Magnesium rescues the morphology of Bacillus subtilis mreB mutants through its inhibitory effect on peptidoglycan hydrolases.

Cell wall homeostasis in bacteria is tightly regulated by balanced synthesis and degradation of peptidoglycan (PG), allowing cells to expand their sacculus during growth while maintaining physical integrity. In rod-shaped bacteria, actin-like MreB proteins are key players of the PG elongation machinery known as the Rod complex. In the Gram-positive model bacterium Bacillus subtilis depletion of the essential MreB leads to loss of rod shape and cell lysis. However, millimolar concentrations of magnesium in the growth medium rescue the viability and morphological defects of mreB mutants by an unknown mechanism. Here, we used a combination of cytological, biochemical and biophysical approaches to investigate the cell surface properties of mreB null mutant cells and the interactions of Mg2+ with the cell wall of B. subtilis. We show that ∆mreB cells have rougher and softer surfaces, and changes in PG composition indicative of increased DL- and DD-endopeptidase activities as well as increased deacetylation of the sugar moieties. Increase in DL-endopeptidase activity is mitigated by excess Mg2+ while DD-endopeptidase activity remains high. Visualization of PG degradation in pulse-chase experiments showed anisotropic PG hydrolase activity along the sidewalls of ∆mreB cells, in particular at the sites of increased cell width and bulging, while PG synthesis remained isotropic. Overall, our data support a model in which divalent cations maintain rod shape in ∆mreB cells by inhibiting PG hydrolases, possibly through the formation of crosslinks with carboxyl groups of the PG meshwork that affect the capacity of PG hydrolases to act on their substrate.

Use of a quartz crystal microbalance platform to study protein adsorption on aluminum hydroxide vaccine adjuvants: Focus on phosphate-hydroxide ligand exchanges.

Aluminum hydroxide (AH) salts are widely used as vaccine adjuvants and controlling antigen-AH interactions is a key challenge in vaccine formulation. In a previous work, we have developed a quartz crystal microbalance (QCM) platform, based on stable AH-coated sensors, to explore the mechanisms of model antigen adsorption. The QCM study of bovine serum albumin (BSA) adsorption at different pH and ionic strength (I) values showed that protein adsorption on AH adjuvant at physiological pH cannot be explained mainly by electrostatic interactions, in contrast with previous reports. Here, we exploit further the developed QCM platform to investigate the role of phosphate-hydroxyl ligand exchanges in the adsorption mechanism of BSA, human serum albumin (HSA) and ovalbumin (OVA) on two commercial AH adjuvants. BSA adsorption decreased on immobilized AH particles previously treated with KH2PO4, highlighting the role of exchangeable sites on AH particles in the adsorption process. BSA and OVA were dephosphorylated by treatment with an acid phosphatase to decrease their phosphate content by about 80% and 25%, respectively. Compared to native BSA, adsorption of dephosphorylated BSA decreased significantly on one AH adjuvant at pH 7. Adsorption of dephosphorylated OVA was comparable to the one of native OVA. Further QCM assays showed that phospho-amino acids (PO4-serine and PO4-threonine) displaced previously adsorbed BSA and OVA from AH particles in conditions that were depending on the protein and the AH. Taken together, these observations suggest that phosphate-hydroxyl ligand exchange is an important adsorption mechanism of proteins on AH. These results moreover confirm that the developed AH-coated QCM sensors offer a new platform for the study of antigen adsorption, to the benefit of vaccine formulation.

Immobilization of Aluminum Hydroxide Particles on Quartz Crystal Microbalance Sensors to Elucidate Antigen-Adjuvant Interaction Mechanisms in Vaccines.

