Effect of cholesterol or phospholipid incorporation on the chemical stability of the muramyldpeptide derivative B30-MDP in mixed vesicles

Application of the muramyldipeptide derivative B30-MDP to liposomal vaccines will aid in the development of improved high immunogenicity vaccines. To give full play to the effectiveness of B30-MDP as a liposomal vaccine, it is important to evaluate the effect of cholesterol, dimyristoylphosphatidylcholine (DMPC) or distearoylphosphatidylcholine (DSPC) incorporation on the chemical stability of B30-MDP and physicochemical properties of B30-MDP/lipid mixed vesicles from the view point of pharmaceutics.The observed degradation rate constants of B30-MDP by hydrolysis in B30-MDP/cholesterol mixed vesicles were increased with increasing concentration of cholesterol, however, those in B30-MDP/DMPC and B30-MDP/DSPC mixed vesicles were unchanged with increasing concentration of DMPC and DSPC. The degradation behavior of B30-MDP was then compared with physicochemical properties of B30-MDP/lipid mixed vesicles, such as membrane fluidity and particle size. It was apparent that the degradation of B30-MDP in B30-MDP/cholesterol mixed vesicles was influenced by the particle size, but not by the fluidity of the membranes. In the case of B30-MDP/phospholipid mixed vesicles, MDP/phospholipid mixed vesicles, the degradation of B30-MDP was not influenced by either the membranes' fluidity or the particle size of the mixed vesicles.It is considered that the degradation of B30-MDP in the mixed vesicles is dependent on the membrane state, and the addition of cholesterol to B30-MDP vesicle inhibits the mutual interaction of MDP regions, whereas the addition of phospholipids hardly influences the mutual interaction of MDP regions, possibly owing to phase separation between B30-MDP and phospholipids.     

Moving blue band caused by cooperation of diffusion and phase separation

Diffusions of Cu(2+) and Fe(3+) in gelatin generate a moving blue band. It is formed by a diffusion of Cu(2+) and a phase separation of gelatin with diffusing Fe(3+). The diffusing Fe(3+) forms Fe(OH)(3) colloids and gathers gelatin molecules from the surroundings. The diffusion of gelatin molecules generates the concentration gradient, resulting in a gel/sol transition in the dilute phase. In the region where the concentration of Fe(3+) is high enough, the gel remains hard, while a sol phase appears under the hard gel. The absorption spectrum of Cu(2+) depends on the concentration ratio of Cu(2+) to gelatin. As a consequence, we can see a blue band in the restricted region between the diffusing front of Cu(2+) and the phase separation front. The movement of the blue band is caused by a coupling of a simple diffusion and the phase separation.     

Effect of pressure on the Prodan fluorescence in bilayer membranes of phospholipids with varying acyl chain lengths

The fluorescence spectra of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) were observed as a function of pressure for the bilayer membrane systems of dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC). The wavelength of the emission maximum, lambdamax, was found to be 480, 430, and 500 nm for the liquid crystalline (Lalpha), ripple gel (P'beta), and pressure-induced interdigitated gel (LbetaI) phase, respectively. Since the lambdamax reflects the solvent property around the probe molecules, we could speculate on the location of the Prodan molecules in the bilayer membranes; in the Lalpha phase of the lipid bilayer, the Prodan molecules distribute around the phosphate of the lipids (i.e. the polar region). The Lalpha/P'beta phase transition caused the Prodan molecules to move into the less polar region near the glycerol backbone. The fluorescence intensity of the Prodan in the P'beta phase was dependent on the chain length of the lipids and on pressure; the shorter the chain length of the lipid, the stronger the fluorescence intensity of the Prodan. Moreover, for the DLPC bilayer membrane system, the fluorescence intensity at 430 nm increased with increasing pressure, indicating that the partition of Prodan into the DLPC bilayer membrane is promoted by applying pressure. In the case of the DPPC and DSPC bilayers, as the pressure increased further, the pressure-induced interdigitation caused the Prodan molecules to squeeze out of the glycerol backbone region and to move the hydrophilic region near the bilayer surface. The ratio of fluorescence intensity at 480 nm to that at 430 nm, F480/F430, showed a sharp change at the phase-transition pressure. In the case of the DPPC and DSPC bilayers, the values of F480/F430 showed an abrupt increase above a certain pressure higher than the Lalpha/P'beta transition pressure, which corresponds to the interdigitation from the P'beta to the LbetaI phase. The plot of F480/F430 versus pressure is available for recognition of the bilayer phase transitions, especially the bilayer interdigitation.     

