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The oxidation of tyrosine residues of silk fibroin involves the generation of dityrosine and 3,4-dihydroxyphenylalanine (DOPA). However, it remains a challenge to selectively control the reaction pathway to produce dityrosine or DOPA in a selective fashion. Here, silk hydrogels with controllable formation of not only dityrosine and DOPA but also DOPA-Fe 3+ complexes within the cross-linked networks were developed. The use of chitosan particles in the Fenton reaction allowed the interaction of Fe 3+ ions with silk fibroin to be limited through the adsorption of Fe 3+ ions onto chitosan particles by manipulating contact time between the reaction medium and chitosan particles. This led to significant suppression of the premature formation of β-sheet structures that cause steric hindrance to the collisions between tyrosyl radicals and thus enabled higher selectivity toward the formation of dityrosine than DOPA. Remarkably, the addition of ethylenediaminetetraacetic acid (EDTA) to the chitosan particle-assisted Fenton reactions resulted in hydrogels that significantly favored the formation of DOPA over dityrosine due to the increase in the hydroxylation of phenol in the presence of EDTA. Despite the existence of Fe 3+ -EDTA complexes, Raman spectra indicated the DOPA-Fe 3+ complexation in the hydrogels. Mechanistically, the hydrogel networks with small-sized and uniformly distributed β-sheet structures as well as the abundance of DOPA appear to make non-EDTA-chelated Fe 3+ ions more accessible to complexation with DOPA. These findings have important implications for understanding the oxidation of tyrosine residues of silk fibroin by metal-catalyzed oxidation systems with potential benefits for future studies on silk protein-based hydrogels capable of generating intrinsic adhesive features as well as for exploring dual-cross-linked silk hydrogels constructed by chemical cross-linking and metal-coordinate complexation.

As a biomaterial, silk presents unique features with a combination of excellent mechanical properties, biocompatibility, and biodegradability. The biodegradability aspects of silk biomaterials, especially with options to control the rate from short (days) to long (years) time frames in vivo, make this protein-based biopolymer a good candidate for developing biodegradable devices used for tissue repairs and tissue engineering, as well as medical device implants. Silk materials, including native silk fibers and a broad spectrum of regenerated silk materials, have been investigated in vitro and in vivo to demonstrate degradation by proteolytic enzymes. In this Review, we summarize the findings on these studies on the enzymatic degradation of Bombyx mori ( B. mori ) silk materials. We also present a discussion on the factors that dictate the degradation properties of silk materials. Finally, in future perspectives, we highlight some key challenges and potential directions toward the future study of the degradation of silk materials.

Hydrophobic biomolecules realize their functions in vivo in aqueous environments, often through a delicate balance of amphiphilicity and chaperones. Introducing exogenous hydrophobic biomolecules into in vivo aqueous systems is a challenge in drug delivery and regenerative medicine, where labile linkers, carriers, and fusions or chimeric molecules are often designed to facilitate such aqueous interfaces. Here, we utilize naturally derived silk nanofiber shuttles with the capacity to transport hydrophobic cargos directly into aqueous solutions. These nanofibers disperse in organic solvents and in aqueous solutions because of their inherent amphiphilicity, with enriched hydrophobicity and strategically interspersed negatively charged groups. Hydrophobic molecules loaded on these shuttles in organic solvent-water systems separated from the solvent after centrifugation. These concentrated hydrophobic molecule-loaded nanofibers could then be dispersed into aqueous solution directly without modification. These shuttle systems were effective for different hydrophobic molecules such as drugs, vitamins, and dyes. Improved biological stability and functions of hydrophobic cargos after loading on these nanofibers suggest potential applications in drug delivery, cosmetology, medical diagnosis, and related health fields, with a relatively facile process.

Detailed control of nano- and microstructures in porous biomaterial scaffold systems is important for control of interfacial and biological functions. Self-assembled silk protein nanostructured building blocks were incorporated into salt-leached scaffolds to control these features. Controllable concentration and pH were used to induce the formation of amorphous silk nanofibers in solution and also to reduce β-sheet transformation during the more traditional salt-leaching process. These new scaffolds showed nanofibrous-microporous structures, reduced β-sheet content, and tunable mechanical properties. Bone marrow mesenchymal stem cells grew better and showed differentiation behavior on these nanofibrous scaffolds, suggesting cytocompatibility and support for tunable differentiation via the scaffolds. These results suggested a new strategy of designing bioactive silk scaffolds by combining traditional scaffold formation processes with the controllable self-assembly of silk.

