Thermodynamical characteristics and volume compressibility of dipalmitoylphosphatidylcholine liposomes containing bacteriorhodopsin.

The effects of bacteriorhodopsin (BR) interaction with large dipalmitoylphosphatidylcholine (DPPC) liposomes (approx. 100 nm in diameter) were examined at various BR/DPPC ratios, using differential scanning calorimetry (DSC) and ultrasonic velocimetry (USV). On DSC, the lipid phase transition temperature, Tc, and the half-width of the phase transition peak, delta T1/2, showed significant non-monotonic changes with the increasing BR concentration. Two exponential segments could be distinguished in the dependence of the transition enthalpy change per mol of lipid (delta H/nL) on the BR/DPPC ratio: one corresponding to ratios between 0:1 and 1:64, and another corresponding to ratios between 1:44 and 1:16. A maximal value of delta H/nL was observed for BR/DPPC ratio 1:44, probably corresponding to maximal BR-lipid ordering with each BR molecule being surrounded by two layers of lipid molecules. The nonmonotonic changes of thermodynamical parameters suggest long-distance interactions between regions of altered bilayer structure which form around each BR molecule. The results obtained with USV provided support for the above conclusions. The dependence of ultrasound velocity increment A on BR concentration supplies information on relative changes of membrane volume compressibility. Decreasing volume compressibility is reflected in increasing values of parameter A. Within T less than Tc, the values of A increased with the increasing BR concentration; saturation was observed at BR/DPPC ratio 1:500 (A = A(BR/DPPC]. No significant BR-concentration dependent changes of A were observed at T greater than Tc. From these values the average diameter of the distorted region of lipid bilayer was estimated to be approximately 20 nm.     

Analysis of multi-component fluorescence emission by phase-sensitive detection using one modulation frequency

We describe the use of phase-sensitive detection of fluorescence to resolve the lifetimes and fractional intensities from multi-component fluorescence samples, using data obtained at a single modulation frequency. Phase-sensitive spectra of the mixture are recorded at arbitrarily chosen detector phase angles. The steady-state spectrum of each component must be known. The phase-sensitive spectra are fitted, using a nonlinear least-squares algorithm, to obtain the lifetimes and fractional intensities of each fluorophore in the mixture. Simulations for two- and three-component mixtures are presented to illustrate how the resolution is affected by spectral overlap and lifetime separation. Experimentally, we resolved two- and three-component mixtures of protein-like fluorophores (N-acetyl-L-tyrosinamide, N-acetyl- L-tryptophanamide, indole and 2,3-dimethylindole) using data collected at 30 MHz. These fluorophores have closely spaced lifetimes of 1.5, 2.9, 4.5 and 4.3 ns, respectively, and display extensive spectral overlap. These results demonstrate that phase-sensitive spectra, recorded at only one modulation frequency with a standard phase fluorometer, can be used to resolve multi-component emissions.     

The influence of local anesthetics on the gel-liquid crystal phase transition in model dipalmitoylphosphatidylcholine membranes

The influence of local anesthetics (LA): tetracaine, lidocaine, cocaine, dibucaine and heptacaine derivatives on the gel to liquid crystalline phase transition temperature (Tc) of model dipalmitoylphosphatidylcholine (DPPC) membranes was studied using electron spin resonance (ESR) and polarization microscopy methods. The decrease of Tc in the presence of anesthetics (delta Tc) was found to be dependent on the [DPPC]/[H2O] molar ratio at constant [LA]/[DPPC] molar ratio. Hence, the parameter alpha = delta Tc/[( LA]/[DPPC]) in dependence on [H2O]/[DPPC] was extrapolated to zero concentration of water and compared with biological efficiency.     

