Layer-by-layer assembly of chitosan/alginate thin films containing Salmonella enterica bacteriophages for antibacterial applications.

The emergence of antibiotic resistant bacteria and the ineffectiveness of routine treatments inspired development of alternatives to biocides for antibacterial applications. Bacteriophages are natural predators of bacteria and are promising alternatives to antibiotics. This study presents fabrication of a Salmonella enterica bacteriophage containing ultra-thin multilayer film composed of chitosan and alginate and demonstrates its potential as an antibacterial coating for food packaging applications. Chitosan/alginate film was prepared through layer-by-layer (LbL) self-assembly technique. A bacteriophage, which belongs to Siphoviridae morphotype (MET P1-001_43) and infects Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella Enteritidis), was post-loaded into chitosan/alginate film. The LbL growth, stability, and surface morphology of chitosan/alginate film as well as phage deposition into multilayers were analysed through ellipsometry, QCM-D and AFM techniques. The bacteriophage containing multilayers showed antibacterial activity at pH 7.0. In contrast, anti-bacterial activity was not observed at acidic conditions. We showed that wrapping a Salmonella Enteritidis contaminated chicken piece with aluminium foil whose surface was modified with phage loaded chitosan/alginate multilayers decreased the number of colonies on the chicken meat, and it was as effective as treating the meat directly with phage solution.

Biodegradable polymer promotes osteogenic differentiation in immortalized and primary osteoblast-like cells.

Biodegradable polymers have been broadly used as agents that can complex with and deliver osteoinductive agents, but osteoinductivity of the polymers themselves has been rarely studied. Here we report the osteoinductivity of poly(4-hydroxy-L-proline ester) (PHPE), a biodegradable cationic polymer with cell penetrating properties. Under physiological conditions, PHPE degrades into trans-4-hydroxy-L-proline (trans-Hyp), a non-coded amino acid with essential functions in collagen fibril formation and fibril stability. Treatment of SaOS-2 osteoblast-like cells and hFOB 1.19 primary osteoblast cells with PHPE promoted earlier collagen nodule formation and mineralization of the extracellular matrix compared to untreated cells, even when mineralization activators were absent in the growth medium. Our results indicate that PHPE is a potential osteoinductive agent in vitro that can favor bone regeneration. Moreover, this osteoinductive property could be partly attributed to the degradation product trans-Hyp, which could recapitulate some, but not all of the osteogenic activity. The primary findings of this study can be summarized as follows: treatment of cells with PHPE led to (1) the induction of COL1A1 expression, collagen synthesis and secretion in osteoblast-like cells, (2) mineralization of the ECM in both SaOS-2 and hFOB 1.19 primary osteoblasts, and (3) induction of BMP2 gene and protein expression in osteoblast-like cells, which can promote mineralization of the ECM and regeneration of the bone tissue. Our results suggest that PHPE is a non-cytotoxic polymer and can be potentially used to overcome collagenopathies such as osteogenesis imperfecta.

Biologically Functional Ultrathin Films Made of Zwitterionic Block Copolymer Micelles.

We report the preparation of ultrathin coatings of zwitterionic block copolymer micelles and a comparison of their protein adsorption, adhesiveness, and antibacterial properties. Zwitterionic block copolymer micelles were obtained through pH-induced self-assembly of poly[3-dimethyl(methacryloyloxyethyl)ammonium propanesulfonate- b-2-(diisopropylamino)ethyl methacrylate] (ßPDMA- b-PDPA) at pH 7.5. ßPDMA- b-PDPA micelles with zwitterionic ßPDMA-corona and pH-responsive PDPA-core were then used as building blocks to prepare layer-by-layer (LbL) assembled multilayer films together with hyaluronic acid (HA), tannic acid (TA), or poly(sodium 4-styrenesulfonate) (PSS). Protein adsorption tests showed that 3-layer ßPDMA- b-PDPA micelles/HA films were the most effective to reduce the adhesion of BSA, lysozyme, ferritin, and casein. In contrast, ßPDMA- b-PDPA micelles/TA films were the most attractive surfaces for protein adsorption. Bacterial antiadhesive tests against a model Gram-negative bacterium, Escherichia coli, and a model Gram-positive bacterium, Staphylococcus aureus, were in good agreement with the protein adsorption properties of the films. The differences in the antiadhesive properties between these three different film systems are discussed within the context of chemical nature and the functional chemical groups of the polyanions, layer number, and surface morphology of the films. Multilayers were found to lose their antiadhesiveness in the long term. However, by taking advantage of the pH-responsive hydrophobic micellar cores, we show that an antibacterial agent could be loaded into the micelles and multilayers could exhibit antibacterial activity in the long term especially at moderately acidic conditions. In contrast to antiadhesive properties, no significant differences were recorded in the antibacterial properties between the different film types.

