Characterization of the interactions between synthetic nematic LCs and model cell membranes

Differential scanning calorimetry (DSC) was used to characterize interactions of synthetic LCs, 4‐pentyl‐4′‐cyanobiphenyl (5CB) and TL205 (a mixture of cyclohexane‐fluorinated biphenyls and fluorinated terphenyls) with simple mimics of cell membranes. The investigation was motivated by reports that living cells can be placed into contact with TL205 without apparent toxicity, whereas contact of cells with 5CB leads to cell death. The tendency was examined for 5CB and TL205 to spontaneously partition into and influence the organization for model cell membranes composed of phospholipids. Upon contact of an aqueous dispersion of DPPC liposomes with neat LC for 4 h, 5CB partitioned into the liposomes at a weight ratio of 5:1 DPPC:5CB, whereas TL205 partitioned at a ratio of 310:1 DPPC:TL205. DSC endotherms indicated that the 5CB spontaneously partitioned into the liposomes was far more perturbing than TL205. DSC endotherms of DPPC bilayers containing the same concentration of either 5CB or TL205 also revealed 5CB to be more perturbing than TL205. The effect of up to 7.8 wt % of TL205 was small, resulting in a shift in the melting transition from 41.4°C to 40.1°C and a minor change in peak width, indicating only minor effects on the organization of the bilayer. These effects are similar to those caused by cholesterol in DPPC bilayers. In contrast, 5CB shifted the DPPC melting transition from 41.4°C to ～36°C and increased the width of the transition peak by a factor of ten, indicating a destabilization of the ordered phase in the bilayer and a disruption of the cooperative nature of the gel‐to‐LC transition of the phospholipid bilayer. Taken together, the results demonstrate that 5CB and TL205 differ significantly in their interactions with model cell membranes, which suggests one possible origin of their different toxicities toward cells.     

Freeze-fracture electron microscopic and calorimetric studies on microscopic states of surface-modified liposomes with poly(ethylene glycol) chains

The differential scanning calorimetry (DSC) and the freeze-fracture electron microscopy of dipalmitoyl phosphatidylcholine (DPPC) liposomes containing distearoyl-N-monomethoxy poly(ethylene glycol)-succinyl-phosphatidylethanolamines (PEG-DSPE) were carried out. The DSC peak of DPPC liposomes containing PEG-DSPE had a shoulder. The main phase transition temperature of DPPC bilayer membranes containing PEG-DSPE whose molecular weight of PEG is less than 3000 was slightly shifted to a higher temperature, while that containing PEG-DSPE whose molecular weight of PEG is more than 5000 was slightly shifted to a lower temperature. The electron micrographs of freeze-fracture replicas of DPPC liposomes containing PEG-DSPE quenched from 37±2°C exhibited banded and planar textures, suggesting the lateral phase separation in the bilayer membranes.     

Methotrexate interaction with a lipid membrane model of DPPC

Dipalmitoylphosphatidylcholine (DPPC) liposomes were employed as membrane models for the investigation of the interaction occurring between methotrexate (MTX) and bilayer lipid matrix. Liposomes were obtained by hydrating a lipid film with 50 mM Tris buffer (pH 7.4). The differential scanning calorimetry (DSC) evaluation of the thermotropic parameters associated with the phase transitions of DPPC liposomes gave useful information about the kind of drug-membrane interaction. The results showed an electrostatic interaction taking place with the negatively charged molecules of MTX and the phosphorylcholine head groups, constituting the outer part of DPPC bilayers. No interaction with the hydrophobic phospholipid bilayer domains was detected, revealing a poor capability of MTX to cross through lipid membranes to reach the interior compartment of a lipid bounded structure. These findings correlate well within vitro biological experiments on MTX cell susceptibility.     

