PT107426B - 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

DESCRIPTION

SILK FIBROINE DERIVATIVE HYDROGELS: METHODS AND

ITS APPLICATIONS

Object of the invention

The present invention relates to the development of peroxidase crosslinked silk fibroin hydrogels. Methods of preparing concentrated aqueous silk solutions and silk fibroin hydrogels under different conditions are presented. Silk fibroin hydrogels can be formed in a single step with gelation occurring under close physiological conditions. Crosslinking can be performed manually or automatically in situ. Enzymatically crosslinked silk fibroin hydrogels exhibit controlled performance and allow encapsulation of cells and / or therapeutic agents, genetic material, bioactive agents, detection and targeting agents, conductive molecules or inorganic materials, providing versatile systems that can be used as biodegradable matrix in acellular and cellular systems for tissue engineering and regenerative medicine applications or as drug delivery, bioadhesive, filler, coatings systems to improve the operation of a medical device, or other pharmaceutical or medical applications.

Technical Domain

The present invention relates to peroxidase crosslinked silk fibroin hydrogels which are capable of being produced at temperatures and pH close to physiological. Enzymatically cross-linked silk fibroin hydrogels have controlled performance and enable therapeutic and bioactive agents, cell and / or genetic material encapsulation agents, detection and targeting agents, conductive molecules or inorganic materials, providing versatile systems that can be used as a biodegradable matrix in cell and cellular systems for tissue engineering and regenerative medicine applications or as release systems, bioadhesives and filler, coatings for enhancing the function of medical devices, in electronics and optoelectronics, or other pharmaceutical or medical applications. Methods for preparing peroxidase cross-linked silk fibroin hydrogels are presented herein. The system is biocompatible and capable of being applied manually or automatically and for in situ polymerization.

State of art

Hydrogels have attractive properties for their use in tissue engineering, 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 an adjustable biodegradation rate, (v) may be applied by minimally invasive procedures (eg injection). Thus, hydrogels provide a highly hydrated microenvironment, similar to the extracellular matrix. They can be injected and further cross-linked in situ allowing cell encapsulation and preservation / induction of a given phenotype. An injectable hydrogel must also have a clinically suitable sol-gel transition mechanism, ie it should be liquid to facilitate injection and homogeneous cell distribution, while being able to rapidly gel after implantation under physiological conditions ( e.g. pH and temperature).

This unique combination of features, in addition to the ability to adjust their rate of degradation in vivo, also makes them useful in applications assuming controlled release of drugs or as bioadhesives.

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

Recently, enzymatic crosslinking methodologies have emerged as an alternative route to overcome the difficulties associated with more conventional chemical crosslinking methods. Different hydrogels have been obtained by crosslinking reactions using horseradish peroxidase-HRP as a catalyst. HRP is a type b single chain hemoprotein that catalyzes the coupling of phenol or aniline derivatives by decomposition of hydrogen peroxide (H ₂ O ₂ ). Several synthetic and natural polymers functionalized with tyramine, tyrosine (also known as 4hydroxyphenylalanine) and aminophenol side groups are currently under development to form hydrogels in situ using this strategy. Examples include: HRP crosslinked alginate with phenolic groups (Sakai, et al Acta Biomaterialia,

2007, 3: 495-501.), Dextran-tyramine conjugates (Jin et al

Biomaterials, 2007, of floretic acid with chitosan grafts (jin, et al.

Biomaterials, 2009: 25442551), hyaluronic acid tyramine conjugates (Kurisawa, et al. Chemical Communications, 2005, 14: 4312-4314), polyaspartic acid conjugates with each of the three aromatic side groups (Sofia, et al. Journal of Macromolecular Science Part A, 2002, A39: 1151-1181), tyrosine-containing peptides (Gly-Tyr-Gly) (Oudgenoeg, et al.

Journal of Agricultural and Food Chemistry, 2001, 49: 25032510), the food casein protein (Li, et al.

African biotechnology,

2009,

8: 5508-5515), tyrosine containing a peptide marker (Minamihata, et al.

Bioconjugate Chemistry, 2011, 22: 74-81).

These systems have several advantages, such as biocompatibility and easy control of the reaction rate under mildly aggressive conditions, mimicking biological crosslinking processes. In addition, adjustable mechanical properties can be obtained by varying the concentration of H ₂ O ₂ , as has been shown for gelatin-hydroxyphenylpropionic acid hydrogels (Wang, et al. Biomaterials, 2010, 31: 8608-8616) where The stiffness varies as a function of the H ₂ O ₂ concentration without changing the polymer content in the precursor solution. The hydrogels developed in this study were able to withstand cell adhesion and proliferation and in a stiffness dependent manner. In addition, it has been reported that by controlling the phenolic group content in the carboxymethylcellulose (CMC) -tyramine 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 cellular adhesiveness after gelation by crosslinking phenolic groups. Protein-polysaccharide conjugated hydrogels from alginate and protein derivatives (gelatin and albumin) have also been developed (Sakai, et al, 2010, Biomacromolecules 11: 1370-1375.), All having portions of phenol groups via a reaction. catalyzed by HRP. In another study, both in situ hydrogelization and RGD conjugation of a tyramine conjugate with a PPO-PEO block copolymer could be achieved simultaneously (Park, et al. Soft Matter, 2011, 7: 986-992). The effect of conjugated RGDs on cell adhesion and proliferation was superior and can be adjusted in a concentration dependent manner. According to previously published literature, some protein-based materials (such as gelatin, peptides gave rise to hydrogels or conjugates using et al African

Journal of

Biotechnology, 2009, 8: 5508-5515; Minamihata, et al Bioconjugate chemistry, 2011, 22: 74-81; Wang, et al

Biomaterials, 2010, 31: 8608-8616). To date, the production of peroxidase-mediated silk fibroin hydrogels and a peroxide (e.g. hydrogen peroxide) has not been described as the oxidizing agent. Only casein has been reported by Li et al. as having been cross-linked via peroxidase / hydrogen peroxide, and the resulting cross-linked casein did not yield a hydrogel (Li, et al. African Journal of Biotechnology, 2009, 8: 5508-5515). Casein is mainly derived from milk, and its structure and properties are very different from silk fibroin, which is derived mainly from 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, etc. (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). Chitosan and gelatin conjugates were developed using tyrosinase (Chen, et al Biometerials, 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 gelatin / chitosan hydrogel showed poor stability (Moreira Teixeira et al. Biomaterials,

2012, 33: 1281-1290). Similar studies suggest that conjugates prepared by tyrosinase can 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 silk fibroin and chitosan conjugates have also been reported (Kang et al Macromolecular Research, 2004,12: 5347

539; Anghileri, et al. Journal of Biotechnology, 2007, 127: 508-519; Freddi, et al. Biotechnology Journal, 2006, 125: 281-294). In these studies, pure silk fibroin hydrogels or tyrosinase crosslinked pure silk fibroin materials were not reported. Only silk fibroin / chitosan or sericin / chitosan conjugates were considered. Further, the mechanism of formation of tyrosinase-mediated silk / chitosan and sericin / chitosan fibroin conjugates is different from that of a peroxidase-mediated tyrosine-containing polymer conjugate. Silk fibroin (or sericin) conjugates to chitosan by reacting silk-activated quinones with amino groups in chitosan. In turn, tyrosine-containing polymers form conjugates through reactions between tyrosine groups (Moreira Teixeira et al. Biomaterials, 2012, 33: 1281-1290).

There are studies in the literature on tyrosyase-mediated silk / chitosan fibroin hydrogels or conjugates.

