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Poly(ethylene glycol) (PEG) hydrogels have been extensively used as scaffolds for tissue engineering applications, owing to their biocompatibility, chemical versatility, and tunable mechanical properties. However, their bio-inert properties require them to be associated with additional functional moieties to interact with cells. To circumvent this need, we propose here to reticulate PEG molecules with poly(L-lysine) dendrigrafts (DGL) to provide intrinsic cell functionalities to PEG-based hydrogels. The physico-chemical characteristics of the resulting hydrogels were studied in regard of the concentration of each component. With increasing amounts of DGL, the cross-linking time and swelling ratio could be decreased, conversely to mechanical properties, which could be tailored from 7.7 ± 0.7 to 90 ± 28.8 kPa. Furthermore, fibroblasts adhesion, viability, and morphology on hydrogels were then assessed. While cell adhesion significantly increased with the concentration of DGL, cell viability was dependant of the ratio of DGL and PEG. Cell morphology and proliferation; however, appeared mainly related to the overall hydrogel rigidity. To allow cell infiltration and cell growth in 3D, the hydrogels were rendered porous. The biocompatibility of resulting hydrogels of different compositions and porosities was evaluated by 3 week subcutaneous implantations in mice. Hydrogels allowed an extensive cellular infiltration with a mild foreign body reaction, histological evidence of hydrogel degradation, and neovascularization.

The scope of this paper is to present a gold standard method to evaluate functional activity of antibody (Ab)-based materials during the different phases of their development, after their exposure to forced degradations or even during routine quality control. Ab-based materials play a central role in the development of diagnostic devices, for example, for screening or therapeutic target characterization, in formulation development, and in novel micro(nano)technology approaches to develop immunosensors useful for the analysis of trace substances in pharmaceutical and food industries, clinical and environmental fields. A very important aspect in diagnostic device development is the construction of its biofunctional surfaces. These Ab surfaces require biocompatibility, homogeneity, stability, specificity and functionality. Thus, this work describes the validation and applications of a unique ligand binding assay to directly perform the quantitative measurement of functional Ab binding sites immobilized on the solid surfaces. The method called Antibody Anti-HorseRadish Peroxidase (A2HRP) method, uses a covalently coated anti-HRP antibody (anti-HRP Ab) and does not need for a secondary Ab during the detection step. The A2HRP method was validated and gave reliable results over a wide range of absorbance values. Analyzed validation criteria were fulfilled as requested by the food and drug administration (FDA) and European Medicines Agency (EMA) guidance for the validation of bioanalytical methods with 1) an accuracy mean value within +15% of the nominal value; 2) the within-assay precision less than 7.1%, and 3) the inter-day variability under 12.1%. With the A2HRP method, it is then possible to quantify from 0.04 × 1012 to 2.98 × 1012 functional Ab binding sites immobilized on the solid surfaces. A2HRP method was validated according to FDA and EMA guidance, allowing the creation of a gold standard method to evaluate Ab surfaces for their resistance under laboratory constraints. Stability testing was described through forced degradation studies after exposure of Ab-surfaces to storage, pH and aqueous-organic solvent mixture stresses.

Antibody-coated surfaces (Ab surfaces) play a key role in bioanalytical tools developed for biosensors and diagnostics. Therefore, a high and well-defined functional activity of the Ab surface is required. The functional activity of the Ab surface depends on its available binding sites i.e. "the active sites" that are able to capture antigen (Ag). The number of active binding sites strongly depends on the immobilization strategy used to fix the Ab on the solid surface. Determination of layer thickness or surface topology are often used to characterize the Ab surfaces but there is no gold standard method for the study of the functionality of the Ab surfaces. For that purpose, we aim at developing an assay allowing to determine the performances of Ab surfaces. In the present study, anti-horseradish peroxidase antibody (anti-HRP Ab) is used as capture Ab covalently bound to the surface and enzyme HRP as Ag. This direct assay permits, in one-step, to generate a signal utilizing the catalytic properties of HRP. The signal is directly proportional to the amount of HRP bound on the Ab surface, and therefore to the active binding sites of immobilized Ab. The HRP/anti-HRP Ab interactions may be a useful indicator to construct accurate and reproducible active Ab surfaces and also to improve their performances in term of stability and sensitivity. Optimization of the assay parameters and quality of the results are presented. A good repeatability and an acceptable inter-day precision (RSD10%) are reported.