Preparation of liposomes containing Ceramide 3 and their membrane characteristics

Liposomes composed of Ceramide 3, [2S,3S,4R-2-stearoylamide-1,3,4-octadecanetriol], and L-alpha-dipalmitoylphosphatidylcholine (DPPC) were prepared by varying the amount of Ceramide 3, and the effects of Ceramide 3 on the liposome formation, particle size, dispersibility, microviscosity and phase transition temperature were examined by means of a microscopy, a dynamic light scattering method, a fluorescence polarization method, a differential scanning calorimetry (DSC) and so on. All the DPPC was able to contribute to the formation of liposomes up to 0.130 mol fraction of Ceramide 3. The particle size of liposomes was almost unaffected by the addition of Ceramide 3. The dispersibility of liposomes containing Ceramide 3 was maintained for at least 15 days. The microviscosity of liposomal bilayer membranes in the liquid crystalline state was increased with increasing the mole fraction of Ceramide 3, while that in the gel state was independent of the mole fraction of Ceramide 3. The phase transition temperature from gel to liquid crystalline states of DPPC bilayer membranes was shifted upwards with the addition of Ceramide 3, indicating a cooperative interaction between DPPC and Ceramide 3 molecules. However, a sharp DSC peak became broad and split at higher mole fractions of Ceramide 3, suggesting a phase separation in the mixed DPPC/Ceramide 3 liposomal bilayer membranes. These phenomena were suggested to be related to the previously observed fact for the mixed DPPC/Ceramide 3 monolayers that Ceramide 3 interacts with DPPC in the liquid-expanded phase with consequent phase separation accompanied with domain formation.     

Structural effects of lipophilic methotrexate conjugates on model phospholipid biomembranes

Lipophilic conjugates of the antitumor drug methotrexate (MTX) with lipoamino acids (LAAs) have been previously described as a tool to enhance MTX passive entrance into cells, overcoming a form of transport resistance which makes tumour cells insensitive to the antimetabolite. A knowledge of the mechanisms of interaction of such lipophilic derivatives with cell membranes could be useful for planning further lipophilic MTX derivatives with an optimal antitumour activity. To this aim, a calorimetric study was undertaken using a biomembrane model made from synthetic 1,2-dipalmitoyl-glycero-3-phosphocholine (DPPC) multilamellar liposomes. The effects of MTX and conjugates on the phase transition of liposomes were investigated using differential scanning calorimetry.

The interaction of pure MTX with the liposomes was limited to the outer part of the phospholipid bilayers, due to the polar nature of the drug. Conversely, its lipophilic conjugates showed a hydrophobic kind of interaction, perturbing the packing order of DPPC bilayers. In particular, a reduction of the enthalpy of transition from the gel to the liquid crystal phase of DPPC membranes was observed. Such an effect was related to the structure and mole fraction of the conjugates in the liposomes.

The antitumour activity of MTX conjugates was evaluated against cultures of a CCRF–CEM human leukemic T-cell line and a related MTX resistant sub-line. The in vitro cell growth inhibitory activity was higher for bis(tetradecyl) conjugates than for both the other shorter- and longer-chain derivatives. The biological effectiveness of the various MTX derivatives correlated very well with the thermotropic effects observed on the phase transition of DPPC biomembranes.     

Effects of lipid chain unsaturation and headgroup type on molecular interactions between paclitaxel and phospholipid within model biomembrane

