Off-the-shelf silk sericin, methods and uses thereof

Abstract

The present disclosure relates to a method of processing a silk sericin protein, to obtain a sterile, off-the-shelf powder. Hydrogels comprising said silk sericin are also disclosed.

Description

D E S C R I P T I O NOFF-THE-SHELF SI LK SERICIN, M ETHODS AND USES THEREOFTECH NICAL FIELD

[0001] The present disclosure relates to a method of processing a silk sericin protein, to obtain a sterile, off-the-shelf powder. Hydrogels comprising said silk sericin are also disclosed.BACKGROU ND

[0002] Silk sericin (SS) is a natural polymer that can be recovered from the silk textile industry and has increasingly gained interest from the scientific and industrial community due to its bioactive properties [1], At the industrial scale silk is obtained from genetically stable cocoons from silkworms, being the most common specie the Bombyx mori. SS is a glue-like protein that maintains the structural integrity of the silk fibres and represents 20 - 30 % of their total weight [2],

[0003] This protein is comprised of 18 amino acids, predominantly: serine, glycine, and aspartic acid, thus turning it into a water-soluble protein [1], Sericin mainly adopts the form of an amorphous random spiral and may also present the form of a |3-sheet organized structure. The random spiral easily acquires |3-sheet conformation as a consequence of moisture absorption and mechanical elongation, forming a denser, organized, crystalline, and less soluble structure [2],

[0004] Over the last few years, increasing literature demonstrates that sericin has promising properties for the field of tissue engineering such as biodegradability, biocompatibility, antioxidant behaviour and regenerative potential [3], [4], Owing to this fact, sericin finds usage in a multitude of industries such as biomedical engineering and cosmetics with commercially available products [5], [6], [7],

[0005] Natural-based polymers, such as SS, are considerably affected by the processing methods used to extract, purify, and fabricate the final device. Thus, when working with this material it is essential to assure quality consistency and batch-to-batchreproducibility [8], Genetically stable Bombyx mori silk is a reliable source which can be industrially produced following good practices. Sericin can be naturally extracted in boiling water in the so-called degumming process, resulting in molecular weights that range from 20-400 KDa. To ensure high yields there are several other chemical or enzymatic methods to extract sericin

[0015] , However, these often lead to a decrease of the bioactive properties of the material. Boiling in water under ambient or increased pressure has the advantage of not requiring any chemicals, introducing no impurities, and being the simplest method [9], Furthermore, sericin can be used directly after extraction without any further purification steps

[0017] ,

[0006] To regenerate sericin into 3D structures such as scaffolds and hydrogels, this protein is usually concentrated after extraction. As the sericin concentration increases, its rate of gelation accelerates, and hydrogel mechanical properties increase significantly

[0011] , Several techniques such as concentration using the rotavapor

[0012] , lyophilization

[0013] dialysis

[0014] and evaporation [3] have been reported. Evaporation relies in temperature to conduct concentration. Heating at high temperatures can cause some change in the sericin molecule

[0015] , However, major protein degradation peaks are only detectable when heated around 216 °C

[0016] , Although evaporation in a heating plate is challenging to control, this technique is simple and allows to obtain concentrations up to 14 % [3], Lyophilization has been used in different works, where sericin is dried and further redissolved to improve control over the concentrations used

[0017] ,

[0018] , However, the drying process is reported to induce 0-sheet conformation which causes water insolubility

[0019] ,

[0020] , Rotavapor is also used to concentrate silk-sericin, however, studies do not usually report all the relevant experimental parameters used (temperature, rotation, pressure, and final concentration) while temperatures between 85 - 60 °C and "low pressures" are often indicated

[0011] ,

[0012] ,

[0021] , Similarly, only limited information is available about dialysis, a time-consuming process which can be carried out using a PEG solution

[0022] ,

[0023] , Although these techniques have been reported in the development of sericin-based materials, there is no consensus on the best available technique, and published studies lack information regarding its advantages, challenges or even regarding the most adequate final applications.

[0007] An additional very relevant issue in the application of sericin for the development of biomaterials is the sterilization method used. Conventional sterilization methods such as ethylene oxide (EtO), gamma radiation, electron beam, steam, hydrogen peroxide plasma and UV radiation are very harsh for protein-based materials and lead to changes in its structure and characteristics

[0024] , These drawbacks severely limit the utility of these sterilization techniques for sericin hydrogels which have high water content and are sensitive to temperature and radiation

[0025] , In this context, supercritical CO2 (scCCh), is gaining increasing attention as a sterilization agent able to inactivate vegetative forms of a wide range of Gram-positive and Gram-negative bacteria

[0026] , The mild operating conditions, the low content of additives required and the excellent scCO2 permeability are clear advantages for the sterilization of biomaterials for biomedical applications

[0027] , The sterilization capability can be enhanced with the incorporation of low quantities of additives such as H2O2. Previous results demonstrated that this technology can successfully be applied to silk proteins

[0028] ,

[0008] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.GENERAL DESCRIPTION

[0009] The present disclosure relates to a method of processing a silk sericin protein, to obtain a sterile, off-the-shelf powder. Hydrogels comprising said silk sericin are also disclosed.

[0010] The present disclosure also relates to a method based on cryo-lyophilization and scCOz for obtaining sterile silk sericin.

[0011] In an embodiment, the resulting off-the-shelf material was fully characterized in terms of protein and molecular structure, crystallinity, morphology and rheology.

[0012] In an embodiment, the sericin of the present disclosure and Hydrogels comprising said silk sericin may be use in medicine and regenerative medicine, namely may be used in wound healing applications, this application was assessed in vitro using human dermal fibroblasts (HDFs).