Aluminum hydroxide (AH) salts are the most widely used adjuvants in vaccine formulation. They trigger immunogenicity from antigenic subunits that would otherwise suffer from a lack of efficiency. Previous studies focusing on antigen-AH interaction mechanisms, performed with model proteins, suggested that electrostatic interactions and phosphate-hydroxyl ligand exchanges drive protein adsorption on AH. We however recently evidenced that NaCl, used in vaccine formulation, provokes AH particle aggregation. This must be taken into account to interpret data related to protein adsorption on AH. Here, we report on the successful development and use of a stable AH-coated surface to explore the mechanisms of protein adsorption by means of ultrasensitive surface analysis tools. Bovine serum albumin (BSA) adsorption was studied at different pHs and ionic strengths (I) using quartz crystal microbalance. The results show that protein adsorption on the AH adjuvant cannot be explained solely by electrostatic interactions and ligand exchanges. Hence, a higher adsorption was observed at pH 3 compared to pH 7, although AH and BSA respectively undergo repulsive and attractive electrostatic interactions at these pH values. Almost no effect of I on adsorption was moreover noted at pH 7. These new developments and observations not only suggest that other mechanisms govern protein adsorption on AH but also offer a new platform for the study of antigen adsorption in the context of vaccine formulation. Immobilizing particles on QCM sensors also enriches the range of applications for which QCM can be exploited, especially in colloid science.

NaCl strongly modifies the physicochemical properties of aluminum hydroxide vaccine adjuvants.

The immunostimulation capacity of most vaccines is enhanced through antigen adsorption on aluminum hydroxide (AH) adjuvants. Varying the adsorption conditions, i.e. pH and ionic strength (I), changes the antigen adsorbed amount and therefore the ability of the vaccine to stimulate the immune system. Vaccine formulations are thus resulting from an empirical screening of the adsorption conditions. This work aims at studying the physicochemical effects of adjusting the ionic strength of commercial AH adjuvant particles suspensions with sodium chloride (NaCl). X-ray photoelectron spectroscopy data show that AH particles surface chemical composition is neither altered by I adjustment with NaCl nor by deposition on gold surfaces. The latter result provides the opportunity to use AH-coated gold surfaces as a platform for advanced surface analysis of adjuvant particles, e.g. by atomic force microscopy (AFM). The morphology of adjuvant particles recovered from native and NaCl-treated AH suspensions, as studied by scanning electron microscopy and AFM, reveals that AH particles aggregation state is significantly altered by NaCl addition. This is further confirmed by nitrogen adsorption experiments: I adjustment to 150mM with NaCl strongly promotes AH particles aggregation leading to a strong decrease of the developed specific surface area. This work thus evidences the effect of NaCl on AH adjuvant structure, which may lead to alteration of formulated vaccines and to misinterpretation of data related to antigen adsorption on adjuvant particles.

Proteins dominate in the surface layers formed on materials exposed to extracellular polymeric substances from bacterial cultures.

The chemical compositions of the surface conditioning layers formed by different types of solutions (from isolated EPS to whole culture media), involving different bacterial strains relevant for biocorrosion were compared, as they may influence the initial step in biofilm formation. Different substrata (polystyrene, glass, steel) were conditioned and analyzed by X-ray photoelectron spectroscopy. Peak decomposition and assignment were validated by correlations between independent spectral data and the ubiquitous presence of organic contaminants on inorganic substrata was taken into account. Proteins or peptides were found to be a major constituent of all conditioning layers and polysaccharides were not present in appreciable concentrations; the proportion of nitrogen which may be due to DNA was lower than 15%. There was no significant difference between the compositions of the adlayers formed from different conditioning solutions, except for the adlayers produced with tightly bound EPS extracted from D. alaskensis.

Chitosan-coated electrospun nanofibers with antibacterial activity.

Charged nanofibers were prepared by electrospinning (ESP) poly(ε-caprolactone) with a copolymer bearing carboxylic acid functions. The presence of these functions allowed exposing some negative charges on the fiber surface, by dipping the fibers in a phosphate buffer. A layer of chitosan, a polycation in acidic medium, was then deposited on the nanofiber surface, thanks to electrostatic attraction. Fibers were characterized at each step of the process and the influence of the copolymer architecture on chitosan deposition was discussed. The antibacterial activity of the resulting fibers was finally assessed.

Understanding and controlling type I collagen adsorption and assembly at interfaces, and application to cell engineering.

