turbidity was accompanied with the gradual increase in apparent partition coefficient, suggesting the formation of mixed micelles. The freeze-fracture electron micrographs confirmed the transformation of saturated bilayers to wormlike mixed micelles. When a further increase in TC (D _w≥3.53 or R _e≥0.59) was applied, the turbidity and apparent particle sizes became small. Transformation of the wormlike structure to ellipsoid mixed micelles was observed. The ESR spectra showed that TC/EggPC mixed micelles still retain a somewhat orderly structure.     

Membrane properties of cationic liposomes composed of dipalmitoylphosphatidylcholine and dipalmityldimethylammonium bromide

Cationic liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmityldimethylammmonium bromide (DPAB) were prepared by the Bangham method and the effect of DPAB on the membrane properties was examined in terms of liposomal shape, particle size, trapping efficiency, surface potential and dispersibility. The dispersibility of the mixed DPPC/DPAB liposomes (the mole fraction of DPAB (X_DPAB)  0.05) was excellent and the dispersibility was maintained for 6 months, since the zeta-potential of the mixed liposomes was approximately +40 mV. The trapping efficiency of the mixed DPPC/DPAB liposomes (X_DPAB = 0.05) was 10 times greater than that of the DPPC liposomes, and the value was largest among the mixed liposomes (X_DPAB = 0–1.0). Freeze-fracture electron micrographs indicated that the shape of the mixed DPPC/DPAB liposomes (X_DPAB = 0.05) was that of large unilamellar vesicles (LUVs) with a diameter of approximately 2 μm, while the shape of the DPPC liposomes was that of multilamellar vesicles (MLVs). The mixed liposomes had, therefore, a high trapping efficiency. Furthermore, the shape of the mixed DPPC/DPAB liposomes (X_DPAB = 0.75) was also that of LUVs with a diameter of approximately 2 μm and these had a high trapping efficiency. Whereas, the particle size (500 nm) of the mixed DPPC/DPAB liposomes (X_DPAB = 0.25) was smaller than that of the former and had the minimum trapping efficiency. The phase transition temperature of the liposomal bilayer membranes indicated a maximum value at 0.25–0.30 mole fractions of DPAB. These facts were considered to be due to the fact that DPPC and DPAB, whose molar ratio was 7.5:2.5, were tightly packed in the liposomal bilayer membranes and that the curvature of the liposomal particle was resultantly large. Nevertheless, LUVs having a high trapping efficiency were easily obtained by mixing a small amount of DPAB with the DPPC.     

Influence of the hydrophobic tail of alkyl glucosides on their ability to solubilize stratum corneum lipid liposomes

The lytic interactions of a series of alkyl glucosides (alkyl chain lengths ranging from C₈ to C₁₂) with liposomes formed by a mixture of lipids modeling the stratum corneum (SC) lipid composition were investigated. The surfactant-to-lipid molar ratios (Re) and the normalized bilayer/aqueous phase partition coefficients (K) were determined by monitoring the changes in the static light-scattering (SLS) of the system during solubilization. The fact that the free surfactant concentrations were always similar to their critical micelle concentrations indicates that the liposome solubilization was mainly ruled by the formation of mixed micelles. At the two interaction levels studied (100 and 0% SLS) the nonyl glucoside showed the highest ability to saturate and to solubilize liposomes (lowest Re values), whereas the dodecyl glucoside showed the highest degree of partitioning into liposomes or affinity with these structures (highest K values). Comparison of the data for octyl glucoside with that reported for the interaction of this surfactant with phosphatidylcholine (PC) liposomes shows that whereas the SC lipid liposomes were more resistant to the action of this surfactant (higher Re values), its degree of partitioning into SC bilayers was both in the saturation and solubilization of liposomes similar to that exhibited in PC vesicles (similar K values). Received: 27 November 2000/Accepted: 19 February 2001     

Membranotropic properties of fullerene-containing amphiphilic (co)polymers of <Emphasis Type="Italic">N</Emphasis>-vinylpyrrolidone

The effect of fullerene-containing amphiphilic (co)polymers of N-vinylpyrrolidone (C₆₀/PVP) differed in composition and topology of the polymer chains on the membrane structure of phosphatidylcholine liposomes was studied. Using the method of fluorescent probes it was found that the C₆₀/PVP structures under study are localized in the phosphatidylcholine membrane in the region of glycerol fragments and fatty acid chains of phospholipids. The C₆₀/PVP copolymers form complexes with 2,7-dibromoproflavin and pyrene probes. The effective equilibrium constants of the probe–C₆₀/PVP complexes in aqueous solutions and in the liposome membrane were determined. It is most likely that the fullerene-containing (co)polymers of N-vinylpyrrolidone form complexes not only with fluorescent probes but also with phospholipids of the liposome membrane, which reduces the stability of the probe–C₆₀/PVP complex.     