Partitioning of dual-lipidated peptides into membrane microdomains: lipid sorting vs peptide aggregation

The lateral membrane organization and phase behavior of the lipid mixture DMPC(di-C(14))/DSPC(di-C(18))/cholesterol (0-33 mol %) with and without an incorporated fluorescence-labeled palmitoyl/farnesyl dual-lipidated peptide, BODIPY-Gly-Cys(Pal)-Met-Gly-Leu-Pro-Cys(Far)-OMe, which represents a membrane recognition model system for Ras proteins, was studied by two-photon excitation fluorescence microscopy. Measurements were performed on giant unilamellar vesicles (GUVs) over a large temperature range, ranging from 30 to 80 degrees C to cover different lipid phase states (all-gel, fluid/gel, liquid-ordered, all-fluid). At temperatures where the fluid-gel coexistence region of the pure binary phospholipid system occurs, large-scale concentration fluctuations appear. Incorporation of cholesterol levels up to 33 mol % leads to a significant increase of conformational order in the membrane system and a reduction of large domain structures. Adding the peptide leads to dramatic changes in the lateral organization of the membrane. With cholesterol present, a phase separation is induced by a lipid sorting mechanism owing to the high affinity of the lipidated peptide to a fluid, DMPC-rich environment. This phase separation leads to the formation of peptide-containing domains with high fluorescence intensity that become progressively smaller with decreasing temperature. As a result, the local concentration of the peptide increases steadily within the confines of the shrinking domains. At the lowest temperatures, where the acyl-chain order parameter of the membrane has already drastically increased and the membrane achieves a liquid-ordered character, an efficient lipid sorting mechanism is no longer supported and aggregation of the peptide into small clusters prevails. We can conclude that palmitoyl/farnesyl dual-lipidated peptides do not associate with liquid-ordered or gel-like domains in phase-separated bilayer membranes. In particular, the study shows the interesting ability of the peptide to induce formation of fluid microdomains at physiologically relevant cholesterol concentrations, and this effect very much depends on the concentration of fluid vs ordered lipid molecules.     

Dynamical and morphological studies on the adsorption and penetration of human serum albumin into phospholipid monolayers at the air/water interface

The dynamic adsorption and penetration of human serum albumin (HSA) into the monolayers of five biologically important surfactants—DSPC, DPPC, DMPC, DMPE and DMPA—were systematically studied using Brewster angle microscopy, film balance and pendent drop techniques. Isotherms after different adsorption times show that the presence of HSA changed the monolayer phase behavior (e.g. the shifts of the LE→LC phase transition in the mixed phospholipid/HSA monolayers). Apparent inhomogeneous phases—‘honey-comb’ (J. Mol. Liq., 2001, 90, 149), ‘block’ or ‘stripe’ shape phases are formed due to the adsorption and penetration of HSA into these phospholipid monolayers at the air/water interface. Both the phase behavior changes and the morphological changes were confirmed by our recent structure studies in DPPA/HSA and DPPS/HSA monolayers using X-ray diffraction at grazing incidence, which directly shows that HSA penetration can change the tilt angle of phospholipids. It was found that the adsorption and penetration of HSA strongly depends on the phospholipid head-group structure and the physical state of the phospholipid films. The latter played a dominant role by providing enough space for the penetration of HSA and affecting the hydrophobic interactions of HSA with the aliphatic chains of phospholipids in monolayers at the air/water interface. In general, HSA penetrates more efficiently and quickly into monolayers of phospholipids in liquid state (e.g. DMPC compared to DSPC) and with unprotected charges (e.g. PA compared to PE and PC).     

