Medical silk fibroin solution grafted with polysaccharide as well as preparation method and application of medical silk fibroin solution

Abstract

The invention relates to a polysaccharide-grafted medical silk fibroin solution as well as a preparation method and application thereof, and belongs to the technical field of medical materials. The preparation method comprises the following steps: performing protease enzymolysis on silk, simplifying the process, obtaining high beta-folded silk fibroin precipitate, treating the silk fibroin precipitate with a low-concentration second organic solvent, and keeping space shielding of internal amino groups while terminal amino groups are exposed; meanwhile, triazoline diketone is used for activating carboxyl of the polysaccharide, and polysaccharide containing high-activity anhydride is obtained; the medical silk fibroin solution grafted with polysaccharide can be obtained by reacting silk fibroin with polysaccharide containing high-activity anhydride. The medical silk fibroin solution can be stably stored for a long time and kept in a solution state, has good performance and is widely applied to preparation of tissue engineering materials.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical materials, in particular to a grafted polysaccharide medical silk fibroin solution and a preparation method and application thereof. BACKGROUND

[0002] Silk is composed of about 70% silk fibroin and about 25% sericin. Silk fibroin is a natural polymer material extracted from silk, and its molecular structure is composed of highly ordered beta-sheet crystalline regions and loose disordered amorphous regions. Among them, the crystalline region is mainly composed of small molecular amino acid residues such as glycine, alanine and serine, which are repeatedly arranged to form a tightly packed antiparallel beta-sheet structure, giving silk fibroin excellent mechanical strength and stability; while the amorphous region is rich in large side chain amino acids such as phenylalanine, tyrosine and tryptophan, which makes the material have certain flexibility and functional modification.

[0003] The conventional enzymatic hydrolysis of silk fibroin starts from the regenerated silk fibroin solution, and the regenerated silk fibroin solution is obtained by degumming and drying the silk to form degummed silk, dissolving and purifying the degummed silk to form the regenerated silk fibroin solution, and then adding protease to the regenerated silk fibroin solution to obtain silk fibroin. This process includes multiple steps, which not only has a complex process, but also consumes a large amount of chemical reagents and generates a large amount of industrial wastewater.

[0004] After enzymatic hydrolysis of silk fibroin, the connection sites between the amorphous region and the crystalline region are cut off, and the silk fibroin with high beta-sheet content of repeated GAGAGS (G represents glycine, A represents alanine, and S represents serine) is precipitated due to its large molecular weight. These precipitates have unique application advantages in the fields of materials science and biomedical science. However, due to the enzymatic hydrolysis, a large number of hydrophobic regions are exposed, which significantly reduces the hydrophilicity of the silk fibroin surface and affects its solubility. To improve the solubility of silk fibroin, polysaccharides are often used to graft and modify silk fibroin to improve its hydrophilicity. In this process, silk fibroin and polysaccharides form a gel through cross-linking or electrostatic interaction, thereby improving the solubility of silk fibroin. However, these modification techniques all target gel as the final product, which has poor stability, and a large amount of beta-sheets are lost during the preparation process. Therefore, the solid material prepared from the gel state silk fibroin product has insufficient rigidity and limited application.

[0005] Therefore, it is urgent to seek a new preparation method to simplify the process flow while obtaining a silk fibroin solution with good solution stability and mechanical properties. SUMMARY

[0006] Therefore, the technical problem to be solved by the present application is to overcome the problem of lack of a silk fibroin solution with good stability, simple preparation process and good mechanical properties in the prior art.

[0007] To solve the above technical problems, the application provides a grafted polysaccharide medical silk fibroin solution and a preparation method and application thereof. In the preparation process of silk fibroin, the silk fibroin is usually separated by enzymolysis of silk. The traditional enzymolysis technology starts from the regenerated silk fibroin solution (i.e. the silk fibroin solution after degumming of silk), consumes a large amount of chemical reagents, and produces a large amount of industrial wastewater. The application optimizes the process, directly dissolves the silk and then enzymolyzes, improves the process efficiency, and reduces the pollution of reagents and wastewater. At the same time, the enzymolysis of silk under the condition of not degumming can obtain silk fibroin precipitate with high beta-sheet structure. In addition, in the prior art, the hydrophobic region of silk fibroin with high beta-sheet structure is exposed, resulting in poor solubility of the silk fibroin precipitate in water, which is difficult to be directly used as a raw material. The application grafts the polysaccharide to the terminal amino group of the silk fibroin without affecting the internal amino group, which can increase the water solubility of the silk fibroin and avoid excessive crosslinking of the silk fibroin and the polysaccharide to form a gel. Specifically, the application treats the silk fibroin precipitate with a low-concentration solvent to ensure the exposure of the terminal amino group while maintaining the spatial shielding of the internal amino group. At the same time, the carboxyl group of the polysaccharide is activated by triazolinone, and the carboxyl group is converted into a highly reactive anhydride. The intermediate is easy to react with the silk fibroin containing active amino groups, and the terminal amino group of the silk fibroin forms an amide bond with the anhydride of the polysaccharide to enhance the hydrophilicity of the silk fibroin and effectively inhibit the occurrence of self-crosslinking, thereby realizing long-term stable storage of the solution. In addition, the grafted polysaccharide medical silk fibroin solution of the application can promote cell proliferation and has good mechanical properties, and has potential application value in the preparation of tissue engineering materials.