International audience; Antibody-based analytical instruments are under development to detect signatures of life on planetary bodies.Antibodies are molecular recognition reagents able to detect their target at sub-nanomolar concentrations, withhigh affinity and specificity. Studying antibody binding performances under space conditions is mandatory toconvince space agencies of the adequacy of this promising tool for planetary exploration.To complement previous ground-based experiments on antibody resistance to simulated irradiation, weevaluate in this paper the effects of antibody exposure to real space conditions during the EXPOSE-R2 missionoutside the International Space Station. The absorbed dose of ionizing radiation recorded during the 588 days ofthis mission (220 mGy) corresponded to the absorbed dose expected during a mission to Mars. Moreover,samples faced, at the same time as irradiation, thermal cycles, launch constraints, and long-term storage. Amodel biochip was used in this study with antibodies in freeze-dried form and under two formats: free orcovalently grafted to a solid surface.We found that antibody-binding performances were not significantly affected by cosmic radiation, and morethan 40% of the exposed antibody, independent of its format, was still functional during all this experiment. Weconclude that antibody-based instruments are well suited for in situ analysis on planetary bodies. Key Words:Astrobiology—Cosmic rays—Biochip—Antibody—Planetary exploration. Astrobiology 19, 1053–1062.

International audience; Some microarray-based instruments that use bioaffinity receptors such as antibodies or aptamers are under development to detect signatures of past or present life on planetary bodies. Studying the resistance of such instruments against space constraints and cosmic rays in particular is a prerequisite. We used several ground-based facilities to study the resistance of aptamers to various types of particles (protons, electrons, neutrons, and carbon ions) at different energies and fluences. We also tested the resistance of aptamers during the EXPOSE-R2 mission outside the International Space Station (ISS). The accumulated dose measured after the 588 days of this mission (220 mGy) corresponds to the accumulated dose that can be expected during a mission to Mars. We found that the recognition ability of fluorescently labeled aptamers was not significantly affected during short-term exposure experiments taking into account only one type of radiation at a time. However, we demonstrated that the same fluorescent dye was significantly affected by temperature variations (−21°C to +58°C) and storage throughout the entirety of the ISS experiment (60% of signal loss). This induced a large variability of aptamer signal in our analysis. However, we found that >50% of aptamers were still functional after the whole EXPOSE-R2 mission. We conclude that aptamer-based instruments are well suited for in situ analysis on planetary bodies, but the detection step requires additional investigations.

The anti-horseradish peroxidase (HRP) antibody is conventionally used in immunohistochemistry. More recently, it has been used as the key element in a gold standard method to evaluate the functionality of antibody-based materials. However, few information are available about its melting temperature and its stability after exposition to laboratory stress conditions including freeze-drying and freeze-thawing cycles. The aim of this study was to evaluate the effects of these environmental constraints on the anti-HRP antibody in order to further use it as a reference in quality control and in the development of new antibody-based materials. In the developed method, the anti-HRP antibody is covalently immobilized onto a solid surface. After the direct recognition of its antigen HRP, the signal is proportional to the number of antibody active binding sites. The method was successfully utilized to accurately evaluate the anti-HRP antibody melting temperature (Tm was 73.5 ± 0.2 °C). The method is a rapid and reliable tool with minimal cost for studying the anti-HRP antibody stability to solvent stress, freeze-thawing cycles, and freeze-drying process. The obtained information may be useful for routine analysis or in the development of antibody-based materials. This can be also proposed as an easy way to control antibody freeze-drying process.

International audience; This work aims at studying the potentialities of an on-line capillary electrophoresis (CE)-based digestion methodology for evaluating polymer–drug conjugates degradability in the presence of free trypsin (in-solution digestion). A sandwich plugs injection scheme with transverse diffusion of laminar profile (TDLFP) mode was used to achieve on-line digestions. Electrophoretic separation conditions were established using poly-l-Lysine (PLL) as reference substrate. Comparison with off-line digestion was carried out to demonstrate the feasibility of the proposed methodology. The applicability of the on-line CE-based digestion methodology was evaluated for two PLL-drug conjugates and for the four first generations of dendrigraft of lysine (DGL). Different electrophoretic profiles presenting the formation of di, tri, and tetralysine were observed for PLL-drug and DGL. These findings are in good agreement with the nature of the linker used to link the drug to PLL structure and the predicted degradability of DGL. The present on-line methodology applicability was also successfully proven for protein conjugates hydrolysis. In summary, the described methodology provides a powerful tool for the rapid study of biodegradable polymers.

