Effect of lipid molecule headgroup mismatch on non steroidal anti-inflammatory drugs induced membrane fusion

Membrane fusion is an essential process guiding many important biological events, which most commonly requires the aid of proteins and peptides as fusogenic agents. Small drug induced fusion at low drug concentration is a rare event. Only three drugs, namely, meloxicam (Mx), piroxicam (Px), and tenoxicam (Tx), belonging to the oxicam group of non steroidal anti-inflammatory drugs (NSAIDs) have been shown by us to induce membrane fusion successfully at low drug concentration. A better elucidation of the mechanism and the effect of different parameters in modulating the fusion process will allow the use of these common drugs to induce and control membrane fusion in various biochemical processes. In this study, we monitor the effect of lipid headgroup size mismatch in the bilayer on oxicam NSAIDs induced membrane fusion, by introducing dimyristoylphosphatidylethanolamine (DMPE) in dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles (SUVs). Such headgroup mismatch affects various lipid parameters which includes inhibition of trans-bilayer motion, domain formation, decrease in curvature, etc. Changes in various lipidic parameters introduce defects in the membrane bilayer and thereby modulate membrane fusion. SUVs formed by DMPC with increasing DMPE content (10, 20, and 30 mol %) were used as simple model membranes. Transmission electron microscopy (TEM) and differential scanning calorimetry (DSC) were used to characterize the DMPC-DMPE mixed vesicles. Fluorescence assays were used to probe the time dependence of lipid mixing, content mixing, and leakage and also used to determine the partitioning of the drugs in the membrane bilayer. How the inhibition of trans-bilayer motion, heterogeneous distribution of lipids, decrease in vesicle curvature, etc., arising due to headgroup mismatch affect the fusion process has been isolated and identified here. Mx amplifies these effects maximally followed by Px and Tx. This has been correlated to the enhanced partitioning of the hydrophobic Mx compared to the more hydrophilic Px and Tx in the mixed bilayer.     

Effect of PIP2 on Bilayer Structure and Phase Behavior of DOPC: An X‐ray Scattering Study

Phosphatidylinositol 4,5‐bis‐phosphate (PIP₂) is an important lipid in regulation of several cellular processes, particularly membrane fusion. We use X‐ray diffraction from solid‐supported multilamellar 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC)/PIP₂ samples to study changes in bilayer structure and the lyotropic phase behavior induced by physiologically relevant concentrations of PIP₂. Electron‐density profiles reconstructed from X‐ray reflectivity measurements indicate that PIP₂ strongly affects structural parameters such as lipid head‐group width, bilayer thickness, and lamellar repeat spacing of DOPC bilayer stacks. In addition, at lower degrees of hydration, a few molar per cent of PIP₂ facilitates stalk‐phase formation and also leads to formation of a hexagonal phase, which is not observed in pure DOPC. These results indicate that the role of PIP₂ in membrane fusion could be, in part, due to its effect on the properties of the lipid bilayer matrix. Furthermore, coexistence of two lamellar phases with different lattice constants is observed in single‐component PIP₂ samples.     

Temperature-dependence of cationic lipid bilayer intermixing: possible role of interdigitation

In this work, we investigated the properties of a fusogenic cationic lipid, diC14-amidine, and show that this lipid possesses per se the capacity to adopt either an interdigitated structure (below and around its transition temperature) or a lamellar structure (above the transition temperature). To provide experimental evidence of this lipid bilayer organization, phospholipids spin-labeled at different positions of the hydrocarbon chain were incorporated into the membrane and their electron spin resonance (ESR) spectra were recorded at different temperatures. For comparison, similar experiments were performed with dimyristoyl phosphatidylcholine, a zwitterionic lipid (DMPC) which adopts a bilayer organization over a broad temperature range. Lipid mixing between diC14-amidine and asolectin liposomes was more efficient below (10-15 °C) than above the transition temperature (above 25 °C). This temperature-dependent "fusogenic" activity of diC14-amidine liposomes is opposite to what has been observed so far for peptides or virus-induced fusion. Altogether, our data suggest that interdigitation is a highly fusogenic state and that interdigitation-mediated fusion occurs via an unusual temperature-dependent mechanism that remains to be deciphered.     

