Decrease of elastic moduli of DOPC bilayers induced by a macrolide antibiotic, azithromycin

The elastic properties of membrane bilayers are key parameters that control its deformation and can be affected by pharmacological agents. Our previous atomic force microscopy studies revealed that the macrolide antibiotic, azithromycin, leads to erosion of DPPC domains in a fluid DOPC matrix [A. Berquand, M. P. Mingeot-Leclercq, Y. F. Dufrene, Real-time imaging of drug-membrane interactions by atomic force microscopy, Biochim. Biophys. Acta 1664 (2004) 198-205.]. Since this observation could be due to an effect on DOPC cohesion, we investigated the effect of azithromycin on elastic properties of DOPC giant unilamellar vesicles (GUVs). Microcinematographic and morphometric analyses revealed that azithromycin addition enhanced lipid membranes fluctuations, leading to eventual disruption of the largest GUVs. These effects were related to change of elastic moduli of DOPC, quantified by the micropipette aspiration technique. Azithromycin decreased both the bending modulus (k_c, from 23.1 ± 3.5 to 10.6 ± 4.5 k_BT) and the apparent area compressibility modulus (K_app, from 176 ± 35 to 113 ± 25 mN/m). These data suggested that insertion of azithromycin into the DOPC bilayer reduced the requirement level of both the energy for thermal fluctuations and the stress to stretch the bilayer. Computer modeling of azithromycin interaction with DOPC bilayer, based on minimal energy, independently predicted that azithromycin (i) inserts at the interface of phospholipid bilayers, (ii) decreases the energy of interaction between DOPC molecules, and (iii) increases the mean surface occupied by each phospholipid molecule. We conclude that azithromycin inserts into the DOPC lipid bilayer, so as to decrease its cohesion and to facilitate the merging of DPPC into the DOPC fluid matrix, as observed by atomic force microscopy. These investigations, based on three complementary approaches, provide the first biophysical evidence for the ability of an amphiphilic antibiotic to alter lipid elastic moduli. This may be an important determinant for drug: lipid interactions and cellular pharmacology.     

Effect of potassium perfluorooctanesulfonate, perfluorooctanoate and octanesulfonate on the phase transition of dipalmitoylphosphatidylcholine (DPPC) bilayers

Perfluorooctanesulfonic acid (PFOS) is a persistent environmental pollutant that may cause adverse effects by inhibiting pulmonary surfactant. To gain further insights in this potential mechanism of toxicity, we investigated the interaction of PFOS potassium salt with dipalmitoylphosphatidylcholine (DPPC) - the major component of pulmonary surfactant - using steady-state fluorescence anisotropy spectroscopy and DSC (differential scanning calorimetry). In addition, we investigated the interactions of two structurally related compounds, perfluorooctanoic acid (PFOA) and octanesulfonic acid (OS) potassium salt, with DPPC. In the fluorescence experiments a linear depression of the main phase transition temperature of DPPC (T_m) and an increased peak width was observed with increasing concentration of all three compounds, both using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) as fluorescent probes. PFOS caused an effect on T_m and peak width at much lower concentrations because of its increased tendency to partition onto DPPC bilayers, i.e., the partition coefficients decrease in the K(PFOS) > K(PFOA) >> K(OS). Similar to the fluorescence anisotropy measurements, all three compounds caused a linear depression in the onset of the main phase transition temperature and a significant peak broadening in the DSC experiments, with PFOS having the most pronounced effect of the peak width. The effect of PFOS and other fluorinated surfactants on DPPC in both mono- and bilayers may be one mechanism by which these compounds cause adverse biological effects.     

The effects of various peptides on the thermotropic properties of phosphatidylcholine bilayers