Decreased lipid order induced by microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in model membranes: fluorescence and electron spin resonance studies

Cytochrome P-450 and NADPH-cytochrome P-450 reductase were reconstituted in unilamellar lipid vesicles prepared by the cholate dialysis technique from pure dimyristoylphosphatidylcholine (DMPC), pure dipalmitoylphosphatidylcholine (DPPC), pure dioleoylphosphatidylcholine (DOPC), and phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine (PC/PE/PS) (10:5:1). As probes for the vesicles' hydrocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) and spin-labeled PC were used. The steady-state and time-resolved fluorescence parameters of DPH were determined as a function of temperature and composition of liposomes. Incorporation of either protein alone or together increased the steady-state fluorescence anisotropy (rs) of DPH in DOPC and PC/PE/PS (10:5:1) liposomes. In DMPC and DPPC vesicles, the proteins decreased rs significantly below the transition temperature (Tc) of the gel to liquid-crystalline phase transition. Time-resolved fluorescence measurements of DPH performed in reconstituted PC/PE/PS and DMPC proteoliposomes showed that the proteins disorder the bilayer both in the gel and in the liquid-crystalline phase. Little disordering by the proteins was observed by a spin-label located near the mid-zone of the bilayer 1-palmitoyl-2-(5-doxylstearoyl)-3-sn-phosphatidylcholine (8-doxyl-PC), whereas pronounced disordering was detected by 1-palmitoyl-2-(8-doxylpalmitoyl)-3-sn-phosphatidylcholine (5-doxyl-PC), which probes the lipid zone closer to the polar part of the membrane. Fluorescence lifetime measurements of DPH indicate an average distance of greater than or equal to 60 A between the heme of cytochrome P-450 and DPH.     

Thermodynamic properties and characterization of proteoliposomes rich in microdomains carrying alkaline phosphatase

Tissue-nonspecific alkaline phosphatase (TNAP) is associated to the plasma membrane via a GPI-anchor and plays a key role in the biomineralization process. In plasma membranes, most GPI-anchored proteins are associated with "lipid rafts", ordered microdomains enriched in sphingolipids, glycosphingolipids and cholesterol. In order to better understand the role of lipids present in rafts and their interactions with GPI-anchored proteins, the insertion of TNAP into different lipid raft models was studied using dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), sphingomyelin (SM) and ganglioside (GM1). Thus, the membrane models studied were binary systems (9:1 molar ratio) containing DPPC:Chol, DPPC:SM and DPPC:GM1, ternary systems (8:1:1 molar ratio) containing DPPC:Chol:SM, DPPC:Chol:GM1 and DPPC:SM:GM1 and finally, a quaternary system (7:1:1:1 molar ratio) containing DPPC:Chol:SM:GM1. Calorimetry analysis of the liposomes and proteoliposomes indicate that lateral phase segregation could be noted only in the presence of cholesterol, with the formation of cholesterol-rich microdomains centered above Tc=41.5°C. The presence of GM1 and SM into DPPC-liposomes influenced mainly ΔH and Δt(1/2) values. The gradual increase in the complexity of the systems decreased the activity of the enzyme incorporated. The presence of the enzyme also fluidifies the systems, as seen by the intense reduction in ?H values, but do not alter Tc values significantly. Therefore, the study of different microdomains and its biophysical characterization may contribute to the knowledge of the interactions between the lipids present in MVs and its interactions with TNAP.     

Diffusion and Partitioning of Fluorescent Lipid Probes in Phospholipid Monolayers

The pressure-dependent diffusion and partitioning of single lipid fluorophores in DMPC and DPPC monolayers were investigated with the use of a custom-made monolayer trough mounted on a combined fluorescence correlation spectroscopy (FCS) and wide-field microscopy setup. It is shown that lipid diffusion, which is essential for the function of biological membranes, is heavily influenced by the lateral pressure and phase of the lipid structure. Both of these may change dynamically during, e.g., protein adsorption and desorption processes. Using FCS, we measured lipid diffusion coefficients over a wide range of lateral pressures in DMPC monolayers and fitted them to a free-area model as well as the direct experimental observable mean molecular area. FCS measurements on DPPC monolayers were also performed below the onset of the phase transition (Π < 5 mN/m). At higher pressures, FCS was not applicable for measuring diffusion coefficients in DPPC monolayers. Single-molecule fluorescence microscopy and differential scanning calorimetry clearly showed that this was due to heterogeneous partitioning of the lipid fluorophores in condensed phases. The results were compared with dye partitioning in giant lipid vesicles. These findings are significant in relation to the application of lipid fluorophores to study diffusion in both model systems and biological systems.     

