Chronic wound healing: A specific antibiofilm protein-asymmetric release system.

Chronic infection is a major cause of delayed wound-healing. It is recognized to be associated with infectious bacterial communities called biofilms. Currently used conventional antibiotics alone often reveal themselves ineffective, since they do not specifically target the wound biofilm. Here, we report a new conceptual tool aimed at overcoming this drawback: an antibiofilm drug delivery system targeting the bacterial biofilm as a whole, by inhibiting its formation and/or disrupting it once it is formed. The system consists of a micro/nanostructured poly(butylene-succinate-co-adipate) (PBSA)-based asymmetric membrane (AM) with controlled porosity. By the incorporation of hydrophilic porogen agents, polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG), we were able to obtain AMs with high levels of porosity, exhibiting interconnections between pores. The PBSA-PEG membrane presented a dense upper layer with pores small enough to block bacteria penetration. Upon using such porogen agents, under dry and wet conditions, membrane's integrity and mechanical properties were maintained. Using bovine serum albumin (BSA) as a model protein, we demonstrated that protein loading and release from PBSA membranes were affected by the membrane structure (porosity) and the presence of residual porogen. Furthermore, the release curve profile consisted of a fast initial slope followed by a second slow phase approaching a plateau within 24 h. This can be highly beneficial for the promotion of wound healing. Cross-sectional confocal laser scanning microscopy (CLSM) images revealed a heterogeneous distribution of fluorescein isothiocyanate (FITC) labeled BSA throughout the entire membrane. PBSA membranes were loaded with dispersin B (DB), a specific antibiofilm matrix enzyme. Studies using a Staphylococcus epidermidis model, indicate significant efficiency in both inhibiting or dispersing preformed biofilm (up to 80 % eradication). The asymmetric PBSA membrane prepared with the PVP porogen (PBSA-PVP) displayed highest antibiofilm activity. Moreover, in vitro cytotoxicity assays using HaCaT and reconstructed human epidermis (RHE) models revealed that unloaded and DB-loaded PBSA-PVP membranes had excellent biocompatibility suitable for wound dressing applications.

Designing Biodegradable PHA-Based 3D Scaffolds with Antibiofilm Properties for Wound Dressings: Optimization of the Microstructure/Nanostructure.

One major factor inhibiting natural wound-healing processes is infection through bacterial biofilms, particularly in the case of chronic wounds. In this study, the micro/nanostructure of a wound dressing was optimized in order to obtain a more efficient antibiofilm protein-release profile for biofilm inhibition and/or detachment. A 3D substrate was developed with asymmetric polyhydroxyalkanoate (PHA) membranes to entrap Dispersin B (DB), the antibiofilm protein. The membranes were prepared using wet-induced phase separation (WIPS). By modulating the concentration and the molecular weight of the porogen polymer, polyvinylpyrrolidone (PVP), asymmetric membranes with controlled porosity were obtained. PVP was added at 10, 30, and 50% w/w, relative to the total polymer concentration. The physical and kinetic properties of the quaternary nonsolvent/solvent/PHA/PVP systems were studied and correlated with the membrane structures obtained. The results show that at high molecular weight (Mw = 360 kDa) and high PVP content (above 30%), pore size decreased and the membrane became extremely brittle with serious loss of physical integrity. This brittle effect was not observed for low molecular weight PVP (Mw = 40 kDa) at comparable contents. Whatever the molecular weight, porogen content up to 30% increased membrane surface porosity and consequently protein uptake. Above 30% porogen content, the pore size and the physical integrity/mechanical robustness both decreased. The PHA membranes were loaded with DB and their antibiofilm activity was evaluated against Staphylococcus epidermidis biofilms. When the bacterial biofilms were exposed to the DB-loaded PHA membrane, up to 33% of the S. epidermidis biofilm formation was inhibited, while 26% of the biofilm already formed was destroyed. These promising results validate our approach based on the development of bioactive-protein-loaded asymmetric membranes for antibiofilm strategies in situations where traditional antibiotic therapies are ineffective.

Surface composition XPS analysis of a plasma treated polystyrene: Evolution over long storage periods.

