Hydrogels, methods of making and uses thereof

Abstract

The present application relates to a kind of hydrogel and its preparation method and application.Hydrogel is formed by the cross-linking of structural protein, wedge reagent and cross-linking agent, and the wedge reagent is the same or similar to the hydrophobic crystalline region of structural protein in hydrophobic property and forms hydrogen bond interaction with the hydrophobic crystalline region of structural protein.The present application can effectively inhibit the self-assembly of structural protein molecules by cross-linking wedge reagent and structural protein molecules to form hydrogel, so that the hydrogel maintains excellent light transmission and mechanical properties.

Description

TECHNICAL FIELD

[0001] The present application relates to a hydrogel and a preparation method and application thereof. BACKGROUND

[0002] Hydrogel is a kind of high water absorption and high water retention material formed by chemical cross-linking or physical cross-linking of high molecular materials, which is widely used in drug delivery, tissue regeneration materials, medical diagnosis and other fields. Physical cross-linking is the combination of intramolecular or intermolecular through hydrogen bond, polar bond and other physical forces to form a network structure, while chemical cross-linking is the formation of new polymer structure by chemical bond or chemical reaction between two or more compounds. These high molecular polymers can be divided into two categories: natural and synthetic. Natural high molecular polymers have higher biological safety and biocompatibility due to their green source and green processing, and are paid more and more attention in the field of health care. The physical cross-linking process is difficult to control, and the gel has high hardness but insufficient toughness; the chemical cross-linking mechanism has strong designability, and the cross-linking process is controllable and the gel has excellent elasticity. However, some structural proteins, such as silk protein, collagen, fibrin and keratin, can form secondary structure cross-linking through intramolecular or intermolecular matrix, which can destroy the excellent properties of chemical cross-linking hydrogel, such as light transmittance and elasticity. Xiaolin Cui et al. (Rapid Photocrosslinking of Silk Hydrogels with High Cell Density and Enhanced Shape Fidelity) improved the efficiency of bis-tyrosine cross-linking reaction by a new type of photocatalysis, thereby improving the stability of enzyme-catalyzed (HRP / H2O2) silk fibroin structure. Taowang et al. (CN113549227A, Biomaterials, 2022:121611. Intraarticularly injectable silk hydrogel microspheres with enhanced mechanical and structural stability to attenuate osteoarthritis.) greatly improved the types of amino acids in silk fibroin molecules involved in cross-linking reaction by a new type of BDDE cross-linking reaction, which greatly improved the structural stability of silk fibroin chemical cross-linking hydrogel. However, after the hydrogel was soaked in 80% ethanol for 72 hours to accelerate aging, the content of β-sheet secondary structure was significantly increased, and the mechanical properties and light transmittance of the hydrogel were decreased.

[0003] The structural protein hydrogel prepared by enzyme cross-linking, photo cross-linking, diglycidyl ether and the like cannot maintain high light transmittance, mechanical stability and secondary structure stability for a long time. For example, in Advanced healthcare materials, 2020, 9(4): 1901667. Rapid photocrosslinking of silk hydrogels with high cell density and enhanced shape fidelity, the compression modulus of the silk fibroin hydrogel cross-linked by HRP / H2O2 is increased from 18 KPa to 166 KPa (P<0.0001) after two weeks, and the beta-sheet content of the hydrogel is increased from 30.7±3.3% to 42.7±2.7% after 35 days of cross-linking. Or after treatment by ethanol incubation, methanol incubation, high temperature treatment and the like, the light transmittance, mechanics and secondary structure content of the hydrogel will change significantly. For example, in Intraarticularly injectable silk hydrogel microspheres with enhanced mechanical and structural stability to attenuate osteoarthritis, the beta-sheet content of the silk fibroin hydrogel cross-linked by HRP / H2O2 is increased from 14.31±2.25% to 49.23±6.75% after 80% methanol incubation for 60 min. This phenomenon will affect the use of the structural protein hydrogel in the field of tissue engineering and regenerative medicine, such as artificial cornea, ok mirror and the like, which require the hydrogel to have high light transmittance and be able to maintain high light transmittance for a long time; and the like, which require the hydrogel to have stable mechanics. The secondary structure will directly affect the size of the mechanics and light transmittance, and high beta-sheet content will cause the hydrogel to have large hardness, poor light transmittance and slow degradation rate, and the hardness of the hydrogel will affect cell activity, such as cell differentiation, hydrophilicity and availability of cell binding sites in the hydrogel network. SUMMARY

[0004] The purpose of the present application is to improve a hydrogel with good stability and a preparation method and application thereof.

[0005] To achieve the above purpose, the technical solution adopted by the present application is:

[0006] A hydrogel is formed by cross-linking a structural protein, a wedge reagent and a cross-linking agent, wherein the wedge reagent is a substance with the same or similar hydrophilic or hydrophobic properties as the hydrophobic crystalline region of the structural protein and forms a hydrogen bond with the hydrophobic crystalline region of the structural protein.

[0007] The existing structural protein hydrogel has the following problems: first, the light transmittance of the structural protein hydrogel is low or the light transmittance stability is poor, for example, the light transmittance decreases with time, and for another example, the light transmittance decreases obviously after alcohol or high temperature treatment; second, the mechanical stability of the structural protein hydrogel is poor (compression modulus, shear modulus, elastic modulus, etc.); third, the secondary structure stability of the structural protein hydrogel is poor. In the present application, the wedge reagent is used, which has the same or similar hydrophilic or hydrophobic properties as the core region of the secondary structure of the structural protein, and forms a hydrogen bond interaction with the molecular fragments in the region, which can effectively inhibit the molecular assembly of the core region of the secondary structure of the structural protein; and the wedge reagent is cross-linked with the structural protein molecules to form a composite hydrogel, which can effectively inhibit the self-assembly of the structural protein molecules, so that the hydrogel still maintains excellent light transmittance and mechanical properties after alcohol treatment (accelerated aging), and has a wider application prospect in various fields such as drug delivery, tissue regeneration materials, medical diagnosis, etc.

[0008] Preferably, the crystal size of the hydrogel is less than or equal to 5 nm after soaking in deionized water for 3 days, and the crystal size of the hydrogel is less than or equal to 7 nm after soaking in 80% ethanol for 3 days.

[0009] Preferably, the light transmittance of the hydrogel is more than 75% of the initial light transmittance after dialysis in deionized water for 3 days and then soaking in 80% ethanol for 3 days, and the compression modulus loss rate is less than or equal to 35% after 1000 cycles of compression at a compression deformation of 60%.

[0010] Further preferably, the light transmittance of the hydrogel is more than 85% of the initial light transmittance after dialysis in deionized water for 3 days and then soaking in 80% ethanol for 3 days, and the compression modulus loss rate is less than or equal to 25% after 1000 cycles of compression at a compression deformation of 60%.

[0011] In some preferred embodiments, the initial light transmittance of the hydrogel with a thickness of 1 mm is not less than 60%, further not less than 70%, and more further not less than 80%.

[0012] In some preferred embodiments, the initial compression modulus loss rate of the hydrogel is less than or equal to 20% after 1000 cycles of compression at a compression deformation of 60%, for example, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, etc. Wherein, the initial compression modulus loss rate can be understood as the compression modulus loss rate of the newly prepared hydrogel.

[0013] Preferably, the water contact angle of the wedge reagent is θ1, and the water contact angle of the structural protein is θ2, and the range of θ1 is (θ2-25.5°)~(θ2+25.5°).

[0014] Preferably, the wedge agent is selected from one or more of the following: cellulose, high molecular polymer, inorganic substance, modified material.

[0015] In some specific embodiments, the cellulose includes one or more of the following: methyl cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, lignocellulose. The molecular weight of the cellulose is not specifically limited, and preferably, the molecular weight of the cellulose is 100-1000 KDa.

[0016] In some specific embodiments, the high molecular polymer includes one or both of the following: polyvinyl alcohol, poly-L-glutamate sodium.

[0017] In some specific embodiments, the inorganic substance includes carbon nanotube.

[0018] In some specific embodiments, the modified material includes one or more of the following: lignin sulfonate, hydrophobically modified hyaluronic acid, hydrophobically modified hydroxyethyl cellulose, hydrophobically modified gelatin. In the absence of specific instructions, the hydrophobic modification is modified according to conventional methods or the modified material can be obtained by purchase. Among them, the lignin sulfonate includes sodium lignosulfonate; the hydrophobically modified hyaluronic acid includes one or both of adipic acid dihydrazide grafted hyaluronic acid, methacrylated hyaluronic acid; the hydrophobically modified hydroxyethyl cellulose includes dodecyl succinic anhydride hydrophobically modified hydroxyethyl cellulose; the hydrophobically modified gelatin includes methacrylated gelatin.