Influence of phospholipid composition on the adjuvanticity and protective efficacy of liposome-encapsulated Leishmania donovani antigens

In this study, we evaluate the effect of phospholipid on the adjuvanicity and protective efficacy of liposome vaccine carriers against visceral leishmaniasis (VL) in a hamster model. Liposomes prepared with distearyol derivative of L-alpha-phosphatidyl choline (DSPC) having liquid crystalline transition temperature (Tc) 54 C were as efficient as dipalmitoyl (DPPC) (Tc 41 C) and dimyristoyl (DMPC) (Tc 23 C) derivatives in their ability to entrap Leishmania donovani membrane antigens (LAg) and to potentiate strong antigen-specific antibody responses. However, whereas LAg in DPPC and DMPC liposomes stimulated inconsistent delayed type hypersensitivity (DTH) responses, strong DTH was observed with LAg in DSPC liposomes. The heightened adjuvant activity of DSPC liposomes corresponded with 95% protection, with almost no protectivity with LAg in DPPC and DMPC liposomes, 4 mo after challenge with L. donovani. These data demonstrate the superiority of DSPC liposomes for formulation of L. donovani vaccine. In addition, they demonstrate a correlation of humoral and cell-mediated immunity with protection against VL in hamsters.     

Location and dynamics of anthracyclines bound to unilamellar phosphatidylcholine vesicles

We have exploited the intrinsic fluorescence properties of the anthracycline antitumor antibiotics to study the dependence on drug structure of relative drug location and dynamics when the anthracyclines were bound to sonicated dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) vesicles at 27.5 degrees C. Iodide quenching experiments at constant ionic strength were used to evaluate the relative accessibilities of the bound fluorophores to membrane-impermeable iodide. Iodide was found to quench the fluorescence of anthracyclines in free solution by both static and dynamic mechanisms, whereas quenching of membrane-bound fluorophores was predominantly due to the dynamic mechanism. Modified Stern-Volmer plots of anthracyclines bound to fluid-phase DMPC bilayers were linear, and the biomolecular rate constant (kq) values ranged from 0.6 X 10(9) to 1.3 X 10(9) M-1 s-1. Modified Stern-Volmer plots of anthracyclines bound to solid-phase DPPC bilayers were curved, indicative of a heterogeneous-bound drug population. A strong correlation between drug hydrophobicity and penetration of the fluorophore into the bilayer was observed for the daunosamine-containing anthracyclines. Steady-state fluorescence anisotropy measurements under iodide quenching conditions were used to investigate the diffusive motions of anthracyclines in isotropic solvent and in fluid-phase DMPC bilayers. Anthracycline derivatives free in solution exhibited limiting anisotropy (alpha infinity) values which decayed to zero at times long compared to the excited-state lifetime, in contrast to anthracyclines bound to fluid-phase DMPC bilayers, which showed nonzero alpha infinity values. Steady-state anisotropies of membrane-bound anthracyclines were found to be governed principally by alpha infinity and not by the mean rotational rate (R).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of pyrethroids with phosphatidylcholine bilayers: comparisons in liposomal systems exhibiting large or small radii of curvature

Pyrethroid interactions with dipalmitoyl phosphatidylcholine (DPPC) vesicles have been characterized in bilayers having large and small radii of curvature. The abilities of pyrethroids to alter the gel-fluid phase transition profiles were determined by steady state fluorescence anisotropy and phase-modulation lifetime techniques using the fluorescent probes cis- and trans-parinaric acid. Using the geometric isomers of parinaric acid as membrane probes, pyrethroids were found to lower the phase transition temperature (Tc) of DPPC large multilamellar vesicles with the same order of comparative effectiveness as previously reported using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Permethrin had a greater depressive effect upon the Tc of DPPC in the small unilamellar vesicle (SUV) system than in the large multilamellar system. Conversely, allethrin was less effective in reducing the Tc of DPPC SUVs. The enhanced effect of permethrin in decreasing the Tc of DPPC SUVs was greatest in regions of more rigid lipid packing, as determined by trans-parinaric acid fluorescence parameters. The results indicate that changes in lipid packing configuration caused by differing bilayer radii of curvature may alter the interactive characteristics of pyrethroids with lipid membranes.     

