Membrane fusion between liposomes composed of acidic phospholipids and neutral phospholipids induced by melittin: a differential scanning calorimetric study

Melittin-induced membrane fusion between neutral and acidic phospholipids was examined in liposome systems with a high-sensitivity differential scanning calorimeter. Membrane fusion could be detected by calorimetric measurement by observing thermograms of mixed liposomal lipids. The roles of hydrophobic and electrostatic interactions were investigated in membrane fusion induced by melittin. Melittin, a bee venom peptide, is composed of a hydrophobic region including hydrophobic amino acids and a positively charged region including basic amino acids. When phosphatidylcholine liposomes were prepared in the presence of melittin, reductions in the phase transition enthalpies were observed in the following order; dimyristoylphosphatidylcholine (DMPC) > dipalmitoylphosphatidylcholine (DPPC) > distearoylphosphatidylcholine (DSPC) > dielaidoylphosphatidylcholine (DEPC). The plase transition enthalpy of an acidic phospholipid, dipalmitoylphosphatidylserine (DPPS), was raised by melittin at low concentrations, then reduced at higher concentrations. DPPC liposomes prepared in melittin solution were fused with DPPS liposomes when the liposomal dispersions were mixed and incubated. Similar fusion was observed between dipalmitoylphosphatidylcholine and dimyristoylphosphatidic acid (DMPA) liposomes. These results indicate that a peptide including hydrophobic and basic regions can mediate membrane fusion between neutral and acidic liposomes by hydrophobic and electrostatic interactions.     

Selective partitioning of cholesterol and a model drug into liposomes of varying size

The resistance of a lipid bilayer with respect to a bending deformation generally depends on the presence of membrane additives such as sterols, cosurfactants, peptides, and drugs. As a consequence, the partitioning of membrane additives into liposomes becomes selective with respect to liposome size; i.e., membrane rigidification depletes the membrane additives in the smaller (more strongly curved) liposomes. We have measured this liposome size-selective partitioning for two membrane additives - cholesterol and the porphyrin-based photosensitizer temoporfin - using asymmetrical flow field-flow fractionation (AF4) of liposomes and radioactive labeling of the membrane additive and lipid. The method yields either the molar cholesterol-to-lipid or the temoporfin-to-lipid ratio as a function of liposome size, from which we calculate the corresponding change of the membrane bending stiffness. For small unilamellar fluid-phase liposomes composed of palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylglycerol (POPG), we find that cholesterol rigidifies the host membrane in a manner consistent with previously reported measurements. In contrast, temoporfin softens this membrane. Partitioning results for gel-phase liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) are also curvature-sensitive but cannot be interpreted on the basis of the bending stiffness alone.     

Effects of phospholipid hydrolysis on the aggregate structure in DPPC/DSPE-PEG2000 liposome preparations after gel to liquid crystalline phase transition

Upon storage of phospholipid liposome samples, lysolipids, fatty acids, and glycerol-3-phosphatidylcholine are generated as a result of acid- or base-catalyzed hydrolysis. Accumulation of hydrolysis products in the liposome membrane can induce fusion, leakage, and structural transformations of the liposomes, which may be detrimental or beneficial to their performance depending on their applications as, e.g., drug delivery devices. We investigated in the present study the influence of phospholipid hydrolysis on the aggregate morphology of DPPC/DSPE-PEG₂₀₀₀ liposomes after transition of the phospholipid membrane from the gel phase to liquid crystalline phase using high performance liquid chromatography (HPLC) in combination with static light scattering, dynamic light scattering, and cryo-transmission electron microscopy (cryo-TEM). The rates of DPPC hydrolysis in DPPC/DSPE-PEG₂₀₀₀ liposomes were investigated at a pH of 2, 4, or 6.5 and temperatures of 22 °C or 4 °C. Results indicate that following phase transition, severe structural reorganizations occurred in liposome samples that were partially hydrolyzed in the gel phase. The most prominent effect was an increasing tendency of liposomes to disintegrate into membrane discs in accordance with an increasing degree of phospholipid hydrolysis. Complete disintegration occurred when DPPC concentrations had decreased by, in some cases, as little as 3.6%. After extensive phospholipid hydrolysis, liposomes and discs fused to form large bilayer sheets as well as other more complex bilayer structures apparently due to a decreased ratio of lysolipid to palmitic acid levels in the liposome membrane.     

