Qiang Lu

Water-Insoluble Silk Films with Silk I Structure

Water-insoluble regenerated silk materials are normally produced by increasing the beta-sheet content (silk II). In the present study water-insoluble silk films were prepared by controlling the very slow drying of Bombyx mori silk solutions, resulting in the formation of stable films with a predominant silk I instead of silk II structure. Wide angle X-ray scattering 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 by slow drying had a globule-like structure at the core surrounded by nano-filaments. The core region was composed of silk I and silk II, surrounded by hydrophilic nano-filaments containing random turns and alpha-helix secondary structures. The insoluble silk films prepared by slow drying had unique thermal, mechanical and degradative properties. Differential scanning calorimetry results revealed that silk I crystals had stable thermal properties up to 250 degrees C, without crystallization above the T(g), but degraded at lower temperatures than silk II structure. Compared with water- and methanol-annealed films the films prepared by slow drying had better mechanical ductility and were more rapidly enzymatically degraded, reflecting 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 by the present approach of very slow drying, mimicking the natural process. The results also point to a new mode of generating new types of silk biomaterials with enhanced mechanical properties and increased degradation rates, while maintaining water insolubility, along with a low beta-sheet content.

Silk nanospheres and microspheres from silk/pva blend films for drug delivery.

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. Spindle-shaped 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 microm. 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.

Degradation Mechanism and Control of Silk Fibroin

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

Stabilization of Enzymes in Silk Films

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 10 months retained significant activity, even when stored at 37 degrees 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 an extent, without the need for cryoprotectants, emulsifiers, covalent immobilization, or other treatments. Further, these systems are amenable to optical applications and characterization, environmental distribution without refrigeration, are ingestible, and offer potential use in vivo, because silk materials are biocompatible and FDA approved, degradable with proteases, and currently used in biomedical devices.

Insoluble and Flexible Silk Films Containing Glycerol

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.

Electrogelation for Protein Adhesives

Adv. Mater. 2010, 22, 711–715 2010 WILEY-VCH Verlag Gm IC A T IO N Adhesives are common in biology as critical elements in motility, adhesion, and survival for many land and sea creatures. 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. 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, mediatedmost often by solvents andmechanical 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, 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 VDC is applied over a 3min 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; initially high current draw drops exponentially to a minimal milliampere level. The actual current amplitudes depend on many factors, including applied voltage level, electrode area and spacing, and conductivity of the silk solution. The decay in current draw is likely related to the electrical insulating effect of the growing volume of silk gel and the bubbles that become trapped near the positive electrode surface. Silk gel formed through electrogelation has a highly viscous (soft) consistency and is very tacky, bearing a resemblance to thick mucus. Remarkably, we observed that after the electric field was turned off, the adhesive gel state was retained, thus, the structural state of the protein formed under e-gel conditions was sufficiently stable to retain material functions in the absence of the applied electric field. Yet, the gel formed can be returned to the solution state through a reverse electrical process (Fig. 1). If the electrode polarity is reversed and 25 VDC reapplied, the gel disappears, while fresh gel is formed on the newly created positive electrode. Electrogelation and reversal back to silk solution can be cycled many times. If an electrogelation process is performed for an extended period of time, or at high voltage, the gel nearest the positive electrode persists and reversal of the electric field no longer drives the gel back to solution form. Alternatively, after intermediate electrogelation times, heating the gel up to ca. 60 8C led to the disappearance of the gel-like material and transitioning back to the solution state. When cooled back to room temperature, e-gel reformed as a highly viscous and tacky material, thus, the gelation is reversible over several cycles. Consequently, the process is controllable in terms of gel features, reversibility, or permanency, such as by temperature. The changes in mechanical characteristics due to electrogelation of silk solutions were investigated by dynamic oscillatory shear rheology. For silk solutions, liquidlike, viscous behavior measured by the loss modulus (G00) dominated the mechanical response within the probed frequency range (v) with G00 v (Fig. 2A). On the other hand, the mechanical response of e-gels resembled that of a soft–solidlike, physical gel. There was a significant increase in the elastic response, measured by the storage modulus (G0). The frequency dependence of G0 was weak but finite (G0 v), while the apparent minimum in G00 suggested a possible G0, G00 crossover at even lower frequencies due to eventual relaxation of temporary, physical crosslinks. To investigate the significance of increased proton concentration in the mechanism of e-gel formation at the positive electrode, we titrated the solution to control the pH, termed pH-gels (Fig. 2A).

Stabilization and release of enzymes from silk films.