Steady-state oxidation of cholesterol catalyzed by cholesterol oxidase in lipid bilayer membranes on platinum electrodes

Cholesterol oxidase is immobilized in electrode-supported lipid bilayer membranes. Platinum electrodes are initially modified with a self-assembled monolayer of thiolipid. A vesicle fusion method is used to deposit an outer leaflet of phospholipids onto the thiolipid monolayer forming a thiolipid/lipid bilayer membrane on the electrode surface. Cholesterol oxidase spontaneously inserts into the electrode-supported lipid bilayer membrane from solution and is consequently immobilized to the electrode surface. Cholesterol partitions into the membrane from buffer solutions containing cyclodextrin. Cholesterol oxidase catalyzes the oxidation of cholesterol by molecular oxygen, forming hydrogen peroxide as a product. Amperometric detection of hydrogen peroxide for continuous solution flow experiments are presented, where flow was alternated between cholesterol solution and buffer containing no cholesterol. Steady-state anodic currents were observed during exposures of cholesterol solutions ranging in concentration from 10 to 1000 μM. These data are consistent with the Michaelis-Menten kinetic model for oxidation of cholesterol as catalyzed by cholesterol oxidase immobilized in the lipid bilayer membrane. The cholesterol detection limit is below 1 μM for cholesterol solution prepared in buffered cyclodextrin. The response of the electrodes to low density lipoprotein solutions is increased upon addition of cyclodextrin. Evidence for adsorption of low density lipoprotein to the electrode surface is presented.     

'Boomerang'-like insertion of a fusogenic peptide in a lipid membrane revealed by solid-state 19F NMR

Solid state (19)F NMR revealed the conformation and alignment of the fusogenic peptide sequence B18 from the sea urchin fertilization protein bindin embedded in flat phospholipid bilayers. Single (19)F labels were introduced into nine distinct positions along the wild-type sequence by substituting each hydrophobic amino acid, one by one, with L-4-fluorophenylglycine. Their anisotropic chemical shifts were measured in uniaxially oriented membrane samples and used as orientational constraints to model the peptide structure in the membrane-bound state. Previous (1)H NMR studies of B18 in 30% TFE and in detergent micelles had shown that the peptide structure consists of two alpha-helical segments that are connected by a flexible hinge. This helix-break-helix motif was confirmed here by the solid-state (19)F NMR data, while no other secondary structure (beta-sheet, 3(10)-helix) was compatible with the set of orientational constraints. For both alpha-helical segments we found that the helical conformation extends all the way to the respective N- and C-termini of the peptide. Analysis of the corresponding tilt and azimuthal rotation angles showed that the N-terminal helix of B18 is immersed obliquely into the bilayer (at a tilt angle tau approximately 54 degrees), whereas the C-terminus is peripherally aligned (tau approximately 91 degrees). The azimuthal orientation of the two segments is consistent with the amphiphilic distribution of side-chains. The observed 'boomerang'-like mode of insertion into the membrane may thus explain how peptide binding leads to lipid dehydration and acyl chain perturbation as a prerequisite for bilayer fusion to occur.     

