Effects of silver nanoparticles on the fluidity of bilayer in phospholipid liposome

This paper describes the formation and characterization of liposome entrapping the silver nanoparticles in bilayer. Silver nanoparticles were entrapped in the bilayer of dipalmitoylphosphatidylcholine (DPPC) liposome, named as silver-loaded liposome. Specifically, above the gel to liquid-crystalline phase transition temperature of this lipid (i.e., 41 degrees C), it was observed that membrane fluidities of silver-loaded liposomes were increased, and fluorescence anisotropy values were reduced from 0.114 to 0.097. This might be due to the structural modifications and interactions between DPPC molecules and silver nanoparticles within the bilayer. It was also confirmed that silver nanoparticles were entrapped in hydrophobic region of lipid bilayer with transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) measurements.     

Purification and analysis of mono‐PEGylated HSA by hydrophobic interaction membrane chromatography

We discuss the purification of mono‐PEGylated HSA by hydrophobic interaction membrane chromatography. The hydrophobicity difference between the different fractionated species was induced by the addition of a lyotropic salt that caused phase transition of PEG (hydrophilic under normal condition) to a mildly hydrophobic form. The HSA PEGylation reaction mixture was mixed with lyotropic salt and passed through a stack of hydrophilized polyvinylidene fluoride membrane discs. Unmodified HSA was obtained in the flow through, while the PEGylated forms of the protein bound to the membrane and could be eluted by reducing the salt concentration. Among the three major PEGylated forms of HSA present in the feed (i.e. mono–, di–, and tri–), mono‐PEGylated HSA was eluted first and could be resolved from the others. The purified material was analyzed by SDS‐PAGE, dynamic light scattering, and SEC combined with multi‐angle light scattering. All these analytical techniques indicated the presence of species that has a molar mass consistent with mono‐PEGylated HSA. A scaled‐down version of the membrane chromatographic methods could be used for the rapid and sensitive analysis of PEGylated proteins.     

Vertical and directional insertion of helical peptide into lipid bilayer membrane

A novel helical hexadecapeptide carrying a poly(ethylene glycol) (PEG) chain at the N terminal was synthesized. The N and C terminals of the compound are labeled with a fluorescein isothiocyanate (FITC) group and an N-ethylcarbazolyl group (ECz), respectively. An octapeptide carrying the same groups and a hexadecapeptide without a PEG chain were also synthesized and used as control. A mixture of the peptide and dimyristoylphosphatidylcholine was sonicated in a buffer to prepare the liposome. The orientation as well as direction of the helical segment in the lipid bilayer were analyzed by quenching experiments of the FITC and the ECz fluorescence. The results clearly indicated that the helical segment of the peptide penetrated into the lipid bilayer with vertical orientation in both the gel and liquid crystalline states of the lipid bilayer. Notably, the bulky N terminal was left behind in the outer aqueous phase of liposome, meaning that the C terminal of the peptide points to the inner aqueous phase of liposome. The insertion mode of the helical peptide into a bilayer membrane is therefore well-regulated in terms of the orientation and the directionality by designing the balance between the PEG chain and the helix length. The methodology presented here will initiate a way to construct artificial functional molecular systems that can induce vectorial transport phenomena as seen in biological systems.     

Interaction of a solid supported liquid-crystalline phospholipid membrane with physical vapor deposited metal atoms

A metal (Ag, Au, Pd, Sn, Bi, In, Co, Al) layer directly deposited onto a liquid-crystalline lipid membrane resulted in different morphologies, ranging from a monolayer of discrete particles to a continuous film, depending on the metal's oxidation tendency.     

