PT106041A - SILK FIBROIN DERIVATIVE HYDROGENS: METHODS AND THEIR APPLICATIONS - Google Patents

Abstract

This invention relates to a Silicate Fibroin Hydrogel intersected by a peroxide derivative and suitable compositions for application of tissue engineering and regenerative medicine or as systems for the release of therapeutic agents. THE SILK FIBROIN AQUEOUS SOLUTIONS ARE ABLE TO FORM HYDROGES IN THE PRESENCE OF PEROXIDASE AND PEROXIDE AT MODERATE TEMPERATURES / INTERVAL OF PH. The gelation process allows the combination of human and animal cells and / or therapeutic agents, reagents, bioactive agents and combinations thereof. THE PHYSICAL-CHEMICAL AND BIOLOGICAL PERFORMANCE OF THE MATRIX MAY BE ADJUSTED FOR SPECIFIC APPLICATIONS, USING DIFFERENT FORMULATIONS AND REACTION CONDITIONS. IN STRUCTURAL TERMS THE MATRIX CAN PRESENT DIFFERENT SPACIAL CONFORMATIONS THROUGH IMMERSION IN ORGANIC SOLVENTS OR OTHER TREATMENTS. THE MATRIX MAY BE USED AS WELL AS A CELLULAR OR CELLULAR SYSTEM AND MANUALLY OR AUTOMATICALLY PRODUCED THROUGH INJECTION, STILL THE RETICULATION PROCEDURE MAY OCCUR DIRECTLY AT THE APPLICATION SITE. MAY ALSO BE PROCESSED IN DIFFERENT FORMS / STATE, SUCH AS BIOADHESES, HYDROGEES, FIBERS, DISCS, MEMBRANES AND MICRO / NANO PARTICLES.

Description

1

DESCRIPTION

SILK FIBROIN DERIVATIVE HYDROGENS: METHODS AND THEIR APPLICATIONS

OBJECT OF THE INVENTION The present invention relates to the development of silicone fibroin based hydrogels, cross-linked by peroxidase. Methods of preparing concentrated aqueous solutions of silk and silk fibroin hydrogels under different conditions are given. The silk fibroin hydrogels can be formed in a single step and the gelation takes place under conditions close to the physiological ones. The crosslinking can be performed manually or automatically in-situ. The enzymatically crosslinked silk fibroin hydrogels exhibit controlled performance and permit the encapsulation of therapeutic cells and / or agents, genetic material, bioactive agents, detection and targeting agents, conducting molecules or inorganic materials, providing versatile systems which can be used as biodegradable matrix in acellular and cellular systems for tissue engineering and regenerative medicine applications or as drug release, bioadhesive, fillers, coatings for improving the functioning of a medical device, or other pharmaceutical or medical applications.

State of art

Hydrogels have attractive properties for use in tissue engineering applications, regenerative medicine, and controlled drug delivery systems as they: (i) swell and retain large amounts of water, (ii) mimic natural tissues; (iii) may have adjustable mechanical properties, (iv) have a biodegradable rate of adjustment, (v) can be applied by minimally invasive (e.g., injection) procedures. In this way, hydrogels provide a highly hydrated microenvironment similar to the extracellular matrix. They can be injected and then crosslinked in situ allowing the encapsulation of cells and preservation / induction of a given phenotype. An injectable hydrogel should also have a suitable sol-gel transition mechanism for clinical purposes, i.e. it should be liquid to facilitate homogenous injection and distribution of cells, and at the same time be able to readily gel upon implantation under physiological conditions ( for example, pH and temperature). This unique combination of characteristics, in addition to the ability to adjust their rate of degradation in vivo, also makes them useful in applications assuming controlled drug release or as bioadhesives.

Heretofore, various methods have been used to prepare injectable hydrogels, such as: (i) physical crosslinking systems (such as those based on ionic interactions, stereo complexation and hydrophobic interactions as in the case of thermosensitive hydrogels), and ) chemical crosslinking systems (using photopolymerization and enzymatic reactions). While physical crosslinking systems have several advantages, such as the formation of a hydrogel under non-aggressive conditions, they also have major drawbacks, such as poor stability and poor mechanical properties. Chemical crosslinking systems have several advantages, including high mechanical properties and high stability of the hydrogel. However, the toxic residues of photoinitiators or organic solvents used are often responsible for reduced biocompatibility and cytotoxicity of the hydrogels.

Recently, enzymatic crosslinking methodologies have emerged as an alternative route to overcome the difficulties associated with more conventional methods of chemical crosslinking. Different hydrogels have been obtained by crosslinking reactions using horseradish peroxidase (horseradish peroxidase-HRP) as the catalyst. HRP is a single chain hemoprotein type b that catalyzes the coupling of phenol or aniline derivatives by the decomposition of hydrogen peroxide (H202). Various synthetic and natural polymers functionalized with tyramine, tyrosine (also known as 4-hydroxyphenylalanine) and aminophenol side groups are currently under development to form hydrogels in situ using this strategy. Examples include: HRP cross-linked alginate with phenolic groups (Sakai, et al. Acta Biomaterialia, 2007, 3: 495-501.), Dextran-tyramine conjugates (Jin et al. Biomaterials, 2007, 28: 2791-2800), (Kurisawa, et al., Chemical Communications, 2005, 14: 4312-4314), and the compounds of the general formula conjugates of polyaspartic acid with each of the three aromatic side groups (Sofia, et al., Journal of Macromolecular Science Part A, 2002, A39: 1151-1181), peptides containing tyrosine (Gly-Tyr-Gly) (Oudgenoeg, et al. Journal of Agricultural and Food Chemistry, 2001, 49: 2503-2510), food casein protein (Li et al., African Journal of Biotechnology, 2009, 8: 5508-5515), tyrosine containing a peptidic marker (Minamihata, et al. al. Bioconjugate Chemistry, 2011, 22: 74-81). These systems have several advantages, such as biocompatibility and easy control of the reaction rate under less aggressive conditions, mimicking the biological processes of cross-linking. In addition, adjustable mechanical properties can be obtained by varying the H202 concentration, as has been demonstrated for gelatine-hydroxyphenylpropionic acid hydrogels (Wang, et al., Biomaterials, 2010, 31: 8608-8616) where rigidity varies as a function of the H2 O2 concentration, without altering the polymer content in the precursor solution. The hydrogels developed in this study are able to withstand cell adhesion and proliferation and in a rigidity dependent manner. Furthermore, it has been reported that by controlling the content of phenolic groups in the carboxymethylcellulose (CMC) -thramine conjugated hydrogel it was possible to effectively control the resulting cell adhesion and proliferation (Sakai, et al. Acta biomaterialia, 2009, 5: 554-559 ). Notably, a hydrogel obtained from CMC, which is known to be an anti-adhesive biopolymer, showed cell adhesiveness after gelation by cross-linking phenolic groups. Protein-polysaccharide conjugated hydrogels from alginate derivatives and proteins (gelatin and albumin) have also been developed (Sakai, et al, 2010, Biomacromolecules 11: 1370-1375.), All having moieties of phenol groups through a reaction enzyme catalyzed by HRP. In another study, the in situ hydrogelification and the RGD conjugation of a tyramine conjugate with a PPO-PEO block copolymer (Park, et al., Soft Matter, 2011, 7: 986-992) were simultaneously achievable. The effect of conjugated RGDs on cell adhesion and proliferation was higher and could be adjusted in a concentration-dependent manner. According to the previously published literature, some protein based materials (such as gelatin, peptides, and casein) gave rise to hydrogels or conjugates using peroxidase (Li et al., African Journal of Biotechnology, 2009, 8: 5508 Minamihata, et al. Bioconjugate chemistry, 2011, 22: 74-81; Wang, et al. Biomaterials, 2010, 31: 8608-8616). The production of peroxidase-mediated silk fibroin hydrogels and a peroxide (e.g., hydrogen peroxide) as the oxidizing agent have not been reported so far. only casein was reported by Li et al. as crosslinked by peroxidase / hydrogen peroxide, and the resulting cross-linked casein did not give rise to a hydrogel (Li, et al., African Journal of Biotechnology, 2009, 8: 5508-5515). Casein is mainly a derivative of milk, and its structure and properties are very different from silk fibroin which derive primarily from the cocoon. Minamihata et al. synthesized a recombinant tyrosine-containing peptide using Escherichia coli, which is a completely different silk fibroin in terms of protein structure, sequence, molecular weight, and the like. (Minamihata, et al., Bioconjugate chemistry, 2011, 22: 74-81). Gelatin is a collagen-derived protein whose amino acid composition, molecular structure and properties are different from those of silk fibroin. Tyrosinase is an enzyme also used to crosslink tyrosine-containing polymers (Moreira Teixeira et al. Biomaterials, 2012, 33: 1281-1290). Conjugates of chitosan and gelatin were developed using tyrosinase (Chen, et al. Biometries, 2003, 24: 2831-2841; Chen, et al. Biopolymers, 2002, 64: 292-302). In these studies, gelatin was not able to form a hydrogel without chitosan. On the other hand, the gelatine / chitosan hydrogel presented little stability (Moreira Teixeira et al. Biomaterials, 2012, 33: 1281-1290). Similar studies suggest that the conjugates prepared by tyrosinase may only form a "glue-like" matrix (Yamada, et al., 2000, Biomacromolecules 1: 252-258, Demolliens, et al. Bioconjugate Chemistry, 2008, 19: 1849 -1854). Tyrosinase-mediated silkworm and chitosan conjugates have also been reported (Kang et al Macromolecular Research, 2004, 12: 534-539; Anghileri, et al., Journal of Biotechnology, 2007, 127: 508-519; al. Biotechnology Journal, 2006, 125: 281-294). In these studies, pure silk fibroin hydrogels or pure silk fibroin materials cross-linked by tyrosinase have not been reported. Only silk fibroin / chitosan or sericin / chitosan conjugates were considered. In addition, the mechanism of formation of tyrosinase-mediated silk / chitosan and sericin / chitosan conjugates is different from that of a peroxidase-mediated tyrosine-containing polymer conjugate. Silk fibroin (or sericin) is conjugated to chitosan by the reaction of activated quinones on the silk with the amine groups in the chitosan. In turn, tyrosine-containing polymers form conjugates through reactions between tyrosine groups (Moreira Teixeira et al., Biomaterials, 2012, 33: 1281-1290).