However, no records were found referring to peroxidase crosslinked silk fibroin hydrogels. As mentioned above, the mechanisms of action of tyrosinase and peroxidase are different.

It is proposed here to use an injectable silk hydrogel based system for application in tissue engineering and regenerative medicine and controlled drug release, using peroxidase as a strategy for crosslinking. Silk fibroin (FS), which is a natural protein composed mainly of glycine, alanine, serine and tyrosine, has been increasingly studied for new biomedical applications due to its biocompatibility, slow degradability and remarkable mechanical properties. The ability to control 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-SerGly-Ala-Gly. FS also contains tyrosine in the semicrystalline or amorphous region. The percentage of this aromatic amino acid is about 5 mol% FS. Thus, FS presents the most suitable chemistry to promote peroxidase mediated crosslinking. This is an advantage over the above described polymeric systems which must be conjugated to phenol or aniline derivatives prior to the HRP catalyzed enzyme reaction. Although FS cross-linking reactions have been widely reported (such as the use to prepare silk / chitosan tyrosinase conjugates), to date no studies have been reported on the use of peroxidase in FS cross-linking to produce a stable hydrogel, in particular. that gelation can occur under physiological conditions.

Several patents have been granted 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 a method for obtaining silk fibroin gel with direct current application ie by electrogelification. Silk fibroin gel can be reversibly converted to a liquid by applying reverse current. The invention also extends to a method for obtaining adhesive silk gels by reducing the pH of the silk solution.

US Patent 2011/0129531 of June 2, 2011 refers to the use of silk fibroin hydrogel and its compositions as a dermal filler. The compositions comprise from about 1 to 10% (w / v) silk fibroin and a resilin-like polypeptide. Silk precipitate is obtained using a 23RGD / ethanol accelerator or dehydration medium. To obtain the gels a gelation time is required 24 hours.

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

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

WO 2011/022771 of March 3, 2011 refers to methods of producing silk extract (comprises about

0.5% to about 15% silk protein) for application in the production of personal care products, plastics, textiles and biomedical products. Silk proteins are obtained from cell-producing cells and are solubilized by contact with a surfactant (eg sodium dodecyl sulfate (SDS), ammonium lauryl sulfate) or an ionic liquid (eg anions, such as chloride and bromide; cation such as 3-4chloropyridinium and dimethylaminopyridinium) and concentrating the proteins to produce silk extract.

WO 2011/026101 of March 3, 2011 refers to electronic and optoelectronic devices made with silk fibroin or blends 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 (insoluble state formation). The invention relates to the repair and replacement of bone and teeth.

WO Patent2011 / 013145 of February 3, 2011, describes

a process to improve the time in gelation in fibroin regenerated silk using per example TiO2 or FeO2SiN3 as the agents gelling agents. 0 composite in fibroin Silk / Silica is proposed as biomaterial in

support for tissue engineering applications.

WO 2011/011347 of January 27, 2011 comprises compositions and methods of binding 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 2011/0008436 of 13 January 2011 relates to methods of purifying silk fibroin, fibroin conjugates with bioactive molecules, and production of silk fibroin hydrogels and formulations with up to 6% by weight fibroin concentration . 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 refers to a vortex treatment of a silk fibroin solution that allows obtaining gels. The method allows control of the rate of formation of β-shaped coverslips and the speed of gelling. Bioactive agents and cells may be encapsulated in hydrogels during vortex treatment.

WO 2011/006133 of January 13, 2011 describes a silk based delivery system that releases nucleic acids from silk complexes. Silk complexes are intended to be applied for transfection as they exhibit high release efficiency, cell selectivity and controlled release of nucleic acids.

WO 2010/141133 of December 9, 2010 relates to methods of preparing silk fibroin / antibiotic (s) 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 purification and functionalization of silk fibroin. The invention describes methods for obtaining hydrogels by combining with 23RGD / ethanol to find tissue engineering applications, coatings to improve the operation of a medical device, and controlled release of drugs.

EP Patent 2010/2211876, August 4, 2010; US Patent 2010/0178304 of July 15, 2010; MX Patents 2009/012978, April 23, 2010; KR Patents 2010/1020100029217 of March 16, 2010; Patents and WO 2008/150861 of December 11, 2008 provide a process for producing silk fibroin gels by ultrasound. The gelling time can be controlled to occur within two hours after ultrasound treatment and utilizes additional salt solution treatment. Bioactive agents and cells may be encapsulated in hydrogels during the gelling process.

EP Patent 2010/2210971 of July 28, 2010 relates to a silk fibroin woven structure comprising an outer concentric tubular sheath and an inner core. The silk fibroin used to weave the structure of the present invention is comprised of a silk thread intended for ligament regeneration applications, 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 (PEG) functionalized silk fibroin. PEG-linked silk is intended for applications such as non-stick and anti-thrombogenic materials. Because it has different surface properties within the matrix, the association with

tissue agents, Bioactives can improve on the side of the adherent matrix. growth internal in Patent 2009/7635755, of 22 in December of 2009; EP Patent 2006/1613796 January 11 in 2006, and Patent WO

2005/012606 of 10 February 2005 refers to the preparation of highly concentrated silk fibroin solutions. The methods described for obtaining the solutions do not use organic solvents, direct additives and harsh chemicals. Highly concentrated silk solutions (less than 30% by weight) are proposed for the development of fibers, membranes, foams, support structures and hydrogels. Processing methods for obtaining silk hydrogels are based on increasing temperature, pH, decreasing and increasing salt concentration or a mixture with other polymers.

WO 2009/133532 of November 5, 2009 relates to a two-step method for obtaining a regenerated silk fibroin solution, that is, the use of an ionic reagent and degumming of silk or woven silk cocoons. . The invention also extends to silk fibroin solution (s), and an implant fibroin material that can be used for cartilage repair.

WO 2009/018610 of February 12,

2009 refers to silk proteins that can be used to produce 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 discloses a method of producing an electroactive material including a polymer, a substrate, pouring the polymer over the substrate, and enzymatically polymerizing an organic compound to generate a conductive polymer between the supplied polymer. and the substrate. The polymer may be silk which have tyrosines to provide a molecular level interface between the biopolymer supplied and a substrate or other conductive layer by enzyme tyrosine polymerization. The result is a carbon carbon structure that provides conjugated polymeric wires 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 models. The microspheres are intended to find applications such as controlled release of drugs.

WO 2008/106485 of September 4, 2008 refers to a layer of lamellar tissue comprising a grooved silk fibroin substrate containing tissue specific cells. Silk fibroin lamella substrates are designed to meet tissue engineering applications.

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

WO 2007/141131 of December 13, 2007 relates to a microfluidic device and method for controlling phase separation of one or a mixture of two or more spider silk proteins which may then allow protein association ( s) silk for the production of spheres, nanofibrils, yarns, 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 along the lumen axis of the tubular body. The invention extends to a method of manufacturing the medical device comprising forming the tubular body and introducing silk elements into the lumen of the tubular body. The device is intended for nerve regeneration applications.

WO 2007/020449 of February 22, 2007 and WO 2006/030182 of March 23, 2006 describe a cartilage tissue repair device with a resorbable, biocompatible three-dimensional silk or other fiber, and a substantially porous biocompatible silk. , resorbable or a hydrogel partially or substantially filling the fiber interstices, with or without a firm anchoring means of the device to the patient's bone.

The 3D silk support structure is obtained by lyophilization and the hydrogel by gelation in the presence of a PBS (buffered phosphate saline) - 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 refers to different silk-based drug delivery systems. The invention extends to methods for producing such drug systems and 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 grafting, wherein the silk is intended to promote in vivo adhesion of the stent to vessel walls, or otherwise induces or accelerates an in vivo fibrotic reaction causing the stent to adhere. to the toilet wall.

