Bilayer Membrane Systems Research Articles

Phase Association and Binding Energetics of Swcnts into Phospholipid Langmuir Monolayers

Single-walled carbon nanotubes (SWCNTs) have been identified as promising candidates for targeted drug delivery due to their low toxicity and ability to be functionalized using various bioactive groups. It is currently undetermined what mechanical and biological mechanism(s) are responsible for uptake into cells. We have used dispersed pristine SWCNTs to study mechanisms responsible for cellular uptake using cellular systems and in vitro model membrane systems. Fluorescence lifetime imaging microscopy (FLIM) of RhoB-GFP, a marker of endocytosis, shows functional spatial and temporal localization of endosomes with SWCNTs. Results demonstrate an increase in the number of endosomes after SWCNTs exposure indicating a stimulation of endocytosis. Measurements of SWCNT on artificial tethered bilayer membrane systems have shown no change in membrane capacitance indicating no penetration or disruption of membranes by SWCNTs. Preliminary results with Langmuir monolayers of lipids exposed to SWCNTs shows no significant increase in surface pressure from the control also indicating no significant uptake of SWCNTs. In sum, results support cellular mediated endocytosis of SWCNTs as the preferred mechanism for cellular uptake.

Atomic Force Microscopy Force Mapping in the Study of Supported Lipid Bilayers

Investigating the structural and mechanical properties of lipid bilayer membrane systems is vital in elucidating their biological function. One route to directly correlate the morphology of phase-segregated membranes with their indentation and rupture mechanics is the collection of atomic force microscopy (AFM) force maps. These force maps, while containing rich mechanical information, require lengthy processing time due to the large number of force curves needed to attain a high spatial resolution. A force curve analysis toolset was created to perform data extraction, calculation and reporting specifically in studying lipid membrane morphology and mechanical stability. The procedure was automated to allow for high-throughput processing of force maps with greatly reduced processing time. The resulting program was successfully used in systematically analyzing a number of supported lipid membrane systems in the investigation of their structure and nanomechanics.

Flow Induced Multilamellar Vesicle Formation of Bilayer Membrane Systems

Flow Induced Multilamellar Vesicle Formation of Bilayer Membrane Systems

Study of the electrical properties of bi-layer ion-exchange membrane systems

The electrical properties of ion-exchange membrane systems with a bi-layer distribution of the fixed-charge inside the membrane, have been analyzed by using the network simulation method. The system under study is constituted by an inhomogeneous membrane with a bi-layer distribution of the fixed-charge and two diffusion boundary layers on both sides of the membrane, the ionic transport of a symmetric 1:1 electrolyte being described by the Nernst-Planck and Poisson equations. The steady-state, transient and small-amplitude ac responses of the membrane system, have been obtained. The influence of the degree of inhomogeneity of the membranes on the steady-state current–voltage characteristic, the electrochemical impedance and the chronoamperometric response of the system, has been analyzed and discussed.

Probing Cholesterol Dependent Lipid Properties Using Molecular Dynamics Simulations of BODIPY-PC in Explicit DMPC and DPPC Membranes

Dunn and co-workers have used polarized total internal reflection fluorescence microscopy to measure the tilt angle of a single molecule (BODIPY-PC) relative to the membrane normal to characterize the membrane properties. Recently, they found that increasing quantities of cholesterol in monolayer and bilayer membrane systems leads to a much smaller tilt angle of the BODIPY-PC molecule. To better understand the influence of cholesterol on membrane properties and its relation to BODIPY-PC tilting, we have performed molecular dynamics (MD) simulations of BODIPY-PC in explicit dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphocholine (DMPC) monolayer and bilayer membrane systems with systematically increasing concentrations of cholesterol (0, 5, 10, 15, 20, 33, 50%). After 4 ns of equilibration, each system has been simulated for another 16 ns. Preliminary results indicate that the BODIPY-PC tilt angle decreases as a function of increasing cholesterol concentration until 10% cholesterol after which there appears to be cholesterol saturation and the rigidifying affect ceases. Other points of interest include confirming the applicability of monolayer data for bilayer systems and explaining any quantifiable differences between the DPPC and DMPC membrane systems. We will present the orientation (azimuthal and rotational angle distribution) of BODIPY-PC, the BODIPY-PC tilting energetics, various membrane properties, and the spatial distribution of the cholesterol as a function of cholesterol concentration.