Cholesterol location and orientation in aqueous suspension of large unilamellar vesicles of phospholipid revealed by intermolecular nuclear overhauser effect

The locational and orientational structure and the dynamics of cholesterol in the bilayer membrane were studied by using the solution-state NMR. The intermolecular nuclear Overhauser effect (NOE) was analyzed for large unilamellar vesicles (100 nm in diameter) composed of dimyristoylphosphatidylcholine (DMPC) and cholesterol at cholesterol concentrations of 9-33 mol %. The DMPC headgroups show (1)H-{(1)H}-NOEs with the methyl groups at the hydrophobic terminals of both cholesterol and DMPC, illustrating the significant fluctuation of the bilayer membrane in the vertical (bilayer normal) direction. Cholesterol was found to keep the hydroxyl (OH) group toward the outer water pool on the basis of the following observations: (1) the cross correlation between the DMPC headgroup and the cholesterol terminal methyl group is weaker than those between the DMPC headgroups and (2) the methyl group at the hydrophobic terminal of cholesterol shows strong correlation with the terminal group of the DMPC chain portion. The OH group plays a crucial role in orienting cholesterol with its OH group outward, since cholestane, which has a molecular structure similar to that of cholesterol except for the absence of the OH group, was found to have no orientational preference in the bilayer membrane. The dynamic slowdown at high cholesterol concentrations is demonstrated on the basis of the correlation times for NOE as well as the broadening of the proton linewidths.     

Modification of membrane heterogeneity by antipsychotic drugs: an X-ray diffraction comparative study

Lipid mixtures are used to mimic biological membranes as they allow characterization of lipid lateral domains defined by their specific lipid molecular organization. Therapeutic agents such as antipsychotic drugs (AP) that may interact with lipids arrangement are likely to modify membrane biological properties. The present study describes the effect of 2 typical and 5 atypical antipsychotic drugs on an aqueous co-dispersion of a lipid mixture made of egg phosphatidylcholine (PC)/brain sphingomyelin (SM)/cholesterol (1/1/1 mol/mol/mol). Lamellar liquid-ordered (Lo) and liquid-disordered (Ld) phase coexistence was identified in the control and antipsychotic-added mixtures at 37 degrees C using synchrotron small-angle X-ray scattering methods (XRD). Intensity of the Bragg peaks was used to generate electron density profiles (EDP) allowing bilayer thickness calculation. All antipsychotic except from amisulpride induced a Lo phase bilayer thickness (d(pp)) decrease. Chlorpromazine, haloperidol, amisulpride and 9-0H-risperidone induced a Ld d(pp) increase while ziprazidone, risperidone and clozapine induced a Ld d(pp) decrease, indicating that antipsychotic atypicality is not associated with a specific d(pp) modification on our lipid model mixture. Results are discussed in terms of competition of antipsychotic compounds with cholesterol and mode of reorganization of lateral domains. A pharmacological relevance of these changes is also discussed.     

Effects of cholesterol component on molecular interactions between paclitaxel and phospholipid within the lipid monolayer at the air-water interface

Cholesterol is a main component of the cell membrane and could have significant effects on drug-cell membrane interactions and thus the therapeutic efficacy of the drug. It also plays an important role in liposomal formulation of drugs for controlled and targeted delivery. In this research, Langmuir film technique, atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) are employed for a systematic investigation on the effects of cholesterol component on the molecular interactions between a prototype antineoplastic drug (paclitaxel) and 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) within the cell membrane by using the lipid monolayer at the air-water interface as a model of the lipid bilayer membrane and the biological cell membrane. Analysis of the measured surface pressure (pi) versus molecular area (a) isotherms of the mixed DPPC/paclitaxel/cholesterol monolayers at various molar ratios shows that DPPC, paclitaxel and cholesterol can form a non-ideal miscible system at the air-water interface. Cholesterol enhances the intermolecular forces between paclitaxel and DPPC, produces an area-condensing effect and thus makes the mixed monolayer more stable. Investigation of paclitaxel penetration into the mixed DPPC/cholesterol monolayer shows that the existence of cholesterol in the DPPC monolayer can considerably restrict the drug penetration into the monolayer, which may have clinical significance for diseases of high cholesterol. FTIR and AFM investigation on the mixed monolayer deposited on solid surface confirmed the obtained results.     