In the literature, there are studies on tyrosine-mediated hydrogels or silk fibroin / chitosan conjugates. However, no records were found referring to peroxidase-mediated silk fibroin hydrogels. As mentioned above, the mechanisms of action of tyrosinase and peroxidase are different.

Here we propose the use of a system based on an injectable silk hydrogel with application in tissue engineering and regenerative medicine and controlled release of drugs, using peroxidase as a strategy to obtain the cross-linking. Silk fibroin (FS), a natural protein composed mainly of glycine, alanine, serine and tyrosine, has been increasingly studied for new biomedical applications because of its biocompatibility, slow degradability and remarkable mechanical properties. The ability to control the molecular structure and morphology through processing and the possibility of functionalizing its surface, expanded the applications of this protein. Its molecular weight is about 300-350 kDa. The repeating unit in the crystalline region of SF is known as Ala-Gly-Ser-Gly-Ala-Gly. FS also contains tyrosine in the semi-crystalline or amorphous region. The percentage of this aromatic amino acid is about 5 mol% FS. Thus, FS presents the most appropriate chemistry to promote peroxidase-mediated cross-linking. This is an advantage over the above-described polymer systems which must be conjugated to phenol or aniline derivatives prior to the HRP-catalyzed enzymatic reaction. Although crosslinking reactions of FS have been widely reported (such as the use to prepare silk tyrosinase / chitosan conjugates), to date there have been no reported studies on the use of peroxidase in FS crosslinking to produce a stable hydrogel in that gelation may occur under physiological conditions. Several patents were awarded based on the use of silk fibroin and its solutions for different applications. The following examples should be taken into account for their relevance in the area (s) of the present invention:

US Patent 2011/0171239 of July 14, 2011 and WO 2010/036992 of April 1, 2010 describes an 8 method for obtaining silk fibroin gel with direct current application ie through electrogelification. The silk fibroin gel can be reversibly converted to a ligand by the application of reverse current. The invention also extends to a method for obtaining adhesive silk geis by reducing the pH of the silk solution. US Patent 2011/0129531 of June 2, 2011 relates to the use of silk fibroin hydrogel and its compositions as dermal filler. The compositions comprise from about 1 to 10% (w / v) silk fibroin and a resilin-like polypeptide. The silk precipitate is obtained using a 23RGD accelerator / ethanol or dehydration medium. For obtaining the geis a gel time of 24 hours is required.

US Patent 2011/0111031 of May 12, 2011 comprises the use of sericin-depleted silk fibroin hydrogel and compositions thereof for use as a drug delivery system. A solution of sericin-free silk fibroin is used to improve the gelling time. A dehydration medium is used to produce the gels, such as solutions of methanol, ethanol, acetone, or salt.

WO patent 2011/041395 of April 7, 2011 describes methods of preparing nano- and microparticle silks for a variety of biomedical and pharmaceutical applications, such as tissue engineering or controlled drug delivery. The method uses polyvinyl alcohol (PVA) and a phase separation technique to obtain particles. No organic solvent is used at any stage of production. 9

WO 2011/022771 of March 03, 2011 relates to methods of producing silk extract (comprises about 0.5% to about 15% silk proteins) for application in the production of personal care products, plastics, textiles and biomedical products. Silk proteins are obtained from cells producing them, being solubilized by contact with a surfactant (for example, sodium dodecyl sulfate (SDS), ammonium lauryl sulfate) or an ionic liquid (for example anions, such as chloride and bromide, cation such as 3-4-chloropyridinium and dimethylaminopyridinium) and concentrating the proteins to produce the silk extract.

WO patent 2011/026101 of March 03, 2011 relates to electronic and optoelectronic devices made with silk fibroin or mixtures with other biopolymers, their manufacturing methods and methods for using the silk-based electronic and optoelectronic system . These sedam-based devices can be used in vitro and in vivo for biomedical applications.

US Patent 2011/0046686 of February 24, 2011 describes methods and compositions comprising mixtures of silk fibroin and hydroxyapatite (HA). Silk fibroin and hydroxyapatite are mixed before gelation occurs (formation of insoluble state). The invention relates to the repair and replacement of bone and teeth. WO Patent2011 / 013145 dated 03 February 2011, discloses a process for improving the gelation time of regenerated silk fibroin using for example TiO2 or Fe02 SiN3 as the gelling agents. The silk / silica fibroin composite is proposed as a support biomaterial for tissue engineering applications.

WO 2011/011347 of January 27, 2011 comprises compositions and methods of attaching silk fibroin to active agents through the specific interaction between avidin and biotin. Functionalized silk-based biomaterials can be linked to antibodies and growth factors for use in various biomedical applications.

US patent application 2011/0008436 of January 13, 2011 relates to methods of purification of silk fibroin, conjugates of fibroin with bioactive molecules and production of silk fibroin hydrogels and formulations with fibroin concentration of up to 6% by weight . The invention comprises gels obtained by mixing silk fibroin and peptides, which are intended for applications as fillers, controlled drug release and tissue engineering.

WO 2011/005381 of January 13, 2011 relates to a vortexing treatment of a solution of silk fibroin which enables the production of gels. The method allows the control of the rate of β-shaped lamella formation and the rate of gelation. Bioactive agents and cells may be encapsulated in the hydrogels during vortexing.

WO 2011/006133 of January 13, 2011 describes a silk-based delivery system which liberates nucleic acids from silk complexes. Silk complexes are intended to be applied in transfection as they exhibit high release efficiency, cell selectivity, and controlled release of the nucleic acids. WO 2010/141133 of December 09, 2010 relates to methods of preparing silk / antibiotic (s) fibroin support structures. The present invention describes silk fibroin compositions based on compositions comprising antibiotic (s) for the prevention of bacterial infections, and for medical implants, tissue engineering, drug delivery systems, or other pharmaceutical or medical applications.

WO 2010/123945; WO 2010/123946, and WO 2010/123947 of October 28, 2010 describe methods for purifying and functionalizing silk fibroin. The invention describes methods for obtaining hydrogels by combining with 23RGD / ethanol to find applications in tissue engineering, coatings for improving the functioning of a medical device, and controlled release of drugs. EP patent 2010/2211876, of August 4, 2010; US Patent 2010/0178304 of July 15, 2010; MX Patents 2009/012978, of April 23, 2010; KR Patents 2010/1020100029217 of 16 March 2010; Patents and WO 2008/150861 of December 11, 2008 provide a process for the production of silk fibroin gels by ultrasound. The gelation time can be controlled to occur within two hours after the ultrasonic treatment and uses an additional treatment with a salt solution. Bioactive agents and cells can be encapsulated in the hydrogels during the gelation process. EP patent 2010/2210971 of July 28, 2010 relates to a silk fibroin tissue structure comprising an outer concentric tubular sheath and an inner core. The silk fibroin used to weave the structure of the present invention consists of a silk thread for regenerative applications of ligaments, particularly the anterior cruciate ligament, tendons, muscles and blood vessels.

WO 2010/057142 of May 20, 2010 discloses compositions and methods for the production of polyethylene glycol functionalized (PEG) silk fibroin. Silk attached to PEG is intended for applications as anti-adherent and anti-thrombogenic materials. As it exhibits different surface properties within the matrix, the association with bioactive agents can improve the internal growth of tissue, on the side of the adherent matrix.

U.S. Patent 2009/7635755 of December 22, 2009; EP Patent 2006/1613796 January 11, 2006, and WO 2005/012606 of February 10, 2005 refer to the preparation of highly concentrated silk fibroin solutions. The methods described for obtaining the solutions do not use organic solvents, direct additives and aggressive chemicals. Highly concentrated silk solutions (less than 30% by weight) are proposed for the development of fibers, membranes, foams, support structures and hydrogels. The processing methods for obtaining silk hydrogels are based on increasing temperature, pH, decreasing and increasing the concentration of salt or a blend with other polymers.

WO 2009/133532 of 05 November 2009 relates to a two-step method for obtaining a solution of regenerated silk fibroin, i.e. the use of an ionic reagent and degumming of silk or silk cocoons woven. The invention also extends to the silk fibroin solution (s), and a fibroin material from an implant that can be used for cartilage repair.

WO 2009/018610 of February 12, 2009 relates to silk proteins which may be used for the production of silk with a trans-beta structure, as well as nucleic acids encoding such proteins. The invention also comprises recombinant cells and / or organisms capable of synthesizing silk proteins.

WO 2008/140562 of November 20, 2008 describes a method of producing an electroactive material which includes a polymer, a substrate, leaking the polymer onto the substrate, and enzymatically polymerizing an organic compound to generate a conductive polymer between the polymer supplied and the substrate. The polymer may be silk containing tyrosines to provide a molecular interface between the supplied biopolymer and a further substrate or conductive layer through a tyrosine-enzyme polymerization. The result is a carbon-carbon structure that provides conjugates " yarn " for use in polymeric and biopolymer optical devices.

WO 2008/118133 of October 2, 2008 provides a method for preparing silk fibroin microspheres using lipid vesicles as templates. Microspheres are intended to find applications as controlled drug delivery. 14

WO 2008/106485 of September 4, 2008 relates to a layer of woven lamella comprising a silk fibroin substrate having grooves containing tissue-specific cells. Silicone fibroin lamella substrates are intended to find applications in tissue engineering.