Study of gramicidin A--phospholipid interactions in Langmuir monolayers: analysis of their mechanical, thermodynamical, and electrical properties

The mechanisms of interactions between gramicidin A (gA) and dimyristoylphosphatidylcholine (DMPC) in monolayers formed at the air-water interface were studied by analyzing their mechanical, thermodynamical, and electrical properties evaluated from measurements of pressure-area isotherms and of Maxwell displacement currents (MDC). A contactless method of recording MDC enabled us to monitor changes in the charge state of the monolayer-constituting molecules and to find the relation between a phase state of the monolayer and structural transitions of gA. The peptide-lipid interactions were quantified in terms of the excess of Gibbs free energy, excess entropy, as well as the molecular dipole moments at various gA/DMPC molar ratios, at various temperatures (in the gel phase and also in the liquid-crystalline phase of DMPC molecule), and at various surface pressures. It was found that the strongest interactions between gA and DMPC took place at the gA/DMPC molar ratio at around 0.25. At this monolayer composition, the phospholipids, via their carbonyl moieties, dominantly interact with the single helical gA, which mostly stands upright on the surface and is anchored by its C-terminus to the water surface, and prevent the formation of the intertwined helical gA dimers. The optimum ratio was confirmed also by anomalous electrical behavior of electrical dipole moments derived from MDC measurements.     

Investigation of Factors Affecting Controlled Release from Photosensitive DMPC and DSPC Liposomes

An investigation of liposomes comprised of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) lipids with cholesterol and zinc phthalocyanine (ZnPC) revealed that several fundamental liposome properties are influenced by composition and by lipid-specific features. DMPC and DSPC liposomes were synthesized, and their compositional changes, encapsulation capacities, morphologies, and release properties were evaluated. In this research, liposome degradation, lysis, and content release were initiated by photolysis, i.e., rupture induced by exposure to light. A controlled release mechanism was created through the introduction of photosensitizers (i.e., ZnPC) embedded within the cholesterol-stabilized liposome membrane. The light wavelength and light exposure time accelerated photodegradation properties of DMPC liposomes compared to DSPC liposomes, which exhibited a slower release rate. Morphological changes in the liposomes were strongly influenced by light wavelength and light exposure time. For both the DMPC and DSPC liposomes, visible light with wavelengths in the red end of the spectrum and broad spectrum ambient lighting (400?C700?nm) were more effective for lysis than UV-A light (365?nm). Heating liposomes to 100?°C decreased the stability of liposomes compared to liposomes kept at room temperatures. In addition, the optimal lipid-to-cholesterol-to-photoactivator ratio that produced the most stable liposomes was determined.     

Effects of lipid chain length on molecular interactions between paclitaxel and phospholipid within model biomembranes

Molecular interactions between an anticancer drug, paclitaxel, and phosphatidylcholine (PC) of various chain lengths were investigated in the present work by the Langmuir film balance technique and differential scanning calorimetry (DSC). Both the lipid monolayer at the air-water interface and lipid bilayer vesicles (liposomes) were employed as model biological cell membranes. Measurement and analysis of the surface pressure versus molecular area curves of the mixed monolayers of phospholipids and paclitaxel under various molar ratio showed that phospholipids and paclitaxel formed a nonideal miscible system at the interface. Paclitaxel exerted an area-condensing effect on the lipid monolayer at small molecular surface areas and an area-expanding effect at large molecular areas, which could be explained by the intermolecular forces and geometric accommodation between the two components. Paclitaxel and phospholipids could form thermodynamically stable monolayer systems: the stability increased with the chain length in the order DMPC (C14:0)>DPPC (C16:0)>DSPC (C18:0). Investigation of paclitaxel penetration into the pure lipid monolayer showed that DMPC had a higher ability to incorporate paclitaxel and the critical surface pressure for paclitaxel penetration also increased with the chain length in the order DMPC>DPPC>DSPC. A similar trend was testified by DSC studies on vesicles of the mixed paclitaxel/phospholipids bilayer. Paclitaxel showed the greatest interaction with DMPC while little interaction could be measured in the paclitaxel/DSPC liposomes. Paclitaxel caused broadening of the main phase transition without significant change at the peak melting temperature of the phospholipid bilayers, which demonstrated that paclitaxel was localized in the outer hydrophobic cooperative zone of the bilayer. The interaction between paclitaxel and phospholipid was nonspecific and the dominant factor in this interaction was the van der Waals force or hydrophobic force. As the result of the lower net van der Waals interaction between hydrocarbon chains for the shorter acyl chains, paclitaxel interacted more readily with phospholipids of shorter chain length, which also increased the bilayer intermolecular spacing.     