Fusogenic tilted peptides induce nanoscale holes in supported phosphatidylcholine bilayers

Tilted peptides are known to insert in lipid bilayers with an oblique orientation, thereby destabilizing membranes and facilitating membrane fusion processes. Here, we report the first direct visualization of the interaction of tilted peptides with lipid membranes using in situ atomic force microscopy (AFM) imaging. Phase-separated supported dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers were prepared by fusion of small unilamellar vesicles and imaged in buffer solution, in the absence and in the presence of the simian immunodeficiency virus (SIV) peptide. The SIV peptide was shown to induce the rapid appearance of nanometer scale bilayer holes within the DPPC gel domains, while keeping the domain shape unaltered. We attribute this behavior to a local weakening and destabilization of the DPPC domains due to the oblique insertion of the peptide molecules. These results were directly correlated with the fusogenic activity of the peptide as determined using fluorescently labeled DOPC/DPPC liposomes. By contrast, the nontilted ApoE peptide did not promote liposome fusion and did not induce bilayer holes but caused slight erosion of the DPPC domains. In conclusion, this work provides the first direct evidence for the production of stable, well-defined nanoholes in lipid bilayer domains by the SIV peptide, a behavior that we have shown to be specifically related to the tilted character of the peptide. A molecular mechanism underlying spontaneous insertion of the SIV peptide within lipid bilayers and the subsequent removal of bilayer patches is proposed, and its relevance to membrane fusion processes is discussed.     

Evaluation of 1-naphthol as a convenient fluorescent probe for monitoring ethanol-induced interdigitation in lipid bilayer membrane.

In this work we have tried to evaluate the usefulness of 1-naphthol as an excited state proton transfer fluorescent probe for studying the ethanol-induced interdigitation in lipid bilayer membranes. When ethanol concentration in lipisome is progressively increased, the neutral form fluorescence of 1-naphthol is found to decrease with corresponding increase in the anionic form intensity. This behavior is in contrast to that observed in the absence of lipid where a reverse effect is noticed. Modification of lipid bilayer is known to occur in the presence of ethanol, which increases the packing density of the membrane. Due to this induction of interdigitated gel phase, redistribution of naphthol between the inner core and interfacial region of the lipid bilayer takes places, accounting for the reduction in neutral form fluorescence intensity. The partition coefficient values and the quenching studies also support the redistribution of 1-naphthol in the liposome membrane. The neutral form fluorescence of 1-naphthol successfully monitors the shift in phase transition temperature due to ethanol-induced interdigitation. It also explains the prevention of interdigitation in lipid bilayer at high cholesterol concentration.     

Partitioning of dual-lipidated peptides into membrane microdomains: lipid sorting vs peptide aggregation

The lateral membrane organization and phase behavior of the lipid mixture DMPC(di-C(14))/DSPC(di-C(18))/cholesterol (0-33 mol %) with and without an incorporated fluorescence-labeled palmitoyl/farnesyl dual-lipidated peptide, BODIPY-Gly-Cys(Pal)-Met-Gly-Leu-Pro-Cys(Far)-OMe, which represents a membrane recognition model system for Ras proteins, was studied by two-photon excitation fluorescence microscopy. Measurements were performed on giant unilamellar vesicles (GUVs) over a large temperature range, ranging from 30 to 80 degrees C to cover different lipid phase states (all-gel, fluid/gel, liquid-ordered, all-fluid). At temperatures where the fluid-gel coexistence region of the pure binary phospholipid system occurs, large-scale concentration fluctuations appear. Incorporation of cholesterol levels up to 33 mol % leads to a significant increase of conformational order in the membrane system and a reduction of large domain structures. Adding the peptide leads to dramatic changes in the lateral organization of the membrane. With cholesterol present, a phase separation is induced by a lipid sorting mechanism owing to the high affinity of the lipidated peptide to a fluid, DMPC-rich environment. This phase separation leads to the formation of peptide-containing domains with high fluorescence intensity that become progressively smaller with decreasing temperature. As a result, the local concentration of the peptide increases steadily within the confines of the shrinking domains. At the lowest temperatures, where the acyl-chain order parameter of the membrane has already drastically increased and the membrane achieves a liquid-ordered character, an efficient lipid sorting mechanism is no longer supported and aggregation of the peptide into small clusters prevails. We can conclude that palmitoyl/farnesyl dual-lipidated peptides do not associate with liquid-ordered or gel-like domains in phase-separated bilayer membranes. In particular, the study shows the interesting ability of the peptide to induce formation of fluid microdomains at physiologically relevant cholesterol concentrations, and this effect very much depends on the concentration of fluid vs ordered lipid molecules.     