Interaction of N,N,N-trialkylammonioundecahydro-closo-dodecaborates with dipalmitoyl phosphatidylcholine liposomes

N,N,N-Trialkylammonioundecahydrododecaborates (1-), a novel class of compounds of interest for use as anions in ionic liquids, interact with DPPC liposomes. Increasing compound concentration causes an increasing negative ζ potential. Dissociation constants demonstrate that the binding capacity increases strongly with longer chain length. N,N,N-Trialkylammonioundecahydrododecaborates with longer alkyl chains show a detergent-like behavior: the compounds incorporate into the liposome membrane and differential scanning calorimetric experiment show already low concentrations cause a complete disappearance of the peak representing the gel-to-liquid crystalline phase transition. In contrast, compounds with shorter alkyl chains only interact with the headgroups of the lipids. Investigations by means of cryo-TEM reveal that all derivatives induce significant morphological changes of the liposomes. N,N,N-Trialkylammonioundecahydrododecaborates with short alkyl chains produce large bilayer sheets, whereas those with longer alkyl chains tend to induce the formation of open or multi-layered liposomes. We propose that the binding of N,N,N-trialkylammonioundecahydrododecaborates is mainly due to electrostatic interactions between the doubly negatively charged cluster unit and the positively charged choline headgroup; the positively charged ammonium group might be in contact with the deeper-lying negatively charged phosphate. For N,N,N-trialkylammonioundecahydrododecaborates with longer alkyl chains hydrophobic interactions with the non-polar hydrocarbon part of the membrane constitute an additional important driving force for the association of the compounds to the lipid bilayer.     

Phospholipid-cationic lipid interactions: influences on membrane and vesicle properties

Liposomes composed of synthetic dialkyl cationic lipids and zwitterionic phospholipids such as dioleoylphosphatidylethanolamine have been studied extensively as vehicles for gene delivery, but the broader potentials of these cationic liposomes for drug delivery have not. An understanding of phospholipid-cationic lipid interactions is essential for rational development of this potential. We evaluated the effect of the cationic lipid DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium) on liposome physical properties such as size and membrane domain structure. DSC (differential scanning calorimetry) showed progressive decrease and broadening of the phase transition temperature of dipalmitoylphosphatidylcholine (DPPC) with increasing fraction of DOTAP, in the range of 0.4-20 mol%. Laurdan (6-dodecanolyldimethylamino-naphthalene), a fluorescent probe of membrane domain structure, showed that DOTAP and DPPC remained miscible at all ratios tested. DOTAP reduced the size of spontaneously-forming PC-containing liposomes, regardless of the acyl chain length and degree of saturation. The anionic lipid DOPG (dioleoylphosphatidylglycerol) had similar effects on DPPC membrane fluidity and size. However, DOTAP/DOPC (50/50) vesicles were taken up avidly by OVCAR-3 human ovarian tumor cells, in contrast to DOPG/DOPC (50/50) liposomes. Overall, DOTAP exerts potent effects on bilayer physical properties, and may provide advantages for drug delivery.     

Effects of phospholipid hydrolysis on the aggregate structure in DPPC/DSPE-PEG2000 liposome preparations after gel to liquid crystalline phase transition

Upon storage of phospholipid liposome samples, lysolipids, fatty acids, and glycerol-3-phosphatidylcholine are generated as a result of acid- or base-catalyzed hydrolysis. Accumulation of hydrolysis products in the liposome membrane can induce fusion, leakage, and structural transformations of the liposomes, which may be detrimental or beneficial to their performance depending on their applications as, e.g., drug delivery devices. We investigated in the present study the influence of phospholipid hydrolysis on the aggregate morphology of DPPC/DSPE-PEG2000 liposomes after transition of the phospholipid membrane from the gel phase to liquid crystalline phase using high performance liquid chromatography (HPLC) in combination with static light scattering, dynamic light scattering, and cryo-transmission electron microscopy (cryo-TEM). The rates of DPPC hydrolysis in DPPC/DSPE-PEG2000 liposomes were investigated at a pH of 2, 4, or 6.5 and temperatures of 22 degrees C or 4 degrees C. Results indicate that following phase transition, severe structural reorganizations occurred in liposome samples that were partially hydrolyzed in the gel phase. The most prominent effect was an increasing tendency of liposomes to disintegrate into membrane discs in accordance with an increasing degree of phospholipid hydrolysis. Complete disintegration occurred when DPPC concentrations had decreased by, in some cases, as little as 3.6%. After extensive phospholipid hydrolysis, liposomes and discs fused to form large bilayer sheets as well as other more complex bilayer structures apparently due to a decreased ratio of lysolipid to palmitic acid levels in the liposome membrane.     