Probing the interplay between chain diffusion and polymer crystal growth under nanoscale confinement: a study by single molecule fluorescence microscopy

Motions of single poly(ε-caprolactone) (PCL) molecules during the formation of the dendrite crystals in ultrathin films are captured by single molecule fluorescence microscopy. The relationship of single molecule diffusion coefficient with the crystal growth rate, together with radius curvature, side-branch spacing of dendrite crystal and morphology are examined. The results support Mullins-Sekerka (MS) instability as the origin of lamellar branching induced by a diffusion field generated by a gradient of polymer segment density ahead of the crystal. Further analysis of the molecular trajectories has recognized different types of motions, depending on the distance to the crystal front: Fickian diffusion in regions far away from the crystal, sub-diffusion in regions adjacent to the crystal, and directed motion between these two regions. Anti-correlation of successive steps is discovered accompanying the sub-diffusion, providing a clear signature of macromolecule crowding at the crystal growth front. This anomalous diffusion process in polymer ultrathin films presents a new insight into the understanding of the retarded dynamics of interfacial mass transport towards the crystal front. It is considered to play a decisive role in controlling the crystal growth and evolution of crystal morphology.     

Liposome modified with Mn-porphyrin complex can simultaneously induce antioxidative enzyme-like activity of both superoxide dismutase and peroxidase

An antioxidative liposome catalysis that mimics both superoxide dismutase (SOD) and peroxidase (POD) activities has been developed by using the liposomes modified with lipophilic Mn-(5,10,15,20-tetrakis[1-hexadecylpyridium-4-yl]-21H,23H-porphyrin) (Mn-HPyP). The SOD- and POD-like activities of the Mn-HPyP-modified liposome were first investigated by varying the type of phospholipid, such as 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC). Higher SOD-like activity was obtained in the case of DLPC and DMPC liposomes, in which the ligands were well-dispersed on the membrane in the liquid crystalline phase. The POD-like activity was maximal in the case of DMPC liposome, in which the Mn-HPyP complex was appropriately clustered on the membrane in the gel phase. On the basis of the above results, the co-induction of the SOD and POD activities to eliminate the superoxide and also hydrogen peroxide as a one-pot reaction was finally performed by using the Mn-HPyP-modified DMPC liposome, resulting in an increase in the efficiency of the elimination of both superoxide and hydrogen peroxide.     

Diffusion of glycosylphosphatidylinositol (GPI)-anchored bovine prion protein (PrP) in supported lipid membranes studied by single-molecule and complementary ensemble methods

In this work cellular bovine prion protein (PrP^c) was incorporated in supported lipid membranes and its lateral diffusion was studied by single-dye tracking (SDT) and a complementary ensemble method, fluorescence recovery after photobleaching (FRAP). PrP^c was purified from calf brain with its native glycosylphosphatidylinositol (GPI) anchor and reconstituted into DMPC lipid vesicles. Homogeneous spreading on solid supports over macroscopic areas was confirmed with fluorescence microscopy. FRAP results demonstrated very high mobile fractions of up to 94%, confirming that most of the GPI-anchored PrP^c are freely diffusive in the fluid supported membrane matrix. Moreover, the lateral diffusivity of PrP^c significantly depends on the pH of the buffer, suggesting that the conformation of PrP^c and thus the frictional drag exerted to the protein molecule (and thus the effective hydrodynamic radius) is influenced by the effective net charge. To complement the ensemble results obtained by FRAP, the statistical variation of lateral diffusion coefficients of individual PrP^c molecules in the supported membranes were measured with SDT. Simulation-based statistical analysis indicated that in addition to the expected statistical scatter there is a significant spread of diffusion coefficients, while the average of the diffusion coefficients of individual proteins obtained by SDT is in excellent agreement with those measured by ensemble FRAP. In further experiments, PrP^c was laterally concentrated in the membrane by the application of tangential electric fields (membrane electrophoresis). However, the equilibrium concentration profile reached after 20 min was different from an exponential gradient. This finding suggests that PrP^c purified from bovine brain possesses non-uniform net charges. As the lateral diffusion coefficient of proteins in two-dimensional lipid membranes sensitively depends upon the frictional drag, the combination of SDT, ensemble FRAP, and membrane electrophoresis can be used as a powerful tool to gain insights into protein–protein binding and oligomer formation that would play a crucial role in infectious protein transmitted diseases such as BSE.     