WO 2004/041845 of May 21, 2004 describes a method of preparing a fibrous protein hydrogel by solvent method. The method consists of pouring an aqueous solution of fibrous protein into a container which

understand a solvent that is not miscible with water; O container aging at temperature is prohibited environment, and collecting the fibrous protein hydrogel resulting and letting it dry. The invention extends to a

smmal hydrogel obtained by the solvent method.

WO 2003/022909 of March 20, 2003 and EP 2004/1436346 of July 14, 2004 describe a process for the production of silk fibroin hydrogels by treating a solution of fibroin with soluble polymers. water and / or with acid solutions of pH less than 4 and / or polar solvents and / or with cross-linking compounds, followed by washing of the resultant.

The invention described herein for developing silk fibroin cross-linked hydrogels and their peroxidase-mediated cross-linked compositions are completely different from the above-mentioned patents.

For example, US Patent 2011/0171239 of July 14, 2011 and WO 2010/036992 April 2010 describe methods for the preparation of silk hydrogels utilizing direct application of electric current. In this patent (WO Patent silk fiber was degummed for 20 minutes, purified silk fibroin dissolved in lithium bromide at 60 ° C

C for hours, (3) silk fibroin was dialyzed into cassettes (molecular weight (MWCO) 3500), (4) silk fibroin solution contained no saline buffer, (5) silk fibroin hydrogel was formed using electric field, (6) the formed silk fibroin hydrogel has silk-I 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. Longer 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, that is, the higher temperature may accelerate the dissolution of silk fibroin, and the shorter heating time may decrease silk fibroin degradation, (3) the silk fibroin was dialyzed to in a benzoyl dialysis tube (MWCO 2000), lower molecular weight (MWCO). A dialysis tubing can hold more protein inside the tube and reduce possible contamination, (4) a saline ion buffer was introduced into the silk fibroin during dialysis, this allows the pH value of the solution to be adjusted. silk fibroin close to 7.4, which allows the incorporation of cells and / or bioactive agents, (5) the silk fibroin hydrogel is formed by chemical cross-linking by peroxidase (enzymatic reaction), (6) the hydrogel of silk fibroin formed mediated by conformation peroxidase is amorphous (random spiral), (7) the formed hydrogels are stable and can be processed into different shapes and modulated their biodegradability.

In another patent WO Patent 2003/022909 of March 20, 2003 and patent EP 2004/1436346 of July 14, 2004 describe methods for preparing a silk hydrogel by treating a solution of fibroin with water-soluble polymers. and / or with acid solutions of pH less than 4 and / or polar solvents and / or with the crosslinked compounds. In this patent (Patent 2003/022909), it was stated that (1) silk fibroin was dissolved in solvents of 20-60 ° C, (2) the concentration of dialysed silk fibroin solution was between 1-20% by weight. Weight; (3) the polyethylene glycol solution is 50% molecular weight 1,000 and (4) the hydrogel was prepared by dialysis against acid solution silk fibroin solution (pH less than 4), or water soluble polymers, or solvents. (5) the pH of the silk fibroin solution was not controlled prior to the formation of the hydrogel, (6) when a cross-linking agent is used to form hydrogels, the crosslinking of the hydrogels occurs. hydroxyl and amine groups, (7) silk fibroin hydrogels prepared by different methods were all formed inside the dialysis tube, (8) the hydrogels should be washed before use, (9) the FTIR spectrum revealed that The hydrogels prepared by all methods mentioned in this patent were beta conformable, as indicated by the strong absorbance around 1620 cm ^-1 (Figure 6). There are major 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, higher temperature may accelerate the dissolution of silk fibroin, and the shorter heating time may decrease the degradation of silk fibroin, (2) the concentration of dialysed silk fibroin solution was between 2-30% by weight, the high concentration of silk fibroin solution. silk fibroin allows the formation of rigid silk hydrogels, (3) the polyethylene glycol solution for silk fibroin solution concentration was 20% and molecular weight was 20,000, the lowest PEG concentration made the solution more stable, and higher molecular weight PEG did not penetrate the dialysis membrane to contaminate the silk fibroin solution, (4) hydrogels were prepared by direct mixing of peroxidase / hydrogen peroxide and a silk fibroin solution, and this step allowed the incorporation of cells and / or bioactive agents, (5) the pH value of the silk fibroin solution was under control before gelation, between 7.07.4 (physiological pH) , (6) peroxidase-mediated crosslinking mechanisms acted on tyrosine groups in silk fibroin, (7) hydrogel may be formed at the site of application, such as tissue filling, (8) hydrogels may 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 major peaks at 1620 cm -1 (Figure 3b).

The present invention provides methods for the production of peroxidase mediated silk fibroin cross-linked hydrogels. Gelling occurs independently of the presence of culture media and cells or body fluids and is therefore clinically relevant as it allows homogenization of cells prior to hydrogel formation and then in situ gelation. 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 applications, bioadhesives, and drug delivery systems.

The present invention also has shape memory properties of peroxidase mediated silk fibroin cross-linked hydrogels, which are stimulated by ionic strength. For example, after preparation, silk fibroin hydrogels may retain their shape (initial form) in phosphate buffered saline (PBS). The hydrogel will swell as the ionic strength of the solution decreases. The maximum swelling ratio in ultrapure water is reached. Thereafter, the shape of the hydrogels will be recovered to some extent from their initial shape by immersing the hydrogels in solutions of different ionic strength. This enhances its application as a bioadhesive biomaterial, filler, coating to improve the operation of a medical device, or other pharmaceutical or medical applications. The silk fibroin hydrogel prepared by peroxidase mediated crosslinking 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 dipping in organic solvents. The layered hydrogel has beta conformation in the outer layer and dominant amorphous conformation in the inner layer. Other shaping combinations may also be formed by other treatments which enable their application as a structured system (carrier layers) for application in electronic and optoelectronic devices.

Brief description of the figures:

For a better understanding of the present invention, the figures are attached, but are not limited to the present since:

Figure 1 is a schematic illustration of HRP enzyme-mediated silk fibroin cross-linking. (1) HRP / H2O2, (2) PBS, (A) Silk Fibroin, (B) Silk Fibroin Hydrogel.

Figure 2 depicts the formation of silk fibroin hydrogels from HRP-mediated cross-linked hydrogels developed and the transparency properties. Silk fiber 16% wt. Solution, 0.84 mg / ml HRP solution in PBS, and 0.18 wt.% H2O2 in PBS were used (HRP / wt. Silk 1.10 wtú) (a): pure silk fibroin solution without PBS, (b): silk fibroin solution in PBS, (a) and (b): hydrogen peroxide was used as an oxidizing agent, ( c): calcium peroxide was used as oxidizing agent.

Figure 3 represents the results obtained from Fourier Transform Infrared Spectroscopy (FTIR) analysis of silk fibroin solution, with or without PBS, and silk / HRP / H2O2 mixtures, before and after gelation. Hydrated samples were tested by attenuated total reflectance (ATR). (a) A silk fibroin solution, with or without PBS: 1. Silk fibroin solution without PBS, 2. Silk fibroin solution with PBS.

(b) PBS silk fibroin solution was used to obtain the 3 spectra: 1. Silk / HRP / H2O2: After gelation, 2. Silk / HRP / H2O2: Before gelation, 3. Silk / HRP / H2O2: After gelation.

