LβI Phase Research Articles

Induction of the Interdigitated Gel Phase of Hydrated Dipalmitoylphosphatidylcholine Bilayers by the Artificial Sweetener Sucralose.

Recent research indicates that high doses of sucralose content can weaken the immune response in mice. To better understand the interaction between cell membranes and sucralose, we studied model biomembranes composed of dipalmitoylphosphatidylcholine bilayers in a sucralose solution. Calorimetry measurements showed that the effect of sucralose on the phase behavior is biphasic. Pretransitions and main transitions are decreased at low sucralose concentrations, while the main transition is increased at high concentrations. Pretransitions cannot be detected above the concentration at which the direction of change in the main transition temperature reverses. X-ray diffraction measurements revealed that sucralose at concentrations higher than 0.2 M induces the interdigitated gel (LβI) phase below the main transition temperature. Fluorescence Prodan measurements suggested that the sucralose solution is slightly more hydrophobic than the sucrose solution. This could be one reason why sucralose induces the LβI phase.

Stretch-Induced Interdigitation of a Phospholipid/Cholesterol Bilayer.

The interdigitated gel (LβI) phase is one of the membrane phases of phospholipid molecules in which the hydrophobic tails of the phospholipid molecules penetrate the opposite leaflet of the bilayer. Recent molecular dynamics (MD) simulations have shown that interdigitation can take place as a phase transition from the liquid-ordered (Lo) phase to the LβI phase under stretching. However, there is still no conclusive experimental evidence for this process, so its existence remains controversial. In this study, to explain the transition from energy balance, we propose a free-energy model. The model consists of three energy components: the elastic deformation energy, surface energy at the bilayer-water interface, and interphase boundary energy. To determine the parameters of the model, we perform MD simulations of a stretched 1,2-dipalmitoyl- sn-glycero-3-phosphocholine/cholesterol bilayer. The phase diagrams from our model are in good agreement with those obtained from MD simulations. The energy balance among the components in the stretched bilayer quantitatively explains the stretch-induced transition. In the model, increasing the system size to that used in experiments shows that interdigitation is favorable for rigid bilayers under stretching or in alcohol solutions. These results suggest that the stretch-induced interdigitation might be observed in microscopic experiments.

Ethanol- and trifluoroethanol-induced changes in phase states of DPPC membranes. Prodan emission-excitation fluorescence spectroscopy supported by PARAFAC analysis

It has been shown that Prodan emission-excitation fluorescence spectroscopy supported by Parallel Factor (PARAFAC) analysis is a fast, simple and sensitive method used in the study of the phase transition from the noninterdigitated gel (Lβ′) state to the interdigitated gel (LβI) phase, triggered by ethanol and 2,2,2-trifluoroethanol (TFE) molecules in dipalmitoylphosphatidylcholines (DPPC) membranes. The relative contribution of lipid phases with spectral characteristics of each pure phase component has been presented as a function of an increase in alcohol concentration. It has been stated that both alcohol molecules can induce a formation of the LβI phase, but TFE is over six times stronger inducer of the interdigitated phase in DPPC membranes than ethanol molecules. Moreover, in the TFE-mixed DPPC membranes, the transition from the Lβ′ to LβI phase is accompanied by a formation of the fluid phase, which most probably serves as a boundary phase between the Lβ′ and LβI regions. Contrary to the three phase-state model of TFE-mixed DPPC membranes, in ethanol-mixed DPPC membranes only the two phase-state model has been detected.

Azide-Modified Membrane Lipids: Synthesis, Properties, and Reactivity.

In the present work, we describe the synthesis and the temperature-dependent behavior of photoreactive membrane lipids as well as their capability to study peptide/lipid interactions. The modified phospholipids contain an azide group either in the middle part or at the end of an alkyl chain and also differ in the linkage (ester vs ether) of the second alkyl chain. The temperature-dependent aggregation behavior of the azidolipids was studied using differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and small-angle X-ray scattering (SAXS). Aggregate structures were visualized by stain and cryo transmission electron microscopy (TEM) and were further characterized by dynamic light scattering (DLS). We show that the position of the azide group and the type of linkage of the alkyl chain at the sn-2 position of the glycerol influences the type of aggregates formed as well as their long-term stability: P10AzSPC and r12AzSHPC show the formation of extrudable liposomes, which are stable in size during storage. In contrast, azidolipids that carry a terminal azido moiety either form extrudable liposomes, which show time-dependent vesicle fusion (P15AzPdPC), or self-assemble in large sheet-like, nonextrudable aggregates (r15AzPdHPC) where the lipid molecules are arranged in an interdigitated orientation at temperatures below Tm (LβI phase). Finally, a P10AzSPC:DMPC mixture was used for photochemically induced cross-linking experiments with a transmembrane peptide (WAL-peptide) to demonstrate the applicability of the azidolipids for the analysis of peptide/lipid interactions. The efficiency of photo-cross-linking was monitored by attenuated total reflection infrared (ATR-IR) spectroscopy and mass spectrometry (MS).