Differential permeation. Part II: Behaviors of polyvinylalocohol films in transient permeation

The variations of the permeation rate of ethanol in ethanol-water mixtures through poly(vinylalcohol) membranes of different crystallinities were studied in the transient regime. We observed an anomalous two-wave kinetics, which was accounted for in terms of a model postulating two diffusion-pathways for ethanol molecules in the polymer. One wave corresponds to the permeation of ethanol through the amorphous polymer matrix, which occurs whatever the water content in the mixture. The other wave appears only when the water content in the mixture exceeds a threshold. The higher the crystallinity, the higher the value of this threshold. This wave is attributed to the diffusion of ethanol molecules through clusters formed with the water molecules sorbed on the hydroxyl sites.     

Electrochemical and PM-IRRAS studies of the effect of cholesterol on the properties of the headgroup region of a DMPC bilayer supported at a Au(111) electrode

Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) was employed to investigate the interaction of cholesterol with the headgroups of dimyristoylphosphatidycholine (DMPC) molecules under a static electric field. DMPC/cholesterol (7:3 molar ratio) mixtures form a bilayer on a Au(111) electrode surface by fusion and spreading of small unilamellar vesicles. PM-IRRAS experiments provided detailed information concerning the conformation and hydration of headgroups of DMPC bilayers in the presence and absence of 30% cholesterol. The presence of 30% cholesterol increases the space between the headgroups of DMPC molecules and hence increases the hydration of the DMPC/cholesterol mixed bilayer. The conformational state of the headgroups of DMPC molecules in the mixed bilayer is also significantly changed. The phosphate group is closer to the surface compared with the pure DMPC bilayer. The conformation of the -O-C-C-N moiety changes from gauche to trans in the presence of cholesterol.     

Isomerization dynamics of photochromic spiropyran molecular switches in phospholipid bilayers

Transient absorption spectroscopy was used to investigate the dynamics of the photochromic indolinobenzospiropyran reaction in toluene solution and in phosphatidylcholine bilayers (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)). After excitation with UV light, colorless (R/S)-2-(3',3'-dimethyl-6-nitro-3'H-spiro[chromene-2,2'-indol]-1'-yl)ethanol derivatives are converted to colored merocyanine products in high yield; Phi = 0.45 in DMPC liposomes. We find that the reaction occurs in the bilayer aliphatic region in the gel (P(beta)(')) and liquid (L(alpha)) phases. The Arrhenius activation energy for the isomerization in DMPC bilayers was approximately 3.5 times larger in the liquid phase (L(alpha), E(a) = 26.0 +/- 1.0 kJ mol(-1)) than that in the gel phase (P(beta)('), E(a) = 7.3 +/- 1.6 kJ mol(-1)). Analysis of the isomerization rate constant temperature dependence allows an estimation of the bilayer viscosity and free volume properties in the L(alpha) phase.     

Maxwell displacement current allows to study structural changes of gramicidin A in monolayers at the air-water interface

We applied methods of measurement Maxwell displacement current (MDC) pressure-area isotherms and dipole potential for analysis of the properties of gramicidin A (gA) and mixed gA/DMPC monolayers at an air-water interface. The MDC method allowed us to observe the kinetics of formation of secondary structure of gA in monolayers at an air-water interface. We showed, that secondary structure starts to form at rather low area per molecule at which gA monolayers are in gaseous state. Changes of the MDC during compression can be attributed to the reorientation of dipole moments in a gA double helix at area 7 nm(2)/molecule, followed by the formation of intertwined double helix of gA. The properties of gA in mixed monolayers depend on the molar fraction of gA/DMPC. At higher molar fractions of gA (around 0.5) the shape of the changes of dipole moment of mixed monolayer was similar to that for pure gA. The analysis of excess free energy in a gel (18( ) degrees C) and in a liquid-crystalline phase (28( ) degrees C) allowed us to show influence of the monolayer structural state on the interaction between gA and the phospholipids. In a gel state and at the gA/DMPC molar ratio below 0.17 the aggregates of gA were formed, while above this molar ratio gA interacts favorably with DMPC. In contrast, for DMPC in a liquid-crystalline state aggregation of gA was observed for all molar fractions studied. The effect of formation ordered structures between gA and DMPC is more pronounced at low temperatures.     

