Phase behavior of model lipid bilayers

We investigated the phase behavior of double-tail lipids, as a function of temperature, headgroup interaction and tail length. At low values of the head-head repulsion parameter a(hh), the bilayer undergoes with increasing temperature the transitions from the subgel phase L(c) via the flat gel phase L(beta) to the fluid phase L(alpha). For higher values of a(hh), the transition from the L(c) to the L(alpha) phase occurs via the tilted gel phase L(beta)(') and the rippled phase P(beta)('). The occurrence of the L(beta)(') phase depends on tail length. We find that the rippled structure (P(beta)(')) occurs if the headgroups are sufficiently surrounded by water and that the ripple is a coexistence between the L(c) or L(beta)(') phase and the L(alpha) phase. The anomalous swelling, observed at the P(beta)(') --> L(alpha) transition, is not directly related to the rippled phase, but a consequence of conformational changes of the tails.     

Structural study of BSA/poly(ethylene glycol) lipid conjugate complexes

In this work we report the structural characteristics of bovine serum albumin/poly(ethylene glycol) lipid conjugate (BSA/PEG(2000)-PE) complexes under physiological conditions (37 degrees C and pH 7.4) for particular fractions of BSA to PEG-lipid concentration, c(BSA)/c(PEG)(2000)-PE. Ultraviolet fluorescence spectroscopy (UV) results shown that PEG(2000)-PE is associated to BSA, leading to protein unfolding for fixed c(BSA) = 0.01 wt % and variable c(PEG)(2000)-PE = 0.0015-0.6 wt %. Tryptophan groups on the BSA surface are in contact with the PEG-lipid at c(PEG)(2000)-PE = 0.0015 wt %, while they are exposed to water at c(PEG)(2000)-PE > 0.0015 wt %. Dynamic and static light scattering (DLS and SLS) and small-angle neutron scattering (SANS) point out the existence of individual BSA/PEG-lipid complexes in the system for fixed c(BSA) = 1 wt % and variable c(PEG)(2000)-PE = 0.15-2 wt %. DLS shows that there is only one BSA molecule per protein/PEG-lipid complex, while SLS shows that the PEG-lipid associates to the BSA without promoting aggregation between adjacent protein/polymer-lipid conjugate complexes. SANS was used to show that BSA/PEG(2000)-PE complexes adopt an oblate ellipsoidal shape. Partially unfolded BSA is contained in the core of the oblate ellipsoid, which is surrounded by an external shell containing the PEG(2000)-PE.     

Reduction of cytochrome c at a lipid bilayer modified electrode

The reduction of horse heart cytochrome c has been investigated at a platinum electrode modified with a lipid bilayer membrane (BLM) which immobilized vinyl ferrocene as an electron mediator. The current—voltage curves show that the direct electrochemistry of cytochrome c at the metal electrode occurs quite efficiently. An adsorption equilibrium constant for cytochrome at the BLM surface, as well as an electron transfer rate constant between the protein and the modified electrode have been estimated from these results. The values of both parameters are much higher than those reported with other types of electrode modifications, indicating that a lipid bilayer-modified platinum electrode system using vinyl ferrocene as a mediator provides substantial improvements in electrochemical activity of cytochrome c at metal electrodes. The potential for modifying and utilizing this new class of “biomembrane-like” electrode surface for metalloprotein electrochemistry is briefly discussed.     

Relationship between densitometric peak area and concentration of lipid in the quantitative analysis of a lipid mixture separated by thin-layer chromatography.

A mixture of the neutral lipids squalene, cetyl stearate, triolein, oleic acid and cholesterol was separated by thin-layer chromatography and the components were measured quantitatively by photodensitometry. Several empirical expressions relating densitometric peak area (A) and lipid concentration (C) were subjected to statistical analysis, and the relationship A=k-C-B is linear in the logarithmic form log A=B-log c -0g k within 95% confidence limits on the observed results. Exponent B, representing the proportionality factor in the expression relating the relative differential changes in peak area and concentration, is constant for lipids studied on the same chromatoplate, whereas the constant k is lipid dependent. All standard measurements per plate are therefore utilized for estimating a common B value, which gives greater accuracy than in the individual cases. The plate capacity for measurements of unknown lipid concentrations may consequently be increased, as a common B value, based on a reduced number of standard measurements, may be estimated with the same accuracy as an individual B value, based upon the non-reduced number of standard measurements. Equations are given for the calculation of B and k and for the determination of unknown concentrations with appropriate confidence intervals.     