二分子膜系ラメラ相における流動誘起多層膜ベシクル構造形成

Shear-induced instability of bilayer membrane systems has attracted much attention. One of the most interesting shear effects is a shear-induced structural transition from the lamellar to the multilamellar vesicle. In this review, we show two topics related to the non-equilibrium structural formation phenomena under shear; the influence of the triblock copolymer on the shear-induced multilamellar vesicle formation, and the shear quench-induced discontinuous size change of the multilamellar vesicle. In the first topic, we study the effect of the triblock copolymer on the shear-induced lamellar-to-multilamellar vesicle formation. Shear-induced multilamellar vesicle formation behavior is quite sensitive to the interaction between grafted polymer chains and surfactant bilayer membranes. Entropy based contribution to the modulus of bilayer controls the shear-induced multilamellar vesicle formation behavior. In the second topic, we study how the kinetic pathway on the size change process of the multilamellar vesicle is decided under shear. These studies will give us a hint how the soft matter systems change their structure under non-equilibrium states.

Simulations of the Morphological Evolution of Lipid Bilayer Membranes Using a Phase-Field Method

We present a continuum-level simulation method for modeling phase separation and morphological evolution of multicomponent lipid bilayer membranes. Our objective is to investigate how various physical parameters input into the model, such as spontaneous curvature, phase fraction, and interleaflet coupling strength, affect the dynamics and equilibrium morphological phases formed in two-phase lipid bilayer membrane systems. The model applies to membranes with planar and spherical background geometries, simulating a nearly planar portion of membrane or entire vesicle, respectively. The compositions and shape of the membrane are coupled through a modified Helfrich free energy. The planar model treats the composition of each leaflet, and thus includes a term coupling these compositions across the bilayer. The compositional evolution is modeled using a phase-field method and is described by a Cahn-Hilliard-type equation, while shape changes are described by relaxation dynamics. For nearly planar bilayer systems with each leaflet having the same phase fraction, we find that domains in both leaflets align to reduce the interaction energy, as expected. When the coupling effect is stronger, this alignment occurs more quickly and more precisely, showing that the coupling affects the dynamics. This leaflet coupling is found to heavily influence morphological evolution; in some cases the equilibrium morphological phase observed is very different from what was observed with our simpler monolayer model using similar conditions. We construct a phase diagram of equilibrium morphological phases in the composition space for a few values of the strength of the leaflet coupling. This model has been able to reproduce results found in lipid bilayer experiments probing interleaflet interactions, including the effect of domain registration across leaflets. For the vesicle model, we investigate how an ellipsoidal geometry imposed in the initial conditions affects the phase and morphological evolution.

New BODIPY lipid probes for fluorescence studies of membranes

Many fluorescent lipid probes tend to loop back to the membrane interface when attached to a lipid acyl chain rather than embedding deeply into the bilayer. To achieve maximum embedding of BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore into the bilayer apolar region, a series of sn-2 acyl-labeled phosphatidylcholines was synthesized bearing 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me(4)-BODIPY-8) at the end of C(3)-, C(5)-, C(7)-, or C(9)-acyl. A strategy was used of symmetrically dispersing the methyl groups at BODIPY ring positions 1, 3, 5, and 7 to decrease fluorophore polarity. Iodide quenching of the phosphatidylcholine probes in bilayer vesicles confirmed that the Me(4)-BODIPY-8 fluorophore was embedded in the bilayer. Parallax analysis of Me(4)-BODIPY-8 fluorescence quenching by phosphatidylcholines containing iodide at different positions along the sn-2 acyl chain indicated that the penetration depth of Me(4)-BODIPY-8 into the bilayer was determined by the length of the linking acyl chain. Evaluation using monolayers showed minimal perturbation of <10 mol% probe in fluid-phase and cholesterol-enriched phosphatidylcholine. Spectral characterization in monolayers and bilayers confirmed the retention of many features of other BODIPY derivatives (i.e., absorption and emission wavelength maxima near 498 nm and approximately 506-515 nm) but also showed the absence of the 620-630 nm peak associated with BODIPY dimer fluorescence and the presence of a 570 nm emission shoulder at high Me(4)-BODIPY-8 surface concentrations. We conclude that the new probes should have versatile utility in membrane studies, especially when precise location of the reporter group is needed.