Fusion of lipid vesicles with planar lipid bilayers induced by a combination of peptides

We studied the peptide-induced membrane fusion process between small unilamellar vesicles (SUVs) and supported planar bilayers (SPBs) with the aim of developing a method for incorporating membrane components into SPBs. As fusogenic peptides, two analogues of the N-terminal region of an influenza membrane fusion protein hemaggulutinin, anionic E5 and cationic K5, were synthesized, and the membrane fusion was investigated using SPB and SUVs composed of phosphatidylcholine from egg yolk (EggPC). We directly visualized the process of lipid transfer from SUVs to SPB by total internal reflection fluorescence (TIRF) microscopy. The transfer of fluorescent lipids was effectively induced only by the combination of two peptides. The TIRF microscopy observations of single SUV fusion events also revealed that lipid membranes from SUV could completely fuse into the SPB. However, the presence of single peptide (either E5 or K5) rather inhibited the lipid transfer, presumably due to the electrostatic repulsion between SUVs and SPB. The opposite effects induced by the peptides indicate the possibility for a designed application of two peptides as a means to control the membrane fusion spatially and temporally.     

Designing a Useful Lipid Raft Model Membrane for Electrochemical and Surface Analytical Studies

A model biomimetic system for the study of protein reconstitution or drug interactions should include lipid rafts in the mixed lipid monolayer, since they are usually the domains embedding membrane proteins and peptides. Four model lipid films composed of three components: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol (Chol) and sphingomyelin (SM) mixed in different molar ratios were proposed and investigated using surface pressure measurements and thermodynamic analysis of the monolayers at the air–water interface and imaged by Brewster angle microscopy. The ternary monolayers were transferred from the air–water onto the gold electrodes to form bilayer films and were studied for the first time by electrochemical methods: alternative current voltammetry and electrochemical impedance spectroscopy and imaged by atomic force microscopy. In excess of DOPC, the ternary systems remained too liquid for the raft region to be stable, while in the excess of cholesterol the layers were too solid. The layers with SM in excess lead to the formation of Chol:SM complexes but the amount of the fluid matrix was very low. The equimolar content of the three components lead to the formation of a stable and well-organized assembly with well-developed raft microdomains of larger thickness, surrounded by the more fluid part of the bilayer. The latter is proposed as a convenient raft model membrane for further physicochemical studies of interactions with drugs or pollutants or incorporation of membrane proteins.     

pH-induced destabilization of lipid bilayers by a peptide from the VP3 protein of the capsid of hepatitis A virus.

The membrane destabilizing and fusogenic properties of the synthetic peptide VP3(110-121), corresponding to an immunogenic sequence of the hepatitis A virus (HAV) VP3 capsid protein, were studied. By tryptophan fluorescence and acryalmide quenching it was demonstrated that the peptide binds liposomes of POPC-SM-DPPE (47 + 39 + 14) and POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) and penetrates the membrane, at both neutral and acidic pH (POPC = 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine; SM = sphingomyelin; DPPE = 1,2-dipalmitoylphosphatidylethanolamine; DOTAP = 1,2-dioleoyl-3-trimethylammoniumpropane). VP3(110-121) did not have membrane-destabilizing properties at neutral pH. Acid-induced destabilization of the vesicles was demonstrated by fluorescence techniques and dynamic light scattering. VP3(110-121) induced aggregation of POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) vesicles, lipid mixing and leakage of vesicle contents, all consistent with fusion of vesicles. In POPC-SM-DPPE (47 + 39 + 14) vesicles, at acidic pH, VP3(110-121) induced membrane destabilization with leakage of contents but without aggregation of vesicles or lipid mixing. The peptide only showed fusogenic properties when bound to the vesicles at neutral pH before acidification to pH below 6.0, and no effect was seen if the peptide was added to vesicles already set at acidic pH. These results may have physiological significance in the mechanism of infection of host hepatic cells by HAV.     