Chain-melting phase transition and short-range molecular interactions in phospholipid foam bilayers

Occurrence of two-dimensional chain melting phase transition in foam bilayers was established for the first time. Microscopic horizontal foam bilayers [Newton black films (NBF)] were investigated by the microinterferometric method of Scheludko-Exerowa. The foam bilayers were formed from water-ethanol solutions of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) and egg phosphatidylcholine (Egg PC) and samples of amniotic fluid (AF) at different temperatures. The influence of temperature on the foam bilayer thickness h(w) and on the critical concentration Cc for formation of foam bilayer was studied. It was shown that in the range of the main phase transition the temperature dependence of h(w) and C(c) changed specifically in the case of DMPC and DPPC foam bilayers. The thickness of the foam bilayers increased with decreasing temperature in the range of the main phase transition due to the melting of hydrocarbon tails of phospholipid molecules. These changes took place at the temperatures of the bulk chain-melting phase transitions, as determined by differential scanning calorimetry (DSC) for both aqueous, and water/ethanol DMPC, DPPC, and DPPC dispersions. An effect of the 'disperse medium' on h(w) was found for foam bilayers from DPPC. The results that foam bilayers could have different thickness at different temperatures disproved the current concept that NBF acquired constant thickness at concentrations higher than C(el,cr). The data for Cc were analysed on the basis of the hole-nucleation theory of bilayer stability of Kashchiev and Exerowa. This theory considered the amphiphile bilayer as a two-dimensional ordered system with short-range molecular interactions between the first neighbour molecules (as in a crystal). The short-range molecular interactions were presented by the parameter binding energy Q of an amphiphile molecule in the bilayer. The binding energy Q of two neighbouring phospholipids was calculated for the gel (30-60 kT) and liquid crystalline state (16-18 kT) of the bilayers from DMPC, DPPC, Egg PC, AF. Concentration/temperature phase diagram of DPPC foam bilayers that defined regions of gaseous (ruptured), gel and liquid crystalline foam bilayers were drawn. The values of Q obtained for various samples were very close and vary from 5.3 x 10(-20) to 9.4 x 10(-20) (approx. 13-22 kT) which indicated that in all cases the foam bilayers were in liquid-crystalline state. This is an important result since the parameter studied-threshold concentration (threshold dilution) is crucial for a very successful assessment of the risk for respiratory distress syndrome (RDS) in newborns and could be employed in medicine for assessment of other respiratory disturbances. It is to be expected that foam bilayers from phospholipids could be used as a model for investigation of short-range forces in biological structures, of interaction between membranes, etc.     

Interaction of a poly(acrylic acid) oligomer with dimyristoylphosphatidylcholine bilayers

We studied the influence of 5 kDa poly(acrylic acid) (PAA) on the phase state, thermal properties, and lateral diffusion in bilayered systems of dimyristoylphosphatidylcholine (DMPC) using (31)P NMR spectroscopy, differential scanning calorimetry (DSC), (1)H NMR with a pulsed field gradient, and (1)H nuclear Overhauser enhancement spectroscopy (NOESY). The presence of PAA does not change the lamellar structure of the system. (1)H MAS NOESY cross-peaks observed for the interaction between lipid headgroups and polyion protons demonstrated only surface PAA-biomembrane interaction. Small concentrations of PAA (up to ～4 mol %) lead to the appearance of a new lateral phase with a higher main transition temperature, a lower cooperativity, and a lower enthalpy of transition. Higher concentrations lead to the disappearance of measurable thermal effects. The lateral diffusion coefficient of DMPC and the apparent activation energy of diffusion gradually decreased at PAA concentrations up to around 4 mol %. The observed effects were explained by the formation of at least two types of PAA-DMPC lateral complexes as has been described earlier (Fujiwara, M.; Grubbs, R. H.; Baldeschwieler, J. D. J. Colloid Interface Sci., 1997, 185, 210). The first one is characterized by a stoichiometry of around 28 lipids per polymer, which corresponds to the adsorption of the entire PAA molecule onto the membrane. Lipid molecules of the complex are exchanged with the "pure" lipid bilayer, with the lifetime of the complex being less than 0.1 s. The second type of DMPC-PAA complex is characterized by a stoichiometry of 6 to 7 lipids per polymer and contains PAA molecules that are only partially adsorbed onto the membrane. A decrease in the DMPC diffusion coefficient and activation energy for diffusion in the presence of PAA was explained by the formation of a new cooperative unit for diffusion, which contains the PAA molecule and several molecules of lipids.     