Structure and characteristics of the complexes between polyampholites and anionic liposomes

Polyampholites are synthesized by the alkylation of poly-4-vinylpyridine with ω-bromocarboxylic acids, and their interaction with the negatively charged bilayer lipid vesicles (liposomes) is studied. In the above polymers, quaternized pyridine units are zwitterion (betaine) groups, in which cationic and anionic groups are linked by the -(CH₂) _n -bridges. Via the methods of fluorescence, laser scattering, and DSC, the length of the ethylene spacer in the betaine group is shown to control the ability of the polymer to interact with anionic liposomes and induce structural rearrangements in the liposomal membrane. At n = 1, polybetaine is not linked to anionic liposomes. At n = 2, polybetaine is sorbed on the membrane, but it causes no dramatic structural rearrangements in the bilayer. At n = 3, the adsorption of polybetaine triggers the lateral segregation of lipids in the outer membrane layer. At n = 5, adsorption of polymer is accompanied by the lateral segregation and flip-flop of lipid molecules; as a result, all anionic membrane lipids are involved in the microphase separation. This evidence is of evident interest for the controlled design of polymers and related complexes and conjugates for biomedical applications.     

Solubilities of polypeptides with hydrophobic and negatively charged side-chains into cationic artificial membrane vesicles

The influence of relatively low molecular weight polypeptides on the structure of membrane vesicles composed of distearyldimethylammoniumchloride (DSACl) was investigated by means of calorimetric, fluorescence spectroscopic and fluorescence polarization measurements, and correlated with the degree of hydrophobicity and/or dissociation of the polypeptide side chains. The polypeptides used were poly(-methyl L-glutamate) (PMLG,M _v =4400), copoly(methyl L-glutamate L-glutamic acid) containing 20 mol % of L-glutamic acid (80/20 MG/GA,M _v =4200) and poly(L-glutamic acid) (PLGA,M _v =9200). The hydrophobic polypeptide, PMLG, was readily incorporated into the DSACl membrane vesicles to form membrane-spanning helices, resulting in a decrease in the microviscosity of the hydrophobic region of the membrane phase. The partially charged hydrophobic polypeptide, 80/20 MG/GA, was almost insoluble into the membrane below the phase transition temperature of the DSACl vesicle,T _c 40.4 °C, however, the solubility of the copolymer into the membrane was drastically increased aboveT _c . The negatively charged polypeptide, PLGA, can hardly penetrate through the membrane vesicle and formed crosslinking between the positively charged DSACl vesicles. It was also confirmed that aggregation or clustering of the hydrophobic PMLG-helices progressed in the membrane phase belowT _c .     

Protective effect caused by the exopolymer excreted by Pseudoalteromonas antarctica NF3 on liposomes against Triton X-100

The capacity of the glycoprotein (GP) excreted by Pseudoalteromonas antarctica NF₃ to protect phosphatidylcholine (PC) liposomes against the action of Triton X-100 was studied in detail. Increasing amounts of GP assembled with liposomes resulted in a linear increase in the effective surfactant-to-PC molar ratios needed to produce the same alterations in liposomes and in a linear fall in the surfactant partitioning between the bilayer and the aqueous phase. Thus, the higher the proportion of GP assembled with liposomes the lower the surfactant ability to alter the permeability of vesicles and the lower its affinity with these bilayer structures. In addition, increasing GP proportions resulted in a progressive increase in the free surfactant concentration (S _W) for the same surfactant–liposome interaction step. The fact that S _W was always lower than the surfactant critical micelle concentration indicates that the interaction was mainly ruled by the action of surfactant monomers, regardless of the amount of GP assembled. Received: 4 May 1999 Accepted in revised form: 6 July 1999     

Interaction between phospholipid vesicular structures with long chain zwitterionic surfactants

Solubilization of different zwitterionic phospholipid vesicles structures such as L--phosphatidylcholine (PC) and 1,2-didecanoyl-sn-glycero-3-phosphocholine (DPC) have been studied in aqueous bulk by using zwitterionic surfactant dimethylhexadecylammoniopropanesulfonate (HPS). This has been done by studying the aggregation of HPS in pure water and in the presence of 7–36 M of fixed concentrations of each lipid with the help of pyrene fluorescence intensity (I ₁/I ₃) measurements. The fluorescence measurements showed that HPS monomers undergo two kinds of aggregation process, which were identified by the three breaks in a plot of pyrene fluorescence versus HPS concentration. The first two breaks, C ₁ and C ₂, indicate the onset and completion of vesicle solubilization respectively, upon incorporation of HPS monomers into the vesicles and led to solubilization in the form of mixed micelles. This process was not clearly visible at low lipid concentration. We evaluated the partition coefficient (K), which defines the degree of partitioning of surfactant monomers into the vesicles with respect to the aqueous medium. A high K value of HPS-lipid aggregates indicates the stronger interactions between surfactant and lipid vesicles. The K values evaluated for PC and DPC are quite close to each other, which indicates that K values were independent of phospholipid chain length.     