Solid-supported biomimetic membranes with tailored lipopolymer tethers

Stable lipid membranes with controlled substrate-membrane spacing can be prepared using well-defined lipopolymers as a tether. Based on the living cationic ring-opening polymerization of 2-methyl- or 2-ethyl-2-oxazoline, lipopolymers can be synthesized bearing a lipid head group as well as a silanol reactive coupling end group. Using a “grafting onto” procedure these polymers can form dense, brush like monolayers, whose layered structures can be obtained by x-ray reflectivity measurements. By transfer of a pre-organized monolayer that is followed by vesicle fusion, stable polymer supported lipid membranes can be prepared. The substrate-membrane spacing can be controlled via the degree of polymerization, while the lateral diffusion of lipids within the membrane depends on the density of polymer tethers. Preliminary experiments implied that the membrane with long (N = 40) polymer tethers could reside trans-membrane receptors homogeneously, suggesting a large potential of this strategy.     

Modification of Lipid Bilayer Structure by Diacylglycerol: A Comparative Study of Diacylglycerol and Cholesterol

Diacylglycerols (DAGs) are important second messengers in biomembranes, and they can activate protein kinase C and many other enzymes and receptors. However, their interactions with cholesterol and other lipids have not been previously studied using molecular dynamics (MD) simulation. In this study, nine independent atomistic MD simulations were performed to specifically investigate the interactions between di16:0DAG, 16:0,18:1-phosphatidylcholine (POPC), and cholesterol. Despite of their substantial differences in chemical structure, DAG and cholesterol produce some very similar effects in POPC bilayers: increasing acyl chain order and bilayer thickness, reducing volume-per-lipid, and decreasing lateral diffusion of molecules. More significantly, DAG also produces a strong "condensing effect" in PC bilayers. In comparison, cholesterol is more effective than DAG in producing the above effects. The driving force for the condensing effect is their molecular shape: DAG and cholesterol both have small polar headgroups and large hydrophobic bodies. In a lipid bilayer, in order to avoid the unfavorable exposure of their hydrophobic parts to water, neighboring phospholipid headgroups move toward cholesterol or DAG to provide cover. Thus, seemingly complex interactions between DAG, cholesterol and phospholipid can be clearly explained using the Umbrella Model. Our simulations confirmed the hypothesis that DAG increases the spacing between phospholipid headgroups, which is important for activating protein kinase C and other enzymes. Interestingly, our simulations also show that the conventional wisdom that the spacing created by a DAG is directly above the DAG molecule is incorrect; instead, the largest spacing usually occurs between the first and the second nearest-neighbor PC headgroups from a DAG, due to the umbrella effect.     

Fluorescent lipids: functional parts of fusogenic liposomes and tools for cell membrane labeling and visualization

In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system.     