Absence of clustering of phosphatidylinositol-(4,5)-bisphosphate in fluid phosphatidylcholine

Phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] plays a key role in the modulation of actin polymerization and vesicle trafficking. These processes seem to depend on the enrichment of PI(4,5)P(2) in plasma membrane domains. Here, we show that PI(4,5)P(2) does not form domains when in a fluid phosphatidylcholine matrix in the pH range of 4.8-8.4. This finding is at variance with the spontaneous segregation of PI(4,5)P(2) to domains as a mechanism for the compartmentalization of PI(4,5)P(2) in the plasma membrane. Water/bilayer partition of PI(4,5)P(2) is also shown to be dependent on the protonation state of the lipid.

Self‐Assembled Nanofibrillar Aggregates with Amphiphilic and Lipophilic Molecules

AbstractSummary: This article gives a review on self‐assembled nanofibrillar aggregates such as helical, twisted ribbon‐like and tubular forms, those are produced in aqueous bilayer membrane and organogel systems. Two common features necessary for the chemical structure that yields special morphology are a chiral carbon atom and moieties feasible for intermolecular interactions although there are some exceptions. In aqueous systems, a hydrophobic effect is also an essential driving force for molecular aggregates in aqueous solution systems but almost disappear in organic media. More positive intermolecular interactions play an important role in molecular aggregation in organic media. Hydrogen bonding interaction is especially effective and many organogelators are classified into this category. Some lipophilic peptides have been investigated not only as organogelators but also with respect to their self‐assembling behaviors. This latter property gives them distinct advantages compared with conventional gel systems because the gels include highly‐ordered structures supramolecular functions like aqueous lipid membranes through molecular orientation. This article also introduces applicability of the organogel system.

A synthesis and properties of new 4,4-difluoro-3a,4a-diaza-s-indacene (BODIPY)-labeled lipids

A series of fluorescently labeled fatty acids of various chain lengths with 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me4-BODIPY-8) residue in the omega-position were synthesized. These acids were used to prepare new fluorescently labeled phosphatidylcholines, sphingomyelin, and galactosyl ceramide. Taking into account the symmetry of the Me4-BODIPY-8-fluorophore, one can presume that, in most bilayer membrane systems, this fluorophore is would be embedded into the bilayer.

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, λ max, was found to be 480, 430, and 500 nm for the liquid crystalline (L α), ripple gel ( P ′ β ), and pressure-induced interdigitated gel (L βI) phase, respectively. Since the λ max reflects the solvent property around the probe molecules, we could speculate on the location of the Prodan molecules in the bilayer membranes; in the L α phase of the lipid bilayer, the Prodan molecules distribute around the phosphate of the lipids (i.e. the polar region). The L α/ P ′ β 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 ′ β 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, F 480/ F 430, showed a sharp change at the phase-transition pressure. In the case of the DPPC and DSPC bilayers, the values of F 480/ F 430 showed an abrupt increase above a certain pressure higher than the L α/ P ′ β transition pressure, which corresponds to the interdigitation from the P ′ β to the L βI phase. The plot of F 480/ F 430 versus pressure is available for recognition of the bilayer phase transitions, especially the bilayer interdigitation.

Topological Relaxation of a Shear-Induced Lamellar Phase to Sponge Equilibrium and the Energetics of Membrane Fusion

We report time-resolved small angle neutron scattering (t-SANS) measurements of the topological relaxation of Couette shear-induced stacked L(alpha) lamellar states to their multiconnected isotropic L3 sponge equilibrium phases in a surfactant bilayer membrane system. Comparison of this structural relaxation time to the interval between diffusive membrane contacts, as determined from dynamic light scattering or estimated from the shear rates required for L(alpha) saturation, allows us to determine the activation energy barrier to the membrane fusion process reestablishing the solution channel handles that characterize the sponge phase.