[0013] An aspect of the present disclosure relates to a method to obtain a sterile silk sericin powder, the method comprising the following steps: extracting the silk sericin in boiling water; flash-freezing the silk sericin in liquid nitrogen; freeze-drying the frozen silk sericin under vacuum to obtain a silk sericin powder; placing the silk sericin powder in a sealed reactor; heating the sealed reactor to 31 to 60 °C and pressurizing it from 8 to 60 MPa; depressurizing the sealed reactor until atmospheric pressure.

[0014] In an embodiment, the sealed reactor is heated to 37 to 40 °C.

[0015] In an embodiment, the sealed reactor is pressurized to 14 to 18 MPa.

[0016] In an embodiment, the heating and pressurizing step takes 2 to 2.5h.

[0017] In an embodiment, the sealed reactor is pressurized at a constant rate ranging from 45 g / min to 55 g / min.

[0018] Another aspect of the present disclosure relates to a sterile silk sericin obtainable by a cryo-lyophilization method as described in any of the previous claims.

[0019] In an embodiment, the silk sericin is in a powder form.

[0020] Another aspect of the present disclosure relates to a hydrogel comprising the sterile silk sericin of the present disclosure.

[0021] In an embodiment, the hydrogel is chemically crosslinked.

[0022] In an embodiment, the hydrogel is crosslinked by a peroxidase, in particular a horseradish peroxidase.

[0023] In an embodiment, the hydrogel is enzymatically crosslinked by peroxidases in the presence of peroxide solutions, in particular hydrogen peroxide or calcium peroxide.

[0024] In an embodiment, the hydrogel is further crosslinked by a polyphenol molecule, in particular by tannic acid.

[0025] In an embodiment, the concentration of silk sericin ranges from 1 to 16 wt.%, preferably from 2 to 6 wt.%.

[0026] In an embodiment, the hydrogel of the present disclosure may be use in medicine. Preferably, for use in tissue engineering and regenerative medicine.

[0027] In an embodiment, the hydrogel of the present disclosure may be use as replacement biomaterials, drug delivery platform, cell delivery platform, platforms for 3D cell culture and disease modelling, cell culture platform, cell encapsulation, organ development and tissue growth, artificial extracellular matrices, bioinks, scaffolds, or other biomedical and biological applications.

[0028] Another aspect of the present disclosure relates to a composition comprising the silk sericin described in the present disclosure and / or the hydrogel described in the present disclosure.

[0029] Bioink, artificial extracellular matrix, patch or scaffold comprising the silk sericin described in the present disclosure and / or the hydrogel described in the present disclosure.BRI EF DESCRIPTION OF TH E DRAWINGS

[0030] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0031] Figure 1: Schematic representation of the silk sericin (SS) solution concentration methodologies: evaporation (SS.E), Rotavapor (SS.RV), lyophilization (SS.L) and dialysis (SS.D).

[0032] Figure 2: Representation of an embodiment of circular dichroism (CD) of sericin solution after extraction in boiling water (SS) and after concentration using different methods (SS.E, SS.RV, SS.L, or SS.D).

[0033] Figure 3: Representation of an embodiment of A) X-Ray diffraction analysis (XRD) spectra of the sericin solutions (SS, SS.E, SS.RV, SS.L, or SS.D), where the arrows represent the peak width; and B) Fast protein liquid chromatography chromatogram of molecular weight (Mw) distribution, for the same samples.

[0034] Figure 4: Representation of an embodiment of a fourier-transform infrared spectroscopy (FTIR) spectra of sericin-based formulations, after extraction in boiling water (SS) and after concentration using different methods (SS.E, SS.RV, SS.L, or SS.D).

[0035] Figure 5: Representation of an embodiment of a Raman spectrum for the SS- solutions, after extraction in boiling water (SS) and after concentration using different methods (SS.E, SS.RV, SS.L, or SS.D).

[0036] Figure 6: Representation of an embodiment of A)1H NMR spectrum of a 2% wt. SS.L / D2O solution. B)13C CP / MAS spectrum of SS.L.

[0037] Figure 7: Representation of an embodiment of Cryo-SEM images for silk-sericin after extraction and concentration (samples: SS.E, SS.RV, SS.L, or SS.D; scale bar: 30pm).

[0038] Figure 8: Representation of an embodiment of the rheological properties of silk- sericin solutions (SS, SS.E, SS.RV, SS.L, or SS.D; final concentration of lwt.%): A) G' and tan 6 and B) viscosity measured at 37 °C.

[0039] Figure 9: Embodiment of results relating to A) Turbidity tests after 12 days for 1: sterilized B. pumilus, 2: sterilized B. atrophaeus, 3: sterilized B. stearothermophilus and 4: non-sterilized B. pumilus (positive control); B) Culture of the medium collected from the turbidity tests and incubated during 2 days in TSA plates (1: B. atrophaeus, 2: B. pumilus, 3: B. stearothermophilus); C) FTIR spectra of lyophilized sericin before and after SCO2 sterilization, in which amide I peak position was maintained; D) sericin raw material in sterilization pouches before SCO2 and after dissolution in PBS (2% wt.).

[0040] Figure 10: Embodiment of results of cell viability of HDFs cultured using extracts of SS.L1 (1 wt%) and SS.L2 (0.5 wt%) collected after 1 and 7 days of culture.

[0041] Figure 11: Embodiment of results of fluorescence images collected for the wound healing assay after 3h, 20h, 40h and lOOh for SS.L1, SS.L2 and control groups . Scale bar: 500pm.

[0042] Figure 12: Embodiment of rheology analysis of a solution of lwt.% of silk sericin obtained by cryo-lyophilization, at a controlled temperature of 37 °C and with a coneplate cross-geometry (20-mm diameter).

[0043] Figure 13: Embodiment of dissolution of SS.L (1 wt.%) and respective intrinsic gelling properties.

[0044] Figure 14: Embodiment of dissolution of commercial silk sericin (1 wt.%) and respective aspect after dissolution, where no gel is formed.

[0045] Figure 15: Embodiment of FITIR analysis performed in commercial silk sericin and silk sericin obtained by cryo-lyophilization.

