High-strength regenerated silk fibroin material and preparation method thereof

Abstract

The application belongs to the technical field of biomedicine and specifically relates to a high-strength regenerated silk fibroin material and a preparation method thereof, and the preparation method is specifically as follows: gradient alcohol treatment and vacuum drying treatment are sequentially performed on chemical crosslinking silk fibroin hydrogel, and the high-strength regenerated silk fibroin material is obtained, wherein the gradient alcohol treatment refers to stage-by-stage treatment by using ethanol aqueous solution, the concentration of the ethanol aqueous solution in each stage is fixed, and the concentration of the ethanol aqueous solution in different stages becomes larger as the stage number becomes larger; and the finally prepared high-strength regenerated silk fibroin material has a double crosslinking network structure, and the double crosslinking network structure is formed by mutual intertwining of a silk fibroin chemical crosslinking network and a beta-sheet structure physical crosslinking network. The preparation method is simple and easy to implement, and significantly improves the compression mechanical properties (compression modulus is greater than or equal to 0.2 GPa) of the regenerated silk fibroin material.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to a high-strength regenerated silk fibroin material and a preparation method thereof. BACKGROUND

[0002] Silk fibroin (SF) is a natural amino acid polymer with excellent physicochemical properties, and has excellent biocompatibility, degradability, processability and flexibility, and has a wide application prospect in the field of biomedical. The raw material of silk fibroin is usually obtained by degumming the cocoon to remove the sericin covered on the surface, and then is regenerated into various regenerated silk fibroin (RSF) materials for use, such as silk fibroin microspheres, fibers, films, hydrogels, three-dimensional scaffolds and the like. However, due to the influence of the thinking set of the soft performance of natural silk fibroin, and the limitation of the conventional regeneration process and technology, the developed RSF materials often cannot obtain high mechanical strength (such as compression modulus). This leads to its difficulty in meeting the repair and treatment needs of load-bearing tissues, and further limits the application range of RSF materials in the field of biomedicine and the like to a certain extent.

[0003] In recent years, a small number of frontier reports have carried out exploratory research in related fields. Because a considerable part of regenerated silk fibroin materials has obvious water-soluble properties at the initial stage, it is usually necessary to have certain pretreatment to give it the property of "water insoluble" in order to facilitate subsequent use in aqueous environment. The strategy that comes to mind is to first use high-concentration alcohol treatment to make it "water insoluble", at the same time, such treatment methods will also bring a certain degree of mechanical property improvement. For example, patent (publication number: CN109999227A) by using 100% ethanol to treat silk fibroin and chitin composite electrospun fibers first, silk fibroin-based nanofiber scaffold that can be used stably in aqueous environment is obtained. Literature (Enhancing Mechanical Properties of Silk Fibroin Hydrogel through Restricting the Growth of β-Sheet Domains[J]. ACS Applied Materials & Interfaces, 2017, 9(20): 17489-17498.) by using 90% ethanol to treat peroxidase cross-linked silk fibroin hydrogel, silk fibroin hydrogel with excellent strength is prepared, and the compression modulus can reach 3MPa. Similarly, literature (Engineering biomimetic silk fibroin hydrogel scaffolds with "organic-inorganic assembly" strategy for rapid bone regeneration[J]. Bioactive Materials, 2024, 40:541-556.) by using 100% ethanol to treat methacrylated silk fibroin chemical cross-linked hydrogel, silk fibroin hydrogel material with compression modulus of 2.33MPa is obtained.The compression modulus of the material obtained by such processing methods (literature (Scaffolds for bone-tissue engineering[J]. Matter, 2022, 5(9): 2722-2759), literature (Scaffold Fabrication Technologies and Structure / Function Properties in Bone Tissue Engineering[J]. Advanced Functional Materials, 2021, 31: 2010609.), literature (Biomimetic porous scaffolds for bone tissue engineering[J]. Materials Science and Engineering: R: Reports, 2014, 80: 1-36.)) is obviously improved compared with the RSF material prepared by the traditional method, but it is still difficult to meet the ideal demand (0.1-2GPa) of load-bearing tissue repair and treatment. In addition, the literature (Thermoplastic moulding of regenerated silk[J]. Nature Materials, 2020, 19: 102-108.) prepared a high-strength silk fibroin material by high-temperature combined with ultra-high pressure molding method, that is, using silk fibroin grinding powder raw material to prepare a high-strength silk fibroin material with a compression modulus of up to 3GPa by hot pressing molding under high temperature and ultra-high pressure (145℃, 632MPa). However, the processing conditions of such regenerated silk fibroin material are extremely harsh, which brings a significant increase in processing cost, and is not conducive to large-scale production and promotion.

[0004] In summary, there is still a lack of a simple and easy-to-use method for preparing high-strength regenerated silk fibroin material to meet the needs of its application in the field of load-bearing tissue repair and treatment. SUMMARY

[0005] The purpose of the present application is to overcome the problems existing in the prior art, and to provide a high-strength regenerated silk fibroin material and a preparation method thereof.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is:

[0007] The application discloses a preparation method of a high-strength regenerated silk fibroin material.

