Differential scanning calorimetric study of the effect of sterol side chain length and structure on dipalmitoylphosphatidylcholine thermotropic phase behavior.

We have investigated the thermotropic phase behavior of dipalmitoylphosphatidylcholine (DPPC) bilayers containing a series of cholesterol analogues varying in the length and structure of their alkyl side chains. We find that upon the incorporation of up to approximately 25 mol % of any of the side chain analogues, the DPPC main transition endotherm consists of superimposed sharp and broad components representing the hydrocarbon chain melting of sterol-poor and sterol-rich phospholipid domains, respectively. Moreover, the behavior of these components is dependent on sterol side chain length. Specifically, for all sterol/DPPC mixtures, the sharp component enthalpy decreases linearly to zero by 25 mol % sterol while the cooperativity is only moderately reduced from that observed in the pure phospholipid. In addition, the sharp component transition temperature decreases for all sterol/DPPC mixtures; however, the magnitude of the decrease is dependent on the sterol side chain length. With respect to the broad component, the enthalpy initially increases to a maximum around 25 mol % sterol, thereafter decreasing toward zero by 50 mol % sterol with the exception of the sterols with very short alkyl side chains. Both the transition temperature and cooperativity of the broad component clearly exhibit alkyl chain length-dependent effects, with both the transition temperature and cooperativity decreasing more dramatically for sterols with progressively shorter side chains. We ascribe the chain length-dependent effects on transition temperature and cooperativity to the hydrophobic mismatch between the sterol and the host DPPC bilayer (see McMullen, T. P. W., Lewis, R. N. A. H., and McElhaney, R. N. (1993) Biochemistry 32:516-522).(ABSTRACT TRUNCATED AT 250 WORDS)     

Micelle-vesicle transition in phospholipid-bile salt mixtures. A study by precision scanning calorimetry

A systematic study of the micelle-vesicle transformation in phospholipid-bile salt mixtures using differential scanning calorimetry (DSC) indicates that the lipid undergoes a variety of changes in its thermal properties as mixed micellar solutions are diluted to concentrations at which vesicles form. In the experiments, micellar solutions of 50 mg/mL total lipid, containing sodium taurocholate (TC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), are diluted to concentrations corresponding to differing extents of aggregation of the TC with phospholipid. Turbidity and equilibrium dialysis measurements are used to establish boundaries between where micelles persist and where vesicles are formed and to determine the extent of aggregation of the TC with DPPC. At molar ratios Re of bound TC to DPPC greater than 0.3, micellar solutions are formed, while at Re less than 0.15 vesicles are evident upon dilution. As the transformation from micelles to vesicles occurs, the thermal transitions in the lipid change from broad, low Cp (max) peaks in the micelle region to multiple peaks of high cooperativity in regions of composition where lamellar structures and vesicles form. The DSC curves show that in the composition region corresponding to where bilayer micelles exist a new thermal phase forms, which has a melting transition near 32 degrees C, if the solutions are allowed to equilibrate for 48 h at 21 degrees C. Furthermore, at compositions between Re = 0 and 0.25, there is metastability in the lipid when equilibrated at 21 degrees C, but heating the lipid through the thermal transitions leads to reversible behavior.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of triglycerides with phospholipids: incorporation into the bilayer structure and formation of emulsions

Interactions of carbonyl 13C-enriched triacylglycerols (TG) with phospholipid bilayers [egg phosphatidylcholine (PC), dipalmitoylphosphatidylcholine (DPPC), and an ether-linked phosphatidylcholine] were studied by 13C NMR spectroscopy. Up to 3 mol % triolein (TO) or tripalmitin (TP) was incorporated into DPPC vesicles by cosonication of the TG and DPPC at approximately 50 degrees C. NMR studies were carried out in a temperature range (30-50 degrees C) in which pure TO is a liquid whereas pure TP is a solid. In spectra of DPPC vesicles with TG at 40-50 degrees C, both TO and TP had narrow carbonyl resonances, indicative of rapid motions, and chemical shifts indicative of H bonding of the TG carbonyls with solvent (H2O) at the aqueous interfaces of the vesicle bilayer. Below the phase transition temperature of the DPPC/TG vesicles (approximately 36 degrees C), most phospholipid peaks broadened markedly. In DPPC vesicles with TP, the TP carbonyl peaks broadened beyond detection below the transition, whereas in vesicles with TO, the TO carbonyl peaks showed little change in line width or chemical shift and no change in the integrated intensity. Thus, in the gel phase, TP solidified with DPPC, whereas TO was fluid and remained oriented at the aqueous interfaces. Egg PC vesicles incorporated up to 2 mol % TP at 35 degrees C; the TP carbonyl peaks had line-width and chemical shift values similar to those for TP (or TO) in liquid-crystalline DPPC. TO incorporated into ether-linked PC had properties very similar to TO in ester-linked PC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative calorimetric and spectroscopic studies of the effects of lanosterol and cholesterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes

We carried out comparative DSC and Fourier transform infrared spectroscopic studies of the effects of cholesterol and lanosterol on the thermotropic phase behavior and organization of DPPC bilayers. Lanosterol is the biosynthetic precursor of cholesterol and differs in having three rather than two axial methyl groups projecting from the β-face of the planar steroid ring system and one axial methyl group projecting from the α-face, whereas cholesterol has none. Our DSC studies indicate that the incorporation of lanosterol is more effective than cholesterol is in reducing the enthalpy of the pretransition. Lanosterol is also initially more effective than cholesterol in reducing the enthalpies of both the sharp and broad components of the main phase transition. However, at sterol concentrations of 50 mol %, lanosterol does not abolish the cooperative hydrocarbon chain-melting phase transition as does cholesterol. Moreover, at higher lanosterol concentrations (~30–50 mol %), both sharp and broad low-temperature endotherms appear in the DSC heating scans, suggestive of the formation of lanosterol crystallites, and of the lateral phase separation of lanosterol-enriched phospholipid domains, respectively, at low temperatures, whereas such behavior is not observed with cholesterol at comparable concentrations. Our Fourier transform infrared spectroscopic studies demonstrate that lanosterol incorporation produces a less tightly packed bilayer than does cholesterol, which is characterized by increased hydration in the glycerol backbone region of the DPPC bilayer. These and other results indicate that lanosterol is less miscible in DPPC bilayers than is cholesterol, but perturbs their organization to a greater extent, probably due primarily to the rougher faces and larger cross-sectional area of the lanosterol molecule and perhaps secondarily to its decreased ability to form hydrogen bonds with adjacent DPPC molecules. Nevertheless, lanosterol does appear to produce a lamellar liquid-ordered phase in DPPC bilayers, although this phase is not as tightly packed as comparable cholesterol/DPPC mixtures.     

High-sensitivity scanning calorimetric study of mixtures of cholesterol with dimyristoyl- and dipalmitoylphosphatidylcholines

A highly sensitive and stable scanning microcalorimeter is employed in a reinvestigation of the effect of cholesterol on multilamellar suspensions of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC). Below 20 mol % cholesterol the DPPC mixtures give heat-capacity curves each of which can be resolved into a narrow and a broad peak, suggesting the coexistence of two immiscible solid phases; above 20 mol % only the broad peak is observed and this disappears at about 50 mol %. The DMPC mixtures show a more complicated behavior; from about 13.5 to 20 mol % cholesterol the observed curves appear to be the sum of three component peaks. As with the DPPC mixtures, only a single broad peak is observed above 20 mol % cholesterol, and this broad peak becomes undetectable above about 50 mol %. These results are discussed.     

A calorimetric and spectroscopic comparison of the effects of lathosterol and cholesterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes

We performed differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopic measurements to study the effects of lathosterol (Lath) on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine (DPPC) bilayer membranes and compared our results with those previously reported for cholesterol (Chol)/DPPC binary mixtures. Lath is the penultimate intermediate in the biosynthesis of Chol in the Kandutsch-Russell pathway and differs from Chol only in the double bond position in ring B, which is between C7 and C8 in Lath and between C5 and C6 in Chol. Our DSC studies indicate that the incorporation of Lath is more effective than Chol in reducing the temperature and enthalpy of the DPPC pretransition. At lower sterol concentrations (≤10 mol %), incorporation of both Lath and Chol decreases the temperature, enthalpy, and cooperativity of the sharp component of the main phase transition of DPPC to a similar extent, but at higher sterol concentrations, Lath is more effective at decreasing the phase transition temperature, enthalpy, and cooperativity than Chol. These results indicate that at higher concentrations, Lath is more disruptive of DPPC gel-state bilayer packing than Chol is. Moreover, incorporation of Lath decreases the temperature of the broad component of the main phase transition of DPPC, whereas Chol increases it; this difference in the direction and magnitude of the temperature shift is accentuated at higher sterol concentrations. Although at sterol concentrations of ≤20 mol % Lath and Chol are almost equally effective at reducing the enthalpy and cooperativity of the broad component of the main phase transition, at higher sterol levels Lath is less effective than Chol in these regards and does not completely abolish the cooperative hydrocarbon chain melting phase transition at 50 mol %, as does Chol. These latter results indicate that Lath both is more disruptive with respect to the low-temperature state of the sterol-enriched domains of DPPC bilayers and has a lower lateral miscibility in DPPC bilayers than Chol. Our FTIR spectroscopic studies suggest that Lath incorporation produces a less tightly packed bilayer than does Chol at both low (gel state) and high (liquid-crystalline state) temperatures, which is characterized by increased H-bonding between water and the carbonyl groups of the fatty acyl chains in the DPPC bilayer. Overall, our studies indicate that Lath and Chol incorporation can have rather different effects on the thermotropic phase behavior and organization of DPPC bilayers and thus that the position of the double bond in ring B of a sterol molecule can have an appreciable effect on the physical properties of sterol molecules.     

Application of pressure-modulated differential scanning calorimetry to the determination of relaxation kinetics of multilamellar lipid vesicles

We report an extension of the recently published PMDSC method that permitted synchronous determination of heat capacity and expansibility when using slow, defined pressure formats in a DSC scan. Here we applied continuously opposing pressure changes that are fast compared to the time constants of the DSC instrument to study relaxation kinetics of phospholipids. Investigations of multilamellar vesicles of DPPC or DSPC in water revealed for both lipids relaxation times of about 30 s at the maximum of the main transition peak and about 15 s at the maximum of the pretransition. The relaxation times in the transition range are proportional to heat capacity of main- and pretransition. The molecular origin of the relaxation processes appears to stem from pressure-induced water fluxes between the interbilayer region and the bulk water phase.     

Molecular characteristics of astaxanthin and beta-carotene in the phospholipid monolayer and their distributions in the phospholipid bilayer.

The molecular characteristics of the monolayers of astaxanthin with polar group on the beta-ionone ring in the molecule and beta-carotene without polar group and their interactions in mixed carotenoid-phospholipid monolayers and the effects of carotenoids on the phase behavior of the phospholipid bilayers were examined by the monolayer technique and differential scanning calorimetry (DSC). We found from the monolayer study that beta-carotene had an amphiphilic nature. The molecular assembly of astaxanthin in the monolayer at the hydrophobic/hydrophilic interface was more stable than that of beta-carotene. Dimyristoylphosphatidylcholine (DMPC) in the monolayer was miscible with astaxanthin in the range of 0-0.4 mol fractions of astaxanthin, but not fully miscible with beta-carotene even at low concentrations below 0.1 mol fraction of beta-carotene. Surface potential and compression/expansion cycles of beta-carotene monolayer indicated the formation of molecular aggregates by itself. DSC study showed that when small amount of astaxanthin was added, the transition temperature of dipalmitoylphosphatidylcholine (DPPC) was markedly shifted to lower temperatures and that the transition peak was asymmetrically broadened, indicative of a significant depression in cooperativity of the gel to liquid-crystalline transition. The asymmetric DSC endothermic bands of DPPC incorporating small amounts of astaxanthin were well fit by deconvolution into two to three domains containing different concentrations of astaxanthin. On the contrary, the incorporation of beta-carotene resulted in a small depression of the main transition temperature with a slight broadening of the transition peak, suggesting a small miscibility of beta-carotene with the phospholipid bilayer or a formation of aggregates of beta-carotene in the membranes. These results suggest that there would be a high localized concentration in the phase separated membrane for astaxanthin or beta-carotene to function effectively as scavenger.     

Properties of ganglioside GM1 in phosphatidylcholine bilayer membranes.