The effects of an amino acid derivative (N-benzoyl-l-argininamide), four small peptides (Phe-Gly-Phe-Gly, gastrin-related peptide (Trp-Met-Arg-Phe-NH₂), tetragastrin (Trp-Met-Asp-Phe-NH₂), pentagastrin (Boc-βAla-Trp-Met-Asp-Phe-NH₂)) and one medium-sized peptide. glucagon (29 residues), on the gel-to-liquid crystalline transition of a multilamellar suspension of dimyristoylphosphatidylcholine have been studied by means of high-sensitivity differential scanning calorimetry. At low concentrations of added solutes, the temperature at which the excess apparent specific heat in the gel-to-liquid crystalline phase transition of the lipid is maximal is lowered by an amount proportional to the total concentration of the peptide, with proportionality constants ranging from ?0.018 K mM^?1 for Phe-Gly-Phe-Gly to ?3.1 K mM^?1 for the gastrin-related peptide. The lipid mixtures involving the first two solutes listed above exhibited approximately symmetrical curves of excess apparent specific heat vs. temperature. The curves for the other solutes were asymmetric, and could be well represented as the sum of either two or three two-state curves. The asymmetry, which was especially pronounced in the cases of pentagastrin and glucagon, thus appeared to be due to the presence of components having lower and/or higher transition temperatures than that of the lipid. Pentagastrin and glucagon (R.M. Epand and J.M. Sturtevant, Biochemistry 20 (1981) 4603) have much smaller effects on the gel-to-liquid crystalline phase transition of dipalmitoylphosphatidylcholine than on that of the dimyristoyl analog.     

Effect of progesterone on DPPC membrane: evidence for lateral phase separation and inverse action in lipid dynamics

Interactions of progesterone with zwitterionic dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes were investigated as a function of temperature and progesterone concentration by using three non-invasive techniques namely Fourier transform infrared spectroscopy, turbidity at 440 nm, and differential scanning calorimetry. The results reveal that progesterone changes the physical properties of DPPC bilayers by decreasing the main phase-transition temperature, abolishing the pre-transition, broadening the phase-transition profile, disordering the system both in gel and liquid crystalline phase, increasing the dynamics at low concentrations whereas stabilizing the membrane at high concentrations, and inducing phase separation. Progesterone does not change the hydration of the CO groups, while it strengthens the hydrogen bonding between the PO2- groups of lipids and the water molecules around.     

The chain conformational order of ergosterol- or cholesterol-containing DPPC bilayers as modulated by Amphotericin B: a FTIR study

Amphotericin B (AmB) is the most widely used antibiotic to treat systemic fungal infections. However, the molecular mechanism of its activity is still not completely understood. In the present work we have used FTIR spectroscopy to investigate the conformational state of the aliphatic chains of DPPC liposomes using the 2850 cm(-1) band, associated with the methylene symmetric stretching mode. The liposomes were either binary mixtures of the lipid with AmB, cholesterol or ergosterol, or ternary systems of these constituents. The two sterols contribute to an ordering of the aliphatic chains of the lipid, this ordering being slightly more important with ergosterol. In the gel state, AmB does not change the conformational order of DPPC even at high concentration. In the fluid phase, however, the drug clearly structures its lipid environment. Our results show that AmB can initiate a redistribution of the ergosterol in the plane of the membrane, but not of the cholesterol molecules, which might constitute an additional mechanism to explain the activity of the antibiotic.     

Effect of the nature of the 3beta-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, 3beta-hydroxy-manoyl oxide (1) and 3beta-acetoxy-manoyl oxide (2) have been shown to exhibit in vitro antimicrobial and cytotoxic activity, while N-imidazole-3 beta-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 degrees 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.     

The role of the anticancer drug vinorelbine in lipid bilayers using differential scanning calorimetry and molecular modeling

Differential scanning calorimetry (DSC) has been employed to investigate the thermal changes caused by the anticancer alkaloid drug vinorelbine in dipalmytoylphosphatidylcholine (DPPC) bilayers. The total enthalpy change was increased by the presence of the drug molecule, indicating a partial interdigitation of the lipid alkyl chains. The presence of cholesterol in DPPC bilayers including vinorelbine induced an obstruction of the interdigitation, since cholesterol interrupts the upraise of enthalpy change. Vinorelbine's interdigitation ability and stabilizing properties with the active site of the receptor have been compared with those of similar in structure amphipathic and bulky alkaloid vinblastine. The obtained results may in part explain their similar mechanism of action but different bioactivity.     

The influence of NBD fluorescent probe on model membranes containing POPC and DPPC

To investigate the effect of fluorescent probe on the properties of membranes, we studied model membranes composed of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl 2-oleoyl-sn-glycero-3-phosphocholine (POPC) in the presence and absence of fluorescent probe. The morphology of giant unilamellar vesicles (GUVs) has been observed as a function of temperature and composition by fluorescence microscopy using NBD-DOPE or C₆-NBD-PC as the probe. The phase behavior of model membranes containing no fluorescent probe was investigated by ²H-NMR spectroscopy. We found that the bright phase observed on GUVs was the fluid phase enriched in POPC and the dark phase was the gel phase enriched in DPPC. NBD-DOPE and C₆-NBD-PC preferentially participated in the fluid-phase domains when GUVs were in the gel?+?fluid phase coexistence. Inclusion of both fluorescent probes (1?mol%) lowered the transition temperature of POPC/DPPC membranes. In addition, C₆-NBD-PC exhibited a stronger effect than NBD-DOPE, which was considered to be associated with the structures of fluorescent molecules.     