Interactions of VIP with rigid phospholipid bilayers: implications for vasoreactivity

The purpose of this study was to determine whether vasoactive intestinal peptide (VIP), a pleiotropic amphipathic peptide, interacts with rigid liposomes composed of gel phase phospholipids. We found that incubation of VIP with small unilamellar gel phase liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and egg phosphatidylglycerol (ePG) for 2h at room temperature had no significant effects on VIP secondary structure. Moreover, suffusion of VIP (0.01, 0.1 and 1.0nmol) incubated in saline or with DPPC/ePG liposomes (size, 30 and 100nm) for 2h at room temperature or 4 degrees C onto the intact hamster cheek pouch microcirculation elicited a similar concentration-dependent vasodilation except for 0.01nmol VIP (P<0.05). By contrast, incubation of VIP with gel phase liposomes overnight at 4 degrees C significantly potentiated vasodilation evoked by all three concentrations of the peptide in comparison to aqueous VIP (P<0.05). VIP-induced vasodilation was liposome size-independent. The ratio of VIP to phospholipids in DPPC/ePG liposomes was concentration-independent. Collectively, these data indicate that short-term interactions of VIP with rigid phospholipid bilayers are limited resulting in only modest effects on VIP vasoreactivity in vivo.     

Encapsulation of Enrofloxacin in Liposomes I: Preparation and In Vitro Characterization of LUV

Liposomes are effectively used in the treatment of microbial infections. Higher cellular uptake has been reported when antibiotics are encapsulated in liposomes. In this study, enrofloxacin (ENF) was encapsulated in large unilamellar vesicles (LUVs) and the effects of formulation variables on the liposome characteristics were investigated. Liposomes were prepared using dry lipid film method. A number of variables such as molar ratios of phospholipid (DPPC; DL‐α‐phosphatidylcholine dipalmitoyl), cholesterol, ENF and amount of α‐tocopherol and the volumes of internal (chloroform) and external phases [phosphate buffered saline PBS (pH 7.4)] were studied. In vitro characterization of the liposomes including the encapsulation capacity, size and drug release properties were carried out. Using of this method, spherical LUV liposomes with high drug content could be produced. Particle size of liposomes changed between 3.12 and 4.95 µm. The molar ratios of DPPC, cholesterol and ENF affected the size of the liposome (p < 0.05). The drug encapsulation capacities were high and changed between 37.1% and 79.5%. The highest ENF encapsulation was obtained with the highest cholesterol content. An increase in the drug encapsulation capacity of the liposome was found with increasing molar ratios of DPPC, cholesterol and ENF (p < 0.05). Furthermore, the release of ENF from the liposomes decreased as the molar ratios of DPPC, cholesterol and ENF increased (p < 0.05). In conclusion, a convenient colloidal carrier for the controlled release of ENF can be prepared by changing the formulation parameters of LUVs.     

Encapsulation of enrofloxacin in liposomes I: preparation and in vitro characterization of LUV

Liposomes are effectively used in the treatment of microbial infections. Higher cellular uptake has been reported when antibiotics are encapsulated in liposomes. In this study, enrofloxacin (ENF) was encapsulated in large unilamellar vesicles (LUVs) and the effects of formulation variables on the liposome characteristics were investigated. Liposomes were prepared using dry lipid film method. A number of variables such as molar ratios of phospholipid (DPPC; DL-alpha-phosphatidylcholine dipalmitoyl), cholesterol, ENF and amount of alpha-tocopherol and the volumes of internal (chloroform) and external phases [phosphate buffered saline PBS (pH 7.4)] were studied. In vitro characterization of the liposomes including the encapsulation capacity, size and drug release properties were carried out. Using of this method, spherical LUV liposomes with high drug content could be produced. Particle size of liposomes changed between 3.12 and 4.95 microm. The molar ratios of DPPC, cholesterol and ENF affected the size of the liposome (p < 0.05). The drug encapsulation capacities were high and changed between 37.1% and 79.5%. The highest ENF encapsulation was obtained with the highest cholesterol content. An increase in the drug encapsulation capacity of the liposome was found with increasing molar ratios of DPPC, cholesterol and ENF (p < 0.05). Furthermore, the release of ENF from the liposomes decreased as the molar ratios of DPPC, cholesterol and ENF increased (p < 0.05). In conclusion, a convenient colloidal carrier for the controlled release of ENF can be prepared by changing the formulation parameters of LUVs.     