Gangliosides have been shown to function as cell surface receptors, as well as participating in cell growth, differentiation, and transformation. In spite of their multiple biological functions, relatively little is known about their structure and physical properties in membrane systems. The thermotropic and structural properties of ganglioside GM1 alone and in a binary system with 1,2-dipalmitoyl phosphatidylcholine (DPPC) have been investigated by differential scanning calorimetry (DSC) and x-ray diffraction. By DSC hydrated GM1 undergoes a broad endothermic transition TM = 26 degrees C (delta H = 1.7 kcal/mol GM1). X-ray diffraction below (-2 degrees C) and above (51 degrees C) this transition indicates a micellar structure with changes occurring only in the wide angle region of the diffraction pattern (relatively sharp reflection at 1/4.12 A-1 at -2 degrees C; more diffuse reflection at 1/4.41 A-1 at 51 degrees C). In hydrated binary mixtures with DPPC, incorporation of GM1 (0-30 mol%; zone 1) decreases the enthalpy of the DPPC pretransition at low molar compositions while increasing the TM of both the pre- and main transitions (limiting values, 39 and 44 degrees C, respectively). X-ray diffraction studies indicate the presence of a single bilayer gel phase in zone 1 that can undergo chain melting to an L alpha bilayer phase. A detailed hydration study of GM1 (5.7 mol %)/DPPC indicated a conversion of the DPPC bilayer gel phase to an infinite swelling system in zone 1 due to the presence of the negatively charged sialic acid moiety of GM1. At 30-61 mol % GM1 (zone 2), two calorimetric transitions are observed at 44 and 47 degrees C, suggesting the presence of two phases. The lower transition reflects the bilayer gel --> L alpha transition (zone 1), whereas the upper transition appears to be a consequence of the formation of a nonbilayer, micellar or hexagonal phase, although the structure of this phase has not been defined by x-ray diffraction. At > 61 mol % GM1 (zone 3) the calorimetric and phase behavior is dominated by the micelle-forming properties of GM1; the presence of mixed GM1/DPPC micellar phases is predicted.     

Monounsaturated PE does not phase-separate from the lipid raft molecules sphingomyelin and cholesterol: role for polyunsaturation?

We investigated interactions of the lipid raft molecules sphingomyelin (SM) and cholesterol (CHOL) in monolayers and bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylethanolamine (POPE) or 1-palmitoyl-2-docosahexaenoyl-sn-glycerophosphatidylethanolamine (PDPE) at 35 degrees C. Techniques employed were pressure-area (pi-A) isotherms generated from Langmuir-Blodgett films, solid-state (2)H and (31)P NMR spectroscopies, and differential scanning calorimetry (DSC). Condensation calculated from pi-A isotherms and reduction in the enthalpy of the gel-liquid-crystalline transition in DSC scans showed CHOL has a strong affinity for POPE, comparable to that observed between SM-CHOL. Order parameters derived from (2)H NMR spectra of the perdeuterated sn-1 chain of POPE-d(31) increased by >50% upon addition of equimolar CHOL to POPE-d(31)/SM (1:1 mol) bilayers. Close proximity of CHOL to POPE even in the presence of SM is indicated. Chemical shift anisotropy (Deltasigma(csa)) measured from (1)H-decoupled (31)P NMR spectra also implied intimate lipid mixing in POPE/SM/CHOL (1:1:1 mol). In contrast, pi-A isotherms and corroborating DSC studies of PDPE/SM (1:1 mol) indicate phase separation between SM and PDPE, which was maintained in the presence of CHOL. The cholesterol-associated increase in order of the perdeuterated sn-1 chain of PDPE determined by (2)H NMR was 2-fold less for PDPE-d(31)/SM/CHOL (1:1:1 mol) than POPE-d(31)/SM/CHOL (1:1:1 mol). Our findings support the notion that acyl chain dependent lateral phase separation occurs in the presence of a docosahexaenoic acid (DHA)-containing phospholipid (PDPE), but not an oleic acid-containing phospholipid (POPE). We propose that monounsaturated lipids do not promote formation of stable lipid rafts and that polyunsaturation may be important for raft stability.     

Dependence on phospholipid composition of the fraction of cholesterol undergoing spontaneous exchange between small unilamellar vesicles

Spontaneous cholesterol exchange between small unilamellar vesicles comprised of different phospholipids and their binary mixtures has been studied in order to understand the factors involved in the establishment and maintenance of intracellular cholesterol distributions. Exchange was performed from neutral donor vesicles containing different cholesterol concentrations, traces of [3H]cholesterol, and [14C]cholesteryl oleate as a nonexchangeable marker. The acceptor vesicles, in 10-fold excess, had the same composition, but 15 mol % phosphatidylglycerol was included to permit chromatographic separation. Data were best fitted by a single exponential and a base value. In donor vesicles containing only one phospholipid, the kinetic rate constants agreed with data reported previously; however, the base values were larger than the expected equilibrium value of 9.09%. The size of this nonexchangeable pool and the exchange rate were found to depend on the type of phospholipid. In binary phospholipid donor systems, well above the transition temperatures of the lipid components, the exchange parameters were preferentially closer to those of one component according to the order POPC greater than DMPC greater than DPPC greater than bovine brain SPM.     