The molecular interactions of silk materials plasticized using glycerol were studied, as these materials provide options for biodegradable and flexible protein-based systems. Plasticizer interactions with silk were analyzed by thermal, spectroscopic, and solid-state NMR analyses. Spectroscopic analysis implied that glycerol was hydrogen bonded to the peptide matrix, but may be displaced with polar solvents. Solid-state NMR indicated that glycerol induced β-sheet formation in the dried silk materials, but not to the extent of methanol treatment. Fast scanning calorimetry suggested that β-sheet crystal formation in silk-glycerol films appeared to be less organized than in the methanol treated silk films. We propose that glycerol may be simultaneously inducing and interfering with β-sheet formation in silk materials, causing some improper folding that results in less-organized silk II structures even after the glycerol is removed. This difference, along with trace residual glycerol, allows glycerol extracted silk materials to retain more flexibility than methanol processed versions.

Protein hydrogels are an important class of materials for applications in biotechnology and medicine. The fine-tuning of their sequence, molecular weight, and stereochemistry offers unique opportunities to engineer biofunctionality, biocompatibility, and biodegradability into these materials. Here we report a new family of redox-sensitive protein hydrogels with controllable mechanical properties composed of recombinant silk-elastin-like protein polymers (SELPs). The SELPs were designed and synthesized with different ratios of silk-to-elastin blocks that incorporated periodic cysteine residues. The cysteine-containing SELPs were thermally responsive in solution and rapidly formed hydrogels at body temperature under physiologically relevant, mild oxidative conditions. Upon addition of a low concentration of hydrogen peroxide at 0.05% (w/v), gelation occurred within minutes for the SELPs with a protein concentration of approximately 4% (w/v). The gelation time and mechanical properties of the hydrogels were dependent on the ratio of silk to elastin. These polymer designs also significantly affected redox-sensitive release of a highly polar model drug from the hydrogels in vitro. Furthermore, oxidative gelation was performed at other physiologically relevant temperatures, and this resulted in hydrogels with tunable mechanical properties, thus, providing a secondary level of control over hydrogel stiffness. These newly developed injectable SELP hydrogels with redox-sensitive features and tunable mechanical properties may be potentially useful as biomaterials with broad applications in controlled drug delivery and tissue engineering.

As a functional material, silk has been widely used in tissue engineering, drug release, and tissue regeneration. Increasing subtle control of silk hierarchical structures and thus specific functional performance is required for these applications but remains a challenge. Here, we report a novel silk nanofiber solution achieved through tuning solvent systems used to generate the material. Unlike the β-sheet rich silk nanofibers reported previously, these new silk nanofibers are mainly composed of amorphous structures and maintain a solution state in aqueous environments. The amorphous silk nanofibers are stable enough for storage and also metastable, making them easy to use in the further fabrication of materials through various processes. Silk scaffolds, hydrogels, and films were prepared from these silk nanofiber solutions. These silk materials from amorphous nanofiber solutions show different properties and tunable performance features. Therefore, these amorphous silk nanofibers are suitable units or building blocks for designing silk-based materials.

Bombyx mori (BM) silk fibroin is composed of two different subunits: heavy chain and light chain fibroin linked by a covalent disulfide bond. Current methods of separating the two silk fractions is complicated and produces inadequate quantities of the isolated components for the study of the individual light and heavy chain silks with respect to new materials. We report a simple method of separating silk fractions using formic acid. The formic acid treatment partially releases predominately the light chain fragment (soluble fraction) and then the soluble fraction and insoluble fractions can be converted into new materials. The regenerated original (total) silk fibroin and the separated fractions (soluble vs insoluble) had different molecular weights and showed distinctive pH stabilities against aggregation/precipitation based on particle charging. All silk fractions could be electrospun to give fiber mats with viscosity of the regenerated fractions being the controlling factor for successful electrospinning. The silk fractions could be mixed to give blends with different proportions of the two fractions to modify the diameter and uniformity of the electrospun fibers formed. The soluble fraction containing the light chain was able to modify the viscosity by thinning the insoluble fraction containing heavy chain fragments, perhaps analogous to its role in natural fiber formation where the light chain provides increased mobility and the heavy chain producing shear thickening effects. The simplicity of this new separation method should enable access to these different silk protein fractions and accelerate the identification of methods, modifications, and potential applications of these materials in biomedical and industrial applications.

