Silk sericin-based hydrogel, methods and uses thereof

Abstract

The present disclosure relates to a novel sericin-based hydrogel wherein the silk sericin is enzymatically cross-linked for an improved treatment of wound healing, ischemic diseases or cardiovascular diseases, namely chronic wound healing, in particular diabetic wound.

Claims

1. A hydrogel comprising: at least 4% (w/v) of an enzymatically cross-linked silk sericin, wherein the silk sericin is enzymatically cross-linked by an enzyme complex selected from the group consisting of: horseradish peroxidase and hydrogen peroxide, laccase, transglutaminase, and mixtures thereof; wherein a cross-section of said hydrogel has pores with a diameter between 50-100 μm, and; wherein the hydrogel is suitable for medicinal applications, veterinary applications, cosmetic applications or as an in vitro model for cell culture studies.

2. The hydrogel of claim 1, wherein the hydrogel comprises at least 7.5% (w/v) of silk sericin.

3. The hydrogel of claim 1, comprising 4-20% (w/v) of an enzymatically cross-linked silk sericin.

4. The hydrogel of claim 1, comprising 6-7.5% (w/v) of an enzymatically cross-linked silk sericin.

5. The hydrogel of claim 1, wherein the silk sericin is enzymatically cross-linked by horseradish peroxidase and hydrogen peroxide for 10 seconds-5 minutes.

6. The hydrogel of claim 1, wherein the silk sericin is enzymatically cross-linked with 0.1-0.6% (w/v) of horseradish peroxidase and 0.15-0.4% (v/v) of hydrogen peroxide.

7. The hydrogel of claim 1, wherein the silk sericin has a molecular weight of 150-400 kDa.

8. The hydrogel of claim 1, wherein said hydrogel has an antioxidant activity of 0.03-0.05 eq [Asc. Ac.]g/L)/g.

9. The hydrogel of claim 1, further comprising a biological active agent, a therapeutic agent, an additive, a pharmaceutically acceptable excipient, a pharmaceutically acceptable carrier, and mixtures thereof.

10. The hydrogel of claim 9, wherein the additive is a surfactant selected from the following list: polysorbates, cationic molecules or polymers, and mixtures thereof.

11. The hydrogel of claim 9, wherein the additive is polysorbate 20, polysorbate 80 or poly-lysine.

12. The hydrogel of claim 9, wherein the biological active agent or the therapeutic agent is selected from the following list: antibiotic, coagulation agent, cell, stem cell, ligand, growth factor, platelet, and mixtures thereof.

13. The hydrogel of claim 12, wherein the antibiotic is vancomycin, streptomycin, ciprofloxacin, or mixtures thereof.

14. The hydrogel of claim 12, wherein the coagulation agent is thrombin or calcium.

15. The hydrogel of claim 1, wherein the hydrogel is transparent.

16. The hydrogel of claim 1, wherein the hydrogel is an injectable hydrogel or a topical hydrogel.

17. An adhesive or a patch comprising the hydrogel of claim 1.

18. A method for producing the hydrogel of claim 1, comprising the following steps: extracting silk sericin by immersing cocoons in water, at 100° C. for 40-60 min, in a ratio of 1-3% weight cocoons/volume of water; filtering the silk sericin; concentrating the silk sericin to at least 4% (w/v); preparing the hydrogel by adding 0.2% (w/v) of horseradish peroxidase, 0.3% (v/v) hydrogen peroxide and at least 4% (w/v) of silk sericin to water to form a mixture; and mixing the mixture for at least 10 seconds.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of disclosure.

(2) FIG. 1. Schematic illustration of sericin enzymatically crosslinked silk sericin-based hydrogel development and optimization.

(3) FIGS. 2A-2D. SEM micrographs of lyophilized sericin-based hydrogels at different resolutions FIGS. 2A and 2B at 500×; and FIGS. 2C and 2D at 1000×.