Effect of pressure on bilayer phase behavior of N-methylated di-O-hexadecylphosphatidylethanolamines: relevance of head-group modification on the bilayer interdigitation

The phase transitions of N-methylated di-O-hexadecylphosphatidylethanolamines (DHPE, DH-N-methyl-PE (DHMePE) and DH-N,N-dimethyl-PE (DHMe2PE)) were observed by differential scanning calorimetry (DSC) and fluorometry under atmospheric pressure and by light-transmittance measurements under high pressure. The DSC thermograms showed that the N-methylated DHPE bilayers underwent the phase transition from the gel phase to the liquid crystalline (Lα) phase under atmospheric pressure. The gel phase was identified by fluorometry as the lamellar gel (Lβ) phase, and not interdigitated gel (LβI) phase. The gel/Lα transition temperature increased with pressure while decreased stepwise with increasing polar head-group size. This stepwise depression of the transition temperature may be caused by the inverse-proportional hydrogen-bonding capabilities of the head-group to the head-group size. The thermodynamic quantities of the gel/Lα transition were comparable for the N-methylated DHPE bilayers. The pressure-induced LβI phase was not found in these bilayers although the bilayer of di-O-hexadecylphosphatidylcholine (DHPC), which is a kind of N-methylated DHPEs, forms the LβI phase only by hydration under atmospheric pressure. Taking into account that the bilayers of diacyl-homologs of N-methylated DHPEs, N-methylated dipalmitoyl-PEs except for dipalmitoylphosphatidylcholine (DPPC), do not form the LβI phase in the whole pressure range investigated but the DPPC bilayer forms the LβI phase under high pressure, we can say that the interdigitation requires weaker interaction between large-sized head groups like the bulky choline group.

Influence of water concentrations on the phase transformation of a model surfactant/co-surfactant/water system

The influence of water concentrations on phase transformations of a surfactant/co-surfactant/water system is investigated by using all atom molecular dynamics simulations. At higher water concentrations, where surfactant (behenyl trimethyl ammonium chloride, BTMAC) to co-surfactant (stearyl alcohol, SA) ratio is fixed, BTMAC and SA self-assemble into spherical micelles, which transform into strongly interdigitated one dimensional rippled lamellar phases upon decreasing water concentrations. Fragmentation or fusions of spherical micelles of different sizes are evident from the radial distribution functions at different temperatures. However, at lower water concentrations rippled lamellar phase transforms into an LβI phase upon heating. Our simulations reveal that the concentrations of water can influence available space around the head groups which couple with critical thickness to accommodate the packing fraction required for respective phases. This directs towards obtaining a controlling factor to design desired phases important for industrial and medical applications in the future.

Phase transition process in DDAB supported lipid bilayer

We report the results of microscope measurements examining the phase transition process of a cationic lipid, Dimethyldioctadecylammonium bromide (DDAB) supported lipid bilayer (SLB). Due to lateral fluidity and strong electrostatic interaction with DNA, SLB serves as a fluid substrate for assembling 2D lattices of DNA functionalized nanoparticles (DNA-NPs): lipid molecules work as carriers for transporting DNA-NPs. By fluorescence microscopy and atomic force microscopy (AFM), two types of phase transitions, which correspond to liquid crystalline-gel and liquid crystalline-interdigitated gel (LβI) ones, were observed in DDAB SLB during cooling. In thermal equilibrium at room temperature both gel and LβI phases have enough adsorbed amounts of DNA-NPs which indicate that both domains have enough surface charge densities for adsorbing DNA-NPs, however, during phase transition DNA-NPs preferably distributed into LβI phase.