[0019] Compared with the hydrogel without the addition of the wedge agent, the addition of the wedge agent can improve the light transmittance of the hydrogel, and as the proportion of the wedge agent increases, the light transmittance has an upward trend, but when the proportion of the wedge agent further increases, the light transmittance will decrease. Preferably, the wedge agent accounts for 0.05% or more of the total mass of the structural protein and the wedge agent, and the hydrogel has better light transmittance and stability. Further preferably, the wedge agent accounts for 1-50% of the total mass of the structural protein and the wedge agent, such as 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, etc.

[0020] Preferably, the hydrogel is first formed by contacting the wedge agent and the structural protein in the presence of a solution capable of destroying the secondary structure of the structural protein, and then cross-linked with the cross-linking agent.

[0021] Preferably, the structural protein includes one or more of the following: collagen, fibrin, silk fibroin, sericin, keratin. The collagen may, for example, be collagen type I, collagen type II, collagen type III, collagen type IV, collagen type V, collagen type VI. The keratin may, for example, be wool keratin, feather keratin.

[0022] Preferably, the cross-linking agent is one or more of an organic solvent-based cross-linking agent, an enzymatic cross-linking, an activator, a photo-cross-linking agent.

[0023] Further preferably, the organic solvent-based cross-linking agent includes one or more of a diglycidyl ether cross-linking agent, such as butanediol diglycidyl ether; the enzymatic cross-linking includes horseradish peroxidase and hydrogen peroxide; the activator includes one or both of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide; and the photo-cross-linking agent includes glycidyl methacrylate.

[0024] A second object of the present application is to provide a method for preparing the hydrogel as described above, comprising the following steps:

[0025] (1) mixing a wedge reagent and a structural protein with a solution capable of destroying the secondary structure of the structural protein;

[0026] (2) adding a cross-linking agent to the mixture of step (1) to perform a cross-linking reaction to prepare the hydrogel.

[0027] Preferably, the solution capable of destroying the secondary structure of the structural protein includes one or more of a lithium bromide solution, an ionic liquid solution, a urea solution, a guanidine hydrochloride solution, an SDS solution, a calcium chloride-ethanol-water solution, and the like. As a preference, the solution is an aqueous solution.

[0028] Preferably, the mass concentration of the structural protein in the mixture is 0.5-30%. The present inventors have found that as the mass concentration of the structural protein increases, the compression modulus of the hydrogel shows a rising trend, and when the mass concentration of the structural protein is greater than 30%, the hydrogel is fragile.

[0029] In some preferred embodiments, the method for preparing specifically comprises the following steps:

[0030] S1, mixing one of a structural protein and a wedge reagent with a solution capable of destroying the secondary structure of the structural protein to obtain a first mixture;

[0031] S2, mixing the other with the first mixture or mixing the other with the solution capable of destroying the secondary structure of the structural protein and then mixing the other with the first mixture to obtain a second mixture containing the structural protein and the wedge reagent;

[0032] S3, adding a cross-linking agent to the second mixture to perform a cross-linking reaction to prepare the hydrogel.

[0033] Preferably, the mixing temperature of S1 is 4-200°C, further 15-180°C, and more further 20-150°C.

[0034] Preferably, the mixing temperature of S2 is 4-200℃, further 15-180℃, and more further 20-150℃.

[0035] Preferably, the temperature of the cross-linking reaction is -80-120℃, further -80-100℃, and more further -80-80℃.

[0036] Preferably, the time of the cross-linking reaction is 3min-96h, further 5min-72h, and more further 5min-60h.

[0037] In some embodiments, the preparation method specifically comprises the following steps:

[0038] 1) mixing the structural protein with a solution capable of destroying the secondary structure of the structural protein under light-proof condition to obtain a mixed solution A;

[0039] 2) mixing the cross-linking agent with the first mixed solution and then with the wedge reagent under light-proof condition to obtain a mixed solution B;

[0040] 3) adding the photo initiator to the mixed solution B and performing cross-linking reaction under light irradiation to prepare the hydrogel.

[0041] Preferably, the photo initiator is a LAP initiator.

[0042] Preferably, the preparation method further comprises dialysis of the hydrogel in deionized water.

[0043] A third object of the present application is to provide a gel ball formed by cross-linking of a structural protein, a wedge reagent and a cross-linking agent, wherein the structural protein, the wedge reagent and the cross-linking agent are respectively the structural protein, the wedge reagent and the cross-linking agent as described above.

[0044] Preferably, the particle size of the gel ball is 1-1000μm.

[0045] Preferably, the gel ball is formed by cross-linking of the wedge reagent and the structural protein in the presence of a solution capable of destroying the secondary structure of the structural protein, and then with the cross-linking agent.

[0046] A fourth object of the present application is to provide a preparation method of the gel ball as described above, comprising the following steps:

[0047] Step 1, mixing the wedge reagent, the structural protein and a solution capable of destroying the secondary structure of the structural protein;

[0048] Step two, add the cross-linking agent to the mixed solution of step one to obtain a mixed solution containing the cross-linking agent, and drop the mixed solution containing the cross-linking agent into the oil phase in motion to react and prepare the gel ball.

[0049] Preferably, the preparation method further comprises filtering the gel ball from the oil phase, washing the surface with an organic solvent, and then washing with water and filtering.

[0050] A fifth object of the present application is to provide an artificial cornea substitute or auxiliary material formed by cross-linking a structural protein, a wedge agent and a cross-linking agent.

[0051] Preferably, the artificial cornea substitute or auxiliary material is formed by cross-linking the wedge agent and the structural protein after being contacted in the presence of a solution capable of destroying the secondary structure of the structural protein, and then the cross-linking agent.

[0052] A sixth object of the present application is to provide a preparation method of the artificial cornea substitute or auxiliary material as described above, comprising the following steps:

[0053] Step one, mixing a wedge agent, a structural protein and a solution capable of destroying the secondary structure of the structural protein;

[0054] Step two, adding a cross-linking agent to the mixed solution of step one to obtain a mixed solution containing the cross-linking agent, and dropping the mixed solution containing the cross-linking agent into a mold to react and prepare the artificial cornea substitute or auxiliary material.

[0055] A seventh object of the present application is to provide an application of the hydrogel as described above or the gel ball as described above in the field of tissue engineering filling or repair, drug delivery. The tissue engineering filling or repair includes joint lubrication, joint cartilage repair or corneal repair and shaping, or other vision correction or ophthalmic disease treatment compositions.

[0056] Due to the use of the above technical solutions, the present application has the following advantages compared with the prior art:

[0057] The present application can effectively inhibit the self-assembly of structural protein molecules by cross-linking the wedge agent with structural protein molecules to form a hydrogel, so as to maintain excellent light transmission and mechanical properties of the hydrogel. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 The actual photos of the hydrogels made of different cellulose materials after water dialysis for 3 days (top photo) and water dialysis for 3 days + ethanol soaking for 3 days (bottom photo), respectively;

[0059] Figure 2 Contact angle of different cellulose materials with silk fibroin;

[0060] Figure 3 Transmittance of hydrogels made of different celluloses with different addition amounts;

[0061] Figure 4 Transmittance of hydrogels made of different celluloses with different addition amounts after being soaked in 80% ethanol;

[0062] Figure 5 Compression modulus retention rate of hydrogels made of different celluloses with different addition amounts under the conditions of compression rate 60%, loading speed 40 mm / min, and cyclic compression 100 times, wherein the compression modulus retention rates from large to small are L-H 0.05%, L-H 0.1%, SF, L-H 0.5%, L-H 1.0%, and L-H 5.0% in turn;

[0063] Figure 6 Compression modulus retention rate of hydrogels made of different celluloses with different addition amounts after being soaked in 80% ethanol under the conditions of compression rate 60%, loading speed 40 mm / min, and cyclic compression 100 times, wherein the compression modulus retention rates from large to small are L-H 5.0%, L-H 1.0%, L-H 0.5%, L-H 0.1%, L-H 0.05%, and SF in turn;

[0064] Figure 7 Compression modulus of hydrogels made of different concentrations of silk fibroin, wherein SF 15%, SF 20%, and SF 30% respectively refer to silk fibroin solutions with concentrations of 15%, 20%, and 30%, and the corresponding addition amounts of degummed silk are 1.455 g, 1.94 g, and 2.91 g respectively;

[0065] Figure 8 Compression modulus retention rate of different hydrogels, wherein the compression modulus retention rates from large to small are L-H 3%, SF, and CMC 3% in turn;

[0066] Figure 9 Compression modulus retention rate of different hydrogels after being soaked in 80% ethanol for 3 days;

[0067] Figure 10 Transmittance of different hydrogels;

[0068] Figure 11 Secondary structure of different hydrogels;

[0069] Figure 12 Morphology and particle size distribution of gel balls;

[0070] Figure 13Hydrogel XRD and crystal size chart. DETAILED DESCRIPTION

[0071] The application will be further described in conjunction with the following examples. However, the application is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are the conventional conditions in the industry. The technical features involved in each embodiment of the application can be combined with each other as long as there is no conflict between them.