Dynamic light scattering study of the dynamics of a gelled polymeric micellar system

The dynamics of the E(92)B(18)/water system are studied by dynamic light scattering (DLS) in the liquid, soft gel, and hard gel phases. Both the liquid and the soft gel phases are micellar phases, although the structural order is higher in the soft gel phase than in the liquid phase. The hard gel phase corresponds to a face-centered cubic arrangement of micelles. DLS results show that the dilute liquid phase is characterized by a single characteristic time tau(1) associated with the diffusion of the micelles. In addition, a second characteristic time tau(2) associated with the presence of micellar clusters in the system is identified in the concentrated liquid and in the soft gel phases. According to these results, DLS suggests that the structure of the soft gel phase comprises micellar clusters coexisting with micellar fluid, in good agreement with hypotheses from our previous work. The dynamics of the system slows down as the hard gel phase is approached and a plateau is observed in the DLS correlation function. The structure of the hard gel is "softened" upon increasing temperature and/or decreasing concentration.     

大分子拥挤环境中脂肪酸影响磷脂囊泡相变的差示扫描量热研究

脂肪酸诱导的磷脂膜的热力学行为对于认识细胞内复杂的机制有着重要意义,而前人在研究脂肪酸与磷脂膜相互作用时大都在稀溶液中进行;拥挤环境下脂肪酸诱导磷脂膜的相变行为还未见报道。本文以二肉豆蔻酰磷脂酰胆碱(DMPC)构建囊泡模型,采用差示扫描量热法系统地研究了在不同浓度、不同分子量的聚乙二醇(PEG)拥挤环境中不同结构的脂肪酸对DMPC磷脂囊泡相变的影响。研究结果表明,在拥挤环境中,PEG对纯的磷脂囊泡相变的影响与大分子的分子量和浓度相关。对于脂肪酸/磷脂囊泡(FA/DMPC),PEG的存在对囊泡相变产生显著影响。在所考察的分子量和浓度范围内,PEG使FA/DMPC囊泡相变增加。短链饱和脂肪酸、不饱和脂肪酸原本使DPMC囊泡相变降低,但PEG缩小了降低幅度,甚至导致相变增加。进一步的研究表明,在大多数情况下,PEG对FA/DMPC的相变具有协作增强效应,且其影响均与大分子的分子量和浓度相关。另外,随着PEG浓度的升高,磷脂囊泡的协同单位数逐渐降低,表明拥挤环境会影响磷脂双分子层的均一性,使协同发生相变的分子数降低。本文的研究表明,大分子拥挤环境能够对扰动的磷脂双分子层起到一定的修复作用,这一现象在生物膜相关领域不可忽视。     

Correlating anomalous diffusion with lipid bilayer membrane structure using single molecule tracking and atomic force microscopy

Anomalous diffusion has been observed abundantly in the plasma membrane of biological cells, but the underlying mechanisms are still unclear. In general, it has not been possible to directly image the obstacles to diffusion in membranes, which are thought to be skeleton bound proteins, protein aggregates, and lipid domains, so the dynamics of diffusing particles is used to deduce the obstacle characteristics. We present a supported lipid bilayer system in which we characterized the anomalous diffusion of lipid molecules using single molecule tracking, while at the same time imaging the obstacles to diffusion with atomic force microscopy. To explain our experimental results, we performed lattice Monte Carlo simulations of tracer diffusion in the presence of the experimentally determined obstacle configurations. We correlate the observed anomalous diffusion with obstacle area fraction, fractal dimension, and correlation length. To accurately measure an anomalous diffusion exponent, we derived an expression to account for the time-averaging inherent to all single molecule tracking experiments. We show that the length of the single molecule trajectories is critical to the determination of the anomalous diffusion exponent. We further discuss our results in the context of confinement models and the generating stochastic process.