Sulfated glycosaminoglycans (GAGs) are complex, linear polysaccharides that are covalently linked to proteins to form proteoglycans. They are located in the extracellular matrix and at the cell surface and interact with many proteins. More than 400 interactions have been reported for heparin/heparan sulfate and these interactions are involved in numerous biological processes such as development, angiogenesis, tumor growth, host–pathogen interactions and inflammation, extracellular matrix (ECM) assembly, cell–matrix interactions and signaling. The building of GAG–protein interaction networks is required to determine how these individual interactions influence each other in vivo, are coordinated in biological processes, and are altered in diseases. This chapter reports the roadmap designed to build and analyze these interaction networks. New interactions were identified by surface plasmon resonance imaging (SPRi) using a Biacore Flexchip system and were combined with data manually curated from the literature to build a GAG–protein network. The values of equilibrium dissociation constants and of association and dissociation rates, calculated by SPR and biolayer interferometry (BLI), were integrated into the network. The network was then analyzed in silico to determine the biological processes and pathways associated with GAG partners.

Due to the diversity of antibody (Ab)-based biochips chemistries available and the little knowledge about biochips resistance to space constraints, immobilization of Abs on the surface of the biochips dedicated to Solar System exploration is challenging. In the present paper, we have developed ten different biochip models including covalent or affinity immobilization with full-length Abs or Ab fragments. Ab immobilizations were carried out in oriented/non-oriented manner using commercial activated surfaces withN-hydroxysuccinic ester (NHS-surfaces) or homemade surfaces using three generations of dendrimers (dendrigraft of poly L-lysine (DGL) surfaces). The performances of the Ab -based surfaces were cross-compared on the following criteria: (i) analytical performances (expressed by both the surface density of immobilized Abs and the amount of antigens initially captured by the surface) and (ii) resistance of surfaces to preparation procedure (freeze-drying, storage) or spatial constraints (irradiation and temperature shifts) encountered during a space mission. The latter results have been expressed as percentage of surface binding capacity losses (or percentage of remaining active Abs). The highest amount of captured antigen was achieved with Ab surfaces having full-length Abs and DGL-surfaces that have much higher surface densities than commercial NHS-surface. After freeze-drying process, thermal shift and storage sample exposition, we found that more than 80% of surface binding sites remained active in this case. In addition, the resistance of Ab surfaces to irradiation with particles such as electron, carbon ions or protons depends not only on the chemistries (covalent/affinity linkages) and strategies (oriented/non-oriented) used to construct the biochip, but also on the type, energy and fluence of incident particles. Our results clearly indicate that full-length Ab immobilization on NHS-surfaces and DGL-surfaces should be preferred for potential use in instruments for planetary exploration.

PCPE-1 (procollagen C-proteinase enhancer-1) is an extracellular matrix glycoprotein that can stimulate procollagen processing by procollagen C-proteinases such as BMP-1 (bone morphogenetic protein 1). PCPE-1 interacts with several proteins in addition to procollagens and BMP-1, suggesting that it could be involved in biological processes other than collagen maturation. We thus searched for additional partners of PCPE-1 in the extracellular matrix, which could provide new insights into its biological roles. We identified 17 new partners of PCPE-1 by SPR (surface plasmon resonance) imaging. PCPE-1 forms a transient complex with the β-amyloid peptide, whereas it forms high or very high affinity complexes with laminin-111 ( K D=58.8 pM), collagen VI ( K D=9.5 nM), TSP-1 (thrombospondin-1) ( K D1=19.9 pM, K D2=14.5 nM), collagen IV ( K D=49.4 nM) and endostatin, a fragment of collagen XVIII ( K D1=0.30 nM, K D2=1.1 nM). Endostatin binds to the NTR (netrin-like) domain of PCPE-1 and decreases the degree of superstimulation of PCPE-1 enhancing activity by heparin. The analysis of the PCPE-1 interaction network based on Gene Ontology terms suggests that, besides its role in collagen deposition, PCPE-1 might be involved in tumour growth, neurodegenerative diseases and angiogenesis. In vitro assays have indeed shown that the CUB1CUB2 (where CUB is complement protein subcomponents C1r/C1s, urchin embryonic growth factor and BMP-1) fragment of PCPE-1 inhibits angiogenesis. Abbreviations: BiNGO, Biological Networks Gene Ontology; BMP-1, bone morphogenetic protein 1; CUB, complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1; ECM, extracellular matrix; EGF, epidermal growth factor; GO, Gene Ontology; HEK, human embryonic kidney; LTBP-1, latent TGF-β-binding protein-1; MMP-2, matrix metalloproteinase-2; NTR, netrin-like; PCPE, procollagen C-proteinase enhancer; SimCT, Similarity Clustering Tree; SPARC, secreted protein acidic and rich in cysteine; SPR, surface plasmon resonance; TEM-8, tumour endothelial marker 8; TGF-β, transforming growth factor β; TSP-1, thrombospondin-1

