Structural Origins of Silk Piezoelectricity.

Uniaxially oriented, piezoelectric silk films were prepared by a two-step method that involved: (1) air drying aqueous, regenerated silk fibroin solutions into films, and (2) drawing the silk films to a desired draw ratio. The utility of two different drawing techniques, zone drawing and water immersion drawing were investigated for processing the silk for piezoelectric studies. Silk films zone drawn to a ratio of λ= 2.7 displayed relatively high dynamic shear piezoelectric coefficients of d(14) = -1.5 pC/N, corresponding to over two orders of magnitude increase in d(14) due to film drawing. A strong correlation was observed between the increase in the silk II, ß-sheet content with increasing draw ratio measured by FTIR spectroscopy (C(ß)∝ e(2.5) (λ)), the concomitant increasing degree of orientation of ß-sheet crystals detected via WAXD (FWHM = 0.22° for λ= 2.7), and the improvement in silk piezoelectricity (d(14)∝ e(2.4) (λ)). Water immersion drawing led to a predominantly silk I structure with a low degree of orientation (FWHM = 75°) and a much weaker piezoelectric response compared to zone drawing. Similarly, increasing the ß-sheet crystallinity without inducing crystal alignment, e.g. by methanol treatment, did not result in a significant enhancement of silk piezoelectricity. Overall, a combination of a high degree of silk II, ß-sheet crystallinity and crystalline orientation are prerequisites for a strong piezoelectric effect in silk. Further understanding of the structural origins of silk piezoelectricity will provide important options for future biotechnological and biomedical applications of this protein.

Non-equilibrium silk fibroin adhesives.

Regenerated silkworm silk solutions formed metastable, soft-solid-like materials (e-gels) under weak electric fields, displaying interesting mechanical characteristics such as dynamic adhesion and strain stiffening. Raman spectroscopy, in situ electric field dynamic oscillatory rheology and polarized optical microscopy indicated that silk fibroin electrogelation involved intermolecular self-assembly of silk molecules into amorphous, micron-scale, micellar structures and the formation of relatively long lifetime, intermicellar entanglement crosslinks. Overall, the electrogelation process did not require significant intramolecular beta-strand or intermolecular beta-sheet formation, unlike silk hydrogels. The kinetics of e-gel formation could be tuned by changing the field strength and assembly conditions, such as silk concentration and solution pH, while e-gel stiffness was partially reversible by removal of the applied field. Transient adhesion testing indicated that the adhesive characteristics of e-gels could at least partially be attributed to a local increase in proton concentration around the positive electrode due to the applied field and surface effects. A working model of electrogelation was described en route to understanding the origins of the adhesive characteristics.

Injectable solid hydrogel: mechanism of shear-thinning and immediate recovery of injectable ß-hairpin peptide hydrogels.

ß-Hairpin peptide-based hydrogels are a class of injectable hydrogel solids with significant potential use in injectable therapies. ß-hairpin peptide hydrogels can be injected as preformed solids, because the solid gel can shear-thin and consequently flow under a proper shear stress but immediately recover back into a solid on removal of the stress. In this work, hydrogel behavior during and after flow was studied in order to facilitate fundamental understanding of how the gels flow during shear-thinning and how they quickly recover mechanically and morphologically relative to their original, pre-flow properties. While all studied ß-hairpin hydrogels shear-thin and recover, the duration of shear and the strain rate affected both the gel stiffness immediately recovered after flow and the ultimate stiffness obtained after complete rehealing of the gel. Results of structural analysis during flow were related to bulk rheological behavior and indicated gel network fracture into large (>200 nm) hydrogel domains during flow. After cessation of flow the large hydrogel domains are immediately percolated which immediately reforms the solid hydrogel. The underlying mechanisms of the gel shear-thinning and healing processes are discussed relative to other shear-responsive networks like colloidal gels and micellar solutions.

Vortex-induced injectable silk fibroin hydrogels.

A novel, to our knowledge, technique was developed to control the rate of beta-sheet formation and resulting hydrogelation kinetics of aqueous, native silk solutions. Circular dichroism spectroscopy indicated that vortexing aqueous solutions of silkworm silk lead to a transition from an overall protein structure that is initially rich in random coil to one that is rich in beta-sheet content. Dynamic oscillatory rheology experiments collected under the same assembly conditions as the circular dichroism experiments indicated that the increase in beta-sheet content due to intramolecular conformational changes and intermolecular self-assembly of the silk fibroin was directly correlated with the subsequent changes in viscoelastic properties due to hydrogelation. Vortexing low-viscosity silk solutions lead to orders-of-magnitude increase in the complex shear modulus, G*, and formation of rigid hydrogels (G* approximately 70 kPa for 5.2 wt % protein concentration). Vortex-induced, beta-sheet-rich silk hydrogels consisted of permanent, physical, intermolecular crosslinks. The hydrogelation kinetics could be controlled easily (from minutes to hours) by changing the vortex time, assembly temperature and/or protein concentration, providing a useful timeframe for cell encapsulation. The stiffness of preformed hydrogels recovered quickly, immediately after injection through a needle, enabling the potential use of these systems for injectable cell delivery scaffolds.