[0046] Figure 16: Embodiment of differential scanning calorimetry (DSC) analysis performed in commercial silk sericin and silk sericin obtained by cryo-lyophilization

[0047] Figure 17: Embodiment of SEM analysis performed in commercial silk sericin and silk sericin after extraction and dissolution. Scale bar: 30pm

[0048] Figure 18: Embodiment of results on cell viability using hydrogels prepared from SS.L and chemically crosslinked with horseradish peroxidase and / or tannic acid.DETAILED DESCRIPTION

[0049] The present disclosure relates to a method of processing a silk sericin protein, to obtain a sterile, off-the-shelf powder. Hydrogels comprising said silk sericin are also disclosed.

[0050] In an embodiment, Bombyx mori silkworm cocoons were cut into small pieces, cleaned, and immersed in deionized water in a proportion of 1:100 (w / v) at boiling temperature for 60 minutes, for extraction. After extraction, the silk sericin solution was filtered to a final volume of =100 mL (Whatman 1113-150). The obtained silk sericin solution was concentrated using distinct techniques: evaporation (SS.E), Rotavapor (SS.RV), lyophilization (SS.L) or dialysis (SS.D), as illustrated in Figure 1. Obtained SS stock solutions were kept at 4 °C for further experiments.

[0051] In an embodiment of concentration by evaporation, a suitable amount of a solution of SS was placed in an open baker and heated at a fixed temperature of 100 °C, monitored with a thermocouple. A stirring magnet was used at 400 rpm. The initial volume of silk-sericin solution was kept at 20 mL and a final volume of ~ 5 mL of concentrated sericin was obtained.

[0052] In an embodiment of concentration by rotavapor, SS was concentrated in a rotary evaporator (BUCHI Rotavapor™ RII), at a temperature of 60 °C and pressure of 300 mbar. The initial volume of silk-sericin solution was kept at 20 mL and a final volume of ~ 1 mL of concentrated sericin was obtained.

[0053] In an embodiment of concentration by lyophilization, a suitable amount of silk sericin solution was instantaneously frozen in liquid nitrogen (flash-freezing) and stored at -80 °C. Instant freezing was performed in order to reduce or avoid changes in the secondary conformation of the silk protein. Afterwards, the solution was placed in the freeze-drier under vacuum and collected after 24h. The resulting sericin powder was kept in the desiccator. Before usage, the sample was rehydrated by dissolving it in PBS at 100 °C.

[0054] In an embodiment of concentration by dialysis, 50mL of sericin solution, obtained after extraction in boiling water, was placed into a dialysis membrane (Spectra / Por™ dialysis membrane pre-wetted RC tubing MWCO: 3.5 kD) and immersed in a 20 wt.% poly (ethylene glycol) (PEG, Sigma-Aldrich, 20 KDa) PEG solution for 3h. A heating plate at 70 °C and a magnetic stirrer were used to assure that the sericin solution remained fluid.

[0055] In an embodiment, to standardize the determination of sericin concentration and guarantee reproductivity between experiments the concentration of the SS solution was determined by the dry weight method (ISO 638:2008) at 105 °C for 24 h (equation 1)Dry weight (%) =Wd Wcx 100 (equation 1),where Wd is the dry weight of the sample with the container; Ww is the wet weight of the sample with the container, and Wc is the weight of the container.

[0056] Sericin absorbance at 275 nm was used to establish a linear relationship with its concentration (R=0.98) using a Nanodrop (Thermo Scientific NanoDrop One).

[0057] In an embodiment, the secondary structure of the silk sericin (SS) was evaluated using circular dichroism (CD). Triplicates of each sample were analysed. This technique was conducted using a Jasco J-815 spectrometer equipped with a quartz cell having a pathlength of 2 mm at room temperature (25 °C). Measurements were conductedbetween 190 - 250 nm at a scanning speed of 50 nm / min and using a continuous scanning mode and 0.1 nm data pitch. Silk sericin solutions (SS, SS.E, SS.RV, SS.L and SS.D) were diluted with deionized water to a final concentration of approximately 0.4 wt.%. This dilution was carried out immediately after extraction or concentration to precede the formation of a gelled material. In order to reduce noise, the solutions were filtered before analysis (0.2 pm) and the cuvette was cleaned between samples with detergent, methanol and water. After normalization, the spectra obtained were analysed with DichroWeb

[0029] , This software is based on the singular value decomposition method in which the data is reduced to a small number of linearly dependent vectors that form the curves common to all the data. The algorithm interacts through linear combinations of the basis curve until it converges to a curve that best fits the spectra.

[0058] In an embodiment, the circular dichroism (CD) spectra (Figure 2) of the extracted and concentrated silk sericin solutions presented strong negative bands around 200 nm assigned to the random coil conformation and a weak negative band around 220 assigned to the ^-structure (Table 1). The absence of any positive bands at around ~190 nm is associated with a lack of or residual presence of a-helix. The CD peaks obtained are characteristic of silk-sericin after the degumming process. The secondary structure fractions (%) predicted in DichroWeb corroborated the presence of these conformations (Table 1). The data output was calculated through a comparison of the CD raw data (~260 - 190 nm) with a wide range of protein spectra datasets and their associated secondary structure, generating a reconstructed spectrum of the best-fit solution overlying the experimental spectrum.

[0059] The different concentration techniques used did not appear to cause significant changes in the secondary structure of sericin as compared with the initial solution (Table 1). This was also valid for the case of lyophilization treatment, where the water is fully removed. During the process of water freezing, a high propensity for protein selfassembly occurs, leading to |3-sheet structure formation. Hence, structural properties obtained during freeze-drying are mainly regulated by exploiting the kinetics of ice formation and fast freezing, and can prevent protein self-assembly, thus preserving the initial protein self-assembly.