Aims Virus detection has often been difficult due to a low concentration in water. In this study, we developed a new procedure based on concentration of virus particles on an innovative support: poly-l-lysine dendrigrafts (DGL), coupled with directed nucleic acid extraction and real-time PCR quantification. Methods and Results This method was evaluated using the bacteriophage MS2 as a model virus. This virus exhibited the size and structural properties of human pathogenic enteric viruses and has often been used to assess new supports of concentration. Moreover, this bacteriophage is also a faecal contamination indicator. In this study, many water filtration conditions were tested (volume of water, concentration, etc.), and more than 80% of bacteriophage were recovered after filtration on polymer, in most conditions. We demonstrated that the method was linear (slope = 0·99 ± 0·04 and Y intercept when x = −0·02 ± 0·28), valid (as manipulators, tested concentrations, volumes of sample and batch of polymer did not have any influence on concentration) and sensitive (allowing to concentrate up to 16 600-fold 1 l of sample and to detect and quantify down to 750 GC l−1 and 7500 GC l−1, respectively). Conclusions To conclude, this support exhibits high interest to retain viruses and to allow to detect low concentration of virus in water. Significance and Impact of the Study This study gives valuable advance in the methods of concentration and diagnosis of virus in water.

Several instruments based on immunoassay techniques have been proposed for life-detection experiments in the framework of planetary exploration but few experiments have been conducted so far to test the resistance of antibodies against cosmic ray particles. We present several irradiation experiments carried out on both grafted and free antibodies for different types of incident particles (protons, neutrons, electrons and 12C) at different energies (between 9 MeV and 50 MeV) and different fluences. No loss of antibodies activity was detected for the whole set of experiments except when considering protons with energy between 20 and 30 MeV (on free and grafted antibodies) and fluences much greater than expected for a typical planetary mission to Mars for instance. Our results on grafted antibodies suggest that biochip-based instruments must be carefully designed according to the expected radiation environment for a given mission. In particular, a surface density of antibodies much larger than the expected proton fluence would prevent significant loss of antibodies activity and thus assuring a successful detection.

Dynamic control of endothelial cell junctions is essential for vascular homeostasis and angiogenesis. We recently provided genetic evidence that ANGPTL4 is a key regulator of vascular integrity both during developmental and in hypoxia-induced pathological conditions. The purpose of the present study was to decipher the molecular mechanisms through which ANGPTL4 regulates vascular integrity. Using surface plasmon resonance and proximity ligation assays, we show that ANGPTL4 binds integrin αvβ3. In vitro and in vivo functional assays with Angptl4-deficient mice demonstrate that ANGPTL4-αvβ3 interaction is necessary to mediate ANGPTL4 vasoprotective effects. Mechanistically, ANGPTL4-αvβ3 interaction enhances Src recruitment to integrin αvβ3 and inhibits Src signalling downstream of vascular endothelial growth factor receptor 2 (VEFGR2), thereby repressing hypoxia-induced breakdown of VEGFR2-VE-cadherin and VEGFR2-αvβ3 complexes. We further demonstrate that intravitreal injection of recombinant human ANGPTL4 limits vascular permeability and leads to increased adherens junction and tight junction integrity. These findings identify a novel mechanism by which ANGPTL4 counteracts hypoxia-driven vascular permeability through integrin αvβ3 binding, modulation of VEGFR2-Src kinase signalling, and endothelial junction stabilization. We further demonstrate that Angptl4-deficient mice show increased vascular leakage in vivo in a model of laser-induced choroidal neovascularization, indicating that this newly identified ANGPTL4-αvβ3 axis might be a target for pharmaceutical intervention in pathological conditions. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