[0008] As a preferred technical scheme,

[0009] The preparation method of the high-strength regenerated silk fibroin material as described above, wherein the compression modulus of the chemical cross-linking silk fibroin hydrogel is greater than or equal to 1 KPa, and the compression modulus of the high-strength regenerated silk fibroin material is greater than or equal to 0.2 GPa.

[0010] The preparation method of the high-strength regenerated silk fibroin material as described above, wherein the stage treatment is divided into at least two stages, the volume concentration of the ethanol aqueous solution is 10% to 100%, the treatment time of each stage is 1 to 3 hours, and the treatment temperature is 25 to 37 DEG C.

[0011] The preparation method of the high-strength regenerated silk fibroin material as described above, wherein the volume ratio of the chemical cross-linking silk fibroin hydrogel to the ethanol aqueous solution in each stage is 1:10 to 50.

[0012] The preparation method of the high-strength regenerated silk fibroin material as described above, wherein the vacuum drying treatment is carried out at a temperature of 25 to 37 DEG C, an air pressure of less than or equal to 2 KPa, and for a time of 8 to 24 hours.

[0013] The preparation method of the high-strength regenerated silk fibroin material as described above, wherein the preparation process of the chemical cross-linking silk fibroin hydrogel comprises the following steps: adding a LAP (lithium salt of phenyl-2, 4, 6-trimethyl benzoyl phosphinic acid) solution into a SFMA (methacrylated silk fibroin) solution, and then curing the mixture into a gel by ultraviolet light, so as to obtain the chemical cross-linking silk fibroin hydrogel (SFMA hydrogel, silk fibroin is cross-linked into a gel after introducing other functional groups by chemical modification).

[0014] The preparation method of the high-strength regenerated silk fibroin material as described above, wherein the concentration of the SFMA solution is 10 to 20 wt%, the concentration of the LAP solution is 2 wt%, the mass ratio of the SFMA solution to the LAP solution is 1:0.1 to 0.3, the time for curing the mixture into a gel by ultraviolet light is 2 to 5 minutes, the ultraviolet light intensity is 20 to 50 mW / cm 2 , and the curing temperature is 25 to 37 DEG C.

[0015] The preparation method of the high-strength regenerated silk fibroin material as described above, and the preparation process of the chemically cross-linked silk fibroin hydrogel is as follows: after adding Ru (tris (2, 2'-dipyridyl) dichloro ruthenium (II) hexahydrate) solution and SPS (sodium persulfate) solution into the SF (silk fibroin) solution in sequence, visible light curing is carried out to form a gel, and the chemically cross-linked silk fibroin hydrogel (SF hydrogel, functional groups of amino acids of the silk fibroin itself are cross-linked to form a gel) is obtained.

[0016] The preparation method of the high-strength regenerated silk fibroin material as described above, and the preparation process of the chemically cross-linked silk fibroin hydrogel is as follows: after adding Ru (tris (2, 2'-dipyridyl) dichloro ruthenium (II) hexahydrate) solution and SPS (sodium persulfate) solution into the SF (silk fibroin) solution in sequence, visible light curing is carried out to form a gel, and the chemically cross-linked silk fibroin hydrogel (SF hydrogel, functional groups of amino acids of the silk fibroin itself are cross-linked to form a gel) is obtained. 2 The concentration of the SF solution is 10-20wt%, the concentration of the Ru solution is 0.03-0.07mol / L, the concentration of the SPS solution is 0.3-0.7mol / L, the volume ratio of the SF solution to the Ru solution is 20-100:1, the volume ratio of the Ru solution to the SPS solution is 1:0.8-1.2, the visible light curing time is 5-30min, and the visible light intensity is 20-50mW / cm

[0017] The high-strength regenerated silk fibroin material prepared by the preparation method of the high-strength regenerated silk fibroin material as described above has a double cross-linked network structure, and the double cross-linked network structure is formed by the silk chemical cross-linked network and the beta-fold structure physical cross-linked network.

[0018] Invention principle:

[0019] The present application is based on the chemically cross-linked silk fibroin hydrogel, and further uses gradient alcohol treatment and vacuum drying post-processing to realize the preparation of the high-strength regenerated silk fibroin material.

[0020] Firstly, the silk fibroin hydrogel is constructed based on the chemical cross-linking strategy, and the regenerated silk fibroin material with a three-dimensional chemical cross-linked network structure is formed. For example, the characteristic functional groups (such as phenolic hydroxyl groups) remaining in the natural silk fibroin molecules are reacted to form a three-dimensional cross-linked network based on an enzyme / non-enzyme cross-linking system; or the characteristic cross-linking groups (such as carbon-carbon double bonds) are introduced on the natural silk fibroin molecules based on the chemical modification method, and then the corresponding reaction strategy is used to form a three-dimensional cross-linked network structure.