Molecular interactions between paclitaxel, an anticancer drug, and phospholipids of various chain unsaturations and headgroup types were investigated in the present study by Langmuir film balance and differential scanning calorimetry. Both the lipid monolayer at the air-water interface and the lipid bilayer vesicles (liposomes) were employed as model cell membranes. It was found that, regardless of the difference in molecular structure of the lipid chains and headgroup, the drug can form nonideal, miscible systems with the lipids at the air-water interface over a wide range of paclitaxel mole fractions. The interaction between paclitaxel and phospholipid within the monolayer was dependent on the molecular area of the lipids at the interface and can be explained by intermolecular forces or geometric accommodation. Paclitaxel is more likely to form thermodynamically stable systems with 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) and 1,2-dielaidoyl-sn-glycero-3-phosphocholine (DEPC) than with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Investigation of the drug penetration into the lipid monolayer showed that DPPC and DEPC have higher incorporation abilities for the drug than DPPE and DSPC. A similar trend was also evidenced by DSC investigation with liposomes. While little change of DSC profiles was observed for the DPPE/paclitaxel and DSPC/paclitaxel liposomes, paclitaxel caused noticeable changes in the thermographs of DPPC and DEPC liposomes. Paclitaxel was found to cause broadening of the main phase transition without significant change in the peak melting temperature of the DPPC bilayers, which demonstrates that paclitaxel was localized in the outer hydrophobic cooperative zone of the bilayer, i.e., in the region of the C1-C8 carbon atoms of the acyl chain or binding at the polar headgroup site of the lipids. However, it may penetrate into the deeper hydrophobic zone of the DEPC bilayers. These findings provide useful information for liposomal formulation of anticancer drugs as well as for understanding drug-cell membrane interactions.     

Chain-melting phase transition and short-range molecular interactions in phospholipid foam bilayers

Occurrence of two-dimensional chain melting phase transition in foam bilayers was established for the first time. Microscopic horizontal foam bilayers [Newton black films (NBF)] were investigated by the microinterferometric method of Scheludko-Exerowa. The foam bilayers were formed from water-ethanol solutions of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) and egg phosphatidylcholine (Egg PC) and samples of amniotic fluid (AF) at different temperatures. The influence of temperature on the foam bilayer thickness h(w) and on the critical concentration Cc for formation of foam bilayer was studied. It was shown that in the range of the main phase transition the temperature dependence of h(w) and C(c) changed specifically in the case of DMPC and DPPC foam bilayers. The thickness of the foam bilayers increased with decreasing temperature in the range of the main phase transition due to the melting of hydrocarbon tails of phospholipid molecules. These changes took place at the temperatures of the bulk chain-melting phase transitions, as determined by differential scanning calorimetry (DSC) for both aqueous, and water/ethanol DMPC, DPPC, and DPPC dispersions. An effect of the 'disperse medium' on h(w) was found for foam bilayers from DPPC. The results that foam bilayers could have different thickness at different temperatures disproved the current concept that NBF acquired constant thickness at concentrations higher than C(el,cr). The data for Cc were analysed on the basis of the hole-nucleation theory of bilayer stability of Kashchiev and Exerowa. This theory considered the amphiphile bilayer as a two-dimensional ordered system with short-range molecular interactions between the first neighbour molecules (as in a crystal). The short-range molecular interactions were presented by the parameter binding energy Q of an amphiphile molecule in the bilayer. The binding energy Q of two neighbouring phospholipids was calculated for the gel (30-60 kT) and liquid crystalline state (16-18 kT) of the bilayers from DMPC, DPPC, Egg PC, AF. Concentration/temperature phase diagram of DPPC foam bilayers that defined regions of gaseous (ruptured), gel and liquid crystalline foam bilayers were drawn. The values of Q obtained for various samples were very close and vary from 5.3 x 10(-20) to 9.4 x 10(-20) (approx. 13-22 kT) which indicated that in all cases the foam bilayers were in liquid-crystalline state. This is an important result since the parameter studied-threshold concentration (threshold dilution) is crucial for a very successful assessment of the risk for respiratory distress syndrome (RDS) in newborns and could be employed in medicine for assessment of other respiratory disturbances. It is to be expected that foam bilayers from phospholipids could be used as a model for investigation of short-range forces in biological structures, of interaction between membranes, etc.     