High Resolution Structure of the Ripple Phase of DMPC Bilayers

We have obtained the most detailed ripple phase structure of dimyristoylphosphatidylcholine (DMPC) bilayers by taking synchrotron low and wide angle X-ray scattering (LAXS and WAXS) from highly aligned multilamellar samples. Our LAXS data have 52 measured reflections from which a two-dimensional electron density map was obtained at higher resolution than earlier maps obtained from less than half as many reflections. Consistent with the previous lower resolution study, the bilayer has a ripple amplitude of 18.5 A with a thicker and longer major arm and a thinner and shorter minor arm, but the features are sharper allowing better estimates for the modulated bilayer profile and the distribution of headgroups along the aqueous interface. WAXS scattering, employing both grazing incidence and transmission geometry, revealed two sharp Bragg rod reflections with small out-of plane qz components, narrow in-plane qr width consistent with high lateral order, and a breadth in qz consistent with tight coupling of the chains from opposite monolayers. Analysis shows that the major arm hydrocarbon chains are tilted in the ripple plane by 18° with respect to the local bilayer normal, contrary to a previous experimental interpretation. Each chain is tilted toward a next nearest neighbor similarly to the gel LβF phase rather than the more usual LβI phase. There was no clear signature in the WAXS data relating to the minor arm, consistent with those chains being disordered. However, based on the headgroup electron density, we propose that the minor arm is not just like the fluid Lα phase, as often supposed in the literature, because the maximum chain disorder is offset laterally between the upper and lower monolayers. This asynchronous monolayer modulated melting suggests a direction for better theories of the ripple phase.

Membrane order parameters for interdigitated lipid bilayers measured via polarized total-internal-reflection fluorescence microscopy

Incorporating ethanol in lipid membranes leads to changes in bilayer structure, including the formation of an interdigitated phase. We have used polarized total-internal-reflection fluorescence microscopy (pTIRFM) to measure the order parameter for Texas Red DHPE incorporated in the ethanol-induced interdigitated phase (LβI) formed from ternary lipid mixtures comprising dioleoylphosphatidylcholine, cholesterol and egg sphingomyelin or dipalmitoylphosphatidylcholine. These lipid mixtures have 3 co-existing phases in the presence of ethanol: liquid-ordered, liquid-disordered and LβI. pTIRFM using Texas Red DHPE shows a reversal in fluorescence contrast between the LβI phase and the surrounding disordered phase with changes in the polarization angle. The contrast reversal is due to changes in the orientation of the dye, and provides a rapid method to identify the LβI phase. The measured order parameters for the LβI phase are consistent with a highly ordered membrane environment, similar to a gel phase. An acyl-chain labeled BODIPY-FL-PC was also tested for pTIRFM studies of ethanol-treated bilayers; however, this probe is less useful since the order parameters of the interdigitated phase are consistent with orientations that are close to random, either due to local membrane disorder or to a mixture of extended and looping conformations in which the fluorophore is localized in the polar headgroup region of the bilayer. In summary, we demonstrate that order parameter measurements via pTIRFM using Texas Red-DHPE can rapidly identify the interdigitated phase in supported bilayers. We anticipate that this technique will aid further research in the effects of alcohols and other additives on membranes.

Effects of cis- and trans-unsaturated lipids on an interdigitated membrane

The effects of adding cis- and trans-unsaturated lipid to a fully interdigitated membrane were examined using differential scanning calorimetry (DSC) and X-ray diffraction. A monofluorinated analog of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was used as the interdigitated lipid. The single fluorine atom on the end of the sn-2 chain allows 1-palmitoyl-2-(16-fluoropalmitoyl)sn-glycero-3-phosphocholine (F-DPPC) to spontaneously form the interdigitated gel phase (LβI) below the main transition temperature (Tm). The cis 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and equivalent trans lipid 1,2-dielaidoyl-sn-glycero-3-phosphocholine (DEPC) are strongly disfavored to form the LβI phase. Wide-angle X-ray scattering (WAXS) data demonstrate that the unsaturated lipids progressively disrupt the intermolecular packing at higher concentrations. Small-angle X-ray scattering (SAXS) data show that as the ratio of unsaturated lipid increases, the amount of interdigitated lipid decreases. The cis isomer is more disruptive and inhibits interdigitation more effectively than the trans isomer.