Folding, self-assembly, and bulk material properties of a de novo designed three-stranded beta-sheet hydrogel.

A de novo designed three-stranded beta-sheet (TSS1) has been prepared that undergoes temperature-induced folding and self-assembly to afford a network of beta-sheet rich fibrils that constitutes a mechanically rigid hydrogel. Circular dichroism and infrared spectroscopies show that TSS1 folds and self-assembles into a beta-sheet secondary structure in response to temperature. Rheological measurements show that the resulting hydrogels are mechanically rigid [at pH 9, G' = 1750-9000 Pa, and at pH 7.4, G' = 8500 Pa] and that the storage modulus can be modulated by temperature and peptide concentration. Nanoscale structure analysis by transmission electron microscopy and small angle neutron scattering indicate that the hydrogel network is comprised of fibrils that are about 3 nm in width, consistent with the width of TSS1 in the folded state. A unique property of the TSS1 hydrogel is its ability to shear-thin into a low viscosity gel upon application of shear stress and immediately recover its mechanical rigidity upon termination of stress. This attribute allows the hydrogel to be delivered via syringe to a target site with spatial and temporal resolution. Finally, experiments employing C3H10t1/2 mesenchymal stem cells seeded onto the hydrogel and incubated for 24 h indicate that the TSS1 hydrogel surface is noncytotoxic, supports cell adhesion, and allows cell migration.

Silk hydrogels for sustained ocular delivery of anti-vascular endothelial growth factor (anti-VEGF) therapeutics.

Silk hydrogels were formulated with anti-vascular endothelial growth factor (anti-VEGF) therapeutics for sustained ocular drug delivery. Using silk fibroin as a vehicle for delivery, bevacizumab-loaded hydrogel formulations demonstrated sustained release of 3 months or greater in experiments in vitro as well as in vivo using an intravitreal injection model in Dutch-belted rabbits. Using both standard dose (1.25mg bevacizumab/50 µL injection) and high dose (5.0mg bevacizumab/50 µL injection) hydrogel formulations, release concentrations were achieved at day 90 that were equivalent or greater than those achieved at day 30 with the positive standard dose control (single injection (50 µL) of 1.25mg bevacizumab solution), which is estimated to be the therapeutic threshold based on the current dosage administration schedule of 1 injection/month. These gels also demonstrated signs of biodegradation after 3 months, suggesting that repeated injections may be possible (e.g., one injection every 3-6 months or longer). Due to its pharmacokinetic and biodegradation profiles, this delivery system may be used to reduce the frequency of dosing for patients currently enduring treatment using bevacizumab or other anti-VEGF therapeutics.

Influence of Hydrophobic Face Amino Acids on the Hydrogelation of ß-Hairpin Peptide Amphiphiles.

Hydrophobic residues provide much of the thermodynamic driving force for the folding, self-assembly, and consequent hydrogelation of amphiphilic ß-hairpin peptides. We investigate how the identity of hydrophobic side chains displayed from the hydrophobic face of these amphiphilic peptides influences their behavior to expound on the design criteria important to gel formation. Six peptides were designed that globally incorporate valine, aminobutyric acid, norvaline, norleucine, phenylalanine, or isoleucine on the hydrophobic face of the hairpin to study how systematic changes in hydrophobic content, ß-sheet propensity, and aromaticity affect gelation. Circular dichroism (CD) spectroscopy indicates that hydrophobic content, rather than ß-sheet propensity, dictates the temperature- and pH-dependent folding and assembly behavior of these peptides. Transmission electron microscopy (TEM) and small-angle neutron scattering (SANS) show that the local morphology of the fibrils formed via self-assembly is little affected by amino acid type. However, residue type does influence the propensity of peptide fibrils to undergo higher order assembly events. Oscillatory rheology shows that the mechanical rigidity of the peptide gels is highly influenced by residue type, but there is no apparent correlation between rigidity and residue hydrophobicity nor ß-sheet propensity. Lastly, the large planar aromatic side chain of phenylalanine supports hairpin folding and assembly, affording a gel characterized by a rate of formation and storage modulus similar to the parent valine-containing peptide.

Silk-based biomaterials for sustained drug delivery.