Collagen is a large anisotropic and self-assembling extracellular matrix protein. Understanding and controlling its adsorption and assembly at interfaces is expected to increase our general knowledge of protein adsorption as well as to open the way to the development of biointerfaces of interest for biomaterials science and tissue engineering. The work related to type I collagen adsorption performed in our laboratory over the past twenty years is reviewed. Substrate chemical nature and adsorption conditions (collagen concentration, adsorption duration) were shown to affect collagen adsorbed amount and supramolecular organization. Collagen assemblies were formed starting from the interface, and assembly was favored by hydrophobic substrates and high adsorbed amount. Substrates were designed to better control collagen adsorption and assembly. The spatial control of adsorption was ensured by chemically heterogeneous substrates, which also affected collagen assembly when domains with a dimension smaller than the length of the collagen molecule (i.e. 300nm) were prepared. Mixed polymer brushes were used to achieve a temporal control of adsorption: adsorption and desorption were reversibly triggered by changes of pH and ionic strength. Layer-by-layer assembly of collagen in a nanoporous template was used to elaborate collagen-based nanotubes, which were further deposited on ITO glass substrates by electrophoretic deposition. Finally, the evaluation of cell behavior on the created biointerfaces showed that the control of collagen organization can be successfully used to alter cell behavior.

Conditioning materials with biomacromolecules: composition of the adlayer and influence on cleanability.

The influence of substrate hydrophobicity and biomacromolecules (dextran, bovine serum albumin - BSA) adsorption on the cleanability of surfaces soiled by spraying aqueous suspensions of quartz particles (10-30µm size), then dried, was investigated using glass and polystyrene as substrates. The cleanability was evaluated using radial flow cell (RFC). The surface composition was determined by X-ray photoelectron spectroscopy (XPS). The interpretation of XPS data allowed the complexity due to the ubiquitous presence of organic contaminants to be coped with, and the surface composition to be expressed in terms of both the amount of adlayer and the mass concentration of adlayer constituents. When soiled with a suspension of particles in water, glass was much less cleanable than polystyrene, which was attributed to its much lower water contact angle, in agreement with previous observations on starch soil. Dextran was easily desorbed and did not affect the cleanability. The presence of BSA at the interface strongly improved the cleanability of glass while the contact angle did not change appreciably. In contrast, soiling polystyrene with quartz particles suspended in a BSA solution instead of water did not change markedly the cleanability, while the contact angle was much lower and the aggregates of soiling particles were more flat. These observations are explained by the major role of capillary forces developed upon drying, which influence the closeness of the contact between the soiling particles and the substrate and, thereby, the adherence of particles. The capillary forces are proportional to the liquid surface tension and depend in a more complex way on contact angles of the particles and of the substrate. The dependence of cleanability on capillary forces, and in particular on the liquid surface tension, is predominant as compared with its dependence on the size and shape of the soiling aggregates, which influence the efficiency of shear forces exerted by the flowing water upon cleaning.

Influence of poly(ethylene oxide)-based copolymer on protein adsorption and bacterial adhesion on stainless steel: modulation by surface hydrophobicity.

The aim of the present work is to study the adhesion of Pseudomonas NCIMB 2021, a typical aerobic marine microorganism, on stainless steel (SS) substrate. More particularly, the potential effect on adhesion of adsorbed poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer is investigated. Bacterial attachment experiments were carried out using a modified parallel plate flow chamber, allowing different surface treatments to be compared in a single experiment. The amount of adhering bacteria was determined via DAPI staining and fluorescence microscopy. X-ray photoelectron spectroscopy (XPS) was used to characterize the surface chemical composition of SS and hydrophobized SS before and after PEO-PPO-PEO adsorption. The adsorption of bovine serum albumin (BSA), a model protein, was investigated to test the resistance of PEO-PPO-PEO layers to protein adsorption. The results show that BSA adsorption and Pseudomonas 2021 adhesion are significantly reduced on hydrophobized SS conditioned with PEO-PPO-PEO. Although PEO-PPO-PEO is also found to adsorb on SS, it does not prevent BSA adsorption nor bacterial adhesion, which is attributed to different PEO-PPO-PEO adlayer structures on hydrophobic and hydrophilic surfaces. The obtained results open the way to a new strategy to reduce biofouling on metal oxide surfaces using PEO-PPO-PEO triblock copolymer.