Change in volume magnetic susceptibility at the phase transition of dipalmitoylphosphatidylcholine.

We have observed a change at 41 degrees C in the relative volume magnetic susceptibility of an aqueous dispersion containing 13 wt% multilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles. The magnitude of the change is consistent with the known density change of the phospholipid bilayer and the assumption that the mass susceptibility of the system is constant through the transition. The superconducting susceptometer used in this study of the sharp transition of DPPC will be able to detect 1% changes in bilayer density for 10 wt% dispersions even when the transition temperature and transition width of phospholipid vesicle under various experimental conditions.     

Dynamic fluorescence measurements on the main phase transition of dipalmytoylphosphatidylcholine vesicles

The kinetics of the main phase transition in dipalmytoylphosphatidylcholine (DPPC) vesicles have been investigated using our iodine laser-Tjump technique with fluorescence detection. A set of three fluorescent probes has been used to sense different parts of the bilayer hydrocarbon chain region. The well established membrane probes DPH and TMADPH as well as DPHPC, a labelled DPPC molecule. We report three relaxation signals in the s and ms time range, which are detected with all three probes. This result supports our model of the main phase transition in DPPC vesicles.Abbreviations DMPC Dimyristoylphosphatidylcholine - DPPC Dipalmytoylphosphatidylcholine - DPH 1,6-Diphenylhexa-1,3,5-triene - TMADPH 1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene - DPHPC Diphenylhexatriene-phosphatidylcholine - T_m Temperature of the main phase transition     

Effects of cannabinoids in membrane bilayers containing cholesterol.

The thermotropic and dynamic properties of the biologically active Delta(8)-tetrahydrocannabinol (Delta(8)-THC) and its inactive congener O-methyl-Delta(8)-tetrahydrocannabinol (Me-Delta(8)-THC) in DPPC/cholesterol (CHOL) bilayers have been studied using a combination of DSC and solid-state NMR spectroscopy. The obtained results showed differential effects of the two cannabinoids under study. These are summarized as follows: (a) the presence of the active compound fluidizes more significantly the DPPC/CHOL bilayers than the inactive analog as it is revealed by DSC and NMR spectroscopy results; (b) cholesterol seems to play a significant role in the way cannabinoids act in membrane bilayers; (c) the observed additional peaks in (13)C/MAS-NMR spectra which were cannabinoid specific offer an evidence of their different dynamic properties in membranes. In particular, the aromatic part of the inactive cannabinoid appears more mobile than that of the active one. This finding is in agreement with previously obtained X-ray data which locate the inactive cannabinoid in the hydrophobic core of the bilayer while the active one in the polar region; and (d) the observed downfield shift of C-1 carbon in the preparation containing the active cannabinoid is a strong evidence that Delta(8)-THC resides nearby the polar region where also cholesterol is well known to locate itself. Such downfield shift is absent when Me-Delta(8)-THC is resided in the membrane bilayer. These differential effects of the two cannabinoids propose that the phospholipid/cholesterol core of the membrane may play an important role in the mode of cannabinoid action by regulating their thermotropic and dynamic properties.     

Thermotropic behavior of dipalmitoylphosphatidylcholine vesicles reconstituted with the glycoprotein of vesicular stomatitis virus

The vesicular stomatitis virus glycoprotein reconstituted into dipalmitoylphosphatidylcholine (DPPC) vesicles exerts a profound effect upon the DPPC gel to liquid-crystalline phase transition. The glycoprotein was reconstituted into DPPC vesicles by octyl glucoside dialysis. The gel to liquid-crystalline phase transition of these vesicles was monitored by differential scanning calorimetry. Vesicles formed in the absence of glycoprotein (600--2100-A diameter) underwent the phase transition at 41.0 degrees C and had an associated enthalpy change of 8.0 +/- 1.6 kcal/mol. Increasing the mole ratio of glycoprotein to DPPC in the vesicles to 0.15 mol % reduced both the transition temperature and the transition enthalpy change. The enthalpy change as a function of the mole percent glycoprotein could be fit to a straight line by a least-squares procedure. Extrapolation of the results to the glycoprotein concentration where the enthalpy change was zero indicated one glycoprotein molecule bound 270 +/- 150 molecules of DPPC.     