[0072] Example 1

[0073] The wedge reagent material mainly includes the following systems:

[0074] a. Cellulose material system: 10 mL of 9.3 M lithium bromide (referring to aqueous lithium bromide solution unless otherwise specified) was measured, and 1 g of cellulose was gradually added to the lithium bromide under stirring, and was stirred at room temperature (25°C) until completely dissolved to obtain a cellulose lithium bromide solution. After 1 g of degummed silk was fully immersed, it was dissolved at 60°C for 1.5 h to obtain a cellulose lithium bromide silk solution with a concentration of 200 mg / mL. 500 μL of butanediol diglycidyl ether (BDDE) was added and mixed uniformly, and was placed in a 60°C oven for 3 h of reaction. Among them, the cellulose refers to one or several of methylcellulose (MC), carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), hydroxypropyl methylcellulose (HPMC), and lignocellulose.

[0075] b. Polymer material system: 10 mL of 9.3 M lithium bromide was measured, and 1 g of polymer was gradually added to the lithium bromide under stirring, and was stirred at room temperature until completely dissolved to obtain a polymer lithium bromide solution. After 1 g of degummed silk was fully immersed, it was dissolved at 60°C for 1.5 h to obtain a polymer lithium bromide silk solution with a concentration of 200 mg / mL. 500 μL of butanediol diglycidyl ether (BDDE) was added and mixed uniformly, and was placed in a 60°C oven for 3 h of reaction. Among them, the polymer refers to one or several of polyvinyl alcohol (PVA), poly-L-glutamic acid sodium (PGA), and hyaluronic acid (HA).

[0076] c. Inorganic material system: 10 mL of 9.3 M lithium bromide was measured, 1 g of inorganic material was gradually added into lithium bromide under stirring, and was stirred at room temperature until completely dissolved to obtain an inorganic material lithium bromide solution. After 1 g of degummed silk for 10 min was fully immersed, it was dissolved at 60 °C for 1.5 h to obtain an inorganic material lithium bromide silk solution with a concentration of 200 mg / mL. 500 μL of butanediol diglycidyl ether (BDDE) was added and mixed uniformly, and was placed in a 60 °C oven for 3 h of reaction. The inorganic material refers to one or more of carbon nanotubes and graphene.

[0077] d. Modified material system:

[0078] 10 mL of 9.3 M lithium bromide was measured, 1 g of modified material was gradually added into lithium bromide under stirring, and was stirred at room temperature until completely dissolved to obtain a modified material lithium bromide solution. After 1 g of degummed silk for 10 min was fully immersed, it was dissolved at 60 °C for 1.5 h to obtain a modified material lithium bromide silk solution with a concentration of 200 mg / mL. 500 μL of butanediol diglycidyl ether (BDDE) was added and mixed uniformly, and was placed in a 60 °C oven for 3 h of reaction. The modified material refers to one or more of sodium lignosulfonate, adipic acid dihydrazide grafted HA (HA-ADH), HA modified with methacrylic anhydride into methacrylated HA (HA-MA), and dodecyl succinic anhydride hydrophobically modified hydroxyethyl cellulose (HEC-DDSA).

[0079] e. 10 mL of 9.3 M lithium bromide was measured, and 2 g of degummed silk for 10 min was fully immersed and dissolved at 60 °C for 1.5 h to obtain a lithium bromide silk solution with a concentration of 200 mg / mL. 500 μL of butanediol diglycidyl ether (BDDE) was added and mixed uniformly, and was placed in a 60 °C oven for 3 h of reaction.

[0080] The gel prepared in the reaction system was first placed in pure water for 3 days of dialysis, and the water was changed every 6 h. The gel was prepared into a gel block with a thickness of 1 mm, clamped on a sample table, and the light transmittance of the gel at a wavelength of 550 nm was observed. The Carry 5000 ultraviolet spectrophotometer (Agilent, USA) was used for light transmittance test. Then it was immersed in 80% ethanol for 3 days. The gel was prepared into a gel block with a thickness of 1 mm, clamped on a sample table, and the light transmittance of the gel at a wavelength of 550 nm was observed. The Carry 5000 ultraviolet spectrophotometer (Agilent, USA) was used for light transmittance test. If not specified, the light transmittance of the gel in the following examples refers to the light transmittance of the gel with a thickness of 1 mm.

[0081] The above-mentioned group a is cellulose as a wedge reagent in a reported lithium bromide BDDE reaction system; group b is a polymer material as a wedge reagent in a reported lithium bromide BDDE reaction system; group c is an inorganic material as a wedge reagent in a reported lithium bromide BDDE reaction system; group d is a modified material as a wedge reagent in a reported lithium bromide BDDE reaction system; and group e is a blank control in a reported lithium bromide BDDE reaction system.

[0082] Compression modulus retention rate test: The above-mentioned wedge reagents can be used to prepare hydrogels, wherein, as observed from Table 1 below, when the wedge reagent is a modified material (HEC-DDSA or sodium lignosulfonate (water contact angle 54.26±2.66°)), an inorganic material (carbon nanotube (water contact angle 97.81±3.45°)), excellent mechanical stability (>90%, specifically referring to maintaining more than 90% of the initial compression modulus) can be maintained under the conditions of a compression rate of 60%, a compression rate of 40 mm / min, and a cyclic compression of 100 times, whether treated with deionized water or treated with 80% ethanol; and the control group (e) has relatively excellent mechanical stability after deionized water treatment, but the compression modulus after 80% ethanol treatment is greater than the range of the texture analyzer (25N), and thus the mechanical stability is poor.

[0083] Table 1

[0084]

[0085] It can be seen from Figure 1 that in the lithium bromide silk dissolving system, the wedge reagent material used in the silk fibroin wedge reagent hydrogel is different, and the effect of improving transparency is different. For example, when CMC and HPMC are used as wedge reagents, whether or not they are soaked in 80% ethanol (accelerated treatment), they can have good transparency, and when HEC and HA are used as wedge reagents, they have good transparency after water dialysis for 3 days, but the transparency is poor after being soaked in 80% ethanol for 3 days. Therefore, the wedge reagent material has a significant effect, and some have no effect.

[0086] The inventor carried out experiments on one or various combinations of substances with secondary structures such as type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, fibrin, silk fibroin, silk sericin, wool keratin, feather keratin, etc. according to the experimental scheme of Example 1, and obtained similar experimental phenomena and conclusions to Example 1.

[0087] The inventors performed experiments on one or various combinations of cross-linking agents such as organic solvents (diglycidyl ethers: butanediol diglycidyl ether), enzymatic cross-linking (HRP / H2O2), activated agent cross-linking (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) (EDC) / N-hydroxysuccinimide (NHS), photo-cross-linking agents (glycidyl methacrylate), etc. according to the experimental protocol of Example 1, and obtained similar experimental phenomena and conclusions to those of Example 1.

[0088] The inventors performed experiments on one or several of special materials capable of destroying substances having secondary structures such as lithium bromide solution, ionic liquid solution, urea solution, guanidine hydrochloride solution, SDS solution, calcium chloride-ethanol-water solution, etc. according to the experimental protocol of Example 1, and obtained similar experimental phenomena and conclusions to those of Example 1.

[0089] Example 2

[0090] The inventors performed orthogonal tests on a solution mixing temperature, b solution mixing temperature, cross-linking temperature, and cross-linking time according to the experimental protocol of Example 1.

[0091] a solution mixing temperature: temperature at which one of the structural protein and the wedge reagent is mixed with a solution capable of destroying the secondary structure of the structural protein, to obtain a first mixed solution;

[0092] b solution mixing temperature: temperature at which the other is mixed with the first mixed solution or the other is mixed with a solution capable of destroying the secondary structure of the structural protein and then mixed with the first mixed solution, to obtain a second mixed solution containing the structural protein and the wedge reagent, wherein the temperature at which the other is mixed with the solution capable of destroying the secondary structure of the structural protein is the same as the a solution mixing temperature, and the temperature at which the other is mixed with the first mixed solution is the b solution mixing temperature.

[0093] (1) Control b solution mixing temperature (25°C), cross-linking temperature, and cross-linking time, and change a solution mixing temperature

[0094] The inventors selected CMC as the wedge reagent and listed a solution mixing temperatures of 4°C, 15°C, 20°C, 50°C, 100°C, 150°C, 180°C, and 200°C according to the experimental protocol of Example 1, and analyzed the light transmittance after the prepared gels were soaked in water for 3 days and then in 80% ethanol for 3 days.