[0008] The first object of the application is to provide a preparation method of a grafted polysaccharide medical silk fibroin solution, which comprises the following steps: S1, dissolving silk in a first organic solvent with a concentration of 6-10M, then adding a protease solution for incubation, inactivating the protease after incubation, centrifuging to obtain a silk fibroin precipitate, dissolving the silk fibroin precipitate in a second organic solvent with a concentration of 4-6M to obtain an enzyme-cut silk fibroin solution; S2, mixing and reacting a polysaccharide containing at least one carboxyl group with triazolinone in the presence of a third organic solvent to obtain an activated polysaccharide; S3, mixing and reacting the enzyme-cut silk fibroin solution in S1 with the activated polysaccharide in S2, dialyzing to obtain a grafted polysaccharide medical silk fibroin solution.

[0009] Further, the first organic solvent and the second organic solvent in step S1 are independently selected from one or more of lithium bromide, sodium thiocyanate, zinc chloride, calcium chloride-ethanol-water ternary solvent. The concentration of the first organic solution is greater than the concentration of the second organic solvent.

[0010] Further, the protease in step S1 is selected from one or more of α-chymotrypsin, pepsin, trypsin, proteinase K, papain, and alkaline protease.

[0011] Further, the mass ratio of the protease to silk in step S1 is 1: (5-500).

[0012] Further, the polysaccharide in step S2 is selected from one or more of sodium alginate, hyaluronic acid, carboxymethyl chitosan, agarose, chitosan, and carboxymethyl cellulose.

[0013] Further, the third organic solvent in step S2 is selected from one or more of N , N dimethylformamide, dimethyl sulfoxide, N , N dimethylacetamide, N methyl pyrrolidone.

[0014] Further, the mass ratio of the triazolin dione to the polysaccharide in step S2 is (10-1): 1.

[0015] Further, the reaction temperature in step S2 is 4-40℃, and the reaction time is 10-180 min.

[0016] Further, after the polysaccharide and the triazolin dione are mixed in step S2, a reaction mediator is added to terminate the reaction, and the reaction mediator is selected from one or more of β-mercaptoethanol, dithiothreitol, cysteine, and glycine. The reaction mediator can rapidly react with the triazolin dione to form a stable adduct (such as a thioester or a disulfide bond), thereby preventing the triazolin dione from continuously activating the carboxyl group.

[0017] Further, the mass ratio of the precipitated dissolved silk fibroin to the polysaccharide in step S3 is 1: (1-10).

[0018] Further, the mass ratio of the triazolin dione to the third organic solvent is 1 g: (5-10) mL.

[0019] Further, the reaction temperature in step S3 is 2-8℃, and the reaction time is 5-60 min.

[0020] A second object of the present application is to provide a medical silk fibroin solution of the grafted polysaccharide prepared by the above preparation method.

[0021] A third object of the present application is to provide an application of the above medical silk fibroin solution in preparing a tissue engineering material.

[0022] Further, the tissue engineering material includes bone tissue material and cell proliferation material.

[0023] The mechanism of the present application is:

[0024] One-step enzymatic hydrolysis of silk: directly dissolve silk in a first organic solvent, and add protease for incubation and enzymatic hydrolysis. Silk is mainly composed of sericin and fibroin, and the first organic solvent can dissolve sericin and fibroin and make their molecular chains unfold, and protease can cut both of them. Due to the structural differences between sericin and fibroin, only the highly β-sheeted crystalline region of fibroin with repeated GAGAGS fragments precipitates to form fibroin precipitate, and the amorphous region of fibroin and sericin remain in the supernatant.