Observation of the main phase transition of dinervonoylphosphocholine giant liposomes by fluorescence microscopy

The phase heterogeneity of giant unilamellar dinervonoylphosphocholine (DNPC) vesicles in the course of the main phase transition was investigated by confocal fluorescence microscopy observing the fluorescence from the membrane incorporated lipid analog, 1-palmitoyl-2-(N-4-nitrobenz-2-oxa-1,3-diazol)aminocaproyl-sn-glycero-3-phosphocholine (NBDPC). These data were supplemented by differential scanning calorimetry (DSC) of DNPC large unilamellar vesicles (LUV, diameter approximately 0.1 and 0.2 microm) and multilamellar vesicles (MLV). The present data collected upon cooling reveal a lack of micron-scale gel and fluid phase coexistence in DNPC GUVs above the temperature of 20.5 degrees C, this temperature corresponding closely to the heat capacity maxima (T(em)) of DNPC MLVs and LUVs (T(em) approximately 21 degrees C), measured upon DSC cooling scans. This is in keeping with the model for phospholipid main transition inferred from our previous fluorescence spectroscopy data for DMPC, DPPC, and DNPC LUVs. More specifically, the current experiments provide further support for the phospholipid main transition involving a first-order process, with the characteristic two-phase coexistence converting into an intermediate phase in the proximity of T(em). This at least macroscopically homogenous intermediate phase would then transform into the liquid crystalline state by a second-order process, with further increase in acyl chain trans-->gauche isomerization.     

Effect of acyl chain mismatch on the contact mechanics of two-component phospholipid vesicle during main phase transition

It has been recently demonstrated that acyl chain mismatch of phospholipid bilayer composed of a binary lipid mixture induces component formation on the lateral plane of the bilayer [Biophys. J. 83 (2002) 1820-1883]. In this report, the contact mechanics of unilamellar vesicles composed of binary dimyristoyl-phosphatidylcholine (DMPC)/dipalmitoyl-phosphocholine (DPPC) mixtures on fused silica and amino-modified substrates is simultaneously probed by confocal-reflectance interference contrast microscopy (C-RICM) and cross-polarized light microscopy during gel to liquid crystalline transition of the lipid bilayer. C-RICM results indicate that the average degree of vesicle deformation for DMPC-rich and DPPC-rich vesicles adhering on fused silica substrate is increased by 30% and 14%, respectively, in comparison with that in pure DMPC and DPPC vesicles. Also, lateral heterogeneity induced by acyl chain mismatch increases the average magnitude of adhesion energy in DMPC-rich and DPPC-rich vesicles of all sizes by 6.4 times and 2.3 times, respectively. Similar modulation of adhesion mechanics induced by carbon chain difference is obtained on amino-modified substrate. Most importantly, the thermotropic transition of the mixed bilayer from gel (below T(m)) to fluid phase (above T(m)) further exemplifies the effect of acyl chain mismatch on the increases of degree of vesicle deformation and adhesion energy.     

Effects of divalent cations on the ultrasonic absorption coefficient of negatively charged liposomes (LUV) near their phase transition temperature

The ultrasonic absorption coefficient per wavelength (alpha lambda), as a function of temperature and frequency, was determined for large unilamellar vesicles (LUV) in the vicinity of their phospholipid phase transition temperature, using a double crystal acoustic interferometer. (The vesicles were composed of a 4:1 (w/w) mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). It has been found that alpha lambda reaches a maximum (alpha lambda)max at the phase transition temperature (tm) of the phospholipids in the bilayer, at an ultrasonic relaxation frequency of 2.1 MHz. Divalent cations (Ca2+ and Mg2+), added to LUV suspensions, shifted (alpha lambda)max to higher temperatures, dependent upon the concentration of divalent cation. It was also found that the shape of the alpha lambda versus t curve was significantly changed, representing changes in the Van't Hoff enthalpy of the transition, and therefore, the cooperative unit of the transition. This suggests that divalent cations interact individually with the negatively charged phospholipid headgroups of DPPG and with DPPC headgroups, thus decreasing the cooperative unit of the transition. The observed upward shift in tm suggests an interaction that increases the activation energy and, therefore, the temperature of the phase transition. However, alpha lambda as a function of frequency did not change with the addition of divalent cations and, thus, the relaxation time of the event responsible for the absorption of ultrasound is not changed by the addition of divalent cations.     