Silk presents a rare combination of desirable properties for sustained drug delivery, including aqueous-based purification and processing options without chemical cross-linkers, compatibility with common sterilization methods, controllable and surface-mediated biodegradation into non-inflammatory by-products, biocompatibility, utility in drug stabilization, and robust mechanical properties. A versatile silk-based toolkit is currently available for sustained drug delivery formulations of small molecule through macromolecular drugs, with a promise to mitigate several drawbacks associated with other degradable sustained delivery technologies in the market. Silk-based formulations utilize silk's well-defined nano- through microscale structural hierarchy, stimuli-responsive self-assembly pathways and crystal polymorphism, as well as sequence and genetic modification options towards targeted pharmaceutical outcomes. Furthermore, by manipulating the interactions between silk and drug molecules, near-zero order sustained release may be achieved through diffusion- and degradation-based release mechanisms. Because of these desirable properties, there has been increasing industrial interest in silk-based drug delivery systems currently at various stages of the developmental pipeline from pre-clinical to FDA-approved products. Here, we discuss the unique aspects of silk technology as a sustained drug delivery platform and highlight the current state of the art in silk-based drug delivery. We also offer a potential early development pathway for silk-based sustained delivery products.

Silk fibroin rods for sustained delivery of breast cancer therapeutics.

A silk-protein based reservoir rod was developed for zero-order and long-term sustained drug delivery applications. Silk reservoir rod formulations were processed in three steps. First, a regenerated silk fibroin solution, rich in random-coil content was transformed into a tubular silk film with controllable dimensions, uniform film morphology and a structure rich in silk II, ß-sheet content via "film-spinning." Second, the drug powder was loaded into swollen silk tubes followed by tube end clamping. Last, clamped silk tube ends were sealed completely via dip coating. Anastrozole, an FDA approved active ingredient for the treatment of breast cancer, was used as a model drug to investigate viability of the silk reservoir rod technology for sustained drug delivery. The in vitro and in vivo pharmacokinetic data (in a female Sprague-Dawley rat model) analyzed via liquid chromatography-tandem mass spectroscopy indicated zero-order release for 91 days. Both in vitro and in vivo anastrozole release rates could be controlled simply by varying silk rod dimensions. The swelling behavior of silk films and zero-order anastrozole release kinetics indicated practically immediate film hydration and formation of a linear anastrozole concentration gradient along the silk film thickness. The dependence of anastrozole release rate on the overall silk rod dimensions was in good agreement with an essentially diffusion-controlled sustained release from a reservoir cylindrical geometry. In vivo results highlighted a strong in vitro-in vivo pharmacokinetic correlation and a desirable biocompatibility profile of silk reservoir rods. During a 6-month implantation in rats, the apparent silk molecular weight values decreased gradually, while rod dry mass and ß-sheet crystal content values remained essentially constant, providing a suitable timeframe for controlled, long-term sustained delivery applications. Overall, the silk reservoir rod may be a viable candidate for sustained delivery of breast cancer therapeutics.

Materials fabrication from Bombyx mori silk fibroin.

Silk fibroin, derived from Bombyx mori cocoons, is a widely used and studied protein polymer for biomaterial applications. Silk fibroin has remarkable mechanical properties when formed into different materials, demonstrates biocompatibility, has controllable degradation rates from hours to years and can be chemically modified to alter surface properties or to immobilize growth factors. A variety of aqueous or organic solvent-processing methods can be used to generate silk biomaterials for a range of applications. In this protocol, we include methods to extract silk from B. mori cocoons to fabricate hydrogels, tubes, sponges, composites, fibers, microspheres and thin films. These materials can be used directly as biomaterials for implants, as scaffolding in tissue engineering and in vitro disease models, as well as for drug delivery.

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.

Direct Observation of Early-Time Hydrogelation in beta-Hairpin Peptide Self-Assembly.

Triggered hydrogelation of MAX1 peptide, (VK)(4)-V(D)PPT-(KV)(4)-NH(2), proceeds through peptide intramolecular folding into beta-hairpins and concomitant self-assembly into branched clusters of well-defined (uniform, 3 nm cross section), semiflexible, beta-sheet-rich nanofibrils. Cryogenic transmission electron microscopy indicates that dangling fibrils extend from one growing cluster to another and lead to early, intercluster communication in solution. At the apparent percolation threshold, the dynamic shear modulus measured by oscillatory rheology (G'(omega), G''(omega) proportional, variant omega(n)) and the field-intensity autocorrelation function measured by dynamic light scattering (g(1)(tau) proportional, variant tau(-beta')) show power-law behavior with comparable critical dynamic exponents (n approximately 0.47 and beta' approximately 0.45). Finite interpenetration of percolating clusters with smaller clusters, along with permanent intercluster entanglements, increase the network rigidity. The self-assembly of MAX1 peptide was compared and contrasted with the assembly of other biopolymeric networks in literature.