(4) FIGS. 3A-3C. Structural analysis and optical absorbance profile of the sercin hydrogels. FIG. 3A: Macroscopic image of the formed hydrogels; FIG. 3B: colorimetry evaluation; FIG. 3C: ATR-FTIR spectra of the Hydrogen peroxide, HRP, aqueous sericin solution, the mixture of sericin/HRP/H.sub.2O.sub.2 before gelation, and the final formed SHG.

(5) FIG. 4. Thermogram of sercin solution and sericin-based hydrogel.

(6) FIGS. 5A-5B. Oscillatory scans displaying both the elastic modulus (G′) and viscous modulus (G″) of sercin extraction solution (FIG. 5A), and enzymatically crosslinking hydrogel (FIG. 5B).

(7) FIG. 6. Swelling ratio profiles of the sericin-based hydrogel in PBS and Ultrapure water at 37° C.

(8) FIGS. 7A-7B. Degradation response of sericin-based hydrogels: pH degradation during 96 hours (FIG. 7A) and enzymatic degradation with protease XVI during 400 hours (FIG. 7B), both at 37° C.

(9) FIGS. 8A-8B. Ionic strength stimuli response of sericin-based hydrogels. The prepared hydrogel discs were alternatively immersed in: PBS and distilled water and each immersion lasted for 2 h (FIG. 8A) and 0.154M and 2M of sodium chloride solutions (FIG. 8B) (both of pH 7.4).

(10) FIG. 9. UV-Vis absorbance spectra of the materials. Kubelka-Munk function plot analysis used in the determination of band.

(11) FIGS. 10A-10D. Antimicrobial activity against Staphylococcus aureus ATCC 29213 (FIG. 10A), Staphylococcus aureus DSM 11729 (FIG. 10B), Pseudomonas aeruginosa (FIG. 10C) and Escherichia coli ATCC 25922 (FIG. 10D) for sericin solution and Macroscopic image of the well diffusion assay for each microrganism in 6 replicate hydrogels (Scale bar: 9 cm). For each figure (FIGS. 10A-10D), letters represent: (a) negative control, sericin without inoculum, (b) negative control, Muller-Hinton Broth without inoculum, (c) positive control, Muller-Hinton Broth with inoculum and (d) Sericin solution (0.67%) with inoculum.

(12) FIGS. 11A-11C. SEM micrographs of fibroblasts seeding in sericin-based hydrogels at different resolutions at 24 (FIG. 11A), at 48 h (FIG. 11B), at 72 h (FIG. 11C), top micrograph in each figure in 200× resolution and bottom micrographic in each figure in 1000×.

(13) FIG. 12. Micrographs of Live/Dead test, of fibroblasts seeding in sericin-based hydrogels at different resolutions after 72 h f seeding at 200× resolution and down figures in 1000×.

(14) FIG. 13. Micrographs of Live/Dead test, of fibroblasts seeding in sericin-based hydrogels at different resolutions after 72 h f seeding at 200× resolution and down figures in 1000×.

(15) FIG. 14. Effect of SHG on viability of L929 cell lines after 24 h, 48 h and 72 h. Tissue culture polystyrene (TCP) and DMSO were used as positive and negative controls respectively. The results were the mean of 4 replicates, bars represent standard deviation.

DETAILED DESCRIPTION

(16) In an embodiment, Bombyx mori cocoons were supplied by the Portuguese Association of Parents and Friends of Mentally Disabled Citizens (APPACDM, Castelo Branco, Portugal). The other materials and reagents were purchased from Sigma-Aldrich (St Louis, Mo., USA) unless mentioned otherwise.

(17) In an embodiment, the preparation of silk solution and sericin-based hydrogels was conducted by sericin extraction by boiling, filtration and concentration to different weight ratios (i.e. 4, 7.5 and 7.8% (m/v)). Solutions were filtered and optimized at a final concentration of 7.5%. The hydrogel was prepared by adding different volumes and concentrations of HRP and hydrogen peroxide until gelation occurred (FIG. 1). The final formulation was fixed using 0.2% (m/v) HRP, 0.3% (v/v) hydrogen peroxide and 7.5% m/v of sericin, and then mixed for 15 seconds.