Effect of adsorption of bovine serum albumin on liposomal membrane characteristics

The effect of adsorption of bovine serum albumin (BSA) on the membrane characteristics of liposomes at pH 7.4 was examined in terms of zeta potential, micropolarity, microfluidity and permeability of liposomal bilayer membranes, where negatively charged L-alpha-dipalmitoylphosphatidylglycerol (DPPG)/L-alpha-dipalmitoylphosphatidylcholine (DPPC), negatively charged dicetylphosphate (DCP)/DPPC and positively charged stearylamine (SA)/DPPC mixed liposomes were used. BSA with negative charges adsorbed on negatively charged DPPG/DPPC mixed liposomes but did not adsorb on negatively charged DCP/DPPC and positively charged SA/DPPC mixed liposomes. Furthermore, the adsorption amount of BSA on the mixed DPPG/DPPC liposomes increased with increasing the mole fraction of DPPG in spite of a possible electrostatic repulsion between BSA and DPPG. Thus, the adsorption of BSA on liposomes was likely to be related to the hydrophobic interaction between BSA and liposomes. The microfluidity of liposomal bilayer membranes near the bilayer center decreased by the adsorption of BSA, while the permeability of liposomal bilayer membranes increased by the adsorption of BSA on liposomes. These results are considered to be due to that the adsorption of BSA brings about a phase separation in liposomes and that a temporary gap is consequently formed in the liposomal bilayer membranes, thereby the permeability of liposomal bilayer membranes increases by the adsorption of BSA.     

Effects of poly(ethylene glycol) (PEG) chain length of PEG-lipid on the permeability of liposomal bilayer membranes

The effects of poly(ethylene glycol) (PEG) chain length of PEG-lipid on the membrane characteristics of liposomes were investigated by differential scanning calorimetry (DSC), freeze-fracture electron microscopy (FFEM), fluorescence polarization measurement and permeability measurement using carboxyfluorescein (CF). PEG-liposomes were prepared from mixtures of dipalmitoyl phosphatidylcholine (DPPC) and distearoyl phosphatidylethanolamines with covalently attached PEG molecular weights of 1000, 2000, 3000 and 5000 (DSPE-PEG). DSC and FFEM results showed that the addition of DSPE-PEG to DPPC in the preparation of liposomes caused the lateral phase separation both in the gel and liquid-crystalline states. The fluidity in the hydrocarbon region of liposomal bilayer membranes was not significantly changed by the addition of DSPE-PEG, while that in the interfacial region was markedly increased. From these results, it was anticipated that the CF leakage from PEG-liposomes is accelerated compared with DPPC liposomes. However, CF leakage from liposomes containing DSPE-PEG with a 0.060 mol fraction was depressed compared with regular liposomes, and the leakage decreased with increasing PEG chain length. Furthermore, the CF leakage from liposomes containing DSPE-PEG with a 0.145 mol fraction was slightly increased compared with that of liposomes containing DSPE-PEG with a 0.060 mol fraction. It is suggested that the solute permeability from the PEG-liposomes was affected by not only properties of the liposomal bilayer membranes such as phase transition temperature, phase separation and membrane fluidity, but also the PEG chain of the liposomal surface.     

Effects of lipid chain length on molecular interactions between paclitaxel and phospholipid within model biomembranes

Molecular interactions between an anticancer drug, paclitaxel, and phosphatidylcholine (PC) of various chain lengths were investigated in the present work by the Langmuir film balance technique and differential scanning calorimetry (DSC). Both the lipid monolayer at the air-water interface and lipid bilayer vesicles (liposomes) were employed as model biological cell membranes. Measurement and analysis of the surface pressure versus molecular area curves of the mixed monolayers of phospholipids and paclitaxel under various molar ratio showed that phospholipids and paclitaxel formed a nonideal miscible system at the interface. Paclitaxel exerted an area-condensing effect on the lipid monolayer at small molecular surface areas and an area-expanding effect at large molecular areas, which could be explained by the intermolecular forces and geometric accommodation between the two components. Paclitaxel and phospholipids could form thermodynamically stable monolayer systems: the stability increased with the chain length in the order DMPC (C14:0)>DPPC (C16:0)>DSPC (C18:0). Investigation of paclitaxel penetration into the pure lipid monolayer showed that DMPC had a higher ability to incorporate paclitaxel and the critical surface pressure for paclitaxel penetration also increased with the chain length in the order DMPC>DPPC>DSPC. A similar trend was testified by DSC studies on vesicles of the mixed paclitaxel/phospholipids bilayer. Paclitaxel showed the greatest interaction with DMPC while little interaction could be measured in the paclitaxel/DSPC liposomes. Paclitaxel caused broadening of the main phase transition without significant change at the peak melting temperature of the phospholipid bilayers, which demonstrated that paclitaxel was localized in the outer hydrophobic cooperative zone of the bilayer. The interaction between paclitaxel and phospholipid was nonspecific and the dominant factor in this interaction was the van der Waals force or hydrophobic force. As the result of the lower net van der Waals interaction between hydrocarbon chains for the shorter acyl chains, paclitaxel interacted more readily with phospholipids of shorter chain length, which also increased the bilayer intermolecular spacing.     