Electrostriction of biotin-streptavidin-modified bilayer lipid membranes on a metal support

Using the electrostriction method we have studied the elasticity modulus perpendicular to the membrane plane, E⊥, electrical capacitance, C, coefficient of dynamic viscosity, η, and membrane potential difference δф_m of supported bilayer lipid membranes (s-BLM) modified by biotin-streptavidin, as a function of d.c. voltage applied to the membrane. Binding of streptavidin to biotin-modified s-BLM resulted in a slight decrease of membrane capacitance, increase of E_⊥ and increase of η, while δф_m did not change. The val of E_⊥ of unmodified membranes was found to change considerably with increasing d.c. voltage and the rate of voltage change. Modification of s-BLM by streptavidin leads to reduced changes of E_⊥ with the rate of d.c. voltage change, and it made s-BLM extremely stable even at an external d.c. voltage of 2 V. Our results indicate that streptavidin considerably stabilized s-BLM by means of the formation of a complex with biotin-modified phospholipids.     

An overview of lipid membrane supported by colloidal particles

In recent years, original hybrid assemblies composed of a particle core surrounded by a lipid shell emerged as promising entities for various biotechnological applications. Their broadened bio-potentialities, ranging from model membrane systems or biomolecule screening supports, to substance delivery reservoirs or therapeutic vectors, are furthered by their versatility of composition due to the possible wide variation in the particle nature and size, as well as in the lipid formulation. The synthesis, the characteristics, and the uses of these Lipid/Particle assemblies encountered in the literature so far are reviewed, and classified according to the spherical core size in order to highlight general trends. Moreover, several criteria are particularly discussed: i) the interactions involved between the particles and the lipids, and implicitly the assembly elaboration mechanism, ii) the most suited techniques for an accurate characterization of the entities from structural and physicochemical points of view, and iii) the remarkable properties of the solid-supported lipid membrane obtained.     

Mesomorphic complexes of DNA with the mixtures of a cationic surfactant and a neutral lipid

Polyanionic DNA binds to cationic lipids to form electrostatic complexes exhibiting rich self-assembled structures. These types of complexes have been considered as a nonviral carrier in gene therapy and as a template for nanostructure construction. For the latter application where biocompatibility is not the key issue, replacement of cationic lipid by cationic surfactant is advantageous due to the wide availability of surfactant. Here we report the self-assembly behavior of the complexes of DNA with a cationic surfactant, dodecyltrimethylammonium bromide (DTAB), mixed with a neutral lipid, dioleoylphosphatidylethanolamine (DOPE), in fully hydrated state as a function of DTAB-to-DNA base pair molar ratio (x), DOPE-to-DTAB molar ratio (m) and temperature. The binary complexes of DNA with DTAB microphase separated to form hydrophilic and hydrophobic domains without long-range order. Incorporating DOPE into the complexes effectively strengthened the hydrophobic interaction and hence promoted the formations of long-range ordered mesophases, including a condensed multilamellar phase (L(alpha)(c)) at small to intermediate m (m approximately 6). The lyotropic mesophase transition with respect to the change of m was properly predicted by a formula for calculating the packing parameter of amphiphile mixture. In addition to the lyotropic transition, an unusual thermotropic order-order transition (OOT) between L(alpha)(c) and H(II)(c) phases was revealed for the isoelectric complex with m = 3. This OOT was thermally reversible and was postulated to be driven by the reduction of the effective headgroup area due to the release of trapped water molecules.     

Photodynamic lipid peroxidation in biological systems

Oxidative degradation of cell membrane lipids in the presence of molecular oxygen, a sensitizing agent and exciting light is termed photodynamic lipid peroxidation (photoperoxidation). Like other types of lipid peroxidation, photoperoxidation is detrimental to membrane structure and function, and could play a role in many of the toxic as well as therapeutic effects of photodynamic action. Recent advances in our understanding of photoperoxidation and its biomedical implications are reviewed in this article. Specific areas of interest include (a) reaction mechanisms; (b) methods of detection and quantitation; and (c) cellular defenses (enzymatic and non-enzymatic).     

