Tuna Yucel

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.

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.

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).

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 silks 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.

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.

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.