[0095] (2) Control a solution mixing temperature (25°C), cross-linking temperature, and cross-linking time, and change b solution mixing temperature

[0096] The inventors selected CMC as the wedge reagent according to the experimental protocol of Example 1, and listed the b solution mixing temperatures of 4°C, 15°C, 20°C, 50°C, 100°C, 150°C, 180°C, and 200°C. The prepared gels were soaked in water for 3 days, and then soaked in 80% ethanol for 3 days, and the transmittance was analyzed.

[0097] Table 2

[0098]

[0099] Therefore, the a solution mixing temperature and the b solution mixing temperature have good effects at 4-200°C, further at 15-180°C, and more further at 20-150°C.

[0100] (3) Controlling the a solution mixing temperature (room temperature), the b solution mixing temperature (room temperature), and the crosslinking time (30 min), and changing the crosslinking temperature

[0101] The inventors selected CMC as the wedge reagent according to the experimental protocol of Example 1, and listed the crosslinking temperatures of -80°C,

[0102] -40°C, -20°C, 20°C, 60°C, 80°C, 100°C, and 120°C. The prepared gels were soaked in water for 3 days, and then soaked in 80% ethanol for 3 days, and the transmittance was analyzed.

[0103] (4) Controlling the a solution mixing temperature, the b solution mixing temperature, and the crosslinking temperature (60°C), and changing the crosslinking time

[0104] The inventors selected CMC as the wedge reagent according to the experimental protocol of Example 1, and listed the crosslinking times of 3 min, 5 min, 30 min, 1 h, 30 h, 60 h, 72 h, and 96

[0105] h. The prepared gels were soaked in water for 3 days, and then soaked in 80% ethanol for 3 days, and the transmittance was analyzed.

[0106] Table 3

[0107]

[0108] As can be seen above, when the crosslinking temperature is -80-120°C, the transmittance of the hydrogel after 80% ethanol soaking can be as high as 75% or more, further -80-100°C, and more further -80-80°C.

[0109] When the crosslinking time is 3 min-96 h, the transmittance of the hydrogel after 80% ethanol soaking can be as high as 75% or more, further 5 min-72 h, and more further 5 min-60 h.

[0110] Example 3

[0111] Regenerated silk fibroin preparation: 60 g of raw silk and 25.44 g of anhydrous sodium carbonate were weighed and prepared for use, 12 L of deionized water was measured into a stainless steel barrel and heated with an electromagnetic oven. When the deionized water was about to boil, the weighed anhydrous sodium carbonate was added, and heating and stirring were continued until boiling to ensure that the anhydrous sodium carbonate was fully dissolved. Then the weighed raw silk was added, and boiling was maintained for 10 min (i.e., degumming for 10 min), and stirring was performed every 5 min to dissolve the sericin on the surface of the raw silk. The degummed raw silk was kneaded with deionized water for 4 times to fully remove the sericin on the surface of the raw silk, and finally the degummed silk (degummed silk) was wrung dry and dried in a fume hood overnight. If not specified, the degummed silk used in the examples of the present application was prepared by this method.

[0112] 10 g of dried degummed silk was taken into 40 mL of 9.3 M lithium bromide and stirred uniformly with a glass rod. After mixing the lithium bromide solution and the degummed silk, it was heated in an oven at 60°C for 4 hours to promote the dissolution of the degummed silk. Next, the completely dissolved silk fibroin solution was poured into a dialysis bag (molecular weight cut-off 3500 Da) and sealed. The dialysis bag containing the silk fibroin solution was placed in a container containing 5 L of deionized water, and a magnetic stirrer was used to continuously stir at the bottom of the container to dilute the leaked lithium bromide. The dialysis time was 3 days, and the total water change was 7-8 times. After complete desalination, the silk fibroin solution was placed in a centrifuge bottle and repeatedly centrifuged at a speed of 9000 rpm and a low temperature of 4°C. Finally, a clean silk fibroin solution was obtained and stored in a 4°C refrigerator.

[0113] The concentration of the silk fibroin solution was further determined by weighing method, i.e., the weighing dish was weighed and marked as W, 1 mL of silk fibroin solution was added to the dish and weighed and marked as W1, the dish containing the silk fibroin solution was placed in an oven and dried for 24 hours, and then weighed and marked as W2. The concentration (w / w) of the silk fibroin solution was calculated according to the formula:

[0114] Concentration = (W2-W) / (W1-W) x 100%.

[0115] The concentration of the silk fibroin solution was adjusted to 50 mg / mL, and CMC, HPMC (high, medium, and low viscosity), HEC, and HA were respectively configured into 50 mg / mL solutions with deionized water. 4 mL of each solution was placed in a common petri dish with a diameter of 35 mm, and the petri dish was placed in a fume hood for natural air drying to obtain silk fibroin membranes, CMC membranes, high-viscosity HPMC membranes, medium-viscosity HPMC membranes, low-viscosity HPMC membranes, HEC membranes, and HA membranes.

[0116] The water contact angle of the membranes was observed, and the water contact angle was tested using a contact angle measuring instrument (DSA100, Kruss, Germany). As shown inFigure 2 As shown, the contact angles of silk fibroin film (SF), CMC film, high viscosity HPMC film (H-H), medium viscosity HPMC film (M-H), and low viscosity HPMC film (L-H) are relatively close, being 72.60±4.86°, 72.07±1.65°, 56.63±3.79°, 58.00±2.82°, and 55.53±6.90°, respectively, while the contact angles of HEC film and HA film are 46.50±3.21° and 43.23±4.71°, respectively, which are smaller than the contact angle of silk fibroin film and are more hydrophilic. In Example 1, the transparency of HEC / silk fibroin hydrogel is reduced after immersion in 80% ethanol, and the transparency of HA / silk fibroin hydrogel is also reduced after immersion in 80% ethanol. Therefore, the wedge reagent with similar hydrophilicity and hydrophobicity (±25.5°) to the material with secondary structure can stabilize the secondary structure.

[0117] From Examples 1 and 3, it can be inferred that the gel formed by crosslinking of silk fibroin with cellulose / polymer / modification materials with similar hydrophilicity and hydrophobicity to silk fibroin can maintain high transparency after dialysis in deionized water for 3 days and then immersion in 80% ethanol for 3 days (for example, CMC and HPMC). The gel formed by crosslinking of silk fibroin with more hydrophilic cellulose / polymer / modification materials appears whitening after dialysis in deionized water for 3 days and then immersion in 80% ethanol for 3 days (for example, HEC and HA). Therefore, cellulose / polymer / modification materials with similar hydrophilicity and hydrophobicity to silk fibroin have specificity in maintaining the structural stability of silk fibroin hydrogel.

[0118] Preferably, the wedge reagent is one or more of cellulose, polymer material, inorganic material, and modification material with similar hydrophilicity and hydrophobicity to silk fibroin.

[0119] Example 4

[0120] HRP / H2O2 crosslinked, photocrosslinked silk fibroin wedge reagent hydrogel

[0121] HRP / H2O2: 40 mL of lithium bromide solution in Example 3 was first mixed with 0 g (control group), 0.3 g of CMC (total solid content 3%) uniformly, and then dissolved silk according to the experimental scheme in Example 3 (silk weight was 10 g and 9.7 g, respectively), and dialysis was performed.

[0122] The concentration of the regenerated silk fibroin solution was adjusted to 5%, and 20 μL of HRP (900 U / mL) and 20 μL of H2O2 (0.5% v / v) were added to 1 mL of the regenerated silk fibroin solution. After the mixture was incubated at 37°C for 30 min, an HRP / H2O2 cross-linked silk fibroin wedge reagent hydrogel was formed. After the hydrogel was placed in deionized water for 3 days, its light transmittance at 550 nm was detected, and then the hydrogel was placed in 80% ethanol for 3 days, and its light transmittance at 550 nm was detected.

[0123] Photo-crosslinking: (1) Preparation of a methacrylated silk fibroin solution: degummed silk was prepared according to Example 3. 10 g and 9.7 g of degummed silk were added to 40 mL of a 9.3 M LiBr solution, respectively, and dissolved at 60°C for 1 h to obtain a silk fibroin solution, followed by the addition of 12 mL of glycidyl methacrylate to the silk fibroin solution, and reaction at 60°C for 3 h, with the whole process being carried out in the dark. The reacted solution was added to 0 g (control group) and 0.3 g of CMC (total solid content 3%) and dissolved uniformly, and then placed in a dialysis bag and dialyzed against deionized water for 7 days, with the whole process being carried out in the dark. After dialysis, the solution was filtered and centrifuged, and concentrated to obtain a methacrylated CMC silk fibroin solution with a mass fraction of about 25%.