[0021] Further gradient alcohol treatment is performed on the chemical cross-linking formed silk fibroin hydrogel, so that the relatively loose alpha helix conformation in the silk fibroin material is converted into a dense beta sheet conformation (crystalline structure). Unlike single high concentration ethanol treatment, the present patent innovatively introduces gradient alcohol treatment (from low concentration to high concentration) to make the conformation conversion process of silk fibroin molecules relatively mild, and the conformation conversion occurs relatively synchronously and uniformly from the inside to the outside. Further, it ensures that the internal cross-linking network structure (including chemical cross-linking network and beta sheet conformation formed physical cross-linking network) of the hydrogel is more uniform, and the overall shrinkage of the hydrogel is more uniform, which ultimately leads to good shape retention of the appearance and morphology characteristics of the material, and significantly improves the overall mechanical properties of the material. The specific principles include: when the hydrogel is soaked in a lower concentration alcohol aqueous solution, the water molecules in the alcohol aqueous solution first enter the hydrogel to swell the hydrogel, so that the alcohol molecules are more conducive to entering the interior of the hydrogel, thus the internal silk fibroin molecule chains can also be in full contact with the alcohol, ensuring the uniformity of the internal and external microenvironment; the low concentration alcohol treatment in the early stage does not cause rapid conformation conversion of silk fibroin, thus providing conditions for the entry of alcohol molecules under medium or high concentration conditions in the later stage; when the hydrogel is gradually soaked in an alcohol aqueous solution with a higher alcohol concentration, the conformation conversion of silk fibroin molecules will gradually accelerate. However, due to the relatively uniform internal and external microenvironment of the material, this will lead to relatively uniform conformation conversion of different parts of the material, and the shrinkage of different parts of the material becomes more consistent. On the contrary, if the hydrogel is directly soaked in a high concentration alcohol aqueous solution, the high concentration alcohol will cause the surface part of the hydrogel to rapidly undergo conformation conversion and generate a relatively dense beta sheet crystalline structure, which will inevitably block the further entry of alcohol molecules, thus leading to uneven conformation conversion and material shrinkage, which is not conducive to the substantial improvement of the mechanical properties of the material.

[0022] Subsequently, the material is subjected to further vacuum drying post-treatment, so that a large number of alcohol molecules and a small amount of water on the surface and inside are removed relatively uniformly under relatively mild external conditions. In this process, further uniform shrinkage of different parts of the material occurs, and a high-strength regenerated silk fibroin material is finally prepared. The specific principle is as follows: the vacuum drying post-treatment is carried out at a relatively mild room temperature. In addition, the vacuum condition is conducive to the simultaneous removal of alcohol molecules and a small amount of water on the surface and inside of the material under relatively uniform conditions, thereby ensuring the uniformity of solvent removal and shrinkage of the material in different parts. If high-temperature drying or room temperature drying is used for post-treatment, since the ethanol on the surface of the material is easily removed first, the rapid removal of the surface solvent may cause the surface of the material to shrink rapidly and become denser, which will increase the difficulty of internal solvent removal and cause uneven shrinkage and solvent evaporation in different parts of the material. Thus, the anisotropy of the material is increased and the mechanical strength of the material is decreased. If the cold drying method is directly used for post-treatment, since the formation and disappearance of "ice crystals" are involved in the freezing and sublimation process, the anisotropy of the internal pore structure will be significantly increased, thereby significantly reducing the mechanical strength of the material.

[0023] In summary, on the basis of the formation of a three-dimensional chemical cross-linking network of silk fibroin material, the gradient alcohol treatment and vacuum drying post-treatment further jointly ensure the formation of a regular and uniform β-sheet crystal structure in the material and the uniform shrinkage of different parts of the material. The formation of the structure and the reduction of the anisotropy of the material comprehensively ensure the preparation of high-strength silk fibroin material. In addition, it can be predicted that by introducing reinforcing fillers or other substances that bring about enhanced chemical or physical cross-linking networks into such silk fibroin systems using related process flows, composite modified materials with better comprehensive mechanical properties are expected to be obtained.

[0024] Advantages:

[0025] (1) The preparation method of the high-strength regenerated silk fibroin material of the present application has simple and easy-to-operate preparation process and low cost. In the preparation process, the gradient alcohol treatment and vacuum drying post-treatment processes are used, which is conducive to the formation of a relatively regular and uniform crystal structure, can effectively avoid the occurrence of anisotropic structure and anisotropic shrinkage of the silk fibroin material to a great extent, and significantly improves the compressive mechanical properties (compressive modulus ≥ 0.2 GPa) of the regenerated silk fibroin material.

[0026] (2) The high-strength regenerated silk fibroin material of the present application simultaneously has excellent biocompatibility and biodegradability, and has good application prospect in the field of hard tissue biomedical materials. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1Macroscopic comparison of regenerated silk fibroin materials obtained after single concentration alcohol treatment (Comparative Example 1) and gradient alcohol treatment (Example 1), where (a) is Comparative Example 1, (b) is Example 1, and it can be seen that gradient alcohol treatment + vacuum drying post-treatment can obtain uniform material shrinkage;

[0028] Figure 2 Internal structure comparison of regenerated silk fibroin materials obtained after single concentration alcohol treatment (Comparative Example 1) and gradient alcohol treatment (Example 1), where (a) is Comparative Example 1, (b) is Example 1, and it can be seen that gradient alcohol treatment + vacuum drying post-treatment can obtain regenerated silk fibroin materials with more compact and uniform internal structure. DETAILED DESCRIPTION

[0029] The application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the application and not to limit the scope of the application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the application after reading the content taught by the application, and these equivalent forms also fall within the scope of the appended claims.