WO 2008/069919 of June 12, 2008 and U.S. Patent 2008/0131509 of June 5 describe methods for the treatment of tissue comprising self-reinforced composite matrices. The matrix may be a gelling system composed of two components based on fibrin glue. In some formulations the silk proteins or the functionalized silk proteins may be combined with the two component gelling system by methods known in the art.

WO 2007/141131 of December 13, 2007 relates to a microfluidic device and method for controlling the phase separation of one or a mixture of two or more spider silk proteins which may then allow the association of protein ( s) of silk for the production of spheres, nanofibrils, threads, etc. EP Patent 2007/1789103 of May 30, 2007 provides a medical device comprising a tubular body having a lumen and a longitudinal axis, and a plurality of silk elements placed substantially parallel to each other along the axis of the lumen of the tubular body. The invention extends to a method of manufacturing the medical device which comprises forming the tubular body and the silk introducing members into the lumen of the tubular body. The device is intended for applications in nerve regeneration. WO 2007/020449 of February 22, 2007 and WO 2006/030182 of March 23, 2006 disclose a cartilage tissue repair device with a biocompatible, biocompatible or other fiber silk, and a substantially porous silk biocompatible, or a hydrogel partially or substantially filling the interstices of the fiber, with or without a means of firmly anchoring the device to the patient's bone. The silk 3D support structure is obtained by lyophilization and the hydrogel by gelling in the presence of a PBS (Phosphate Buffer Solution) ionic gelation. A mineralizing silk gel is obtained by combining the silk hydrogel solution with a calcium chloride solution.

WO 2005/123114 of December 29, 2005 relates to different silk-based drug delivery systems. The invention extends to methods for the production of such systems and drug formulations.

WO 2004/060424 of July 22, 2004 describes silk-containing stents comprising an endoluminal stent and a graft. The invention extends to methods for the production of grafts, wherein the silk is intended to promote in vivo adhesion of the stent to the vessel walls, or otherwise induces or accelerates a fibrotic reaction in vivo leading to the stent graft to adhere to the wall of the vessel.

WO 2004/041845 of May 21, 2004 describes a method of preparing a fibrous protein hydrogel by means of a solvent method. The method is to pour an aqueous solution of fibrous protein into a vessel which comprises a solvent which is not water miscible; the aging vessel is sealed at ambient temperature, and collecting the resulting fibrous protein hydrogel and allowing it to dry. The invention extends to a hydrogel obtained by the solvent method.

WO 2003/022909 of March 20, 2003 and EP-A-2004/1436346 of July 14, 2004 disclose a process for the production of silk fibroin hydrogels by treatment of a solution of fibroin with water and / or acid solutions having a pH lower than 4 and / or polar solvents and / or cross-linking compounds, followed by washing the resultant. The invention described herein for developing silicon fibroin cross-linked hydrogels and compositions thereof, which underwent peroxidase-mediated cross-linking, are completely different from the above-mentioned patents. For example, U.S. Patent 2011/0171239, issued July 14, 2011 and WO 2010/036992 April 2010 describes methods for the preparation of silk hydrogels using the direct application of electric current. In that patent (WO Patent 2010/036992), (1) silk fiber was degummed for 20 minutes, (2) purified silk fibroin dissolved in lithium bromide at 60 ° C for 4 hours, (3) silk was dialyzed in cassettes (MWCO) 3500), (4) the silk fibroin solution did not contain any saline buffer, (5) the silk fibroin hydrogel was formed using electric field, (6) the hydrogel of shaped silk fibroin has I-silk conformation; (7) the silk fibroin formed hydrogels were similar to a glue. There are many differences between the above-mentioned patent (WO 2010/036992) and the present invention, namely: (1) silk fibroin has been degummed for 1 hour. Further degumming time may improve the purity of silk fibroin to allow the silk solution to be more stable in the aqueous state, (2) purified silk fibroin was dissolved in lithium bromide at 70 ° C for 1 hour , ie the higher temperature may accelerate the dissolution of silk fibroin, and the shorter heating time may decrease the degradation of silk fibroin, (3) the silk fibroin was dialyzed into a benzoylated dialysis tube (MWCO 2000) , of lower molecular weight (MWCO). A dialysis tubing may maintain more protein inside the tube and decrease possible contamination, (4) a saline ion buffer was introduced into the silk fibroin during dialysis, this allows adjustment of the pH value of the solution of (5) the silk fibroin hydrogel is formed by peroxidase-mediated chemical cross-linking (enzymatic reaction), (6) the silicon fibroin, which allows the incorporation of cells and / or bioactive agents, (7) the formed hydrogels are stable and can be processed in different forms and modulated their biodegradability.

In another WO patent 2003/022909 of March 20, 2003 and patent EP 2004/1436346 of July 14, 2004 there are described methods for preparing a silk hydrogel by treatment of a solution of fibroin with water-soluble polymers and / or acid solutions having a pH of less than 4 and / or the polar solvents and / or the crosslinked compounds. In that patent (Patent 2003/022909), it was reported that (1) silk fibroin was dissolved in solvents of 20-60øC, (2) the concentration of the dialysed silk fibroin solution was between 1-20% in Weight; (3) the polyethylene glycol solution is 50% and the molecular weight is 1,000, and (4) the hydrogel was prepared by dialysis against acid fibroin solution solution (pH less than 4), or water soluble polymers, or polar solvents, or solutions containing cross-linking agents, (5) the pH of the silk fibroin solution was not controlled prior to the formation of the hydrogel, (6) when a crosslinking agent is used to form hydrogels, crosslinking of the hydroxyl and amine groups, (7) the silk fibroin hydrogels prepared by different methods were all formed within the dialysis tube, (8), the hydrogels should be washed prior to use, (9) the FTIR spectrum revealed that the hydrogels prepared by all methods mentioned in this patent were of beta conformation, as indicated by the strong absorbance around 1620 cm -1 (Figure 6). There are large differences between the above-mentioned patent (Patent 2003/022909) and the present invention, namely: (1) purified silk fibroin was dissolved in lithium bromide at 70 ° C for 1 hour, the higher temperature can accelerate the dissolution of the silk fibroin, and the shorter heating time may decrease the degradation of silk fibroin, (2) the concentration of the silk fibroin dialysate solution was between 2-30% by weight, the high concentration of the silk solution (3), the polyethylene glycol solution for the concentration of the silk fibroin solution was 20% and the molecular weight was 20,000, the lower PEG concentration made the solution more stable, and the higher molecular weight PEG did not penetrate the dialysis membrane to contaminate the silk fibroin solution, (4) the hydrogels were prepared by direct mixing of the peroxidase / hydrogen peroxide and a (5) the pH value of the silk fibroin solution was under control prior to gelation, between 7.0-7.4 (pH physiological), (6) the peroxidase-mediated mechanisms of cross-linking act on the tyrosine groups in silk fibroin, (7) the hydrogel may be formed at the site of application, such as tissue filling, (8) hydrogels can be used directly after gelation or during gelation, and (9) the conformation of the silk fibroin hydrogel of this invention is amorphous, confirmed by FTIR spectra with the major peaks at 1620 cm -1 (Figure 3b). The present invention provides methods for the production of peroxidase-mediated silk fibroin crosslinked hydrogels. Gelling occurs independently of the presence of culture media and cells or body fluids and is thus clinically relevant as it allows the homogenization of the cells prior to formation of the hydrogel and then gelation in situ. The ability to control gelation time, mechanical and structural properties of hydrogels, bioadhesiveness and degradation rate are relevant aspects in tissue engineering and regenerative medicine, bioadhesive applications, and drug delivery systems. The present invention also features shape memory properties of peroxidase-mediated silk fibroin crosslinked hydrogels, which are stimulated by ionic strength. For example, after preparation, the silk fibroin hydrogels can retain their shape (initial form) in phosphate buffered saline (PBS). The hydrogel will swell with the decrease in the ionic strength of the solution. The maximum swelling ratio in ultrapure water is attained. Subsequently, the shape of the hydrogels will be recovered to some extent to their initial shape by immersing the hydrogels in solutions of different ionic strength. This potentiates its application as a bioadhesive biomaterial, filler, and coating to enhance the functioning of a medical device, or other pharmaceutical or medical applications. Silk fibroin hydrogel prepared by peroxidase-mediated cross-linking has amorphous conformation. This invention provides methods for the preparation of silk fibroin hydrogels with adjustable spatial conformations. For example, layered silk fibroin hydrogels may be formed by immersion in organic solvents. The layered hydrogel has beta conformation in the outer layer and dominant amorphous conformation in the inner layer. Other combinations of conformation can also be formed by other treatments that allow its application as a structured system (transport layers) for application in electronic and optoelectronic devices.