In this work, capillary isotachophoresis (ITP) was used to determine the effective charge of the first five generations of dendrigraft poly-l-lysines. This approach, which is based on the linear dependence of ITP zone length of the solute on its concentration and effective charge, offers a simple and straightforward method for effective charge determination. The cationic ITP system employed in this work yields good linearity, repeatability and sharp zones. The value of effective charge number per one lysine residue obtained for long linear poly-l-lysine is in a good agreement with the Manning theoretical value (0.5). Results obtained for dendrigraft poly-l-lysines show a dramatic decrease in the effective charge number per lysine residue with increasing generation number, from 0.84 for short oligolysines (generation 1) down to 0.08 for the fifth generation. This decrease in effective charge is due to the proximity of charged groups in the dendrigraft structure of higher generation number.

Collagen V is the defective product in most cases of classical Ehlers-Danlos syndrome (EDS), a connective tissue disorder typically characterized by skin fragility and abnormal wound healing. Collagen V assembles into diverse molecular forms. The predominant α1(V)(2)α2(V) heterotrimer controls fibrillogenesis in skin and other tissues. The α1(V)(3) minor form is thought to occur in skin, but its function is unknown. To elucidate its role, we generated transgenic mice that overexpress the human α1(V)(3) homotrimer in the epidermis. The transgene-derived product is deposited as thin unstriated fibrillar material in the basement membrane zone of embryonic and perinatal epidermis and hair follicles. Accumulation of α1(V)(3)-containing fibrils leads to ultrastructural modifications at the epidermis-dermis interface and provokes changes in biomechanical properties, although not statistically significant. Using superparamagnetic immunobeads to isolate authentic suprastructures and protein-binding assays, we demonstrate that the homotrimer is part of a protein network containing collagen IV, laminin-111, and the dermal collagen VI. Our data show that the homotrimer serves as a bridging molecule that contributes to the stabilization of the epidermal-dermal interface. This finding strongly suggests that collagen V may be expressed in skin as different subtypes with important but distinct roles in matrix organization and stability.

Endostatin is an endogenous inhibitor of angiogenesis. Although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanism of action is not fully elucidated. We used surface plasmon resonance assays to characterize interactions between endostatin, integrins, and heparin/heparan sulfate. alpha5beta1 and alphavbeta3 integrins form stable complexes with immobilized endostatin (KD=approximately 1.8x10(-8) M, two-state model). Two arginine residues (Arg27 and Arg139) are crucial for the binding of endostatin to integrins and to heparin/heparan sulfate, suggesting that endostatin would not bind simultaneously to integrins and to heparan sulfate. Experimental data and molecular modeling support endostatin binding to the headpiece of the alphavbeta3 integrin at the interface between the beta-propeller domain of the alphav subunit and the betaA domain of the beta3 subunit. In addition, we report that alpha5beta1 and alphavbeta3 integrins bind to heparin/heparan sulfate. The ectodomain of the alpha5beta1 integrin binds to haparin with high affinity (KD=15.5 nM). The direct binding between integrins and heparin/heparan sulfate might explain why both heparan sulfate and alpha5beta1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.

International audience; Angiopoietin-like 4 (ANGPTL4) is involved in angiogenesis and lipid metabolism. It is secreted by liver and adipose tissues and cleaved to generate circulating coiled-coil domain (CCD) and fibrinogen-like domain (FLD) fragments. The full-length ANGPTL4 produced by hypoxic endothelial cells interacts with the extracellular matrix (ECM). The ECM-bound and soluble forms of ANGPTL4 have antiangiogenic properties. We carried out a structure-function analysis to investigate the regulation of ANGPTL4 bioactivity in endothelial cells. We found that the recombinant CCD binds to the ECM, whereas the FLD is released into the medium. The CCD, like the full-length ANGPTL4, binds to heparan and dermatan sulfates in surface plasmon resonance assays and inhibits endothelial cell adhesion, motility, and tubule-like formation. In endothelial cells, ANGPTL4 is processed in the secretion medium after release from the ECM. This processing is altered by the proprotein convertases inhibitor alpha1-PDX and abolished by the mutation of the (161)RRKR(164) cleavage site without modification of the ECM binding and release. These data suggest that the full-length form, which interacts with heparan sulfate proteoglycans via its CCD, is protected from proteolysis by proprotein convertases and constitutes the major active pool of ANGPTL4 in hypoxic endothelial cells.