Erratum to: “Effects of Gamma Irradiation on the Physical Stability of Liposomal Phospholipids” [J. Lipos. Res. 7, (1997) 503-528]

Abstract

The effects of liposome composition and gamma irradiation on the phase transition, size, zeta potential and pH were investigated using factorial designs. In addition, the effect of irradiation on the leak-in rate of calcein was evaluated for one of the liposome composition. The liposomes were stored for 6 months in order to reveal any possible long term effects. The phospholipids used were dipalmitoyl phosphatidyl choline (DPPC) or egg phosphatidyl choline (egg PC). Charge was introduced to the liposomal bilayers by the addition of 10% dipalmitoyl phosphatidyl glycerol (DPPG) or egg phosphatidyl glycerol (egg PG). The liposome-suspensions were obtained by the extrusion method. After gamma irradiation changes in the phase transition, zeta potential and pH of the liposomes were observed. The size of the liposomes was not affected by the irradiation, but the irradiation prevented the neutral DPPC-liposomes from aggregation. This was confirmed by cryo-electron microscopy. No change in the leak-in rate was observed. During storage, a significant increase in size was observed only for the non-irradiated egg PC-liposomes. For all the liposome-suspensions composed of unsaturated phospholipids, a significant drop in pH and an increased zeta potential (more negative) was measured. Changes in the phase transition for the neutral DPPC-liposomes (non-irradiated and irradiated) were observed during gamma irradiation.     

Effect of the Nature of the 3β-Substitution in Manoyl Oxides on the Thermotropic Behavior of DPPC Lipid Bilayer and on DPPC Liposomes

Functionalized manoyl oxide derivatives have been proved over the years to evoke several biological responses. Among them, 3β-hydroxy-manoyl oxide (1) and 3β-acetoxy-manoyl oxide (2) have been shown to exhibit in vitro antimicrobial and cytotoxic activity, while N-imidazole-3 β-thiocarbonyl ester of manoyl oxide (3) was found to exhibit potent cytotoxic effect. Their partitioning into phospholipid bilayers may lead to membrane structure modifications that are crucial in liposome development as they may influence their maintenance and integrity. DSC was used to study the modifications induced in DPPC bilayers by incorporating increasing concentrations of the three manoyl oxide derivatives. All derivatives were found to strongly affect the bilayer structural organization in terms of a decrease of the cooperativity, the fluidization and partially destabilization of the gel phase and the induction of a lateral phase separation in clustering domains. Derivatives 1 and 3 were incorporated into DPPC liposomes and their physicochemical stability was monitored at 4°C. The stability of liposomes was strongly influenced by the presence of 1 and 3 at any molar ratio studied. DPPC/1 liposomes were found to retain its stability for 48 h at low concentration of 10% mol, while at higher concentrations up to 30% mol they collapsed into aggregated material. In all cases DPPC/3 liposomes were found unstable and sticky aggregated structures precipitated from the bulk suspension.     