(18) In an embodiment, the sericin quantification by dry weight was performed as follows: dry weights were determined by placing sample aliquots of sericin solution and hydrogel at 105° C. to a constant weight. In the end, the total solids were calculated according the equation:

(19) $\mathrm{Dry}\mathrm{weight}\left(\%\right)=\frac{\left(\mathrm{Wd}-\mathrm{Wc}\right)}{\left(\mathrm{Ww}-\mathrm{Wc}\right)}\times 100$

(20) In equation, Wd refers to the dry weight of sample with container; Ww means wet weight of sample with container and We refers to the weight of container.

(21) In an embodiment, the sericin quantification by dry weight was determined and values of 0.67±0.05 and 7.54±0.50(%) were obtained for both, extracted sericin solution and final hydrogel formulation, respectively.

(22) In an embodiment, the microstructure evaluation and porosity analysis of the sericin-based hydrogel were performed by scanning electron microscopy (SEM). For this purpose, hydrogels were cast in moulds and immersed in liquid nitrogen to avoid the development of ice crystals, before freeze-drying. SEM was operated at low vacuum mode, using a spot size of 27-28 and a potential of 30 kV. All analyses were performed at room temperature, in particular at 20° C.

(23) In an embodiment, the morphology and microstructure of the sericin-based hydrogel were examined by SEM at different resolutions (FIGS. 2A-2D). Prior to the analyses, the specimens were immersed in nitrogen to avoid the formation of ice crystals and then lyophilized. Among the cross-section of the sericin hydrogels were observed pores, showing the potential permeability of the hydrogels. From the obtained images, pores were noted with approximately 50 μm.

(24) In an embodiment, the structure and chemical analysis of sericin-based hydrogel were performed: sericin solution, HRP, hydrogen peroxide solution and the formed sericin-based hydrogels were further analysed by Infrared spectroscopy analysis in a Spectrum Series, Perkin Elmer FTIR spectrometer (ABB, Switzerland) equipped with attenuated total reflectance (ATR) sampling accessory (PIKE Technologies, USA), and a diamond/ZnSe crystal. All spectra were acquired using 16 scans and a 4 cm.sup.−1 resolution in the region of 4000-700 cm.sup.−1. In addition, baseline—point adjustment and spectra normalization was performed. PBS solution was used as background in the FTIR. All samples used were and run in triplicate, and the data presented were the average of the three measurements.

(25) In an embodiment, sericin-based hydrogel was successfully developed via HRP mediated cross-linking in physiological condition and presented transparent appearance, as showed in FIG. 3A. The hydrogel color was evaluated (FIG. 3B) using a portable Chroma meter CR-400 (from Minolta Chroma, Osaka, Japan). A CIELab color scale was employed to measure the degree of lightness (L), redness (+a) or greenness (−a), and yellowness (+b) or blueness (−b) of the films, under D65 (daylight). Hydrogel specimens were measured on the surface of the white standard plate, with color coordinates Lstandard=97.6, astandard=0.01 and bstandard=1.60. The color of the hydrogel was expressed as the total difference in color (DE), three samples were taken e and, on each hydrogel piece, four readings were made on each side, as previously described. Apparently as it can be seen in (FIG. 3A) sericin-based hydrogel were transparent, flexible and homogeneous. Their surfaces appeared smooth, without visible pores or cracks.

(26) The ATR-FTIR spectra (FIG. 3C) demonstrated that one of the absorbance peaks of the hydrogels was at 1549 cm.sup.−1, at the same position with the ones from the sericin solution and the mixed solution of sericin/HRP/H.sub.2O.sub.2, which were located also at 1549 cm.sup.−1. Additionally, all these three spectra presented also absorbance peaks at 3383 cm.sup.−1. The results are in accordance with previous similar works based on different sericin hydrogel or considering other silk based-hydrogels.