[0025] The fibroin precipitate after enzymatic hydrolysis has a highly repeated GAGAGS secondary structure, and a part of the amino groups are located at the terminal, and a part are located on the internal side chain, especially on the lysine side chain. The conventional way to dissolve the fibroin precipitate is to use a high concentration of a second organic solvent for dissolution, which leads to the exposure of the ε-amino group of the internal lysine of fibroin, and further leads to the excessive crosslinking and gelation of polysaccharides. While the present application uses a low concentration of a second organic solvent to expose only the terminal amino groups of fibroin, but not the internal amino groups; at the same time, the triazolin dione is used to convert the carboxyl groups of polysaccharides into highly reactive anhydrides, and the terminal amino groups of fibroin form amide bonds with the anhydrides of polysaccharides to enhance the hydrophilicity of fibroin and effectively inhibit the occurrence of self-crosslinking phenomenon, thereby realizing the long-term stable storage of its solution.

[0026] The beneficial effects of the present application are:

[0027] (1) The process is more simplified. The traditional method is to form amide bonds between polysaccharides and terminal amino groups of fibroin to obtain silk fibroin solution with directional grafted polysaccharides. The enzymatic hydrolysis of silk fibroin needs to go through the steps of degumming, dissolution and purification to obtain regenerated silk fibroin solution, and then protease is added to the regenerated silk fibroin solution for enzymatic hydrolysis. The present application adopts one-step enzymatic hydrolysis, directly dissolves silk and adds protease for enzymatic hydrolysis, which omits the steps of degumming, dissolution and purification, greatly reduces chemical reagents and industrial pollutants, simplifies the process, improves production efficiency and reduces cost.

[0028] (2) The solution has high stability. The silk fibroin solution with grafted polysaccharides prepared by the present application has good stability, and no precipitate is observed within 90 days, which not only exceeds the stability of traditional regenerated silk fibroin solution and EDC / NHS modified silk fibroin solution, but also avoids the excessive crosslinking of polysaccharides to form gel, which is beneficial to the further development and use.

[0029] (3) The oriented grafting of polysaccharide endows the silk fibroin solution with good performance. The second organic solvent with low concentration is used in the application, only part of the amino groups of the silk fibroin is exposed, especially the terminal amino groups, but the amino groups of the internal side chains are not exposed, so that the polysaccharide is oriented and grafted to the terminal amino groups of the silk fibroin, and the by-products and gelation caused by excessive cross-linking are avoided.

[0030] (4) The mechanical property is excellent, and the application prospect is wide. The film prepared from the grafted polysaccharide medical silk fibroin solution of the application has good mechanical property and cell proliferation effect, and has a wide application prospect in the preparation of tissue engineering materials, especially bone tissue engineering materials and cell proliferation materials. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is a process flow chart of the grafted polysaccharide medical silk fibroin solution of the application and the process flow chart of the silk fibroin product of comparative example 1;

[0032] Figure 2 is a silk gum residue determination result chart of the application example 1-6 and comparative example 1;

[0033] Figure 3 is an infrared spectrum chart of the polysaccharide grafted silk fibroin before and after the application example 1. DETAILED DESCRIPTION

[0034] The application will be further described below in combination with the drawings and specific examples, so that those skilled in the art can better understand the application and implement it, but the examples are not as a limitation on the application.

[0035] The experimental methods used in the following examples are conventional methods, and the materials, reagents, etc. used are commercially available, unless otherwise specified.

[0036] The sources and item numbers of the reagents are as follows.