[0030] The compression modulus detection method in the following text: using INSTRON / 5969 electronic universal material testing machine to perform static compression test on the test material, and the specific test method is: adjusting the upper and lower clamps to make the clamps just contact the test material, and then compressing the test material at a compression rate of 10 mm / min until the test material breaks, obtaining the stress-strain data of the test material at different time points, and selecting the region with strain of 0%-5% in the stress-strain curve for linear fitting, and calculating to obtain the compression modulus of the test material.

[0031] Example 1

[0032] A preparation method of a high-strength regenerated silk fibroin material, and the specific steps are as follows:

[0033] (1) Preparation of raw materials:

[0034] SFMA solution: concentration of 15wt%, solvent is deionized water;

[0035] LAP solution: concentration of 2wt%, solvent is deionized water;

[0036] Ethanol aqueous solution;

[0037] (2) Preparation of chemically cross-linked silk fibroin hydrogel:

[0038] After adding the LAP solution (mass ratio of 1:0.1) to the SFMA solution, the solution was irradiated under ultraviolet light with an intensity of 30 mW / cm 2, and the chemical cross-linked silk fibroin hydrogel with a compression modulus of 3 KPa is obtained after UV curing for 5 min at 25℃;

[0039] (3) Preparation of high-strength regenerated silk fibroin:

[0040] The chemical cross-linked silk fibroin hydrogel is treated by using ethanol aqueous solution in stages, and the volume ratio of the chemical cross-linked silk fibroin hydrogel to the ethanol aqueous solution in each stage is 1:50, and the concentration of the ethanol aqueous solution in each stage is fixed:

[0041] The volume concentration of the ethanol aqueous solution in stage one is 20%, the treatment time is 1.5 h, and the treatment temperature is 25℃;

[0042] The volume concentration of the ethanol aqueous solution in stage two is 40%, the treatment time is 1.5 h, and the treatment temperature is 25℃;

[0043] The volume concentration of the ethanol aqueous solution in stage three is 60%, the treatment time is 1.5 h, and the treatment temperature is 25℃;

[0044] The volume concentration of the ethanol aqueous solution in stage four is 80%, the treatment time is 1.5 h, and the treatment temperature is 25℃;

[0045] The volume concentration of the ethanol aqueous solution in stage five is 100%, the treatment time is 3 h, and the treatment temperature is 25℃;

[0046] After the treatment in stages, vacuum drying treatment is performed at an ambient pressure of 2 KPa and a temperature of 25℃ for 12 h, and the high-strength regenerated silk fibroin material is obtained.

[0047] The compression modulus of the finally obtained high-strength regenerated silk fibroin material is 0.3 GPa, and the high-strength regenerated silk fibroin material has a double cross-linked network structure formed by the silk chemical cross-linked network and the beta-fold structure physical cross-linked network interwoven with each other.

[0048] Comparative Example 1

[0049] A method for preparing a regenerated silk fibroin material is basically the same as that in Example 1, except that the volume concentration of the ethanol aqueous solution used in stages one to five in the comparative example is the same, and is 100%.

[0050] The compression modulus of the finally obtained regenerated silk fibroin material is 152 MPa, and the regenerated silk fibroin material has a double cross-linked network structure formed by the silk chemical cross-linked network and the beta-fold structure physical cross-linked network interwoven with each other.

[0051] Compared with Example 1, it can be seen that the compression modulus of the regenerated silk fibroin material prepared in Comparative Example 1 decreases significantly. The reason for this difference is that the chemical crosslinking silk fibroin hydrogel in Comparative Example 1 is directly immersed in high-concentration pure ethanol, while Example 1 adopts a gradient immersion method. Due to direct immersion in high-concentration ethanol, the chemical crosslinking silk fibroin hydrogel in Comparative Example 1 rapidly undergoes a conformational transition on the surface, forming a dense structure. This rapidly formed dense structure hinders the further penetration of alcohol molecules into the interior of the chemical crosslinking silk fibroin hydrogel, resulting in an uneven conformational transition of the hydrogel material as a whole (as shown in Figure 1 、 Figure 2 indicated). This uneven conformational transition increases the anisotropy of the internal structure, thereby making the compression modulus of the finally prepared regenerated silk fibroin material less improved than Example 1.

[0052] Comparative Example 2

[0053] A method for preparing a regenerated silk fibroin material, which is basically the same as Example 1, except that Comparative Example 2 uses natural drying instead of vacuum drying.

[0054] The compression modulus of the finally prepared regenerated silk fibroin material is 79 MPa, and it has a double crosslinking network structure composed of a silk chemical crosslinking network and a β-sheet structure physical crosslinking network intertwined with each other.

