Gel-liquid Crystal Transition Temperature Research Articles

Role of lipopolysaccharides and lipoteichoic acids on C-Chrysophsin-1 interactions with model Gram-positive and Gram-negative bacterial membranes.

Antimicrobial peptides (AMPs) are attractive as biomaterial coatings because they have broad spectrum activity against different microbes, with a low likelihood of incurring antimicrobial resistance. Direct action against the bacterial membrane is the most common mechanism of action (MOA) of AMPs, with specific MOAs dependent on membrane composition, peptide concentration, and environmental factors that include temperature. Chrysophsin-1 (CHY1) is a broad spectrum salt-tolerant AMP that is derived from a marine fish. A cysteine modification was made to the peptide to facilitate attachment to a surface, such as a biomedical device. The authors used quartz crystal microbalance with dissipation monitoring to study how temperature (23 and 37 °C) and lipid composition influence the MOA of cysteine-modified peptide (C-CHY1) with model membranes comprised of supported lipid bilayers (SLBs). These two temperatures were used so that the authors could better understand the differences in behavior between typical lab temperatures and physiologic conditions. The authors created model membranes that mimicked properties of Gram-negative and Gram-positive bacteria in order to understand how the mechanisms might differ for different types of bacterial systems. SLB models of Gram-positive bacterial membranes were formed using combinations of phosphatidylcholine, phosphatidylglycerol (PG), and S. aureus-derived lipoteichoic acid (LTA). SLB models of Gram-negative bacterial membranes were formed using combinations of phosphatidylethanolamine (PE), PG, and E. coli-derived lipopolysaccharides (LPS). The molecules that distinguish Gram-positive and Gram-negative membranes (LTA and LPS) have the potential to alter the MOA of C-CHY1 with the SLBs. The authors' results showed that the MOA for the Gram-positive SLBs was not sensitive to temperature, but the LTA addition did have an effect. Specifically, similar trends in frequency and dissipation changes across all overtones were observed, and the same mechanistic trends were observed in the polar plots at 23 and 37 °C. However, when LTA was added, polar plots showed an association between C-CHY1 and LTA, leading to SLB saturation. This was demonstrated by significant changes in dissipation, while the frequency (mass) was not increasing after the saturation point. For the Gram-negative SLBs, the composition did not have a significant effect on MOA, but the authors saw more differences between the two temperatures studied. The authors believe this is due to the fact that the gel-liquid crystal transition temperature of PE is 25 °C, which means that the bilayer is more rigid at 23 °C, compared to temperatures above the transition point. At 23 °C, a significant energetic shift would be required to allow for additional AMP insertion. This could be seen in the polar plots, where there was a steep slope but there was very little mass addition. At 37 °C, the membrane is more fluid and there is less of an energetic requirement for insertion. Therefore, the authors observed greater mass addition and fewer changes in dissipation. A better understanding of C-CHY1 MOA using different SLB models will allow for the more rational design of future therapeutic solutions that make use of antimicrobial peptides, including those involving biomaterial coatings.

Stabilized liposomes with phospholipid polymers and their interactions with blood cells

To stabilize a phospholipid liposome, addition of various water-soluble polymers into a liposomal aqueous suspension was investigated. The water-soluble polymers were poly(ethylene oxide) (PEO), poly( N-vinyl pyrrolidone) (PVPy) and poly[2-methacryloyloxyethyl phosphorylcholine(MPC)], and poly[MPC-co- n-butyl methacrylate(BMA)]. The gel–liquid crystal transition temperature ( T c) of the diparmitoylphosphatidylcholine (DPPC) liposome was not changed by addition of these polymers significantly. However, membrane fluidity of DPPC liposome treated with water-soluble polymers, which was measured with fluorescence probe, depended on the chemical structure of the water-soluble polymers. In the case of PEO and PVPy, the temperature dependence of membrane fluidity was the same as that of the original DPPC liposome, on the other hand, poly(MPC) and poly(MPC-co-BMA) induced a rise in the temperature where an increase in the membrane fluidity was observed. The release of carboxy fluorescein from the DPPC liposome was suppressed by the addition of the MPC polymers. The liposomes in the MPC polymer solution were stable compared with those in water when plasma was added into the suspension. Interactions with stabilized liposome with blood cells such as platelets and erythrocytes were evaluated. Activation of platelets in contact with liposome covered with poly(MPC) or poly(MPC-co-BMA) was less than PEO-stabilized liposome. On the other hand, no hemolysis of erythrocytes was observed when every polymer-treated liposome was added in the suspension of erythrocytes. Based on these results, the MPC polymers could interact with the liposome surface, adsorb on the liposomes and stabilize them, and had no adverse effect to the blood cells even when they were in a physiological environment.

