Plasmalogens protect unsaturated lipids against UV-induced oxidation in monolayer

Oxidative stress results from the attack by free radicals of several cellular targets (proteins, DNA and lipids). The cell equilibrium is a direct consequence of the pro-/antioxidant balance. In order to understand the physiological processes involved in oxidative stress, we followed oxidation of unsaturated lipids using a biomimetic system: Langmuir monolayers. The oxidation mode chosen was UV-irradiation and the lipid model was a polyunsaturated phospholipid: 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC). The monomolecular film technique was used to measure membrane rheology before and after UV-irradiation. We showed that the UV-irradiation of a DLPC monomolecular film led to a molecular area and surface elasticity modulus decrease that attests to the apparition of new molecular species at the air-water interface. The antioxidant effect of a synthetic plasmalogen (1-O-(1′-(Z)-hexadecenyl)-2-O-oleoyl-sn-glycero-3-phosphocholine or P_PLMOPE) was tested on the oxidation of DLPC. Indeed, for about 25% mol P_PLMOPE in mixed DLPC/P_PLMOPE monolayers, a complete inhibition of the molecular area and the surface elasticity modulus decreases was observed in our experimental conditions. Lower P_PLMOPE quantities delayed but did not prevent the DLPC oxidation in mixed monolayers.     

The natural antioxidant rosmarinic acid spontaneously penetrates membranes to inhibit lipid peroxidation in situ

Exogenous molecules from dietary sources such as polyphenols are very efficient in preventing the alteration of lipid membranes by oxidative stress. Among the polyphenols, we have chosen to study rosmarinic acid (RA). We investigated the efficiency of RA in preventing lipid peroxidation and in interacting with lipids. We used liposomes of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) to show that RA was an efficient antioxidant. By HPLC, we determined that the maximum amount of RA associated with the lipids was ~1 mol%. Moreover, by using Langmuir monolayers, we evidenced that cholesterol decreases the penetration of RA. The investigation of transferred lipid/RA monolayers by atomic force microscopy revealed that 1 mol% of RA in the membrane was not sufficient to alter the membrane structure at the nanoscale. By fluorescence, we observed no significant modification of membrane permeability and fluidity caused by the interaction with RA. We also deduced that RA molecules were mainly located among the polar headgroups of the lipids. Finally, we prepared DLPC/RA vesicles to evidence for the first time that up to 1 mol% of RA inserts spontaneously in the membrane, which is high enough to fully prevent lipid peroxidation without any noticeable alteration of the membrane structure due to RA insertion.     

Characterization of two oxidatively modified phospholipids in mixed monolayers with DPPC

The properties of two oxidatively modified phospholipids viz. 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) and 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), were investigated using a Langmuir balance, recording force-area (pi-A) isotherms and surface potential psi. In mixed monolayers with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) a progressive disappearance of the liquid expanded-liquid condensed transition and film expansion was observed with increasing content of the oxidized phospholipids. The above is in agreement with fluorescence microscopy of the monolayers, which revealed an increase in the liquid expanded region of DPPC monolayers. At a critical pressure pi(s) approximately 42 mN/m both Poxo- and PazePC induced a deflection in the pi-A isotherms, which could be rationalized in terms of reorientation of the oxidatively modified acyl chains into aqueous phase (adaptation of the so-called extended conformation), followed upon further film compression by solubilization of the oxidized phospholipids into the aqueous phase. Surface potential displayed a discontinuity at the same value of area/molecule, corresponding to the loss of the oxidized phospholipids from the monolayers. Our data support the view that lipid oxidation modifies both the small-scale structural dynamics of biological membranes as well as their more macroscopic lateral organization. Accordingly, oxidatively modified lipids can be expected to influence the organization and functions of membrane associated proteins.     

Enzyme-catalyzed oxidation of cholesterol in mixed phospholipid monolayers reveals the stoichiometry at which free cholesterol clusters disappear.