Interaction of preosteoblasts with surface-immobilized collagen-based nanotubes.

In a previous work, we demonstrated the successful use of electrophoretic deposition (EPD) to immobilize collagen-based nanotubes onto indium-tin-oxide-coated glass (ITO glass), leading to the creation of biointerfaces with protein-based chemistry and topography [1]. In this work, we present a first study of preosteoblasts behavior in contact with surface-immobilized collagen-based nanotubes. Changes in cell morphology after their interaction with ITO glass modified with collagen-based nanotubes were studied using fluorescence microscopy and compared to those observed on virgin ITO glass as well as on ITO glass on which a collagen layer was simply adsorbed. Scanning electron microscopy (SEM) was used to study interactions of cell filopodias with the deposited nanotubes. Cytotoxicity of these biointerfaces was examined as well in short term cultures, using Alamar blue assay. Cells showed particular morphologies on ITO glass coated with nanotubes compared to virgin ITO glass or collagen adsorbed layer on ITO glass. High resolution SEM images suggest that apart from cell morphology, length and thickness of filopodias seem to be significantly affected by surface modification with collagen-based nanotubes. Moreover, nanotube-coated ITO glass did not show any obvious cytotoxicity in short term culture, opening new perspectives for the surface modification of biomaterials. We show the versatility of the proposed surface modification procedure by tailoring biointerfaces with a mixture of micro- and nanometer-scale collagen-based tubes.

Colloidal lithography using silica particles: improved particle distribution and tunable wetting properties.

Colloidal lithography rests on the adhesion of colloids in a relatively ordered pattern on a charged surface owing to electrostatic interactions. However, due to capillary forces, the colloids tend to form aggregates during the drying process. These capillary forces are especially strong for large hydrophilic particles. In this paper, different experimental approaches are explored to limit the aggregation of large (500 nm) silica particles deposited on glass substrates that were previously treated with polyallylamine (PAH), a polycation. These approaches consist in the addition of smaller colloids between the large ones, or of a layer of macromolecules (PAH or albumin) on top of the deposit. Scanning electron microscopy observations show that the addition of PAH and even better of albumin on top of the adherent colloids efficiently limits the formation of aggregates. Interestingly, the water contact angle of the surface obtained after silica colloid deposition and albumin adsorption is very high (~95°), while very hydrophilic surfaces are obtained after calcination. This is discussed in light of the Wenzel and Cassie-Baxter models. In conclusion, the proposed method allows a nanoscale topographic pattern with tunable wettability to be created on large surface areas using a soft and inexpensive technique.

Nano-organized collagen layers obtained by adsorption on phase-separated polymer thin films.

The organization of adsorbed type I collagen layers was examined on a series of polystyrene (PS)/poly(methyl methacrylate) (PMMA) heterogeneous surfaces obtained by phase separation in thin films. These thin films were prepared by spin coating from solutions in either dioxane or toluene of PS and PMMA in different proportions. Their morphology was unraveled combining the information coming from X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and water contact angle measurements. Substrates with PMMA inclusions in a PS matrix and, conversely, substrates with PS inclusions in a PMMA matrix were prepared, the inclusions being either under the form of pits or islands, with diameters in the submicrometer range. The organization of collagen layers obtained by adsorption on these surfaces was then investigated. On pure PMMA, the layer was quite smooth with assemblies of a few collagen molecules, while bigger assemblies were found on pure PS. On the heterogeneous surfaces, it appeared clearly that the diameter and length of collagen assemblies was modulated by the size and surface coverage of the PS domains. If the PS domains, either surrounding or surrounded by the PMMA phase, were above 600 nm wide, a heterogeneous distribution of collagen was found, in agreement with observations made on pure polymers. Otherwise, fibrils could be formed, that were longer compared to those observed on pure polymers. Additionally, the surface nitrogen content determined by XPS, which is linked to the protein adsorbed amount, increased roughly linearly with the PS surface fraction, whatever the size of PS domains, suggesting that adsorbed collagen amount on heterogeneous PS/PMMA surfaces is a combination of that observed on the pure polymers. This work thus shows that PS/PMMA surface heterogeneities can govern collagen organization. This opens the way to a better control of collagen supramolecular organization at interfaces, which could in turn allow cell-material interactions to be tailored.

