The BSMA division is active in the field of (bio-)functional organic coatings. Our efforts in this area are directed to the development and characterization of surfaces and interfaces using a variety of strategies and advanced tools. Our main projects deal with the engineering of antibacterial surfaces, nanostructured and biomimetic surfaces to control cell behavior, stimuli-responsive surfaces for the control of protein adsorption or cell stimulation and multifunctional surfaces exhibiting a catalytic activity.
Bioactive surfaces
Antibacterial surfaces, microparticles
Group of Karine Glinel
Prevention of bacterial adhesion and biofilm formation on material surfaces is a topic of a major medical and societal importance. In this context, we develop new coatings based on polymer chains immobilized on solid substrates and grafted with antibacterial peptides. These natural substances offer an attractive alternative to conventional bactericidal products such as antibiotics or metal derivatives since they show a large spectrum of activity while not inducing a phenomenon of resistance.
The immobilization of the antibacterial peptides on the polymer chains is achieved through bioconjugation techniques which preserve their activity. Depending on the application, these antibacterial coatings are deposited on flat substrates or microparticles of varying nature. The methodology used to produce these antibacterial layers is also developed from stimuli-responsive polymers in order to fabricate surfaces showing antibacterial properties on demand.
Biomimetic surfaces based on extracellular matrix proteins for the control of cell-material interactions
Group of Christine Dupont, Alain M. Jonas, Bernard Nysten, Sophie Demoustier
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. Over the past fifteen years, we have investigated type I collagen adsorption and designed collagen-based biointerfaces.
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 (ie 300 nm) 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. The evaluation of cell behavior on the created biointerfaces showed that the control of collagen organization can be successfully used to alter cell behavior.
Nano/micropatterned surfaces to control bacteria and mammalian cell behavior
Group of Christine Dupont, Karine Glinel, Alain M. Jonas
Surfaces patterned at the nano/micrometer scale are developed to address locally a specific (bio)chemical or physical cue to cells in order to direct their behavior. Different surface properties are patterned such as the topography, the (bio)chemistry and the stiffness. To prepare these platforms, we combine nanofabrication techniques such as colloidal lithography, nanoimprint lithography, soft templating and photolithography with surface functionalization techniques such as surface-initiated polymerization, electropolymerization, layer-by-layer assembly, polymer demixing, silanation and thiolisation.
These surfaces multi-engineered at the nano/micrometer scale allow the complex interactions between cells and material surfaces to be elucidated.
Nanostructured 2D and 3D biointerfaces using self-assembled ECM-like nanotubes
Group of Sophie Demoustier, Christine Dupont
Bottom-up template-based construction is combined with the versatile layer-by-layer (LbL) deposition technique to produce ECM-like multilayered nanotubes with highly regular dimensions. Based on this method, free collagen-based nanotubes are successfully prepared, and efficiently collected by electrophoretic deposition to create 2D nanostructured biointerfaces coated with tunable density of protein nanotubes. The possibility of elaborating nanostructured 3D biointerfaces based on self-assembled nanotubes using relevant ECM components as building blocks and including bioactive substances is currently explored.
Such LbL assemblies nanostructured into 2D or 3D networks that mimic certain features of natural ECM offer new opportunities for studying cell-material interactions at the nanoscale. From a more applied point of view, the ability to engineer such biointerfaces is very advantageous for various biomedical applications, in particular for the successful regeneration of tissues.
Surface-modified 3D printed environment for the control of stem cell behavior
Group of Christine Dupont – in collaboration with Anne des Rieux (LDRI, UCL)
Additive manufacturing techniques, commonly known as 3D printing, attract more and more interest in the field of tissue engineering. Among them, Fused Deposition Modeling (FDM) is a low-cost and easy-to-use technique, based on the layer-by-layer deposition of thermoplastic polymer in a semi-liquid state along an extrusion path. It can be applied to a large variety of polymers, and allows the production of patient-personalized scaffolds with a precise control of the geometry. Beside, stem cells offer promising possibilities in this field because they can be differentiated into the desired cell type before implanting the cell-scaffold construct. Their differentiation is known to be influenced, among others, by the physical environment provided by the substrate to which cells adhere.
The first aim of the project is to optimize the printing parameters of a poly(lactic acid) (PLA) complex object at a scale close to the resolution limit of fused deposition modeling. The gyroid design is used as a mesh for the 3D printing of scaffolds due to its remarkable properties, including large pore size, high interconnectivity, high porous volume and isotropy. The effect of mesh design, polymer crystallinity, and printing layer resolution on mechanical properties and degradation rate will be assessed. The second aim of the work is to study how a complex 3D-printed scaffold coated with a polymer with tunable mechanical properties will impact the behavior of adipose-derived mesenchymal stem cells (AMSC).