In this study, we have used cholesterol oxidase as a probe to study cholesterol/phospholipid interactions in mixed monolayers at the air/water interface. Mixed monolayers, containing a single phospholipid class and cholesterol at differing cholesterol/phospholipid molar ratios, were exposed to cholesterol oxidase at a lateral surface pressure of 20 mN/m (at 22 degrees C). At equimolar ratios of cholesterol to phospholipid, the average rate of cholesterol oxidation was fastest in unsaturated phosphatidylcholine mixed monolayers (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and egg yolk phosphatidylcholine), intermediate in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and slowest in sphingomyelin monolayers (egg yolk or bovine brain sphingomyelin). The average oxidation rate in mixed monolayers was not exclusively a function of monolayer packing density, since egg yolk and bovine brain sphingomyelin mixed monolayers occupied similar mean molecular areas even though the measured average oxidation rate was different with these two phospholipids. This suggests that the phospholipid acyl chain composition influenced the oxidation rate. The importance of the phospholipid acyl chain length on influencing the average oxidation rate was further examined in defined phosphatidylcholine mixed monolayers. The average oxidation rate decreased linearly with increasing acyl chain lengths (from di-8:0 to di-18:0). When the average oxidation rate was examined as a function of the cholesterol to phospholipid (C/PL) molar ratio in the monolayer, the otherwise linear function displayed a clear break at a 1:1 stoichiometry with phosphatidylcholine mixed monolayers, and at a 2:1 C/PL stoichiometry with sphingomyelin mixed monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane permeabilizing activity of amphotericin B is affected by chain length of phosphatidylcholine added as minor constituent

The effect of acyl-chain length of phospholipid on the membrane permeabilizing activity of amphotericin B (AmB) was examined using egg phosphatidylcholine (eggPC) liposomes containing 5% or 20% phosphatidylcholine with various lengths of fatty acyl chains from C(10) to C(18); 1,2-dicapryloyl-sn-glycero-3-phosphocholine (DCPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The membrane activity of AmB was evaluated by two methods; the drug was added to a liposome suspension (added-via-aqua), or mixed with lipids prior to liposome preparation (mixed-with-lipid). In both cases, K(+) influx by AmB was measured as pH change inside liposomes by 31P-NMR. The C(10) and C(12) acyl phospholipids markedly enhanced the activity of AmB, the C(14) and C(16) lipids virtually showed no effect, and the C(18) lipid was inhibitory to the AmB's action. Clear distinction between the C(12) and C(14) lipids, which differ only in acyl chains by two carbons, implies that molecular interaction between phospholipid and AmB is partly due to the matching of their hydrophobic length.     

Spatial variation in the molecular tilt orientational order within the solid domains of phase-separated, mixed dialkylphosphatidylcholine monolayers

The miscibility of the solid-phase-forming distearoylphosphatidylcholine (DSPC) and the fluid-phase-forming dilauroylphosphatidylcholine (DLPC) at the air/water interface was investigated by the Langmuir film balance. Surface pressure-area isotherms suggest that mixtures containing 25.0-62.5-mol% DLPC (range of composition investigated) are phase-separated. The lateral structure of the DSPC/DLPC monolayers was imaged by Brewster angle microscopy (BAM) as a function of the surface pressure. Quasi-circular condensed domains appeared at pressures between 0 and 0.5mN m(-1), and these structures were already fully developed at approximately 1mN m(-1). Further compression of the monolayers above 1mN m(-1) merely brought the domains closer together. The mixed monolayers consisted of solid domains of DSPC, approximately 3-20 micro in size, in a fluid matrix of DLPC. BAM and the phase contrast mode of intermittent-contact atomic force microscopy (AFM) revealed that the quasi-circular DSPC domains are divided into segments of different reflectivities (BAM) or phase shift (AFM) that arise from abrupt changes in the long-range orientational order of the tilted hydrocarbon chains. The DSPC domains in DSPC/DLPC internally exhibited star and cardioid textures that were heretofore only reported for single-component lipid monolayers in the phase coexistence region.     

Flunitrazepam-membrane non-specific binding and unbinding: two pathways with different energy barriers

The effect of molecular packing on flunitrazepam's ability to interact with bio-membranes was studied using dipalmitoylphosphatidylcholine monomolecular layers at the air-water interface as a model membrane. Flunitrazepam penetrated from the subphase into monolayers at lateral pressures below 44.8 mN/m and induced their concentration-dependent expansion. As inferred from the values of compressibility modulus, the elasticity of the liquid-condensed phase decreased in the presence of flunitrazepam. Although this drug hardly penetrated into high-packed monolayers, it was easily incorporated in the low-packed ones at an extent sufficient to reach the partition equilibrium. Below a molecular area of 75 A(2), contrary to what would be expected, the drug surface concentration increased as a function of surface pressure, suggesting that after its penetration in disordered phases, it became energetically or physically trapped in newly-formed liquid condensed clusters. The phenomenon of flunitrazepam penetration and release would have different energy barriers depending on the membrane phase-state.     

