Water-insoluble regenerated silk materials are normally achieved by increasing β-sheet content (silk II). In the present study, water-insoluble silk films were prepared by controlling very slow drying of B. mori silk solutions, resulting in the formation of stable films with dominating silk I instead of silk II structure. Wide angle x-ray scattering (WAXS) indicated that the silk films stabilized by slow drying were mainly composed of silk I rather than silk II, while water-and methanol-annealed silk films had a higher silk II content. The silk films prepared through slow drying had a globule-like structure in the core with nano-filaments. The core region was composed of silk I and silk II, and these regions are surrounded by hydrophilic nano-filaments containing random, turns, and α-helix secondary structures. The insoluble silk films prepared by slow drying had unique thermal, mechanical and degradative properties. DSC results revealed that silk I crystals had stable thermal properties up to 250°C, without crystallization above the Tg, but degraded in lower temperature than silk II structure. Compared with water-and methanol-annealed films, the films prepared through slow drying achieved better mechanical ductility and more rapid enzymatic degradation, reflective of the differences in secondary structure achieved via differences in post processing of the cast silk films. Importantly, the silk I structure, a key intermediate secondary structure for the formation of mechanically robust natural silk fibers, was successfully generated in the present approach of very slow drying, mimicking the natural process. The results also point to a new mode to generate new types of silk biomaterials, where mechanical properties can be enhanced, and degradation rates increased, yet water insolubility is maintained along with low beta sheet content.

Silk fibroin protein-based micro-and nanospheres provide new options for drug delivery due to their biocompatibility, biodegradability and their tunable drug loading and release properties. In the present study, we report a new aqueous-based preparation method for silk spheres with controllable sphere size and shape. The preparation was based on phase separation between silk fibroin and polyvinyl alcohol (PVA) at a weight ratio of 1/1 and 1/4. Water-insoluble silk spheres were easily obtained from the blend in a three step process: (1) air-drying the blend solution into a film, (2) film dissolution in water and (3) removal of residual PVA by subsequent centrifugation. In both cases, the spheres had approximately 30% beta-sheet content and less than 5% residual PVA. Spindleshaped silk particles, as opposed to the spherical particles formed above, were obtained by stretching the blend films before dissolving in water. Compared to the 1/1 ratio sample, the silk spheres prepared from the 1/4 ratio sample showed a more homogeneous size distribution ranging from 300 nm up to 20 μm. Further studies showed that sphere size and polydispersity could be controlled either by changing the concentration of silk and PVA or by applying ultrasonication on the blend solution. Drug loading was achieved by mixing model drugs in the original silk solution. The distribution and loading efficiency of the drug molecules in silk spheres depended on their hydrophobicity and charge, resulting in different drug release profiles. The entire fabrication procedure could be completed within one day. The only chemical used in the preparation except water was PVA, an FDA-approved ingredient in drug formulations. Silk micro-and nanospheres reported have potential as drug delivery carriers in a variety of biomedical applications.

We directly prepared insoluble silk films by blending with glycerol and avoiding the use of organic solvents. The ability to blend a plasticizer like glycerol with a hydrophobic protein like silk and achieve stable material systems above a critical threshold of glycerol is an important new finding with importance for green chemistry approaches to new and more flexible silk-based biomaterials. The aqueous solubility, biocompatibility, and well-documented use of glycerol as a plasticizer with other biopolymers prompted its inclusion in silk fibroin solutions to assess impact on silk film behavior. Processing was performed in water rather than organic solvents to enhance the potential biocompatibility of these biomaterials. The films exhibited modified morphologies that could be controlled on the basis of the blend composition and also exhibited altered mechanical properties, such as improved elongation at break, when compared with pure silk fibroin films. Mechanistically, glycerol appears to replace water in silk fibroin chain hydration, resulting in the initial stabilization of helical structures in the films, as opposed to random coil or beta-sheet structures. The use of glycerol in combination with silk fibroin in materials processing expands the functional features attainable with this fibrous protein, and in particular, in the formation of more flexible films with potential utility in a range of biomaterial and device applications.

Silkworms and spiders generate fibres that exhibit high strength and extensibility. The underlying mechanisms involved in processing silk proteins into fiber form remain incompletely understood, resulting in the failure to fully recapitulate the remarkable properties of native fibers in vitro from regenerated silk solutions. In the present study, the extensibility and high strength of regenerated silks were achieved by mimicking the natural spinning process. Conformational transitions inside micelles, followed by aggregation of micelles and their stabilization as they relate to the metastable structure of silk are described. Subsequently, the mechanisms to control the formation of nanofibrous structures were elucidated. The results clarify that the self-assembly of silk in aqueous solution is a thermodynamically driven process where kinetics also play a key role. Four key factors, molecular mobility, charge, hydrophilic interactions and concentration underlie the process. Adjusting these factors can balance nanostructure and conformational composition, and be used to achieve silk-based materials with properties comparable to native fibers. These mechanisms suggest new directions to design silk-based multifunctional materials.