Brief description of the figures:

BRIEF DESCRIPTION OF THE DRAWINGS For a better understanding of the present invention, the figures are set forth in, but not limited to, the following, because: Figure 1 is a schematic illustration of HRP enzyme-mediated silk fibrotic crosslinking. Figure 2 depicts the formation of the silk fibroin hydrogels of the hydrogels developed by HRP-mediated cross-linking and the transparency properties. Silicone fibroin solution of 16 wt%, 0.84 mg / ml HRP solution in 21 PBS, and 0.18 wt% H202 in PBS were used (HRP / silk in weight 0.26 V), H2 O2 / Silk 1.10 wtVo) (a): pure silk fibroin solution without PBS, (b): Silk fibroin solution in PBS, (a) and (b): hydrogen peroxide was used as the source of H2 O2 , (c): calcium peroxide was used as the source of H2O2. Figure 3 depicts the results obtained in the Fourier transform infrared spectroscopy (FTIR) analysis of the silk fibroin solution, with or without PBS, and silk / HRP / H202 mixtures, before and after gelation. The hydrated samples were tested by attenuated total reflectance (ATR). (a) A silk fibroin solution, with or without PBS, (b) silk fibroin solution with PBS was used to obtain the 3 spectra. Figure 4 shows the absorbance of the silk fibroin solution obtained at 550 nm, and silk / HRP / H202 mixture, before and after gelation. Silk fibroin solution 16 wt%, 0.84 mg / ml HRP solution in PBS, and 0.18 wt% H202 in PBS were used (HRP / silk 0.26 wtVo, H202 / silk 1.10 wtVo ). For each group, 50 ul of solution was placed on a 96-well TCPS plate and measured on a microplate reader at 550 nm. Absorbance was determined before and after gelation using the same sample in the well. Figure 5 depicts the microstructure of the crosslinked hydrogel of silk fibroin by HRP and lyophilized. Silk fibroin solution 16 wt%, 0.84 mg / ml HRP solution in PBS, and 0.18 wt% H202 in PBS were used (HRP / silk 0.26 wt., H202 / Silk 1.10 wt. . Figure 6 depicts the influences of HRP and H202 on the gelation time of HRP cross-linked silk fibroin hydrogel, determined at 37øC. Silk-10, 22

Silk-12, Silk-16 corresponds to silk hydrogels prepared from 10 wt%, 12 wt%, and 16 wt% silk fibroin solutions, respectively. (a) H202 / silk: 1.10 wt%, (b) HRP / silk: 0.26 wt%. The gel time was determined using a vial tilt method. Each condition was tested in triplicate. Figure 7 depicts the influences of HRP and H202 on the storage modulus of the crosslinked mediated silk fibroin peroxidase hydrogel, measured by a model rheometer (strain: 0.1%, frequency: 0.5 Hz) at 37ø W. Silk-10, Silk-12, Silk-16 corresponds to silk hydrogels prepared from 10 wt%, 12 wt%, and 16 wt% silk fibroin solutions, respectively. (a) H202 / silk: 1.10 wt., (b) HRP / silk: 0.26 wt. Values were determined after the module reached a linear value and became stable for 1 minute. Each condition was tested in triplicate. Figure 8 shows the influence of HRP on the mechanical properties of HRP crosslinked silk fibroin hydrogels, in particular on the modulus. The modulus was determined on a rotation model rheometer at 37 ° C. Silk-10, Silk-12, Silk-16 corresponds to silk hydrogels prepared from 10 wt%, 12 wt%, and 16 wt% silk fibroin solutions, respectively. H2C> 2 / silk was fixed at 1.1 wtls for all formulations, (a), (b), and (c): The analysis frequency was set at 1 Hz. ): The analysis frequency was set at 0.1% voltage. Each analysis was measured after the module of the silk fibroin hydrogel reached a plateau for 5 minutes. Figure 9 depicts the influence of H202 on the mechanical properties of the crosslinked silk fibroin hydrogels by HRP, namely on the modulus. The modulus was determined on a rotation model rheometer at 37 ° C. Silk-10, Silk-12, Silk-16 corresponds to silk hydrogels prepared from 10 wt%, 12 wt%, and 16 wt% silk fibroin solutions, respectively. HRP / silk was fixed at 0.26 wtVo for all formulations, (a), (b), and (c): The analysis frequency was set at 1 Hz. (D), (e) and (f): The analysis frequency was determined at 0.1% voltage. Each analysis was measured after the module of the silk fibroin hydrogel reached a plateau for 5 minutes. Figure 10 represents the percent swelling of HRP-crosslinked silk fibroin hydrogel in distilled water (a), and PBS (b). Figure 11 depicts the enzymatic degradation profiles of the HRP crosslinked silk fibroin hydrogel at 37øC. Silk-10, Silk-12, Silk-16 corresponds to silk hydrogels prepared from 10 wt%, 12 wt%, and 16 wt% silk fibroin solutions, respectively. For each test, about 500 mg of silk fibroin hydrogel was immersed in 5 ml of PBS containing 0.025 U Protease XIV obtained from Streptomyces griseus (0.005 U / ml). Figure 12 depicts the shape memory properties of the crosslinked silk fibroin hydrogel by HRP. The concentration of silk fibroin was 16 wt%, HRP / silk was 0.26 wt%, H202 / silk was 1.1 wt%. The silk fibroin hydrogel was first prepared in a template (a), and then the hydrogel was removed from the template (b). 24

Silk fibroin hydrogel after immersion in distilled water for 12 hours (c). The diameter of the silk fibroin hydrodel was recovered after immersion in PBS over the 12 hour period (d). Figure 13 depicts the variation of the diameter of the HRP crosslinked silk fibroin hydrogel when alternately immersed in distilled water and PBS. Silk-10, Silk-12, Silk-16 corresponds to silk hydrogels prepared from 10 wt%, 12 wt%, and 16 wt% silk fibroin solutions, respectively. (a) HRP / silk is 0.26 wt%, H202 / silk is 1.1 wtko, (b) HRP / silk is 0.26 wt VO, H202 / silk is 1.4 wt%. Figure 14 depicts photographs of HRP-crosslinked silk fibroin hydrogel prior to (a), and after (b) immersion in methanol for 5 minutes. Figure 15 shows the cross-sectional images of HRP-crosslinked silk fibroin hydrogel after immersion in methanol for the period of: (a) 0, (b) 1, (c) 3, (d) 5, and (e) 10 minutes. The concentration of silk fibroin is 16 wt%, HRP / silk is 0.26 wtVo, H2 C> 2 / silk is 1.1 wt%. The multilayers of the silk fibroin hydrogel prepared by immersing the hydrogel in methanol can be seen. Figure 16 depicts the thickness of the outer layer of the silk fibroin hydrogel, prepared by immersing the hydrogel in methanol for different periods of time. The concentration of silk fibroin is 16 wt%. * Indicates significant difference between the two formulations of the silk fibroin hydrogel immersed in methanol over the same period of time. Figure 17 depicts the multicast ATR spectra of the HRP crosslinked silk fibroin hydrogel and immersed in methanol for 5 minutes. The concentration of silk fibroin is 16 wt%, HRP / silk is 0.2 wt%, H202 / silk is 1.1 wtVo. Figure 18 depicts the enzymatic degradation profile of the outer layer " skin " of HRP-crosslinked silk fibroin hydrogel after immersion in methanol for different periods of time. The test was performed at 37 ° C. The concentration of silk fibroin is 16 wt%, HRP / silk is 0.26 wt%, H202 / silk is 1.1 wt%. In each test, about 100 mg outer layer of silk fibroin hydrogel was immersed in 5 ml of PBS containing 1 U Protease XIV obtained from Streptomyces griseus (0.2 U / ml). Figure 19 depicts the enzymatic degradation profile of the " inner portion " of HRP-crosslinked silk fibroin hydrogel after immersion in methanol for 5 minutes. The test was performed at 37 ° C. The concentration of silk fibroin is 16 wt%, HRP / silk is 0.26 wt%, H202 / silk is 1.1 wt%. In each test, about 300 mg of the inner layer of hydrogel was immersed in 5 ml of PBS containing 0.025 U Protease XIV obtained from Streptomyces griseus (0.005 U / ml). Figure 20 represents cell viability (MTS assay). ATDC-5 cells were encapsulated in the hydrogel in vitro. The concentration of silk fibroin is 16 wt%, HRP / silk is 0.26 wt%, H202 / silk is 1.1 wtVo or 1.45 wtVo. The cell density used is 1 million cells / ml of hydrogel. Cells were encapsulated in the AIDS gel containing HRP and H202, and then 50 ul of the mixture was placed on a TCPS coverslip until gel formation under sterile conditions. Silk fibroin cells / hydrogels were cultured in alpha-MEM medium and cell viability was tested using the MTS assay, after different time periods. Figure 21 shows the ATDC-5 cells encapsulated in the silk fibroin hydrogel after immersion in medium containing MTS for 3 hours. The concentration of silk fibroin is 16 wt%, HRP / silk is 0.26 wtVo, H202 / silk is 1.1 wt%. ATDC-5 cells were cultured in the silk fibroin hydrogel for 1 day. Figure 22 depicts the fluorescence microscopy images of the ATDC-5 cells encapsulated in the silk fibroin hydrogel after 11 days in culture. Calcein AM test (green color shows live cells) and propidium iodide (red color shows dead cells). Silk fibroin hydrogels were prepared by 16 wt. The concentration of silk fibroin is 16 wt%, HRP / silk is 0.26 wt%, H202 / silk is 1.1 wt%.

Detailed Description of the Invention:

It is to be understood that this invention is not limited to the specific protocols, method (s), reagent (s) etc. described herein, and as such, there may be changes. The terminology used herein is for the purpose of describing only particular embodiments, and is not intended to limit the scope of the present invention, which is defined solely in the claims.

All patents and other publications identified herein are expressly incorporated herein by reference for the purpose of disclosure and disclosure. For example, the method (s) described in such publications and reports may be related to the present invention. These papers are published prior to the submission date of the present invention. Nothing in this context shall be construed as an admission that inventors are not entitled to antedate such disclosure by virtue of a previous invention or for any other reason. All statements regarding the date or representation on the content of these documents are based on the information available to the candidates and do not constitute any admission as to the accuracy of the dates or contents of these documents. Unless defined otherwise, all terms used herein have the same meaning as those generally understood by one skilled in the art to which this invention relates.