Thermal transitions in the bilayer assembly of dimyristoylphosphatidylglycerol sodium salt dispersion in water: a new phase transition through an intermediate state

The thermal properties of the dispersion of sodium salt of dimyristoylphosphatidylglycerol (NaDMPG) in water have been investigated as functions of incubation temperature and aging time by DSC, XRD, sodium ion activity, pH, ζ-potential, and IR measurements. The DSC charts for NaDMPG dispersions incubated below 30°C showed an endothermic peak at 31.7°C with a small shoulder peak at T m (gel–liquid crystal transition temperature: 23.5°C). The temperature of 31.7°C coincides with the T* temperature at which a high-order transition in the NaDMPG bilayer assembly has been found to occur in our previous studies. However, no peak was observed for the dispersions incubated above 32°C. These results indicate that thermal properties of NaDMPG bilayers definitely differ below and above the T* temperature. The dispersion which had been once incubated at 40°C for 24 h never showed the endothermic peak at T* even after the further aging at 3°C for 12-day. Namely, the NaDMPG bilayer assembly exhibits an intensive thermohysteresis. The XRD charts for the NaDMPG dispersions incubated at 25°C showed a sharp X-ray diffraction pattern corresponding to the repeat distance of d=4.75 nm regardless of their aging time, while the dispersions incubated at 40°C had no diffraction peak until 9-day elapsed. After 10-day aging at 40°C, however, a diffraction peak corresponding to d=5.55 nm clearly appeared. In the DSC measurements for the dispersion incubated at 40°C, a few endothermic peaks began to appear between T m and T* after approximately 7-day aging. Then, they shifted toward higher temperatures and finally converged into a single peak at 40–42°C after 14-day aging. These XRD and DSC peaks observed after a long period of aging time above T* suggest that conformations of the hydrophilic groups and the hydrocarbon chains in the NaDMPG bilayers take a more tight and closer arrangement very slowly via an intermediate state above T*, and a new gel phase of the bilayers is consequently formed, the transition temperature ( T I temperature) of which is 40–42°C. A molecular interpretation for such transition processes in the bilayer assembly of NaDMPG dispersions has been proposed on the basis of pH, sodium ion activity, ζ-potential, IR data, etc.

Solubilization of hydrocarbons into a bilayer assembly of dimyristoylphosphatidylcholine in water

Solubilization of octane, hexadecane and perfluorononane into a bilayer assembly of dimyristoylphosphatidylcholine (DMPC) in water has been studied at 25 and 35°C as a function of mole fraction ( X) of these solubilizates. In order to determine the occurrence of solubilization, a change of the gel–liquid crystal transition temperature ( T m) of the DMPC bilayer assembly was monitored by differential scanning calorimetry measurement. Solubilization of octane into the DMPC bilayer assembly was observed both at 25 and 35°C. On the other hand, solubilization of hexadecane took place above X=0.40 at 25°C, while at 35°C solubilization did not occur at any mole fraction. Solubilization of perfluorononane into the DMPC bilayer assembly was not observed either at 25 or 35°C. Saturated solubilization of octane or hexadecane into the DMPC bilayer assembly was achieved at X=0.67, where the mole ratio is 1(DMPC):2(oil), i.e. the ratio of the number of hydrocarbon chains of DMPC to that of oil is 1:1. The heat of gel–liquid crystal transition (Δ H m) of the DMPC bilayers saturated with octane at 25°C is 16.5 J g −1 at T m=14.8°C, and that of the DMPC bilayers saturated with hexadecane at 25°C is 22.0 J g −1 at T m=31.4°C; whereas the Δ H m value for the pure DMPC bilayers is 27.0 J g −1 at T m=23.7°C. X-ray diffraction data indicate that the hydrocarbon solubilizates are located in the palisade layers of the DMPC bilayers. It has been also revealed that the surface of emulsion droplets of octane or hexadecane are covered with multibilayers of DMPC at 25°C, and with a monolayer of DMPC at 35°C. Such differences in solubilization and emulsion stabilization mechanisms between 25 and 35°C are attributable to the higher order transition of the DMPC bilayers at T* (29.0°C).

Effects of Sonication on the Lamellar Structures of L-.ALPHA.-Dipalmitoyl Phosphatidylcholine(DPPC)/Saccharide/Water Systems.