[0060] The results shown in Table 1 indicate that the content of |3-sheet in the initial solution as compared with the regenerated solution after freezing with liquid nitrogen (-196 °C) followed by lyophilization process, does not significantly increase (from 41.7 to 42.2%), potentially increasing the gel permeability and reducing mechanical constraints.

[0061] Table 1. Secondary structure fractions (%) of the different SS solutions using DichroWeb, from CD analysis of sericin solution after extraction in boiling water (SS) and after concentration using different methods (SS.E: evaporation, SS.RV: rota- evaporation; SS.L: lyophilization; SS.D: dialysis).0062] * Turn: combination of beta turns, bends and bridges.

[0063] *NRMS: normalized standard deviation ("goodness-of-fit parameter": indicates the correspondence between the experimental input spectrum and the back-calculated best-fit spectrum derived from the analysis).

[0064] In an embodiment, X-ray diffraction (XRD) of the different SS solutions was conducted to analyse the crystalline structure of silk sericin (Figure 3A). Sample preparation consisted in filling an aluminium mold with 1 mL of solution (1 wt.% of silk sericin, concentrated using the different testing methods). Analyses were performed on a Bruker D8 Advance instrument which works with CuKa radiation (nA. = 1.542 A) at a 0.02 step size and at 0.5 s per step. A diffraction peak with broad intensity at 20 = 28 was verified for all samples, which is attributed to a |3 -sheet crystalline structure. In addition, a shoulder at approximately 38-42 degrees indicates the low crystallinity of the sericin-solutions, characteristic of random coil conformations and the presence of high polar amino acids (Figure 3A). While the XRD spectra of sericin after extraction and of sericin obtained through concentration using rotavapor and lyophilization (SS.RV and SS.L) are identical, the intensities of the conditions attributed to dialysis (SS.D) andevaporation (SS.E) are slightly distinct (this comparison is possible since the same quantity of sericin was analysed). For SS.D, the presence of a more intense and well- defined peak can indicate an increase in crystallinity when compared to the other concentration methods. On the other hand, SS.E has a narrower distribution which can indicate that the sample is more amorphous.

[0065] In an embodiment, the molecular weight distribution of silk sericin in solution was determined by Protein Profile by Fast Protein Liquid Chromatography (FPLC) (AKTA pure) (Figure 3B). Diluted samples (500 pL) of silksericin solutions, as extracted and after the concentration processes, were analysed using a Superdex 200 10 / 300 GL column coupled to an FPLC AKTA-purifier system (GE). The eluent used was 0.05 M phosphate- buffered saline (PBS) (pH 7.0), containing 0.15 M sodium chloride (for ionic strength) and 0.2 g / L of sodium azide (as preservative) at a flow rate of 0.5 mL / min. Elution was monitored at 280 nm, and the approximated molecular weight (Mw) of silk sericin solutions was determined with standard protein kits provided by GE Healthcare. One of the most influenced parameters by the extraction method used is the molecular weight, which for boiling water extraction ranges between 20 - 400 KDa, mostly used for the development of functional medical biomaterials. Figure 3B depicts an embodiment of the chromatograms of the different experimental conditions. The resulted curves are identical amongst the different tested conditions, with the exception of SS.D, which in addition to having an additional peak (10 KDa) that can suggest protein degradation, also has a band around 30 KDa (9 mL elution volume), characteristic of agglomerates.

[0066] Figure 4 shows an embodiment of the FTIR spectra for the silk-sericin solutions tested. Typical absorption bands in sericin protein such as amide I (1600-1700 cm'1), II (1504-1582 cm'1), and III (1200-1300 cm'1) were detected for all samples at around 1649 cm1(C=O stretching in carbonyl group within amidic backbone), 1528 cm1and 1238 cm'1(phenolic hydroxyl functionalities of tyrosine residues of sericin), respectively. The peaks at 1395 cm-1can be attributed to C=O symmetry stretching, and peaks at 1067 cm-1can be attributed to C-O-C and C-0 stretching vibrations. When analysing the difference in ratios between amide I and amide II, proportional to the amount of hydrogen bonding that occurs in the carbonyl to the secondary amine structures of the peptide repeats and associated with a change in secondary structure it is visible that thisratio changes for SS.D (Table 2). As a result of Amide I peak deconvolution, the presence of both random coil and p-sheet characteristic peaks in all SS solutions studied was verified.

[0067] In an embodiment, the chemical structure of the sericin gels was further evaluated using Raman spectroscopy. The Raman spectrum was measured using a Renishaw InVia Reflex Raman system (Renishaw, New Mills, Gloucestershire UK). The Raman scattering is excited using a diode laser at a wavelength of 785 nm. The laser beam is focused on the sample with a 0.85 xlOO microscope objective coupled to the system. The exposure time and the number of accumulations for the Raman measurements correspond to 10 seconds and 10 respectively. In an embodiment, the results retrieved from Raman spectroscopy (Figure 5) also evidenced the presence of |3- sheet structures, in which the typical wavenumbers are 1674-1672 cm1and 1242-1227 cm1for amide I and amide III modes.

[0068] Table 2. Peak identification of silks sericin samples after extraction in boiling water (SS) and after concentration using different methods (SS.E: evaporation, SS.RV: rota-evaporation; SS.L: lyophilization; SS.D: dialysis).

[0069] In an embodiment, the chemical structure of the SS.Lwas further evaluated using Nuclear Magnetic Resonance (NMR) (Figure 6).1H NMR measurement have been performed on a JEOL JNM-ECZ400R operating at 400 MHz at 45 °C in pre-saturated conditions. SS.L was dissolved at a concentration of 2% wt in deuterium oxide (D2O, Merck KGaA, Darmstadt, Germany). Carbon-13 nuclear magnetic resonance (CP / MAS 13C NMR) measurement was conducted on a Bruker Avance TM400WB equipped with a wide-mouth superconducting magnet (89 mm) operating at 9.4 T. (Larmor frequencl00.61MHz). Sample were milled after immersion in liquid nitrogen andcontained in a cylindrical ceramic rotor.13C NMR spectra were acquired at ambient temperature and a spinning speed of 7 kHz, 3.50 ms width 90 pulses with 3 s repetition time, cross-polarization contact time of 1 ms. Spectra accumulation of 2000 scans and referenced to adamantane. Spectral analysis was analysed using MestreNova 12 Software.