[0124] (2) Under room temperature and in the dark, 5 mL of the methacrylated CMC silk fibroin solution was added with a LAP initiator at a final concentration of 0.03 wt%, and stirred to obtain a precursor solution. The precursor solution was rapidly crosslinked under ultraviolet light (wavelength 365 nm) within 5 seconds, and a hydrogel was obtained. After the hydrogel was placed in deionized water for 3 days, its light transmittance at 550 nm was detected, and then the hydrogel was placed in 80% ethanol for 3 days, and its light transmittance at 550 nm was detected.

[0125] The results are shown in Table 4:

[0126] Table 4

[0127]

[0128] The experimental results were consistent with those of Examples 1 and 2, indicating that changing the crosslinking agent can also improve the light transmittance and stability.

[0129] The inventors performed experiments according to the experimental scheme of Example 1, using one or various combinations of crosslinking agents such as organic solvents (diglycidyl ether: butanediol diglycidyl ether), activators (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) (EDC) / N-hydroxysuccinimide (NHS), and obtained similar experimental phenomena and conclusions to Example 1.

[0130] The effect of the CMC that was originally excellent but was modified to be hydrophilic became worse.

[0131] Take 10 mL of 9.3 M lithium bromide, gradually add 0.06 g of polyacrylamide modified carboxymethyl cellulose (CMC-PAM) to the lithium bromide under stirring, and stir at room temperature until completely dissolved to obtain a CMC-PAM lithium bromide solution. After the 10 min degumming of the silk is fully soaked, it is dissolved at 60°C for 1.5 h to obtain a CMC-PAM lithium bromide silk solution with a concentration of 200 mg / mL. 500 μL of butanediol diglycidyl ether (BDDE) is added and mixed uniformly, and then placed in a 60°C oven for 3 h of reaction.

[0132] The prepared gel is first placed in deionized water for dialysis for 3 days, with water changed every 6 h, and then soaked in 80% ethanol for 3 days. The light transmittance of the gel at a wavelength of 550 nm is observed, wherein the Carry 5000 ultraviolet spectrophotometer (Agilent, USA) is used for light transmittance testing.

[0133] The light transmittance after soaking in 80% ethanol for 3 days is significantly lower than that after dialysis in deionized water for 3 days, and the water contact angle of CMC-PAM is 41.17 ± 4.20°. Therefore, it is further verified that the wedge reagent in Example 1 has similar hydrophilic and hydrophobic properties as substances with secondary structures, which can achieve the excellent effect of the present application.

[0134] Example 6

[0135] The inventors carried out orthogonal tests on different amounts of cellulose, silk protein concentrations, and different celluloses.

[0136] Different amounts of cellulose: The amount of cellulose added is divided into the following categories according to whether it is cross-linked with a diglycidyl ether cross-linking agent: cross-linked: 0.001%, 0.05%, 0.1%, 0.5%, 1%, 3%, 5%, 30%, 50%. For example, 0.001% refers to the mass percentage of cellulose in the total solid content, which is the total mass of cellulose and degummed silk. Unless otherwise specified, the total solid content is 2 g.

[0137] Silk protein concentration: 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%.

[0138] Different celluloses: CMC: 175 KDa, 300-450 KDa, 500-600 KDa; HEC: 300-450 KDa, 550-600 KDa; HPMC: 400-500 KDa, 550-600 KDa, 650 KDa; MC: 200-400 KDa, 450-600 KDa

[0139] (1) In a fixed reaction system, the concentration of silk fibroin and the condition of cellulose, the amount of cellulose was changed.

[0140] a) The inventor gradually added 0 g (control group), 0.001 g (0.05% of total solid content), 0.002 g (0.1%), 0.01 g (0.5%), 0.02 g (1%), 0.06 g (3%), 0.1 g (5%) carboxymethyl cellulose (CMC) into 10 mL 9.3 M lithium bromide solution during stirring, and placed it in room temperature for stirring until completely dissolved, to obtain a cellulose lithium bromide solution. Then 2 g, 1.999 g, 1.998 g, 1.99 g, 1.98 g, 1.94 g, 1.9 g degummed silk (if not specified, the degummed silk is the degummed silk described in Example 1) was dissolved in the cellulose lithium bromide solution respectively to obtain a cellulose lithium bromide silk solution with a concentration of 200 mg / mL. It was placed in an oven at 60°C for incubation for 1.5 hours. 500 μL BDDE was added to the reaction system, and it was placed in an oven at 60°C for reaction for 3 hours. The obtained gel was dialyzed in deionized water for 3 days, and then soaked in 80% ethanol for 3 days. The transmittance and compression modulus stability of the reaction system were observed. The Carry 5000 (Agilent) ultraviolet spectrophotometer was used to test the transmittance at a wavelength of 550 nm, and the texture analyzer (TMS-PRO, USA) was used to test the compression modulus.

[0141] b) The difference from a) above is that high-viscosity hydroxypropyl methyl cellulose (H-H), medium-viscosity hydroxypropyl methyl cellulose (M-H) and low-viscosity hydroxypropyl methyl cellulose (L-H) were used to replace CMC respectively.

[0142] From Figure 3The results show that when the cellulose addition was 0g (control group), 0.001g (0.05%), 0.002g (0.1%), 0.01g (0.5%), 0.02g (1%), 0.06g (3%), and 0.1g (5%), the transmittance after dialysis with deionized water for 3 days was 53.02±3.53% (0g / SF), 63.87±2.48% (CMC) / 53.23±2.82% (LH) (0.05%), 64.11±6.44% (CMC) / 50.20±1.74% (LH) (0.1%), and 69.24±2.66% (FPS). The transmittance was observed to be improved to some extent when cellulose was added, even at a concentration of 0.05%. When the cellulose content was 1%, the transmittance was significantly improved compared to the gel without added cellulose.

[0143] The present invention further increases the amount of cellulose added, as shown in Table 5 below. As the amount of CMC added continues to increase, the light transmittance shows a decreasing trend. However, when the addition amount does not exceed 50%, the light transmittance is higher than that of gels without added cellulose. When the addition amount exceeds 50%, the light transmittance of the gel is poor. The inventors speculate that this is mainly due to the nature of the CMC material itself; the higher the CMC content, the more uneven the gel surface, leading to a decrease in light transmittance.

[0144] Table 5

[0145]

[0146] After soaking in 80% ethanol, as Figure 4 As shown, the transmittance of the gel after ethanol soaking can be maintained to a certain extent when cellulose is added. Among them, when the amount of cellulose added is 1%, the gel performance is significant, and the transmittance of the gel is 69.12±2.49% (CMC) / 72.12±2.49% (LH), which is much higher than the transmittance of the gel without added cellulose (45.23±2.96%).

[0147] from Figure 5As can be seen from the table, when the cellulose addition amount is 0 g (control group), 0.001 g (0.05%), 0.002 g (0.1%), 0.01 g (0.5%), 0.02 g (1%) or 0.1 g (5%), after dialysis in deionized water for 3 days, all the gels are punched into cylinders with a diameter of 10 mm and a height of 8 mm, and under the conditions of a compression rate of 60%, a loading speed of 40 mm / min and a cyclic compression of 100 times, the initial compression modulus of the gels can be maintained at 90% or above.

[0148] As can be seen from the table, when the cellulose addition amount is 0 g (control group), 0.001 g (0.05%), 0.002 g (0.1%), 0.01 g (0.5%), 0.02 g (1%) or 0.1 g (5%), after dialysis in deionized water for 3 days, all the gels are punched into cylinders with a diameter of 10 mm and a height of 8 mm, and under the conditions of a compression rate of 60%, a loading speed of 40 mm / min and a cyclic compression of 100 times, the initial compression modulus of the gels can be maintained at 90% or above. Figure 6 As can be seen from the table, when the cellulose addition amount is 0 g (control group), 0.001 g (0.05%), 0.002 g (0.1%), 0.01 g (0.5%), 0.02 g (1%) or 0.1 g (5%), after dialysis in deionized water for 3 days, all the gels are punched into cylinders with a diameter of 10 mm and a height of 8 mm, and under the conditions of a compression rate of 60%, a loading speed of 40 mm / min and a cyclic compression of 100 times, the initial compression modulus of the gels can be maintained at 90% or above.

[0149] As a preferred embodiment, the addition amount of the wedge reagent is 0.05% to 50%, and further preferably 1% to 50%.

[0150] (2) The addition amount of the fiber and the concentration of the silk protein under the condition of the cellulose are changed.