The effects of sonication, conducted prior to dehydration by heat drying, on the multilamellar vesicles of L-α-dipalmitoyl phosphatidylcholine (DPPC), DPPC/glucose, DPPC/trehalose or DPPC/maltose systems were examined by differential scanning calorimetry (DSC) and powder X-ray diffraction (XRD). The results were compared with those for the corresponding unsonicated and DPPC systems without saccharide. In the DPPC/glucose system, no clear differences between the unsonicated and sonicated systems were found because glucose did not prevent fusion of vesicles by dehydration. DSC showed one sharp peak at the gel-liquid crystal transition temperature (Tc) of 43°C, indicating that glucose was distributed homogeneously between the DPPC bilayers of the vesicles. Subcells formed by hydrocarbon chains of DPPC changed from the hexagonal gel (Lβ) to the hexagonal liquid crystal (Lα) form at Tc with an increase in temperature, essentially as noted for DPPC systems except for differences in Tc. In the DPPC/disaccharide system, the unsonicated and sonicated systems were clearly different. DSC and XRD of the unsonicated system consistently showed transition from a gel to a liquid crystal state over a wide temperature range, while for the sonicated system, there was only a sharp peak on the DSC curve. The thermal behavior of DPPC/disaccharide systems may be explained as follows. Although disaccharide is distributed homogeneously between the bilayers of multilamellar vesicles, interactions with DPPC depend on the surface curvature of the bilayer. Heating of multilamellar vesicles may possibly result in transition from a gel to a liquid crystal phase since multilamellar vesicles consist of many bilayers differing considerably in their surface curvature, in contrast to sonicated unilamellar vesicles which possess a definite curvature.

Relationship between Hydrophobic Index of Saccharide and Gel-Liquid Crystal Transition Temperature of the L-.ALPHA.-Dipalmitoyl Phosphatidylcholine (DPPC)/Saccharide/Water System.

An examination of the relationship between the hydrophobic index of saccharide and gel-liquid crystal transition temperatures in L-α-dipalmitoyl phosphatidylcholine (DPPC)/saccharide systems at a water content of 10 wt% and 70wt% was performed using differential scanning calorimetry. Saccharide at 10 wt% water was found to interact with phospholipids hydrophobically via the hydrophobicity-rich side of the pyranose ring. Molar ratios (pyranose ring/DPPC) were determined as 2 for the DPPClrhamnose system with 8.2 wt% water and for the DPPC/trehalose system at 15.8 wt%. Pyranose would thus appear to form a 2:1 complex with lipid that faces the hydrophobicity-rich side of the pyranose ring. Saccharide with 70 wt% water interacted with the phospholipid hydrophilically through hydration of the saccharide.

Effect of water on lamellar structure of DPPC/sugar systems

The ability of two monosaccharides, four disaccharides and one trisaccharide, to lower the transition temperature of l-α-dipalmitoyl phosphatidylcholine (DPPC) was investigated using differential scanning calorimetry (DSC) and the ability of these sugars to change the lateral packing of the acyl chains of DPPC was investigated using wide-angle X-ray diffraction. The sugars affected the gel–liquid crystal transition temperature ( T c) of DPPC when the water content of the DPPC/sugar systems was less than 20 wt.%. Specifically, T c of the DPPC without sugar increased to approximately 106°C, the T c of the DPPC/monosaccharide system remained almost constant at 43°C and of the DPPC/disaccharide or trisaccharide systems decreased to approximately 24°C. In the dehydrated state, di- and trisaccharides caused looser packing of the DPPC hydrocarbon chains than the monosaccharides did, and the sugars affected the packing mode in different ways. The addition of water caused this difference in the sugars' effects on the packing mode to disappear and further addition of water caused the effect of sugar to almost disappear. Thus, the addition of water to a DPPC/sugar system weakens the interaction between the sugar and lipid and strengthens the DPPC chain packing.

Thermal Properties of Hexadecane Emulsion Stabilized with Dimyristoylphosphatidylcholine.

Emulsification of hexadecane with dimyristoylphosphatidylcholine (DMPC) in the (L α + H 2 O)phase was investigated as a function of temperature. The particle size of the emulsion prepared under the same condition was about 800 nm in the temperature range from the gel-liquid crystal transition temperature, T M (23.5°C) to about 28°C, but sharply decreased and reached about 390 nm above the critical temperature, T* (29.0°C), at which a higher order transition occurred in the DMPC bilayer. The changes in emulsion concentration by creaming were observed, as expected fromthe temperature dependence of the emulsion particle size. The zeta-potential of the emulsion surface was also shifted around T* from -36 mV to -15 mV. On the other hand, the interfacial tension at the hexadecane-DMPC dispersion interface decreased with increasing temperature up to T*, and then became almost a constant value less than 1 mN m -1 above T * . These novel phenomena were explained by the concept that thermal characters of the DMPC dispersion used as an emulsifier changed not only at T M , but also at T*. Furthermore, by taking into account the amount of emulsifier, the particle size of emulsion, and the molecular area of DMPC in bilayer, it was found that the surface of hexadecane emulsion was covered with the amount of DMPC corresponding to the triple layers of DMPC below T*, but to the monolayer above T*.