(27) In an embodiment, the differential scanning calorimetry analysis of the sericin-based hydrogel was conducted: thermograms were obtained using a DSC (DSC-60, Shimadzu, Columbia, USA). Hydrogels were prepared and kept at refrigerated conditions (4° C.) for 24 h, than 5.0 mg of the hydrogel were crimped in a standard aluminium pan and heated from 20° C. to 350° C. at a heating constant rate of 10° C./min under constant purging of nitrogen at 20 mL/min. All samples were run in triplicate and data presented were the average of the three measurements.

(28) In an embodiment, the similar thermal behavior was observed in both thermograms of sericin-based hydrogel and sericin solution (FIG. 4), respectively. In the sericin solution curve, an endothermic peak of 51.7° C. was observed, while in the sericin-based hydrogel an endothermic peak of 61.5° C. was shown. In the sericin solution one peak at low temperature (round 75° C.), may be due to the evaporation of water. In the same curve it was not observed at high temperatures (below 200° C.) any peak like it has been described in literature (endo peak at 212° C.), attributed to thermal decomposition of sericin with oriented n-sheet configuration. This confirms the amorphous status of sericin in the extraction solution. The thermal difference (of 10° C.) in sericin solution and sericin based hydrogel may be due to the enzymatically crosslinking effect, that turns this final formulation more thermal stable.

(29) In an embodiment, mechanical tests of the sericin-based hydrogel was performed. The compression and perforation properties of hydrogels, were measured according to the Texture Analyser TXT plus from Stable Micro Systems (UK). The Exponent software was used for Ottawa cell with 6.5 mm holes, and 2 mm Dia cylinder stainless method. The hydrogels samples were prepared in a 24 wells plate (diameter of 1.5 cm and thickness of 3 mm). At least three disks of each hydrogel sample were analysed.

(30) Regarding the compression and perforation mechanical tests, sericin-based hydrogel were compared with well-described agar hydrogels at 1%. For the same distance (1.286 mm), and for 6 replicates of each formulation.

(31) This result clearly opens a new lack of opportunities for this novel hydrogel considering applications such as wound dressings, or other medical applications that requires resistant hydrogels, with good mechanical proprieties.

(32) In an embodiment, the rheology assessment of the sericin-based hydrogel was conducted. Oscillatory shear rheological tests were performed using a controlled stress rheometer (CS-50, Bohlin Instruments, Cranbury N.J., USA), with a cone-and-plate geometry (diameter 40 mm and angle 2°) fitted at 115 μm gap. The temperature of the bottom plate was controlled with a Peltier system. Immediately after preparation approximately 1.5 mL of the solution was transferred to the rheometer equilibrated at 25° C. Testing was then performed at low strain amplitude (1%) and low frequency (1 Hz), within the linear viscoelastic range—as assessed by stress and frequency sweep experiments, respectively. The gel development was assessed by measuring the storage modulus (G′) and the loss modulus (G″) through time sweep experiments (25° C., 1 Hz and 1%) carried out for approximately 1 h.

(33) In an embodiment, in vitro swelling ratio and degradation profile of the sericin-based hydrogel was performed. The prepared sericin-based hydrogel discs in 96 wells microplate, with 100 μL in each well, were used for the swelling and degradation study. The swelling ratios of the hydrogels were tested in both ultrapure water and PBS at 37° C. Each piece of hydrogel was placed in an eppendorf with 1 mL PBS or ultrapure water (0.55 μS/cm) prepared by a ultrapure water system (Millipore Q, Advantage A10, Germany), subsequently the samples were placed in a thermostatic water bath (Dubinoff bath BSD/D, Cambridgeshire, UK) at 37° C. The wet weight of the sample was measured at 1, 2, 4, 6, and 24 hours. Before weighting, surface liquid in the hydrogels were absorbed by tissue. The swelling ratio at each time point was calculated as following equation 1:

(34) $\mathrm{Swelling}\mathrm{ratio}\left(\%\right)=\frac{\mathrm{wt}-\mathrm{wd}}{\mathrm{wd}}\times 100$

(35) In Equation 1, wt referred to the wet weight of the sample tested in different time point, and wd is the dry weight of the sample. It was assumed that the dry weight of each specimen was constant during the tested time-period.