Influence of Cis and Trans Unsaturated Lipids on an Interdigitated Membrane

We have examined the effects of adding cis- and trans-unsaturated lipid to a fully interdigitated membrane using differential scanning calorimetry (DSC) and x-ray diffraction. For the interdigitated lipid, we used a monofluorinated analog of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The single fluorine atom on the end of the sn-2 chain allows 1-palmitoyl-2-(16-fluoropalmitoyl)sn-glycero-3-phosphocholine (F-DPPC) to spontaneously form the interdigitated gel phase (LβI) below the main transition temperature (Tm). Both the cis 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and the equivalent trans lipid 1,2-dielaidoyl-sn-glycero-3-phosphocholine (DEPC) are strongly disfavored to form the LβI phase. There is a large degree of phase segregation between interdigitated and non-interdigitated lipid in DOPC/F-DPPC and DEPC/F-DPPC. The DSC thermograms reveal low miscibility and that both unsaturated lipids broaden and lower the main transition corresponding to the F-DPPC-rich component. Our WAXS data demonstrate that the unsaturated lipids progressively disrupt the intermolecular packing at higher concentrations. Furthermore, the SAXS data show that as the ratio of unsaturated lipid increases, the amount of interdigitated lipid decreases. The interdigitated gel phase formed by F-DPPC is resilient in the sense that the interdigitated phase disappears only at very high fractions of the unsaturated lipid. However, at the same concentration of unsaturated lipid, a greater percentage of the membrane remains interdigitated with DEPC than with DOPC. Therefore, the cis isomer is more disruptive and inhibits interdigitation more effectively than the trans isomer. This behavior supports the general conclusion that lipids with trans fatty acids have properties that are intermediate between saturated and cis-unsaturated lipid.

Influence of the interdigitated gel phase in mixtures of ether-linked and monofluorinated ester-linked phospholipids

To evaluate the influence of the hydrocarbon chain linkage on the thermodynamic phase behavior of spontaneously interdigitating lipids, mixtures of ether-linked 1,2-di-O-hexadecyl-phosphocholine (DHPC) and ester-linked 1-palmitoyl-2-[16-fluoropalmitoyl]sn-glycero-3-phosphocholine (F-DPPC) were studied. A combination of differential scanning calorimetry (DSC), fluorescence spectroscopy, and transmittance spectrophotometry was used. Small amounts of F-DPPC increase the pretransition temperature (Tp) between the interdigitated gel phase (LβI) and the ripple gel phase (Pβ′). There are some signs of immiscibility where the non-interdigitated lipid is present in the phase diagram. However, at around 20mol.% F-DPPC, the pretransition merges with the main transition and is no longer detectable as a separate entity. Additionally, the Tm hysteresis increases steadily with higher mole fractions of F-DPPC. These results support that incorporating F-DPPC progressively stabilizes the LβI phase of DHPC until the membrane is fully interdigitated below the main transition temperature (Tm). The mixtures of F-DPPC and DHPC are miscible once the membrane is entirely interdigitated in the gel phase. Therefore, the ability of both lipids to interdigitate is an important factor controlling gel phase miscibility. Our results also demonstrate that the gel phase behavior of DHPC is highly sensitive to changes in its environment.

Effects of cholesterol on phospholipid membranes: Inhibition of the interdigitated gel phase of F-DPPC and F-DPPC/DPPC

Unlike the parent phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), the monofluorinated analog, 1-palmitoyl-2-(16-fluoropalmitoyl)sn-glycero-3-phosphocholine (F-DPPC), spontaneously forms an interdigitated gel phase (LβI) below the main transition temperature (Tm). We have examined the effects of introducing cholesterol to F-DPPC and 1:1 F-DPPC/DPPC membranes using a combination of DSC, optical density, fluorescence intensity and polarization, 31P NMR, and X-ray diffraction techniques. Cholesterol increases the fluidity of the gel phase, broadens the main transition, and decreases the main transition enthalpy. However, these results also reveal that there is an unusually large degree of phase coexistence between the LβI and non-interdigitated gel phases when cholesterol is added. Cholesterol encourages this phase segregation by partitioning into the thicker non-interdigitated domains. At higher cholesterol concentrations, the majority or all of the LβI phase of F-DPPC and 1:1 F-DPPC/DPPC is eliminated and is replaced by a non-interdigitated liquid-ordered (lo) phase with properties similar to DPPC/cholesterol. Consequently, cholesterol mitigates the influence the CF moiety has on the thermodynamic phase behavior of F-DPPC. Our findings demonstrate that there are multiple characteristics of cholesterol-rich membranes that disfavor interdigitation.

Effect of Polymer Grafting on the Bilayer Gel to Liquid-Crystalline Transition

Grafted polymers on the surface of lipid membranes have potential applications in liposome-based drug delivery and supported membrane systems. The effect of polymer grafting on the phase behavior of bilayers made up of single-tail lipids is investigated using dissipative particle dynamics. The bilayer is maintained in a tensionless state using a barostat. Simulations are carried out by varying the grafting fraction, G(f), defined as the ratio of the number of polymer molecules to the number of lipid molecules, and the length of the lipid tails. At low G(f), the bilayer shows a sharp transition from the gel (L(beta)) to the liquid-crystalline (L(alpha)) phase. This main melting transition temperature is lowered as G(f) is increased, and above a critical value of G(f), the interdigitated L(betaI) phase is observed prior to the main transition. The temperature range over which the intermediate phases are observed is a function of the lipid tail length and G(f). At higher grafting fractions, the presence of the L(betaI) phase is attributed to the increase in the area per head group due to the lateral pressure exerted by the polymer brush. The areal expansion and decrease in the melting temperatures as a function of G(f) were found to follow the scalings predicted by the self-consistent mean field theories for grafted polymer membranes. Our study shows that the grafted polymer density can be used to effectively control the temperature range and occurrence of a given bilayer phase.