Surfactant protein SP-B induces ordering at the surface of model membrane bilayers

The effects of bovine pulmonary surfactant-associated protein B (SP-B) on molecular packing of model membrane lipids (7:1 DPPC/DPPG) were studied by fluorescence anisotropy. The bilayer surface was markedly ordered by SP-B below the gel to fluid phase transition temperature (Tc) while it was only slightly ordered above this temperature as indicated by surface-sensitive probes 6-NBD-PC and 6-NBD-PG. The effects of SP-B on fluorescence anisotropy were concentration dependent, reaching maximal activity at 1-2% protein to phospholipid by weight. Anisotropy measurements of interior-selective fluorescent probes (cis-parinaric acid and DPH) imply that addition of SP-B into the phospholipid shifted the Tc of the model membrane but did not alter lipid order at the membrane interior. Since fluorescence anisotropy studies with trans-parinaric acid, an interior-sensitive probe with high affinity for gel-phase lipids, did not detect any changes in lipid packing or Tc, it is likely that SP-B resides primarily in fluid-phase domains. Fluorescence lifetime measurements indicated that two conformers of the NBD-PC probe exist in this DPPC/DPPG model membrane system. Fluorescence intensity measurements generated with NBD-PC and NBD-PG, in conjunction with information from lifetime measurements, support the concept that SP-B increases the distribution of the short-lifetime conformer in the gel phase. In addition, the anisotropy and intensity profiles of NBD-PG in the model membrane indicate that bovine SP-B interacts selectively with phosphatidylglycerol.     

Control of a redox reaction on lipid bilayer surfaces by membrane dipole potential

Nitro-2,1,3-benzoxadiazol-4-yl (NBD) group is a widely used, environment-sensitive fluorescent probe. The negatively charged dithionite rapidly reduces the accessible NBD-labeled lipids in liposomes to their corresponding nonfluorescent derivatives. In this study both the phospholipid headgroup and acyl chain NBD-labeled L-alpha-1,2-dipalmitoyl-sn-glycero-3-phospho-[N-(4-nitrobenz-2-oxa-1,3-diazole)-ethanolamine] (DPPN) and 1-acyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (NBD-PC), respectively, were employed. The correlation of both the rate coefficient k(1) of the redox reaction and the fluorescence properties of the two probes with the membrane dipole potential Psi in fluid dipalmitoylglycerophosphocholine (DPPC) liposomes is demonstrated. When Psi of the bilayer was varied (decreased by phloretin or increased by 6-ketocholestanol), the value for k1 decreased for both DPPN and NBD-PC with increasing Psi. For both fluorophores a positive correlation to Psi was evident for the relative fluorescence emission intensity (RFI, normalized to the emission of the fluorophore in a DPPC matrix). The relative changes in emission intensity as a function of Psi were approximately equal for both NBD derivatives. Changes similar to those caused by phloretin were seen when dihexadecylglycerophosphocholine (DHPC) was added to DPPC liposomes, in keeping with the lower dipole potential for the former lipid compound compared with DPPC. These effects of Psi on NBD fluorescence should be taken into account when interpreting data acquired using NBD-labeled lipids as fluorescent probes.     

Interactions of pyrethroids with phosphatidylcholine liposomal membranes

Interactions of several pyrethroids with membrane lipids in the form of dipalmitoylphosphatidylcholine (DPPC) liposomes have been studied using fluorescent membrane probes. Fluorescence anisotropy values and lifetimes (determined by phase-shift and demodulation techniques) of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene, were decreased in gel phase liposomes by pyrethroids at concentrations on the order of 10 microM. The pyrethroids containing a cyano substituent were also observed to cause collisional quenching of diphenylhexatriene fluorescence. Pyrethroids differed in their effectiveness at lowering the phase transition temperature of DPPC, and in their ability to broaden the temperature range of this transition. The fluorescence intensity of DPPC-incorporated chlorophyll a was used to monitor the pretransition of DPPC and the lateral diffusion of a membrane component located in the polar headgroup region. Permethrin did not affect chlorophyll a fluorescence intensity at any temperature. It may be concluded from these results that pyrethroids are preferentially located in the interior hydrophobic regions of the lipid bilayer, and that these compounds can disorder hydrocarbon packing in the bilayer core. However, polar headgroups were not disordered, and diffusion of membrane components in the polar headgroup region was not altered.     