Cardiovascular ischemic injuries are associated with vascular damage induced by loss of endothelial junction integrity. Deciphering the mechanisms involved in the regulation of vascular integrity is of major interest in order to develop relevant therapeutic cardioprotective approaches to achieve tissue protection. Our team identified angiopoietin-like 4 (ANGPTL4) as a hypoxiainduced target and a key regulator of vascular integrity by protecting interendothelial cell junctions. However ANGPTL4 receptors and downstream signaling pathways which mediate vasoprotective effect remain poorly investigated. Using both in vitro binding (Surface Plasmon Resonance, SPR) and functional in vivo assays, we show that ANGPTL4 binds αvβ3 integrin and that this interaction is necessary to mediate vasoprotective effects. In addition, ANGPTL4 induces phosphorylation of Tyr773 of β3 integrin subunit and reorganization of focal adhesions in endothelial cells. Mechanistically, binding of ANGPTL4 to αvβ3 leads to Src recruitment and its sequestration away from VEGFR2 combined to a diminished Src signaling downstream VEGFR2, thereby inducing stabilization of both VEGFR2/VE-cadherin and VEGFR2/ αvβ3 complexes. Thus, ANGPTL4 strengthens maturation of adherens junctions, characterized by a transition from a zipper to linear organization of VEcadherin organization. Altogether, our results identify a novel mechanism by which ANGPTL4 counteracts hypoxia-driven vascular permeability through αvβ3 binding, modulation of VEGFR2/Src kinase signaling and endothelial junction stabilization.

International audience; Dendrigraft poly(L-lysine) (DGL) polyelectrolytes, obtained by iterative polycondensation of N-trifluoroacetyl-L-lysine-N-carboxyanhydride, constitute very promising candidates in many biomedical applications. In order to get a better understanding of their structure-property relationships in these applications, their absolute average molecular weights have to be accurately measured. Size-exclusion chromatography coupled to a multi-angle laser-light-scattering detector (SEC-MALLS) is known to be the most appropriate analytical tool. These measurements require the determination of the refractive index increment, dn/dc, of these highly branched polycationic macromolecules in aqueous solution. This optical property has to be measured in the same aqueous conditions as SEC-MALLS eluents. Consequently, data are determined and discussed as a function of different aqueous SEC-MALLS eluents, as well as different counter-ions of the many ammonium groups of DGL (generation 3, DGL-3, used as a model herein). The resulting number-average molecular weights, (M) over bar (n), are found to be very dissimilar when the measured dn/dc values are directly considered. In contrast, very close (M) over bar (n) values are obtained (average (M) over bar (n) = 18 700, standard error of 1110 g mol(-1)) with a low coefficient of variation for such data (ca.6% for six analyses), when the dn/dc are corrected by the exact lysine amount (measured by the total Kjeldahl nitrogen method).

A straightforward method for the preparation of hybrid bioorganic–inorganic materials is reported. Common strategies to synthesize such promising materials require special surface modifications of silica followed by grafting of the organic moiety via chemoselective ligation. In this context, we set up a general and bottom-up strategy relying on modified peptides functionalized with a trialkoxysilane group. Used in mixtures with TEOS and a surfactant as the structure directing agent, these hybrid building blocks allow one step direct synthesis of bioorganic–inorganic hybrid materials. Two examples were chosen to demonstrate our general approach. (1) An antifouling surface was prepared by dip coating of a sol containing an antibacterial silylated peptide. (2) Organized mesoporous silica displaying a peptide catalyst in the pores was prepared in one step and tested.

International audience; The present work describes a new environmental friendly strategy for the development of surfaces with high amine density via the grafting of native or modified poly-L-lysine dendrigraft (DGL G3) onto plasma activated polypropylene (PP), polystyrene (PS), polyimide, and polytetrafluoroethylene (PTFE) surface. Modified DGL G3 was prepared by replacement of few peripheral amines by various functionalities. Grafting efficiency was determined by wettability measurements, IRTF, XPS, AFM, and by colorimetry using optimized Coomassie Brilliant Blue method tailored for surface analysis. It was shown that a 4-7 nm DGL G3 monolayer with 4 1014 amine cm 2 was covalently grafted onto various surfaces. Immobilization of adenosine triphosphate on the DGL-g-PP material from dilute solution was studied by bioluminescence and proved the ability of the material to interact with polyanionic biological compounds: 1 ATP complex with 5 amine groups. So, this material has a potential use in diagnostic and more widely for biotechnology due to its high capacity for biomolecule immobilization.