Thermal Properties of Dimyristoylphosphatidylglycerol Sodium Salt Dispersed in Water

Thermal properties of smectic liquid crystals of dimyristoylphosphatidylglycerol sodium salt (DMPG) in water were investigated as a function of temperature above the gel-liquid crystal transition temperature, Tm, by X-ray diffraction analysis of the repeat distance of the bilayer, differential scanning calorimetry (DSC) for phase transition, the hyperfine splitting constant in electron spin resonance (ESR) method with spin probes and equilibrium spreading pressure. The data obtained were found to differ remarkably below and above the critical temperature T* (=31.7°C) for DMPG bilayers. The weakening of hydration of terminal glycerol moiety in DMPG at T*, with consequently enhanced microflexibility of DMPG bilayers above this temperature may be explanation for these differences. The effects of dehydration of the terminal glycerol moiety on surface activity was confirmed from the temperature dependence of equilibrium spreading pressure of DMPG.

DODAC in bidimensional states monolayers, Langmuir—Blodgett films and vesicles

The spreading isotherms of DODAC at the water—air interface were determined as a function of temperature. We verified that a study of the spreading film is the starting point for a better understanding of the most suitable conditions to obtain more complex aggregates, such as Langmuir-Blodgett (LB) films or vesicles. When the DODAC monolayer was in a liquid-condensed phase, this resulted in the optimum conditions for obtaining LB multilayers. In contrast, when the monolayer was in a liquid-expanded phase, multi- and unilamellar vesicles were obtained after sonication. Vesicles were also obtained when the system was below the gel-liquid crystal transition temperature ( T c=37°C), but a greater fraction of multilamellar vesicles were formed, as indicated by turbidimetric data.

Calcium ion influence on thermotropic behaviour of dipalmitoylphosphatidylcholine-vitamin D 3 systems

An analysis of Ca 2+ influence on the thermotropic behaviour of different phosphatidylcholine-vitamin D 3 mixtures was carried out by differential scanning calorimetry technique (DSC). A competitive effect between Ca 2− and vitamin D 3 on the membrane fluidity was detected. The observed shifts of the gel-liquid crystal transition temperature were correlated with the mole fraction of vitamin D 3 dissolved within the lipid bilayer as well as with the Ca 2+ concentration in the surrounding medium. These shifts were rationalized on the ground of a simple microscopic model through the calculation of the internal pressure exerted by the adsorbed Ca 2− on the lipid matrix by the Clapeyron equation. The experimental results and the obtained equations accord with each other and support the idea of micro-domain formation richer in one lipid component. The extent of such lateral phase separation of the lipid components seems to be favoured by the adsorption of Ca 2+ at the membrane-water interface.

Equilibrium studies of phospholipid bilayer assembly. Coexistence of surface bilayers and unilamellar vesicles

To understand how single lipid bilayers might form in cells, we have examined the equilibrium phase diagram of dilute dimyristoylphosphatidylglycerol (DMPG) dispersions in water. Using surface pressure measurements, quasielastic light scattering and phase-contrast microscopy, unilamellar vesicle formation has been observed at a critical temperature, T*, the identical temperature where single bilayers form in films of the lipid at the air–water surface. T* is ca. 8 °C higher than the gel–liquid-crystal transition temperature, Tm, for DMPG dispersions. At T T*, DMPG dispersions form multilamellar vesicles. The solubility of DMPG in water, Xs, was measured over the temperature interval of 8–40 °C. The function ∂lnXs/∂T was found to be discontinuous at Tm, but was continuous in the temperature interval that encompasses T*.

Interaction of the serum amyloid A proteins with phospholipid.