Controlling the degradation process of silk is an important and interesting subject in biomaterials field. In the present study, silk fibroin films with different secondary conformations and nanostructures were used to study the degradation behavior. Silk fibroin films with highest β-sheet content achieved highest degradation rate, different from the previous studies. A new degradation mechanism revealed that degradation behavior of silk fibroin was related to not only crystal content, but also hydrophilic interaction and crystal-noncrystal alternant nanostructures. The hydrophilic blocks of silk were firstly degraded. Then, the hydrophobic crystal blocks which were formerly surrounded and immobilized by hudrophilic blocks, became free particles and moved into solution. Based on the mechanism, which enables the process more controllable and flexible, controlling the degradation behavior of silk fibroin without sacrificing other performances such as mechanical or hydrophilic properties become feasible, and this would greatly expand the applications of silk as a biomedical material.

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.

Material systems are needed that promote stabilization of entrained molecules, such as enzymes or therapeutic proteins, without destroying their activity. We demonstrate that the unique structure of silk fibroin protein, when assembled into the solid state, establishes an environment that is conducive to the stabilization of entrained proteins. Enzymes (glucose oxidase, lipase and horseradish peroxidase) entrapped in these films over ten months retained significant activity, even when stored at 37°C, and in the case of glucose oxidase did not lose any activity. Further, the mode of processing of the silk protein into the films could be correlated to the stability of the enzymes. The relationship between processing and stability offers a large suite of conditions within which to optimize such stabilization processes. Overall, the techniques reported here result in materials that stabilize enzymes to a remarkable extent, without the need for cryoprotectants, emulsifiers, covalent immobilization or other treatments. Further, these systems are amenable to optical characterization, environmental distribution without refrigeration, are ingestible, and offer potential use in vivo, since silk materials are biocompatible and FDA approved, degradable with proteases and currently used in biomedical devices.

Adhesives are common in biology as critical elements in motility, adhesion, and survival for many land and sea creatures.[1] Despite many attempts to mimic such features with natural or synthetic polymers, this has proven to be challenging due to the subtle and metastable state of the polymeric material properties that are required to control the functional attributes of such systems including during storage, processing, adhesion, and release. The viscoelastic behavior also limits the types of material systems that can be exploited for biomimetic approaches to this important material behavior. Most often, modified polysaccharides are found associated with mucoadhesives from biological systems, due to their hydration and charge density. [2] We report the discovery of a novel, electrically mediated adhesive formed from silkworm silk. This process, termed electrogelation provides a protein-based adhesive that offers biomimetic features when used in conjunction with devices. Further, we report on the solution behavior, morphology, and structural features of electrogels (e-gels), to demonstrate the mechanisms involved in the process. The adhesion can be controlled via electrical inputs. Most importantly, and quite unexpectedly, this is a reversible process, depending on voltage, time, and conditions used. This finding is very novel, as silkbased protein systems in particular are usually considered irreversible in terms of polymer transitions from the solution to solid state, mediated most often by solvents and mechanical shear forces.The basis for the current discovery comes from recent observations where aqueous solutions of silkworm silk were exposed to direct current (DC). Under certain electric fields, the solution began to gel on the positive electrode (Fig. 1). This observation prompted further investigation into the conditions and responses of the solution under different electric fields. While electrospinning of polymers, including silks, is performed at voltage potentials as high as >30 kV, [3] the utilization of low DC voltages to generate a controlled volume of silk gel is novel. In the basic setup (Fig. 1), electrodes are immersed in an aqueous solution of silk protein and 25 V DC is applied over a 3 min period to a pair of mechanical pencil leads. As the process progresses, bubbles evolve on both electrodes. Since the silk solution has a high water content, electrolysis occurs during electrogelation. The bubbles reflect the generation of oxygen gas at the positive electrode and hydrogen gas at the negative electrode during electrolysis. Within seconds of the application of the voltage, a visible gel forms at the positive electrode, locking in some oxygen bubbles at the electrode surface as the gel emanates outward. While the gel appears to have formed symmetrically about the electrode in Figure 1, it is typical that the forming gel front is directed toward the negative electrode.When silk electrogelation is executed in a voltage-controlled format, the current draw in the process follows a repeated trend; ...