Benoît Liberelle

Quantification of Primary Amine Groups Available for Subsequent Biofunctionalization of Polymer Surfaces

Biocompatible polymers are commonly functionalized with specific moieties such as amino groups to modify their surface properties and/or to attach bioactive compounds. A reliable method is usually required to characterize amino group surface densities. In this study, aminated polyethylene terephthalate (PET) films were generated via an aminolysis reaction involving either ethylenediamine molecules (EtDA), in order to vary easily the amino group density on PET surfaces, or 25 kDa polyvinylamine (PVAm) as an alternative reagent preventing bulk damages resulting from the aminolysis reaction. Among commonly used dyes for amino group quantification, Orange II and Coomassie Brillant Blue (CBB) were selected to quantify the extent of amine grafting resulting from these derivatization procedures. Rapid and convenient colorimetric assays were compared to surface atomic compositions obtained from X-ray photoelectron spectroscopy (XPS) measurements. Orange II was found to be the most appropriate dye for quantifying primary amine groups in a reliable and specific way. Due to its unique negative charge and low steric hindrance compared to CBB, the Orange II dye was very sensitive and provided reliable quantification over a wide range of amino group surface densities (ca. 5 to at least 200 pmol/mm(2)). In order to further validate the use of the Orange II dye for amino group quantification, a heterobifunctional linker reacting with amino groups was then grafted on modified PET surfaces. Interestingly, the good correlation between the densities of adsorbed Orange II and covalently grafted linkers suggests that the Orange II method is a relevant, reliable, easy, and inexpensive method to predict the amount of amino groups available for subsequent functionalization of polymer surfaces.

Friction and normal interaction forces between irreversibly attached weakly charged polymer brushes.

Polyelectrolyte brushes were built on mica by anchoring polystyrene-poly(acrylic acid) (PS-b-PAA) diblock copolymers at a controlled surface density in a polystyrene monolayer covalently attached to OH-activated mica surfaces. Compared to physisorbed polymer brushes, these irreversibly attached charged brushes allow the polymer grafting density to remain constant upon changes in environmental conditions (e.g., pH, salt concentration, compression, and shear). The normal interaction and friction forces as a function of surface separation distance and at different concentrations of added salt (NaCl) were investigated using a surface forces apparatus. The interaction force profiles were completely reversible both on loading and receding and were purely repulsive. For a constant polymer grafting density, the influence of the polyelectrolyte charges and the Debye screening effect on the overall interaction forces was investigated. The experimental interaction force profiles agree very well with scaling models developed for neutral and charged polymer brushes. The variation of the friction force between two PAA brushes in motion with respect to each other as a function of surface separation distance appeared to be similar to that observed with neutral brushes. This similarity suggests that the increase in friction is associated with an increase in mutual interpenetration upon compression as observed with neutral polymers. The effect of the PAA charges and added ions was more significant on the repulsive normal forces than on the friction forces. The reversible characteristics of the normal force profiles and friction measurements confirmed the strong attachment of the PAA brushes to the mica substrate. High friction coefficients (ca 0.3) were measured at relatively high pressures (40 atm) with no surface damage or polymer removal.

Stability of Silanols and Grafted Alkylsilane Monolayers on Plasma-Activated Mica Surfaces

We investigated the effect of physical and chemical modifications of mica surfaces induced by water vapor-based plasma treatments on the stability of silanols and grafted alkylsilane monolayers. The plasma-activated substrates were characterized using XPS, TOF-SIMS, and contact angle measurements. They revealed a large surface coverage of silanol groups (Si-OH) and a loss of aluminum atoms compared to freshly cleaved mica surfaces. The stability of plasma-induced silanol groups was investigated by contact angle measurements using ethylene glycol as a probe liquid. The Si-OH surface coverage decreased rapidly under vacuum or thermal treatment to give rise to hydrophobic dehydrated surfaces. The stability of end-grafted monofunctionalized n-alkylsilanes was investigated in different solvents and at different pH using water contact angle measurements. The degrafting of alkylsilanes from the activated mica was promoted in acidic aqueous solutions. This detachment was associated with the hydrolysis of covalent bonds between the alkylsilanes and the mica surface. The monolayer stability was enhanced by increasing the length of the alkyl chains that probably act as a hydrophobic protective layer against hydrolysis reactions. Stable alkylsilane monolayers in water with pH greater than 5.5 were obtained on mica surfaces activated at low plasma pressure. We attributed this stability to the loss of surface Al atoms induced by the plasma treatment.