This invention provides methods for the preparation of peroxidase-crosslinked silk fibroin hydrogels and their use in the field of tissue engineering and regenerative medicine as bioadhesive material or controlled release of bioactive agents. This invention proposes a novel hydrogel and its formulations which can be used as a minimally invasive system for tissue regeneration. This system can be used in conjunction with or loaded with cells and / or other combinations to restore, maintain or enhance tissue functions. In addition, this system allows conjugation with other molecules, and can be used for controlled release of bioactive molecules. The enzymatically crosslinked silk fibroin hydrogels allow the incorporation of genetic material, bioactive agents, the detection and targeting agents, conductive molecules and inorganic materials, providing versatile and layered systems which can be used as fillers as well as in coatings to enhance the function of a medical device, or in electronics or optoelectronics or other pharmaceutical and medical applications.

Silk Fibroin is a versatile biodegradable biomaterial that contains a large amount of glycine-alanine-glycine-alanine-glycine-serine (GAGAGS) repeat sequences, allowing the formation of an anti-parallel β structure that gives the silk fibroin superior resistance . There are also several reactive amino acids in silk fibroin that allow chemical modification to adapt this protein to desired applications, such as serine, threonine, aspartic and glutamic acid, and tyrosine. Silk fibroin contains about 5 mol% tyrosine groups, which may be oxidized by peroxidase / hydrogen peroxide and, subsequently, the cross-linked to form a three-dimensional network. The silk fibroin hydrogels developed in the present invention are formed due to the crosslinked groups of tyrosine in silk fibroin. The silk fibroin solution was prepared as described above, but with important modifications (Sofia et al, J. Biomed Mater Res A 2001, 54: 139-148, .... WO 2010/036992, and WO 2003/036992, 022909 2003/022909), so as to control the final properties of the silk solution, such as pH and ion content (s). Aqueous solutions of silk fibroin at high concentration (up to 30 wt%) and containing PBS were prepared. PBS allowed the regulation of the pH value of the silk fibroin solution. The concentrated silk fibroin solution was diluted in different concentrations. Hydrogels were prepared by adding different amounts of peroxidase (for example, horseradish peroxidase) and peroxide (such as hydrogen peroxide) to the silk fibroin solutions (Figure 1). The crosslinking process may be carried out over a wide temperature range. The developed hydrogels can be degraded by proteolysis in vivo or in vitro. The gelation time and mechanical performance of the silica fibroin formed hydrogels were investigated. The biological performances of the hydrogels were tested in vitro.

The physical-chemical and biological performances of the hydrogels can be adapted in a wide range of different formulations and reaction conditions. For example, mechanical performance can be adjusted using silk fiber solutions of different concentrations or using different amounts of peroxidase. The gelation time of the aqueous solutions of PBS containing silk fibroin can also be controlled by the concentration of silk fibroin, the type and concentration of the oxidant (e.g., peroxide), light (e.g. exposure to UV light), pH and temperature. Other performances of the silk hydrogels, such as durability, absorption capacity and degradation rate can also be modulated over a wide range depending on the preparation conditions. The performance control of the silk fibroin hydrogels produced in the present invention may satisfy different requirements in the field of tissue engineering and regenerative medicine when applied thereto as an injectable matrix for tissue regeneration or a bioactive agent delivery system or other applications as mentioned above. 30

The properties of prepared peroxidase-crosslinked silk fibroin hydrogels can be modified by post-treatments. The shape of the prepared hydrogels can be adjusted by immersion in solutions of different ionic strength. The size of the hydrogels will increase after immersion in solutions of low ionic strength. And its size can be recovered, to a certain extent, to the original size, increasing the ionic strength of the solutions. Peroxidase crosslinked silk fibroin hydrogels can be used as a platform to prepare a matrix with controllable spatial conformations. As examples, a layered structure may be formed by immersing the fresh preparation or expanded hydrogel by immersion in organic solvents, mixtures of organic solvents, mixtures of water and organic solvents, or by means of electric field treatment, or other treatments, or any combination thereof. Any combination of conformation can also be developed using peroxidase cross-linked silk fibroin hydrogels with other treatments. The shape memory property and / or the layered structure of peroxidase-crosslinked silk fibroin hydrogels opens up unlimited possibilities for their application in tissue engineering and regenerative medicine, controlled release of bioactive agents, and as fill material as well as as in coatings to enhance the function of medical devices or other pharmaceutical or medical applications.

These freshly prepared or posttreatened modified peroxidase crosslinked silk fibroin hydrogels, used alone or in combination with inorganic detection and conducting materials, may also have potential applications in electronics or optoelectronics. With respect to their application in these fields, these materials may be applied by manual methods or by using an automatic dispensing device, being cross-linked during or after deposition. The disclosure of the present invention is supplemented by means of examples which are intended to provide a better understanding thereof, although these examples should not be treated with a restrictive character.

The silk fibroin solution was prepared as described above, but with modifications (Sofia et al, J. Biomed Mater Res A 54: 139-148, 2001, WO Patent 2010/036992, and WO Patent 2003/022909 2003/022909) in order to control the final properties of the silk solution, such as the pH value and ion content. Bombyx Mori silk fibroin was boiled from a solution of sodium carbonate (0.02 M, Sigma, USA) by boiling for one hour and then washed extensively with distilled water. Purified silk fibroin was dissolved in 9.3 M lithium bromide solution (Sigma, USA) at 70 ° C for 1 hour. Then, the solution was dialyzed with distilled water using a benzoylated dialysis tube (cut molecular weight: 2000, Sigma, USA) for the period of 2 days. Then, the aqueous solution of silk fibroin was dialyzed against a dilute saline solution of phosphate buffer (PBS, Sigma, USA), a solution (0.2 wt.% In distilled water) for 24 hours. Then, the dialysed solution of silk fibroin was concentrated by dialysis against a 20 wt% (20,000 g / mol, Sigma, USA) polyethylene glycol solution for 9 hours. Finally, the dialysis tube was carefully washed with distilled water and the silk fibroin solution was collected into a vial. The pH value of the concentrated fibroin silk solution can be adjusted using solutions of different concentrations of PBS during the above-mentioned dialysis process. The final concentration of concentrated silk fibroin was determined by measuring the dry weight of the silk fibroin solutions. The prepared silk fibroin solution was stored at 4Â ° C until use. The PBS content in the silk fibroin solution was tested by thermal gravimetric analysis (TGA), and the results showed that the PBS content in the silk fibroin solution can be controlled between 1.5-2% (PBS / silk, in% by weight). And the pH of the silk fibroin solution without the PBS was in the range of 5.6-6.2. After incorporation of PBS into the silk fibroin solution, the pH of the solution can be controlled in the range of 7.0-7.4, which is suitable for the incorporation of cells or bioactive factors.

Example 2: Hydrogel Formation-Method 1 In order to produce peroxy crosslinked silk fibroin hydrogels, silk fibroin solutions having different concentrations (2-30 wt.%) Were prepared by diluting with a solution of PBS. Horseradish peroxidase (HRP, 260 unit / mg, Sigma, USA) and hydrogen peroxide (H2O2, Sigma, USA), the solutions were prepared by dissolving in PBS. Then the HRP and H202 solutions were added to the silk fibroin solutions in different concentrations, ranging from 1 to 1000æg / ml and 0.5 to 50 mmol / ml, respectively. The mixture was gently shaken and then gelling was carried out in a water bath or in the oven at different temperatures, ranging from 5 to 50 ° C. The hydrogel can also be processed in different ways using different molds and placing them in a furnace at the required temperature. A silica fibroin hydrogel disk may be prepared from a 7.5 mm diameter die (Figure 2b), using 16% by weight silk fibroin solution with PBS (the PBS content in the silk solution was 1.7%, and the pH was 7.2) along with 0.84 mg / ml HRP in PBS and 0.18 wt. % H2 O2 in PBS (HRP / Silica 0.26 ° C, H202 / Silk 1.10%). Not only the silk fibroin solution with PBS can be used for the preparation of peroxidase-crosslinked hydrogels, as the same solution without PBS was also able to form hydrogel under the same method. For example, the silk fibroin hydrogel can be prepared in a flask using 16 wt% pure silk fibroin solution along with 0.84 mg HRP / ml solution in PBS and 0.18 wt% H202 in PBS (HRP / silk 0.26 wt. fc, H202 / silk 1.10 wt.h) in a 37 ° C bath (Figure 2a). In the following studies, Silk-10, Silk-12, Silk-16 were corresponding to 10 wt.%, 12 wt.%, And 16 wt.% Silk fibroin solution respectively. In addition, if not mentioned in the examples below, the concentrations of H 2 O 2 and HRP were 0.84 mg / ml and 0.18 wt%, and the silk fibroin solution was PBS (PBS silica solution content was 1.7%, and the pH was 7.2.

Example 2: Hydrogel-Method 2 Formation Method In order to produce peroxidase-crosslinked silk fibroin hydrogels, silk fiber solutions of different concentrations (2 to 20 wt.%) Were prepared by diluting the concentrated silk fibroin solution with PBS. Horseradish peroxidase solution (HRP, 260 unit / mg, Sigma, USA) was prepared by dissolving in PBS. Calcium peroxide (75% purity, Sigma, USA) was dissolved in 0.1 M HCl, and then the pH of the solution increased to 5.0. HRP and calcium peroxide solutions were added to the silk fibroin solutions at different concentrations, ranging from 1 to 1000æg / ml and 0.5 to 50 mmol / ml, respectively. The mixture was gently stirred and then gelling was carried out at different temperatures, ranging from 5 ° C to 50 ° C. The hydrogel can be processed in different forms using various molds and placed in an oven at the required temperature. Here, calcium peroxide was used as a source of peroxide, assuming that 1 mol of calcium peroxide will give rise to one mole of hydrogen peroxide in the present study. According to the calculations, the concentration of the calcium peroxide solution of 0.51 wt% is equal to 0.18% H2O2 by weight in solution in this study. For example, the silk fibroin hydrogel disk can be prepared by this method using 16 wt.% Silk fibroin solution, with PBS (PBS content in silk solution was 1.7%, and pH was 7.2) along with 0.84 mg / ml of HRP solution in PBS and 0.51 wt. CaO2% in distilled water (HRP / Silk in weight 0.26 V, H202 / Silk 1.10 wt. B) at 37 ° C (Figure 2c).