Alterations in the Detergent-Induced Membrane Permeability and Solubilization of Saturated Phosphatidylcholine/Cholesterol Liposomes: Effects of Poly(ethylene glycol)-Conjugated Lipid

We have investigated the effects of two bile salts, chenodeoxycholate (CDC) and ursodeoxycholate (UDC), and a widely used detergent, Triton X-100 (T(X-100)), on normal and poly(ethylene glycol)-modified liposomes (PEGylated liposomes). We tested various lipid compositions, including hydrogenated soybean phosphatidylcholine/cholesterol/PEG-conjugated lipid (HSPC/PEG-lipid). Alterations in permeability were determined by the rate of drug release from the liposomes and solubilization was assessed by measuring the particle size of liposomes. In addition, we attempted to observe interactions between the detergents and lipid bilayers by using surface plasmon resonance (SPR). CDC induced drug release from liposomes in a dose-dependent manner, and the PEGylated liposomes tended to be susceptible to CDC. While UDC did not strongly induce drug release from liposomes, UDC exhibited a similar tendency with CDC. In case of T(X-100), there were significant differences in the percentage of released drug between normal and PEGylated liposomes, and the percentage of T(X-100)-induced drug release further increased with an increased ratio of PEG-lipid. SPR analysis revealed that the lipid bilayer including PEG-lipid was selectively solubilized by T(X-100), correlating with the drug release data. These results suggest that the effect of detergents on the lipid bilayer of liposomes depends on both the kind of detergent and the lipid composition, including the presence or absence of PEG-lipid. Moreover, the effects of T(X-100) on the lipid bilayers of the PEGylated liposomes significantly differed from those on the lipid bilayers of the normal liposomes.     

Cholesterol slows down the lateral mobility of an oxidized phospholipid in a supported lipid bilayer

We investigated the mobility and phase-partitioning of the fluorescent oxidized phospholipid analogue 1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N-Alexa647-ethanolamine (PGPE-Alexa647) in supported lipid bilayers. Compared to the conventional phospholipid dihexadecanoylphosphoethanolamine (DHPE)-Bodipy we found consistently higher diffusion constants. The effect became dramatic when immobile obstacles were inserted into the bilayer, which essentially blocked the diffusion of DHPE-Bodipy but hardly influenced the movements of PGPE-Alexa647. In a supported lipid bilayer made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), the differences in probe mobility leveled off with increasing cholesterol content. Using coarse-grained molecular dynamics simulations, we could ascribe this effect to increased interactions between the oxidized phospholipid and the membrane matrix, concomitant with a translation in the headgroup position of the oxidized phospholipid: at zero cholesterol content, its headgroup is shifted to the outside of the DOPC headgroup region, whereas increasing cholesterol concentrations pulls the headgroup into the bilayer plane.     

Membrane disordering induced by chloroform and carbon tetrachloride

We studied effects of chloroform and carbon tetrachloride on bilayer membranes of dimyristoyl-phosphatidylcholine (DMPC) and egg yolk phosphatidylcholine (Egg-PC) by birefringence, dynamic light scattering and fluorescence methods. It is shown that interference light due to the membrane birefringence considerably decreases by addition of the organohalogen compounds for both lipid membranes, indicating a significant decrease in membrane order. In addition, results of dynamic light scattering and turbidity measurements show a rupture of multilamellar DMPC vesicles induced by addition of chloroform at concentrations above 0.2 v/v%. No rupture of the vesicles is observed within the limit of solubility of carbon tetrachloride in water, but excessive addition of carbon tetrachloride (above 0.2 v/v%) induces the vesicle rupture. Chain orientational order was estimated from the interference light intensity at low concentrations of the organohalogen compounds without the occurrence of the vesicle rupture. The estimation shows a monotonic decrease in the chain order with increasing the concentration. The decreases in DMPC chain order by chloroform and by carbon tetrachloride are about 17% at 0.2 v/v% and 23% at 0.05 v/v%, respectively. The reduction in the chain order is correlated with an increase in the membrane fluidity observed by excimer fluorescence of pyrene incorporated to the membrane. Behavior of membrane disordering of Egg-PC is approximately similar with that of DMPC. This implies the strong interaction between the organohalogen compounds and the lipid chains, whether or not the bilayer has the vacancy resulted from unsaturated double bonds and different chains in length. The results of this work suggest that damages of biological membranes by chloroform and tetrachloride are not only induced by a direct attack on proteins but also by a significant membrane disorder.