[0070] The assignment of 1H-NMR peaks was based on previous assignments of silk fibroin and small peptides. In the spectrum, the chemical shifts of the main amino acid residues can be observed at the following ppm values: 7.6 and 7.4 ppm forTyr(() residues, 4.1, 4.4, and 4.5 ppm for C“H in Gly, Ala, and Ser residues respectively, 3.7, 3.5, and 3.3 ppm for Ser, Asp, and Ala residues respectively, and 1.9 ppm for Arg and (1.6 and 1.5) ppm for Lys. These chemical shifts closely resemble the1H chemical shifts of random coil structures, as determined by different authors

[0033] ,

[0034] , Similarly, in the CP / MAS spectra, the corresponding peaks for Ser Ca, Ser CP, and Gly Ca were observed at 173.3, 55.4, 61.1, and 42.6 ppm respectively. This indicates that sericin maintains a largely random coil structure even after lyophilization

[0035] ,

[0071] In an embodiment, the morphology of sericin obtained from the different methods was analysed by scanning electron microscopy with energy dispersive spectroscopy (SEM / EDS). The examination was performed using a high-resolution Scanning Electron Microscope with X-Ray Microanalysis and CryoSEM experimental facilities: JEOL JSM 6301F / Oxford INCA Energy 350 / Gatan Alto 2500. The specimens were rapidly cooled (plunging it into sub-cooled nitrogen-slush nitrogen) and transferred under vacuum to the cold stage of the preparation chamber. After that, the different sericin samples were fractured, sublimated ('etched') for 300 sec at -90 °C, and coated with Au / Pd by sputtering for 50 s. The samples were then transferred into the SEM chamber and studied at a temperature of -150 °C.

[0072] In an embodiment, the hydrogels were prepared by concentrating sericin using the different methods known in the art. Namely, evaporation, rota-vapor, lyophilization, dialysis, among others.

[0073] In an embodiment, the hydrogels were prepared by concentrating sericin using evaporation. Evaporation to reach the desired concentration was based on the work of Baptista-et al., [5], Briefly, a given solution of SS in an open baker was heated andprocess control was achieved by fixing the temperature at 100 °C and monitoring with a thermocouple. A stirring magnet was used at 400 rpm. The initial volume of silk-sericin solution was kept at 20 mL and a final volume of ~ 5 mL of concentrated sericin was obtained.

[0074] In an embodiment, the hydrogels were prepared by concentrating sericin using rota-vapor. SS was also concentrated in a rotary evaporator (BUCHI Rotavapor™ RII). Initially, the influence of the temperature (40, 50, 60, 90 °C), rotation (120, 240 rpm), and pressure (250, 500 mbar) (Vacuum controller CVC 3000) was studied. Thereafter, the experimental condition which led to a higher sericin concentration was selected to perform further studies. The initial volume of silk-sericin solution was kept at 20 mL and a final volume of ~ 1 mL of concentrated sericin was obtained.

[0075] In an embodiment, the hydrogels were prepared by concentrating sericin using lyophilization. A given sericin solution was instantaneously frozen in liquid nitrogen and stored at -80 °C. Instant freezing was performed to reduce or avoid changes in the secondary conformation. Afterwards, the solution was placed in the freeze-drier under vacuum and collected after 24h. The resulting sericin powder was kept in the desiccator. Before usage, the sample was rehydrated by dissolving in PBS at 100 °C.

[0076] In an embodiment, the hydrogels were prepared by concentrating sericin using dialysis. Sericin solution was concentrated by a method adapted from the literature

[0073] , Briefly, 50mL of sericin solution after extraction was placed into a dialysis membrane (Spectra / Por™ dialysis membrane pre-wetted RC tubing MWCO: 3.5 kD) and immersed in a 20 wt.% poly (ethylene glycol) (PEG, Sigma-Aldrich, 20 KDa) PEG solution for 3h. A heating plate at 70 °C and a magnetic stirrer were used to assure that the sericin solution remained fluid

[0039] ,

[0077] Figure 7 depicts an embodiment of the morphology of silk sericin as analysed by Cryo-SEM. All protein-based hydrogel structures can generate porosity after water removal, which increases with a decrease in protein content (SS.E, SS.RV, SS.D and SS.L).

[0078] In an embodiment, rheology experiments were performed using triplicates of SS samples at a concentration of 1 wt.% at a controlled temperature of 37 °C, using a rheometer (ARG2, TA Instruments, New Castle, DE, USA) equipped with a cone-platecross-geometry (20-mm diameter). Storage modulus (G') and loss modulus (G") were recorded at a frequency of 0.1 - 1000 Hz and 1% strain. Complex viscosity (also represented as q*) is a measure of the total resistance to flow as a function of angular frequency (co) and is given by the quotient of the maximum stress amplitude and maximum strain rate amplitude. Viscosities were also determined by rotational shear measurements at an increasing shear rate from 0.1 to 10000 s-1.