Binding of Lipid Vesicles to Protein-Coated Solid Polymer Surfaces: A Model for Cell Adhesion to Artificial Biocompatible Materials

The adhesion of lipid vesicles (liposomes) having controlled chemical and physical structure to polymer supported human serum albumin (HSA) thin layers was investigated by a spectrofluorimetric technique. The vesicle lipid bilayer was labeled with a small amount of an apolar fluorescent probe (diphenylexathriene) and the vesicle suspension was set in contact with the protein film. After washing and drying, the adhering vesicles containing sample was dissolved in chloroform and the homogeneous solution was analyzed by standard spectrofluorimetric techniques. Different parameters of the lipid bilayer, suspending solution, and protein film were varied and their influence on the liposome binding was investigated. Concerning the lipid bilayer, we studied the effect of liposome surface charge by using different mixtures of neutral (dipalmitoyl-phosphatidylcholine) and charged (dipalmitoyl-phosphatidic acid) phospholipids and the fluid or gel nature of the lipid bilayer (switched on and off by temperature variation). Variations of the local environment involve Ca(2+) and H(+) changes in the millimolar range as well as different hydrodynamical flows (in the range 0.1-10 cm/s). Preliminary measurements using different protein layers were also performed. Results show: (a) negligible adhesion without the protein layer, (b) the presence of a maximum for the liposome adhesion vs ion concentration (depending on the liposome composition and kind of the adsorbed ions), (c) a much stronger adhesion for vesicles in the fluid phase (overcoming the entropy-driven desorption increase with temperature), and (d) a dramatic lowering of the adhesion capability under hydrodynamic flow. Points a-c have been interpreted on the basis of a simple mechanoelectrical model. Copyright 2000 Academic Press.     

Langmuir balance investigation of superoxide dismutase interactions with mixed-lipid monolayers

Higher than theoretical encapsulation efficiencies in liposomes of the cytoplasmic protein, superoxide dismutase (SOD), were previously observed. The high encapsulation of SOD led to the consideration of lipid-protein interactions and the embedding of SOD in the lipid bilayer. Difficulty in other methods such as dynamic scanning calorimetry due to cholesterol obscuring the measurements brought about the interest for a modified Langmuir monolayer relaxation study. A novel method was devised to distinguish between different lipid compositions that formed either a favorable or an unfavorable environment for SOD. Normalized monolayer relaxations with SOD were compared between mixed-lipid compositions containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol (Chol). Lipid-monolayer relaxation with and without SOD in the subphase was plotted over 30 min to determine if the protein was altering the lipid-monolayer relaxation. The monolayer relaxation with SOD was normalized to the monolayer relaxation without SOD over the 30 min period. The results indicated that lipid length and mole percent of cholesterol were important parameters that must be adjusted in order to support a favorable environment for SOD interaction with the lipid. It was determined that hydrophobic interactions were dominant over electrostatic forces; thus, SOD was embedding into the lipid monolayer. Additionally, this study was correlated to a previous liposome study and proved that lipid-protein interactions were the reason for the higher encapsulation efficiencies. The significance of this method is that it (1) provides a connection between lipid-protein interactions observed in monolayers and bilayers and (2) establishes a simple and effective manner to test lipid compositions for lipid-protein interaction that will aid in optimization of liposome encapsulation efficiency.     

Interactions of complementary PEGylated liposomes and characterization of the resulting aggregates

The interaction of complementary liposomes bearing both recognizable and protective ligands at their external surface has been investigated. Aggregation of hydrogenated phosphatidyl choline/cholesterol (2:1 molar ratio) based liposomes was mediated by the molecular recognition of the complementary phosphate and guanidinium groups incorporated in separate unilamellar liposomes. The phosphate group was incorporated in the bilayer employing dihexadecyl phosphate, while the guanidinium moiety was introduced in the membrane through the incorporation of various guanidinium lipids. For the latter, anchoring ability and primarily introduction of a spacer group between their lipophilic part and the guanidinium group was found to affect the ability for molecular recognition. Also, poly(ethylene glycol) (PEG) introduced in both types of liposomes at various concentrations and up to 15% with respect to cholesterol modifies the interaction effectiveness and morphology of the obtained aggregates. Interaction of these complementary liposomes leads to large precipitating aggregates or fused liposomes, as shown by phase contrast microscopy and dynamic light scattering. Specifically, fusion of liposomes takes place under a nonleaking process involving lipid mixing, as demonstrated by calcein entrapment and resonance energy transfer experiments. Calorimetric parameters also correlate with the processes of aggregation and fusion. The interactions of non-PEGylated liposomes involve exothermic processes of higher enthalpic content than those of the PEGylated counterparts.     

