Phase Transition Of Model Membranes Research Articles

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer.

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits versus other methods: DIBs are stable and often long-lasting, bilayer area can be reversibly tuned, leaflet asymmetry is readily controlled via droplet compositions, and tissue-like networks of bilayers can be obtained by adjoining many droplets. Forming DIBs requires spontaneous assembly of lipids into high density lipid monolayers at the surfaces of the droplets. While this occurs readily at room temperature for common synthetic lipids, a sufficient monolayer or stable bilayer fails to form at similar conditions for lipids with melting points above room temperature, including some cellular lipid extracts. This behavior has likely limited the compositions-and perhaps the biological relevance-of DIBs in model membrane studies. To address this problem, an experimental protocol is presented to carefully heat the oil reservoir hosting DIB droplets and characterize the effects of temperature on the lipid membrane. Specifically, this protocol shows how to use a thermally conductive aluminum fixture and resistive heating elements controlled by a feedback loop to prescribe elevated temperatures, which improves monolayer assembly and bilayer formation for a wider set of lipid types. Structural characteristics of the membrane, as well as the thermotropic phase transitions of the lipids comprising the bilayer, are quantified by measuring the changes in electrical capacitance of the DIB. Together, this procedure can aid in evaluating biophysical phenomena in model membranes over various temperatures, including determining an effective melting temperature (TM) for multi-component lipid mixtures. This capability will thus allow for closer replication of natural phase transitions in model membranes and encourage the formation and use of model membranes from a wider swath of membrane constituents, including those that better capture the heterogeneity of their cellular counterparts.

Capacitive Detection of Low-Enthalpy, Higher-Order Phase Transitions in Synthetic and Natural Composition Lipid Membranes.

In-plane lipid organization and phase separation in natural membranes play key roles in regulating many cellular processes. Highly cooperative, first-order phase transitions in model membranes consisting of few lipid components are well understood and readily detectable via calorimetry, densitometry, and fluorescence. However, far less is known about natural membranes containing numerous lipid species and high concentrations of cholesterol, for which thermotropic transitions are undetectable by the above-mentioned techniques. We demonstrate that membrane capacitance is highly sensitive to low-enthalpy thermotropic transitions taking place in complex lipid membranes. Specifically, we measured the electrical capacitance as a function of temperature for droplet interface bilayer model membranes of increasing compositional complexity, namely, (a) a single lipid species, (b) domain-forming ternary mixtures, and (c) natural brain total lipid extract (bTLE). We observed that, for single-species lipid bilayers and some ternary compositions, capacitance exhibited an abrupt, temperature-dependent change that coincided with the transition detected by other techniques. In addition, capacitance measurements revealed transitions in mixed-lipid membranes that were not detected by the other techniques. Most notably, capacitance measurements of bTLE bilayers indicated a transition at ∼38 °C not seen with any other method. Likewise, capacitance measurements detected transitions in some well-studied ternary mixtures that, while known to yield coexisting lipid phases, are not detected with calorimetry or densitometry. These results indicate that capacitance is exquisitely sensitive to low-enthalpy membrane transitions because of its sensitivity to changes in bilayer thickness that occur when lipids and excess solvent undergo subtle rearrangements near a phase transition. Our findings also suggest that heterogeneity confers stability to natural membranes that function near transition temperatures by preventing unwanted defects and macroscopic demixing associated with high-enthalpy transitions commonly found in simpler mixtures.

Thermotropic phase transitions in model membranes of the outer skin layer based on ceramide 6

The lipid intercellular matrix stratum corneum of the outer skin layer is a multilayer membrane consisting of a complex mixture of different lipids: ceramides, fatty acids, cholesterol, and its derivatives. The basis of the multilayer membrane is the lipid bilayer, i.e., a two-dimensional liquid crystal. Currently, it is known that the main way of substance penetration through the skin is the lipid matrix. The complexity of the actual biological system does not allow reliable direct study of its properties; therefore, system modeling is often used. Phase transitions in the lipid system whose composition simulates the native lipid matrix are studied by the X-ray synchrotron radiation diffraction method.