Investigating fatty acids inserted into magnetically aligned phospholipid bilayers using EPR and solid-state NMR spectroscopy

This is the first time 2H solid-state NMR spectroscopy and spin-labeled EPR spectroscopy have been utilized to probe the structural orientation and dynamics of a stearic acid incorporated into magnetically aligned phospholipid bilayers or bicelles. The data gleaned from the two different techniques provide a more complete description of the bilayer membrane system. Both methods provided similar qualitative information on the phospholipid bilayer, high order, and low motion for the hydrocarbon segment close to the carboxyl groups of the stearic acid and less order and more rapid motion at the end towards the terminal methyl groups. However, the segmental order parameters differed markedly due to the different orientations that the nitroxide and C–D bond axes transform with the various stearic acid acyl chain conformations, and because of the difference in dynamic sensitivity between NMR and EPR over the timescales examined. 5-, 7-, 12-, and 16-doxylstearic acids spin-labels were used in the EPR experiments and stearic acid-d 35 was used in the solid-state NMR experiments. The influence of the addition of cholesterol and the variation of temperature on the fatty acid hydrocarbon chain ordering in the DMPC/DHPC phospholipid bilayers was also studied. Cholesterol increased the degree of ordering of the hydrocarbon chains. Conversely, as the temperature of the magnetically aligned phospholipid bilayers increased, the order parameters decreased due to the higher random motion of the acyl chain of the stearic acid. The results indicate that magnetically aligned phospholipid bilayers are an excellent model membrane system and can be used for both NMR and EPR studies.

Der Stellenwert von Integra1 als Dermisäquivalent in der primären und sekundären Verbrennungschirurgie

mary and Secondary Burn Surgery. Since introduction of Integra in 1980 as a synthetic bilayer membrane system for skin replacement, there is an increasing number of publications concerning the indications. First used as a temporary coverage after excised deep burns, the product was established as a dermal equivalente in primary burn surgery and particular in secondary reconstruction of scars. Besides these indications, there is an extended application of Integra according chronic wounds. The mechanical and biological features of the artificial dermis protect the tissue from fluid loss and bacterial invasion in open wounds. The Integra-induced formation of a neodermis through a straight matrix leads to an evident functionally and aesthetically highly acceptable scar.

Analysis of antimicrobial peptide interactions with hybrid bilayer membrane systems using surface plasmon resonance

The lipid binding behaviour of the antimicrobial peptides magainin 1, melittin and the C-terminally truncated analogue of melittin (21Q) was studied with a hybrid bilayer membrane system using surface plasmon resonance. In particular, the hydrophobic association chip was used which is composed of long chain alkanethiol molecules upon which liposomes adsorb spontaneously to create a hybrid bilayer membrane surface. Multiple sets of sensorgrams with different peptide concentrations were generated. Linearisation analysis and curve fitting using numerical integration analysis were performed to derive estimates for the association ( k a) and dissociation ( k d) rate constants. The results demonstrated that magainin 1 preferentially interacted with negatively charged dimyristoyl- l-α-phosphatidyl- dl-glycerol (DMPG), while melittin interacted with both zwitterionic dimyristoyl- l-α-phosphatidylcholine and anionic DMPG. In contrast, the C-terminally truncated melittin analogue, 21Q, exhibited lower binding affinity for both lipids, showing that the positively charged C-terminus of melittin greatly influences its membrane binding properties. Furthermore the results also demonstrated that these antimicrobial peptides bind to the lipids initially via electrostatic interactions which then enhances the subsequent hydrophobic binding. The biosensor results were correlated with the conformation of the peptides determined by circular dichroism analysis, which indicated that high α-helicity was associated with high binding affinity. Overall, the results demonstrated that biosensor technology provides a new experimental approach to the study of peptide-membrane interactions through the rapid determination of the binding affinity of bioactive peptides for phospholipids.

Artificial skin, split-thickness autograft and cultured autologous keratinocytes combined to treat a severe burn injury of 93% of TBSA

Despite refinements in burn shock resuscitation, improvements in surgical techniques, advances in intensive care medicine and the presence of very expert surgeons, the treatement of patients with severe burns exceeding 60% TBSA remains a big challenge. A major problem in the treatment of severe burn injuries is the lack of autologous skin. In selected cases cultured epidermal autograft (CEA) may be used. However, they are available only 2–3 weeks after biopsy, thus requiring a temporary wound closure after necrosectomy. A new option is Integra TM, an artificial skin consisting of a bilayer membrane system. The three-dimensional porous matrix from bovine tendon collagen and a glycosaminoglycan layer is covered by a silicon sheet. The latter prevents fluid loss from the wounds and serves as a barrier against germ invasion. After adequate vascularisation of the dermal template, the silicon layer is removed and replaced by a thin autograft. We present a 26-year old male who sustained a 93% TBSA burn injury (60% full-thickness burn, 33% partial-thickness burn). He was treated with artificial skin, split-thickness autograft and CEA in combination. The clinical history and the follow-up of approx. 1 year are presented and the results discussed. We consider the survival of this patient being a result of the therapeutic progress of the recent decades.