Sterol orientations in phosphatidylcholine liposomes as determined by deuterium NMR

Deuterium magnetic resonance spectra (55.26 MHz) of cholesterol-3 alpha-d1 and epicholesterol-3 beta-d1 in dipalmitoylglycerophosphocholine (DPPC) liposomes were measured as a function of sterol-to-phospholipid ratio below (24 degrees C) and above (60 degrees C) the phase transition temperature of DPPC. From the quadrupolar splittings delta vq, the molecular order parameters S describing the motions of the sterols in the bilayer were calculated, and the most probable angle of tilt alpha 0 of the molecular axis of the sterols relative to the bilayer normal was determined. We observed that the molecular axis of cholesterol in DPPC liposomes at both 24 and 60 degrees C is tilted at an angle of 16-19 degrees with the 3 beta-hydroxyl group projecting parallel to the bilayer normal into the aqueous interface. In contrast, at 24 degrees C, epicholesterol is aligned parallel (0 degrees) to the bilayer normal, placing the 3 alpha-hydroxyl group essentially perpendicular to the bilayer normal along the aqueous interface. At 60 degrees C, the average angle of epicholesterol (16-18 degrees) is similar to that of cholesterol, which can project the 3 alpha-hydroxyl group into the hydrophobic bilayer region. On the basis of the observed tilt angles of the two isomeric sterols in DPPC liposomes, a model is proposed that can rationalize the differential effects of cholesterol and epicholesterol on membrane properties.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid domain structure correlated with membrane protein function in placental microvillus vesicles

Membrane fluidity properties of placental microvillus membrane vesicles (MVV) were determined from fluorescence anisotropy (r), dynamic depolarization, and lifetime heterogeneity studies of diphenylhexatriene (DPH), trimethylamino-DPH (TMA-DPH), and cis- and trans-parinaric acids (c-PnA and t-PnA). Plots of r against temperature for DPH and TMA-DPH in MVV had slope discontinuities at 26 degrees C (Tc, transition temperature); however, analysis of r in terms of probe rotational rate (R), limiting anisotropy (r infinity), and lifetime (tau) revealed that DPH reported a phase transition because of changes in r infinity, whereas the phase transition observed by TMA-DPH occurred primarily because of changes in R. Heterogeneity analysis using phase and modulation lifetimes at three frequencies showed that DPH and TMA-DPH lifetimes were homogeneous in MVV. Both long (greater than 25 ns) and short (less than 6 ns) lifetime components were detected for c-PnA and t-PnA in MVV, corresponding to the probes in solid and fluid lipid phases. The fractional amplitude of the long lifetimes (solid phase) decreased from 0.86 to 0.12 with increasing temperature (5-55 degrees C) as the membrane passed through the phase transition, with 50% of the change occurring at 27 degrees C (c-PnA) or 33 degrees C (t-PnA). The activation energies for alkaline phosphatase, aminopeptidase M, and sodium-proton antiporter activities all showed discontinuities in the temperature range 27-31 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential polarized phase fluorometric investigations of diphenylhexatriene in lipid bilayers. Quantitation of hindered depolarizing rotations.