Surface film pressure of actin: interactions with lipids in mixed monolayers

The interactions of actin with neutral lipid films made from DLPC, and with positively charged films built from DLPC and stearylamine (SA), have been characterized by the monolayer technique. Injection of actin underneath an expanded lipid film produces an increase in the surface pressure that is consistent with a penetration of the lipid molecules by actin. This adsorption of actin to the lipid is more pronounced either with positively charged films or with Mg(2+) present in the sub-phase, suggesting that the mechanism involves an electrostatic attraction. During compression, the actin molecules are squeezed out into the sub-phase, carrying along some lipid molecules; this suggests a strong affinity of the lipids for actin. An analysis of the dilational modulus shows that when actin is found as monomers at the interface, the mixed actin-lipid film undergoes three phase changes upon compression. On the other hand, when actin is polymerized at the interface, the actin and the lipid form a rigid film for which the compressibility is mostly dominated by actin.     

Behavior of a GPI-anchored protein in phospholipid monolayers at the air-water interface

The interaction between alkaline phosphatase (AP), a glycosylphosphatidylinositol (GPI)-anchored protein (AP-GPI), and phospholipids was monitored using Langmuir isotherms and PM-IRRAS spectroscopy. AP-GPI was injected under C16 phospholipid monolayers with either a neutral polar head (1,2-dipalmitoyl-sn-glycero-3-phosphocholine monohydrate (DPPC)) or an anionic polar head (1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS)). The increase in molecular area due to the injection of protein depended on the surface pressure and the type of phospholipid. At all surface pressures, it was highest in the case of DPPS monolayers. The surface elasticity coefficient E, determined from the pi-A diagrams, allowed to deduct that the AP-GPI-phospholipid mixtures presented a molecular arrangement less condensed than the corresponding pure phospholipid films. PM-IRRAS spectra suggested different protein-lipid interactions as a function of the nature of the lipids. AP-GPI modified the organization of the DPPS deuterated chains whereas AP-GPI affected only the polar group of DPPC at low surface pressure (8 mN/m). Different protein hydration layers between the DPPC and DPPS monolayers were suggested to explain these results. PM-IRRAS spectra of AP-GPI in the presence of lipids showed a shape similar to those collected for pure AP-GPI, indicating a similar orientation of AP-GPI in the presence or absence of phospholipids, where the active sites of the enzyme are turned outside of the membrane.     

Characterization of the binary mixed monolayers of α-tocopherol with phospholipids at the air-water interface

The mixed Langmuir monolayers composed of model constituents of biological membranes, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), were investigated to provide information on the intermolecular interactions between these membrane components and the physiologically active vitamin E–α-tocopherol (TF), as well as on the phase behavior of these mixed systems. Additionally, topography of these monolayers transferred onto the mica support was investigated by the inverted metallurgical microscope. Morphological characteristics were directly observed by Brewster angle microscopy (BAM). From the surface pressure–area isotherms and the analysis of physicochemical parameters (compressibility and mean molecular area at the maximum compressibility) it was found that depending on the acyl chains saturation degree, TF has different effect on the phospholipids. In the case of DPPC, the addition of TF to the phospholipid film causes destabilization of the ordered hydrocarbon chains, while in the POPC/DOPC–TF systems, the attractive interactions are responsible for the monolayer increased stability. Thus, the results of these studies confirm the hypothesis that α-tocopherol may play a role in the stabilization of biological membranes.     

Effect of externally applied electrostatic fields on the surface topography of ceramide-enriched domains in mixed monolayers with sphingomyelin

Lipid and protein molecules anisotropically oriented at a hydrocarbon-aqueous interface configure a dynamic array of self-organized molecular dipoles. Electrostatic fields applied to lipid monolayers have been shown to induce in-plane migration of domains or phase separation in a homogeneous system. In this work, we have investigated the effect of externally applied electrostatic fields on the distribution of the condensed ceramide-enriched domains in mixed monolayers with sphingomyelin. In these monolayers, the lipids segregate in different phases at all pressures. This allows analyzing by epifluorescence microscopy the effect of the electrostatic field at all lateral pressure because coexistence of lipid domains in condensed state are always present. Our observations indicate that a positive potential applied to an electrode placed over the monolayer promotes a repulsion of the ceramide-enriched domains which is rather insensitive to the film composition, depends inversely on the lateral pressure and exhibits threshold dependence on the in-plane elasticity.     