Relationships between equilibrium spreading pressure and phase equilibria of phospholipid bilayers and monolayers at the air-water interface

The intricate interplay between the bilayer and monolayer properties of phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) phospholipids, in relation to their polar headgroup properties, and the effects of chain permutations on those polar headgroup properties have been demonstrated for the first time with a set of time-independent bilayer-monolayer equilibria studies. Bilayer and monolayer phase behavior for PE is quite different than that observed for PC and PG. This difference is attributed to the characteristic biophysical PE polar headgroup property of favorable intermolecular hydrogen-bonding and electrostatic interactions in both the bilayer and monolayer states. This characteristic hydrogen-bonding ability of the PE polar headgroup is reflected in the condensed nature of PE monolayers and a decrease in equilibrium monolayer collapse pressure at temperatures below the monolayer critical temperature, T(c) (whether above or below the monolayer triple point temperature, T(t)). This interesting phenomena is compared to equilibrated PC and PG monolayers which collapse to form bilayers at 45 mN/m at temperatures both above and below monolayer T(c). Additionally, it has been demonstrated by measurements of the equilibrium spreading pressure, pie, that at temperatures above the bilayer main gel-to-liquid-crystalline phase-transition temperature, T(m), all liquid-crystalline phospholipid bilayers spread to form monolayers with pie around 45 mN/m, and spread liquid-expanded equilibrated monolayers collapse at 45 mN/m to form their respective thermodynamically stable liquid-crystalline bilayers. At temperatures below bilayer T(m), PC and PG gel bilayers exhibit a drop in bilayer pi(e) values < or =0.2 mN/m forming gaseous monolayers, whereas the value of pic of spread monolayers remains around 45 mN/m. This suggests that spread equilibrated PC and PG monolayers collapse to a metastable liquid-crystalline bilayer structure at temperatures below bilayer T(m) (where the thermodynamically stable bilayer liquid-crystalline phase does not exist) and with a surface pressure of 45 mN/m, a surface chemical property characteristically observed at temperatures above bilayer T(m) (monolayer T(c)). In contrast, PE gel bilayers, which exist at temperatures below bilayer T(m) but above bilayer T(s) (bilayer crystal-to-gel phase-transition temperature), exhibit gel bilayer spreading to form equilibrated monolayers with intermediate pie values in the range of 30-40 mN/m; however, bilayer pie and monolayer pic values remain equal in value to one another. Contrastingly, at temperatures below bilayer T(s), PE crystalline bilayers exhibit bilayer pie values < or =0.2 mN/m forming equilibrated gaseous monolayers, whereas spread monolayers collapse at a value of pic remaining around 30 mN/m, indicative of metastable gel bilayer formation.     

Asymmetry of lipid bilayers induced by monovalent salt: atomistic molecular-dynamics study

Interactions between salt ions and lipid components of biological membranes are essential for the structure, stability, and functions of the membranes. The specific ionic composition of aqueous buffers inside and outside of the cell is known to differ considerably. To model such a situation we perform atomistic molecular-dynamics (MD) simulations of a single-component phosphatidylcholine lipid bilayer which separates two aqueous reservoirs with and without NaCl salt. To implement the difference in electrolyte composition near two membrane sides, a double bilayer setup (i.e., two bilayers in a simulation box) is employed. It turns out that monovalent salt, being in contact with one leaflet only, induces a pronounced asymmetry in the structural, electrostatic, and dynamical properties of bilayer leaflets after 50 ns of MD simulations. Binding of sodium ions to the carbonyl region of the leaflet which is in contact with salt results in the formation of "Na-lipids" complexes and, correspondingly, reduces mobility of lipids of this leaflet. In turn, attractive interactions of chloride ions (mainly located in the aqueous phase close to the water-lipid interface) with choline lipid groups lead to a substantial (more vertical) reorientation of postphatidylcholine headgroups of the leaflet adjoined to salt. The difference in headgroup orientation on two sides of a bilayer, being coupled with salt-induced reorientation of water dipoles, leads to a notable asymmetry in the charge-density profiles and electrostatic potentials of bilayer constitutes of the two leaflets. Although the overall charge density of the bilayer is found to be almost insensitive to the presence of salt, a slight asymmetry in the charge distribution between the two bilayer leaflets results in a nonzero potential difference of about 85 mV between the two water phases. Thus, a transmembrane potential of the order of the membrane potential in a cell can arise without ionic charge imbalance between two aqueous compartments.     