Figure 4 represents the absorbance of silk fibroin solution obtained at 550 nm, and silk / HRP / H2O2 mixture, before and after gelation. Silk fibroin solution 16 wt%, 0.84 mg / ml HRP solution in PBS, and 0.18 wt% H2O2 in

PBS were used (HRP / 0.26 wtb silk, H ₂ O ₂ / 1.10 wtb silk). For each group, 50 µl of solution was placed in a 96-well TCPS plate and measured in a 550 nm microplate reader. Absorbance was determined before and after gelation using the same sample in the well. (A) Before gelation, (B) After gelation, 1. Silk solution, 2. Silk / HRP / Η ₂ Ο ₂ , 3. White.

Figure 5 depicts the microstructure of the freeze-dried HRP crosslinked silk fibroin hydrogel. 16 wt% silk fibroin solution, 0.84 mg / ml HRP solution in PBS, and 0.18 wt% H ₂ O ₂ in PBS were used (HRP / 0.26wth silk, H2O2 / silk 1, 10 wth).

Figure 6 represents the influences of HRP and H2O2 on the gelation time of HRP crosslinked silk fibroin hydrogel determined at 37 ° C. Silk-10, Silk-12, Silk-16 correspond to silk hydrogels prepared from 10 wt%, 12 wt%, and 16 wt% silk fibroin solutions, respectively. (a) H ₂ O ₂ / silk: 1.10 wth, (b) HRP / silk: 0.26 wth. Gelling time was determined using a vial tilt method. Each condition was tested in triplicate (A) Silk-10, (B) Silk-12, (C) Silk-16.

Figure 7 represents the influences of HRP and H ₂ O ₂ on the storage modulus of the cross-linked mediated silk fibroin peroxidase hydrogel, measured by a one-rotation rheometer (strain: 0.1%, frequency: 0.5 Hz) model, at 37 ° C. Silk-10, Silk-12, Silk-16 correspond to silk hydrogels prepared from 10 wt%, 12 wt%, and 16 wt% silk fibroin solutions, respectively. (a) H ₂ O ₂ / silk: 1.10 wt%, (b) HRP / silk: 0.26 wth. Values were determined after the modulus reached a linear value and became stable for 1 minute. Each condition was tested in triplicate. (A) Silk-10, (B) Silk-12, (C) Silk-16.

Figure 8 represents the influence of HRP on the mechanical properties of HRP cross-linked silk fibroin hydrogels, namely on the modulus. 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 (wt.h): 0.13, 0.26, 0.39, 0.52. H ₂ O ₂ / silk was set at 1.1 wtbo for all formulations, (a) Silk-10, (b) Silk-12 and (c) Silk-16: The analysis frequency was set to 1 Hz. (D ) Silk-10, (e) Silk-12, and (f) Silk-16: The frequency of analysis was set at a voltage of 0.1%. Each analysis was measured after the modulus of the silk fibroin hydrogel reached a plateau for 5 minutes.

Figure 9 represents the influence of H2O2 on the mechanical properties of HRP crosslinked silk fibroin hydrogels, namely on the modulus. 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. H ₂ O ₂ / Silk (wt.h): 0.80, 0.95, 1.10, 1.25, 1.45. HRP / silk was set at 0.26 wtbo for all formulations. (a) Silk-10, (b) Silk-12 and (c) Silk-16: The analysis frequency has been set to 1 Hz.

(d) Silk-10, (e) Silk-12, and (f) Silk-16: The frequency of analysis was set at 0.1% voltage. Each analysis was measured after the modulus of the silk fibroin hydrogel reached a plateau for 5 minutes.

Figure 10 represents the percentage swelling of cross-linked silk fibroin hydrogel by HRP in distilled water (a) and PBS (b). (A) HRP / Silk: 0.26 wt.u; H ₂ O ₂ / Silk: 1.10 wt.u (B) HRP / Silk: 0.52 wt.u; H ₂ O ₂ / Silk: 1.10 wt.h (C) HRP / Silk: 0.26 wt.h; H ₂ O ₂ / Silk: 1.45 wt.h.

Figure 11 shows the enzymatic degradation profiles of HRP cross-linked silk fibroin hydrogel at 37 ° C. Silk-10, Silk-12, Silk-16. 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 silk fibroin hydrogel was immersed in 5 ml PBS containing 0.025 U Protease XIV obtained from Streptomyces griseus (0.005 U / ml). (a) HRP / Silk: 0.26 wt.u; H2O2 / Silk: 1.10 wt.u (b) HRP / Silk: 0.52 wt.u; H ₂ O ₂ / Silk: 1.10 wt.u (c) HRP / Silk: 0.26 wt.u; H ₂ O ₂ / Silk: 1.45 wt.u.

Figure 12 depicts the shape memory properties of HRP crosslinked silk fibroin hydrogel. The concentration of silk fibroin was 16 wt%, HRP / silk was 0.26 wtú, H ₂ O ₂ / silk was 1.1 wtú. Silk fibroin hydrogel was first prepared in a mold (a), and then the hydrogel was removed from mold (b). Silk fibroin hydrogel after soaking in distilled water for 12 hours (c). The diameter of the silk fibroin hydrodel was recovered after immersion in PBS during the 12 hour period (d).

Figure 13 represents the diameter variation of the crosslinked silk fibroin hydrogel alternately in distilled water and PBS.

HRP when immersed

Silk-10, Silk-12,

Silk-16.

Silk-10,

Silk-12, Silk-16 corresponds to silk hydrogels prepared from 10 wt%, wt%, and 16 wt% silk fibroin solutions

1, 1 wt to,

W t 00 · (A)

HRP / silk is from

0.26 wtt, H ₂ O ₂ / silk is (b) HRP / silk is 0.26 wtt,

H ₂ O ₂ / silk is of

1.45 hours in PBS.

Figure 14 depicts photographs of HRP cross-linked silk fibroin hydrogel before (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 silk fibroin concentration is 16 wt%, HRP / silk is 0.26 wth, H ₂ O ₂ / silk is 1.1 wth. The multilayers of the silk fibroin hydrogel prepared by immersion of the hydrogel in methanol can be observed.

Figure 16 represents the thickness of the outer layer of silk fibroin hydrogel prepared by immersing the hydrogel in methanol over different time periods. The silk fibroin concentration is 16 wt%. (A) HRP / silk and 0.26 wtt, H ₂ O ₂ / silk and 1.45 wtt, (B) HRP / silk 6 is 0.2 wtt, H ₂ O ₂ / silk is 1, 1 wtt.

* Indicates significant difference between the two formulations of silk fibroin hydrogel immersed in methanol over the same time period.

Figure 17 depicts the ATR spectra of the HRP-crosslinked silk fibroin hydrogel multicolayers immersed in methanol for 5 minutes. The silk fibroin concentration is 16 wt%, HRP / silk is 0.26 wtk, H ₂ O ₂ / silk is 1.1 wtk. (1) Outer layer, (2) Silk solution, (3) Inner layer.

Figure 18 depicts the enzymatic degradation profile of the outer skin layer of HRP-crosslinked silk fibroin hydrogel after immersion in methanol for different time periods. (1) 3 min, (2) 5 min, (3) 10 min. The test was performed at 37 ° C. The silk fibroin concentration is 16 wt%, HRP / silk is 0.26 wtú, H ₂ O ₂ / silk is 1.1 wtk. In each test, about 100 mg of the outer layer of silk fibroin hydrogel was immersed in 5 ml PBS containing 1 U Protease XIV obtained from Streptomyces griseus (0.2 U / ml).