AFM study of the thermotropic behaviour of supported DPPC bilayers with and without the model peptide WALP23

Temperature-controlled Atomic Force Microscopy (TC-AFM) in Contact Mode is used here to directly image the mechanisms by which melting and crystallization of supported, hydrated DPPC bilayers proceed in the presence and absence of the model peptide WALP23. Melting from the gel L_β′ to the liquid-crystalline L_α phase starts at pre-existing line-type packing defects (grain boundaries) in absence of the peptide. The exact transition temperature is shown to be influenced by the magnitude of the force exerted by the AFM probe on the bilayer, but is higher than the main transition temperature of non-supported DPPC vesicles in all cases due to bilayer–substrate interactions. Cooling of the fluid L_α bilayer shows the formation of the line-type defects at the borders between different gel-phase regions that originate from different nuclei. The number of these defects depends directly on the rate of cooling through the transition, as predicted by classical nucleation theory.The presence of the transmembrane, synthetic model peptide WALP23 is known to give rise to heterogeneity in the bilayer as microdomains with a striped appearance are formed in the DPPC bilayer. This striated phase consists of alternating lines of lipids and peptide. It is shown here that melting starts with the peptide-associated lipids in the domains, whose melting temperature is lowered by 0.8–2.0 °C compared to the remaining, peptide-free parts of the bilayer. The stabilization of the fluid phase is ascribed to adaptations of the lipids to the shorter peptide. The lipids not associated with the peptide melt at the same temperature as those in the pure DPPC supported bilayer.     

Molecular studies of the gel to liquid-crystalline phase transition for fully hydrated DPPC and DPPE bilayers

Molecular dynamics simulations were used for a comprehensive study of the structural properties of saturated lipid bilayers, DPPC and DPPE, near the main phase transition. Though the chemical structure of DPPC and DPPE are largely similar (they only differ in the choline and ethanolamine groups), their transformation process from a gel to a liquid-crystalline state is contrasting. For DPPC, three distinct structures can be identified relative to the melting temperature (T_m): below T_m with “mixed” domains consisting of lipids that are tilted with partial overlap of the lipid tails between leaflet; near T_m with a slight increase in the average area per lipid, resulting in a rearrangement of the lipid tails and an increase in the bilayer thickness; and above T_m with unhindered lipid tails in random motion resulting in an increase in %gauche formed and increase in the level of interdigitation between lipid leaflets. For DPPE, the structures identified were below T_m with “ordered” domains consisting of slightly tilted lipid tails and non-overlapping lipid tails between leaflets, near T_m with minimal rearrangement of the lipids as the bilayer thickness reduces slightly with increasing temperature, and above T_m with unhindered lipid tails as that for DPPC. For DPPE, most of the lipid tails do not overlap as observed to DPPC, which is due to the tight packing of the DPPE molecules. The non-overlapping behavior of DPPE above T_m is confirmed from the density profile of the terminal carbon atoms in each leaflet, which shows a narrow distribution near the center of the bilayer core. This study also demonstrates that atomistic simulations are capable of capturing the phase transition behavior of lipid bilayers, providing a rich set of molecular and structural information at and near the transition state.     