Surface characterization, collagen adsorption and cell behaviour on poly(L-lactide-co-glycolide).

Poly(L-lactide-co-glycolide) (PLG) was modified through the adsorption of collagen to improve the behaviour of fibroblasts and osteoblasts. As reference materials cell-resistant polystyrene (PS) and cell-conductive tissue-culture polystyrene (TCPS) were also evaluated. The physicochemical surface properties of the materials were studied by X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and water contact angle measurements. The morphology of cells was examined using optical microscopy, while their growth was evaluated by both crystal violet and MTT tests. Nitric oxide level and protein concentration were tested in cell supernatants. The results showed that the adsorbed amount and the organization of the adsorbed collagen were influenced by surface hydrophobicity. Cell culture experiments on native substrates revealed that cell attachment, spreading and growth enhanced, depending on the substrate, in the following order: PS

Putrescine differently influences the effect of salt stress on polyamine metabolism and ethylene synthesis in rice cultivars differing in salt resistance.

Effects of salt stress on polyamine metabolism and ethylene production were examined in two rice (Oryza sativa L.) cultivars [I Kong Pao (IKP), salt sensitive; and Pokkali, salt resistant] grown for 5 d and 12 d in nutrient solution in the presence or absence of putrescine (1 mM) and 0, 50, and 100 mM NaCl. The salt-sensitive (IKP) and salt-resistant (Pokkali) cultivars differ not only in their mean levels of putrescine, but also in the physiological functions assumed by this molecule in stressed tissues. Salt stress increased the proportion of conjugated putrescine in salt-resistant Pokkali and decreased it in the salt-sensitive IKP, suggesting a possible protective function in response to NaCl. Activities of the enzymes ornithine decarboxylase (ODC; EC 4.1.1.17) and arginine decarboxylase (ADC; EC 4.1.1.19) involved in putrescine synthesis were higher in salt-resistant Pokkali than in salt-sensitive IKP. Both enzymes were involved in the response to salt stress. Salt stress also increased diamine oxidase (DAO; 1.4.3.6) and polyamine oxidase (PAO EC 1.5.3.11) activities in the roots of salt-resistant Pokkali and in the shoots of salt-sensitive IKP. Gene expression followed by reverse transcription-PCR suggested that putrescine could have a post-translational impact on genes coding for ADC (ADCa) and ODC (ODCa and ODCb) but could induce a transcriptional activation of genes coding for PAO (PAOb) mainly in the shoot of salt-stressed plants. The salt-resistant cultivar Pokkali produced higher amounts of ethylene than the salt-sensitive cultivar IKP, and exogenous putrescine increased ethylene synthesis in both cultivars, suggesting no direct antagonism between polyamine and ethylene pathways in rice.

Competitive adsorption of fibrinogen and albumin and blood platelet adhesion on surfaces modified with nanoparticles and/or PEO.