[0079] Figure 8 depicts an embodiment of the viscous properties of the sericin-gels as assessed through rheological testing. The curves obtained are representative profiles of a hydrogel matrix in which tan 6 < 1, indicating that G' > G" (Figure 8A), consistent with a material that behaves like an elastic solid. Sericin's gelation phenomenon can explain this behaviour and consists of an interesting property for the development of naturalbased hydrogels due to its self-assembly nature. According to the frequency sweep tests, G' is frequency independent up to approximately 50 rad / s. In addition, the G' of SS.D is higher, indicating the presence of a stiffergel (less viscous flow behaviour) (Figure 8A), which can be linked to a more organized structure as exhibited by XRD (Figure 3A). The viscosity profile (Figure 8B) reveals a shear thinning behaviour for all SS solutions analysed, with viscosity decreasing under shear strain. This profile is expected of concentrated polymer solutions. SS after dialysis (SS.D) also exhibits higher viscosity. The opposite occurs for SS.E, with lower G' and viscosity, which is also in agreement with the XRD results. The longer concentration period during dialysis has favoured the molecular self-assembly of silk sericin, which explains the differences registered. According to the different characterization techniques performed, the gel obtained from SS.D has higher 0-sheet content, which results in a material with higher crystallinity and more rigidity. On the other hand, the lyophilization approach (SS.L), using a freezing temperature of - 196°C, was able to maintain silk amorphous structure, thus validating the proposed cryolyophilization methodology. Dissolution tests on the dried sericin material confirmed the presence of random coil conformation, p-sheet structures contribute to reduced solubility, due to its higher crystallinity, which would hinder the gel preparation.

[0080] These findings were supported by the physicochemical assessment conducted since the complete dissolution of the material was achieved in both water and PBS (considering that the abovementioned techniques performed with the solutions in gel-state were post-dissolution, ensuring that the intrinsic gelling properties are preserved, which is not the case when the dissolution of the sericin powder is partial).

[0081] Rotavapor and evaporation can also be implemented to obtain an SS- concentrated solution with preserved characteristics, with the condition that the resulting material must be used after processing to keep the protein stable and as a whole. However, the final concentration is more difficult to control, and an adjustment step, such as dilution, may have to be added. In contrast, the sericin-dried powder obtained from SS.L can be used as needed to prepare a SS solution with the desired concentration.

[0082] Thus, surprisingly, SS.L allowed to obtain a ready-to-use amorphous dry material which is easy to re-dissolve. Moreover, lyophilized materials have a longer shelf-life and do not require refrigeration, as compared with their solutions. Besides being easier to transport, handle and process than a jellified material or solution, the use of sericin powder also allows greater control over the final concentration of the material, reducing variability and allowing the scale-up.

[0083] To evaluate the possibility of creating an off-the-shelf sericin dry powder for further regeneration and use, lyophilized sericin samples were sealed in sterilization pouches (3M™ Steri-Dual™) and placed inside a 2L-stainless steel autoclave (Eurotechnica GmbH, Bargteheide, Germany) equipped with an agitation system (600 rpm), including 1200 ppm of H2O2. The reactor was heated to 39 °C and pressurized until 14 MPa at a constant pressurization rate of 50 g / min. The system was maintained in batch mode for 2.5 h. Finally, the reactor was manually depressurized until atmospheric pressure in 30 min.

[0084] In an embodiment, turbidity tests were performed to assess the sterility of the process and therefore, of the sericin powder, before and after sterilization. Commercial spore strips with 106 spores of three different biological indicators were used. Bacillus stearothermophilus (B. stearothermophilus ATCC 7953, Valencia, Spain) and Bacillus pumilus (B. pumilus ATCC 27142 Valencia, Spain) were purchased from Sigma-Aldrich (Madrid, Spain). Bacillus atrophaeus (B. atrophaeus cell line ATCC 9372, Catalog number: 0953) spores were obtained from Crosstex International (Rush, NY, USA). The treated spore strips were incubated in 6 mL of Tryptic Soy Broth (TSB) liquid medium atthe optimal growth temperatures (37 °C, B. pumilus and B. atrophaeus; 60 °C, B. stearothermophilus) and the bacterial growth was visually evaluated after 7 and 14 days. In addition, after 7 and 14 days of incubation, 1 mL of the culture medium was seeded in trypticase soy agar (TSA) plates and incubated for 1 day to determine the presence of viable microorganisms.

[0085] The turbidity tests showed that after 12 days of incubation, all biological indicators used were clean (indicated by the clear solutions), as presented in Figure 9A. The change in colour registered in the tube 3 can be attributed to the type of spore tested. In the positive control (Tube 4), the medium is turbid which indicates the growth of bacteria. After further culture of the medium collected from the turbidity tests in TSA plates (Figure 9B), the results obtained were identical, with no indication of contamination for the sterilized specimens. Hence, according to the results, the sterilization method was terminal and can be used with silk sericin.

[0086] Regarding protein conformation, through the analysis of the chemical structure of lyophilized sericin, it was possible to verify that the scCCh sterilization did not change the protein structure since there is no shift in the Amide I peak (Figure 9C). This was further supported by the preservation of sericin shape and colour after sterilization; gelling properties after dissolution were also not affected (Figure 9D).

[0087] In an embodiment, cell culture assays were performed using human dermal fibroblasts (HDFs) (Innoprot, P10856). For the in vitro biological evaluation, cells were cultured in a basal medium consisting of Dulbecco's modified Eagle's medium (DMEM) - low glucose enriched with 110 mg / L of sodium bicarbonate and supplemented with 20% v:v of fetal bovine serum (FBS, Gibco), 200mM L-glutamine, 100 units / mL penicillin and 100 mg / mL streptomycin. Cells were maintained under a humidified 5% CO2 atmosphere at 37 °C.

[0088] In an embodiment, the extracts were obtained by placing DMEM in contact with the sericin gel for 24h to allow the release of residues. This medium was collected and placed in contact with confluent cells to test their behaviour.