An improved NMR study of liposomes using 1-palmitoyl-2-oleoyl-sn-glycero-3-phospatidylcholine as model

In this paper we report a comparative characterization of Small Unilamellar Vesicles (SUVs), Large Unilamellar Vesicles (LUVs) and Multilamellar Vesicles (MLVs) prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phospatidylcholine (POPC), carried out using two NMR techniques, namely High Resolution NMR in solution and High Resolution-Magic Angle Spinning (HR-MAS). The size and size distributions of these vesicles were investigated using the dynamic light scattering technique. An improved assignment of the (1)H-NMR spectrum of MLVs is also reported.     

Solubilization of phospholipid bilayers by C14-alkyl betaine/anionic mixed surfactant systems

The mechanisms governing the solubilizing interactions between zwitterionic/anionic mixed surfactant systems at different molar fractions of the zwitterionic surfactant (X _zwitter) and unilamellar liposomes were investigated. Solubilization was detected as a decrease in static lightscattering of liposome suspensions. Three parameters were regarded as corresponding to the effective surfactant/lipid molar ratios (Re) at which the surfactant system a) saturated the liposomesRe _sat; b) resulted in 50% solubilization of liposomesRe _50%, and c) led to a complete solubilization of these structuresRe _sol. These parameters corresponded to theRe at which light scattering stars to decrease, reaches 50% of the original value and shows no further decrease. From these parameters the surfactant partition coefficients for these three steps (K _sat,K _50% andK _sol) were also determined. The mixed systems were formed byN-tetradecyl-N, N-dimethylbetaine (C₁₄-Bet) and sodium dodecyl sulphate (SDS) in PIPES buffer at pH 7.20. Liposomes were formed by egg phosphatidylcholine and phosphatidic acid (9:1 molar ratio). When the range ofX _zwitter was about 0.4–0.6Re andK parameters showed a maximum, whereas the critical micelle concentration (CMC) of these systems exhibited a minimum. Given that the ability of the surfactant systems to solubilize liposomes is inversely related toRe _sol, this capacity appeared to be directly correlated with the CMC of the systems. The progressive rise ofK during the process indicates that an increasing surfactant partition equilibrium governs the interaction process from the saturation until the solubilization of vesicles, the free surfactant concentration remaining almost constant with similar values to the CMC for each mixed system studied.     

Reactivity of singlet oxygen generated by the photosensitization of tetraphenylporphyrin in liposomes

A hydrophobic porphyrin derivative, tetraphenylporphyrin (TPP), was used as a sensitizer, and an anionic dye, methyl orange (MO), was employed as a substrate of photooxidation. TPP was incorporated into the hydrophobic environment of phosphatidylcholine (PC) bilayer membranes, liposomes. When oxygen was purged out of the liposome suspension by nitrogen bubbling, the degradation of MO was completely inhibited. A specific superoxide scavenger, superoxide dismutase, had no effect on the MO degradation. The replacement of H₂O by D₂O resulted in a 10 times enhancement in the photodegradation of MO. These results suggested that singlet oxygen was generated by the TPP photosensitization and worked as the mediator of the photoreaction from TPP. Trisulphonated TPP,-phenyl-,, -tri(p-sulphonyl)porphyrin (TPPS), is soluble in aqueous solution. The light irradiation to an aqueous solution of TPPS gave rise to the rapid bleaching (decomposition) of the sensitizer itself. On the other hand, TPP in the hydrophobic environment of liposomes was stable during light irradiation and worked as a sensitizer for the continuous photoreaction. Maximum reactivity was observed at the PC/TPP mole ratio of 50. When TPP molecules were incorporated into liposomes at larger concentrations (PC/TPP<50), a part of the excitation energy of the sensitizer molecules was nonradiatively converted into the lattice energy by the resonance between the closely located TPP molecules. This led to lower efficiency for the photoactivation of oxygen. On the other hand, the increase in liposome concentration resulted in the enhancement of the MO binding to lipid membranes and the retardation of MO degradation. Also, the electrostatic attraction and repulsion between the membrane and the substrate influenced the reaction rate greatly. The oxidative degradations of the substrate by singlet oxygen were considered to be much faster in the polar environment than in the less polar environment. The charge transfer or the polarized transition complex of singulet oxygen and MO are presumed to be stabilized in the polar environment. The distribution of substrate between the less polar membrane surface and the polar bulk aqueous solution was another important factor in the photooxidation.     