(work of Loïc Germain, PhD student)
Protein-polyelectrolyte complexes to improve the biological activity of proteins in layer-by-layer assemblies
Group of Christine Dupont
Biofunctionalization is a cornerstone for biosensing, biocatalysis, cell and virus manipulations, biobanking, delivery, etc. A staggering number of biointerfaces were thus proposed with a common key target: keeping the protein biologically active. In the search for simple, versatile and efficient modification methods, a standard method based on the use of protein-polyelectrolyte complexes (PPCs) as building blocks for layer-by-layer (LbL) assembly is proposed. The LbL assembly which consists in the alternate adsorption of oppositely charged polyelectrolyte, is indeed achievable in soft condition and is versatile towards surface geometry and chemistry. By alternate adsorption of PPCs and polyelectrolytes, thicker multilayers, with a higher polyelectrolyte fraction are obtained compared to single protein molecules integration. In the case of lysozyme-poly(styrene sulfonate) complexes, the specific activity is higher compared to the one obtained for multilayers based on lysozyme alone. This is attributed to the more hydrated state of the assemblies. This new method of protein immobilization opens up perspectives for biotechnologies and biomedical applications. (work of Aurélien vander Straeten, PhD student)
Biomolecules at interfaces
Characterizing proteins and other biomolecules at interfaces
Group of Arnaud Delcorte, Christine Dupont
Advanced surface characterization techniques are combined in order to investigate adsorbed protein layers. Parameters governing adsorption (including substrate surface properties, medium composition and adsorption procedure) are varied to unravel adsorption mechanisms and gain a better control of adsorption. The main tools used for adsorbed proteins characterization are: atomic force microscopy (AFM), time-of-flight secondary ion mass spectrometry (ToF-SIMS), X-ray photoelectron spectroscopy (XPS), quartz crystal microbalance (QCM) and radio- or fluorescent labeling. AFM gives access to the protein layer organization with nanometer-scale resolution and in physiological conditions.
ToF-SIMS being sensitive to the extreme surface layer (probed depth ~1nm), it may deliver information related to protein orientation and,or conformation. Advanced statistical treatment of the mass spectra is required to identify the useful peaks. These ToF-SIMS data are advantageously combined to XPS results, which give access to a quantitative evaluation of the thickness and,or the surface coverage of the substrate by the protein layer. QCM allows adsorption to be monitored in situ and in real time. Other labeling methods are useful to evaluate adsorbed amount. In most cases, it is necessary to couple several of these characterization methods to decipher protein layer organization.
Interactions between antigens and adjuvant particles in vaccines
Group of Christine Dupont
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. Controlling antigen-AH interactions is a key challenge in vaccine formulation. Previous studies focusing on protein-AH interaction mechanisms suggested that electrostatic interactions and phosphate-hydroxyl ligand exchanges drive protein adsorption. We however 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. We have successfully developed and used a stable AH-coated interface to explore the mechanisms of protein adsorption by means of ultra-sensitive surface analysis tools (Figure 1). By studying bovine serum albumin (BSA) adsorption at different pH and ionic strength (I) via Quartz Crystal Microbalance, we show that protein adsorption on AH adjuvant cannot be explained solely by electrostatic interactions and ligand exchanges. Almost no effect of I on adsorption was indeed noted at pH 7, in conditions of attractive electrostatic interactions. Moreover, a higher adsorption is observed at pH 3, in conditions of repulsive electrostatic interactions, compared to pH 7. These new developments and observations not only suggest that other mechanisms than electrostatic interactions regulate protein adsorption on AH, but also offer a new platform for the study of antigen adsorption, to the benefit of vaccine formulation.
(work of Jean-François Art, PhD student)
Stimuli responsive and other functional surfaces
Responsive interfaces for the control of protein adsorption
Group of Sophie Demoustier, Christine Dupont
Interfaces are tailored with a view to better control protein adsorption. In particular, coatings incorporating stimuli-responsive polymers are designed to trigger protein adsorption or desorption by changing environmental conditions such as pH, temperature or ionic strength (I).
For instance, mixed brushes of poly(ethylene oxide) (PEO) and poly(acrylic acid) (PAA) were assembled on gold substrates. Depending on pH and I conditions, protein adsorbed amount can be very high compared to the one observed on naked gold, or can be very low. This behavior can be related to the polymer chain conformation, and is reversible, opening the possibility to trigger successive adsorption/desorption cycles. Suitable working conditions were identified for proteins with very different characteristics, pointing to the versatility of the approach.