(36) FIG. 6 showed the swelling behavior of the sericin hydrogel in PBS and ultrapure water. The sericin hydrogel reached the maximum swelling profile after 4 hours immersion in ultrapure water higher than in PBS. Sericin hydrogel presented a high swelling ratio of 21% in water and 15% when immersed in PBS. Higher swelling profiles are documented in other silk-based hydrogels. Nonetheless, the sericin hydrogel are more stable in water and PBS, regarding physiological conditions for long periods of time than other silk-based hydrogels, as it can be described in enzymatic degradation profile (FIG. 7B).

(37) In an embodiment, for degradation assay hydrogels were tested in both PBS and PBS with Protease XIV from Streptomyces griseus at physiological concentration of 3.2 U/mg, according to previous works (30). Each piece of hydrogel was placed in an Eppendorf with 1 mL of each solution and then samples were placed in a thermostatic water bath (Dubinoff bath BSD/D, Cambridgeshire, UK) at 37° C. The wet weight of the sample was measured at 1, 2, 4, 6, 24, 48, 72, 96, 168, 240 and 312 hours. The degradation ratio at each time point was calculated as following equation 2:

(38) $\mathrm{Weight}\mathrm{loss}\left(\%\right)=\frac{\mathrm{wi}-\mathrm{wt}}{\mathrm{wt}}\times 100$

(39) In equation 2, wi means the initial wet weight of the hydrogel, and wt is the wet weight tested at each point.

(40) In an embodiment, the pH responsiveness of the sericin-based hydrogels was performed. Sericin-based hydrogel discs in 96 wells microplate, with 100 μL in each well were immersed in 0.154 M NaCl solution (pH 7.4) (Panreac) at 37° C. overnight. The wet weights of the hydrogels were measured and then the hydrogels were immersed in 1 mL of NaCl solutions at different pH values (37° C.): 2.5, 5.5, 6.5 and 8.5. These pH values were selected according the pH values of healthy skin or injured skin. Healthy skin or acute wounds exhibit slightly acidic pH (pH values of 5.5-6.5) and chronic wounds have pH values higher than 7.4 caused by the microbial proliferation. Occasionally, the irregular vascularization of chronic wound is responsible for a heterogeneous dissemination of infection in the wound bed, causing drastic pH variations (31). The pH values were adjusted by addition of NaOH 1M (Merck, Darmstad, Germany) or HCl 1M (Merck, Darmstad, Germany). As control, the hydrogels were also immersed in methanol to undergo n-sheet conversion. After 2 h, 24 h, 96 h the wet weights of the hydrogels were recorded again after removing surface liquid. The wet weight variation was calculated as equation 3.

(41) $\mathrm{Wet}\mathrm{weight}\mathrm{variation}\left(\%\right)=\frac{\mathrm{wt}-\mathrm{wi}}{\mathrm{wi}}\times 100$

(42) In equation 3, wi means initial weight of the hydrogel after overnight immersion in 0.154 M sodium chloride solution, and wt refers to the wet weight tested during alternating immersion. The prepared discs were also immersed in methanol for 3 h to undergo 0 sheet conversion and then the opaque hydrogels were used as control for the response test as well as for swelling ratio and degradation tests.