Interaction of selected anthocyanins with erythrocytes and liposome membranes

Anthocyanins are one of the main flavonoid groups. They are responsible for, e.g., the color of plants and have antioxidant features and a wide spectrum of medical activity. The subject of the study was the following compounds that belong to the anthocyanins and which can be found, e.g., in strawberries and chokeberries: callistephin chloride (pelargonidin-3-O-glucoside chloride) and ideain chloride (cyanidin-3-O-galactoside chloride). The aim of the study was to determine the compounds’ antioxidant activity towards the erythrocyte membrane and changes incurred by the tested anthocyanins in the lipid phase of the erythrocyte membrane, in liposomes composed of erythrocyte lipids and in DPPC, DPPC/cholesterol and egg lecithin liposomes. In particular, we studied the effect of the two selected anthocyanins on red blood cell morphology, on packing order in the lipid hydrophilic phase, on fluidity of the hydrophobic phase, as well as on the temperature of phase transition in DPPC and DPPC/cholesterol liposomes. Fluorimetry with the Laurdan and Prodan probes indicated increased packing density in the hydrophilic phase of the membrane in the presence of anthocyanins. Using the fluorescence probes DPH and TMA-DPH, no effect was noted inside the hydrophobic phase of the membrane, as the lipid bilayer fluidity was not modified. The compounds slightly lowered the phase transition temperature of phosphatidylcholine liposomes. The study has shown that both anthocyanins are incorporated into the outer region of the erythrocyte membrane, affecting its shape and lipid packing order, which is reflected in the increasing number of echinocytes. The investigation proved that the compounds penetrate only the outer part of the external lipid layer of liposomes composed of erythrocyte lipids, DPPC, DPPC/cholesterol and egg lecithin lipids, changing its packing order. Fluorimetry studies with DPH-PA proved that the tested anthocyanins are very effective antioxidants. The antioxidant activity of the compounds was comparable with the activity of Trolox®.     

The Localization of Phenolic Compounds in Liposomal Bilayers and Their Effects on Surface Characteristics and Colloidal Stability

The interactions with and effects of five chemically distinct, bioactive phenolic compounds on the lipid bilayers of model dipalmitoylphosphatidylcholine (DPPC) liposomes were investigated. Complementary analytical techniques, including differential scanning calorimetry (DSC) and phosphorus and proton nuclear magnetic resonance spectroscopy (NMR), were employed in order to determine the location of the compounds within the bilayer and to correlate location with their effects on bilayer characteristics and liposomal stability. As compared to the phenolic compounds localized in the glycerol region of the DPPC head group within the bilayer, which enhanced the colloidal stability of the liposomes, compounds located closer to the center of the bilayer reduced vesicle stability as a function of time. Molecules present in the upper region of liposomal DPPC acyl chains (C₁–C₁₀) inhibited liposomal aggregation and size increase, perhaps due to tighter packing of adjoining DPPC molecules and increased surface exposure of DPPC phosphate head groups. These data may be useful for designing liposomal systems containing hydrophobic phenols and other small molecules, selecting appropriate analytical methods for determining their location within liposomal bilayers, and predicting their effects on liposome characteristics early in the liposome formulation development process.     

Comparative calorimetric and spectroscopic studies of the effects of cholesterol and epicholesterol on the thermotropic phase behaviour of dipalmitoylphosphatidylcholine bilayer membranes

We carried out comparative differential scanning calorimetric and Fourier transform infrared spectroscopic studies of the effects of cholesterol (Chol) and epicholesterol (EChol) on the thermotropic phase behaviour and organization of dipalmitoylphosphatidylcholine (DPPC) bilayers. EChol is an epimer of Chol in which the axially oriented hydroxyl group of C3 of Chol is replaced by an equatorially oriented hydroxyl group, resulting in a different orientation of the hydroxyl group relative to sterol fused ring system. Our calorimetric studies indicate that the incorporation of EChol is more effective than Chol is in reducing the enthalpy of the pretransition of DPPC. EChol is also initially more effective than Chol in reducing the enthalpies of both the sharp and broad components of the main phase transition of DPPC. However, at higher EChol concentrations (~ 30-50 mol%), EChol becomes less effective than Chol in reducing the enthalpy and cooperativity of the main phase transition, such that at sterol concentrations of 50 mol%, EChol does not completely abolish the cooperative hydrocarbon chain-melting phase transition of DPPC, while Chol does. However, EChol does not appear to form a calorimetrically detectable crystallite phase at higher sterol concentrations, suggesting that EChol, unlike Chol, may form dimers or lower order aggregates at higher sterol concentrations. Our spectroscopic studies demonstrate that EChol incorporation produces more ordered gel and comparably ordered liquid-crystalline bilayers compared to Chol, which are characterized by increased hydrogen bonding in the glycerol backbone region of the DPPC bilayer. These and other results indicate that monomeric EChol is less miscible in DPPC bilayers than is Chol at higher sterol concentrations, but perturbs their organization to a greater extent at lower sterol concentrations, probably due primarily to the larger effective cross-sectional area of the EChol molecule. Nevertheless, EChol does appear to produce a lamellar liquid-ordered phase in DPPC bilayers.     