[0151] The inventors prepared a cellulose lithium bromide solution containing 3% cellulose by adding 1.455 g, 1.94 g and 2.91 g of degummed silk into 9.3 M lithium bromide solution containing 3% cellulose to prepare a solution of silk in cellulose lithium bromide with a concentration of 150 mg / mL, 200 mg / mL and 300 mg / mL, respectively, and incubated the solution in an oven at 60°C for 1.5 hours. Then, 500 μL of BDDE was added to the reaction system, and the reaction was carried out in an oven at 60°C for 3 hours. The obtained gel was dialyzed in deionized water for 3 days.

[0152] As can be seen from the table, when the cellulose addition amount is 0 g (control group), 0.001 g (0.05%), 0.002 g (0.1%), 0.01 g (0.5%), 0.02 g (1%) or 0.1 g (5%), after dialysis in deionized water for 3 days, all the gels are punched into cylinders with a diameter of 10 mm and a height of 8 mm, and under the conditions of a compression rate of 60%, a loading speed of 40 mm / min and a cyclic compression of 100 times, the initial compression modulus of the gels can be maintained at 90% or above. Figure 7 As can be seen from the table, when the cellulose addition amount is 0 g (control group), 0.001 g (0.05%), 0.002 g (0.1%), 0.01 g (0.5%), 0.02 g (1%) or 0.1 g (5%), after dialysis in deionized water for 3 days, all the gels are punched into cylinders with a diameter of 10 mm and a height of 8 mm, and under the conditions of a compression rate of 60%, a loading speed of 40 mm / min and a cyclic compression of 100 times, the initial compression modulus of the gels can be maintained at 90% or above.

[0153] As a preferred embodiment, the concentration of the silk protein is greater than or equal to 0.5%, and further preferably 1% to 30%.

[0154] (3) The addition amount of the fiber and the concentration of the silk protein under the condition of the cellulose are changed.

[0155] The inventors listed the following: 1g of CMC (175kDa) and HPMC (650kDa) were gradually added to 10mL of 9.3M lithium bromide solution while stirring. The mixture was stirred at room temperature until completely dissolved to obtain a cellulose lithium bromide solution. Then, 1g of degummed filaments were dissolved in the cellulose lithium bromide solution (200mg / mL) and incubated in a 60℃ oven for 1.5 hours. 500μL of BDDE was added to the reaction system and the mixture was incubated in a 60℃ oven for 3 hours.

[0156] The obtained gels were dialyzed in deionized water for 3 days, and then soaked in 80% ethanol for 3 days. The changes in transmittance in the reaction system were observed, and the water contact angle was tested. The study found that the gels with added CMC and HPMC maintained high transparency after dialyzing in deionized water for 3 days, while the gels with added CMC and HPMC showed excellent transmittance after soaking in 80% ethanol for 3 days.

[0157] Example 7

[0158] The cellulose / silk protein hydrogel prepared in Example 6 (reaction system: lithium bromide concentration 9.3M, cellulose (CMC / LH) addition 0% (control group), silk protein concentration 200 mg / mL; lithium bromide concentration 9.3M, cellulose (CMC) addition 3% (total solids 2g), silk protein concentration 194 mg / mL; lithium bromide concentration 9.3M, cellulose (LH) addition 3% (total solids 2g), silk protein concentration 194 mg / mL; reaction temperature 60℃ and reaction time 3 hours, lithium bromide to BDDE ratio 10 mL: 500 μL) was cut into cylinders with a diameter of 10 mm and a height of 8 mm using a punch. The cylinders were then evaluated using a texture analyzer (25N, TMS-PRO, USA). The loading speed was 40 mm / min, and the compression variation was 60%. When the gel is compressed to 40% of its original height, compression is stopped and the pressure is released. When the pressure sensor returns to the starting position, compression is repeated, and this compression and release cycle is repeated 1000 times.

[0159] like Figure 8 As shown, the study found that after dialysis in deionized water for 3 days, hydrogels with 0% and 3% (2g) cellulose additions both exhibited good mechanical stability, maintaining more than 80% of their initial compressive modulus. Figure 9 As shown, after soaking in 80% ethanol for 3 days, the hydrogel with 3% cellulose added exhibited good mechanical stability, maintaining more than 75% of its initial compressive modulus, while the hydrogel with 0% cellulose added exceeded the maximum range of the texture analyzer and became brittle. This indicates that cellulose (CMC / LH) can act as a wedge agent to maintain the mechanical stability of silk protein hydrogels.

[0160] The inventors performed experiments on one or several of the hydrogels prepared by the wedge reagent of polymer / modifying material / cellulose / inorganic material according to the experimental protocol of Example 7, and obtained similar experimental phenomena and conclusions to Example 6.

[0161] The inventors performed experiments on one or several of the materials with secondary structure such as collagen type I, collagen type II, collagen type III, collagen type IV, collagen type V, collagen type VI, fibroin, silk fibroin, sericin, wool keratin, feather keratin, etc. according to the experimental protocol of Example 7, and obtained similar experimental phenomena and conclusions to Example 6.

[0162] The inventors performed experiments on one or several of the cross-linking agents such as organic solvent type (diglycidyl ether type: butanediol diglycidyl ether), enzyme-mediated cross-linking (HRP / H2O2), activated agent cross-linking (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) (EDC) / N-hydroxysuccinimide (NHS), photo-crosslinking agent (glycidyl methacrylate), etc. according to the experimental protocol of Example 7, and obtained similar experimental phenomena and conclusions to Example 6.

[0163] The inventors performed experiments on one or several of the special materials capable of destroying materials with secondary structure such as lithium bromide solution, ionic liquid solution, urea solution, guanidine hydrochloride solution, SDS solution, calcium chloride-ethanol-water solution, etc. according to the experimental protocol of Example 7, and obtained similar experimental phenomena and conclusions to Example 7.

[0164] Example 8

[0165] The inventors verified the principle of the wedge reagent acting on materials with secondary structure, and tested the gel transmittance.

[0166] Wedge reagent: one or several of CMC, HPMC, carbon nanotube, PVA.

[0167] Wedge reagent addition amount: 0% (total solid content 2g), 1%, 3%, 5%, 10%, 50%, 100%.

[0168] Material with secondary structure: one or several of silk fibroin, collagen, wool keratin, feather keratin.

[0169] Material destroying secondary structure: one or several of urea, guanidine hydrochloride, sodium hydroxide, DNA helicase, etc.

[0170] The inventors used CMC as wedge reagent, the addition amount was 1% (total solid content 2g), silk fibroin, urea for principle verification. 8M urea solution, 8M urea / CMC 1% solution, CMC 1% solution were prepared. The gels were all formed in 48-well plates (100 μL).

[0171] a. Measure 10 mL of 9.3 M lithium bromide, gradually add 0.06 g of carboxymethyl cellulose to the lithium bromide during stirring, stir at room temperature until completely dissolved, obtain a lithium bromide cellulose solution, immerse 1.94 g of degummed silk in it, dissolve at 60°C for 1.5 h, obtain a silk solution of lithium bromide with a concentration of 200 mg / mL, add 500 μL of butanediol diglycidyl ether (BDDE), mix uniformly, place in a 60°C oven, and stand for 3 h of reaction.

[0172] b. Measure 10 mL of 9.3 M lithium bromide, immerse 2 g of degummed silk in it, dissolve at 60°C for 1.5 h, obtain a silk solution of lithium bromide with a concentration of 200 mg / mL, add 500 μL of butanediol diglycidyl ether (BDDE), mix uniformly, place in a 60°C oven, and stand for 3 h of reaction.

[0173] c. Dialyze a in deionized water for 2 days, part of the gel made from b is dialyzed in deionized water for 2 days (b1), and part of the gel made from b is dialyzed in 8M urea for 2 days (b2).

[0174] d. Continue to dialyze the newly prepared gel of a in deionized water for 1 day, immerse part of b1 in 8M urea / CMC 1% solution for 1 day (2), and immerse part of b1 in 8M urea solution for 1 day (3); immerse part of b2 in 8M urea / CMC 1% solution for 1 day (4), and immerse part of b2 in CMC 1% aqueous solution for 1 day (5).

[0175] e. Water-wash (2-2) part of (2), and do not treat the other part (2-1), water-wash (3-2) part of (3), and do not treat the other part (3-1), water-wash (4-2) part of (4), and do not treat the other part (4-1), water-wash (5-2) part of (5), and do not treat the other part (5-1); place the gels of a, (2-1), (2-2), (3-1), (3-2), (4-1), (4-2), (5-1), (5-2) dialyzed in deionized water for 3 days in a 60°C oven, and fumigate with 95% ethanol for 1 day.

[0176] Group a was the pre-added cellulose system; Group b was the blank control group; Group c were all newly prepared groups; Group d were all different introduction groups; Group e were all structure-inducing groups; the transmittance and secondary structure of the newly prepared groups, different introduction groups, and structure-inducing groups were observed and analyzed.