A new method for determining phospholipase D activity using the monomolecular film technique

A versatile and continuous assay for phospholipase D (PL D) activity was developed using the monomolecular film technique. For this purpose, a two-step enzymatic reaction was used. First, PL D hydrolysis of stable 1,2-diacyl-sn-glycero-3-phosphocholine (PC) films by PL D generated a stable 1,2-diacyl-sn-glycero-3-phosphate (PA) film and water-soluble choline. Secondly, the latter acidic phospholipid, in contrast to the initial PC molecule, was further hydrolysed under the action of porcine pancreatic lipase (PPL) in order to give rise to lysophosphatidic acid and fatty acid, which were rapidly desorbed from the interface. With this new procedure, it is possible to obtain continuous and accurate kinetic measurements of the PL D-catalyzed reaction with phospholipid monolayers as substrates. The PLD kinetics were linear with time and the velocities recorded were directly dependent upon the amount of PL D used. In a preliminary study, we investigated the effects of the surface pressure on the PL D activity.     

Interfacial interactions of ceramide with dimyristoylphosphatidylcholine: impact of the N-acyl chain

The mixing behavior of dimyristoylphosphatidylcholine (DMPC) with either N-palmitoyl-sphingosine (C16:0-ceramide) or N-nervonoyl-sphingosine (C24:1-ceramide) was examined using monomolecular films. While DMPC forms highly elastic liquid-expanded monolayers, both neat C16:0-ceramide and C24:1-ceramide yield stable solid condensed monomolecular films with small areas and low interfacial elasticity. Compression isotherms of mixed C16:0-ceramide/DMPC films exhibit an apparent condensation upon increasing X(cer16:0) at all surface pressures. The average area isobars, coupled with the lack of a liquid-expanded to condensed phase transition as X(cer16:0) is increased, are indicative of immiscibility of the lipids at all surface pressures. In contrast, isobars for C24:1-ceramide/DMPC mixtures show surface pressure-dependent apparent condensation or expansion and surface pressure-area isotherms show a composition and surface pressure-dependent phase transition. This suggests miscibility, albeit non-ideal, of C24:1-ceramide and DMPC in both liquid and condensed surface phases. The above could be verified by fluorescence microscopy of the monolayers and measurements of surface potential, which revealed distinctly different domain morphologies and surface potential values for the DMPC/C16:0- and DMPC/C24:1-ceramide monolayers. Taken together, whereas C16:0-ceramide and DMPC form immiscible pseudo-compounds, C24:1-ceramide and DMPC are partially miscible in both the liquid-expanded and condensed phases, and a composition and lateral pressure-dependent two-phase region is evident between the liquid-expanded and condensed regimes. Our results provide novel understanding of the regulation of membrane properties by ceramides and raise the possibility that ceramides with different acyl groups could serve very different functions in cells, relating to their different physicochemical properties.     

Fluid-phase chain unsaturation controlling domain microstructure and phase in ternary lipid bilayers containing GalCer and cholesterol

We report the microstructure and phase behavior of three ternary mixtures each containing a long-chain saturated glycosphingolipid, galactosylceramide (GalCer), and cholesterol at room temperature. The unsaturation level of the fluid-phase component was varied by lipid choice, i.e., saturated 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), singly unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or doubly unsaturated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). GalCer was used because of its biological significance, for example, as a ligand in the sexual transmission of HIV and stimulator of natural killer T-cells. Supported lipid bilayers of the ternary mixtures were imaged by atomic force microscopy and GalCer-rich domains were characterized by area/perimeter ratios (A/P). GalCer domain phase transitions from solid (S) to liquid (L) phase were verified by domain behavior in giant unilamellar vesicles, which displayed two-dimensional microstructure similar to that of supported lipid bilayers. As cholesterol concentration was increased, we observed approximately 2.5, approximately 10, and approximately 20-fold decreases in GalCer domain A/P for bilayers in L-S phase coexistence containing DOPC, POPC, and DLPC, respectively. The transition to L-L phase coexistence occurred at approximately 10 mol % cholesterol for bilayers containing DOPC or POPC and was accompanied by maintenance of a constant A/P. L-L phase coexistence did not occur for bilayers containing DLPC. We systematically relate our results to the impact of chain unsaturation on the interaction of the fluid-phase lipid and cholesterol. Physiologically, these observations may give insight into the interplay of fatty acid chain unsaturation, sterol concentration, and lipid hydrophobic mismatch in membrane phenomena.     

