Bioselectivity of silk protein-based materials and their bio-inspired applications.

Adhesion to material surfaces is crucial for almost all organisms regarding subsequent biological responses. Mammalian cell attachment to a surrounding biological matrix is essential for maintaining their survival and function concerning tissue formation. Conversely, the adhesion and presence of microbes interferes with important multicellular processes of tissue development. Therefore, tailoring bioselective, biologically active, and multifunctional materials for biomedical applications is a modern focus of biomaterial research. Engineering biomaterials that stimulate and interact with cell receptors to support binding and subsequent physiological responses of multicellular systems attracted much interest in the last years. Further to this, the increasing threat of multidrug resistance of pathogens against antibiotics to human health urgently requires new material concepts for preventing microbial infestation and biofilm formation. Thus, materials exhibiting microbial repellence or antimicrobial behaviour to reduce inflammation, while selectively enhancing regeneration in host tissues are of utmost interest. In this context, protein-based materials are interesting candidates due to their natural origin, biological activity, and structural properties. Silk materials, in particular those made of spider silk proteins and their recombinant counterparts, are characterized by extraordinary properties including excellent biocompatibility, slow biodegradation, low immunogenicity, and non-toxicity, making them ideally suited for tissue engineering and biomedical applications. Furthermore, recombinant production technologies allow for application-specific modification to develop adjustable, bioactive materials. The present review focusses on biological processes and surface interactions involved in the bioselective adhesion of mammalian cells and repellence of microbes on protein-based material surfaces. In addition, it highlights the importance of materials made of recombinant spider silk proteins, focussing on the progress regarding bioselectivity.

Free-standing spider silk webs of the thomisid Saccodomus formivorus are made of composites comprising micro- and submicron fibers.

Our understanding of the extraordinary mechanical and physico-chemical properties of spider silk is largely confined to the fibers produced by orb-weaving spiders, despite the diversity of foraging webs that occur across numerous spider families. Crab spiders (Thomisidae) are described as ambush predators that do not build webs, but nevertheless use silk for draglines, egg cases and assembling leaf-nests. A little-known exception is the Australian thomisid Saccodomus formivorus, which constructs a basket-like silk web of extraordinary dimensional stability and structural integrity that facilitates the capture of its ant prey. We examined the physical and chemical properties of this unusual web and revealed that the web threads comprise microfibers that are embedded within a biopolymeric matrix containing additionally longitudinally-oriented submicron fibers. We showed that the micro- and submicron fibers differ in their chemical composition and that the web threads show a remarkable lateral resilience compared with that of the major ampullate silk of a well-investigated orb weaver. Our novel analyses of these unusual web and silk characteristics highlight how investigations of non-model species can broaden our understanding of silks and the evolution of foraging webs.

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-Silica Hybrid Scaffolds with Drug-Releasing Properties for Tissue Engineering Applications.

Fabricating biomaterials with antimicrobial activity to prevent the growth of detrimental microorganisms is of great scientific and practical interest. Here, composite materials comprising recombinant spider silk proteins and mesoporous silica nanoparticles (MSN) loaded with selected antibiotics and antimycotics are fabricated into films and hydrogels. The derived composite materials exhibit excellent antimicrobial properties with sustained release of antibiotics over the course of 15 days. Furthermore, antibiotics/antimycotics inclusion does not impair the cytocompatibility of the composite materials, all of which promote fibroblast cell adhesion and proliferation. Finally, processing of spider silk-MSN composite hydrogels using 3D printing is shown to enable the fabrication of patient-specific antimicrobial implants to prevent infection in the near future.

Recombinant Spider Silk Hydrogels for Sustained Release of Biologicals.

Therapeutic biologics (i.e., proteins) have been widely recognized for the treatment, prevention, and cure of a variety of human diseases and syndromes. However, design of novel protein-delivery systems to achieve a nontoxic, constant, and efficient delivery with minimal doses of therapeutic biologics is still challenging. Here, recombinant spider silk-based materials are employed as a delivery system for the administration of therapeutic biologicals. Hydrogels made of the recombinant spider silk protein eADF4(C16) were used to encapsulate the model biologicals BSA, HRP, and LYS by direct loading or through diffusion, and their release was studied. Release of model biologicals from eADF4(C16) hydrogels is in part dependent on the electrostatic interaction between the biological and the recombinant spider silk protein variant used. In addition, tailoring the pore sizes of eADF4(C16) hydrogels strongly influenced the release kinetics. In a second approach, a particles-in-hydrogel system was used, showing a prolonged release in comparison with that of plain hydrogels (from days to week). The particle-enforced spider silk hydrogels are injectable and can be 3D printed. These initial studies indicate the potential of recombinant spider silk proteins to design novel injectable hydrogels that are suitable for delivering therapeutic biologics.

Structural Insights into Water-Based Spider Silk Protein-Nanoclay Composites with Excellent Gas and Water Vapor Barrier Properties.