[0177] Hydrogels from each step were prepared in 48-well plates (100 μl / well), with a gel thickness of approximately 0.7 mm. The transmittance of the gels of this thickness was measured using a microplate reader, and the secondary structure was determined using a Nicolet 5700 Fourier Transform Infrared Spectrophotometer (FTIR, Nicolet, USA).

[0178] like Figure 10 , 11 As shown, the transmittance of the newly prepared groups was observed, group a ( Figure 10 (As shown in the box) The transmittance reached 86.71±1.79%, and the β-sheet content in the secondary structure was 14.61±0.68%. The transmittance of group b was 59.23±5.38%, similar to b1, and the β-sheet content was 23.32±1.30%. The transmittance of b2 reached 84.96±3.10%, and the β-sheet content was 15.95±0.10%, close to group a. It has been reported that urea can disrupt hydrogen bonds, thereby affecting the secondary structure and transmittance. The fact that group a and group b2 have similar β-sheet content and transmittance indicates that the addition of the wedge reagent forms hydrogen bonds, which affects the secondary structure and transmittance.

[0179] Observing the transmittance of different groups, groups (2), (3), (4), and (5) had better transmittance, while the β-sheet content of groups a and (4) was significantly lower than that of the other groups, further proving that urea’s disruption of hydrogen bonds has an impact on transmittance and secondary structure, and also indicating that the wedge reagent can form hydrogen bonds.

[0180] Observing the structure-inducing groups, group a maintained a transmittance of 78.86±1.52% and a β-sheet content of 34.25±1.9% after 1 day of fumigation with 95% ethanol. Group b and the four groups (2-2), (3-2), (4-2), and (5-2) after water washing all had transmittances below 60% and β-sheet contents above 49%. However, the four groups (2-1), (3-1), (4-1), and (5-1) without water washing also maintained relatively good transmittance and β-sheet contents below 45%. This indicates that silk fibroin can maintain high transmittance and stable secondary structure under the action of wedge reagent and butylene glycol diglycidyl ether (BDDE). Furthermore, butylene glycol diglycidyl ether (BDDE) can make the hydrogen bonding between the wedge reagent and silk fibroin more stable.

[0181] In summary, the wedge reagent can interact with the substance with secondary structure through hydrogen bond, and the diglycidyl ether crosslinking agent can make the hydrogen bond more durable, so that the prepared hydrogel still has high light transmittance and stable secondary structure after structure induction.

[0182] Example 9 Microspheres

[0183] The parameters for preparing the hydrogel in Example 6 (lithium bromide concentration of the reaction system 9.3 M, carboxymethyl cellulose (CMC) addition amount 3% (total solid content 2 g), silk protein concentration 194 mg / mL, and the ratio of lithium bromide to BDDE 10 mL:500 μL) were used to configure the CMC / silk protein solution / silkworm protein / BDDE mixed solution, and the mixed solution was immediately added as the water phase to the oil phase which had been preheated to 60 degrees and was in motion, the rotation speed in the oil phase was 1000 rpm, after the oil phase and the water phase were mixed uniformly, it was placed in a 60°C oven for reaction for 3 hours, and a stable structure of the bone joint lubricating gel ball was obtained. The gel ball was filtered out of the oil phase, the surface oil was washed off with an organic solvent, then washed with water, the collected gel ball was filtered through a 500 mesh screen, the particle size of the gel ball was photographed by a fluorescence microscope, and analyzed. As shown in Figure 12 The particle size of the gel ball was concentrated in 43.40±15.64 μm.

[0184] The rotation speed of the stationary oil phase was 500 rpm, and the oil-water ratio was changed to 1:1, 2:1, 5:1, 10:1, 100:1, and 500:1. After the oil phase and the water phase were mixed uniformly, it was placed in a 60°C oven for reaction for 3 hours, and whether spherical gel was formed in the reaction system was observed. The results showed that when the oil-water ratio was 1:1 and 2:1, the gel showed a block shape. Because the volume of the water phase was too large, the spacing between the round gel balls formed in the moving oil phase was too small, and the gel ball aggregates which were not completely crosslinked gradually crosslinked to form block-shaped gel, rather than crosslinked gel balls. When the oil-water ratio was 5:1, 10:1, 100:1, and 500:1, gel balls were formed. Considering the granulation efficiency, the inventors considered that the oil-water ratio of 10:1 was the best reaction condition.

[0185] The ratio of the fixed oil phase to the water phase was 10:1, and the stirring speed of the oil phase was varied: 50 rpm, 100 rpm, 500 rpm, 1000 rpm, 5000 rpm, and 10000 rpm. After the oil phase and the water phase were mixed uniformly, the mixture was placed in a 60°C oven for 3 hours, and whether gel balls were formed in the reaction system was observed. The results showed that when the stirring speed was 50 rpm, the movement of the oil phase was slow, the movement amplitude and frequency of the gel balls in the oil phase were too small, the gel ball aggregates that were not completely crosslinked were gradually crosslinked to form block-shaped gels, and uncrosslinked gel balls were formed. When the stirring speed of the oil phase was 100 rpm, 500 rpm, 1000 rpm, 5000 rpm, or 10000 rpm, gel balls were formed, and the particle size of the gel balls could be controlled in the range of 1-1000 μm.

[0186] The gel balls were filtered out of the oil phase, washed with an organic solvent to remove the oil, washed with water, and filtered through sieves with different mesh sizes to collect gel balls with a particle size of more than 300 μm, 100-300 μm, and less than 100 μm, respectively. The collected gel balls were freeze-dried, weighed, and the yield was calculated. The results showed that in the range of 100-10000 rpm, ideal silk fibroin hydrogel microspheres could be obtained. In the range of an oil-water ratio of greater than 1:1, ideal silk fibroin hydrogel microspheres could be obtained. In the range of an oil-water ratio of 2:1-500:1, the performance of the silk fibroin hydrogel microspheres was more excellent.

[0187] Optimizing the oil-water ratio and the stirring speed of the oil phase can control the particle size in the range of 1-1000 μm.

[0188] Example 10 XRD crystal characterization

[0189] The prepared and / or treated silk fibroin hydrogel was freeze-dried using a freeze dryer, the freeze-dried gel was ground into a powder, large particles were filtered out using a 200-mesh sieve, and X-ray single crystal diffractometry (XRD, Germany, Bruker D8 VENTURE) was used for testing. Cu-Kα mode was selected, the voltage was 50 kV, the current was 25 mA, the diffraction angle θ was 5°-50°, and after testing, the curve was processed using Jade software, the half-peak width was measured, and the particle size was further analyzed.

[0190] The Scherrer formula is as follows:

[0191] L = 0.94λ / βcosθ

[0192] In the formula, when Cu-Kα radiation is used, λ is β is the half-peak width, and θ is the angle.

[0193] For example,Figure 13 A shows the XRD pattern of silk fibroin hydrogel, and after fitting, calculation and analysis, the crystallite size of the silk fibroin hydrogel soaked in deionized water for 3 days is 9.8 nm, and the crystallite size of the silk fibroin wedge reagent hydrogel with CMC as the wedge reagent is only 4.55 nm. Figure 13 B).

[0194] And the crystallite size of the silk fibroin hydrogel soaked in 80% ethanol for 3 days reaches 10.98 nm, but the crystallite size of the silk fibroin wedge reagent hydrogel with CMC as the wedge reagent is only 6.38 nm.

[0195] Therefore, we speculate that the silk fibroin wedge reagent hydrogel may limit the size of the induced β-sheet domain and help the uniform distribution thereof, so that the silk fibroin wedge reagent hydrogel has better light transmittance, mechanical properties and structural stability.

[0196] The inventors carried out one or more combinations of Example 1 according to the experimental scheme of Example 10 to obtain similar experimental results as Example 10.

[0197] Preferably, the crystallite size of the silk fibroin wedge reagent hydrogel after dialysis in deionized water for 3 days is less than 5 nm; and the crystallite size of the silk fibroin wedge reagent hydrogel after soaking in 80% ethanol for 3 days is less than 7 nm.

[0198] Example 11 Artificial cornea

[0199] A. Methacryl modification of gelatin

[0200] Prepare the instruments needed for the experiment, wash the round-bottom flask, beaker and magnetic stirrer used for the reaction and place them in the oven for drying. Add 4 g of Type A pigskin gelatin to the round-bottom flask, add 200 ml of deionized water, stir at 40°C, and when it is completely dissolved into a clear and transparent solution, add a small amount of NaOH solid to adjust the pH to 7.4. Add 132 ml of N,N-dimethylformamide and 145 μL of methacrylic anhydride to the reaction solution, and after 2 hours of reaction at 40°C, load a dialysis bag with a molecular weight of 3500, dialyze for 3 days, and then freeze-dry.