The serum amyloid A proteins (SAA) are transported in plasma in association with the high density lipoproteins. We have studied the solution properties of two of the polymorphic forms of SAA, SAA1 and SAA4, and compared the lipid-binding properties of SAA4 to those of the well characterized apolipoproteins, apo-A-I, apo-A-II, and apo-C-III. SAA4 was monomeric at pH 2.9 but considerable self-association was demonstrated at pH 8.2, even in the presence of 1.0 M guanidine HCl. SAA4 differed from the apolipoproteins in its ability to disrupt multilamellar dimyristoylphosphatidylcholine (DMPC) liposomes and generate bilayer discs. Apo-A-I, apo-A-II, and apo-C-III reduced the turbidity of DMPC dispersions at protein:lipid molar ratios of 1:200. SAA4, however, increased turbidity at molar ratios of 1:250 and 1:100 even when preincubated in guanidine HCl before addition to liposomes. Optical density decreased only at ratios of 1:50 and 1:25. At an SAA4:DMPC ratio of 1:50, discoidal particles (long axis, 28.1 nm; short axis, 4.4 nm) were formed which were similar to those produced by apo-C-III. Lipid binding induced changes in SAA4 conformation similar to those observed in the apolipoproteins. The alpha-helical content and intrinsic tryptophanyl fluorescence were increased and quenching of tryptophanyl fluorescence by acrylamide was reduced in the presence of DMPC. In addition, SAA4 as well as the apolipoproteins broadened the range and increased the temperature of the gel-liquid crystal transition temperature of DMPC.

Liposome filtration. Dependence on transition temperature

Liposomes formed by vortexing and passed through polycarbonate surface retention membranes showed appreciable differences in filtration behavior depending on the temperature of filtration relative to the liposome gel-liquid crystal transition temperature. Below transition, liposomes were filterable and size distributions could be determined; the cumulative volume distributions were log-normal. Above transition, liposomes were not filterable: smaller liposomes were formed until a limiting size was reached. These results suggest that liquid crystal liposome size distributions cannot be determined by filtration. This filtration behavior is a physical property of liposomes, related to the gel-liquid crystal transition, not previously reported. This property could be exploited as a new method for controlling liposome size distributions, but the implications for lipid membranes, including biological membranes, are general.

Equilibrium Studies of Lecithin-Cholesterol Interactions: I. Stoichiometry of Lecithin-Cholesterol Complexes in Bulk Systems

The maximum molar ratio of lecithin:cholesterol in aqueous dispersions has been reported to be 2:1, 1:1, or 1:2. The source of the desparate results has been examined in this study by analyzing (a) the phase relations in anhydrous mixtures (from which most dispersions are prepared) and (b) various methods of preparing aqueous dispersions, with the purpose of avoiding the formation of metastable states that may be responsible for the variability of the lecithin-cholesterol stoichiometry. Temperature-composition phase diagrams for anhydrous mixtures of cholesterol (CHOL) with dimyristoyl (DML) and with dipalmitoyl (DPL) lecithin were obtained by differential scanning calorimetry (DSC). Complexes form with molar ratios for lecithin:CHOL of 2:1 and 1:2; they are stable up to 70 degrees C. When x(CHOL) < 0.33, two phases coexist: complex (2:1) plus pure lecithin; when 0.33 < x(CHOL) < 0.67 complexes (2:1) and (1:2) coexist as separate phases. The corresponding phase diagram in water for these mixtures was determined by DSC and isopycnic centrifugation in D(2)O-H(2)O gradients. Aqueous dispersions were prepared by various methods (vortexing, dialysis, sonication) yielding identical results except as noted below. The data presented supports the following phase relations. When x(CHOL) < 0.33, two lipid phases coexist: pure lecithin plus complex (2:1) where the properties of the lecithin phase are determined by whether the temperature is below or above T(c), the gel-liquid crystal transition temperature. Therefore, complex (2:1) will coexist with gel state below T(c) and with liquid crystal above T(c). The densities follow in the order gel > complex (2:1) > liquid crystal. The density of complex (2:1) is less sensitive to temperature in the range 5 degrees -45 degrees C compared to the temperature dependence for DML and DPL where large changes in density occur at T(c). When x(CHOL) > 0.33, CHOL phase coexists with complex (2:1); anhydrous complex (1:2) is apparently not stable in H(2)O. The results are independent of the method and temperature used for preparing the lipid dispersions. However, when dispersions are prepared by sonication or with solvents at T > T(c), an apparent 1:1 complex is formed. Evidence suggests the 1:1 complex is metastable.

Equilibrium and metastable states in lecithin films

We have considered whether lecithin surface films below the gel-liquid crystal transition temperature, Tc, are in unique physical states. In general, below Tc, equilibrium films do not exist when surface pressures, pi, exceed about 0.1 dyn/cm. Since surface pressure-surface area isotherms of lecithin films below Tc always encompass pi's much greater than 0.1 dyn/cm, the film states are metastable. We show that the film properties under these conditions depend strongly on the history of the film, particularly the method of film formation. Lecithin surface films below Tc are thus in arbitrary metastable states, so that pi-area isotherms are difficult to interpret. The physical significance of such isotherms remains to be determined. The utility of pure lecithin surface layers below Tc as models for biological systems is also challenged by our results.