(43) The hydrogels were submitted at different pH values similar to acute and chronic wounds (FIG. 7A). At pH values between 2.5 and 6.5, it was observed a slight gel shrinking during the time tested, at pH value of 8.5 was detected an higher reduction (nearly 30%). However, in all cases only a reduction between 7-12% was observed after 24 h. These results demonstrated that the hydrogel is pH resistant comparing to other studies made with other silk hydrogels. Our pH responsiveness assay was performed for 4 days, being superior to previous studies. Therefore, sericin gel demonstrated to be a promising biomaterial for acute as well as for chronic wounds.

(44) The FIG. 7B shows the enzymatic degradation profiles of sericin-based hydrogel. It was observed that these hydrogels degraded over 72 h in physiological protease concentration (3.5 U/mg) degraded only 40% at 37° C. Moreover and in PBS at 37° C. the formulations were stable for 16 days with a degradation of 91% (w/w). These results were higher than similar silk fibroin hydrogels also mediated by HRP/H.sub.2O.sub.2, in which the complete degradation was over 6 h, considering a smaller concentration of protease (e.g. 0.0005 U/mL).

(45) The FIG. 7B also shows the enzymatic degradation profile of sericin-based hydrogel, simulating acute and chronic wounds with a protease concentration 3× times higher (10.5 U/mg) than the physiological described in literature (of 3.5 U/mg). It was observed that these hydrogels degraded completely only in 6 hours even in this acute enzyme concentration. This results stills notable regarding other crosslinked silk hydrogels.

(46) The results of the ionic strength responsive are presented in FIGS. 8A-8B. According these data, the weight of sericin-based hydrogels varied with the osmotic pressures of these specimens in water and PBS. In water, it was observed a weight increase, which is in accordance to the swelling ratios. The variations in the weight were reversible in this system. In order to also test the influence of different ions, hydrogels were alternately immersed in a solution of 0.154 M and 2.0 M NaCl. The weight of hydrogel diminished around 9% when the specimens were changed of a solution of 2M to a 0.154 M. Moreover, after this weight variation, the hydrogel remained stable even with the molarities alterations, which showed that the system was not reversible in this ionic strength disparity.

(47) In an embodiment, the ionic strength responsiveness of the sericin-based hydrogel was performed. Sericin-based hydrogel discs in 96 wells microplate, with 100 μL in each well were used for the ionic strength response assay. Firstly, the hydrogels were immersed in 1 mL of PBS at 37° C. overnight and the wet weight of each hydrogel was measured. After this procedure, hydrogels were alternately placed in deionized water and PBS every 2 h during 8 h and the wet weights of the hydrogels were recorded. For the second part of the test, the hydrogels were immersed at 37° C. overnight in 1 mL 0.154M sodium chloride solution (pH 7.4, adjusted by 1.0 M NaOH) and the wet weight of each hydrogel was noted. Then, hydrogels were alternately immersed in 1 mL 2 M sodium chloride (pH 7.4) and 0.154 M sodium chloride solution (pH 7.4) every 2 h during 8 h. Before every change of solution, the wet weights of the hydrogels were measured. All the samples were maintained in the thermostatic water bath at 37° C. The wet weight variation ratio was calculated according to previous equation 3.

(48) In an embodiment, the UV assays were performed using UV-vis spectra were taken using a Shimadzu UV 3100 spectrometer equipped with an integrating sphere, covering a wavelength range between 250 and 850 nm (0.2 nm step-size, BaSO.sub.4 as the reference).

(49) In an embodiment, in order to investigate hydrophilic/hydrophobic nature of hydrogels, the water contact angle (WCA) was measured using an Optical Contact Angle (Dataphysics, Germany). WCA was performed using a sessile drop with a volume of 3 μL and measured with the OCA 20 software according Young-Laplace model.

(50) In an embodiment, sericin being more hydrophilic than silk fibroin (due to higher content of polar amino acids including serine, aspartic acid and glutamic acid), silk films become more hydrophilic with increasing sericin content, which results in lower contact angles (i.e. 33.23±4.48 degrees).