Nature reveals a great variety of inorganic-organic composite materials exhibiting good mechanical properties, high thermal and chemical stability, and good barrier properties. One class of natural bio-nanocomposites, e.g. found in mussel shells, comprises protein matrices with layered inorganic fillers. Inspired by such natural bio-nanocomposites, the cationic recombinant spider silk protein eADF4(κ16) was processed together with the synthetic layered silicate sodium hectorite in an all-aqueous setup. Drop-casting of this bio-nanocomposite resulted in a thermally and chemically stable film reflecting a one-dimensional crystal. Surprisingly, this bio-nanocomposite coating was, though produced in an all-aqueous process, completely water insoluble. Analyzing the structural details showed a low inner free volume due to the well-oriented self-assembly/alignment of the spider silk proteins on the nanoclay surface, yielding high oxygen and water vapor barrier properties. The here demonstrated properties in combination with good biocompatibility qualify this new bio-nanocomposite to be used in packaging applications.

Enhanced cellular uptake of engineered spider silk particles.

Drug delivery systems allow tissue/cell specific targeting of drugs in order to reduce total drug amounts administered to an organism and potential side effects upon systemic drug delivery. Most drug delivery systems are polymer-based, but the number of possible materials is limited since many commercially available polymers induce allergic or inflammatory responses or lack either biodegradability or the necessary stability in vivo. Spider silk proteins represent a new class of (bio)polymers that can be used as drug depots or drug delivery systems. The recombinant spider silk protein eADF4(C16), which can be processed into different morphologies such as particles, films, or hydrogels, has been shown to fulfil most criteria necessary for its use as biomaterial. Further, eADF4(C16) particles have been shown to be well-suited for drug delivery. Here, a new method was established for particle production to reduce particle size and size distribution. Importantly, cellular uptake of these particles was shown to be poor in HeLa cells. Therefore, variants of eADF4(C16) with inversed net charge or incorporated cell penetrating peptides and receptor interacting motifs were tested, showing much better cellular uptake. Interestingly, uptake of all silk variant particles was mainly achieved by clathrin-mediated endocytosis.

Mimicking biopolymers on a molecular scale: nano(bio)technology based on engineered proteins.

Proteins are ubiquitous biopolymers that adopt distinct three-dimensional structures and fulfil a multitude of elementary functions in organisms. Recent systematic studies in molecular biology and biotechnology have improved the understanding of basic functional and architectural principles of proteins, making them attractive candidates as concept generators for technological development in material science, particularly in biomedicine and nano(bio)technology. This paper highlights the potential of molecular biomimetics in mimicking high-performance proteins and provides concepts for applications in four case studies, i.e. spider silk, antifreeze proteins, blue mussel adhesive proteins and viral ion channels.

Structure-function relationships of the plant cuticle and cuticular waxes - a smart material?

The cuticle is the main interface between plants and their environment. It covers the epidermis of all aerial primary parts of plant organs as a continuous extracellular matrix. This hydrophobic natural composite consists mainly of the biopolymer, cutin, and cuticular lipids collectively called waxes, with a high degree of variability in composition and structure. The cuticle and cuticular waxes exhibit a multitude of functions that enable plant life in many different terrestrial habitats and play important roles in interfacial interactions. This review highlights structure-function relationships that are the subjects of current research activities. The surface waxes often form complex crystalline microstructures that originate from self-assembly processes. The concepts and results of the analysis of model structures and the influence of template effects are critically discussed. Recent investigations of surface waxes by electron and X-ray diffraction revealed that these could be assigned to three crystal symmetry classes, while the background layer is not amorphous, but has an orthorhombic order. In addition, advantages of the characterisation of formation of model wax types on a molecular scale are presented. Epicuticular wax crystals may cause extreme water repellency and, in addition, a striking self-cleaning property. The principles of wetting and up-to-date concepts of the transfer of plant surface properties to biomimetic technical applications are reviewed. Finally, biomechanical studies have demonstrated that the cuticle is a mechanically important structure, whose properties are dynamically modified by the plant in response to internal and external stimuli. Thus, the cuticle combines many aspects attributed to smart materials.

Tomato (Lycopersicon esculentum Mill.) fruit growth and ripening as related to the biomechanical properties of fruit skin and isolated cuticle.

The control of growth rate and the mechanical integrity of the tomato (Lycopersicon esculentum Mill.) fruit has been attributed to the exocarp. This study focused on the biomechanics of the fruit skin (FS) comprising cuticle, epidermis and a few subdermal cell layers, and the enzymatically isolated cuticular membrane (CM) during fruit growth and ripening. Morphology and mechanical properties of the FS and the CM of three cultivars were analysed separately at three distinct ripening stages by scanning electron microscopy (SEM) and one-dimensional tension testing, respectively. Both were subject to significant cultivar-specific changes. Thickness of the CM increased during ripening from 7.8-8.6 to 9.9-15.7 microm and exceeded by far that of the epidermal cell wall. The mechanical properties, such as modulus of elasticity, strength, and failure strain, were highest in the FS for all cultivars at any stage, with only one exception; however, the cuticle largely mirrored these properties throughout fruit maturation. Stiffness of both isolated CM and FS increased from immature to fully ripe fruits for all cultivars, while failure stress and failure strain displayed a tendency to decrease for two of them. Stress-strain behaviour of the CM could be described as strain softening, mostly linear elastic throughout, and strain hardening, and was subject to growth-related changes. The FS displayed strain hardening throughout. The results indicate evidence for the cuticle to become increasingly important as a structural component for the integrity of the tomato fruit in addition to the epidermis. A supplementary putative model for tomato fruit growth is proposed.