In order to evaluate the respective influence of surface nanotopography and chemical composition on blood compatibility, plasma protein adsorption (fibrinogen - Fg and albumin - HSA, quantified simultaneously by dual radioassays) and platelet adhesion were investigated on a range of materials. Reference surfaces were glass, polystyrene and poly(vinyl chloride), as well as pieces of commercial blood bags. Colloidal lithography with 65 and 470 nm polystyrene latex particles was used to prepare nanostructured surfaces with either one layer of colloids or with bimodal roughness. The surfaces were further conditioned by adsorption of poly(ethylene oxide) (PEO)-containing compounds (Pluronic F 68 and PLL-g-PEG). Study of the simultaneous adsorption of Fg and HSA on reference substrates demonstrated that the Fg/HSA adsorbed amount ratio decreases as the substrate hydrophobicity increases, the lower ratio being obtained with commercial blood bag. This is due to the higher resistance of HSA adsorbed on hydrophobic substrates to displacement by proteins from the solution. Such higher resistance was also shown to occur in the case of displacement by constituents of non-diluted blood plasma. Nanostructured substrates gave about the same Fg/HSA ratio as polystyrene and poly(vinyl chloride). Surface conditioning with Pluronic F 68 reduced the adsorption of Fg in competition with HSA on all substrates except glass, while PLL-g-PEG decreased the adsorbed amount of both Fg and HSA on glass but not on the other substrates. Positive correlations between the amount of adhering blood platelets and both the Fg/HSA ratio and the absolute amount of Fg adsorbed in competition with HSA were found for all substrates (reference and nanostructured, as such or after PEO conditioning, except native glass which had to be discarded due to the formation of clots in the liquid phase). These quantities were also related to the state of activation of adhering platelets. This supports the concept that blood compatibility of materials is primarily governed by the presence of Fg in the adsorbed phase, as a result of the competition with other plasma proteins. This is in turn strongly influenced by surface hydrophobicity. Surface nanostructuration as performed here (relief in the range of 50-500 nm) did not affect significantly the relationship between Fg adsorption and platelet adhesion.

Surface spectroscopy of adsorbed proteins: input of data treatment by principal component analysis.

X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectroscopy (ToF-SIMS), two surface-sensitive spectroscopic methods, are commonly used to characterize adsorbed protein layers. Principal component analysis (PCA) is a statistical method which aims at reducing the number of variables in complex sets of data while retaining most of the original information. The aim of this paper is to review work carried out in our group regarding the use of PCA with a view to facilitate and deepen the interpretation of ToF-SIMS or XPS spectra acquired on adsorbed protein layers. ToF-SIMS data acquired on polycarbonate membranes after albumin and, or insulin adsorption were treated with PCA. The results reveal the preferential exposure of particular amino acids at the outermost surface depending on the adsorption conditions (nature of the substrate and of the proteins involved, concentration in solution), giving insight into the adsorption mechanisms. PCA was applied on XPS data collected on three different substrates after albumin or fibrinogen adsorption, followed in some cases by a cleaning procedure with oxidizing agents. The results allow samples to be classified according to the nature of the substrate and to the adsorbed amount and, or the level of surface coverage by the protein. Chemical shifts of particular interest are also identified, which may facilitate further peak decomposition. It is useful to recall that the outcome of PCA strongly depends on data selection and normalisation.

Principal component analysis: a versatile method for processing and investigation of XPS spectra.

Given the relevance of principal component analysis (PCA) to the treatment of spectrometric data, we have evaluated potentialities and limitations of such useful statistical approach for the harvesting of information in large sets of X-ray photoelectron spectroscopy (XPS) spectra. Examples allowed highlighting the contribution of PCA to data treatment by comparing the results of this data analysis with those obtained by the usual XPS quantification methods. PCA was shown to improve the identification of chemical shifts of interest and to reveal correlations between peak components. First attempts to use the method led to poor results, which showed mainly the distance between series of samples analyzed at different moments. To weaken the effect of variations of minor interest, a data normalization strategy was developed and tested. A second issue was encountered with spectra suffering of an even slightly inaccurate binding energy scale correction. Indeed, minor shifts of energy channels lead to the PCA being performed on incorrect variables and consequently to misleading information. In order to improve the energy scale correction and to speed up this step of data pretreatment, a data processing method based on PCA was used. Finally, the overlap of different sources of variation was studied. Since the intensity of a given energy channel consists of electrons from several origins, having suffered inelastic collisions (background) or not (peaks), the PCA approach cannot compare them separately, which may lead to confusion or loss of information. By extracting the peaks from the background and considering them as new variables, the effect of the elemental composition could be taken into account in the case of spectra with very different backgrounds. In conclusion, PCA is a very useful diagnostic tool for the interpretation of XPS spectra, but it requires a careful and appropriate data pretreatment.