Interaction of multicomponent anionic liposomes with cationic pyridylphenylene dendrimer: Does the complex behavior depend on the liposome composition?

Dendrimers are individual macromolecular compounds having a great potential for biomedical application. The key step of the cell penetration by dendrimers is the interaction with lipid bilayer. Here, the interaction between cationic pyridylphenylene dendrimer of third generation (D₃⁵⁰⁺) and multicomponent liquid (CL/POPC), solid (CL/DPPC) and cholesterol-containing (CL/POPC/30% Chol) anionic liposomes was investigated by dynamic light scattering, fluorescence spectroscopy, conductometry, calorimetric studies and molecular dynamic (MD) simulations. Microelectrophoresis and MD simulations revealed the interaction is electrostatic and reversible with only part of pyridinium groups of dendrimers involved in binding with liposomes. The ability of dendrimer molecules to migrate between liposomes was discovered by the labeling liposomes with Rhodamine B. The phase state of the lipid membrane and the incorporation of cholesterol into the lipid bilayer were found to not affect the mechanism of the dendrimer - liposome complex formation. Rigid dendrimer adsorption on liposomal surface does not induce the formation of significant defects in the lipid membrane pave the way for possible biological application of pyridylphenylene dendrimers.     

Influence of phospholipid composition on the adjuvanticity and protective efficacy of liposome-encapsulated Leishmania donovani antigens

In this study, we evaluate the effect of phospholipid on the adjuvanicity and protective efficacy of liposome vaccine carriers against visceral leishmaniasis (VL) in a hamster model. Liposomes prepared with distearyol derivative of L-alpha-phosphatidyl choline (DSPC) having liquid crystalline transition temperature (Tc) 54 C were as efficient as dipalmitoyl (DPPC) (Tc 41 C) and dimyristoyl (DMPC) (Tc 23 C) derivatives in their ability to entrap Leishmania donovani membrane antigens (LAg) and to potentiate strong antigen-specific antibody responses. However, whereas LAg in DPPC and DMPC liposomes stimulated inconsistent delayed type hypersensitivity (DTH) responses, strong DTH was observed with LAg in DSPC liposomes. The heightened adjuvant activity of DSPC liposomes corresponded with 95% protection, with almost no protectivity with LAg in DPPC and DMPC liposomes, 4 mo after challenge with L. donovani. These data demonstrate the superiority of DSPC liposomes for formulation of L. donovani vaccine. In addition, they demonstrate a correlation of humoral and cell-mediated immunity with protection against VL in hamsters.     

Ultrasonic absorption and permeability for liposomes near phase transition

The specific ultrasonic absorption coefficient per wavelength as a function of temperature in the vicinity of the phase transition of liposomes, composed of a 4:1 mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG), of different sizes was determined using an acoustic interferometer. Small unilamellar vesicles (SUV) and multilamellar vesicles (MLV) yielded results similar to those in the literature, viz., an absorption maximum at the transition temperature. Seven intermediate sizes including several size distributions of large unilamellar vesicles (LUV) were studied, yielding information on size dependencies of the temperatures at which the peaks occur, the widths at half peak amplitude, and the peak amplitudes. All liposome sizes except the SUV exhibited approximately the same transition temperature as did the largest MLV. The widths of the peaks were inversely related to liposome size, with a strong dependence for the smallest vesicles and an approach to independence for the largest vesicles. The amplitudes of the peaks exhibited a general increase with size with two exceptions, viz., the SUV and the vesicles with average diameters of 90-100 nm. It was also found that the membrane permeability increased near the transition temperature. The temperature dependencies of ultrasonic absorption and membrane permeability are compared.     