Thermolabile Liposomes with a High Fusion Efficacy at 42°C can be Made of Dipalmitoylphosphatidylcholine/Fatty Alcohol Mixtures in the Molar Ratio of 1/2

Abstract

Liposomes made of dipalmitoylphosphatidylcholine (DPPC²), dipalmitoyl-phosphatidylglycerol (DPPG), and different long-chain fatty alcohols were investigated with respect to their colloidal stability, chain-melting phase transition temperature, and temperature dependent inter-vesicle fusion. In particular, the practical usefulness of the stoichiometric 1/2 (mol/mol) mixtures of the phospholipids and fatty alcohols, mainly elaidoyl alcohol (EL-OH) were studied. The mole fraction of DPPG in the bilayers of such vesicles affects crucially the colloidal stability of the resulting lipid suspensions; at least 15 mol-% of DPPG (relative to DPPC) must be incorporated into the bilayers in order to make the liposome suspension colloidally sufficiently stable at room temperature. The corresponding DPPC/DPPG/EL-OH (0.85/0.15/2) mixed lipid vesicles undergo a lamellar-gel to inverted hexagonal (H_IT) phase transition at 52.7°C, however, and then fuse and aggregate massively. The related phase transition temperature of the DPPC/DPPG/palmitelaidoyl alcohol (0.85/0.15/2) mixture is 48.4°C. This indicates that the chain-melting phase transition temperature of the investigated lipid mixtures is rather sensitive to the alcohol chain-length. This transition temperature is independent, however, of the bulk proton concentration in the pH region between 4.9 and 7.2. Stoichiometric 1/2 mixtures of phospholipids and EL-OH have a high propensity for the inter-vesicle fusion at 42°C and neutral pH. The reason for such fusion 10°C below the lamellar-to-nonlamellar phase transition temperature are the defects that are generated during the chain-melting of the (partly segregated) phospholipid component at 42°C; the proximity of the lamellar to non-lamellar phase transition temperature of the phospholipid/fatty alcohol (1/2) complex at 52°C also plays an important role.     

Stages of the bilayer-micelle transition in the system phosphatidylcholine-C12E8 as studied by deuterium- and phosphorous-NMR, light scattering, and calorimetry.

The perturbation of phospholipid bilayer membranes by a nonionic detergent, octaethyleneglycol mono-n-dodecylether (C12E8), was investigated by 2H- and 31P-NMR, static and dynamic light scattering, and differential scanning calorimetry. Preequilibrated mixtures of the saturated phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), and 1,2-dilauroyl-sn-glycero-3-phosphorylcholine (DLPC) with the detergent were studied over a broad temperature range including the temperature of the main thermotropic phase transition of the pure phospholipids. Above this temperature, at a phospholipid/detergent molar ratio 2:1, the membranes were oriented in the magnetic field. Cooling of the mixtures below the thermotropic phase transition temperatures of the pure phospholipids led to micelle formation. In mixtures of DPPC and DMPC with C12E8, a narrow calorimetric signal at the onset temperature of the solubilization suggested that micelle formation was related to the disorder-order transition in the phospholipid acyl chains. The particle size changed from 150 nm to approximately 7 nm over the temperature range of the bilayer-micelle transition. The spontaneous orientation of the membranes at high temperatures enabled the direct determination of segmental order parameters from the deuterium spectra. The order parameter profiles of the phospholipid acyl chains could be attributed to slow fluctuations of the whole membrane and to detergent-induced local perturbations of the bilayer order. The packing constraints in the mixed bilayers that eventually lead to bilayer solubilization were reflected by the order parameters of the interfacial phospholipid acyl chain segments and of the phospholipid headgroup. These results are interpreted in terms of the changing average shape of the component molecules. Considering the decreasing cross sectional areas in the acyl chain region and the increasing hydration of the detergent headgroups, the bilayer-micelle transition is the result of an imbalance in the chain and headgroup repulsion. A neutral or pivotal plane can be defined on the basis of the temperature dependence of the interfacial quadrupolar splittings.