Photoreduction behavior of cytochrome c by zinc porphyrin in lipid media

To elucidate the role of cardiolipin (CL) on redox behavior of cytochrome c (cyt c (III)), the photoreduction of cyt c using the photosensitization of zinc tetraphenylporphyrin in presence of triethanolamine (TEOA) as a sacrificial electron-donating reagent in various lipid media were studied. The initial rate of cyt c (III) photoreduction in various lipid, CL, l-alpha-phosphatidic acid (PA), dimethyldipalmitoylammonium bromide (DMPA) and Triton X-100 media were 1.0, 0.73, 0.80 and 0.67 micromol dm-3 min-1, respectively. The cyt c (III) photoreduction rate slightly increased by the addition of CL.     

Theoretical modeling of hydroxyl-radical-induced lipid peroxidation reactions

The OH-radical-induced mechanism of lipid peroxidation, involving hydrogen abstraction followed by O2 addition, is explored using the kinetically corrected hybrid density functional MPWB1K in conjunction with the MG3S basis set and a polarized continuum model to mimic the membrane interior. Using a small nonadiene model of linoleic acid, it is found that hydrogen abstraction preferentially occurs at the mono-allylic methylene groups at the ends of the conjugated segment rather than at the central bis-allylic carbon, in disagreement with experimental data. Using a full linoleic acid, however, abstraction is correctly predicted to occur at the central carbon, giving a pentadienyl radical. The Gibbs free energy for abstraction at the central C11 is approximately 8 kcal/mol, compared to 9 kcal/mol at the end points (giving an allyl radical). Subsequent oxygen addition will occur at one of the terminal atoms of the pentadienyl radical fragment, giving a localized peroxy radical and a conjugated butadiene fragment, but is associated with rather high free energy barriers and low exergonicity at the CPCM-MPWB1K/MG3S level. The ZPE-corrected potential energy surfaces obtained without solvent effects, on the other hand, display considerably lower barriers and more exergonic reactions.     

Dissipation-enhanced quartz crystal microbalance studies on the experimental parameters controlling the formation of supported lipid bilayers

We report on the investigations of the transformation of spherically closed lipid bilayers to supported lipid bilayers in aqueous media in contact with SiO(2) surfaces. The adsorption kinetics of small unilamellar vesicles composed of dimyristoyl- (DMPC) and dipalmitoylphosphatidylcholine (DPPC) mixtures on SiO(2) surfaces were investigated using a dissipation-enhanced quartz crystal microbalance (QCM-D) as a function of buffer (composition and pH), lipid concentration (0.01-1.0 mg/mL), temperature (15-37 degrees C), and lipid composition (DMPC and DMPC/DPPC mixtures). The lipid mixtures used here possess a phase transition temperature (T(m)) of 24-33 degrees C, which is close to the ambient temperature or above and thus considerably higher than most other systems studied by QCM-D. With HEPES or Tris.HCl containing sodium chloride (150 mM) and/or calcium chloride (2 mM), intact vesicles adsorb on the surface until a critical density ((c)) is reached. At close vesicle contact the transformation from vesicles to supported phospholipid bilayers (SPBs) occurs. In absence of CaCl(2), the kinetics of the SPB formation process are slowed, but the passage through (c) is still observed. The latter disappears when buffers with low ionic strength were used. SPB formation was studied in a pH range of 3-10, yet the passage through (c) is obtained only for pH values above to the physiological pH (7.4-10). With an increasing vesicle concentration, (c) is reached after shorter exposure times. At a vesicle concentration of 0.01-1 mg/mL, vesicle fusion on SiO(2) proceeds with the same pathway and accelerates roughly proportionally. In contrast, the pathway of vesicle fusion is strongly influenced by the temperature in the vicinity of T(m). Above and around the T(m), transformation of vesicles to SPB proceeds smoothly, while below, a large number of nonruptured vesicles coexist with SPB. As expected, the physical state of the membrane controls the interaction with both surface and neighboring vesicles.     

Real-time atomic force microscopy reveals cytochrome c-induced alterations in neutral lipid bilayers

The interaction of cytochrome c (cyt c) with supported lipid membranes was investigated on the nanoscale by real-time atomic force microscopy. Cyt c promoted the formation and the expansion of depressed areas in the fluid parts of the bilayer. When the depressions reached the gel domains, they induced the thickening of their edges. According to the step-height differences, cyt c was able to remove neutral lipids in the fluid phase and then to reside on the mica surface. Concerning gel phases, cyt c might insert between the two lipid leaflets, or it might intercalate between the mica and the bilayer.     