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.     

Thermotropic phase transition in soluble nanoscale lipid bilayers

The role of lipid domain size and protein-lipid interfaces in the thermotropic phase transition of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) bilayers in Nanodiscs was studied using small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and generalized polarization (GP) of the lipophilic probe Laurdan. Nanodiscs are water-soluble, monodisperse, self-assembled lipid bilayers encompassed by a helical membrane scaffold protein (MSP). MSPs of different lengths were used to define the diameter of the Nanodisc lipid bilayer from 76 to 108 A and the number of DPPC molecules from 164 to 335 per discoidal structure. In Nanodiscs of all sizes, the phase transitions were broader and shifted to higher temperatures relative to those observed in vesicle preparations. The size dependences of the transition enthalpies and structural parameters of Nanodiscs reveal the presence of a boundary lipid layer in contact with the scaffold protein encircling the perimeter of the disc. The thickness of this annular layer was estimated to be approximately 15 A, or two lipid molecules. SAXS was used to measure the lateral thermal expansion of Nanodiscs, and a steep decrease of bilayer thickness during the main lipid phase transition was observed. These results provide the basis for the quantitative understanding of cooperative phase transitions in membrane bilayers in confined geometries at the nanoscale.     

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.     

Phase Transitions and Structural Changes in DPPC Liposomes Induced by a 1-Carba-Alpha-Tocopherol Analogue

Steady-state emission spectroscopy of 1-anilino-8- naphthalene sulfonate (ANS) and 1,6-diphenyl-1,3,5-hexatriene (DPH), fluorescence anisotropy, and DSC methods were used to characterize the interactions of the newly synthesized 1-carba-alpha-tocopherol (CT) with a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membrane. The DSC results showed significant perturbations in the DPPC structure for CT concentrations as low as 2 mol%. The main phase transition peak was broadened and shifted to lower temperatures in a concentration-dependent manner, and pretransition was abolished. Increasing CT concentrations induced the formation of new phases in the DPPC structure, leading to melting at lower temperatures and, finally, disruption of the ordered DPPC structure. Hydration and structural changes of the DPPC liposomes using ANS and DPH fluorescent probes, which are selectively located at different places in the bilayer, were studied. With the increased concentration of CT molecules in the DPPC liposomes, structural changes with the simultaneous formation of different phases of such mixture were observed. Temperature studies of such mixtures revealed a decrease in the temperature of the main phase transition and fluidization at decreasing temperatures related to increasing hydration in the bilayer. Contour plots obtained from concentration–temperature data with fluorescent probes allowed for identification of different phases, such as gel, ordered liquid, disordered liquid, and liquid crystalline phases. The CT molecule with a modified chromanol ring embedded in the bilayer led to H-bonding interactions, expelling water molecules from the interphase, thus introducing disorder and structural changes to the highly ordered gel phase.     