(51) In an embodiment, the antioxidant activity was determined, in particular the free radical-scavenging activity was determined by 2,2-azinobis-3-ethylbenzo thiazoline-6-sulphonic acid (ABTS) radical decolourization assay. The radical cation was produced by reacting ABTS with potassium persulfate. The antioxidant potential was measured according to the percentage of inhibition (PI), to be between 20% and 80%, to guarantee a linear response of the analytical method, after 6 min of reaction with 1 mL of diluted ABTS+ solution. Calibration curve was prepared with ascorbic acid in the range of 0.018-0.125 mg/mL and all the determinations accomplished in triplicate. In this assay, sericin solution after extraction and sericin-based hydrogel under physiological protease (3.5 U/mg) degradation after 24 h at 37° C., were used to evaluate the antioxidant potential. The total antioxidant capacity was expressed as mg ascorbic acid equivalent/mL.

(52) In an embodiment, the determination of the antioxidant capacity was performed by ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic) acid) diammonium salt test (SigmaAldrich, St. Louis, Mo., USA). The antioxidant potential was measured according to the percentage of inhibition (PI), to be between 20% and 80%, to guarantee a linear response of the analytical method, after 6 min of reaction with 1 mL of diluted ABTS•+ solution. The total antioxidant capacity was expressed in (eq [Asc. Ac.]g/L)/g extract. For this purpose sericin solution and sericin-based hydrogel were monitored to evaluate the antioxidant potential after sericin extraction, and after 24 h at 37° C. under physiological protease (3.5 U/mg) degradation.

(53) In an embodiment, the results have shown that even considered only a 20% of hydrogel degradation (according to the in vitro degradation profile, mentioned before) the sericin hydrogel showed antioxidant activity values of 0.053±0.002 (eq [Asc. Ac.]g/L)/g extract). In the other hand sericin solution showed antioxidant activity values of 0.032±0.008 (eq [Asc. Ac.]g/L)/g extract). There results clearly highlight the high antioxidant activity potential of sericin and of these novel crosslinked sericin hydrogels.

(54) In an embodiment, Staphylococcus aureus DSM 11729, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa, and Escherichia coli ATCC 25922 were obtained by collection culture of CBQF, Catholic University of Portugal. The isolates were growth aerobically on Nutrient agar (Merck, Darmstad, Germany) at 37° C. for 24 h.

(55) In an embodiment, the antimicrobial activity of sericin solution was determined using an inoculum of 0.5 MacFarland (1.5×10.sup.8 CFU/mL) of each bacteria. Muller-Hinton broth (Biokar) 180 μL with sericin solution with 0.67% was inoculated with 20 μL of each bacteria. Three controls were simultaneously assessed, one with 0.67% of sericin solution without inoculum, other with the Muller-Hinton broth with the inoculum and without the inoculum, each in triplicate. The absorbance was measured in a microplate reader (Fluostar Optima, BMG Laptech) at 620 nm during 24 h at 37° C.

(56) In an embodiment, the screening of antimicrobial activity of sericin-based hydrogels was performed by well diffusion assay. Plates of Nutrient Agar were seeded with an inoculum of 0.5 MacFarland (1.5×10.sup.8 CFU/mL) of each bacteria. Wells with a diameter of 4 mm were punctured into the plates and filled with 20 μL of hydrogel. Plates were incubated for 24 h at 37° C. The presence or absence of translucent halo zones around the wells were considered as positive or negative for antimicrobial activity, respectively. Control negative was made with Ringer's solution and assays were done in triplicate.