Effects of cholesterol component on molecular interactions between paclitaxel and phospholipid within the lipid monolayer at the air-water interface

Cholesterol is a main component of the cell membrane and could have significant effects on drug-cell membrane interactions and thus the therapeutic efficacy of the drug. It also plays an important role in liposomal formulation of drugs for controlled and targeted delivery. In this research, Langmuir film technique, atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) are employed for a systematic investigation on the effects of cholesterol component on the molecular interactions between a prototype antineoplastic drug (paclitaxel) and 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) within the cell membrane by using the lipid monolayer at the air-water interface as a model of the lipid bilayer membrane and the biological cell membrane. Analysis of the measured surface pressure (pi) versus molecular area (a) isotherms of the mixed DPPC/paclitaxel/cholesterol monolayers at various molar ratios shows that DPPC, paclitaxel and cholesterol can form a non-ideal miscible system at the air-water interface. Cholesterol enhances the intermolecular forces between paclitaxel and DPPC, produces an area-condensing effect and thus makes the mixed monolayer more stable. Investigation of paclitaxel penetration into the mixed DPPC/cholesterol monolayer shows that the existence of cholesterol in the DPPC monolayer can considerably restrict the drug penetration into the monolayer, which may have clinical significance for diseases of high cholesterol. FTIR and AFM investigation on the mixed monolayer deposited on solid surface confirmed the obtained results.     

Forming Lipid Bilayer Membrane Arrays on Micropatterned Polyelectrolyte Film Surfaces

A novel method of forming lipid bilayer membrane arrays on micropatterned polyelectrolyte film surfaces is introduced. Polyelectrolyte films were fabricated by the layer‐by‐layer technique on a silicon oxide surface modified with a 3‐aminopropyltriethoxysilane (APTES) monolayer. The surface pK_a value of the APTES monolayer was determined by cyclic voltammetry to be approximately 5.61, on the basis of which a pH value of 2.0 was chosen for layer‐by‐layer assembly. Micropatterned polyelectrolyte films were obtained by deep‐UV (254 nm) photolysis though a mask. Absorbed fluorescent latex beads were used to visualize the patterned surfaces. Lipid bilayer arrays were fabricated on the micropatterned surfaces by immersing the patterned substrates into a solution containing egg phosphatidylcholine vesicles. Fluorescence recovery after photobleaching studies yielded a lateral diffusion coefficient for probe molecules of 1.31±0.17 μm² s^?1 in the bilayer region, and migration of the lipid NBD PE in bilayer lipid membrane arrays was observed in an electric field.     

In situ formation and characterization of poly(ethylene glycol)-supported lipid bilayers on gold surfaces

Inclusion of a polymer cushion between a lipid bilayer membrane and a solid surface has been suggested as a means to provide a soft, deformable layer that will allow for transmembrane protein insertion and mobility. In this study, mobile, tethered lipid bilayers were formed on a poly(ethylene glycol) (PEG) support via a two-step adsorption process. The PEG films were prepared by coadsorbing a heterofunctional, telechelic PEG lipopolymer (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-2000-N-[3-(2-(pyridyldithio)propionate]) (DSPE-PEG-PDP) and a nonlipid functionalized PEG-PDP from an ethanol/water mixture, as described in a previous paper (Munro, J. C.; Frank, C. W. Langmuir 2004, 20, 3339-3349). Then a two-step lipid adsorption strategy was used. First, lipids were adsorbed onto the PEG support from a hexane solution. Second, vesicles were adsorbed and fused on the surface to create a bilayer in an aqueous environment. Fluorescence recovery after photobleaching experiments show that this process results in mobile bilayers with diffusion coefficients on the order of 2 microm2/s. The mobility of the bilayers is decreased slightly by increasing the density of tethered lipids. The formation of bilayers, and not multilayer structures, is also confirmed by surface plasmon resonance, which was used to determine in situ film thickness, and by fluorimetry, which was used to determine quantitatively the fluorescence intensity for each 18 by 18 mm sample. Unfortunately, fluorescence microscopy also shows that there are large defects on the samples, which limits the utility of this system.     