[0089] In an embodiment, cytotoxicity was first analysed using the Alamar Blue (AB, BIO-RAD BUF012B) bioassay. HDFs were seeded in 96-well plates (Fisher Scientific) at adensity of lxlO5cells / well in 100 mL of basal medium and incubated for 24 h. Afterwards, media were removed and replaced by 100 mL of basal medium containing extracts from SS concentrated by lyophilization at different concentrations (SS.L1: lwt.% and SS.L2: 0.5wt.%).These extracts were obtained by placing DMEM in contact with the sericin gel for 24h and 7 days to allow the release of residues. This medium was collected and placed in contact with confluent cells to test their behaviour. And after 24 h of incubation with the cells, the extracts were collected and 100 mL of the AB solution prepared in warm DM EM -low glucose phenol red-free (10% v:v) (Gibco) was added to each well and the plates were incubated at 37 °C for 3 h. After this period, fluorescence was measured at 590 nm after excitation at 560 nm using a microplate reader (Synergy HT). Relative cellular viability was calculated after subtracting the fluorescence of the blank (AB solution in an empty well) from the fluorescence of the samples and normalizing it to the data of untreated cultures, which were given an arbitrary value of 100. Data were analysed as mean ± standard deviation with n=6 for each condition. GraphPad Software (version 8.0) was used to perform statistical analysis by implementing the 2way ANOVA test followed by Turkey's method as a Multiple Comparison post-test (significance level was *p<0.05, **p<0.01).

[0090] The results from the in vitro tests using HDFs demonstrated that SS.L is biocompatible, with cell viability superior to 98% for extracts from 1 and 7 days (Figure 10). Furthermore, the differences between SS.L1 (1 wt%) and SS.L2 (0.5 wt%) are not statistically significant (p > 0.05), suggesting that both concentrations result in similar biological behaviour.

[0091] In an embodiment, SS.L1 and SS.L2 24h extracts ( n=3) were used to conduct the wound healing assay in 24 well plates with a high level of confluence (90%), seeded with 500 pL of cell suspension at 1.5xl05cells / mL After removing the medium and washing with PBS, a marker was used to do a line across all wells to serve as a reference in order to assure that the pictures of the wounds were performed on the same side. Afterwards, a scratch was done perpendicular to the black line, using a 200 pL pipette tip. After dyeing (CellTrace Calcein Green, AM; Invitrogen), the HDFs were incubated with the extracts of the SS.L gels at 37 °C and 5% CO2. The scratches were documented under the fluorescence microscope after pre-determined time intervals (3h, 20h, 40h and lOOh).

[0092] The wound-healing assay confirmed the excellent biological properties of the collected extracts, which did not hinder cell proliferation (Figure 11). Over time, the scratch simulating the wound decreased until it filled at the end of 100 h. Over this time the migration of cells from one side of the wound to the other was evidenced. Since sericin dissolves in aqueous media when it is not cross-linked, it was released to the culture medium without compromising cell growth.

[0093] Surprisingly, the results show that concentration by dialysis leads to the formation of a stiffer structure with a higher |3-sheet amount. On the other hand, the so-called cryo-lyophilization does not change the properties of the protein while maintaining the amorphous conformation which is found in solution, thus, this methodology is suitable to generate an off-the-shelf, ready-to-use powder that can be easily processed and applied for the further development of sericin-based biomaterials. Additionally, the scCCh sterilization of this sericin system was validated as an alternative to standard sterilization procedures, which use harsh processing conditions. In vitro tests using HDFs demonstrated the high biocompatibility of the obtained lyophilized powder.

[0094] In an embodiment, silk sericin powder obtained by cryo-lyophilization (SS.L) and sterilized by scCCh was compared with a commercial silk sericin powder (Merck, S5201). Surprisingly, SS.L retained its intrinsic gelling properties after dissolution of lwt.% in PBS, confirmed by rheologic analysis (Figure 8), as opposed to the commercial silk sericin that lost its gelling properties after dissolution using the same conditions (Figure 12). This phenomenon was also noticed following the dissolution of the sericin samples and allowing them to stabilize for 1 hour. Sericin obtained from cryo-lyophilization formed a viscous complex that maintained its structure when poured into a mold (Figure 13), whereas commercially available sericin remained entirely liquid (Figure 14). From the FTIR analysis, it is possible to observe that the secondary structure is identical for all studied conditions, with a clear overlap of Amide I peaks (Figure 15). Therefore, the protein resulting from the disclosed method has a different structure from the commercially available one, allowing its gelation after dissolution. The DSC Metoanalysis reveals similar thermal profiles for the lyophilized sericin and commercial sericin samples. The lyophilized sericin exhibits a minor peak at approximately 82°C.Conversely, the commercial sericin demonstrates a minor peak at approximately 102°C and another at 205°C (Figure 16). The SEM analysis further indicates that the surface of the lyophilized sericin exhibits greater roughness compared to the commercial sericin (Figure 17). This characteristic could be advantageous as it has the potential to enhance cellular attachment, thereby providing a beneficial property.

[0095] In an embodiment, the thermal analysis of Silk Sericin was performed to confirm the crosslinking effect through the increase of thermal stability of the Lyophilized Sericin and Commercial Sericin powder. For this purpose, a differential scanning calorimetry (DSC; model DSC-60, Mettler Toledo, Shimadzu, USA), was used. The samples were prepared and kept at 37°C overnight, then 2 / 3 mg of the Silk Sericin was crimped in a standard aluminum pan and heated from 30 to 350 °C at a constant rate of 5 °C / min, under constant purging of nitrogen at 250 mL / min. All the samples were run and the data that are presented were the average of the three measurements.

[0096] In a further embodiment, sterile SS.L, as obtained by the disclosed method, was enzymatically crosslinked with a peroxidase leading to enzymatically crosslinked hydrogels. In particular, SS.L was crosslinked with horseradish peroxidase (SS / HRP hydrogels), prepared based in a enzymatically crosslinked sericin hydrogel, in which HRP (0.2% m / v) and hydrogen peroxide (H2O2)(l%v / v) prepared in PBS were used. In another embodiment, SS.L was mixed with tannic acid (TA) to examine the potential of TA as a binder to induce the gelation of a homogeneous SS hydrogels. In yet another embodiment, SS.L was crosslinked with both TA and HRP / H2O2. Table 3 lists the formulations used.