Interaction of propyl paraben with dipalmitoyl phosphatidylcholine bilayer: a differential scanning calorimetry and nuclear magnetic resonance study

The influence of the preservative, propyl paraben (PPB) on the biophysical properties of dipalmitoyl phosphatidyl choline (DPPC) vesicles, both in multilamellar vesicle (MLV) and unilamellar vesicle (ULV) forms, has been studied using DSC and (¹H and ³¹P) NMR. The mechanism by which PPB interacts with DPPC bilayers was found to be independent of the morphological organization of the lipid bilayer. Incorporation of PPB in DPPC vesicles causes a significant depression in the transition temperature and enthalpy of both the pre-transition (PT) and the gel to liquid crystalline transition. The presence of the PPB also reduces the co-operativity of these transitions. However, at high PPB concentration the PT disappears. DSC and NMR findings indicate that: (i) PPB is bound strongly to the lipid bilayer leading to increased headgroup fluidity due to reduced headgroup–headgroup interaction and (ii) the PPB molecules are intercalated between the DPPC polar headgroups with its alkyl chain penetrate into the co-operative region. MLV incorporated with high PPB concentration shows additional transitions whose intensity increases with increasing PPB concentration. This phase segregation observed could probably be due to co-existence of PPB-rich and PPB-poor phospholipid domains within the bilayers. The effect of inclusion of cholesterol in the PPB-free and PPB-doped DPPC dispersion was also studied. Equilibration studies suggest that PPB molecules are very strongly bound and remain intercalated between the polar headgroup for prolonged time.     

Liposome fluidization and melting point depression by pressurized CO2 determined by fluorescence anisotropy

The influence of CO2 on the bilayer fluidity of liposomes, which are representative of model cellular membranes, was examined for the first time at the elevated pressures (up to 13.9 MPa) associated with CO2-based processing of liposomes and microbial sterilization. Fluidization and melting point depression of aqueous dipalmitoylphosphatidylcholine (DPPC) liposomes by pressurized CO2 (present as an excess phase) were studied by steady-state fluorescence anisotropy using the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Isothermal experiments revealed reversible, pressure-dependent fluidization of DPPC bilayers at temperatures corresponding to near-gel (295 K) and fluid (333 K) phases at atmospheric pressure, where the gel-to-fluid phase transition (Tm) occurs at approximately 315 K. Isobaric measurements (PCO2 =1.8, 7.0, and 13.9 MPa) of DPH anisotropy demonstrate substantial melting point depression (DeltaTm = -4.8 to -18.5 K) and a large broadening of the gel-fluid phase transition region, which were interpreted using conventional theories of melting point depression. Liposome fluidity is influenced by CO2 accumulation in the hydrocarbon core and polar headgroup region, as well as the formation of carbonic acid and/or the presence of buffering species under elevated CO2 pressure.     

The interaction of a peptide with a scrambled hydrophobic/hydrophilic sequence (Pro-Asp-Ala-Asp-Ala-His-Ala-His-Ala-His-Ala-Ala-Ala-His-Gly) (PADH) with DPPC model membranes: a DSC study

Depending on their hydrophobicity, peptides can interact differently with lipid membranes inducing dramatic modifications into their host systems. In the present paper, the interaction of a synthetic peptide with a scrambled hydrophobic/hydrophilic sequence (Pro-Asp-Ala-Asp-Ala-His-Ala-His-Ala-His-Ala-Ala-Ala-His-Gly) (PADH) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) model membranes has been investigated by differential scanning calorimetry (DSC), adopting three different experimental approaches. In the first, the peptide is forced to be included into the hydrocarbon region of the lipid bilayer, by codissolving it with the lipid giving rise to mixed multilamellar vesicles–peptide systems; in the second, this system is passed through an extruder, thus producing large unilamellar vesicles–peptide systems; in the third, it is allowed to interact with the external surface of the membrane.