自组装共混制备PEG化基因载体

通过含PEG链段的两亲聚合物的自组装共混, 制备了基于疏水作用力的新型PEG化非病毒基因载体. 分别选用胆固醇-聚乙二醇和聚乙二醇-聚丙二醇-聚乙二醇作为共混改性剂, 研究两亲聚合物的种类对组装体在生理盐溶液中的稳定性及基因转染效率的影响. 结果表明, 疏水驱动力的大小是获得稳定的PEG化基因超分子组装体的关键. 通过对两亲聚合物中疏水链段的选择调控, 可制备稳定的PEG化基因超分子组装体, 提高基因传递体系在生理盐溶液中的稳定性及基因转染效率. 通过自组装共混, 为新型PEG化基因超分子组装体的制备提供了切实可行的新方法.     

Silver nanoparticle monolayer-to-bilayer transition at the air/water interface as studied by the GISAXS technique: application of a new paracrystal model

An original diffraction model for the analysis of grazing-incidence small-angle X-ray scattering (GISAXS) from the nanoparticle Langmuir films was developed. This model relies on the concept of the 2D hexagonal paracrystal and employs the distorted-wave Born approximation that is relevant for GISAXS measurements at the air/water interface when the angle of incidence is close to the critical value. The model comprises the cases of the close-packed nanoparticle monolayer and bilayer with the AB-type layer stacking. In this way, both the lateral (along the interface) and vertical (normal to the interface) correlations of the nanoparticle positions can be analyzed. The model was applied to an in situ GISAXS study of the formation of a silver nanoparticle Langmuir film during compression at the air/water interface in the Langmuir-Blodgett trough. Spherical nanoparticles of 5.8 ± 0.6 nm diameter were employed. Different compression stages starting from the submonolayer up to the monolayer collapse via bilayer formation were analyzed in terms of the mean lateral interparticle distance, degree of paracrystal disorder, interlayer distance, vertical disorder, and layer-stacking type in the bilayer as well as the ratio between the monolayer and bilayer coverage in the final film. The model developed is applicable to any nanoparticle Langmuir film formed at the air/liquid interface to extract structural parameters on the nanoscale. The particular results obtained have direct implications on the preparation of silver plasmonic templates with "hot spots" for surface-enhanced Raman scattering.     

Preparation and characterization of asymmetric planar supported bilayers composed of poly(bis-sorbylphosphatidylcholine) on n-octadecyltrichlorosilane SAMs

Planar supported lipid bilayers (PSLBs) have been widely studied as biomembrane models and biosensor scaffolds. For technological applications, a major limitation of PSLBs composed of fluid lipids is that the bilayer structure is readily disrupted when exposed to chemical, mechanical, and thermal stresses. A number of asymmetric supported bilayer structures, such as the hybrid bilayer membrane (HBM) and the tethered bilayer lipid membrane (tBLM), have been created as an alternative to symmetric PSLBs. In both HBMs and tBLMs, the inner monolayer is covalently attached to the substrate while the outer monolayer is typically composed of a fluid lipid. Here we address if cross-linking polymerization of the lipids in the outer monolayer of an asymmetric supported bilayer can achieve the high degree of stability observed previously for symmetric PSLBs in which both monolayers are cross-linked [E.E. Ross, L.J. Rozanski, T. Spratt, S.C. Liu, D.F. O'Brien, S.S. Saavedra, Langmuir 19 (2003) 1752]. To explore this issue, HBMs composed of an outer monolayer of a cross-linkable lipid, bis-sorbylphosphatidylcholine (bis-SorbPC), and an inner SAM were prepared and characterized. Several experimental conditions were varied: vesicle fusion time, polymerization method, and polymerization time and temperature. Under most conditions, bis-SorbPC cross-linking stabilized the HBM such that its bilayer structure was largely preserved after drying; however these films invariably contained sub-micron scale defects that exposed the hydrophobic core of the HBM. The defects appear to be caused by desorption of low molecular weight oligomers when the film is removed from water, rinsed, and dried. In contrast, poly(bis-SorbPC) PSLBs prepared under similar conditions by Ross et al. were nearly defect free. This comparison shows that formation of a cross-linked network in the outer leaflet of an asymmetric supported bilayer is insufficient to prevent lipid desorption; inter-leaflet covalent linking appears to be necessary to create supported poly(lipid) assemblies that are impervious to repeated drying and rehydration. The difference in stability is attributed to inter-leaflet cross-linking between monolayers which can form in symmetric bis-SorbPC PSLBs.     