A polystyrene surface (PS) was initially treated by cold nitrogen and oxygen plasma in order to incorporate in particular amine and hydroxyl functions, respectively. The evolution of the chemical nature of the surface was further monitored over a long time period (580 days) by chemical assay, XPS and contact angle measurements. Surface density quantification of primary amine groups was performed using three chemical amine assays: 4-nitrobenzaldehyde (4-NBZ), Sulfo succinimidyl 6-[3'(2 pyridyldithio)-pionamido] hexanoate (Sulfo-LC-SPDP) and iminothiolane (ITL). The results showed amine densities were in the range of 2 per square nanometer (comparable to the results described in the literature) after 5min of nitrogen plasma treatment. Over the time period investigated, chemical assays, XPS and contact angles suggest a drastic significant evolution of the chemical nature of the surface within the first two weeks. Beyond that time period and up to almost two years, nitrogen plasma modified substrates exhibits a slow and continuous oxidation whereas oxygen plasma modifed polystyrene surface is chemically stable after two weeks of storage. The latter appeared to "ease of" showing relatively mild changes within the one year period. Our results suggest that it may be preferable to wait for a chemical "stabilization" period of two weeks before subsequent covalent immobilization of proteins onto the surface. The originality of this work resides in the study of the plasma treated surface chemistry evolution over long periods of storage time (580 days) considerably exceeding those described in the literature.

Elucidation of innovative antibiofilm materials.

It is known for roughly a decade that bacterial communities (called biofilms) are responsible for significant enhanced antibiotherapy resistance. Biofilms are involved in tissue persistent infection, causing direct or collateral damage leading to chronic wounds development and impairing natural wound healing. In this study, we are interested in the development of supported protein materials which consist of asymmetric membranes as reservoir supports for the incorporation and controlled release of biomolecules capable of dissolving biofilms (or preventing their formation) and their use as wound dressing for chronic wound treatment. In a first step, polyhydroxyalkanoates (PHAs) asymmetric membranes were prepared using wet phase inversion technique. Scanning microscopy (SEM) analysis has showed the influence of different processing parameters. In a second step, the porous side of the membranes were functionalized with a surface treatment and then loaded with the antibiofilm agent (dispersin B). In a third step, the properties and antibiofilm performance of the loaded-membranes were evaluated. Exposure of Staphylococcus epidermidis biofilms to such systems weakly inhibited biofilm formation (weak preventive effect) but caused their detachment and disaggregation (strong curative effect). These initial results are promising since they open the way to a new generation of effective tools in the struggle against persistent bacterial infections exhibiting enhanced antibiotherapy resistance, and in particular in the case of infected chronic wounds.

Surface assembly on biofunctional magnetic nanobeads for the study of protein-ligand interactions.

One of the major challenges of proteomics today is to increase the power potential for the identification of as many proteins as possible and to characterize their interactions with specific free ligands (interactomics) or present on cell walls (cell marker), in order to obtain a global, integrated view of disease processes, cellular processes and networks at the protein level. The work presented here proposes the development of biofunctionalized magnetic nanobeads that might be used for interactomic investigations. The strategy consisted in immobilizing proteins via a non covalent technique that provides greater possibilities for the advent of faster, cheaper and highly miniaturizable protein analysis systems, in particular in situations where the amount of isolated protein is scarce (trace proteins). The advantage of the immobilization technique proposed here over more conventional covalent binding techniques is that it is versatile and universal (not protein specific) thus applicable to a wide range of proteins, in "mild" conditions that are non deleterious to the native structure and bioactivity of the immobilized protein. The feasibility of the technique was investigated using a model protein (streptavidin). The nanobeads were analyzed in size by light diffusion and transmission electronic spectroscopy, and in quantity of immobilized protein using a bioassay developed in the laboratory. Results are promising in that nanobeads exhibited good colloidal stability and surface concentrations in the monolayer range.

Bioengineering and characterization of DNA-protein assemblies floating on supported membranes.

This chapter describes the design, practical construction, and characterization of P-DNA and their applications in building a new generation of DNA chips. P-DNAs are artificial covalent assemblies involving a histidine tag head able to bind to modified phospholipids, a core protein domain derived from cytochrome b5 by genetic engineering that features specific spectroscopic and electrochemical properties useful for detection, a synthetic linker acting as a spacer, and an oligonucleotide acting as a probe. P-DNA has the property of being able to efficiently self-associate to a supported bilayer including nickel-iminodiacetate-modified phospholipids. The construction of P-DNA and its interaction with a complementary oligonucleotide sequence can be monitored in real time by surface plasmon resonance using a Biacore system or equivalent. P-DNA chips feature unique properties including tunable surface density of probes; very low nonspecific interaction with external DNA; lateral mobility, minimizing-steric interaction; optimization of hybridization efficiency; and, potentially, recognition by multiple probes of a single target and perfectly defined and homogeneous structure, permitting high density up to a compact monolayer. Potential applications of this new device are multiple, including high-sensitivity and high-selectivity chips for DNA-DNA, DNA-RNA, or DNA-protein interactions.