Probing the Interdigitated Phase of a DPPC Lipid Bilayer by Micropipette Aspiration

AbstractWe used micropipette aspiration of giant unilamellar vesicles to directly measure the areal expansion of gel (Lβ′) phase 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine (DPPC) lipid bilayers induced by exposure to ethanol/water mixtures. Areal expansion began in 7 vol% ethanol and increased monotonically as the concentration of ethanol was increased to 15 vol% at which point areal expansion reached a plateau of 50%. This ethanol concentration range is in good agreement with that of the interdigitated phase (LβI) of DPPC, therefore, we believe that this is the first direct measurement of the areal expansion accompanying interdigitation of gel‐phase lipids. Our observations are consistent with the presence of coexisting LβI and Lβ′ phases in ethanol concentrations between 7% and 15 vol% and 100% LβI phase in 15 vol% ethanol and higher. We observed a bimodal distribution of areal expansion (0% and 20%) induced by 7 vol% ethanol indicating that at the threshold concentration, interdigitation is induced in only a portion of DPPC vesicles. Areal expansion could not be easily reversed, consistent with kinetic trapping of the LβI phase. DPPC vesicles exposed to butanol at the known threshold and plateau concentrations for the LβI phase displayed areal expansion behavior consistent with our ethanol observations. However, the area expanded significantly faster for DPPC bilayers exposed to butanol vs. ethanol, which we attribute to enhanced partitioning of the longer‐chained butanol into the lipid headgroups. Ethanol‐induced areal expansion of DPPC bilayers was inhibited by inclusion of 10 mol% and 25 mol% cholesterol in the bilayer. However, areal expansion could be induced by application of tensions (∼8 mN/m) similar to the phenomena of interdigitation induced by high pressure. The presence of 20 vol% ethanol significantly decreased surface cohesion of DPPC bilayers containing 25 mol% cholesterol as evidenced by a decreased area compressibility modulus and lysis tension.

A swollen gel phase of DPPC aqueous solution with small amount of ethanol observed at moderate pressure and temperature

In order to clarify effects of pressure on phospholipids, small-angle neutron scattering and X-ray scattering experiments on an aqueous solution of dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) with small amount of ethanol were carried out. At moderate temperature and pressure (41 ≤ T ≤ 60°C and 10 ≤ P ≤ 80 MPa), among a fluid lamellar (Lα) phase, a ripple gel (Pα′) phase and an interdigitated gel (LβI) phase, we have found a new phase with a single peak at around Q = 0.06 Å−1. This phase was characterized to be a swollen gel phase from the data analysis. The kinetics of the phase transition to the LβI phase was investigated, and the relaxation was much slower than the transition to the other bilayer structures.

Self-consistent field theoryof compressible phospholipidmembranes at ambient pressure

We present a microscopic theory of compressible, fully hydratedphospholipid membranes in equilibrium with excess solvent, apply it toan idealized physical model of these systems, and calculate a broadrange of their thermodynamic and structural properties. An essentialfeature of the theory is the anisotropy of the effective fields actingon each of the hydrocarbon segments, which arises as a naturalconsequence of the hard-core repulsions between segments and theconnectivity of the lipid molecules. These fields, along with theinhomogeneous particle and bond density distributions throughout thebilayer interior, are determined via numerical, self-consistent fieldcalculations. In addition to the gel and liquid crystal phases, themodel naturally includes the fully intercalated LβI phase. Theincorporation of compressibility effects enables us to calculatevarious effects of pressure and the density change at the transitionand in the liquid crystal phase. In this paper, we focus on theproperties of fully hydrated (DPPE) in the liquid crystal phase,as well as properties of the main transition, at atmosphericpressure. To a lesser extent, we also discuss predicted dependencesof several key properties on chain length and head group size. Despiteits simplifications, the model provides a quantitative description ofmany of the measured properties of DPPE, and the correct qualitativetrends for the other lipids. In an accompanying paper [the following article], we examine the effects ofpressure, and obtain quantitative agreement with recent observations onoverall density and bilayer thickness.PACS Nos.: 87.22.Bt, 87.10.+e, 64.60.Cn