[0055] Compared with Example 1, it can be seen that the compression modulus of the regenerated silk fibroin material prepared in Comparative Example 2 decreases significantly. The reason for this difference is that Comparative Example 2 uses a natural drying method. During the drying process, the ethanol on the surface of the silk fibroin material is easily removed quickly, causing the surface of the silk fibroin material to shrink rapidly and become denser. This change increases the difficulty of solvent removal in the interior of the silk fibroin material, resulting in uneven shrinkage and solvent evaporation in different parts of the silk fibroin material, thereby improving the anisotropy of the finally prepared regenerated silk fibroin material and reducing the compression modulus.

[0056] Example 2

[0057] A method for preparing a high-strength regenerated silk fibroin material, the specific steps are as follows:

[0058] (1) Prepare raw materials:

[0059] SFMA solution: concentration of 20wt%, solvent is deionized water;

[0060] LAP solution: concentration of 2wt%, solvent is deionized water;

[0061] Ethanol aqueous solution;

[0062] (2) Preparation of chemically cross-linked silk fibroin hydrogel:

[0063] After adding the LAP solution (mass ratio of 1:0.3) into the SFMA solution, the chemically cross-linked silk fibroin hydrogel with a compression modulus of 27 KPa was obtained by ultraviolet curing for 20 min under the condition of ultraviolet light intensity of 50 mW / cm 2 and temperature of 30℃.

[0064] (3) Preparation of high-strength regenerated silk fibroin:

[0065] The chemically cross-linked silk fibroin hydrogel was treated by ethanol aqueous solution in stages, and the volume ratio of the chemically cross-linked silk fibroin hydrogel to the ethanol aqueous solution in each stage was 1:50, and the concentration of the ethanol aqueous solution in each stage was fixed.

[0066] The volume concentration of the ethanol aqueous solution in stage one was 20%, the treatment time was 2 h, and the treatment temperature was 37℃.

[0067] The volume concentration of the ethanol aqueous solution in stage two was 40%, the treatment time was 2 h, and the treatment temperature was 37℃.

[0068] The volume concentration of the ethanol aqueous solution in stage three was 60%, the treatment time was 2 h, and the treatment temperature was 37℃.

[0069] After the stage-by-stage treatment, vacuum drying treatment was performed at an environmental pressure of 1 KPa and a temperature of 30℃ for 24 h, and the high-strength regenerated silk fibroin material was obtained.

[0070] The compression modulus of the finally obtained high-strength regenerated silk fibroin material was 0.33 GPa, and the material had a double cross-linked network structure composed of a silk chemical cross-linked network and a β-sheet structure physical cross-linked network intertwined with each other.

[0071] Example 3

[0072] A method for preparing a high-strength regenerated silk fibroin material, and the specific steps are as follows:

[0073] (1) Preparation of raw materials:

[0074] SFMA solution: concentration of 15 wt%, solvent is deionized water;

[0075] LAP solution: concentration of 2 wt%, solvent is deionized water;

[0076] Ethanol aqueous solution;

[0077] (2) Preparation of chemically cross-linked silk fibroin hydrogel:

[0078] After adding the LAP solution (mass ratio 1:0.2) into the SFMA solution, the chemical crosslinking silk fibroin hydrogel with a compression modulus of 10 KPa was obtained by ultraviolet light curing for 20 min under the condition of ultraviolet light intensity of 50 mW / cm 2 , temperature of 37℃.

[0079] (3) Preparation of high-strength regenerated silk fibroin:

[0080] The chemical crosslinking silk fibroin hydrogel was treated by ethanol aqueous solution in stages, and the volume ratio of the chemical crosslinking silk fibroin hydrogel to the ethanol aqueous solution in each stage was 1:20, and the concentration of the ethanol aqueous solution in each stage was fixed.

[0081] The volume concentration of the ethanol aqueous solution in stage one was 10%, the treatment time was 2 h, and the treatment temperature was 30℃.

[0082] The volume concentration of the ethanol aqueous solution in stage two was 30%, the treatment time was 2 h, and the treatment temperature was 30℃.

[0083] The volume concentration of the ethanol aqueous solution in stage three was 50%, the treatment time was 2 h, and the treatment temperature was 30℃.

[0084] The volume concentration of the ethanol aqueous solution in stage four was 70%, the treatment time was 2 h, and the treatment temperature was 30℃.

[0085] The volume concentration of the ethanol aqueous solution in stage five was 90%, the treatment time was 2 h, and the treatment temperature was 30℃.

[0086] The volume concentration of the ethanol aqueous solution in stage six was 100%, the treatment time was 2 h, and the treatment temperature was 30℃.

[0087] After the stage-by-stage treatment, vacuum drying treatment was carried out at an ambient pressure of 2 KPa and a temperature of 37℃ for 8 h, and a high-strength regenerated silk fibroin material was obtained.

[0088] The compression modulus of the finally obtained high-strength regenerated silk fibroin material was 0.28 GPa, and the material had a double crosslinking network structure composed of a silk chemical crosslinking network and a β-sheet structure physical crosslinking network intertwined with each other.