Figure 19 shows the enzymatic degradation profile of the internal portion of the HRP crosslinked silk fibroin hydrogel after immersion in methanol for 5 minutes. The test was performed at 37 ° C. The silk fibroin concentration is 16 wt%, HRP / silk is 0.26 wtk, H ₂ O ₂ / silk is 1.1 wtk. In each test, about 300 mg of hydrogel inner layer was immersed in 5 ml 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 silk fibroin concentration is 16 wt%, HRP / silk is

0.26 wtú, (A) H2O2 / silk is 1.1 wtú or (B) 1.45 wtú. THE density The cell phone used is 1 million cells / ml of hydrogel. The cells were encapsulated in the AIDS gel. containing HRP and H2O2, and then 50 ml mixture was

placed on a TCPS coverslip until gel formation under sterile conditions. Silk fibroin cells / hydrogel were cultured in alpha-MEM medium and cell viability was tested using the MTS assay after different time periods.

Figure 21 shows 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 wtú, Ch / silk is 1.1 wtú. ATDC-5 cells were cultured on silk fibroin hydrogel for 1 day.

Figure 22 depicts fluorescence microscopy images of hydrogel encapsulated ATDC-5 cells

of fibroin gives silk after 11 days in culture. (THE) 1 day, (B) 3 days, (W) 7th days, and (D) 11 days THE concentration of fibroin gives silk is of 16 wt%. d ) HRP / silk is : in 0.26 W160 f H ₂ O ₂ / silk is in 1.1 W160 f 5X; (2) HRP / silk is in 0.26 wt 60 f H ₂ O ₂ / silk is in 1.1 w t 60 f 2 0x; (3) HRP / silk is in 0.26 w t 60 f H2O2 / silk is in 1.45 w t 60 f 5X; (4) HRP / silk is in 0.26 w t 60 f H2O2 / silk is in 1.4 5wtú. Test from calcein AM (coloring

green shows living cells) and propidium iodide (red staining shows dead cells). HRP / silk is 0.26 wtú, H2O2 / silk is 1.1 wtú.

Detailed Description of the Invention

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

All patents and other publications expressly incorporated herein as identified are reference for the purpose of describing and disclosing.

Method (s) publications and reports may be related to the present invention. These works are published prior to the date of submission of the present invention. Nothing in this context should be construed as an admission that inventors are not entitled to antedate such disclosure by virtue of an earlier invention or for any other reason.

Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

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 that can be used as a minimally invasive tissue regeneration system. This system may be used in conjunction 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 may be used for controlled release of bioactive molecules. Enzymatically crosslinked silk fibroin hydrogels allow the incorporation of genetic material, bioactive agents, detection and segmentation agents, conductive molecules and inorganic materials, providing versatile and layered systems that can be used as filler as well as coatings to improve 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 number of glycine-alanine-glycine-alanine-glycine-serine repeat sequences (GAGAGS), allowing the formation of an anti-parallel β structure that gives silk fibroin superior strength. . 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 mol% tyrosine groups which can be oxidized by peroxidase / hydrogen peroxide and subsequently crosslinked to form a three dimensional network. Silk fibroin hydrogels developed in the present invention are formed due to the tyrosine cross-linked groups in silk fibroin.

The silk fibroin solution was prepared as described above, but with major modifications (Sofia et al., J. Biomed Mater Res A 2001, 54: 139-148. WO 2010/036992, and WO 2003/022909 2003 / 022.909 ), in order to control the final properties of the silk solution, such as pH and ion content (s). High concentration (up to 30 wt.%) Silk fibroin aqueous solutions containing PBS were prepared. PBS allowed the pH value of the silk fibroin solution to be adjusted. The concentrated silk fibroin solution was diluted at different concentrations. Hydrogels were prepared by adding different amounts of peroxidase (e.g. horseradish peroxidase) and peroxide (such as hydrogen peroxide) to silk fibroin solutions (Figure 1). The crosslinking process can be carried out over a wide range of temperatures. Developed hydrogels may be degraded by proteolysis in vivo or in vitro. The gelation time and mechanical performance of silk fibroin formed hydrogels were investigated. The biological performance of hydrogels was evaluated in vitro.

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

The properties of prepared peroxidase crosslinked silk fibroin hydrogels may be modified by post-treatments. The shape of the prepared hydrogels can be adjusted by immersion in solutions with different ionic strength. The size of the hydrogels will increase after immersion in low ionic strength solutions. And their size can be restored to some extent to their 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 dipping the fresh preparation or expanded hydrogel by dipping in organic solvents, mixtures of organic solvents, mixtures of water and organic solvents, or by electric field treatment or other treatments, or any combination of these.

Any conformation combination can also be developed using peroxidase crosslinked silk fibroin hydrogels with other treatments. The shape memory property and / or layer structure of peroxidase crosslinked silk fibroin hydrogels opens unlimited possibilities for their application in tissue engineering and regenerative medicine, controlled release of bioactive agents, and as a filler as well as as coatings to enhance the function of medical devices or other pharmaceutical or medical applications.

These freshly prepared or post-treatment modified peroxidase crosslinked silk fibroin hydrogels, used alone or in combination with inorganic detection molecules and conductive materials, may also have potential applications in electronics or optoelectronics. With regard to their application in these fields, these materials may be applied by manual methods or using an automatic dispensing device, being crosslinked during or after deposition.

The description of the present invention is complemented by examples which are intended to provide a better understanding thereof, although these examples should not be treated with a restrictive character.

Example 1: Preparation of Silk Fibroin Concentrated Solution

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 pH value and ion content. Bombyx Mori silk fibroin was extracted by boiling the cocoons of a sodium carbonate solution (0.02 M, Sigma, USA) for one hour and then washed thoroughly with distilled water. Purified silk fibroin was dissolved in 9.3 M lithium bromide solution (Sigma, USA) at 70 ° C for 1 hour. The solution was then dialyzed with distilled water using a benzoyl dialysis tube (molecular weight cut: 2000, Sigma, USA) for a period of 2 days. Then, the aqueous silk fibroin solution was dialyzed against a dilute phosphate buffered saline (PBS, Sigma, USA), a solution (0.2 wt% In distilled water) for 24 hours. The dialysed silk fibroin solution was then concentrated by dialysis against a 20 wt% polyethylene glycol solution (20,000 g / mol, Sigma, USA) for 9 hours. Finally, the dialysis tube was carefully rinsed 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 PBS concentrations during the above dialysis process. The final concentration of concentrated silk fibroin was determined by measuring the dry weight of silk fibroin solutions. The prepared silk fibroin solution was stored at 4 ° 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 weight%). And the pH of the silk fibroin solution without PBS was in the range of 5.6-6.2. Following incorporation of PBS into the silk fibroin solution, the pH of the solution may be controlled in the range 7.0-7.4, which is suitable for incorporation of bioactive cells or factors.

Example 2: Hydrogel Formation-Method 1

In order to produce peroxity crosslinked silk fibroin hydrogels, silk fibroin solutions of different concentrations (2-30 wt.%) Were prepared by dilution with a PBS solution. Horseradish peroxidase (HRP, 260 units / mg, Sigma, USA) and hydrogen peroxide (H2O2,

Sigma, USA), solutions were prepared by dissolving in PBS. HRP and H2O2 solutions were then added to silk fibroin solutions at different concentrations, ranging from 1 to 1000 µg / ml and 0.5 to 50 mmol / ml, respectively. The mixture was gently stirred and then gelation was performed in a water bath or oven at different temperatures, ranging from 5 to 50 ° C. The hydrogel can also be processed into different shapes using different molds and placing them in an oven at the required temperature. A silk fibroin hydrogel disc can be prepared from a 7.5 mm diameter mold (Figure 2b) using 16% by weight silk fibroin solution with PBS (the PBS content in the silk solution 1.7%, and pH 7.2), along with 0.84 mg / ml HRP in PBS and 0.18 wt. % H2O2 in PBS (HRP / 0.26 ½ silk H2O2 / Silk 1.10%). Not only could PBS silk fibroin solution be used for the preparation of peroxidase cross-linked hydrogels, but the same solution without PBS was also able to form hydrogel under the same method. For example, silk fibroin hydrogel may be prepared in a flask using 16 wt% pure silk fibroin solution, together with 0.84 mg HRP solution / ml in PBS and 0.18 wt% H2O2 in PBS. (HRP / 0.26 wtk silk, H ₂ O ₂ / 1.10 wtk silk) in a bath at 37 ° C (Figure 2a). In the following studies, Silk-10, Silk-12, Silk-16 corresponded to silk hydrogels prepared with 10 wt%, 12 wt%, and 16 wt% silk fibroin solution, respectively. In addition, if not mentioned in the following examples, H ₂ O ₂ and HRP concentrations were 0.84 mg / ml and 0.18 wt%, and the silk fibroin solution was PBS (PBS solution content Silk content was 1.7%, and the pH was 7.2.