[0201] B. Preparation of bio-ink for 3D printing

[0202] Add 0.06 g of CMC to 10 ml of lithium bromide, and fully dissolve under continuous stirring to obtain a CMC lithium bromide solution. Add 500 mg of methacrylated gelatin and 1.94 g of degummed silk to the CMC lithium bromide solution, and place it in a 60°C oven for 1.5 h. Then add BDDE and mix uniformly to obtain the bio-ink for 3D printing.

[0203] C. Preparation of 3D printed artificial cornea

[0204] The 3D printing bio-ink prepared in step B is put into a digital light processing printer tank, the corneal model is simulated slicing in a computer with a layer height of 50 μm, after the slicing is completed, the exposure time is set to 10 s, the obtained product is soaked in a phosphate buffer for 5 days, the soaking solution is replaced every half day to completely remove the pigment, then the artificial cornea is soaked in 75% alcohol for sterilization, and then soaked in sterile phosphate buffer until the alcohol is completely replaced, i.e. a structure-stable artificial cornea is obtained.

[0205] Example 12 lubrication

[0206] The embodiment of the present application provides a preparation method of an injectable soft tissue filler, which comprises the following steps:

[0207] 1) 2 g of sodium hyaluronate (molecular weight 500 KDa), 0.4 g of transaminic acid and 10 mL of glycerol are added to 100 mL of injection physiological saline, stirred at 400 rpm for 6 h until the sample is completely dissolved. The solution is sterilized at 121 DEG C. for 15 min, i.e. an auxiliary material solution is obtained;

[0208] 2) 20 g of the structure-stable gel ball prepared in example 8 is weighed and added to the above-mentioned sterilized auxiliary material solution, and further stirred at 400 rpm for 4 h to uniformly disperse the gel ball in the auxiliary material solution;

[0209] 3) 1 mL of the above-mentioned mixed solution is accurately taken by a syringe, and the syringe is sealed to obtain a structure-stable tissue filler.

[0210] The tissue filler has excellent water phase dispersibility, the suspension obtained after dispersion is uniform and delicate, has good needle passing property, has the advantages of quick effect, outstanding filling effect, good plasticity, can promote the proliferation of collagen and fibrocyte of surrounding tissues, has few toxic and side effects and the like in the use as a cosmetic filling material.

[0211] The above has described the present application in detail, the purpose is to enable the person skilled in the art to understand the content of the present application and to implement, and cannot limit the protection scope of the present application, any equivalent change or modification according to the spirit and principle of the present application should be covered in the protection scope of the present application.

Claims

1. A hydrogel, characterized by: The hydrogel is formed by cross-linking the wedge reagent and the structural protein after contacting the wedge reagent and the structural protein in the presence of a solution capable of destroying the secondary structure of the structural protein, and then cross-linking with a cross-linking agent, The wedge reagent is a substance with the same or similar hydrophilic or hydrophobic properties as the hydrophobic crystalline region of the structural protein and capable of forming hydrogen bonds with the hydrophobic crystalline region of the structural protein, and the wedge reagent is selected from one or more of cellulose, inorganic substances, and modified materials, wherein the cellulose includes one or more of methyl cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, and lignocellulose; the inorganic substances include carbon nanotubes; and the modified materials include one or more of lignin sulfonate, hydrophobically modified hyaluronic acid, hydrophobically modified hydroxyethyl cellulose, and hydrophobically modified gelatin. The wedge reagent accounts for 0.5% to 25% of the total mass of the structural protein and the wedge reagent.

2. The hydrogel of claim 1, wherein: The crystal size of the hydrogel is less than or equal to 5 nm after being soaked in deionized water for 3 days, and the crystal size is less than or equal to 7 nm after being soaked in 80% ethanol for 3 days.

3. The hydrogel of claim 1, wherein: The light transmittance of the hydrogel is more than 75% of the initial light transmittance after being dialyzed in deionized water for 3 days and then soaked in 80% ethanol for 3 days, and the compression modulus loss rate is less than or equal to 35% after being compressed at a compression deformation of 60% for 1000 cycles.

4. The hydrogel of claim 3, wherein: The initial light transmittance of the hydrogel with a thickness of 1 mm is not less than 80%; and / or The initial compression modulus loss rate of the hydrogel is less than or equal to 20% after being compressed at a compression deformation of 60% for 1000 cycles.

5. The hydrogel of claim 1, wherein: The water contact angle of the wedge reagent is θ1, and the water contact angle of the structural protein is θ2, and the range of θ1 is (θ2-25.5°) to (θ2+25.5°).

6. The hydrogel of claim 1, wherein: The lignin sulfonate includes sodium lignosulfonate; The hydrophobically modified hyaluronic acid includes one or both of adipic acid dihydrazide grafted hyaluronic acid and methacrylated hyaluronic acid; The hydrophobically modified hydroxyethyl cellulose includes dodecylene succinic anhydride hydrophobically modified hydroxyethyl cellulose; The hydrophobically modified gelatin includes methacrylated gelatin.

7. The hydrogel of claim 1, wherein: The structural protein includes one or more of collagen, fibrin, silk fibroin, sericin, and keratin.

8. The hydrogel of claim 1, wherein: The cross-linking agent is one or more of an organic solvent cross-linking agent, an enzymatic cross-linking, an activator, and a photo-crosslinking agent.

9. The hydrogel of claim 8, wherein: The organic solvent cross-linking agent includes one or more of a diglycidyl ether cross-linking agent; The enzymatic cross-linking includes horseradish peroxidase and hydrogen peroxide; The activator includes one or both of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide; The photo-crosslinking agent includes glycidyl methacrylate.

10. A method of preparing a hydrogel, characterized by: The hydrogel is the hydrogel of any one of claims 1 to 9, and the preparation method comprises the following steps: (1) mixing the wedge reagent, the structural protein, and a solution capable of destroying the secondary structure of the structural protein; (2) adding a cross-linking agent to the mixture of step (1) to perform a cross-linking reaction to produce the hydrogel.

11. The method of claim 10, wherein: The solution capable of destroying the secondary structure of structural proteins includes one or more of lithium bromide solution, ionic liquid solution, urea solution, guanidine hydrochloride solution, SDS solution, calcium chloride-ethanol-water solution, etc.; and / or, The mass concentration of the structural proteins in the mixed solution is 0.5-30%.

12. The method of claim 10, wherein: The preparation method specifically includes the following steps: S1, mixing one of the structural proteins and the wedge reagent with a solution capable of destroying the secondary structure of the structural proteins to obtain a first mixed solution; S2, mixing the other with the first mixed solution or mixing the other with the solution capable of destroying the secondary structure of the structural proteins and then mixing with the first mixed solution to obtain a second mixed solution containing the structural proteins and the wedge reagent; S3, adding a cross-linking agent to the second mixed solution to perform a cross-linking reaction to prepare the hydrogel.

13. A gel bead characterized by: The gel balls are formed by cross-linking the structural proteins, the wedge reagent and the cross-linking agent, and the structural proteins, the wedge reagent and the cross-linking agent are respectively the structural proteins, the wedge reagent and the cross-linking agent according to any one of claims 1-9.

14. The gel sphere of claim 13, wherein: The particle size of the gel balls is 1-1000 μm.

15. A method of producing a gel bead as claimed in claim 13 or 14, characterised by: The preparation method includes the following steps: Step one, mixing the wedge reagent, the structural proteins and the solution capable of destroying the secondary structure of the structural proteins; Step two, adding a cross-linking agent to the mixed solution of step one to obtain a mixed solution containing the cross-linking agent, and adding the mixed solution containing the cross-linking agent dropwise to an oil phase in a moving state to perform a reaction to prepare the gel balls.

16. An artificial corneal substitute or assist material, characterized by: The artificial cornea substitute or auxiliary material is formed by cross-linking the structural proteins, the wedge reagent and the cross-linking agent, and the structural proteins, the wedge reagent and the cross-linking agent are respectively the structural proteins, the wedge reagent and the cross-linking agent according to any one of claims 1-9.

17. A preparation method of the artificial cornea substitute or auxiliary material according to claim 16, characterized in that: Step one, mixing the wedge reagent, the structural proteins and the solution capable of destroying the secondary structure of the structural proteins; Step two, adding a cross-linking agent to the mixed solution of step one to obtain a mixed solution containing the cross-linking agent, and adding the mixed solution containing the cross-linking agent dropwise to a mold to perform a reaction to prepare the artificial cornea substitute or auxiliary material.

18. Use of the hydrogel according to any one of claims 1-9 or the gel balls according to claim 13 or 14 in the preparation of tissue engineering filling or repair, drug delivery materials.

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