Example 3: Characterization of peroxidase-crosslinked silk fibroin hydrogels

Fourier Transform Infrared Spectroscopy (FTIR) Structural conformation of the pure solution of silk fibroin, silk fibroin solution with PBS, and silk / HRP / H2C> 2 mixture before and after the gelation was analyzed by FTIR for the use of attenuated total reflectance (ATR) in wet samples. Ultrapure water was used as control. For the liquid sample, 50 μl of sample was used for each assay. After mixing the silk / HRP / H202 to form a gel, a small piece of moist hydrogel was removed for the test. Infrared spectra were recorded at room temperature with a resolution of 4 cm -1 in the range of 4000-600 cm -1 with 40 readings (Figure 3). FTIR spectra showed that all samples had the main peak at about 1650 cm -1, which is the characteristic peak of the random helical conformation. There were no obvious peaks around 1700 cm -1 and 1620 cm -1, the characteristic peaks of beta conformation (Silk-II).

These data indicated that the silk fibroin hydrogel prepared by peroxidase-mediated cross-linking exhibited amorphous (random spiral) conformation, such as the conformation presented by the aqueous solution of silk fibroin (Figure 3a). The silk fibroin solution with PBS also maintained amorphous conformation. Most of the silk fibroin hydrogels previously reported in the literature have beta conformation. Stable hydrogels of amorphous amorphous silk fibroin have rarely been reported. Here, we present stable silk fibroin hydrogels having mostly amorphous (random) conformation. Optical absorbance at 550 nm Optical absorbance at 550 nm was used primarily to evaluate the conformation transition of the silk fibroin solution or the hydrogel by Kaplan et al. When the silk or hydrogel fibroin solution is in the amorphous state, the absorbance at 550 nm is very low. If the silk or hydrogel fibroin solution has beta conformation, the absorbance increases. For this test, 50 ul of solution was used. The silk solution (with PBS) and silk / HRP / H2C> 2 mixture were placed in a 96-well plate, and the absorbance at 550 nm was recorded by a microplate reader. Six wells were read for each group of samples. After the silk / HRP / H202 mixture was formed into a gel, the same well was read again. Figure 4 showed its absorbance at 550 nm, and it was found that there were no differences in absorbance of the silk / HRP / H202 mixture before and after gelation. There was also no significant difference in absorbance between the silk solution and the silk hydrogel.

Combining the data obtained from the low optical absorbance (Figure 4), the appearance of transparency of the silk hydrogel (Figure 2), and the FTIR spectra (Figure 3), it was concluded that the silk fibroin hydrogel prepared by mediated cross-linking by peroxidase is mainly of amorphous conformation. This property makes it different from the previously reported silk hydrogels, most of them exhibiting beta conformation and cloudy and opaque appearance. The transparency properties of the silk fibroin hydrogel prepared herein extend the possibility of its application in ophthalmology or optical devices.

Scanning Electron Microscopy (SEM) The micromorphology of the lyophilized hydrogel of peroxidase-reticulated silk fibroin was observed by SEM. The silk hydrogel was frozen at -80 ° C after gelation, and then lyophilized. The transverse image was observed under SEM after the surface was coated with gold. For example, Figure 5 shows the micromorphology of the silk hydrogel prepared with 16 wt% silk solution, HRP / silk ratio of 0.26 ¾ and H202 / silk ratio of 1.10 ¾. The SEM image shows that the silk fibroin hydrogel exhibits porous structure and interconnected pores being the pore size 50 to 100 Âμm. The porous structure of the silk hydrogel may facilitate transport of nutrients for further cell incorporation studies, or allow the migration of cells into the hydrogels.

Gelling Time The gelling time of the silk hydrogel was tested by a vial tilt method. The absence of flow within 1 minute after flask inversion is considered as the gel state. Each condition was tested in triplicate. During the test, the flask was immersed in a 37 ° C water bath and the influence of the concentration of silk, HRP and H202 were investigated. Figure 6 shows the gelation time profiles of the silk fibroin hydrogels. When the influence of HRP was studied, the H202 / silk ratio was set at 1.10 ¾ weight. When the influence of H202 was studied, HRP / silk ratio was set at 0.26 weight. It was found that increasing the concentration of silk fibroin and HRP content would shorten the gelation time, as with the increase in H2O2 the gelation time increases. For example, in the case of a 16 wt.% Silk solution, when the HRP content increases from 0.13 wt. V. to 0.52 by weight. V0 the gelation time of the silk fibroin hydrogel decreased from ~35 minutes to less than 4 minutes .. It was also found that silk gelling will occur more rapidly at physiological temperatures, compared to temperatures below 37 ° C. The data presented here are examples only, the gelling time interval being not limited to the data presented herein. 38

These data indicated that a rapidly formed silk hydrogel (less than 4 minutes) can be prepared by the method developed herein. The gelation time presented here was much shorter than previously reported for silkworms prepared by other methods, such as stirring, vortexing and ultrasonication. It is to be noted that in the method described herein the gelation may occur under physiological conditions, which allows the in-situ incorporation of cells or bioactive agents.

Mechanical properties The stored energy modulus of the peroxidase-crosslinked silk fibroin hydrogels was evaluated by reometer in a rotary model at 37øC. The silk fibroin solution was mixed with HPR and H202, and then 1 ml of the mixture was immediately transferred to the chamber to perform the test. A cylinder with a conical tip was immersed in the blending solution, and a drop of oil was placed on top of the silk solution to prevent evaporation during the test. For the measurement of the stored energy module, 0.5 Hz frequency and 0.1% voltage were used. The data points were collected every 30 seconds, until the module reached a plateau for 5 minutes. The stored energy module was obtained by averaging the data points in the " plateau " zone. Triplicates were used for each formulation. After the module reached the plateau for 5 minutes, a frequency scan was performed from 0.1 to 10 Hz frequency with 0.1% constant strain for 10 minutes. When the frequency scan ended, a strain scan was also performed from 0.1% to 100% deformation at a constant frequency of 1 Hz for 10 minutes. Figure 7 shows the influence of silk concentration, and HRP and H202 content on the stored energy module of silk fibroin hydrogels. The modulus was found to increase with increasing silk concentration and H202 content. The HRP content had only a minor influence on the module. When the H202 / silk content was 1.45% by weight, the silk-16 hydrogel reached a stored energy module of about 5 kPa. By controlling the H2 O2 content, the mechanical properties of the silk hydrogel can be adjusted over a wide range. Figure 8 shows the influence of HRP on the stored energy module profiles of peroxidase-crosslinked silk fibroin hydrogel, measured by a rheometer in a rotation model at 37øC. Silk-10, Silk-12, Silk-16 correspond to silk hydrogels prepared with 10 wt.%, 12 wt.%, And 16 wt.% Silk fibroin solution, respectively. The H202 / silk ratio was set at 1.1 weight in this figure. The result showed that the silk fibroin hydrogel prepared herein can keep its energy modules stored under pressure of 40% (Figure 8a, b, c), and less than 5 Hz of frequency (Figure 8d, e, f). Figure 9 depicts the influence of H202 on the profiles of the stored energy module of the peroxidase-crosslinked silk fibroin hydrogel measured by a rheometer in a rotation model at 37øC. Silk-10, Silk-12, Silk-16 were matched to silkworms prepared with 10 wt%, 12 wt%, and 16 wt% silk fibroin solution, respectively. The HRP / silk ratio was set at 0.26 wt% for all formulations. The result showed that the silk fibroin hydrogels prepared herein can keep their energy storage modules under pressure of 40% (Figure 9a, b, c), and less than 5 Hz of frequency (Figure 9d, e, f) .

These results indicated that the mechanical properties of the peroxidase-crosslinked silk fibroin hydrogels can be adjusted over a wide range by varying the different reaction parameters and proportions of the reactants. Its mechanical properties have been quite stable for a varied range of frequency and deformation, which is one of its advantages to be used for tissue regeneration in vivo.

Degree of expansion The degree of expansion of these hydrogels in distilled water and PBS were characterized. A silk / HRP / H202 (300æl volume) was used to prepare hydrogels in a 7.5 mm diameter die. After the peroxidase-crosslinked hydrogels were prepared, they were immediately placed in distilled water or PBS in a 37 ° C bath for 12 hours, allowing them to equilibrate. Then, the hydrogels were removed from the liquid, the liquid was absorbed by the surface of a filter paper. The wet weight was recorded on a scale (Mw). The hydrogels were then dried in an oven at 90 ° C for 2 days. Dry weight was recorded (Md). The degree of expansion was calculated as follows:

Expansion Degree = (Mw-Md) / Md * 100% Figure 10 represents the degree of expansion of peroxidase-crosslinked silk fibroin hydrogel in distilled water (a) and PBS (b). These results showed that the rate of increase increased with the decrease of the concentration of silk and increasing the content of H202. The degree of expansion was higher in distilled water compared to PBS. The differences in the rate of expansion induced by silk fibroin concentration and H202 content are related to the mechanical properties. High concentrations of silk solution and H202 content induced a modulus increase. In this way these hydrogels are more stable and do not absorb as much liquid. The ion concentration also affects the rate of expansion. When the silk fibroin hydrogel has been immersed in distilled water (which has a very low ionic strength), the silk molecules will loosen freely. This explains the differences in the rates of expansion of the silk hydrogel observed when immersed in PBS and distilled water. Enzymatic degradation The degradation profile by enzymatic action of the silk fibroin hydrogels was evaluated. Different Silk / HRP / H2C> 2 (V = 500 Âμl) formulations were produced to prepare the various hydrogels, and using a 7.5 mm diameter die. Different solutions of silk fibroin with concentration of 10 wt%, 12 wt%, and 16 wt% were used. In turn, HRP / silk was 0.26 wt%, H2C> 2 / silk was 1.1 wtVo or 1.45 wtVo. In each assay, 500 mg of the silk fibroin hydrogel was immersed in 5 ml of PBS containing 0.025 U Protease XIV obtached from Streptomyces griseus (0.005 U / ml). The hydrogel mass in the hydrated state was recorded as Wi. At the end of each time point, the hydrogels were removed from the solution and its mass (Wt) determined, after absorption of excess water on the surface through filter paper. And then the mass loss ratio was calculated as follows:

Mass loss ratio (%) = (Wi-Wt) / Wi * 100%

The results revealed that the silk fibroin hydrogels presented a degradation profile in the period between 6 and 8 hours (Figure 11). Hydrogels with the highest mechanical proportions have a lower mass loss profile when compared to hydrogels whose mechanical properties are lower. The degraded degradation profile is completely different from those previously reported. This study shows that HRP-crosslinked silk fibroin hydrogels are amorphous, counter to sheet-beta conformation or other crystalline state as reported in previous studies. The degradation profile of the HRP-mediated silk fibroin hydrogels provides a set of new applications in regenerative medicine.

