Immunomodulation with self-crosslinked polyelectrolyte multilayer-based coatings

This study aims to design an optimal polyelectrolyte multilayer film of poly-L-lysine (PLL) and hyaluronic acid(HA) as an anti-inflammatory cytokine release system in order to decrease the implant failure due to any immune reactions. The chemical modification of the HA with aldehyde moieties allows self-cross-linking of the film and an improvement in the mechanical properties of the film. The cross-linking of the film and the release of immunomodulatory cytokine (IL-4) stimulate the differentiation of primary human monocytes seeded on the films into pro-healing macrophages phenotype. This induces the production of antiinflammatory cytokines (IL1-RA and CCL18) and the decrease of proinflammatory cytokines secreted (IL-12, TNF-α, and IL-1β). Moreover, we demonstrate that cross-linking PLL/HA film using HA-aldehyde is already effective by itself to limit inflammatory processes. Finally, this functionalized self-cross-linked PLL/HA-aldehyde films constitutes an innovative and efficient candidate for immunomodulation of any kind of implants of various architecture and properties

New surfaces for molecular recognition activated by stretching

Le procédé par lequel des forces sont transformées en signaux chimiques joue un rôle fondamental dans de nombreux processus biologiques. Ce travail de thèse a permis de mettre au point de nouvelles surfaces fonctionnelles synthétiques permettant de mimer ce comportement. Il s’agit plus précisément de contrôler l’adsorption d’objets biologiques tels que des protéines ou des cellules sur un support élastique modifié par plasma et présentant des récepteurs spécifiques. Ces récepteurs sont masqués par de longues chaînes de poly(éthylèneglycol) (PEG) qui sont également greffées sur la surface. L'étirement de celles-ci permet d'exhiber les sites d’adsorption ou les sites d'adhésion et de rendre ainsi la surface adhérente. Notre méthode est basée sur la polymérisation plasma de l’anhydride maléique. Cette fonctionnalisation permet de greffer à la surface de films silicones des fonctions carboxylique qui servent de points d’ancrage aux chaînes de PEG. Sur le même principe, la biotine ou les peptides d’adhésion (RGD) sont greffés dans un deuxième temps sur ce substrat. Nous montrons, qu’à l’état non étiré, ces ligands ne sont pas accessibles pour leurs récepteurs. Par contre, à l’état étiré, la surface devient spécifiquement adsorbante pour la streptavidine, l’anti-biotine et adhérente pour les cellules. Ces phénomènes sont parfaitement réversibles.The process by which forces are converted into chemical signals play a fundamental role in many biological processes. This thesis has to develop new functional synthetic surfaces to mimic this behavior. It is more precisely to control the adsorption of biological objects such as proteins or cells on an elastic support modified by plasma and presenting specific receptors. These receptors are masked by long chains of poly (ethylene glycol) (PEG) which are also grafted onto the surface. Stretching allows them to exhibit adsorption sites or adhesion sites and thus make the surface adhesive. Our method is based on the plasma polymerization of maleic anhydride. This functionalization can be grafted to the surface of silicone films carboxylic functions which serve as anchors points for the PEG chains. On the same principle, biotin or adhesion peptides (RGD) have been grafted in a second time on this substrate. We show that the non-stretched state, these ligands are not accessible to their receptors. On the other side, in the stretched state, the surface becomes specifically adsorbent to streptavidin, anti-biotin and also adherent for cells. These phenomena are perfectly reversible

Low Biotinyl Glycogen: A Model for Single-Molecule Force Analysis of Branched Biological Macromolecules

International audienceCharacterization of polysaccharides in their native-like forms is of great importance for their subsequent application in the pharmaceuticals and biomedicine fields. Commercial glycogen molecules immobilized on a modified gold surface were characterized using a combination of Single Molecule Force and InfraRed spectroscopies. For this, low biotinylated glycogen was synthetized and subsequently immobilized on a Bovine Serum Albumin/Streptavidin-coated gold modified surface. The presence of glycogen was ascertained by vibrational spectra and confirmed based on the specific interaction of concanavalin A functionalized AFM tips with -glucose residues. Theoretical values of the particle size of this glycogen fitted well with the structural observations. Conformational properties were deduced from the force curves using the FJC model. The studied glycogen showed a compact slightly ramified structure with molecular elongations up to 1000 nm. The investigation of other conformational properties showed similarities with bacterial glycogen, however some differences that are possibly related to the different biosynthesis pathways were observed