Liquid crystalline/gel state phase separation in docosahexaenoic acid-containing bilayers and monolayers

The phase behavior of lipid mixtures containing 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0, 22:6 PC) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was studied with bilayers using differential scanning calorimetry (DSC), and with monolayers monitoring pressure/area isotherms and surface elasticity, and lipid domain formation followed by epifluorescence microscopy. From DSC studies it is concluded that DPPC/18:0, 22:6 PC phase separates into DPPC-rich and 18:0, 22:6 PC-rich phases. In monolayers, phase separation is indicated by changes in pressure-area isotherms implying phase separation where 18:0, 22:6 PC is 'squeezed out' of the remaining DPPC monolayer. Phase separation into lipid domains in the mixed PC monolayer is quantified by epifluorescence microscopy using the fluorescently labeled phospholipid membrane probe, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl). These results further describe the ability of docosahexaenoic acid to participate in lipid phase separations in membranes.     

Mitochondrial H+-ATPase: identification of subunits of the F0 subcomplex contacting with membrane lipids

The subunits of the F0 membrane sector of bovine heart mitochondrial H(+)-ATPase that contact the lipids of the mitochondrial inner membrane were identified with the use of specially synthesized proteoliposomes that contained active mitochondrial H(+)-ATPase and a photoreactive lipid, which was 1-acyl-2-[12-(diazocyclopentadiene-2-carbonylamino)-[12-14C]dodecanoyl]-sn- glycero-3-phosphocholine, 1-acyl-2-[11-([125I]diazoiodocyclopentadiene-2-carbonyloxy)undecanoyl]-sn- glycero-3-phosphocholine, or 1-acyl-2-[12-(diazocyclopentadiene-2-carbonylamino)dodecanoyl]-sn-glycero- 3-phosphocholine, where acyl is a mixture of the residues of palmitic (70%) and stearic (30%) acids. An analysis of the cross-linked products obtained upon the UV-irradiation of these proteoliposomes indicated that subunits c and a of the F0 membrane sector contact the lipids. The cross-linked products were identified by SDS-PAGE and MALDI mass spectrometry.     

Interactions of Alkylphosphocholines with Model Membranes—The Langmuir Monolayer Study

Alkylphosphocholines (APCs) belong to a class of synthetic antitumor lipids, which are new-generation anticancer agents. In contrast to traditional antitumor drugs, they do not attack the cell nucleus but, rather, the cellular membrane; however, their mechanism of action is not fully understood. This work compared the interactions of selected APCs [namely, hexadecylphosphocholine (miltefosine), octadecylphosphocholine and erucylphosphocholine] with the most important membrane lipids [cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)] and examined their influence on a model membrane of tumor and normal cells. As a simple model of membranes, Langmuir monolayers prepared by mixing cholesterol either with a saturated phosphatidylcholine (DPPC), for a normal cell membrane, or with an unsaturated one (POPC), for a tumor cell membrane, have been applied. The APC–lipid interactions, based on experimental surface pressure (π) versus mean molecular area (A) isotherms, were analyzed qualitatively (with mean molecular area values) as well as quantitatively (with the ΔG ^exc function). Strong attractive interactions were observed for mixtures of APCs with cholesterol, contrary to the investigated phosphatidylcholines, for which the interactions were found to be weak with a tendency to separation of film components. In ternary monolayers it has been found that the investigated model systems (cholesterol/DPPC/APC vs cholesterol/POPC/APC) differ significantly as regards the interactions between film-forming molecules. The results demonstrate stronger interactions between the components of cholesterol/POPC/APC monolayers compared to cholesterol/POPC film, mimicking tumor cell membranes. In contrast, the interactions in cholesterol/DPPC/APC films were found to be weaker than those in the cholesterol/DPPC system, serving as a model of healthy cell membranes, thus proving that the incorporation of APCs is, from a thermodynamic point of view, unfavorable for binary cholesterol/DPPC monolayers. It can be concluded that the composition of healthy cell membranes is a natural barrier preventing the incorporation of APCs into normal cells.     

Trehalose maintains phase separation in an air-dried binary lipid mixture

Mixing and thermal behavior of hydrated and air-dried mixtures of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 1,2-distearoyl-d70-sn-glycero-3-phosphocholine (DSPCd-70) in the absence and presence of trehalose were investigated by Fourier transform infrared spectroscopy. Mixtures of DLPC:DSPCd-70 (1:1) that were air-dried at 25 degrees C show multiple phase transitions and mixed phases in the dry state. After annealing at high temperatures, however, only one transition is seen during cooling scans. When dried in the presence of trehalose, the DLPC component shows two phase transitions at -22 degrees C and 75 degrees C and is not fully solidified at -22 degrees C. The DSPCd-70 component, however, shows a single phase transition at 78 degrees C. The temperatures of these transitions are dramatically reduced after annealing at high temperatures with trehalose. The data suggest that the sugar has a fluidizing effect on the DLPC component during drying and that this effect becomes stronger for both components with heating. Examination of infrared bands arising from the lipid phosphate and sugar hydroxyl groups suggests that the strong effect of trehalose results from direct interactions between lipid headgroups and the sugar and that these interactions become stronger after heating. The findings are discussed in terms of the protective effect of trehalose on dry membranes.     