Scanning near-field IR microscopy of proteins in lipid bilayers

We use infrared near-field microscopy to chemically map the morphology of biological matrices. The investigated sample is built up from surface-tethered membrane proteins (cytochrome c oxidase) reconstituted in a lipid bilayer. We have carried out infrared near-field measurements in the frequency range between 1600 and 1800 cm(-1). By simultaneously recording the topography and chemical fingerprint of the protein-tethered lipid bilayer with a lateral resolution of 80 nm × 80 nm, we were able to probe locally the chemical signature of this membrane and to provide a local map of its surface morphology.     

Nonuniform lipid distribution in membranes

The noniform lateral and transbilayer lipid arrangement existing in two-component lipid bilayers are reviewed.

The lateral lipid organization is considered on the basis of the temperature-composition phase diagrams of the lipid binaries. A comparative analysis of the phase diagrams of synthetic phospholipid mixtures is carried out. The various types of the phase diagrams observed are set in a continuous row determined by the increase of the lipid lateral immiscibility. A special emphasis is laid on the appearance of peculiar points in the phase diagrams--triple, critical, and isoconcentration points. Two basic statistical-mechanical methods for simulation of phase diagrams--Bragg-Williams (regular solutions, mean field) and quasichemical--are compared. Stability criteria indicating the regions of lateral phase separation are also given. The main advantage of the quasichemical method is that it also allows the short-range order in the lipid arrangement to be determined.

The physical interactions contributing to an equilibrium lipid asymmetry in mixed lipid bilayers are pointed out. The most important among them are: (i) electrostatic forces induced by differences in the membrane electric double layers; (ii) nonideal lateral mixing of the lipids; (iii) packing restrictions important in curved bilayers.

A unified electrostatic model is presented to calculate the surface charge asymmetry created by any factors affecting the electric double layers of the bilayer (external electric potential, overlapping electric double layers in parallel membranes or in vescicles, etc.).

The transmembrane asymmetry strongly depends on the degree of c corrections may increase up two-three times the asymmetry induced by factors of the order of 1–3 kT. A typical nonideality effect, which may be used in an experimental verification, is the appearance of an extremum in the dependence of the asymmetry on the mole fraction of the components.

As previously shown in other reviews on membrane organization, the packing restrictions are of importance in highly curved bilayers, e.g., in small unilamellar vesicles.

The experimental data on the asymmetry of two-component small unilamellar vesicles are summarized and some general conclusions are formulated.

With a view toward the native membranes, some inferences are drawn about (i) the state of thermodynamic equilibrium and (ii) the lipid organization in multicomponent membranes.     

Modeling the induction of lipid membrane electropermeabilization

Experiments show significant effects of an electric field on lipid membrane, leading to a pore formation when a high intensity field is applied. The phenomenon of electroporation is preceded by the induction and expansion of defects, responsible for the pre-pore excitation. We examine the mechanism of the induction of the field-driven defects by Monte Carlo simulations. The study is based on the improved Pink's model, which includes explicit interactions between the polar heads and energy of interactions between the heads and the field. No anomalous deformation of the molecules is considered. The study, provided for bilayer dipalmitoyl-phosphatidylcholine (DPPC) membrane in the gel (300 K) and fluid (330 K) phases, shows dependence of the membrane conformational and energetical state on the value of the electric field. We observe that the electric field affects the number of molecules in the gel and in the fluid states. In the layer at the negative potential, when the transmembrane voltage is above U(c) approximately 280 mV, lipid heads abruptly reorient and the number of local spots with fluid conformation increases. The other layer slightly tends to tighten its structure, producing additional mechanical stress between layers. Lipids showed complete insensitivity to the electric field within physiological limits, U<70 mV.     