Chondroitin Sulfate Coatings Display Low Platelet but High Endothelial Cell Adhesive Properties Favorable for Vascular Implants

This study highlights the advantages of chondroitin sulfate (CS) as a sublayer combining selective low-fouling properties, low-platelet adhesion and pro-adhesive properties on endothelial cells, making CS promising for vascular graft applications. These properties were evaluated by comparing CS with well-known low-fouling coatings such as poly(ethylene glycol) (PEG) and carboxymethylated dextran (CMD), which were covalently grafted on primary amine-rich plasma polymerized (LP) films. Protein adsorption studies by quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence measurements showed that CS is as effective as PEG in reducing fibrinogen adsorption (~90% reduction). CS also largely reduced adsorption of bovine serum albumin (BSA) as well as fetal bovine serum (FBS) but to a lower extent than PEG and CMD surfaces (72% vs 85% for BSA and 66% vs 89% for FBS). Whole blood perfusion assays indicated that, while LP surfaces were highly reactive with platelets, PEG, CMD, and CS grafted surfaces drastically decreased platelet adhesion and activation to levels significantly lower than polyethylene terephthalate (PET) surfaces. Finally, while human umbilical vein endothelial cell (HUVEC) adhesion and growth were found to be very limited on PEG and CMD, they were significantly increased on CS compared to that on bare PET and reached similar values as those for tissue culture polystyrene positive controls. Interestingly, HUVEC retention during perfusion with blood was found to be excellent on CS but poor on PET. Overall, our results suggest that the CS surface has the advantage of promoting HUVEC growth and resistance to flow-induced shear stress while preventing fibrinogen and platelet attachment. Such a nonthrombogenic but endothelial-cell adhesive surface is thus promising to limit vascular graft occlusion.

Epidermal growth factor tethered through coiled-coil interactions induces cell surface receptor phosphorylation.

We have elaborated and validated a novel approach for the oriented tethering of proteins such as the epidermal growth factor (EGF) on aminated surfaces. The grafting reactions were optimized to generate a dense and homogeneous EGF layer. Impact of EGF orientation on A-431 cellular response was investigated. Our results demonstrate that, in sharp contrast to responses obtained with soluble EGF supply or with randomly grafted EGF, oriented immobilization of EGF via a de novo designed coiled-coil capture system leads to a sustained phosphorylation of A-431 cell surface EGF receptors. Our results thus indicate that oriented protein immobilization via coiled-coil interactions is an efficient and versatile method to control tethering of bioactive molecules for future applications in the field of regenerative medicine and tissue engineering.

Stimulation of cell growth and resistance to apoptosis in vascular smooth muscle cells on a chondroitin sulfate/epidermal growth factor coating

Deficient healing after endovascular aneurysm repair is thought to be related to the pro-apoptotic environment in abdominal aortic aneurysms and inertness of the graft materials. A bioactive coating containing both chondroitin sulfate (CS) and epidermal growth factor (EGF) was developed in order to increase the growth and resistance to apoptosis of vascular smooth muscle cells (VSMC) on biomaterials surfaces. CS and EGF were covalently grafted using carbodiimide chemistry and the coating was characterized and optimized using ellipsometry, static contact angle and ToF-SIMS. Its potential to improve cell adhesion, growth and resistance to apoptosis was assessed in vitro with rat aortic VSMC. Results showed that CS and EGF immobilization allowed for the creation of a uniform coating that increased cell adhesion, growth and resistance to apoptosis in serum-free medium. Overall, CS and EGF possess great potential as bioactive anti-apoptotic mediators for vascular repair.

A non-damaging chemical amination protocol for poly(ethylene terephthalate) – application to the design of functionalized compliant vascular grafts

Bioengineering approaches have been intensively applied to create small diameter vascular grafts using artificial materials. However, a fully successful, high performing and anti-thrombogenic structure has not been achieved yet. In this study, we present the first step of a process aiming at biofunctionalizing previously designed compliant polyethylene terephthalate (PET) scaffolds (Moreno et al., 2011). The main challenge of such a surface modification is to prevent the bulk polymer from any damage, so that it preserves the mechanical properties that the structures have been designed for. In that endeavor, an aminated long-chain polymer (polyvinylamine, PVAm) was used as an aminolysis reagent to get amine (-NH2) moieties only on the very surface of PET. Different reaction conditions were assayed, leading to a large range of amino group densities associated with slight variations of the planar tensile properties. These results were in stark contrast with those generated with a common small diamine substrate (ethylenediamine, EtDA), as the latter yielded a strong degradation of the mechanical properties for comparable amine densities. Tubular mechanical assays were then carried out on PVAm-functionalized PET scaffolds. The latter showed a compliance match with arteries under the chosen reaction conditions, as initially observed for pristine PET tubular scaffolds.