Stimuli-responsive surfactant/phospholipid stabilized colloidal dispersions and their film formation

Methyl methacrylate (MMA) and n-butyl acrylate (nBA) were copolymerized into stable colloidal particles in the presence of micelle forming sodium dioctyl sulfosuccinate (SDOSS) and liposome forming 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) in aqueous media that serve as thermodynamically stable loci for lipophilic monomers and nanostructured templates. These studies show for the first time that hollow colloidal particles may coalesce to form polymeric films and the combination of SDOSS and DLPC dispersing agents provides a stimuli-responsive environment during film formation through which individual surface stabilizing components can be driven to the film-air (F-A) or film-substrate (F-S) interface. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) of p-MMA/nBA colloidal dispersions revealed preferential and enhanced mobility of SDOSS and DLPC lipid rafts to the F-A and F-S interfaces in response to thermal, ionic, and enzymatic stimuli.     

A correlation between lipid domain shape and binary phospholipid mixture composition in free standing bilayers: A two-photon fluorescence microscopy study

Giant unilamellar vesicles (GUVs) composed of different phospholipid binary mixtures were studied at different temperatures, by a method combining the sectioning capability of the two-photon excitation fluorescence microscope and the partition and spectral properties of 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan) and Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (N-Rh-DPPE). We analyzed and compared fluorescence images of GUVs composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DLPC/DPPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DLPC/DSPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-diarachidoyl-sn-glycero-3-phosphocholine (DLPC/DAPC), 1, 2-dimyristoyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DMPC/DSPC) (1:1 mol/mol in all cases), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine/1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPE/DMPC) (7:3 mol/mol) at temperatures corresponding to the fluid phase and the fluid-solid phase coexistence. In addition, we studied the solid-solid temperature regime for the DMPC/DSPC and DMPE/DMPC mixtures. From the Laurdan intensity images the generalized polarization function (GP) was calculated at different temperatures to characterize the phase state of the lipid domains. We found a homogeneous fluorescence distribution in the GUV images at temperatures corresponding to the fluid region for all of the lipid mixtures. At temperatures corresponding to phase coexistence we observed concurrent fluid and solid domains in the GUVs independent of the lipid mixture. In all cases the lipid solid domains expanded and migrated around the vesicle surface as we decreased the temperature. The migration of the solid domains decreased dramatically at temperatures close to the solid-fluid-->solid phase transition. For the DLPC-containing mixtures, the solid domains showed line, quasicircular, and dendritic shapes as the difference in the hydrophobic chain length between the components of the binary mixture increases. In addition, for the saturated PC-containing mixtures, we found a linear relationship between the GP values for the fluid and solid domains and the difference between the hydrophobic chain length of the binary mixture components. Specifically, at the phase coexistence temperature region the difference in the GP values, associated with the fluid and solid domains, increases as the difference in the chain length of the binary mixture component increases. This last finding suggests that in the solid-phase domains, the local concentration of the low melting temperature phospholipid component increases as the hydrophobic mismatch decreases. At the phase coexistence temperature regime and based on the Laurdan GP data, we observe that when the hydrophobic mismatch is 8 (DLPC/DAPC), the concentration of the low melting temperature phospholipid component in the solid domains is negligible. This last observation extends to the saturated PE/PC mixtures at the phase coexistence temperature range. For the DMPC/DSPC we found that the nonfluorescent solid regions gradually disappear in the solid temperature regime of the phase diagram, suggesting lipid miscibility. This last result is in contrast with that found for DMPE/DMPC mixtures, where the solid domains remain on the GUV surface at temperatures corresponding to that of the solid region. In all cases the solid domains span the inner and outer leaflets of the membrane, suggesting a strong coupling between the inner and outer monolayers of the lipid membrane. This last finding extends previous observations of GUVs composed of DPPE/DPPC and DLPC/DPPC mixtures (, Biophys. J. 78:290-305).