Bacterial anti-adhesive and pH-induced antibacterial agent releasing ultra-thin films of zwitterionic copolymer micelles.

UNLABELLED: We report on preparation of substrates with dual function coatings, i.e. bacterial anti-adhesive and antibacterial agent releasing polymer films of zwitterionic block copolymer micelles (BCMs). BCMs were obtained by pH-induced self-assembly of poly[3-dimethyl (methacryloyloxyethyl) ammonium propane sulfonate-b-2-(diisopropylamino)ethyl methacrylate] (ßPDMA-b-PDPA), resulting in BCMs with zwitterionic ßPDMA-coronae and pH-responsive PDPA-core. These zwitterionic BCMs were then used as building blocks to construct mono- and multi-layer films. We found that the number of layers in the film was critical for the anti-adhesive property and 3-layer films were the most anti-adhesive against a model Gram-positive bacterium, Staphylococcus aureus. Antibacterial activity could be introduced to the films by loading Triclosan into ßPDMA-b-PDPA micelles. Triclosan containing films were effective against Triclosan-sensitive Staphylococcus aureus specifically at moderately acidic conditions due to pH-induced disintegration of the micellar core blocks and release of Triclosan from the surface. Three-layer films also exhibited anti-adhesive property at physiological pH against a model Gram-negative bacterium, Escherichia coli. At moderately acidic pH, the coatings showed a contact antibacterial effect against an isolate of Escherichia coli with low sensitivity to Triclosan only when micellar cores were loaded with Triclosan. Such dual function films can be promising to combat biofouling at the non-homogeneous and/or defective parts of an anti-adhesive coating. Moreover, considering the moderately acidic conditions around an infection site, these multilayers can be advantageous due to their property of pH-induced antibacterial agent release. STATEMENT OF SIGNIFICANCE: This study presents preparation of substrates with dual function ultra-thin coatings of zwitterionic block copolymer micelles which show bacterial anti-adhesive properties against a Gram-positive and a Gram-negative bacterium. Such coatings are also capable of releasing antibacterial compounds in response to pH changes. Films were prepared by self-assembly of polymers at the surface. Our findings showed that zwitterionic micellar coronae introduced bacterial anti-adhesive property to the films, whereas pH-responsive micellar cores enabled release of an antibacterial agent from the surface at acidic pH. Considering the moderately acidic conditions around an infection site, such multilayers can be promising for the coating of implants/medical devices.

Bacterial anti-adhesive properties of a monolayer of zwitterionic block copolymer micelles.

We report on bacterial anti-adhesive properties of a monolayer of block copolymer micelles (BCMs) with zwitterionic coronae and pH-responsive cores. BCMs were obtained by pH-induced self-assembly of selectively betainized poly[3-dimethyl (methacryloyloxyethyl) ammonium propane sulfonate-b-2-(diisopropylamino)ethyl methacrylate] (ßPDMA-b-PDPA) in aqueous solution above neutral pH. Monolayer films were self-assembled at pH 7.5 when ßPDMA-b-PDPA was in the micellar form. Bacterial anti-adhesive properties of the zwitterionic micellar coatings were examined against S. aureus through: i) a macroscopic test based on viable cell counting; ii) direct microscopic visualization of adherent bacteria by live/dead staining and iii) crystal violet staining to evaluate surface adherent biomass. 95% reduction in cell adhesion was observed by microscopy indicating the anti-adhesive properties of ßPDMA-b-PDPA micellar monolayer. Results obtained from the viable cell count assay and crystal violet staining showed similar trends and were in good agreement with the microscopy results. Such coatings are promising to impart both anti-adhesive and antimicrobial properties to a surface due to bacterial anti-adhesive properties of zwitterionic coronae and the potential of pH-responsive cores to release antimicrobial agents.