Di‐ and tri‐phenyltin chlorides transfer across a model lipid bilayer

A compound's ability to penetrate the plasma membrane of a cell is the critical parameter that determines its potential to become a biologically potent factor. A well‐known group of organotin compounds that exhibit toxic properties in relation to biological systems are phenyltins. There are as yet no studies that in a direct manner have established whether organotin compounds such as diphenyltin dichloride (DPhT) and triphenyltin chloride (TPhT) diffuse, or not, through the lipid bilayer, although we know that at least some organotins absorb in both liposome and biological membranes. In this paper we present a series of experiments that show transfer of these compounds across the lipid membrane using the stopped‐flow technique. The results obtained demonstrate that DPhT and TPhT first adsorb onto the lipid bilayer surface, in a diffusion‐controlled manner and within a very short time (0.05 s), whereas the membrane crossing was observed to be on the order of a minute. The adsorption process was easily fitted with a single exponential for both the compounds studied, indicating a single process phenomenon. The longer time kinetics (characteristic of membrane crossing) showed a complex dependence on compound concentration and the presence of cholesterol in the membrane. On passing from the outer to the inner surface of the bilayer, organotins undergo desorption and enter the liposome interior, which has been shown in lipid monolayer desorption studies. In conclusion, it can be stated that amphiphilic DPhT and TPhT permeate the liposome membrane. Copyright © 2005 John Wiley & Sons, Ltd.     

Solubility and binding properties of PEGylated lysozyme derivatives with increasing molecular weight on hydrophobic-interaction chromatographic resins

Dynamic binding capacities and resolution of PEGylated lysozyme derivatives with varying molecular weights of poly (ethylene) glycol (PEG) with 5 kDa, 10 kDa and 30 kDa for HIC resins and columns are presented. To find the optimal range for the operating conditions, solubility studies were performed by high-throughput analyses in a 96-well plate format, and optimal salt concentrations and pH values were determined. The solubility of PEG-proteins was strongly influenced by the length of the PEG moiety. Large differences in the solubilities of PEGylated lysozymes in two different salts, ammonium sulfate and sodium chloride were found. Solubility of PEGylated lysozyme derivatives in ammonium sulfate decreases with increased length of attached PEG chains. In sodium chloride all PEGylated lysozyme derivatives are fully soluble in a concentration range between 0.1 mg protein/ml and 10 mg protein/ml. The binding capacities for PEGylated lysozyme to HIC resins are dependent on the salt type and molecular weight of the PEG polymer. In both salt solutions, ammonium sulfate and sodium chloride, the highest binding capacity of the resin was found for 5 kDa PEGylated lysozyme. For both native lysozyme and 30 kDa mono-PEGylated lysozyme the binding capacities were lower. In separation experiments on a TSKgel Butyl-NPR hydrophobic-interaction column with ammonium sulfate as mobile phase, the elution order was: native lysozyme, 5 kDa mono-PEGylated lysozyme and oligo-PEGylated lysozyme. This elution order was found to be reversed when sodium chloride was used. Furthermore, the resolution of the three mono-PEGylated forms was not possible with this column and ammonium sulfate as mobile phase. In 4 M sodium chloride a resolution of all PEGylated lysozyme forms was achieved. A tentative explanation for these phenomena can be the increased solvation of the PEG polymers in sodium chloride which changes the usual attractive hydrophobic forces in ammonium sulfate to more repulsive hydration forces in this hydrotrophic salt.     