In another ongoing project, poly(N-isopropylacrylamide) (PNIPAM)-based coatings are prepared by Layer-by-Layer (LbL) multilayers deposition on planar and nanoporous substrates using PNIPMA-PAA block copolymers as polyanions and poly(allylamine) (PAH) as polycation. The stimuli-responsive properties of the LbL films are examined by monitoring the adsorption of proteins while varying T, pH, ionic strength, or a combination of all these parameters. Interestingly, adsorption/desorption cycles reveal the ability of the films to switch from favorable to unfavorable configuration towards protein adsorption.
Responsive polymer brushes
Group of Karine Glinel, Alain Jonas, Bernard Nysten
Responsive polymer brushes are dense assemblies of end-grafted polymer chains, whose affinity for a solvent abruptly changes depending on a change of external conditions. Such systems offer attractive opportunities for applications when a rapid variation of surface properties is desired. In our groups, we concentrate on brushes grown by atom transfer radical polymerization, able to respond to temperature, pH or light.
We especially investigate the parameters which can be used to tune the behavior of the brushes, including copolymerization, grafting density, thickness, and patterning. As a typical example, the graph below provides a bidimensional map of the degree of swelling of a dual pH- and temperature-responsive brush, obtained from QCM experimental data.
Responsive surfaces for cell stimulation
Group of Sophie Demoustier, Christine Dupont, Karine Glinel, Alain Jonas
Smart responsive-coatings whose surface properties can be triggered by an external stimulus are developed to control mammalian and bacterial cell behavior. Depending on the application, the variation of the bio-adhesiveness or the bioactivity is aimed. For this, polymer chains showing thermo-, redox- or light-responsive properties are immobilized on various solid substrates by grafting or assembling.
The resulting layers are subsequently modified with bioactive groups which can be shown/hidden or released under the application of a given stimulus in order to direct the behavior of cells on demand.
Multifunctional nano-designed catalytic surfaces
For organo-metallic-grafted surfaces: Group of Antony Fernandes, Olivier Riant, Alain Jonas
For enzyme-based catalytic surfaces: Group of Sophie Demoustier, Alain Jonas
We develop surfaces capable to exhibit a catalytic activity using two different strategies. In the first one, we heterogeneize organo-metallic catalysts by coupling them to silane monolayers or in polymer brushes. We are especially interested by synergetic effects which can arise when catalytic centers and co-catalysts are present in the confined environment of surfaces or brushes.
Another route consists of incorporating enzymes in layer-by-layer assemblies (LbL's), which can be grown on flat surfaces or in tubular geometries for improved surface-to-volume ratio. The specific environment provided by hydrophilic LbL's is favorable for a preservation of enzymatic activity, while also offering a more ubiquitous methodology to functionalize surfaces. The image below is a brush of LbL nanotubes incorporating beta-lactamase.
Plasma polymer coatings
Plasma-polymers and plasma-treated organic surfaces
Group of Arnaud Delcorte
“Mild” plasma conditions (i.e. pulsed plasma at low power) allow the formation of polymeric films, so-called plasma-polymers, by injecting the monomers in the plasma phase, where they gain energy and in turn react with a substrate. Copolymerization can also be obtained by the co-injection of two (or more) monomers in the plasma, leading to copolymers with various compositions [1]. Such coatings have applications in various fields ranging from biomaterials technology, medical diagnostic to aerospace and automotive industries.
On the other hand, (atmospheric) plasmas can also simply be used for the surface treatment of polymers (functionalization, hydrophilicity). In general, plasma-polymers and plasma-treated polymers have a complex structure, involving cross-linking, branching, oxidation and unsaturation. Our contribution in this field focuses on gaining a better understanding of the structure and the local chemistry of polymers synthesized or treated in (almost) atmospheric pressure plasmas. Experimentally, we use ToF-SIMS (time-of-flight secondary ion mass spectrometry) characterization, with giant argon clusters for soft sputtering of the surface, in order to combine lateral analysis with in depth molecular information. Theoretically, the growth of plasma-polymers is modelled using a combination of ab initio calculations and classical molecular dynamics simulations. This work is part of a broader interuniversity research program on plasma-surface interactions (http://psi-iap7.ulb.ac.be/).
(a) Molecular depth profiling of a fluorinated aromatic plasma polymer deposited in a dielectric barrier discharge (coll. ULB); (b) Depth profiling of LDPE treated by an atmospheric D2O plasma torch (coll. ULB); (c) MD simulation of the interaction of a styrene gas with a metal substrate; (c) model of an amorphous PS substrate; (e) DFT calculation of the stability of the reaction product of a styrene radical with a styrene monomer.