[0097] Table 3. Silk sericin-based formulations for crosslinking studies.Core material (SS) TA (% wt.) HRP / H2O2 Sample name0.05 SS1 / TA10.1 - SS1 / TA2SSI: 2.5 % 0.2 SS1 / TA3H2O2 (1% v / v) + HRP SS1 / HRP0.1 (0.2% wt.) SS1 / TA2-HRP0.05 SS2 / TA10.1 - SS2 / TA2SS2: 5 % 0.2 SS2 / TA3H2O2 (1% v / v) + HRP SS2 / HRP0.1 (0.2% wt.) SS2 / TA2-HRP

[0098] In an embodiment, SS / HRP materials exhibit better biological properties, with higher cell viability than SS / TA and SS / TA-HRP formulations. When it comes to silk sericin concentration, SS2-based hydrogels exhibit superior rheological properties and are therefore more suitable for applications where materials with structural integrity are preferred. On the other hand, SSI materials have lower retention capacity and lower stiffness and can be applied for surface wound treatment or bioactive agent release.

[0099] In an embodiment, SS.L-based hydrogels with a volume of 300 pL per well were placed in 48 well plates. The HDFs were seeded in the form of a suspension of 5 x 105cells in 100 pL per well. After incubating for 40 min, 300 pL of DMEM was added to each well. The cells were cultured on the hydrogels for 1, 3, and 7 days with DMEM at 37 °C in 5% CO? with medium replacement every 3 days. In a further embodiment, adhesion and metabolic activity of the cells in the SS-based hydrogels were determined by direct assay with the AlamarBlue® reagent. After 1, 3 and 7 of culture, 500 pL of 10% (v:v) AlamarBlue® solution in medium without phenol red were added to each well. After 3 h of incubation, 100 pL of AlamarBlue® solution were transferred to a 96-well cell culture plate (Greiner Bio-one, Frickenhausen, Germany) in triplicate. Fluorescence was measured at an excitation wavelength of 530 / 25 nm and an emission wavelength of 590 / 35 nm using Biotek Synergy HT plates. Alamar Blue data was analysed as mean ± standard deviation with n=8 for each bar. Statistical analysis was performed as previously described. In a further embodiment, cell adhesion and viability were also assessed through Life / Dead assay (LIVE / DEAD™ Cell Imaging Kit (488 / 570), Thermo Fisher). For Life / Dead assay, DMEM was removed from the wells and 80 pl of reagentprepared by adding the probe to DMEM (1:1) was added. After incubating for 15 min, the seeded hydrogels were collected after 7 days with a spatula and placed on a coverslip. A cross section was made with a scalpel to allow visualization of the cells within the hydrogels. In order to prevent the hydrogel from drying out, DMEM was added to the prepared gels using a dropper. SS1 / TA1 and SS1 / TA2-HRP do not influence cell viability over time and can be used in applications where cell growth is not to be promoted, but rather maintained in a steady state (Figure 18).

[0100] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof.

[0101] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above-described embodiments are combinable.

[0102] The following claims further set out particular embodiments of the disclosure.

Claims

C L A I M S1. A method to obtain a sterile silk sericin powder, the method comprising the following steps: extracting the silk sericin in boiling water; flash-freezing the silk sericin in liquid nitrogen; freeze-drying the frozen silk sericin under vacuum to obtain a silk sericin powder; placing the silk sericin powder in a sealed reactor; heating the sealed reactor to 31 to 60 °C and pressurizing it from 8 to 60 MPa; depressurizing the sealed reactor until atmospheric pressure.

2. The method according to the previous claim wherein the sealed reactor is heated to 37 to 40 °C.

3. The method according to any of the previous claims wherein the sealed reactor is pressurized to 14 to 18 MPa.

4. The method according to any of the previous claims wherein the heating and pressurizing step takes 2 to 2.5h.

5. The method according to any of the previous claims wherein the sealed reactor is pressurized at a constant rate ranging from 45 g / min to 55 g / min.

6. A sterile silk sericin obtainable by a cryo-lyophilization method as described in any of the previous claims.

7. The sterile silk sericin according to the previous claims wherein the silk sericin is in a powder form.

8. A hydrogel comprising the sterile silk sericin as described in any of previous claims 6 to 7.

9. The hydrogel according to the previous claim wherein the hydrogel is chemically crosslinked.

10. The hydrogel according to the previous claim wherein the hydrogel is crosslinked by a peroxidase, in particular a horseradish peroxidase.

11. The hydrogel according to the previous claim wherein the hydrogel is enzymatically crosslinked by peroxidases in the presence of peroxide solutions, in particular hydrogen peroxide or calcium peroxide.

12. The hydrogel according to the previous claim 9 wherein the hydrogel is further crosslinked by a polyphenol molecule, in particular by tannic acid.

13. The hydrogel according to any of previous the claims 9-13 wherein the concentration of silk sericin ranges from 1 to 16 wt.%, preferably from 2 to 6 wt.%.

14. The hydrogel according to any of the previous claims 9-14 for use in medicine.

15. The hydrogel according to the previous claim for use in tissue engineering and regenerative medicine.

16. The hydrogel according to any of the previous claims 14-15 for use as replacement biomaterials, drug delivery platform, cell delivery platform, platforms for 3D cell culture and disease modelling, cell culture platform, cell encapsulation, organ development and tissue growth, artificial extracellular matrices, bioinks, scaffolds, or other biomedical and biological applications.

17. Composition comprising the silk sericin as described in any of claims 1-7 or the hydrogel as described in any of previous claims 8-16.

18. Bioink, artificial extracellular matrix, patch or scaffold comprising the silk sericin as described in any of claims 1-7 or the hydrogel as described in any of previous claims

Images