International audience; Poly(L-lysine) (PLL) dendrigrafts (DGLs) are arborescent biosynthetic polymers of regular and controlled structures. They have specific properties such as biocompatibility and non-immunogenicity, and their surface density of NH2 functions can be easily modified and therefore appears as a powerful tool for the functionalization of hydrophobic polymers used in the context of tissue engineering. In this study, we evaluated several criteria of human skin fibroblasts when cultured with DGL of generations 2, 3 and 4, with linear PLL polymer as reference. In aqueous phase, DGLs and PLL displayed a similar cytotoxicity towards fibroblasts. Plastic culture plates grafted with DGLs were further characterized as homogeneous surfaces by atomic force microscopy and surface characterization by amino density estimation by colorimetric assay. Proliferation of fibroblasts was increased when cultured onto PLL and DGLs monolayers when compared with crude plates. Cellular adhesion was increased by 20% on DGLs in comparison to PLL. Integrin α5 subunit protein expression level was increased after 48 h of culture on DGLs, in comparison to control or PLL-coated surfaces. The presence of DGLs did not lead to overexpression or activation of matrix metalloproteinases 2 and 9. Finally, fibroblasts adhesion was increased by 40% on poly-(lactic-co-glycolic acid) matrices functionalized with DGLs when compared to PLL. Overall, these features make DGL promising candidates for the surface engineering of biomaterials in tissue engineering.

International audience; The efficient immobilization of antibodies on monolithic support is one of the most critical steps when preparing immunoaffinity supports. In this work, the ADECA (amino density estimation by colorimetric assay) method was adapted to tridimensional supports (in a dynamic mode) and proved to be efficient to characterize the antibodies grafting efficiency on 15.3 ± 0.9 mg porous glycidyl methacrylate (GMA)-co-ethylene dimethacrylate (EDMA) monolithic columns. The amount of grafted antibodies measured in situ on the monolith by ADECA (8.2 ± 0.2 μg of antibodies per milligram of monolith) was consistent with values obtained by bicinchoninic acid assay (BCA) after crushing the monolith. ADECA was shown to be less time-consuming and more versatile than BCA. The ADECA method was further implemented to thoroughly study and optimize the antibody grafting conditions (influence of pH and kinetics of the grafting step) on GMA-based monoliths and to check the covalent nature of the antibody/surface linking and its stability. Using the total amount of grafted antibodies and the amount of recognized antigen, we found that 65 ± 6% of antibodies were able to capture their antigen. Finally, the grafting of Fab and F(ab′)2 fragments demonstrated that no significant improvement of the global binding capacity of the monolith was obtained.

International audience; A micro-immunoaffinity monolithic column (μIAC) was developed and in-line coupled with capillary zone electrophoresis in a fully automated way with Ochratoxin A as test solute. The in-line micro-immunoaffinity columns based on monolithic methacrylate polymers (EDMA-GMA) were prepared in situ at the inlet end of a PTFE coated fused silica capillary by UV initiated polymerization and subsequently grafted with antibodies. These μIACs were thoroughly characterized. The synthesis of the polymeric support was first demonstrated to be reproducible in terms of permeability, surface properties and efficiency. The antibodies immobilization was then studied by a new original hydrodynamic method (ADECA) allowing the in situ quantitative determination (at a miniaturized scale) of the total amount of immobilized antibodies. The combination of this measurement with the binding capacity of the μIAC allowed, for the first time, the in situ determination of immobilized antibody activity. A total of 260 ± 15 ng (1.6 ± 0.1 pmol) of IgG antibodies/cm in 75 μm i.d. monolithic column (i.e. 18 μgmg(-1)) was obtained with (anti-Ochratoxin A/Ochratoxin A) as antibody/antigen model. 40% of the immobilized antibodies remain active corresponding to a binding capacity of 1.2 ± 0.2 pmol antigen/cm (i.e. 600 pg/cm of our test solute OTA), a very high capacity when dealing with trace analysis and with regard to the detection limits (30 pg and 0.5 pg with UV and LIF detection, respectively). The recovery yields were quantitative with negligible non-specific adsorption and allow analysis of diluted samples (1 ngmL(-1)) for a percolated volume of 10 μL. It was also demonstrated that despite the progressive denaturation of antibodies consecutive to the elution step, the binding capacity of the μIAC remained high enough to implement at least 15 consecutive analyses with the same column and in a fully automated way.