Atomic force microscopy analysis of IgG films at hydrophobic surfaces: A promising method to probe IgG orientations and optimize ELISA tests performance

International audienceIgG films are widely used in the field of immunoassays, especially in (double) antibody-sandwich ELISA tests where capture antibodies are coated on surfaces like polystyrene or hydrophobic self-assembled monolayers (h-SAMs). It is critical to analyze-at a molecular scale and under liquid conditions the structure of the deposited IgG film in order to quantitatively address the efficiency of the ELISA test in terms of antigen detection. In this communication, we report an atomic force microscopy (AFM) analysis evidencing a strong relationship between immunological activities of mouse monoclonal anti-human interleukin-2 (IL-2) and 6 (IL-6) antibodies, thickness and roughness of the IgG monolayer adsorbed onto h-SAMs, and surface concentration of IgG molecules. Indirect information may be further obtained on antibody orientation. Collating the results obtained by AFM and those from ELISA tests leads us to conclude that antibodies like anti-IL-6 forming flat monolayers should be more efficient under ELISA detection conditions. In addition, the concentration of IgG in the coating suspension should be optimized to obtain a monolayer heavily populated by "end-on" adsorbed molecules, an orientation that is desirable for enhancing ELISA tests performance

Stability of Plasma Treated Non-vulcanized Polybutadiene Surfaces: Role of Plasma Parameters and Influence of Additives

International audienceSurface modification studies of non-vulcanized BR elastomers (butadiene rubber) by low-pressure air plasma treatment and the effect on ageing and adhesion performances are presented in this paper. In particular, the influence of discharge power and distance from the glow discharge, and impact of antioxidant molecules in the BR formulation were examined. To characterize the changes to the BR surface, XPS spectroscopy, contact angle measurements, AFM nanoindentation experiments and tack measurements were utilized. Oxidation and crosslinking were the main mechanisms observed on the polymer chains regardless of the plasma conditions used. Beyond a certain threshold of plasma energy (in our case, discharge power of similar to 60 W and exposure time of similar to 30 s), a steady state was reached irrespective of the distance from the glow discharge. The presence of antioxidant molecules considerably reduced crosslinking phenomena while maintaining oxidation processes on polymer chains and increasing the nitrogen content in the near surface region. The mechanisms responsible for these differences have been identified. Interestingly, the COOH/C=O ratio changed according to the balance between oxidation and crosslinking. The hydrophobic recovery rate was mainly driven by temperature-dependent dynamics and varied according to the degree of crosslinking in the surface region. It was found to be lower in air atmosphere in the presence of antioxidant molecules. Finally, the presence of antioxidant molecules in the BR formulation allowed the adhesion performances after plasma exposure to significantly increase

Pili of Lactobacillus rhamnosus GG mediate interaction with β-lactoglobulin

International audienceRecently dairy products like cheese, yogurt or milk proteins are increasingly used to encapsulate probiotic bacteria to promote their survival. The knowledge of interactions occurring between bacteria and milk matrices is essential for the formulation of efficient new products. In this work, Atomic Force Microscopy was used to study interactions between Lactobacillus rhamnosus GG and isolated whey proteins: beta-lactoglobulin (beta-LG), alpha-lactalbumin (alpha-LA) and bovine serum albumin (BSA). For the first time, it was clearly demonstrated that L. rhamnosus GG is able to interact with the beta-LG but not with alpha-LA and BSA. To identify the bacterial surface biomolecules involved in interaction with beta-LG, two of its surface mutants were used: a pili depleted strain (L. rhamnosus GG spaCBA) and an exopolysaccharides (EPS) depleted strain (L. rhamnosus GG welE). Two predictive models, FJC and WLC, were used to describe behaviors of the molecule stretched by providing parameters like contour length (L-c), adhesion force (F-adh), Kuhn length (l(k)) and Persistence length (l(p)). The absence of adhesion events between beta-LG and mutants having no pili reflects the crucial role of this biomolecule for interactions with the protein