Controlled co-immobilization of EGF and VEGF to optimize vascular cell survival

UNLABELLED Growth factors (GFs) are potent signaling molecules that act in a coordinated manner in physiological processes such as tissue healing or angiogenesis. Co-immobilizing GFs on materials while preserving their bioactivity still represents a major challenge in the field of tissue regeneration and bioactive implants. In this study, we explore the potential of an oriented immobilization technique based on two high affinity peptides, namely the Ecoil and Kcoil, to allow for the simultaneous capture of the epidermal growth factor (EGF) and the vascular endothelial growth factor (VEGF) on a chondroitin sulfate coating. This glycosaminoglycan layer was selected as it promotes cell adhesion but reduces non-specific adsorption of plasma proteins. We demonstrate here that both Ecoil-tagged GFs can be successfully immobilized on chondroitin sulfate surfaces that had been pre-decorated with the Kcoil peptide. As shown by direct ELISA, changing the incubation concentration of the various GFs enabled to control their grafted amount. Moreover, cell survival studies with endothelial and smooth muscle cells confirmed that our oriented tethering strategy preserved GF bioactivity. Of salient interest, co-immobilizing EGF and VEGF led to better cell survival compared to each GF captured alone, suggesting a synergistic effect of these GFs. Altogether, these results demonstrate the potential of coiled-coil oriented GF tethering for the co-immobilization of macromolecules; it thus open the way to the generation of biomaterials surfaces with fine-tuned biological properties. STATEMENT OF SIGNIFICANCE Growth factors are potent signaling molecules that act in a coordinated manner in physiological processes such as tissue healing or angiogenesis. Controlled coimmobilization of growth factors on biomaterials while preserving their bioactivity represents a major challenge in the field of tissue regeneration and bioactive implants. This study demonstrates the potential of an oriented immobilization technique based on two high affinity peptides to allow for the simultaneous capture of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF). Our system allowed an efficient control on growth factor immobilization by adjusting the incubation concentrations of EGF and VEGF. Of salient interest, co-immobilizing of specific ratios of EGF and VEGF demonstrated a synergistic effect on cell survival compared to each GF captured alone.

Additive Benefits of Chondroitin Sulfate and Oriented Tethered Epidermal Growth Factor for Vascular Smooth Muscle Cell Survival

An anti-apoptotic coating combining chondroitin sulfate (CS) and coiled-coil-based tethering of epidermal growth factor (EGF) is designed for vascular applications. The oriented tethering strategy enables to reach higher EGF surface densities compared to the commonly used random covalent grafting, while using much lower concentrations of EGF during incubation. It also significantly improves vascular smooth muscle cell (VSMC) survival and resistance to apoptosis in serum-free conditions. The comparison of CS and low-fouling carboxymethylated dextran as a sublayer for growth factors highlights the tremendous benefit of CS thanks to its selective protein resistance and good cell adhesion properties. This approach can be tuned by capturing other growth factors on CS through coiled-coil interactions.

Coiled-coil-mediated grafting of bioactive vascular endothelial growth factor

Chimeric growth factors may represent a powerful alternative to their natural counterparts for the functionalization of tissue-engineered scaffolds and applications in regenerative medicine. Their rational design should provide a simple, readily scalable production strategy while improving retention at the site of action. In that endeavor, we here report the synthesis of a chimeric protein corresponding to human vascular endothelial growth factor 165 being N-terminally fused to an E5 peptide tag (E5-VEGF). E5-VEGF was successfully expressed as a homodimer in mammalian cells. Following affinity purification, in vitro surface plasmon resonance biosensing and cell survival assays confirmed diffusible E5-VEGF ability to bind to its receptor ectodomains, while observed morphological phenotypes confirmed its anti-apoptotic features. Additional surface plasmon resonance assays highlighted that E5-VEGF could be specifically captured with high stability when interacting with covalently immobilized K5 peptide (a synthetic peptide designed to bind to the E5 moiety of chimeric hVEGF). This immobilization strategy was applied to glass substrates and chimeric hVEGF was shown to be maintained in a functionally active state following capture. Altogether, our data demonstrated that stable hVEGF capture can be performed via coiled-coil interactions without impacting hVEGF bioactivity, thus opening up the way to future applications in the field of tissue engineering and regenerative medicine.