Interaction of curcumin with lipid monolayers and liposomal bilayers

Curcumin shows huge potential as an anticancer and anti-inflammatory agent. However, to achieve a satisfactory bioavailability and stability of this compound, its liposomal form is preferable. Our detailed studies on the curcumin interaction with lipid membranes are aimed to obtain better understanding of the mechanism and eventually to improve the efficiency of curcumin delivery to cells. Egg yolk phosphatidylcholine (EYPC) one-component monolayers and bilayers, as well as mixed systems containing additionally dihexadecyl phosphate (DHP) and cholesterol, were studied. Curcumin binding constant to EYPC liposomes was determined based on two different methods: UV/Vis absorption and fluorescence measurements to be 4.26 × 10⁴ M⁻¹ and 3.79 × 10⁴ M⁻¹, respectively. The fluorescence quenching experiment revealed that curcumin locates in the hydrophobic region of EYPC liposomal bilayer. It was shown that curcumin impacts the size and stability of the liposomal carriers significantly. Loaded into the EYPC/DPH/cholesterol liposomal bilayer curcumin stabilizes the system proportionally to its content, while the EYPC/DPH system is destabilized upon drug loading. The three-component lipid composition of the liposome seems to be the most promising system for curcumin delivery. An interaction of free and liposomal curcumin with EYPC and mixed monolayers was also studied using Langmuir balance measurements. Monolayer systems were treated as a simple model of cell membrane. Condensing effect of curcumin on EYPC and EYPC/DHP monolayers and loosening influence on EYPC/DHP/chol ones were observed. It was also demonstrated that curcumin-loaded EYPC liposomes are more stable upon interaction with the model lipid membrane than the unloaded ones.     

Curvature-modulated phase separation in lipid bilayer membranes

Cellular membranes exhibit a variety of controlled curvatures, with filopodia, microvilli, and mitotic cleavage furrows being only a few of many examples. Coupling between local curvature and chemical composition in membranes could provide a means of mechanically controlling the spatial organization of membrane components. Although this concept has surfaced repeatedly over the years, controlled experimental investigations have proven elusive. Here, we introduce an experimental platform, in which microfabricated surfaces impose specific curvature patterns onto lipid bilayers, that allows quantification of mechanochemical couplings in membranes. We find that, beyond a critical curvature value, membrane geometry governs the spatial ordering of phase-separated domain structures in membranes composed of cholesterol and phospholipids. The curvature-controlled ordering, a consequence of the distinct mechanical properties of the lipid phases, makes possible a determination of the bending rigidity difference between cholesterol-rich and cholesterol-poor lipid domains. These observations point to a strong coupling between mechanical bending and chemical organization that should have wide-reaching consequences for biological membranes. Curvature-mediated patterning may also be useful in controlling complex fluids other than biomembranes.     

Electrochemical reactions of redox cofactors in Rhodobacter sphaeroides reaction center proteins in lipid films.

Cyclic voltammetry of thin films made from the lipid dimyristoylphosphatidyl choline and reaction centers from the purple bacterium Rhodobacter sphaeroides on pyrolytic graphite electrodes in bromide-free pH 8 buffers at 4 degrees C revealed an oxidation peak at 0.98 V and a reduction peak at -0.17 V vs. NHE. No reverse CV peaks were found, suggesting chemical irreversibility. The reduction peak disappeared for reaction centers depleted of quinones, suggesting that the peak represents reduction of this cofactor. The oxidation peak showed a catalytic current increase in the presence of small amounts of ferrous cytochrome c, and decreased by 85% when illuminated by visible light, suggesting assignment to the primary donor (P) cofactor. While oxidized primary donor P(+) is destroyed upon electrochemical formation in the film, reaction of ferrous cyt c with P(+) suggests its persistence in the films on the microsecond time scale.     

Simulations of lipid vesicle adsorption for different lipid mixtures

Numerous experimental studies of lipid vesicle adsorption on solid surfaces show that electrostatic interactions play an important role for the kinetics and end result. The latter can, e.g., be intact vesicles or supported lipid bilayers (SLB). Despite an accumulated quite large experimental data base, the understanding of the underlying processes is still poor, and mathematical models are scarce. We have developed a phenomenological model of a vesicle adsorbing on a substrate, where the charge of the surface and the charge and polar state of the lipid headgroup can be varied. With physically reasonable assumptions and input parameters, we reproduce many key experimental observations, clarify the details of some experiments, and give predictions and suggestions for future experiments. Specifically, we have investigated the influence of different lipid mixtures (different charges of the headgroups) in the vesicle on the outcome of a vesicle adsorption event. For different mixtures of zwitterionic lipids with positive and negative lipids, we investigated whether the vesicle adsorbs or not, and--if it adsorbs--to what extent it gets deformed and when it ruptures spontaneously. Diffusion of neutral vesicles on different types of negatively charged substrates was also simulated. The mean surface charge density of the substrate was varied, including or excluding local fluctuations in the surface charge density. The simulations are compared to available experiments. A consistent picture of the influence of different lipid mixtures in the vesicle on adsorption, and the influence of different types of substrates on vesicle diffusion, appear as a result of the simulation data.