Pili of Lactobacillus rhamnosus GG mediate interaction with beta-lactoglobulin

International audienceRecently dairy products like cheese, yogurt or milk proteins are increasingly used to encapsulate probiotic bacteria to promote their survival. The knowledge of interactions occurring between bacteria and milk matrices is essential for the formulation of efficient new products. In this work, Atomic Force Microscopy was used to study interactions between Lactobacillus rhamnosus GG and isolated whey proteins: beta-lactoglobulin (beta-LG), alpha-lactalbumin (alpha-LA) and bovine serum albumin (BSA). For the first time, it was clearly demonstrated that L. rhamnosus GG is able to interact with the beta-LG but not with alpha-LA and BSA. To identify the bacterial surface biomolecules involved in interaction with beta-LG, two of its surface mutants were used: a pili depleted strain (L. rhamnosus GG spaCBA) and an exopolysaccharides (EPS) depleted strain (L. rhamnosus GG welE). Two predictive models, FJC and WLC, were used to describe behaviors of the molecule stretched by providing parameters like contour length (L-c), adhesion force (F-adh), Kuhn length (l(k)) and Persistence length (l(p)). The absence of adhesion events between beta-LG and mutants having no pili reflects the crucial role of this biomolecule for interactions with the protein

Improving adhesion of powder coating on PEEK composite: Influence of atmospheric plasma parameters

International audienceIn aeronautic industries, powder coatings are increasingly used because of environmental considerations. During the deposition of such a coating on a substrate piece, the main objective is to obtain a good coating/substrate adhesion. In this study, the targeted substrate is a Poly-(Ether EtherKetone)-(PEEK) based composite material. Due to the poor surface energy of PEEK, a surface treatment is necessary in order to enhance its adhesion with the coating. In this purpose, atmospheric plasma treatment has been chosen and the influence of plasma parameters has been studied. Four scan speed nozzles and three gases (Air, N2 and Argon) plasma has been tested. The increase of adhesion with increasing wettability, polarity and nanoroughness has been evidenced. A particular study of the type of grafted polar functionalities according to gas nature allowed to better understand the plasma mechanism and the cross-impact of polarity and nanoroughness in adhesion enhancement

DDB2 (damaged-DNA binding 2) protein: a new modulator of nanomechanical properties and cell adhesion of breast cancer cells

International audienceDDB2, known for its role in DNA repair, was recently shown to reduce mammary tumor invasiveness by inducing the transcription of IκBα, an inhibitor of NF-κB activity. Since cellular adhesion is a key event during the epithelial to mesenchymal transition (EMT) leading to the invasive capacities of breast tumor cells, the aim of this study was to investigate the role of DDB2 in this process. Thus, using low and high DDB2-expressing MDA-MB231 and MCF7 cells, respectively, in which DDB2 expression was modulated experimentally, we showed that DDB2 overexpression was associated with a decrease of adhesion abilities on glass and plastic areas of breast cancer cells. Then, we investigated cell nanomechanical properties by atomic force microscopy (AFM). Our results revealed significant changes in the Young's Modulus value and the adhesion force in MDA-MB231 and MCF7 cells, whether DDB2 was expressed or not. The cell stiffness decrease observed in MDA-MB231 and MCF7 expressing DDB2 was correlated with a loss of the cortical actin-cytoskeleton staining. To understand how DDB2 regulates these processes, an adhesion-related gene PCR-Array was performed. Several adhesion-related genes were differentially expressed according to DDB2 expression, indicating that important changes are occurring at the molecular level. Thus, this work demonstrates that AFM technology is an important tool to follow cellular changes during tumorigenesis. Moreover, our data revealed that DDB2 is involved in early events occurring during metastatic progression of breast cancer cells and will contribute to define this protein as a new marker of metastatic progression in this type of cancer