Example 2: Hydrogel Formation Method - Method 2

In order to produce peroxidase crosslinked silk fibroin hydrogels, silk fibroin solutions of different concentrations (2 to 20 wt%) were prepared by diluting the concentrated silk fibroin solution with PBS. Horseradish peroxidase solution (HRP, 260 units / 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 value of the solution increased to 5.0. HRP and calcium peroxide solutions were added to silk fibroin solutions of different concentrations, ranging from 1 to 1000 µg / ml and 0.5 to 50 mmol / ml, respectively. The mixture was gently stirred and then gelation was performed at different temperatures, ranging from 5 ° C to 50 ° C. The hydrogel can be processed into different shapes using various molds and placing them in a greenhouse at the required temperature. Here, calcium peroxide was used as a source of peroxide, assuming that 1 mol of calcium peroxide will yield one mol of hydrogen peroxide in the present study. According to the calculations, the concentration of 0.51 wt% calcium peroxide solution is equal to 0.18% H ₂ O ₂ 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 HRP solution in PBS and 0.51 wt. CaO2% in distilled water (HRP / Silk by weight 0.26h, H ₂ O ₂ / Silk 1.10 wth) at 37 ° C (Figure 2c).

Example 3: Characterization of peroxidase cross-linked silk fibroin hydrogels

Fourier Transform Spectroscopy Analysis (FTIR)

The structural conformation of the infrared solution by pure silk fibroin, PBS silk fibroin solution, and silk / HRP / H ₂ O2 mixture before and after gelation was analyzed by FTIR using attenuated total reflectance (ATR). on wet samples. As control, ultrapure water was used. For the liquid sample, 50 µl of sample was used for each assay. After the silk / HRP / H ₂ O ₂ mixture formed 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 4000-600 cm ^-1 with 40 readings (Figure 3). FTIR spectra showed that all samples showed the main peak at about 1650 cm ^-1 , which is the characteristic peak of 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 crosslinking showed amorphous conformation (random spiral), as did the conformation presented by aqueous silk fibroin solution (Figure 3a). PBS silk fibroin solution also retained amorphous conformation. Most silk fibroin hydrogels previously reported in the literature have beta conformation. Stable amorphous conformal silk fibroin hydrogels have rarely been reported. Here we present stable silk fibroin hydrogels having mostly amorphous (random) conformation.

The optical absorbance at 550 nm

Optical absorbance at 550 nm was primarily used to assess the conformation transition of silk fibroin solution or 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 µl of solution was used. The silk solution (with PBS) and silk / HRP / H ₂ O 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 sample group. After the silk / HRP / H ₂ O ₂ mixture formed 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 the absorbance of the silk / HRP / H2O2 mixture before and after gelation. There was also no significant difference in absorbance between silk solution and silk hydrogel.

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

Scanning electron microscopy (SEM)

The micromorphology of the peroxidase crosslinked freeze-dried silk fibroin hydrogel was observed by SEM. 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 silk hydrogel prepared with 16 wt% silk solution, HRP / silk ratio of 0.26bo and H ₂ O2 / silk ratio of Ι, ΙΟύ. The SEM image shows that the silk fibroin hydrogel has porous structure and interconnected pores with the pore size being from 50 to 100 µm.

The porous structure of the silk hydrogel may facilitate nutrient transport for further cell incorporation studies, or allow cell migration in hydrogels.

Gelation Time

Silk hydrogel gelation time was tested by a bottle tilt method. The absence of flow, after 1 minute after the inversion of the vial, is indicative that qelification occurred. Each condition was tested in triplicate. During the test, the flask was immersed in a 37 ° C water bath and the influence of silk concentration, HRP and H2O2 were investigated. Figure 6 shows the gelation time profiles of silk fibroin hydrogels. When studying the influence of HRP, the H2O2 / silk ratio was set at 1.10 wth. When studying the influence of H2O2, the HRP / silk ratio was set at 0.26 wth. Increasing silk fibroin concentration and HRP content has been found to shorten the gelation time, as with increasing H2O2 content the gelation time increases. For example, in the case of a 16 wt.% Silk solution, when the HRP content increased from 0.13 wth to 0.52 wth, the gelation time of the silk fibroin hydrogel decreased from ~ 35 minutes to less than 4%. minutes It has also been found that silk gelation will occur more rapidly at physiological temperatures compared to temperatures below 37 ° C. The data presented here are examples only, and the gelation time interval is not limited to the data presented here.

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 herein was much shorter than previously reported for silk hydrogels prepared by other methods such as agitation, vortexing and ultrasonication. It is to be noted that in the method described herein gelation may occur under physiological conditions, which allows in situ incorporation of cells or bioactive agents.

Mechanical properties

The stored energy modulus of peroxidase crosslinked silk fibroin hydrogels was evaluated by rheometry in a rotary model at 37 ° C. The silk fibroin solution was mixed with HPR and H2O2, and then 1 ml of the mixture was immediately transferred to the chamber to perform the test. A cylinder with a tapered tip was immersed in the mixing solution, and a drop of oil was placed on top of the silk solution to prevent evaporation at room temperature.

during 0 test. For the measurement of the module power stored, were 0.5 Hz of frequency and 0.1% in tension. The points data were collected every 30 seconds r < until 0 module reached a landing during 5th

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 threshold for 5 minutes, a frequency scan was performed of

0.1 to

Hz frequency with 0.1% constant strain for minutes. When the frequency scan ended, a strain scan was also performed from 0.1% to

100% deformation with a constant frequency of 1 Hz for 10 minutes.

Figure 7 shows the influence of silk concentration, HRP and H2O2 content on the stored energy modulus of silk fibroin hydrogels. Modulus was found to increase with increasing silk concentration and H2O2 content. HRP content had only a minor influence on the module. When the H ₂ O ₂ / silk content was 1.45% by weight, the silk-16 hydrogel reached a stored energy modulus of around 5 kPa. By controlling the H ₂ O _{2 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 modulus profiles of peroxidase crosslinked silk fibroin hydrogel, measured by a rheometer in a 37 ° C rotation model. Silk-10, Silk-12, Silk-16 correspond to silk hydrogels prepared with 10 wt%, 12 wt%, and 16 wt% silk fibroin solution, respectively. The H2 O2 / silk ratio was set at 1.1% by weight in this figure. The result showed that the silk fibroin hydrogel prepared here can maintain the stored energy modulus under 40% pressure (Figure 8a, b, c), and less than 5 Hz frequency (Figure 8d, e, f).