Differential polarized phase fluorometry has been used to investigate the depolarizing rotations of 1,6-diphenyl-1,3,5-hexatriene (DPH) in isotropic solvents and in lipid bilayers. For DPH dissolved in isotropic solvents, there is a precise agreement between the observed and predicted values for maximum differential tangents, indicating that in these media DPH is a free isotropic rotator. In lipid bilayers the tangent defects (i.e., the differences between the calculated and the observed maximum differential tangents) are too large to be explained by anisotropy in the depolarizing rotations but are accounted for by hindered isotropic torsional motions for the fluorophore [Weber, G (1978) Acta Phys. Pol A 54, 173]. This theory describes the depolarizing rotations of the fluorophore by its rotational rate R (in radians/second) and the limiting fluorescence anisotropy (r) at times long compared with the fluorescence lifetime. Through the combined use of both steady-state anisotropy measurements and differential phase measurements, we have demonstrated that one may obtain unique solutions for both R and r. For DPH embedded in vesicles prepared from dimyristoyl-, dipalmitoyl-, and distearoylphosphatidylcholines, the depolarizing motions are highly hindered at temperatures below the transition temperature (Tc) but are unhindered above Tc. The apparent rotational rates of the probe do not change significantly at Tc. These data suggest that the changes observed in the steady-state anisotropy near Tc derive primarily from changes in the degree to which the probe's rotations are hindered, and only to a small extent from changes in rotational rate. For DPH embedded in bilayers that contained 25 mol % cholesterol, no clear transition occurred and the rotations appeared to be hindered at all temperatures. The rotational motions of DPH embedded in dioleolyphosphatidylcholine were found to be far less hindered, but the rotational rates were similar to those obtained in the saturated phosphatidylcholines. Finally, the data show that in an anisotropic environment, such as that of a lipid bilayer, steady-state fluorescence anisotropy measurements alone cannot yield quantitatively meaningful rotational rates. Extrapolation of steady-state aniosotropy data to the quantitation of membrane viscosity is therefore difficult, if not invalid; however, qualitative comparisons can be useful.     

Analysis of excited-state processes by phase-modulation fluorescence spectroscopy

Fluorescence phase shift and demodulation methods were used in the analysis of excited-state reactions and to investigate solvent relaxation around fluorophores in viscous solvents. The chosen samples illustrate the results expected for fluorophores bound to biological macromolecules. These moderately simple samples served to test the theoretical predictions described in the preceding paper (J.R. Lakowicz and A.B. Balter, Biophys. Chem. 16 (1982) 99.) and to illustrate the characteristic features of phase-modulation data expected from samples which display time-dependent spectral shifts. The excited-stale protonation of acridine and exciplex formation between anthracene and diethylaniline provided examples of one-step reactions in which the lifetimes of the initially excited and the reacted species were independent of emission wavelength. Using these samples we demonstrated the following: (I) Wavelength-dependent phase shift and demodulation values can be used to prove the occurrence of an excited-state process. Proof is obtained by observation of phase angles (φ) larger than 90° or by measurement of ratios of m/cos φ > 1, where m is the modulation of the emission relative to that of the excitation. (2) For a two-state process the individual emission spectra of each state can be calculated from the wavelength-dependent phase and modulation data. (3) The phase difference or demodulation factor between the initially excited and the reacted states reveals directly the fluorescence lifetime of the product of the reaction. (4) Phase-sensitive detection of fluorescence can be used to prove that the lifetimes of both the initially excited and the reacted states are independent of emission wavelength. (5) If steady-state spectra of the individual species are known, then phase-sensitive emission spectra can be used to measure the lifetimes of the individual components irrespective of the extent of spectral overtap. (6) Spectral regions of constant lifetime can be identified by the ratios of phase-sensitive emission spectra. In addition, we examined 6-propionyl-2-dimethylaminonaphthalene(PRODAN) and N-acetyl-l-tryptophanamide (NATA) in viscous solvents where the solvent relaxation times were comparable to the fluorescence lifetimes. Using PRODAN in n-butanol we used m/cos φ measurements, relative to the blue edge of the emission, to demonstrate that solvent relaxation requires more than a single step. For NATA in propylene glycol we used phase-sensitive detection of fluorescence to directly record the emission spectra of the initially excited and the solvent relaxed states. These measurements can be easily extended to fluorophores which are bound to proteins and membranes and are likely to be useful in studies of the dynamic properties of biopolymers.     