Phase behavior and nanoscale structure of phospholipid membranes incorporated with acylated C14-peptides

The thermotropic phase behavior and lateral structure of dipalmitoylphosphatidylcholine (DPPC) lipid bilayers containing an acylated peptide has been characterized by differential scanning calorimetry (DSC) on vesicles and atomic force microscopy (AFM) on mica-supported bilayers. The acylated peptide, which is a synthetic decapeptide N-terminally linked to a C14 acyl chain (C14-peptide), is incorporated into DPPC bilayers in amounts ranging from 0-20 mol %. The calorimetric scans of the two-component system demonstrate a distinct influence of the C14-peptide on the lipid bilayer thermodynamics. This is manifested as a concentration-dependent downshift of both the main phase transition and the pretransition. In addition, the main phase transition peak is significantly broadened, indicating phase coexistence. In the AFM imaging scans we found that the C14-peptide, when added to supported gel phase DPPC bilayers, inserts preferentially into preexisting defect regions and has a noticeable influence on the organization of the surrounding lipids. The presence of the C14-peptide gives rise to a laterally heterogeneous bilayer structure with coexisting lipid domains characterized by a 10 A height difference. The AFM images also show that the appearance of the ripple phase of the DPPC lipid bilayers is unaffected by the C14-peptide. The experimental results are supported by molecular dynamics simulations, which show that the C14-peptide has a disordering effect on the lipid acyl chains and causes a lateral expansion of the lipid bilayer. These effects are most pronounced for gel-like bilayer structures and support the observed downshift in the phase-transition temperature. Moreover, the molecular dynamics data indicate a tendency of a tryptophan residue in the peptide sequence to position itself in the bilayer headgroup region.     

Group 3 LEA protein model peptides protect liposomes during desiccation

We investigated whether a model peptide for group 3 LEA (G3LEA) proteins we developed in previous studies can protect liposomes from desiccation damage. Four different peptides were compared: 1) PvLEA-22, which consists of two tandem repeats of the 11-mer motif characteristic of LEA proteins from the African sleeping chironomid; 2) a peptide with amino acid composition identical to that of PvLEA-22, but with its sequence scrambled; 3) poly-l-glutamic acid; and 4) poly-l-lysine. Peptides 1) and 2) protected liposomes composed of 1-palmitoyl 2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) against fusion caused by desiccation, as revealed by particle size distribution measurements with dynamic light scattering. Indeed, liposomes maintain their pre-stress size distribution when these peptides are added at a peptide/POPC molar ratio of more than 0.5. Interestingly, peptide 1) achieved the comparable or higher retention of a fluorescent probe inside liposomes than did several native LEA proteins published previously. In contrast, the other peptides exhibited less protective effects. These results demonstrate that the synthetic peptide derived from the G3LEA protein sequence can suppress desiccation-induced liposome fusion. Fourier transform infrared (FT-IR) spectroscopic measurements were performed for the dried mixture of each peptide and liposome. Based on results for the gel-to-liquid crystalline phase transition temperature of the liposome and the secondary structure of the peptide backbone, we discuss possible underlying mechanisms for the protection effect of the synthetic peptide on dried liposomes.     

Permeability of dimyristoyl phosphatidylcholine/dipalmitoyl phosphatidylcholine bilayer membranes with coexisting gel and liquid-crystalline phases.

The passive permeation of glucose and a small zwitterionic molecule, methyl-phosphoethanolamine, across two-component phospholipid bilayers (dimyristoyl phosphatidylcholine (DMPC)/dipalmitoyl phosphatidylcholine (DPPC) mixtures) exhibit a maximum when gel domains and fluid domains coexist. The permeability data of the two-phase bilayers cannot be fitted to single-rate kinetics, but are consistent with a Gaussian distribution of rate constants. In pure DMPC and DPPC as well as in their mixtures, at the temperature of the maximum excess heat capacity, the logarithm of the average permeability rate constants are linearly correlated with the mole fraction of DPPC in the total system. In addition, in the 50:50 mixture, the excess heat capacity values as well as the apparent fractions of interfacial lipid correlate with the logarithm of the excess permeabilities in the two-phase region. These results suggest that small polar molecules can cross the membrane at the interface between gel and fluid domains at a much faster rate than through the homogeneous phases; the acyl chains located at the domain interface experience lateral density fluctuations that are inversely proportional to their average length, and large enough to allow rapid transmembrane diffusion of the solute molecules. The distribution of the permeability rate constants may reflect temporal and spatial fluctuations of the lipid composition at the phase boundaries.