Silkworm silk has been widely used as a textile fiber, as biomaterials and in optically functional materials due to its extraordinary properties. The β-sheet-rich natural nanofiber units of about 10-50 nm in diameter are often considered the origin of these properties, yet it remains unclear how silk self-assembles into these hierarchical structures. A new system composed of β-sheet-rich silk nanofibers about 10-20 nm in diameter is reported here, where these nanofibers formed into "flowing hydrogels" at 0.5-2% solutions and could be transformed back into the solution state at lower concentrations, even with a high β-sheet content. This is in contrast with other silk processed materials, where significant β-sheet content negates reversibility between solution and solid states. These fibers are formed by regulating the self-assembly process of silk in aqueous solution, which changes the distribution of negative charges while still supporting β-sheet formation in the structures. Mechanistically, there appears to be a shift toward negative charges along the outside of the silk nanofibers in our present study, resulting in a higher zeta potential (above -50 mV) than previous silk materials which tend to be below -30 mV. The higher negative charge on silk nanofibers resulted in electrostatic repulsion strong enough to negate further assembly of the nanofibers. Changing silk concentration changed the balance between hydrophobic interactions and electrostatic repulsion of β-sheet-rich silk nanofibers, resulting in reversible hydrogel-solution transitions. Furthermore, the silk nanofibers could be disassembled into shorter fibers and even nanoparticles upon ultrasonic treatment following the transition from hydrogel to solution due to the increased dispersion of hydrophobic smaller particles, without the loss of β-sheet content, and with retention of the ability to transition between hydrogel and solution states through reversion to longer nanofibers during self-assembly. These reversible solution-hydrogel transitions were tunable with ultrasonic intensity, time, or temperature.

Silk-elastin-like protein polymers (SELPs) combine the mechanical and biological properties of silk and elastin. These properties have led to the development of various SELP-based materials for drug delivery. However, SELPs have rarely been developed into nanoparticles, partially due to the complicated fabrication procedures, nor assessed for potential as an anticancer drug delivery system. We have recently constructed a series of SELPs (SE8Y, S2E8Y, and S4E8Y) with various ratios of silk to elastin blocks and described their capacity to form micellar-like nanoparticles upon thermal triggering. In this study, we demonstrate that doxorubicin, a hydrophobic antitumor drug, can efficiently trigger the self-assembly of SE8Y (SELPs with silk to elastin ratio of 1:8) into uniform micellar-like nanoparticles. The drug can be loaded in the SE8Y nanoparticles with an efficiency around 6.5% (65 ng doxorubicin/μg SE8Y), S2E8Y with 6%, and S4E8Y with 4%, respectively. In vitro studies with HeLa cell lines demonstrate that the protein polymers are not cytotoxic (IC50 > 200 μg/mL), while the doxorubicin-loaded SE8Y nanoparticles showed a 1.8-fold higher cytotoxicity than the free drug. Confocal laser scanning microscopy (CLSM) and flow cytometry indicate significant uptake of the SE8Y nanoparticles by the cells and suggest internalization of the nanoparticles through endocytosis. This study provides an all-aqueous, facile method to prepare nanoscale, drug-loaded SELPs packages with potential for tumor cell treatments.

In this study, we utilize plasma-enhanced chemical vapor deposition (PECVD) for the deposition of nanostructures composed of diphenylalanine. PECVD is a solvent-free approach and allows sublimation of the peptide to form dense, uniform arrays of peptide nanostructures on a variety of substrates. The PECVD deposited d-diphenylalanine nanostructures have a range of chemical and physical properties depending on the specific discharge parameters used during the deposition process.

Derived from Bombyx mori cocoons, regenerated silk fibroin (RSF) exhibits excellent biocompatibility, high toughness, and tailorable biodegradability. Additionally, RSF materials are flexible, optically clear, easily patterned with nanoscale features, and may be doped with a variety bioactive species. This unique combination of properties has led to increased interest in the use of RSF in sustainable and biocompatible electronic devices. In order to explore the applicability of this biopolymer to the development of future bioelectronics, the dielectric breakdown strength (Ebd) of RSF thin films was quantified as a function of protein conformation. The application of processing conditions that increased β-sheet content (as determined by FTIR analysis) and produced films in the silk II structure resulted in RSF materials with improved Ebd with values reaching up to 400 V/μm.