International audience; Finding a general solution for optimizing the grafting of antibody on solid surfaces is difficult due to the variety of material, grafting principles and chemistries or surface formats available (beads, microplates, fibers, etc.). Pre-screening methods able to assess grafting efficiency (GE) and specific activity (SA) are required. In this context, we present here two colorimetric assays that can be used on a wide variety of surface format, chemistry, etc. The first one, ADECA (Amino Density Estimation by Colorimetric Assay) allows a rapid estimation of grafted antibodies and allows calculating the GE. The second one, A2HRP (Antibody Anti-HorseRadish Peroxidase) provides a measure of the amount of active antibody, which, combined to ADECA, is used to determine the SA of grafted antibody. Analytical parameters (limit of detection, repeatability, linearity, etc.) of these two colorimetric assays are presented. Using two commercially available microplates, we demonstrated that, when used in parallel, these rapid and sensitive methods are well adapted to pre-screening of antibody grafting performances.

International audience; The functionalization of surfaces with amino groups is used in many application areas such as in industrial biocatalytic processes for the development of medical biomaterials and in the environment for removing pollutants from water. Amino group density and grafting stability are often related to functionalized material performances; thus, their characterizations are of prime importance. The determination of amino density and grafting stability on polymeric material (e.g. polypropylene, polystyrene and cylco olefin copolymer) is often time consuming and sometimes presents technical constraints, more particularly with non-flat materials. In this paper, we report a novel colorimetric assay using the Coomassie Brilliant Blue dye for both amino density determination and grafting stability measurement. The assay named ADECA for "Amino Density Estimation by Colorimetric Assay" is sensitive, rapid, robust and versatile. We demonstrate that ADECA makes the evaluation of aminated materials performances possible for numerous material compositions, formats and chemistries used for grafting. Our study focuses on dendrigraft of poly-L-lysine and poly(amidoamine) dendrimers dendritic materials.

Endostatin, a C-terminal fragment of collagen XVIII, binds to TG-2 (transglutaminase-2) in a cation-dependent manner. Recombinant human endostatin binds to TG-2 with an affinity in the nanomolar range (Kd=6.8 nM). Enzymatic assays indicated that, in contrast with other extracellular matrix proteins, endostatin is not a glutaminyl substrate of TG-2 and is not cross-linked to itself by the enzyme. Two arginine residues of endostatin, Arg27 and Arg139, are crucial for its binding to TG-2. They are also involved in the binding to heparin [Sasaki, Larsson, Kreuger, Salmivirta, Claesson-Welsh, Lindahl, Hohenester and Timpl (1999) EMBO J. 18, 6240–6248], and to α5β1 and αvβ3 integrins [Faye, Moreau, Chautard, Jetne, Fukai, Ruggiero, Humphries, Olsen and Ricard-Blum (2009) J. Biol. Chem. 284, 22029–22040], suggesting that endostatin is not able to interact simultaneously with TG-2 and heparan sulfate, or with TG-2 and integrins. Inhibition experiments support the hypothesis that the GTP-binding site of TG-2 is a potential binding site for endostatin. Endostatin and TG-2 are co-localized in the extracellular matrix secreted by endothelial cells under hypoxia, which stimulates angiogenesis. This interaction, occurring in a cellular context, might participate in the concerted regulation of angiogenesis and tumorigenesis by the two proteins.

Endostatin is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds to heparin/heparan sulfate and to a number of proteins, but its molecular mechanisms of action are not fully elucidated. We have used surface plasmon resonance (SPR) arrays to identify new partners of endostatin, and to give further insights on its molecular mechanism of action. New partners of endostatin include glycosaminoglycans (chondroitin and dermatan sulfate), matricellular proteins (thrombospondin-1 and SPARC), collagens (I, IV, and VI), the amyloid peptide Abeta-(1-42), and transglutaminase-2. The biological functions of the endostatin network involve a number of extracellular proteins containing epidermal growth factor and epidermal growth factor-like domains, and able to bind calcium. Depending on the trigger event, and on the availability of its members in a given tissue at a given time, the endostatin network might be involved either in the control of angiogenesis, and tumor growth, or in neurogenesis and neurodegenerative diseases.