Novel asparagine-derived lipid enhances distearoylphosphatidylcholine bilayer resistance to acidic conditions

A novel asparagine-derived lipid analogue (ALA(11,17)) bearing a tetrahydropyrimidinone headgroup and two fatty chains (11 and 17 indicate the lengths of linear alkyl groups) was synthesized in high yield and purity. The thin film hydration of formulations containing 5 mol % or greater ALA(11,17) in distearoylphosphatidylcholine (DSPC) generated multilamellar vesicles (MLVs) that remained unaggregated according to optical microscopy, while those formed from DSPC only were highly clustered. The MLVs were processed into unilamellar liposomes via extrusion and were characterized by dynamic light scattering (DLS), zeta potential, turbidity, and scanning electron microscopy (SEM) analysis. Results show that the presence of ALA(11,17) in DSPC liposomes significantly alters the morphology, colloidal stability, and retention of encapsulated materials in both acidic and neutral conditions. The ability of ALA(11,17)-hybrid liposomes to encapsulate and retain inclusions under neutral and acidic conditions (pH < 2) was demonstrated by calcein dequenching experiments. DLS and SEM confirmed that ALA(11,17)/DSPC liposomes remained intact under these conditions. The bilayer integrity observed under neutral and acidic conditions and the likely biocompatibility of these fatty amino acid analogues suggest that ALA(11,17) is a promising additive for modulating phosphatidylcholine lipid bilayer properties.     

Partition coefficients of ω-phenylalkanols between water and liposome membranes of phospholipids

For the liposome of two types of phospholipids (zwitterionic dipalmitoyl-phosphatidylcholine (DPPC) and anionic dimyristoylphosphatidic acid (DMPA)), the partition coefficient K _X for partition of homologous -phenylalkanols (C₆H₅(CH₂) _mp OH; m _p =0–8) between bulk water and the liposome membrane was determined on the basis of the gel to liquid crystalline phase transition temperature T _m of the liposome membrane. The plot of log K _X vs. m _p gave a break at m _p =7 for both phospholipids, and a second break at m _p =4 was observed for DPPC. The local polarity of the surface region and the orderliness of phospholipid molecules in the liposome membrane were estimated from ESR spectra of two spin probes solubilized in the membrane. The results suggest that the hydration of DPPC liposome membrane is relatively restricted to its surface region, but for DMPA the hydration spreads not only along the surface but also to the inside of the membrane. The main factor controlling the partitioning of the alkanols is the local polarity. The higher alkanols (m _p =7, 8) are solubilized not only in the liquid crystalline phase but also in the gel phase, although the other lower alkanols are solubilized in the liquid phase only.     

Factors influencing the distribution pattern of porphyrins in cell membranes

The mechanism of the sensitizer-membrane interactions has been studied by following the distribution properties of selected porphyrins, including haematoporphyrin (HP) and protoporphyrin (PP), into unilamellar liposomes of dipalmitoyl phosphatidylcholine (DPPC). The endomembrane distribution of HP and PP has been checked as a function of the membrane fluidity and composition by fluorescence polarization and quenching techniques. At porphyrin concentrations below 0.5 microM, HP and PP exclusively localize in the inner phospholipid monolayer; at higher concentrations, the outer monolayer also becomes populated. The porphyrin binding sites in liposomes, however, are different for HP and PP: HP preferentially distributes into water-accessible lipid regions, while PP localizes in the most hydrophobic loci of the lipid matrix. A porphyrin redistribution occurs when the fluidity properties of the liposomes are changed by addition of cholesterol or cardiolipin. In DPPC-cholesterol vesicles, all HP molecules dissolve in DPPC-rich regions while all PP molecules partition in cholesterol-rich environments. In DPPC-cardiolipin vesicles both porphyrins preferentially localize in regions accessible to the external medium. The effect of the nature of the carrier on porphyrin distribution in membranes has been studied by following the uptake and photosensitization properties of free and DPPC-incorporated PP and HP with rat liver mitochondria. The porphyrin photosensitizing efficiency has been checked by following the impairment of the respiratory function of mitochondria upon irradiation. Liposome-bound HP is less active than aqueous HP in determining membrane photodamage in mitochondria. On the contrary, aqueous PP is a very poor sensitizer as compared to a DPPC liposome-entrapped drug.(ABSTRACT TRUNCATED AT 250 WORDS)