Shape Memory Property The shape memory property of the HRP crosslinked silkworm hydrogels was evaluated in vitro. The hydrogels were produced, immersed consecutively in distilled water and PBS, and the diameter determined at each 12 hour immersion period (Figure 12). The concentration of the silk fibroin used is 16 wt%, HRP / silk is 0.26 wtVo, H202 / silk is 1.1 wtVo. The silk fibroin hydrogel was first prepared in a mold (Figure 12a), and then the hydrogel was removed from the mold (Figure 12b). Figure 12c shows the silk fibroin hydrogel (hydrated) after immersion in distilled water for 12 hours. It was found that the diameter of the silk hydrogel was recovered to some extent after immersion in PBS for another 12 hours (Figure 12d). Silk fibroin hydrogels 43 were produced from different concentrations of silk fibroin (eg, 10%, 12% and 16%) as reported in Figure 13. All hydrogels were found to have maintained their diameter in PBS , in different periods of immersion. However, the diameter of the hydrogels in PBS was slightly higher than its original diameter (then removed from the mold). And the diameter in distilled water (the second immersion) was recovered quickly after the first immersion in distilled water.

These results indicated that the HRP crosslinked silk fibroin hydrogels respond to physico-chemical stimuli such as reactive ionic strength and pressure. This property has never been described in previous studies involving silk fibroin hydrogels. This property is associated with its amorphous nature. These hydrogels can function as a stable aqueous platform and suitable for studying the effect of different ions and solvents on the conformation of silk fibroin. These properties open up new possibilities for the application of the HRP-crosslinked silk fibroin hydrogels as a controlled drug delivery system (DDS), which responds to slight variations in environmental conditions.

Silk Fibroin Hydrogel (Multi-layered) that Enable the Spatial Control of its Conformation

Different layers of HRP-crosslinked silk fribroin hydrogels were developed by immersion in methanol for different periods of time. Figure 14 shows HRP crosslinked silk fribroin images before (a) and after (b) immersion in methanol over a period of 544 minutes. The concentration of silk fibroin is 16 wt%, HRP / silk is 0.26 wtVo, and H202 / silk is dee 1.1 wtVo. The silica hydrogel was found to have become opaque after immersion in methanol for a few minutes. It has also been found that the methanol-immersed hydrogels have a layered structure, the outer layer being further shown cross-sectional views of HRP-crosslinked silk fribroin hydrogels after immersion in methanol for from 0 to 10 minutes. The concentration of silk fibroin is 16 wt%, HRP / silk is 0.26 wt V, and H202 / silk is 1.1 wt V. These results show that the thickness of the outer layer increases in thickness with increasing immersion time in methanol. Figure 16 shows the results of the studies of the influence of the immersion time of the hydrogels in methanol on the thickness of the outer layer (opaque). The concentration of silk fibroin is 16 wt%, HRP / silk is 0.26 wt%, and H202 / silk is 1.1 wt% or 1.45 wt%. This study showed that hydrogels presenting higher mechanical properties (H202 / silk at 1.45 wt%) presented a less thick outer layer when compared with hydrogels presenting lower mechanical properties (H202 / silk at 1.10 wt%). And the thickness of the layers can be easily controlled, having a thickness of between 200 and 1000 Âμm. The outer layer (opaque) has a stiffness superior to the inner layer (transparent). The conformation of the different layers of the hydrogels was investigated using the infrared technique with attenuated total reflectance (FTIR-ATR), in a hydrated state. For this assay, the concentration of silk fibroin is 16 wt%, HRP / silk is 0.26 wt%, and H202 / silk is 1.1 wtV. After production, the hydrogel was immersed in methanol for 5 minutes and then washed with distilled water. The different layers of the hydrogel were then analyzed by FTIR-ATR. Infrared spectra were recorded at room temperature with a resolution of 4 cm -1 in the range of 4000-600 cm -1 (with 40 analyzes). Figure 17 shows the FTIR spectra of the different layers of the developed silk hydrogel. The outer (opaque) layer was found to have a main peak around 1620 cm-1, and the inner (transparent) layer exhibits a major peak at about 1650 cm-1, confirming that the conformation of the transparent part is essentially amorphous . Additionally, it has been found that the transparent layer of the hydrogel exhibits a small peak at 1620 cm-1, which is indicative that the hydrogel exhibits some beta-sheet conformation in the inner but non-dominant layer. These results corroborate that the conformations of the silk fibroin in the outer and inner layers are different, as soon as it is possible to control the spatial conformation of the silk hydrogels using the method presented herein, i.e. immersion in methanol. Other organic solvents or aqueous salt solutions may also be used to produce multilayers with different conformations and properties.

The degradation profile of the different layers of the silk fibroin hydrogels were determined in vitro. For this assay, the concentration of silk fibroin is 16 wt%, HRP / silk is 0.26 wt%, and H202 / silk is 1.1 wtVo. The silk fibroin hydrogels were immersed in methanol for 3-10 minutes. For each test, about 100 mg (opaque) outer layer of the silk fibroin hydrogel was immersed in 5 ml of PBS containing 1 U Protease XIV obtained from Streptomyces griseus (0.2 U / ml). And about 300 mg of inner layer of the silk fibroin hydrogel (sample obtained from the hydrogel immersed in methanol for 5 minutes) was immersed in 5 ml of PBS containing 0.025 U Protease XIV 46 obtained from Streptomyces griseus (0.005 U / ml). The initial mass of the hydrated hydrogel was recorded as Wi. At the end of each degradation time, the hydrogels were removed from the solution. The hydrogel mass (Wt) was recorded after absorption of excess water using filter paper. And then the mass loss ratio was calculated as follows:

Figure 18 shows the enzymatic degradation profile of the outer layer of HRP crosslinked silk fibroin hydrogel after immersion in methanol for different periods of time (%) = (Wi-Wt) / Wi * 100% of time. The results showed that the outer layer of the silk fibroin hydrogel is more resistant to enzymatic degradation. The layer immersed in methanol for 3 minutes shows a mass loss of 17% after 4 hours of immersion in the degradation solution. The silk fibroin layer immersed in methanol for 5 minutes has a mass loss of 10%, and the layer immersed for 10 minutes has a mass loss of 2%. These results indicated that the silk-exposed (methanol) outer layer of silk fibroin is in the crystalline state (beta sheet), and by increasing the immersion time in methanol it is possible to increase the crystallinity of silk fibroin. Figure 19 shows the enzymatic degradation profile of the inner (transparent) layer of HRP-crosslinked silk fibroin hydrogel after immersion in methanol for 5 minutes. It has been found that the loss of mass of the silk fibroin is about 80%, after 6 hours in degassing solution, and the concentration of the enzyme used in the degradation solution is 40 times lower than that used in the 47 degradation tests performed with the outer layer of the silk fibroin hydrogel. These results corroborate the fact that the inner and outer layers have different conformations. The inner layer exhibits essentially amorphous conformation (Figure 11). Combining the results in Figure 18 and Figure 19, these results indicated that it is possible to control the conformation of different layers of the silk fibroin hydrogels. The development of silk fibroin hydrogels with different layers exhibiting different conformations were first reported here. This unique property may be useful as a new platform for the controlled release of bioactive drug or molecules, fillers, or multilayer materials to find applications in electronics or optoelectronics.