[0089] Example 4

[0090] A method for preparing a high-strength regenerated silk fibroin material, and the specific steps are as follows:

[0091] (1) Preparation of raw materials:

[0092] SFMA solution: concentration of 10 wt%, solvent is deionized water;

[0093] LAP solution: concentration of 2wt%, solvent is deionized water;

[0094] ethanol aqueous solution;

[0095] (2) Preparation of chemically cross-linked silk fibroin hydrogel:

[0096] After adding the LAP solution (mass ratio of 1:0.1) to the SFMA solution, the chemically cross-linked silk fibroin hydrogel with a compression modulus of 1 KPa was obtained by ultraviolet curing for 30 min under the condition of ultraviolet light intensity of 20 mW / cm 2 , temperature of 37℃.

[0097] (3) Preparation of high-strength regenerated silk fibroin:

[0098] The chemically cross-linked silk fibroin hydrogel was treated by ethanol aqueous solution in stages, and the volume ratio of the chemically cross-linked silk fibroin hydrogel to the ethanol aqueous solution in each stage was 1:10, and the concentration of the ethanol aqueous solution in each stage was fixed:

[0099] The volume concentration of the ethanol aqueous solution in stage one was 10%, the treatment time was 3h, and the treatment temperature was 37℃.

[0100] The volume concentration of the ethanol aqueous solution in stage two was 80%, the treatment time was 3h, and the treatment temperature was 37℃.

[0101] After the stage-by-stage treatment, vacuum drying treatment was carried out at an ambient pressure of 2KPa and a temperature of 25℃ for 8h, and a high-strength regenerated silk fibroin material was obtained.

[0102] The compression modulus of the finally obtained high-strength regenerated silk fibroin material was 0.21GPa, and the material had a double cross-linked network structure composed of a silk chemical cross-linked network and a beta-sheet structure physical cross-linked network intertwined with each other.

[0103] Example 5

[0104] A method for preparing a high-strength regenerated silk fibroin material, the specific steps are as follows:

[0105] (1) Preparation of raw materials:

[0106] SF solution: concentration of 15wt%, solvent is deionized water;

[0107] Ru solution: concentration of 0.05mol / L, solvent is deionized water;

[0108] SPS solution: concentration of 0.5mol / L, solvent is deionized water;

[0109] ethanol aqueous solution;

[0110] (2) Preparation of chemically cross-linked silk fibroin hydrogel:

[0111] After adding the Ru solution and the SPS solution into the SF solution successively, the chemically cross-linked silk fibroin hydrogel with a compression modulus of 13 KPa was obtained by visible light curing for 20 min under the condition of a visible light intensity of 30 mW / cm 2 and a temperature of 30℃.

[0112] The volume ratio of the SF solution concentration to the Ru solution was 20:1, and the volume ratio of the Ru solution to the SPS solution was 1:1.

[0113] (3) Preparation of high-strength regenerated silk fibroin:

[0114] The chemically cross-linked silk fibroin hydrogel was treated by ethanol aqueous solution in stages, and the volume ratio of the chemically cross-linked silk fibroin hydrogel to the ethanol aqueous solution was 1:50 in each stage, and the concentration of the ethanol aqueous solution in each stage was fixed.

[0115] The volume concentration of the ethanol aqueous solution in stage one was 20%, the treatment time was 1.5 h, and the treatment temperature was 25℃.

[0116] The volume concentration of the ethanol aqueous solution in stage two was 40%, the treatment time was 1.5 h, and the treatment temperature was 25℃.

[0117] The volume concentration of the ethanol aqueous solution in stage three was 60%, the treatment time was 1.5 h, and the treatment temperature was 25℃.

[0118] The volume concentration of the ethanol aqueous solution in stage four was 80%, the treatment time was 1.5 h, and the treatment temperature was 25℃.

[0119] The volume concentration of the ethanol aqueous solution in stage five was 100%, the treatment time was 3 h, and the treatment temperature was 25℃.

[0120] After the treatment in stages, the vacuum drying treatment was performed for 12 h under the condition of an ambient pressure of 2 KPa and a temperature of 25℃, and the high-strength regenerated silk fibroin material was obtained.

[0121] The compression modulus of the finally obtained high-strength regenerated silk fibroin material was 0.27 GPa, and the high-strength regenerated silk fibroin material had a double cross-linked network structure formed by the silk fibroin chemical cross-linked network and the β-sheet structure physical cross-linked network.

[0122] Example 6

[0123] A preparation method of a high-strength regenerated silk fibroin material, and the specific steps are as follows:

[0124] (1) Preparation of raw materials:

[0125] SF solution: concentration of 10wt%, solvent is deionized water;

[0126] Ru solution: concentration of 0.07mol / L, solvent is deionized water;

[0127] SPS solution: concentration of 0.7mol / L, solvent is deionized water;

[0128] Ethanol aqueous solution;

[0129] (2) Preparation of chemically cross-linked silk fibroin hydrogel:

[0130] After adding Ru solution and SPS solution into SF solution in turn, the chemically cross-linked silk fibroin hydrogel with compression modulus of 12KPa is obtained by visible light curing for 5min under the condition of visible light intensity of 20mW / cm 2 , temperature of 25℃.