Fluorescence studies of the interactions of ubiquinol-10 with liposomes

Ubiquinone-10 plays a central role in energy production and its reduced form, ubiquinol-10 is also capable of acting as a potent radical scavenging antioxidant against membrane lipid peroxidation. Efficiency of this protection depends mostly on its localization in lipid bilayer. The intrinsic fluorescence of ubiquinol-10 and of the exogenous probe, Laurdan, has been used to determine the location of ubiquinol-10 in unilamellar liposomes of egg phosphatidylcholine (EggPC) and dimyristoyl phosphatidylcholine. Laurdan fluorescence moiety is positioned at the hydrophilic-hydrophobic interface of the phospholipid bilayer and its parameters reflect the membrane polarity and microheterogeneity, which we have used to explore the coexistence of microdomains with distinct physical properties. In liquid-crystalline bilayers ubiquinol has a short fluorescence lifetime (0.4 ns) and a high steady-state anisotropy. In a concentration-dependent manner, ubiquinol-10 influences the Laurdan excitation, emission and generalized polarization measurements. In EggPC liposomes ubiquinol-10 induces a decrease in membrane water mobility near the probe, while in dimyristoyl liposomes a decrease in the membrane water content was found. Moreover the presence of ubiquinol results in the formation of coexisting phospholipid domains of gel and liquid-crystalline phases. The results indicate that ubiquinol-10 molecules are mainly located at the polar-lipid interface, inducing changes in the physico-chemical properties of the bilayer microenvironment.     

Effects of ionic strength and surface charge on protein adsorption at PEGylated surfaces

PEGylated Nb2O5 surfaces were obtained by the adsorption of poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) copolymers, allowing control of the PEG surface density, as well as the surface charge. PEG (MW 2 kDa) surface densities between 0 and 0.5 nm(-2) were obtained by changing the PEG to lysine-mer ratio in the PLL-g-PEG polymer, resulting in net positive, negative and neutral surfaces. Colloid probe atomic force microscopy (AFM) was used to characterize the interfacial forces associated with the different surfaces. The AFM force analysis revealed interplay between electrical double layer and steric interactions, thus providing information on the surface charge and on the PEG layer thickness as a function of copolymer architecture. Adsorption of the model proteins lysozyme, alpha-lactalbumin, and myoglobin onto the various PEGylated surfaces was performed to investigate the effect of protein charge. In addition, adsorption experiments were performed over a range of ionic strengths, to study the role of electrostatic forces between surface charges and proteins acting through the PEG layer. The adsorbed mass of protein, measured by optical waveguide lightmode spectroscopy (OWLS), was shown to depend on a combination of surface charge, protein charge, PEG thickness, and grafting density. At high grafting density and high ionic strength, the steric barrier properties of PEG determine the net interfacial force. At low ionic strength, however, the electrical double layer thickness exceeds the thickness of the PEG layer, and surface charges "shining through" the PEG layer contribute to protein interactions with PLL-g-PEG coated surfaces. The combination of AFM surface force measurements and protein adsorption experiments provides insights into the interfacial forces associated with various PEGylated surfaces and the mechanisms of protein resistance.     

In Situ Electrodeposition of an Asymmetric Sol–Gel Membrane Based on an Octadecyltrimethoxysilane Langmuir Film

The unique properties of Langmuir film formation were utilized in assembling a thin skin of an asymmetric membrane. An octadecyltrimethoxysilane (ODTMS) Langmuir monolayer was formed at the air–water interface and served as the substrate for growing a bulky sol–gel polymer in situ. The latter was based on the electrochemical deposition of tetramethoxysilane dissolved in the water subphase by means of horizontal touch electrochemistry. The resultant asymmetric layer that consisted of a thin hydrophobic ODTMS Langmuir film connected to a bulk hydrophilic sol–gel network was studied in situ and ex situ by using various techniques, such as cyclic voltammetry, electrochemical impedance spectroscopy (EIS), scanning electron microscopy, transmission electron microscopy (TEM), and goniometry. We found that a porous hydrophilic film grew on top of a hydrophobic layer as was evident from TEM, contact angle, and EIS analyses. The film thickness and film permeability could be controlled by changing the deposition conditions such as the potential window applied and its duration. Hence, this method offers an alternative approach for assembling asymmetric films for various applications