Effect of local anesthetics on the phase transition temperatures of ether- and ester-linked phospholipid bilayer membranes

The phase-transition temperatures of ester-linked dipalmitoylphosphatidylcholine (DPPC) and ether-linked dihexadecylphosphatidylcholine (DHPC) bilayer membranes were measured as a function of the concentration of local anesthetics, dibucaine (DC·HCl), tetracaine (TC·HCl), bupivacaine (BC·HCl), lidocaine (LC·HCl) and procaine hydrochlorides (PC·HCl). The temperature of the main transition for both lipid bilayers was depressed by all the anesthetics added in a dose-dependent manner; the order of the depression of transition temperature was DC·HCl>TC·HCl>BC·HCl>LC·HCl>PC·HCl. The pretransition temperature of DPPC bilayer membrane from the lamellar gel ( L β ′) to the ripple gel ( P β ′) phase was depressed by the addition of local anesthetics, while the DHPC bilayer membrane undergoes the pretransition from the interdigitated gel ( L β I) to the P β ′ phase and the elevation of pretransition temperature was observed by the addition of local anesthetics. The presence of local anesthetics stabilize the L β I phase of lipid bilayer membranes. When the more hydrophobic anesthetics such as DC·HCl and TC·HCl were added sufficiently to the bilayer membrane system, the interdigitation of bilayers was induced in the DPPC membrane system and the P β ′ phase disappeared in the DHPC membrane system. From the colligative thermodynamic framework, three kinds of differential partition coefficients corresponding to three phase-transitions, K( L α )– K( P β ′), K( P β ′)– K( L β ′) and K( P β ′)– K( L β I), where K( L α ), K( P β ′), K( L β ′) and K( L β I) refer to the partition coefficients of an anesthetic into the L α , P β ′, L β ′ and L β I phases of lipid bilayer membranes, were evaluated from the depression (or elevation) of the phase-transition temperature. The transfer free energy of anesthetics from the aqueous phase to the L α phase of bilayers, Δ G tr ∘=− RTln K( L α ), was well correlated to the local anesthetic potencies.

Evidence for Nonlinear Capacitance in Biomembrane Channel System

The electrophysiological properties of voltage-dependent anion channels from mitochondrial membrane have been studied in a bilayer membrane system. It was observed that the probability of opening of the membrane channel depends on externally applied voltage and the plot is a bell-shaped curve symmetric around probability axis. A scheme of conformational energy levels under varying externally applied voltage was formulated. Assuming that the probability follows Boltzmann distribution, we arrive at an expression of change in energy containing a separate term identical to the energy of a capacitor. This fact indicates the possibility of existence of an added capacitance due to the channel protein. Further it was shown that the aforesaid channel capacitor could be a function of voltage leading to nonlinearity. We have offered a general method of calculating nonlinear capacitance from the experimental data on opening probability of a membrane channel. In case of voltage-dependent anion channel the voltage dependence of the capacitor has a power 0.786. The results have been interpreted in view of the structural organization of the channel protein in the membrane. Our hypothesis is that the phenomenon of capacitor behaviour is a general one for membrane channels.

26] Construction of biologically active protein molecular architecture using self-assembling peptide-amphiphiles

The peptide-amphiphiles described here provide a simple approach for building stable protein structural motifs using peptide head groups. One of the most intriguing features of this system is the possible formation of stable lipid films on solid substrates, or the use of the novel amphiphiles in bilayer membrane systems, where the lipid tail serves not only as a peptide structure-inducing agent but also as an anchor of the functional head group in the lipid assembly. The peptide-amphiphile system potentially offers great versatility with regard to head and tail group composition and overall geometries and macromolecular structures. For building materials with molecular and cellular recognition capacity, it is essential to have a wide repertoire of tools to produce characteristic supersecondary structures at surfaces and interfaces.