Enhanced Antibacterial Activity of Se Nanoparticles Upon Coating with Recombinant Spider Silk Protein eADF4(κ16).

PURPOSE: Selenium nanoparticles (Se NPs) are promising antibacterial agents to tackle the growing problem of antimicrobial resistance. The aim of this study was to fabricate Se NPs with a net positive charge to enhance their antibacterial efficacy. METHODS: Se NPs were coated with a positively charged protein - recombinant spider silk protein eADF4(κ16) - to give them a net positive surface charge. Their cytotoxicity and antibacterial activity were investigated, with negatively charged polyvinyl alcohol coated Se NPs as a control. Besides, these eADF4(κ16)-coated Se NPs were immobilized on the spider silk films, and the antibacterial activity of these films was investigated. RESULTS: Compared to the negatively charged polyvinyl alcohol coated Se NPs, the positively charged eADF4(κ16)-coated Se NPs demonstrated a much higher bactericidal efficacy against the Gram-negative bacteria E. coli, with a minimum bactericidal concentration (MBC) approximately 50 times lower than that of negatively charged Se NPs. Cytotoxicity testing showed that the eADF4(κ16)-coated Se NPs are safe to both Balb/3T3 mouse embryo fibroblasts and HaCaT human skin keratinocytes up to 31 µg/mL, which is much higher than the MBC of these particles against E. coli (8 ± 1 µg/mL). In addition, antibacterial coatings were created by immobilising the eADF4(κ16)-coated Se NPs on positively charged spider silk films and these were shown to retain good bactericidal efficacy and overcome the issue of low particle stability in culture broth. It was found that these Se NPs needed to be released from the film surface in order to exert their antibacterial effects and this release can be regulated by the surface charge of the film, such as the change of the spider silk protein used. CONCLUSION: Overall, eADF4(κ16)-coated Se NPs are promising new antibacterial agents against life-threatening bacteria.

Recombinant spider silk protein eADF4(C16)-RGD coatings are suitable for cardiac tissue engineering.

Cardiac tissue engineering is a promising approach to treat cardiovascular diseases, which are a major socio-economic burden worldwide. An optimal material for cardiac tissue engineering, allowing cardiomyocyte attachment and exhibiting proper immunocompatibility, biocompatibility and mechanical characteristics, has not yet emerged. An additional challenge is to develop a fabrication method that enables the generation of proper hierarchical structures and constructs with a high density of cardiomyocytes for optimal contractility. Thus, there is a focus on identifying suitable materials for cardiac tissue engineering. Here, we investigated the interaction of neonatal rat heart cells with engineered spider silk protein (eADF4(C16)) tagged with the tripeptide arginyl-glycyl-aspartic acid cell adhesion motif RGD, which can be used as coating, but can also be 3D printed. Cardiomyocytes, fibroblasts, and endothelial cells attached well to eADF4(C16)-RGD coatings, which did not induce hypertrophy in cardiomyocytes, but allowed response to hypertrophic as well as proliferative stimuli. Furthermore, Kymograph and MUSCLEMOTION analyses showed proper cardiomyocyte beating characteristics on spider silk coatings, and cardiomyocytes formed compact cell aggregates, exhibiting markedly higher speed of contraction than cardiomyocyte mono-layers on fibronectin. The results suggest that eADF4(C16)-RGD is a promising material for cardiac tissue engineering.

Aqueous electrospinning of recombinant spider silk proteins.

There has been a significant increase in the use of sensitive biological components, e.g., growth factors or enzymes, in implanted scaffolds/devices. To prevent diffusion away from the targeted area and to maximize access of the biological agent to the desired target, it is necessary to provide a supportive substrate to immobilize and protect biological agents from the environment. For this purpose, nanofiber fabrics are highly promising due to their high porosity, capacity for solution flow-through and high surface-to-volume ratio. However, electrospinning often requires harsh processing conditions, such as the use of volatile solutions, which can result in loss of activity of the incorporated biological components. In this study we developed a mild process for electrospinning of eADF4(C16), a recombinant spider silk protein. eADF4(C16) is non-cytotoxic, displays excellent stability against hydrolytic and enzymatic degradation and opens the opportunity for genetic addition of bioactive factors. Therefore, an aqueous spinning dope of eADF4(C16) was loaded with either green fluorescence protein (GFP) or the recombinant fusion protein GFP-eADF4(C16). The fluorescence activity of GFP is dependent on its proper folding, which does not occur in organic solvents, making it an attractive model protein. We were able to demonstrate the usability as well as the significance of the all-aqueous processing conditions for the activity of GFP in electrospun spider silk scaffolds.

Degradable glycine-based photo-polymerizable polyphosphazenes for use as scaffolds for tissue regeneration.

Photo-polymerizable scaffolds are designed and prepared via short chain poly(organo)phosphazene building blocks bearing glycine allylester moieties. The polyphosphazene was combined with a trifunctional thiol and divinylester in various ratios, followed by thiol-ene photo-polymerization to obtain porous matrices. Degradation studies under aqueous conditions showed increasing rates in correlation with the polyphosphazene content. Preliminary cell studies show the non-cytotoxic nature of the polymers and their degradation products, as well as the cell adhesion and proliferation of adipose-derived stem cells.