The interaction of cholesterol with bilayers of phosphatidylethanolamine

The interaction of cholesterol with the glycerol backbone segments of phospholipids was studied in bilayers of phosphatidylethanolamine containing equimolar amounts of cholesterol. Glycerol selectively deuterated at various positions was supplied to the growth medium of Escherichia coli strain 131 GP which is defective in endogeneous glycerol synthesis. The procedure enables the stereospecific labeling of the three glycerol backbone segments of the membrane phospholipids. Phosphatidylethanolamine with wild-type fatty acid composition was purified from E. coli cells and deuterium magnetic resonance spectra were obtained either from dispersions of pure phosphatidylethanolamine or from equimolar mixtures of phosphatidylethanolamine with cholesterol. For comparative purposes 1,2-di[9,10-²H₂]elaidoyl-sn-glycero-3-phosphoethanolamine and [3-α-²H]cholesterol were synthesized in order to monitor the behavior of the fatty acyl chains and of the cholesterol molecule itself. For all deuterated segments the deuterium quadrupole splittings as well as the deuterium spin-lattice (T₁) relaxation times were measured as a function of temperature. The glycerol backbone was found to be a remarkably stable structural element of the phospholipid molecule. The quadrupole splittings of the backbone segments changed only by at most 2 kHz upon incorporation of 50 mol % cholesterol. This was in contrast to the fatty acyl chains where the same amount of cholesterol increased the quadrupole splitting by more than 20 kHz. The glycerol segments exhibited the shortest T₁ relaxation times of all CH₂ segments indicating that the glycerol backbone is the slowest motional moiety of the lipid molecule. Addition of cholesterol has no effect on the backbone motion but the fast reorientation rate of the trans-double bonds in 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine is increased dramatically.     

The influence of headgroup structure and fatty acyl chain saturation of phospholipids on monolayer behavior: a comparative rheological study

This paper compares six phospholipidic monolayers at the water/chloroform interface by performing dilational rheological measurements with a drop tensiometer apparatus. The chosen lipids differ both in their headgroup structure and fatty acyl chain saturation or symmetry. The study concentrated on monolayers formed with DPPC, DPPE, DOPC, DOPE, POPC and POPE. Using a generalized Maxwell rheological model, transposed at the interface, the intimate intermolecular interactions between amphiphilic molecules are studied on and off the monolayer plane. The equilibrium and nonequilibrium phenomena are analyzed and, respectively, correlated with monolayer cohesion and with monolayer/sub-surface interactions. The purpose of this work is to gain further insights into the influences (as slight as they are) of the weak changes in phospholipid structure and on the behavior of the monolayers. The results, widely described, provide further details on nuances existing between very similar molecules, and likewise, on the synergies created between the different effects.     

Effect of 2,4-dichlorophenol on DPPC/water liposomes studied by X-ray and freeze-fracture electron microscopy

The effect of 2,4-dichlorophenol (DCP) was studied on the fully hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)--water liposomes. The structure and the thermotropic phase behaviour of the liposomes was examined in the presence of DCP (DCP/DPPC molar ratio, varied from 2x10(-2) up to 1) using small- and wide-angle X-ray scattering (SAXS, WAXS) and freeze-fracture electron microscopy. The structural behaviour of the DPPC/DCP/water system was strongly dependent on the concentration of the DCP. In the pretransition range the DCP molecules (at 2x10(-2) DCP/DPPC molar ratio) induced the interdigitated phase beside the parent (gel and rippled gel) phases, locally which can be form at higher DCP concentration. When the DCP/DPPC molar ratio was increased the pretransition disappeared and the main transition was shifted to lower temperatures. In the molar ratio range from 2x10(-1) up to 5x10(-1), a coexistence of different phases was observed in the wide temperature range from 20 up to 40 degrees C. With a further increase of the DCP/DPPC molar ratio (6x10(-1) to 1) only the interdigitated gel phase occurred below 25 degrees C. A schematic phase diagram of DPPC/DCP/water system was constructed to summarise the results.     

Calorimetric studies of the effect of cis-carotenoids on the thermotropic phase behavior of phosphatidylcholine bilayers

Carotenoid geometry is a factor that determines their solubility and orientation in the lipid membrane as well as antioxidant capacities and bioavailability. The effects of the cis-isomers of carotenoids (zeaxanthin and β-carotene) on the thermotropic properties of lipid membranes formed with dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) were investigated by means of differential scanning calorimetry. The results were compared with the effects caused by the all-trans-isomer. Both the trans and cis isomers of zeaxanthin shifted the main phase transition temperature to lower values and decreased the cooperativity of the phase transition. The effect of all-trans zeaxanthin on the physical properties of the lipid bilayers has been shown to strongly depend on the hydrocarbon chain length of the membrane. In the case of cis-zeaxanthin this relationship is weaker.     