[0131] The volume ratio of SF solution concentration to Ru solution is 30:1, and the volume ratio of Ru solution to SPS solution is 1:0.8.

[0132] (3) Preparation of high-strength regenerated silk fibroin:

[0133] The chemically cross-linked silk fibroin hydrogel is treated by ethanol aqueous solution in stages, and the volume ratio of chemically cross-linked silk fibroin hydrogel to ethanol aqueous solution in each stage is 1:50, and the concentration of ethanol aqueous solution in each stage is fixed.

[0134] The volume concentration of ethanol aqueous solution in stage one is 20%, the treatment time is 2h, and the treatment temperature is 37℃.

[0135] The volume concentration of ethanol aqueous solution in stage two is 40%, the treatment time is 2h, and the treatment temperature is 37℃.

[0136] The volume concentration of ethanol aqueous solution in stage three is 60%, the treatment time is 2h, and the treatment temperature is 37℃.

[0137] After the treatment in stages, the vacuum drying treatment is carried out under the condition of environmental pressure of 1KPa and temperature of 30℃ for 24h, and the high-strength regenerated silk fibroin material is obtained.

[0138] The compression modulus of the finally obtained high-strength regenerated silk fibroin material is 0.22GPa, and the high-strength regenerated silk fibroin material has a double cross-linked network structure composed of silk chemical cross-linked network and β-fold structure physical cross-linked network.

[0139] Example 7

[0140] A preparation method of a high-strength regenerated silk fibroin material, and the specific steps are as follows:

[0141] (1) Preparation of raw materials:

[0142] SF solution: concentration of 20wt%, solvent is deionized water;

[0143] Ru solution: concentration of 0.07mol / L, solvent is deionized water;

[0144] SPS solution: concentration of 0.7mol / L, solvent is deionized water;

[0145] Ethanol aqueous solution;

[0146] (2) Preparation of chemically crosslinked silk fibroin hydrogel:

[0147] After adding Ru solution and SPS solution into SF solution in turn, the chemically crosslinked silk fibroin hydrogel with compression modulus of 21KPa is obtained by visible light curing for 30min under the condition of visible light intensity of 50mW / cm 2 and temperature of 37℃;

[0148] Wherein, the volume ratio of SF solution concentration to Ru solution is 20:1, and the volume ratio of Ru solution to SPS solution is 1:1.2;

[0149] (3) Preparation of high-strength regenerated silk fibroin:

[0150] The chemically crosslinked silk fibroin hydrogel is treated by ethanol aqueous solution in stages, and the volume ratio of chemically crosslinked silk fibroin hydrogel to ethanol aqueous solution in each stage is 1:20, and the concentration of ethanol aqueous solution in each stage is fixed:

[0151] The volume concentration of ethanol aqueous solution in stage one is 10%, the treatment time is 2h, and the treatment temperature is 30℃;

[0152] The volume concentration of ethanol aqueous solution in stage two is 30%, the treatment time is 2h, and the treatment temperature is 30℃;

[0153] The volume concentration of ethanol aqueous solution in stage three is 50%, the treatment time is 2h, and the treatment temperature is 30℃;

[0154] The volume concentration of ethanol aqueous solution in stage four is 70%, the treatment time is 2h, and the treatment temperature is 30℃;

[0155] The volume concentration of ethanol aqueous solution in stage five is 90%, the treatment time is 2h, and the treatment temperature is 30℃;

[0156] The volume concentration of ethanol aqueous solution in stage six is 100%, the treatment time is 2h, and the treatment temperature is 30℃;

[0157] After the stage treatment, the high-strength regenerated silk fibroin material is obtained by vacuum drying treatment at 2 KPa and 37℃ for 8h.

[0158] The compression modulus of the finally prepared high-strength regenerated silk fibroin material is 0.25 GPa, and the high-strength regenerated silk fibroin material has a double crosslinking network structure formed by intertwining of the chemical crosslinking network and the physical crosslinking network of the beta sheet structure.

[0159] Example 8

[0160] A preparation method of a high-strength regenerated silk fibroin material, and the specific steps are as follows:

[0161] (1) Preparation of raw materials:

[0162] SF solution: concentration of 10wt%, solvent is deionized water;

[0163] Ru solution: concentration of 0.03mol / L, solvent is deionized water;

[0164] SPS solution: concentration of 0.3mol / L, solvent is deionized water;

[0165] Ethanol aqueous solution;

[0166] (2) Preparation of chemically crosslinked silk fibroin hydrogel:

[0167] After adding the Ru solution and the SPS solution into the SF solution in sequence, the chemically crosslinked silk fibroin hydrogel with a compression modulus of 4 KPa is obtained by visible light curing at a visible light intensity of 20mW / cm 2 and a temperature of 37℃ for 5min;

[0168] The volume ratio of the concentration of the SF solution to the Ru solution is 100:1, and the volume ratio of the Ru solution to the SPS solution is 1:0.8;

[0169] (3) Preparation of high-strength regenerated silk fibroin:

[0170] The chemically crosslinked silk fibroin hydrogel is subjected to stage treatment by using the ethanol aqueous solution, and the volume ratio of the chemically crosslinked silk fibroin hydrogel to the ethanol aqueous solution is 1:10, and the concentration of the ethanol aqueous solution in each stage is fixed:

[0171] The volume concentration of the ethanol aqueous solution in the first stage is 10%, the treatment time is 3h, and the treatment temperature is 37℃;

[0172] The volume concentration of the ethanol aqueous solution in the second stage is 80%, the treatment time is 3h, and the treatment temperature is 37℃;

[0173] After the stage treatment, the high-strength regenerated silk fibroin material is obtained by vacuum drying treatment at 1 KPa and 25℃ for 8h.