Fluorescence (Fluidity/Hydration) and Calorimetric Studies of Interactions of Bile Acid–Drug Conjugates with Model Membranes

We have studied the interactions of three bile acid-tamoxifen conjugates, lithocholic acid-tamoxifen (LA-Tam(1)-Am), deoxycholic acid-tamoxifen (DCA-Tam(2)-Am), and cholic acid-tamoxifen (CA-Tam(3)-Am), possessing 1-3 tamoxifen molecules having an amine headgroup with model DPPC membranes and compared with N-desmethylated tamoxifen (TamNHMe) using DPH based fluorescence anisotropy, Prodan based hydration, and differential scanning calorimetry studies. DPH based anisotropy studies showed that bile acid-tamoxifen conjugates increase membrane fluidity, which strongly depends on the number of tamoxifen molecules conjugated to bile acid and the percentage of doping of bile acid-tamoxifen conjugates in the DPPC membranes. The order of membrane fluidity of the coliposomes from bile acid-tamoxifen conjugates and DPPC lipids in gel phase was found to be CA-Tam(3)-Am > DCA-Tam(2)-Am > LA-Tam(1)-Am > TamNHMe. Incorporation of bile acid-tamoxifen conjugates showed an unusual complex behavior of membrane hydration, as evident from Prodan based hydration studies. Temperature dependent study showed incorporation of LA-Tam(1)-Am and DCA-Tam(2)-Am conjugates decreases membrane hydration with an increase in temperature up to the phase transition temperature (T(m)). Differential scanning calorimetry studies showed a decrease in phase transition temperature (T(m)) upon an increase in the percentage of doping of TamNHMe and CA-Tam(3)-Am, whereas LA-Tam(1)-Am and DCA-Tam(2)-Am do not cause a major change in the phase transition temperature (T(m)) of DPPC liposomes. These studies showed the differential behavior of bile acid-tamoxifen conjugates regulating the membrane fluidity, hydration, and phase transition of model membranes depending upon the percentage of doping and tamoxifen conjugation to bile acids.

Assessment of Merocyanine Subpopulations in DPPC Vesicles using Ansitropy and Lifetime Measurements

The purpose of this study was to further investigate the properties of the fluorescent probe merocyanine 540, which has been used frequently for over two decades to assess membrane phase properties under various conditions. Differences in emission spectrum shape at temperatures above and below the thermotropic phase transition of model membranes have been hypothesized to represent changes in the position and orientation of the probe in the bilayer. This hypothesis suggests specific predictions concerning probe mobility in the membrane as a function of temperature and emission wavelength. We tested the hypothesis using measurements of steady state anisotropy and fluorescence lifetimes in dipalmitoylphosphatidylcholine vesicles. Below the lipid phase transition temperature, steady state anisotropy decreased by 0.2 units across the emission spectrum from short to long wavelength. In contrast, anisotropy was more stable as a function of emission wavelength when measured above the transition temperature. Fluorescence lifetimes showed minimal wavelength dependence at either temperature. Anisotropy experiments were repeated at a variety of probe-to-lipid ratios to assess the role of probe aggregation on the observations. The data supported previous findings from measurements of the quantum yield of merocyanine 540: in the gel phase, two separate populations of the probe (monomers and dimers) fluoresce. The monomers, which emit at short wavelengths, are oriented perpendicular to the bilayer surface, and are limited in mobility by neighboring phospholipids. The dimmers, which emit at long wavelengths, are oriented parallel to the bilayer surface, and are localized in a membrane region where motion is less restricted, perhaps in the region between the membrane leaflets. At higher temperatures, only monomers fluoresce but exhibit higher mobility due to the lower order of the membrane phase.

Differences between coumaric and cinnamic acids in membrane permeation as evidenced by time-dependent calorimetry.

We carried out an investigation by differential scanning calorimetry (DSC) on the effect of two structurally similar bioactive plant phenols, cinnamic and p-coumaric acids, on the phase transition of model membranes constituted by dimyristoylphosphatidylcholine (DMPC) vesicles. The aim was to evaluate how pH and molecular substituents influence liposolubility and thereby modify vesicle permeability. A change in permeability would result in modifications to the phase transition for DMPC liposomes such that the transition temperature would be lowered and the enthalpy changes should be little affected. Evidence of differences in permeability was obtained by comparing the effects exerted by the two compounds left in touch with unilamellar and multilamellar vesicles, at two different pHs (4 and 7.4). While the p-coumaric acid was unable to appreciably modify the thermotropic behavior of the model membrane, the cinnamic acid interacted with lipid vesicles at both pH's, even if at acidic pH the effect was greater than neutral. It can be hypothesized that the interaction between the cinnamic acid and the lipidic layers is due to the lack of a hydroxyl group. This interaction is enhanced by an acidic pH, where the carboxylic acid is in a protonated form.