Example 4: In vitro evaluation of the cytotoxicity of HRP crosslinked silk fibroin hydrogels The cytotoxicity of the silk fibroin hydrogel was evaluated by encapsulation and culture of ATDC-5 cells (chondrogenic cell line) in the hydrogel (direct contact ). Cell viability was assessed by the MTS assay and using the reagent (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2 (4-sulfophenyl) -2H-tetrazolium). This is a standard method for evaluating cytotoxicity in vitro. Silk fibroin hydrogels were prepared from solutions at 16 wt%, HRP / silk at 0.26 wt, and H202 / silk at 1.10 wt -1 or 1.45 wtls. ATDC-5 cells were grown in Minimum Essential Medium (α-MEM) monolayer, 10% fetal bovine serum and 1% antibiotic-antimycotic mixture containing 10,000 48 units / ml penicillin G sodium salt, 10,000 pg / ml streptomycin sulfate and 25 pg / ml amphotericin B as Fungizone®, in 0.85% antimycotic saline. The ATDC-5 cells were incubated at 37øC in a humidified atmosphere with 5% CO 2, and the culture medium renewed every three days. The 16 wt% concentration of silk fibroin solution was sterilized by ultraviolet rays. Confluent cells were detached from the polystyrene culture flasks using a solution containing the enzyme trypsin (0.25% trypsin / EDTA solution). A cell suspension containing 1 million cells per ml was prepared. The cell suspension (1 ml) was centrifuged and the supernatant removed, then 1 ml of the silk fibroin solution mixture and the HRP solution containing H2O2 (HRP / 0.26 wt% silk, H2O2 / silk at 1.10 wt.) or 1.45 wt% (i). After homogenization, 50 ul of silk fibroin solution containing the ATDC-5 cells were transferred to a coverslip. The slides were then placed in the bottom of the wells of a polystyrene (24-well) culture dish. In order to promote cross-linking, the plate containing the gel-cell mixture was incubated at 37øC for a few minutes. And then 1.5 ml of medium was transferred to each well. The culture medium was renewed every three days. After each culture period, ie 24 hours, 4 and 7 days, the hydrogels containing the encapsulated cells were removed from the medium. Washing was carried out with a phosphate buffered solution (PBS) and then 500 ul of MTS solution (CellTiter Kit 96 Proliferation Assay) was added. Then, 37 ° C MTS solution was incubated with hydrogel / ATCC-5 at 37 ° C for a period of three hours. Then, 100æl of the solution was transferred to the respective well of a polystyrene (96 wells) culture dish. The absorbance (OD) at 490 nm was determined using a microplate reader. All tests were performed in triplicate. The cell viability results are shown in Figure 20. Cell viability in both groups of hydrogels was shown to increase from day 1 to day 4, and remained viable until day 7. These results demonstrated that hydrogels of silk fibroin allow to encapsulate and maintain viable ATDC-5 cells, in vitro. We can conclude that the developed hydrogels are non-cytotoxic, and support the viability of the cells. Figure 21 shows the fluorescence microscopy images of the ATDC-5 cells encapsulated in the silk fibroin hydrogels grown in culture medium for the period of 3 hours. The concentration of silk fibroin is 16 wt%, HRP / silk is 0.26 wt%, H202 / silk is 1.1 wtl. After 1 day of culture it is possible to verify a homogeneous distribution of cells and that these are viable within the hydrogel. The viability, distribution and morphology of the cells was also evaluated using the " live / Dead " using calcein AM (green staining represents living cells) and propidium iodide (red cells representing dead cells). On days 1, 3, 7, and 11, the silk hydrogels containing the encapsulated ATDC-5 cells were removed from the culture medium. They were washed with PBS. The hydrogels were then immersed in 1 ml of PBS containing 1 mg of calcein AM and 1æg of propidium iodide. The hydrogels were incubated for 15 minutes at 37 ° C and then washed in PBS prior to observation under fluorescence microscopy. Figure 22 shows the images of the living / dead cells when cultured in the hydrogel. The concentration of silk fibroin is 16 wt%, HRP / silk is 0.26 wtVo, H202 / silk is 1.1 wtt. The results show that most of the 50 cells are viable after encapsulated in the silk fibroin hydrogel up to 11 days of culture. It is also possible to observe the homogeneous distribution of the cells inside the hydrogels.

Taking into account the results of in vitro biological evaluation, and comparing with the silk fibroin hydrogels described in previous studies, it can be stated that the HRP crosslinked silk fibroin hydrogel has several advantages, such as allowing it to encapsulate cells in situ, exhibit rapid gelation (within a few minutes) and under physiological conditions, and support cell viability. The method for encapsulating cells is fast and easy to operate.

Anyone with the appropriate qualifications in the field is capable of modifying and introducing variations not described in this application without departing from the scope of the present invention. It is therefore understood that the present invention is not limited to the described embodiments but is intended to cover modifications which are within its inventive concept as defined by the appended claims.

Date: December 3, 2012

Claims (27)

Hydrogel material or its formulations, characterized in that it comprises an aqueous solution of silk fibroin, a peroxidase enzyme and a peroxide. Hydrogel material according to the preceding claim, characterized in that the source of silk fibroin containing tyrosine groups is selected from the group consisting of: Bombyx mori silk fibroin, Antheraea, woven silk, raw silk waste, silk fibroin or mixtures thereof. Hydrogel material according to the preceding claims, characterized in that the aqueous solution of silk fibroin is free of ions or comprises saline ions, with a pH between 6.5-9.0. A hydrogel material according to the preceding claims, characterized in that the aqueous solution of silk fibroin has a concentration of 2-30% by weight. Hydrogel material according to the preceding claims, characterized in that the source of peroxidase is selected from the group: horseradish or other plants, animals, humans and bacteria. A hydrogel material according to the preceding claims, characterized in that the peroxidase is dissolved in an aqueous solution of water or buffered solutions, having a pH ranging from 5 to 9. A hydrogel material according to the preceding claim, characterized in that the concentration of peroxidase is from 0.001 to 5 mg / ml. Hydrogel material according to the preceding claims, characterized in that the peroxide is hydrogen peroxide or calcium peroxide. Hydrogel material according to the preceding claims, characterized in that the peroxide is dissolved in aqueous solution, in water or buffer solution, having a pH between 5 and 9. Hydrogel material according to the preceding claims, characterized in that the peroxide concentration is between 0.001-30% by weight. A hydrogel material according to the preceding claims, characterized in that it is cross-linked. A hydrogel material according to the preceding claims, characterized in that it has a carrier structure in the form of a hydrogel, fibers or open-pore 3D structure, preferably interconnectable. A hydrogel material according to the preceding claims, characterized in that it is in the form of microparticles or nanoparticles. A hydrogel material according to the preceding claims, characterized in that it is biodegradable and modulatable. 3 A hydrogel material according to the preceding claims, characterized in that it comprises an amorphous conformation and is transparent. A hydrogel material according to the preceding claims, characterized in that it has a layered structure. Hydrogel material according to the previous claims, characterized in that it comprises adjustable spatial conformations, further comprising a concentration gradient of structures with beta conformation or with combination of conformations: silk-I, silk-II - beta- and amorphous- spiral. A process for obtaining the hydrogel material or its formulations described in claims 1-17, characterized in that it is carried out in a single step with the mixture of an aqueous solution of silk fibroin, a peroxidase enzyme and a peroxide. A process according to the preceding claim, characterized in that the temperature of the mixture is between 4 ° C and 60 ° C. A process according to claims 18-19, characterized in that the mixture is immersed in organic solvents, or mixtures of organic solvents, or aqueous mixtures of organic solvents, or the electric field treatment. A pharmaceutical composition comprising the silk fibroin hydrogel material described in the claims 1-17 and obtained in the process described in claims 18-20. A pharmaceutical composition according to the preceding claim, further comprising bioactive substances, cells or genetic material, inorganic compounds or combinations thereof. The silk fibroin hydrogel material described in claims 1-17 and the pharmaceutical composition described in claims 21-22, characterized in that they can be injected and dispensed manually or automatically. Silk fibroin hydrogel material and pharmaceutical composition according to the preceding claim, characterized in that they are sterilized and cross-linked directly at the application site, using minimally invasive techniques. Silk fibroin hydrogel material and pharmaceutical composition according to claims 23-24, characterized in that they are in the form of layers and sensitive to stimuli from the surrounding environment, for use in electronics and optoelectronics. Use of the silk fibroin hydrogel material described in claims 1-17, characterized in that it is used as biomedical material, in particular in surgical products, or as a coating of surfaces. Use of the silk fibroin hydrogel material and pharmaceutical composition described in claims 21-22, characterized in that they are used in cell encapsulation, tissue engineering and regenerative medicine, in particular in controlled release systems of agents ) therapeutic. Lisbon, March 28, 2013 1/21 A B Figure 1 2/21 Figure 2 3/21 Absorbance (u.a) Figure 3 4/21 Figure 4 5/21 Figure 5 6/21 Gel time (min) A B C A B C A B C A B C 0.13 0.20 0.39 0.52 c 1 Φ 30 - O ϊ (5 ii R5 O c ABC ABC A B C ABC A B C HRP / Silk (wt.%) 8.80 0.35 1.10 125 1.45bi202 / Silk (wt.%) Figure 6 7/21 0.13 0.2 «Θ.39« .52 HRP / Silk (wt «o) 9.80 0.95 1.10 1.25 1.45 H, 02 / Silk (wt./i) Figure 7 8/21 G '(kPa) G' (kPa) Figure 8 9/21 G '(kPa) G' (kPa) 3.2 - 3.0 - 2.8 - ^ 26 - Q_ 24 'S / 2.2 - τ T --T τ TT 2.0 - (5 W 0.6 - 0.4 - 0.2 - 0.0 - Ϊ 10 100 Deformation (%) b 0.1 1 10 100 Deformation (%) 5.5 · 5.0 · ηΓ 4 · 5 'Q-4.0 · -W 3.5 · O 15: 1.0 · 0.5 · o' ooC Deformation (%) 3.2 · 3.0 · 5.5 · 5.0 · 4.5 · 2.8 · 2.6 · 0.2 · _ 0.1 1 10 3.0 · 1.52 'αϊ' ΐ '...... Vo d Deformation (%) Θ Deformation (%) 1.0 · 0.5 · 1 " "&Quot; 1 " " " " 0.1 1 10f Deformation (%) Figure 9 10/21 Weight loss rate (%) Swelling rate (%) Figure 10 Figure 11 11/21 Figure 12 12/21 Figure 13 13/21 Figure 14 14/21 Figure 15 15/21 Figure 16 16/21 Wave number (crrr1) Figure 17 17/21 Figure 18 18/21 (Ο < / > < /) (Ο ε α> ο (Ο Ο α > α. 19/21 0 0 · 2 · 2 0.3 · (Ο Ο (0 0.1 - 0.0 1 4 7 Time (days) Figure 20 20/21 Figure 21 21/21 Figure 22