A significant challenge remains to protect protein drugs from inactivation during production, storage, and use. In the present study, the stabilization and release of horseradish peroxidase (HRP) in silk films was investigated. Water-insoluble silk films were prepared under mild aqueous conditions, maintaining the activity of the entrapped enzyme. Depending on film processing and post-processing conditions, HRP retained more than 90% of the initial activity at 4 degrees C, room temperature and 37 degrees C over two months. The stability of protein drugs in silk films is attributed to intermolecular interactions between the silk and the enzymes, based on Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The unique structural feature of silk molecules, periodic hydrophobic-hydrophilic domains, enabled strong interactions with proteins. The entrapped protein was present in two states, untrapped active and trapped inactive forms. The ratio between the two forms varied according to processing conditions. Proteolytic degradation and dissolution of the silk films resulted in the release of the bound enzyme which was otherwise not released by diffusion; enzyme recovered full activity upon release. There was a linear relationship between silk degradation/dissolution and the release of entrapped enzyme. Modifying the secondary structure of the silk matrix and the interactions with the non-crystalline domains resulted in control of the film degradation or dissolution rate, and therefore the release rate of the entrapped enzyme. Based on the above results, silk materials are an intriguing carrier for proteins in terms of both retention of activity and controllable release kinetics from the films.

Biomaterials from ultrasonication-induced silk fibroin-hyaluronic acid hydrogels.

We report formation of biocompatible hydrogels using physically cross-linked biopolymers. Gelation of silk fibroin (from B. mori silkworm) aqueous solution was effected by ultrasonication and used to entrap blended, un-cross-linked, hyaluronic acid (HA) without chemical cross-linking. HA was formed into silk/HA blended hydrogels with different mixing ratios, forming homogeneous materials with stable swelling behavior when the HA content was less than 40 wt %. This is a novel approach to HA hydrogel systems, which otherwise require chemical cross-linking. Further, these systems exploit the beneficial material and biological properties of both polymers. Differential scanning calorimetry (DSC), temperature modulated DSC, and thermal gravimetric analysis were used to show that well-blended silk/HA hydrogel systems formed without macrophase separation. Fourier transform infrared spectroscopy was used to determine secondary structures from the amide I region of silk protein by spectral subtraction and Fourier-self-deconvolution. The β-sheet crystal fraction of the silk protein increased with increase of HA content (26-35 wt %), which resulted in stable, crystalline features in the blend hydrogel materials, favorable features to support human mesenchymal stem cell attachment and proliferation. Scanning electron microscopy was used to characterize morphology. β-Sheet content controlled the stability of the silk/HA hydrogel systems, with a minimum crystalline content needed to maintain a stable hydrogel system of ∼26 wt %. This value is close to the β-sheet content in pure silk fibroin hydrogels. These novel nonchemically cross-linked blend hydrogels may be useful for biomedical applications due to biocompatibility and the widespread utility of hydrogel systems. The attributes of HA in combination with the features of silk, offer a useful suite of properties, combining the mechanical integrity and slow degradation of silk with the control of water interactions and biological signaling of HA.

Silk self-assembly mechanisms and control from thermodynamics to kinetics.

Silkworms and spiders generate fibers 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.

Nanofibrous architecture of silk fibroin scaffolds prepared with a mild self-assembly process.

Besides excellent biocompatibility and biodegradability, a useful tissue engineering scaffold should provide suitable macropores and nanofibrous structure, similar to extracellular matrix (ECM), to induce desired cellular activities and to guide tissue regeneration. In the present study, a mild process to prepare porous and nanofibrous silk-based scaffolds from aqueous solution is described. Using collagen to control the self-assembly of silk, nanofibrous silk scaffolds were firstly achieved through lyophilization. Water annealing was used to generate insolubility in the silk-based scaffolds, thereby avoiding the use of organic solvents. The nano-fibrils formed in the silk-collagen scaffolds had diameters of 20-100 nm, similar with native collagen in ECM. The silk-collagen scaffolds dissolved slowly in PBS solution, with about a 28% mass lost after 4 weeks. Following the dissolution or degradation, the nanofibrous structure inside the macropore walls emerged and interacted with cells directly. During in vitro cell culture, the nanofibrous silk-collagen scaffolds containing 7.4% collagen demonstrated significantly improved cell compatibility when compared with salt-leached silk scaffolds and silk-collagen scaffolds containing 20% collagen that emerged less nano-fibrils. Therefore, this new process provides useful scaffolds for tissue engineering applications. Furthermore, the process involves all-aqueous, room temperature and pressure processing without the use of toxic chemicals or solvents, offering new green chemistry approaches, as well as options to load bioactive drugs or growth factors into process.