The whole of the DSC results obtained have shown that the incorporation of the peptide into the lipid bilayer by means of the first method induces a decrease in the enthalpy of the gel–liquid crystal transition of the membrane and a shift of the transition to the lower temperatures, thus resembling, in spite of its prevalently hydrophilic nature, the behavior of transbilayer hydrophobic peptides. The extrusion of these systems creates unilamellar vesicles free of peptides but of smaller size as evidenced by the decreased cooperativity of the transition. The peptide, added externally to the DPPC model membrane, has no effect on the phase behavior of the bilayer.

These findings suggest that the effect of the interaction of scrambled hydrophobic/hydrophilic peptides into lipid bilayers strongly affects the thermotropic behavior of the host membrane depending on the preparation method of the lipid/peptide systems. The whole of the results obtained in the present paper can be useful in approaching studies of bioactive peptides/lipids systems.     

Loading of gold nanoparticles inside the DPPC bilayers of liposome and their effects on membrane fluidities

Gold nanoparticles were loaded in the bilayer of dipalmitoylphosphatidylcholine (DPPC) liposomes, named as gold-loaded liposomes. Above the gel to liquid-crystalline phase transition temperature, membrane fluidities of DPPC liposomes were changed by loading the gold nanoparticles. Compared with liposomes without loading the gold nanoparticles, gold-loaded liposomes showed the lower fluorescence anisotropy values. That is, the membrane fluidities of DPPC bilayer were increased by loading the gold nanoparticles. The membrane fluidities were increased as the amount of gold nanoparticles increased. The existence of gold nanoparticles in the DPPC bilayer was observed by transmission electron microscopy. Through the energy dispersive X-ray spectrometer, the particles in DPPC bilayer were confirmed to be gold nanoparticles.     

Sodium Taurocholate-Induced Lamellar-Micellar Phase Transitions of DPPC

The thermotropic transitions of 1,2-dipalmitoylphosphatidylcholine (DPPC) and the structural changes of its lamellar phases have been studied between 0 and 50°C by both DSC and synchrotron small angle X-ray diffraction/scattering as a function of temperature (XRDT) and sodium taurocholate concentration [TC] in the 0–40 mM range ([DPPC]=50 mM) at pH 7.4. The existence of multiple phase transitions (up to 5 peaks within a 5°C interval) in a narrow domain of temperature between 25 and 42°C depending on the [TC]/[lipid] ratio was observed in the DSC curves. XRDT showed that at low ratios they might correspond to transitions between lamellar phases, the structural characteristics of which are given. At higher ratios a lamellar to micellar transition was observed, and the temperature at which it was observed decreased as a function of the TC content. The relationships with DPPC vesicle bilayer permeabilization and solubilization are discussed.     

Interaction of the antimalarial agents halofantrine and lumefantrine with lipid bilayers

The effects of antimalarial drugs halofantrine and lumefantrine on the fluoresence anisotropy of diphenylhexatriene (DPH)-containing phospholipid vesicles have been examined. Lumefantrine increases DPH anisotropy, indicating a condensing effect on bilayers of dipalmitoylphosphatidyl choline (DPPC), dioleoylphosphatidylcholine (DOPC), egg lecithin and mouse erythrocyte membranes (including membranes isolated from plasmodial-infected mice). Its condensing effect is more pronounced in bilayers of lower microviscosity. In contrast, increases or decreases in DPH anisotropy are observed with halofantrine, depending on the nature of the lipid. Decreases in anisotropy, which reflect a perturbing effect, are observed in bilayers of high microviscosity (for example, gel state of DPPC bilayers). Increases in anisotropy are observed in bilayers of low microviscosity (such as DOPC and egg lecithin bilayers). The perturbing effect of halofantrine is further confirmed by the increases in permeability of calcein-containing DPPC vesicles in the presence of the drug. However the perturbative effects of halofantrine are observed to the same magnitude in uninfected and plasmodial-infected erythrocyte membranes, and may not be relevant to the antimalarial action of the drug. In contrast, the condensing effect of lumefantrine is significantly greater in infected erythrocyte membranes and may contribute to its antimalarial action.