(57) In an embodiment, a mouse fibroblast cell line (L929), acquired from the European Collection of Cell Cultures (ECACC, United Kingdom) and used at passages 30-32. Cells were grown as monolayer cultures in Dulbecco's Modified Eagle's Medium (DMEM; Sigma Aldrich; Germany) supplemented with 10% fetal bovine serum (FBS; Biochrom, Germany) and 1% antibiotic-antimycotic liquid (Gibco, UK). The cells were incubated in a CO.sub.2 incubator under an atmosphere of 5% CO.sub.2 at 37° C., with medium change every two days. At confluence cells were detached from the culture flasks using TrypLE Express (1×) (Life Technologies, Carlsbad, Calif., USA), centrifuged, resuspended in the cell-culture medium, and seeded in the hydrogel at a density of (1.0×10.sup.4 cells/mL). The cell-seeded hydrogel were also incubated at 37° C., 5% CO.sub.2 and 95% humidity, for 1, 2, and 3 days. Tissue culture polystyrene (TCPS; Sarstedt) coverslips and SF membranes were used as control surfaces (33).

(58) In an embodiment, sericin-based hydrogel were prepared in aseptic conditions, in a sterile cabinet and used for the cell encapsulation. A warmed mixture (500 μL) was mixed with the cell pellet (cell suspension containing: 1.0×10.sup.4 cells/mL) and got a homogeneous cell suspension, and every 50 μL of the cell suspension was transferred into one piece of cover slips with 13 mm diameter (Sarstedt, Newton, N.C., USA) in a 24-well suspension cell culture plate. The plate was then placed into the CO.sub.2 incubator for around 10 minutes to allow the gelation. After the gel was formed, 1 mL (DMEM) alpha medium was supplemented into each well, and the medium was changed every two days. The Live/Dead of the incorporated cells was evaluated by Calcein AM and propidium iodide (Molecular Probes*; Life Technologies, Carlsbad, Calif., USA) staining, after culturing for 1, 2 and 3 days. For this assay, the hydrogels with cells were washed by PBS, and then immersed in 1 mL PBS supplemented with 1 μg Calcein AM and 2 μg propidium iodide for 30 minutes. The samples were observed in a transmitted and reflected light microscope (Axio Imager Z1m, Zeiss, Jena, Germany) after washing by PBS (34). The effect of hydrogels on cell viability was measured at selected concentrations using the methylthiazolyldiphenyl-tetrazolium bromide conversion (MTT) assay at 1, 2 and 3 days. For this assay, 100 μL MTT (5 mg/mL in PBS) working solution was added into each well, followed by incubated for 2 hours. The absorbance of 50 μL supernatant from each well was read in a microplate reader (Fluostar Optima, BMG Laptech) at 570 and 690 nm. Hydrogels without cells were used as control. Each treatment was tested in four individual wells. The negative control used, was also DMSO. The plates were shaken on an orbital shaker to solubilize the crystals of formazan. The hydrogels encapsulated with cells were frozen and then lyophilized, after culturing for 3 days, respectively. The morphology of the hydrogels was observed by scanning electron microscopy (SEM). (JEOL-5600 Lv microscope, Japan) observation, the samples after coated with a layer of Au/Pd SC502314B in an evaporator coater (E6700, Quorum Technologies, East Grinstead, UK).

(59) In an embodiment, the MTT test demonstrates biocompatibility and capacity for cell encapsulation of the hydrogel disclosed in the present subject matter.

(60) In an embodiment, the hemocompatibility studies demonstrate excellent blood compatibility, exhibiting low hemolysis ratio, anti-coagulant and anti-thrombogenic activities of the hydrogel disclosed in the present subject matter.

(61) Throughout the description and claims the word “comprise” and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Additional objectives, advantages and features of the solution will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the solution.

(62) 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.

(63) It will be appreciated by those of ordinary skill in the art that unless otherwise indicated herein, the particular sequence of steps described is illustrative only and can be varied without departing from the disclosure. Thus, unless otherwise stated the steps described are so unordered meaning that, when possible, the steps can be performed in any convenient or desirable order.

(64) 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.

(65) The above-described embodiments are combinable.

(66) The following claims further set out particular embodiments of the disclosure.

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