Ephemeral biogels to control anti-biofilm agent delivery: From conception to the construction of an active dressing.

Chronic wound colonization by bacterial biofilms is common and can cause various complications. An anti-biofilm strategy was developed around the co-entrapment of a commercially available antiseptic, PHMB (polyhexamethylene biguanide 4mgmL-1), with EDTA (Ethylen diamine tetra acetic acid, 20mM) in a gelatin gel. The two active compounds act synergistically against bacterial biofilms, but their efficiency is strongly reduced (16-fold) when entrapped inside the 5% gelatin gel, and they weaken the mechanical properties (50-fold) of the gel. Increasing the gelatin concentration to 7% allows for good mechanical properties but large diffusional constraints. An active ephemeral gel, a chemical gel with controlled hydrolysis, was conceived and developed. When the ephemeral gel was solubilized after 48h, PHMB delivery increased, leading to good anti-biofilm activity. The various gels were examined over 24 and 48h of contact with P. aeruginosa and S. aureus biofilms, two types of bacterial biofilms frequently encountered in chronic wounds. The ephemeral gel eradicated the dense biofilms (>6.107CFU·cm-2) produced by either single or mixed strains; a similar efficiency was measured for biofilms from strains of both laboratory and clinical origin. The formulation was then adapted to develop a dressing prototype that is active against biofilms and fulfils the requirements of an efficient wound care system.

Amyloid peptides derived from CsgA and FapC modify the viscoelastic properties of biofilm model matrices.

The bacterial biofilm is a complex environment of cells, which secrete a matrix made of various components, mainly polysaccharides and proteins. An understanding of the precise role of these components in the stability and dynamics of biofilm architecture would be a great advantage for the improvement of anti-biofilm strategies. Here, artificial biofilm matrices made of polysaccharides and auto-assembled peptides were designed, and the influence of bacterial amyloid proteins on the mechanical properties of the biofilm matrix was studied. The model polysaccharides methylcellulose and alginate and peptides derived from the amyloid proteins curli and FapC found in biofilms of Enterobacteriaceae and Pseudomonas, respectively, were used. Rheological measurements showed that the amyloid peptides do not prevent the gelation of the polysaccharides but influence deformation of the matrices under shear stress and modify the gel elastic response. Hence the secretion of amyloids could be for the biofilm a way of adapting to environmental changes.

Genipin-cross-linked layer-by-layer assemblies: biocompatible microenvironments to direct bone cell fate.

The design of biomimetic coatings capable of improving the osseointegration of bone biomaterials is a current challenge in the field of bone repair. Toward this end, layer-by-layer (LbL) films composed of natural components are suitable candidates. Chondroitin sulfate A (CSA), a natural glycosaminoglycan (GAG), was used as the polyanionic component because it promotes osteoblast maturation in vivo. In their native state, GAG-containing LbL films are generally cytophobic because of their low stiffness. To stiffen our CSA-based LbL films, genipin (GnP) was used as a natural cross-linking agent, which is much less cytotoxic than conventional chemical cross-linkers. GnP-cross-linked films display an original combination of microscale topography and tunable mechanical properties. Structural characterization was partly based on a novel donor/acceptor Förster resonance energy transfer (FRET) couple, namely, FITC/GnP, which is a promising approach for further inspection of any GnP-cross-linked system. GnP-cross-linked films significantly promote adhesion, proliferation, and early and late differentiation of preosteoblasts.

Identification of an amyloidogenic peptide from the Bap protein of Staphylococcus epidermidis.

Biofilm associated proteins (Bap) are involved in the biofilm formation process of several bacterial species. The sequence STVTVT is present in Bap proteins expressed by many Staphylococcus species, Acinetobacter baumanii and Salmonella enterica. The peptide STVTVTF derived from the C-repeat of the Bap protein from Staphylococcus epidermidis was selected through the AGGRESCAN, PASTA, and TANGO software prediction of protein aggregation and formation of amyloid fibers. We characterized the self-assembly properties of the peptide STVTVTF by different methods: in the presence of the peptide, we observed an increase in the fluorescence intensity of Thioflavin T; many intermolecular ß-sheets and fibers were spontaneously formed in peptide preparations as observed by infrared spectroscopy and atomic force microscopy analyses. In conclusion, a 7 amino acids peptide derived from the C-repeat of the Bap protein was sufficient for the spontaneous formation of amyloid fibers. The possible involvement of this amyloidogenic sequence in protein-protein interactions is discussed.

Amyloid fiber formation by synthetic peptides derived from the sequence of the protein CsgA of Escherichia coli.

We characterized the formation of amyloid fibers by two peptides derived from the CsgA sequence: R5 (133- 151) corresponding to the whole repeating unit R5 and a truncated form of this peptide called R5T (134-143). In the presence of either of the two peptides: an increase in the fluorescence intensity of Thioflavin T was observed; a shift of the absorbance of Congo red was measured; spontaneous formation of amyloid fibers was observed by polarized light as well asatomic force microscopy imaging. Large-size aggregates were observed with R5 while R5T formed fagots of individualized fibers. The infrared spectroscopy analysis revealed the presence of a greater number of intermolecular bonds for R5. In conclusion, a 10 aminoacids peptide derived from the R5 sequence was sufficient for the spontaneous formation of amyloid fibrils but not to form large-size aggregates of fibers.