Solid-state NMR study of trehalose/1,2-dipalmitoyl-sn-phosphatidylcholine interactions

31P and 2H solid-state NMR studies of dry trehalose (TRE) and 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) mixtures are reported. 31P spectra are consistent with a rigid head group above and below the calorimetric phase transition for both dry DPPC and a dry 2:1 TRE/DPPC mixture. In addition, 2H spectra of DPPC labeled at the 7-position of the sn-2 chain (2[7,7-2H2]DPPC) show exchange-narrowed line shapes with a width of 120 kHz over the temperature range 25-75 degrees C. These line shapes can be simulated with a model involving two-site jumps of the deuteron. In contrast, the 2H NMR spectrum of a dry 2:1 TRE/2[7,7-2H2]DPPC mixture above the phase transition (Tc = 46 degrees C) is narrowed by a factor of approximately 4 to a width of 29 kHz. Simulation of this spectrum requires a model involving four-site jumps of the deuteron and is indicative of highly disordered lipid acyl chains similar to those found in the L alpha-phases of hydrated lipids. Thus, TRE/DPPC mixtures above their transition temperatures exist in a new type of liquid crystalline like phase, which we term a lambda-phase. The observation of the dynamic properties of this new phase indicates the mechanism by which anhydrobiotic organisms maintain the integrity of their membranes upon dehydration.     

Phospholipid-cationic lipid interactions: influences on membrane and vesicle properties

Liposomes composed of synthetic dialkyl cationic lipids and zwitterionic phospholipids such as dioleoylphosphatidylethanolamine have been studied extensively as vehicles for gene delivery, but the broader potentials of these cationic liposomes for drug delivery have not. An understanding of phospholipid-cationic lipid interactions is essential for rational development of this potential. We evaluated the effect of the cationic lipid DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium) on liposome physical properties such as size and membrane domain structure. DSC (differential scanning calorimetry) showed progressive decrease and broadening of the phase transition temperature of dipalmitoylphosphatidylcholine (DPPC) with increasing fraction of DOTAP, in the range of 0.4-20 mol%. Laurdan (6-dodecanolyldimethylamino-naphthalene), a fluorescent probe of membrane domain structure, showed that DOTAP and DPPC remained miscible at all ratios tested. DOTAP reduced the size of spontaneously-forming PC-containing liposomes, regardless of the acyl chain length and degree of saturation. The anionic lipid DOPG (dioleoylphosphatidylglycerol) had similar effects on DPPC membrane fluidity and size. However, DOTAP/DOPC (50/50) vesicles were taken up avidly by OVCAR-3 human ovarian tumor cells, in contrast to DOPG/DOPC (50/50) liposomes. Overall, DOTAP exerts potent effects on bilayer physical properties, and may provide advantages for drug delivery.     

New approaches to the simulation of heat-capacity curves and phase diagrams of pseudobinary phospholipid mixtures.

A simulation program using least-squares minimization was developed to calculate and fit heat capacity (cp) curves to experimental thermograms of dilute aqueous dispersions of phospholipid mixtures determined by high-sensitivity differential scanning calorimetry. We analyzed cp curves and phase diagrams of the pseudobinary aqueous lipid systems 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol/ 1,2-dipalmitoyl-sn-glycero-3phosphatidylcholine (DMPG/DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid/1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DMPA/DPPC) at pH 7. The simulation of the cp curves is based on regular solution theory using two nonideality parameters rho g and rho l for symmetric nonideal mixing in the gel and the liquid-crystalline phases. The broadening of the cp curves owing to limited cooperativity is incorporated into the simulation by convolution of the cp curves calculated for infinite cooperativity with a broadening function derived from a simple two-state transition model with the cooperative unit size n = delta HVH/delta Hcal as an adjustable parameter. The nonideality parameters and the cooperative unit size turn out to be functions of composition. In a second step, phase diagrams were calculated and fitted to the experimental data by use of regular solution theory with four different model assumptions. The best fits were obtained with a four-parameter model based on nonsymmetric, nonideal mixing in both phases. The simulations of the phase diagrams show that the absolute values of the nonideality parameters can be changed in a certain range without large effects on the shape of the phase diagram as long as the difference of the nonideality parameters for rho g for the gel and rho l for the liquid-crystalline phase remains constant. The miscibility in DMPG/DPPC and DMPA/DPPC mixtures differs remarkably because, for DMPG/DPPC, delta rho = rho l -rho g is negative, whereas for DMPA/DPPC this difference is positive. For DMPA/DPPC, this difference is interpreted as being caused by a negative rho g value, indicating complex formation of unlike molecules in the gel phase.