The partition and transport behavior of cytotoxic ionic liquids (ILs) through the DPPC bilayer: Insights from molecular dynamics simulation

A molecular dynamics (MD) simulation with atomistic details was performed to examine the partitioning and transport behavior of moderately cytotoxic ionic liquids (ILs), namely choline bis(2-ethylhexyl) phosphate (CBEH), choline bis(2,4,4-trimethylpentyl) phosphinate (CTMP) and choline O,O-diethyl dithiophosphate (CDEP) in a fully hydrated dipalmitoylphosphatidylcholine (DPPC) bilayer in the fluid phase at 323?K. The structure of ILs was so selected to understand if the role of dipole and dispersion forces in the ILs distribution in the membrane can be possible. Several analyses including mass density, electrostatic potential, order parameter, diffusion coefficients and hydrogen bond formation, was carried out to determine the precise location of the anionic species inside the membrane. Moreover, the potential of the mean force (PMF) method was used to calculate free energy profile for transferring anionic species from the DPPC membrane into the bulk water. While less cytotoxic DEP is located within the bulk water, more cytotoxic TMP and BEH ILs were found to remain in the membrane and the energy barrier for crossing through the bilayer center of BEH was higher. Various ILs have no significant effect on P–N vector. The thickness of lipid bilayer decreased in all systems comprising ILs, while area per lipid increased.     

Effect of counterions on the influence of dodecyltrimethylammonium halides on thermotropic phase behaviour of phosphatidylcholine bilayers

Effects of dodecyltrimethylammonium chloride (DTAC), dodecyltrimethylammonium bromide (DTAB) and dodecyltrimethylammonium iodide (DTAI) on thermotropic phase behaviour of phosphatidylcholine bilayers as well as on 1H NMR spectra were studied. In order to enhance the effect of counterions on water structure two series of experiments were performed. In the first one the surfactants were added to the water phase and in the other one directly to lipid phase (a mixed film was formed). The effects of particular surfactants on the main phase-transition temperature were more pronounced when they were added to the water phase (1st method) instead of the lipid phase (2nd method). Furthermore, in the case of the first method the transitions were found asymmetrical while in the second method nearly symmetrical. It is suggested that surfactant-poor and surfactant-rich domains are formed when surfactants are added to the water phase.     

Effect of pH on the phase behavior of DMPC bilayers

We have studied the effect of acidic pH on the phase behavior of the zwitterionic lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) using differential scanning calorimetry and x-ray scattering. Dispersions of DMPC in HCl solutions of pH = 4 and 3 behave identical to dispersions in water. The main transition temperature increases sharply and the pre-transition disappears at lower pH. An untilted gel phase is observed at pH = 2 and 1, in contrast to the tilted gel phase found at higher pH. The relatively large periodicity of the untilted gel phase, in comparison to that of the tilted gel phase occurring near neutral pH, clearly demonstrates the simultaneous charging and dehydration of the headgroups as the pH approaches the pK of the phosphate group. Headgroup dehydration at low pH also leads to the formation of DMPC crystallites and the inverted hexagonal phase at low and high temperatures, respectively, after a few days of incubation. These results show the significant effect of acidic pH on the phase behavior of zwitterionic lipids.     

Effect of potassium perfluorooctanesulfonate, perfluorooctanoate and octanesulfonate on the phase transition of dipalmitoylphosphatidylcholine (DPPC) bilayers

Perfluorooctanesulfonic acid (PFOS) is a persistent environmental pollutant that may cause adverse effects by inhibiting pulmonary surfactant. To gain further insights in this potential mechanism of toxicity, we investigated the interaction of PFOS potassium salt with dipalmitoylphosphatidylcholine (DPPC) - the major component of pulmonary surfactant - using steady-state fluorescence anisotropy spectroscopy and DSC (differential scanning calorimetry). In addition, we investigated the interactions of two structurally related compounds, perfluorooctanoic acid (PFOA) and octanesulfonic acid (OS) potassium salt, with DPPC. In the fluorescence experiments a linear depression of the main phase transition temperature of DPPC (T(m)) and an increased peak width was observed with increasing concentration of all three compounds, both using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) as fluorescent probes. PFOS caused an effect on T(m) and peak width at much lower concentrations because of its increased tendency to partition onto DPPC bilayers, i.e., the partition coefficients decrease in the K(PFOS)>K(PFOA)>K(OS). Similar to the fluorescence anisotropy measurements, all three compounds caused a linear depression in the onset of the main phase transition temperature and a significant peak broadening in the DSC experiments, with PFOS having the most pronounced effect of the peak width. The effect of PFOS and other fluorinated surfactants on DPPC in both mono- and bilayers may be one mechanism by which these compounds cause adverse biological effects.