Figure 9 represents the influence of H2O2 on the stored energy modulus profiles of the peroxidase crosslinked silk fibroin hydrogel, measured by a rheometer in a 37 ° C rotation model. Silk-10, Silk-12, Silk-16 corresponded to silk hydrogels prepared with 10 wt%, 12 wt%, and 16 wt% silk fibroin solution, respectively. The HRP / silk ratio was set at 0.26 wtk for all formulations. The result showed that silk fibroin hydrogels prepared herein can maintain their stored energy modules under 40% pressure (Figure 9a, b, c), and less than 5 Hz frequency (Figure 9d, e, f).

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

Degree of expansion

The degree of expansion of these hydrogels in distilled water and PBS was characterized.

A silk / HRP / tbCb mixture (volume of in a 7.5 mm diameter mold) was used. After preparation of the peroxidase cross-linked hydrogels, they were immediately placed in distilled water or

PBS in a bath at 37 ° C for 12 hours allowing them to achieve equilibrium. Then the hydrogels were removed from the liquid, the liquid was absorbed on the surface of a filter paper. The wet weight was recorded on a scale (Mw). Then the hydrogels were 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 the peroxidase crosslinked silk fibroin hydrogel in distilled water (a) and PBS (b). These results showed that the expansion rate increased with decreasing silk concentration and increasing H2O2 content. The degree of expansion was higher in distilled water compared to PBS. The observed differences in expansion rate induced by silk fibroin concentration and H2O2 content are related to mechanical properties. Higher concentrations of silk solution and H2O2 content induced an increase in modulus. This way these hydrogels are more stable and do not absorb as much liquid. Ion concentration also affects the rate of expansion. When the silk fibroin hydrogel has been immersed in distilled water (which has very low ionic strength), the silk molecules will distend freely. This explains the differences in silk hydrogel expansion rates observed when immersed in PBS and distilled water.

Enzymatic degradation enzymatic degradation profile of silk fibroin hydrogels was evaluated. Different formulations of Silk / HRP / H2O2 (V = 500 µl) were produced to prepare the various hydrogels using a 7.5 mm diameter mold. Different solutions of silk fibroin with concentration of 10 wt%, 12 wt%, and 16 wt% were used. In turn, HRP / silk was 0.26 wth, H ₂ O ₂ / silk was 1.1 wth or 1.45 wth. In each assay, 500 mg of silk fibroin hydrogel was immersed in 5 ml PBS containing 0.025 U Protease XIV obtained 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 their mass (Wt) determined after absorption of excess surface water through filter paper. And then the mass loss ratio was calculated as follows:

Mass Loss Ratio (%) = (Wi-Wt) / Wi * 100%

The results revealed that silk fibroin hydrogels show a degradation profile in the period from 6 to 8 hours (Figure 11). Hydrogels with higher mechanical proportions have a lower mass loss profile when compared to hydrogels with lower mechanical properties. The observed degradation profile is completely different from those previously reported. This study shows that HRP crosslinked silk fibroin hydrogels are amorphous, contrary to beta-sheet conformation or other crystalline state as reported in previous studies. The degradation profile of HRP action crosslinked silk fibroin hydrogels enables a number of new applications in regenerative medicine.

Shape Memory Property

The shape memory property of HRP crosslinked silk fibroin hydrogels was evaluated in vitro. The hydrogels were produced, consecutively immersed in distilled water and PBS, and the diameter determined at each 12 hour immersion period (Figure 12). The concentration of silk fibroin used is 16 wt%, HRP / silk is 0.26 wth, H ₂ O2 / silk is 1.1 wth. 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 soaking in distilled water for 12 hours. The diameter of the silk hydrogel was found to be recovered to some extent after soaking in PBS for a further 12 hours (Figure 12d). Silk fibroin hydrogels were produced from different concentrations of silk fibroin (eg, 10%, 12% and 16%), as reported in Figure 13. All hydrogels were found to maintain their diameter in PBS, in different periods of immersion. However, the diameter of the hydrogels in PBS was slightly larger than their original diameter (later 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 HRP crosslinked silk fibroin hydrogels respond to physicochemical stimuli such as reactive ionic strength and pressure.

This property had 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 and suitable aqueous platform for studying the effect of different ions and solvents on silk fibroin conformation. These properties open up new possibilities for the application of HRP-crosslinked silk fibroin hydrogels as a controlled drug release system (DDS) which responds to small variations in environmental conditions.

Silk Fibroin Hydrogel (Multilayer) that

Enable Spatial Control of Your Conformation

Different layers of HRP crosslinked silk fribroin hydrogels were developed by a methanol immersion process for different time periods. Figure 14 shows HRP crosslinked images of silk fribroin before (a) and after (b) immersion in methanol for 5

minutes The concentration of fibroin silk is of 16 wt%, HRP / silk is 0.26 wtú, and H ₂ O ₂ / silk is dee 1.1 W t OO · Checked that the hydrogel of silk became if opaque after

immersion in methanol for a few minutes. The methanol-immersed hydrogels have also been found to have a layered structure, with the outermost layer showing cross-sectional images of HRP-crosslinked silk fribroin hydrogel after immersion in methanol for 0-10 minutes. The concentration of silk fibroin is 16 wt%, HRP / silk is 0.26 wtú, and H ₂ O ₂ / silk is 1.1 wth. These results show that the thickness of the outer layer increases in thickness as the time of immersion in methanol increases.

Figure 16 shows the results of the effect of hydrogel immersion time in methanol on the outer (opaque) layer thickness. The concentration of silk fibroin is 16 wt%, HRP / silk is 0.26 wtú, and ^ Ch / silk is 1.1 wtú or 1.45 wtú. This study showed that hydrogels with higher mechanical properties (^ Ch / silk at 1.45 wt%) had a thinner outer layer compared to hydrogels with lower mechanical properties (^ Ch / silk at 1.10 wt%) . And the thickness of the layers can be easily controlled having a thickness of between 200 and 1000 µm. The outer (opaque) layer has a higher stiffness than the inner (transparent) layer.

The conformation of the different hydrogel layers was investigated using the attenuated total reflectance infrared (FTIR-ATR) technique in hydrated state. For this assay, the silk fibroin concentration is 16 wt%, HRP / silk is 0.26 wtú, and ^ Ch / silk is 1.1 wtú. After production, the hydrogel was immersed in methanol for 5 minutes and then washed with distilled water. The different hydrogel layers 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 to a main peak around 1650 cm ^-1 , confirming that the conformation of the transparent part is essentially amorphous. . In addition, the clear hydrogel layer has been found to have a small peak at 1620 cm ^-1 , which indicates that the hydrogel has some beta sheet conformation in the inner but not dominant layer. These results corroborate that the conformations of silk fibroin in the outer and inner layers are different, as it is possible to control the spatial conformation of silk hydrogels using the method presented here, ie immersion in methanol. Other organic solvents or aqueous saline solutions may also be used to produce multilayers with different conformations and properties.