An AFM, XPS and wettability study of the surface heterogeneity of PS/PMMA-r-PMAA demixed thin films.

A series of homopolymer/random copolymer blends was used to produce heterogeneous surfaces by demixing in thin films. The chosen homopolymer is polystyrene (PS) and the random copolymer is poly(methyl methacrylate)-r-poly(methacrylic acid) (PMMA-r-PMAA), whose acidic functions could be used as reactive sites in view of further surface functionalization. The proportion of each polymer at the interface was deduced from X-ray photoelectron spectroscopy (XPS) data using, on the one hand, the O/C ratio, and on the other hand, decomposition of the carbon peak of the blends in two components corresponding to the carbon peaks of PS and PMMA-r-PMAA. Combining the information from XPS with atomic force microscopy (AFM) images, water contact angle measurements and PS selective dissolution, it appears that the surfaces obtained from blends with a high PS content (90/10 to 70/30) display pits with a bottom made of PMMA-r-PMAA, randomly distributed in a PS matrix. On the other hand, the surfaces obtained from blends with a low PS content (30/70 to 10/90) display randomly distributed PS islands surrounded by a PMMA-r-PMAA matrix. The characteristics of the heterogeneous films are thought to be governed by the higher affinity of PMMA-r-PMAA for the solvent (dioxane), which leads to the elevation of the PS phase compared to the PMMA-r-PMAA phase, and to surface enrichment in PMMA-r-PMAA.

Dual radiolabeling to study protein adsorption competition in relation with hemocompatibility.

Human fibrinogen (Fg) and albumin (HSA) were labeled with (3)H and (14)C, respectively. Dual counting allowed the adsorbed amount of the two proteins to be determined simultaneously. Single adsorption, adsorption of the two proteins in competition, but also exchange (substitution by molecules of the same nature) and displacement (desorption under the action of the other protein) experiments were performed on two model surfaces, glass and polystyrene (PS), as well as on pure polyvinylchloride (PVC-s) and on PVC from blood bag (PVC-b). As expected, the adsorbed amount of a single protein is higher on a hydrophobic compared to a hydrophilic surface. When the two proteins are adsorbed in competition, they are found in equal proportion on glass, while HSA is twice more abundant than Fg on PS and PVC-s and about six times more abundant on PVC-b. This trend is related to an increase of the water contact angle of the substrates. For PVC-b, the contact angle is affected by the presence of aliphatic components exposed at the extreme surface, as determined by angle-resolved X-ray photoelectron spectroscopy. In exchange and displacement experiments, the first adsorbed molecules remain dominating on PS while they can be removed from glass. Given the known importance of HSA and Fg adsorption for the fate of materials placed in contact with blood, the method described in this paper may be used as a first approach to orient the design of surfaces with improved hemocompatibility.

Influence of collagen denaturation on the nanoscale organization of adsorbed layers.

Adsorption (at 37 degrees C) of type I collagen, in native and heat-denatured (30 min at 40 and 90 degrees C) forms, on polystyrene was studied using quartz crystal microbalance with energy dissipation monitoring (QCM-D), atomic force microscopy (AFM) in tapping mode and X-ray photoelectron spectroscopy (XPS). The significance of the parameters deduced from QCM-D data was examined by comparing different approaches. The adsorbed layer of native collagen has a complex organization consisting of a thin mat of molecules near the surface, in which fibrils develop depending on concentration and time, and of a thicker overlayer containing protruding molecules or bundles which modify noticeably the local viscosity. As a result of drastic denaturation, the ability of collagen to assemble into fibrils in the adsorbed phase is lost and the protrusion of molecules into the aqueous phase is much less pronounced. The adsorbed layer of denatured collagen appears essentially as a monolayer of flattened coils. At low concentration, this is easily displaced upon drying, leading to particular dewetting figures; at high concentration, aggregates add to the first layer. Moderate denaturation leads to an adsorbed phase which shows properties intermediate between those observed with native and extensively denatured collagen, regarding the ability to form fibrillar structures and the adlayer thickness and viscosity.