[0174] The compression modulus of the finally prepared high-strength regenerated silk fibroin material is 0.2 GPa, and the high-strength regenerated silk fibroin material has a double crosslinking network structure formed by the silk fibroin chemical crosslinking network and the β-sheet structure physical crosslinking network interwoven with each other.

Claims

1. A method for preparing a high-strength regenerated silk fibroin material, characterized by, The high-strength regenerated silk fibroin material is obtained by gradient alcohol treatment and vacuum drying treatment on the chemical cross-linking silk fibroin hydrogel in sequence, wherein the gradient alcohol treatment refers to stage-by-stage treatment with ethanol aqueous solution, the concentration of the ethanol aqueous solution in each stage is fixed, and the concentration of the ethanol aqueous solution in different stages increases with the stage number.

2. The method for preparing a high-strength regenerated silk fibroin material according to claim 1, characterized in that, The compression modulus of the chemical cross-linking silk fibroin hydrogel is greater than or equal to 1 KPa, and the compression modulus of the high-strength regenerated silk fibroin material is greater than or equal to 0.2 GPa.

3. The method of claim 1, wherein the regenerated silk fibroin material has a tensile strength of 100 MPa or more. The stage-by-stage treatment is divided into at least two stages, the volume concentration of the ethanol aqueous solution is 10% to 100%, the treatment time in each stage is 1 to 3 hours, and the treatment temperature is 25 to 37 DEG C.

4. The method of claim 3, wherein the regenerated silk fibroin material has a tensile strength of 100 MPa or more. The volume ratio of the chemical cross-linking silk fibroin hydrogel to the ethanol aqueous solution in each stage is 1:10 to 50.

5. The method for preparing a high-strength regenerated silk fibroin material according to claim 1, characterized in that, The temperature of the vacuum drying treatment is 25 to 37 DEG C, the air pressure is less than or equal to 2 KPa, and the time is 8 to 24 hours.

6. The method for preparing a high-strength regenerated silk fibroin material according to claim 1, characterized in that, The preparation process of the chemical cross-linking silk fibroin hydrogel is as follows: after adding a lithium salt of phenyl-2,4,6-trimethylbenzoyl phosphinic acid solution into a methylacrylated silk fibroin solution, the solution is cured into a gel by ultraviolet light, and the chemical cross-linking silk fibroin hydrogel is obtained.

7. The method of claim 6, wherein the regenerated silk fibroin material has a tensile strength of 100 MPa or more. The concentration of the methacrylated silk fibroin solution is 10-20 wt%, the concentration of the phenyl-2,4,6-trimethylbenzoyl phosphinic acid lithium salt solution is 2 wt%, the mass ratio of the methacrylated silk fibroin solution to the phenyl-2,4,6-trimethylbenzoyl phosphinic acid lithium salt solution is 1:0.1-0.3, the time for ultraviolet light curing is 2-5 min, the ultraviolet light intensity is 20-50 mW / cm 2 , and the curing temperature is 25-37℃.

8. The method of claim 1, wherein the regenerated silk fibroin material has a tensile strength of 100 MPa or more. The preparation process of the chemical cross-linking silk fibroin hydrogel is as follows: after adding a tris(2,2'-bipyridyl)ruthenium(II) dichloride hexahydrate solution and a sodium persulfate solution into a silk fibroin solution in sequence, the solution is cured into a gel by visible light, and the chemical cross-linking silk fibroin hydrogel is obtained.

9. The method of claim 8, wherein the regenerated silk fibroin material has a tensile strength of 100 MPa or more. The concentration of the silk fibroin solution is 10-20 wt%, the concentration of the tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate solution is 0.03-0.07 mol / L, the concentration of the sodium persulfate solution is 0.3-0.7 mol / L, the volume ratio of the silk fibroin solution to the tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate solution is 20-100:1, the volume ratio of the tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate solution to the sodium persulfate solution is 1:0.8-1.2, the time for visible light curing is 5-30 min, the visible light intensity is 20-50 mW / cm 2 , and the curing temperature is 25-37℃.

10. The high-strength regenerated silk fibroin material prepared by the method according to any one of claims 1 to 9, characterized in that, The high-strength regenerated silk fibroin material has a double cross-linking network structure, which is formed by interweaving a silk chemical cross-linking network and a beta-fold structure physical cross-linking network.

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