The degradation profile of the different layers of silk fibroin hydrogels was determined in vitro. For this assay, the silk fibroin concentration is 16 wt%, HRP / silk is 0.26 wth, and H2O2 / silk is 1.1 wth. Silk fibroin hydrogels were immersed in methanol for 3-10 minutes. For each test, about 100 mg outer layer (opaque) of the silk fibroin hydrogel was immersed in 5 ml PBS containing 1 U Protease XIV obtained from Streptomyces griseus (0.2 U / ml). And about 300 mg of inner layer of silk fibroin hydrogel (sample obtained from hydrogel immersed in methanol for 5 minutes),

was immersed in 5 ml PBS containing 0.025 U Protease XIV obtained from Streptomyces griseus (0.005 U / ml). Mass initial Hydrated hydrogel was registered as Wi. At the

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:

Mass Loss Ratio (%) (Wi-Wt) / Wi * 100%

Figure 18 shows the enzymatic degradation profile of the outer layer of HRP-crosslinked silk fibroin hydrogel after immersion in methanol for different time periods. The results showed that the outer layer of silk fibroin hydrogel is more resistant to enzymatic degradation. The methanol-immersed layer for 3 minutes shows a 17% mass loss after 4 hours of immersion in the degradation solution. The 5-minute methanol-immersed silk fibroin layer has a 10% mass loss, and the 10-minute immersed layer has a 2% mass loss. These results indicated that the outer layer (exposed to methanol) of silk fibroin is in the crystalline state (beta sheet), and by increasing the immersion period 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 silk fibroin mass 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 degradation tests performed. with the outer layer of the silk fibroin hydrogel. These results corroborate the fact that the inner and outer layers present different conformations. The inner layer has an 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 silk fibroin hydrogels.

The development of different layered silk fibroin hydrogels having different conformations was first described herein. This unique property may be useful as a new platform for the controlled release of drug or bioactive molecules, filler materials, or multilayer materials to find applications in electronics or optoelectronics.

Example 4: In vitro cytotoxicity assessment of HRP-crosslinked silk fibroin hydrogels

The cytotoxicity of silk fibroin hydrogel was assessed by encapsulating and culturing 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-2yl) -5- (3-carboxymethoxyphenyl) -2 (4-sulfophenyl) -2H-tetrazolium). This is a standard method for the evaluation of cytotoxicity in vitro. Silk fibroin hydrogels were prepared from 16 wt% solutions, 0.26 wtú HRP / silk, and 1.10 wtú or H2 O2 / silk solutions. ATDC-5 cells were cultured in Minimum Essential Medium (α-ΜΕΜ) monolayer with 10% fetal bovine serum and 1% antibiotic-antimycotic mixture containing 10,000 units / ml sodium penicillin G salt, 10,000 pg / ml streptomycin sulfate and 25 pg / ml amphotericin B as Fungizone® in 0.85% antimycotic saline. ATDC-5 cells were incubated at 37 ° C in a humidified 5% CO 2 atmosphere, and the culture medium renewed every three days. The 16 wt% silk fibroin solution was sterilized by ultraviolet rays. Confluent cells were detached from the polystyrene culture flasks using a trypsin enzyme solution (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, and 1 ml of the silk fibroin solution mixture and the HRP solution containing H2O2 (HRP / 0.26 wth silk, H ₂ O2) were subsequently added. / silk at 1.10 wth or 1.45 wth). After homogenization, 50 µl silk fibroin solution containing ATDC-5 cells were transferred to a coverslip. Subsequently the coverslips were placed at the bottom of the wells of a polystyrene culture plate (24 wells). In order to promote crosslinking, the plate containing the gel and cell mixture was incubated at 37 ° C 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, hydrogels containing the encapsulated cells were removed from the medium. A phosphate buffered solution (PBS) was washed, and then 500 µl of MTS solution (CellTiter 96 Proliferation Assay Kit) was added. Thereafter, the hydrogel / ATDC-5 MTS solution was incubated at 37 ° C for three hours. Then 100 µl of the solution was transferred to the respective well of a polystyrene culture plate (96 wells). Absorbance (OD) at 490 nm was determined using a microplate reader. All tests were performed in triplicate. Cell viability results are shown in Figure 20. Cell viability in both groups of hydrogels was found to increase from day 1 to day 4, and maintain viability 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 cells.

Figure 21 shows the fluorescence microscopy images of ATDC-5 cells encapsulated in silk fibroin hydrogels grown in culture medium for a period of 3 hours. The silk fibroin concentration is 16 wt%, HRP / silk is 0.26 wth, H ₂ O2 / silk is 1.1 wth. After 1 day of culture a homogeneous distribution of cells is found to be viable within the hydrogel.

Cell viability, distribution and morphology were also assessed using the Live / Dead staining method using calcein AM (green staining of living cells) and propidium iodide (red cells representing dead cells). On days 1, 3, 7, and 11, silk hydrogels containing the encapsulated ATDC-5 cells were removed from the culture medium. They were washed with PBS. Then the hydrogels were immersed in 1 ml PBS containing 1 mg calcein AM and 1 µg propidium iodide. The hydrogels were incubated for 15 minutes at 37 ° C and then washed in PBS prior to observation under the fluorescence microscope. Figure 22 shows images of live / dead cells when cultured in the hydrogel. Silk fibroin concentration is 16 wt%, HRP / silk is 0.26 wth, H2O2 / silk is 1.1 wth. The results show that most cells are viable after encapsulation in the silk fibroin hydrogel up to 11 days of culture. It is also possible to observe the homogeneous distribution of cells within 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 HRP crosslinked silk fibroin hydrogel

features various benefits, such as O allow encapsulate cells in situ, present an gelation fast (within a few minutes) and in conditions

and support cell viability. The method for encapsulating cells is quick and easy to operate.

Anyone of suitable skill in the art is able to modify and introduce 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 that are within its inventive concept as defined by the appended claims.

Claims (15)

A process for preparing a crosslinked silk fibroin hydrogel comprising the step of adding a peroxidase and hydrogen peroxide to an aqueous tyrosine group containing silk fibroin solution to oxidize said tyrosine groups and subsequently cross-linking wherein The concentration of peroxide ranges from 0.001-30% by weight. Process according to Claim 1, characterized in that the silk fibroin is selected from the following group: Bombyx mori silk fibroin, Antheraea, woven silk, raw silk waste, recombinant silk fibroin, or mixtures thereof. Process according to any one of the preceding claims, characterized in that said aqueous silk fibroin solution is ion-free or comprises saline ions having a pH between 6.5-9.0. Process according to any one of the preceding claims, characterized in that said aqueous silk fibroin solution has a concentration between 2-30% by weight. Process according to any one of the preceding claims, characterized in that the peroxidase source is selected from the following group: horseradish or other plants, animals, humans and bacteria. Process according to any one of the preceding claims, characterized in that said peroxidase is dissolved in an aqueous solution, water or buffered solutions, with a pH ranging from 5 to 9. Process according to the preceding claim, characterized in that the concentration of said peroxidase ranges from 0.001 to 5 mg / ml. Process according to any one of the preceding claims, characterized in that said peroxide is hydrogen peroxide or calcium peroxide. Process according to any one of the preceding claims, characterized in that said peroxide is dissolved in aqueous solution, water or buffer solution, with a pH between 5 and 9. Process according to any one of the preceding claims, characterized in that the temperature of the mixture is between 4 ° C and 60 ° C. Silk fibroin cross-linked hydrogel obtained by the process described in claims 1 to 10, characterized in that it has: - amorphous conformation; - shape memory properties, swelling and reclaiming the initial shape by immersion in different ionic strength solutions; - spatial conformation controlled by immersion in different organic solvents; and - porous structure and interconnected pores. Pharmaceutical composition comprising silk fibroin hydrogel obtained by the process described in any one of claims 1-10 and described in claim 11. Pharmaceutical composition according to Claim 12, characterized in that it further comprises bioactive substances, cells or genetic material, inorganic compounds or combinations thereof. A material for use in electronics and optoelectronics comprising the silk fibroin hydrogel described in claim 11, wherein the controlled spatial conformation is in the form of layers, the core region being predominantly amorphous, while the outer layer is conformal in shape. beta sheet. 15. Material in a deal with the claim 14, featured per understand additionally molecules inorganic, in detection and conductive materials pray, device in distribution automatic or your combinations. Lisbon, November 24, 2014