Flavonoid-biomembrane interactions: A calorimetric study on dipalmitoylphosphatidylcholine vesicles

Flavonoids, a group of naturally occurring benzo-γ-derivatives, have been shown to possess several biological properties, many of which may be related, partially at least, to their capacity to penetrate into the cell membrane and so to affect membrane-dependent processes. In the present paper we report the results of an investigation by differential scanning calorimetry (DSC) on the effects of three flavonoids (quercetin, hesperetin and naringenin) upon the gel-to-liquid crystalline phase transition of model membranes constituted by l-α-dipalmitoylphosphatidylcholine (DPPC) vesicles. All tested flavonoids interacted with DPPC liposomes causing different shifts, towards lower values, of the T m typical for DPPC multilayers; the enthalpy changes (AH), related to the calorimetric peak area, remained nearly constant. Furthermore, their effects on DPPC thermotropic behaviour were greater for high molar concentrations (up to a limit value different for each flavonoid) and were affected by the time. Several hypotheses (drug molecule aggregation into the model membrane, changes in the barrier function of the lipid bilayer, different drug structural features and conformation, possible formation of flavonoid-phospholipid complexes) are presented to explain the nature of the interaction between flavonoids and phospholipid vesicles.

Modifications of lipid structure and their influence on mesomorphism in model membranes: the influence of hydrocarbon chains.

The influence of hydrocarbon chains on the temperature (TG-LC) of the gel to liquid-crystalline phase transition of model membranes has been investigated over an extensive variety of phosphatidylcholines (PC). The TG-LC is dependent upon the length of the hydrocarbon chains, on whether or not the chains are saturated or have been modified in some way, and on the position of any modification along the chain. For PC having two different acyl chains (heteroacid PC) in the sn-1 and sn-2 positions, the TG-LC is dependent on the chain position and on the inequivalence of chain penetration into the bilayer. Positional isomers of PC have different TG-LC. The first two double bonds introduced in each chain of a PC cause a much greater reduction in TG-LC and in the enthalpy change of the transition than does the subsequent introduction of additional double bonds. Dipolyunsaturated PC have uncooperative (broad) transitions that occur at low temperatures and have small enthalpy changes. While each PC has unique transitional characteristics, there are a number of patterns in the TG-LC which emerge on consideration of all the available data. One such pattern may be useful in predicting TG-LC from analytical data on the composition and positions of acyl chains of various lipids.

Fluorescent probes for asymmetric lipid bilayers: synthesis and properties in phosphatidyl choline liposomes and erythrocyte membranes.

We have synthesized three sets of fluorescent probes which we believe will be useful in studies of asymmetric membranes and have studied their interactions with model lipid bilayers and erythrocyte membranes. The probes were designed to partition preferentially into one face of a lipid bilayer with asymmetrically disposed phospholipids and to report lipid transitions in that monolayer. We synthesized more than twenty probes containing anthroyl-, dansyl-, or pyrene rings with acidic, basic, and neutral functional groups and alkyl "spacers" of various lengths. The interactions of these probes with liposomes of phosphatidyl choline and with erythrocyte membranes were characterized to determine whether probe insertion was asymmetric, how deeply the probe penetrated the bilayer, and whether the probe reflected thermotropic phase transitions in model membranes. The set of variously charged anthroyl esters, analogs of local anaesthetics, appears to be promising for studies of asymmetric membranes. Fluorescent probes have been used extensively to provide information on the lipid regions of biological membranes. Membrane fluidity, a composite of molecular packing and motion of acyl chains in lipid bilayers, has been assessed with a variety of fluorescent probes, the fluorescence of which undergoes some measurable change at the temperature of the membrane's thermotropic phase transition. A large number of fluorescent probes have been used for this purpose. Bashford, Morgan and Radda (Bashford, C.L., Morgan, C.G., Radda, G.K. 1976; Biochim. Biophys. Acta 426:157) and Thulborn and Sawyer (Thulborn, K.R., Sawyer, W. H. 1978; Biochim. Biophys. Acta 511:125) synthesized several fatty acid derivatives in which an anthracene group is attached (in ester linkage) along the acyl chain at various positions, and have shown that this set of probes may be useful in probing membrane fluidity at different depths within the bilayer. This report describes the synthesis and properties of several sets of amphipathic fluorescent probes, which may partition unequally into the two faces of an asymmetric lipid bilayer, and may therefore provide information about membranes complementary to that obtainable with existing probes.