[0037] Lithium bromide (LiBr) is purchased from Aldrich, and the item number is L108934; Sodium thiocyanate (NaSCN) is purchased from Jinan Hui Fengda, and the item number is HFD-248; Zinc chloride (ZnCl2) is purchased from the State Pharmaceutical, and the item number is H1L0491; Calcium chloride (CaCl2)-ethanol-water ternary solvent, wherein the CaCl2 is purchased from Jiyesheng Chemical, and the item number is JYS1545; Ethanol is purchased from Bao'elai Bo, and the item number is L11024-VZU; Trypsin is purchased from Aldrich, and the item number is T274333; Alpha-chymotrypsin was purchased from Merck, with the item number C804761; Pepsin was purchased from Aladdin, with the item number P110928; Protease K was purchased from Shanghai Yekexing Chemical Technology Co., Ltd., with the item number JK-038; Papain was purchased from Sigma, with the item number P3250; Alkaline protease was purchased from Beijing Biaoleibo, with the item number QN0397; Sodium alginate was purchased from Xi'an Jinxiang Pharmaceutical Auxiliary Material Co., Ltd., with the item number 0045; Hyaluronic acid was purchased from Solarbio, with the item number S7020; Carboxymethyl chitosan was purchased from Sigma, with the item number 926043; Agarose was purchased from Sigma, with the item number A9045; Chitosan was purchased from Beijing Biaoleibo, with the item number QN0147; Carboxymethyl cellulose was purchased from Aladdin, with the item number C104977; N , N Dimethylformamide was purchased from Merck, with the item number N807509; Dimethyl sulfoxide was purchased from Merck, with the item number D806645; N , N Dimethylacetamide was purchased from Merck, with the item number N807171; N Methyl pyrrolidone was purchased from Merck, with the item number M814045; Beta-mercaptoethanol was purchased from Merck, with the item number M828395; Dithiothreitol was purchased from Merck, with the item number D806827; Cysteine was purchased from Merck, with the item number D831312; Glycine was purchased from Sigma, with the item number G8790; Phosphate buffer solution (PBS buffer solution) was purchased from Biomed, with the item number bzW2106f; Triazoline dione was purchased from STANDARDS, with the item number ZC68005; Sodium carbonate (Na2CO3) was purchased from National Pharmaceutical, with the item number 10019260.

[0038] Example 1

[0039] S1. One-step enzymatic hydrolysis of silk: Dissolve 10 g of silk in 100 mL of 9.3 M lithium bromide solution. Filter and mix with equal volumes of 3 mg / g trypsin solution (the mass ratio of trypsin to silk is 3:100). Incubate at 37°C for 20 h, heat at 100°C for 10 min to inactivate the enzyme, and centrifuge at 5000 rpm for 10 min. Remove the lower layer and wash to obtain the silk fibroin precipitate.

[0040] The silk fibroin precipitate was dissolved in 10 mL of 4.0 M lithium bromide solution to obtain the enzymatically cleaved silk fibroin solution.

[0041] S2. Activate polysaccharide carboxyl groups with triazolinedione: Dissolve 2 g of triazolinedione in 10 mL N , N -dimethylformamide, add 2 g of sodium alginate and mix and dissolve, the mass ratio of triazolinedione to sodium alginate is 1:1, react at 4 ° C for 180 minutes, and then add β-mercaptoethanol to terminate the reaction.

[0042] S3. Directed grafting of polysaccharide onto silk fibroin: The enzymatically cleaved silk fibroin solution in S1 and the activated polysaccharide solution in S2 were mixed, with the mass ratio of silk fibroin precipitate to polysaccharide being 1:1, and the mixture was reacted at 2°C for 60 min.

[0043] The reaction mixture was placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed with an aqueous medium for 24 h to obtain a medical silk fibroin solution grafted with polysaccharide.

[0044] Example 2

[0045] S1. One-step enzymatic hydrolysis of silk: 50 g of silk was dissolved in 100 mL of 6.0 M sodium thiocyanate solution. After filtration, the mixture was mixed with equal volumes of 1 mg / g α-chymotrypsin solution (the mass ratio of α-chymotrypsin to silk was 1:500). The reaction was carried out at 37°C for 24 h, and the enzyme was inactivated by heating at 100°C for 15 min. The solution was centrifuged at 6000 rpm for 5 min, and the lower layer was removed and washed to obtain the silk fibroin precipitate.

[0046] The silk fibroin precipitate was dissolved in 50 mL of 4.5 M sodium thiocyanate solution to obtain the enzymatically cleaved silk fibroin solution.

[0047] S2. Activation of polysaccharide carboxyl groups by triazolinedione: Dissolve 10 g of triazolinedione in 50 mL of dimethyl sulfoxide, add 1 g of hyaluronic acid and mix and dissolve. The mass ratio of triazolinedione to hyaluronic acid is 10:1. Mix the two and react at 15°C for 120 min. Then add dithiothreitol to terminate the reaction.

[0048] S3. Directed grafting of polysaccharide onto silk fibroin: The enzymatically cleaved silk fibroin solution in S1 and the activated polysaccharide solution in S2 were mixed, with the mass ratio of silk fibroin precipitate to polysaccharide being 1:10, and the mixture was reacted at 4°C for 15 min.

[0049] The reaction mixture was placed in a dialysis bag with a molecular weight cutoff of 14 kDa and dialyzed with PBS buffer for 18 h to obtain a medical silk fibroin solution grafted with polysaccharide.

[0050] Example 3

[0051] S1. One-step enzymatic hydrolysis of silk: 30 g of silk was dissolved in 100 mL of 8 M zinc chloride solution. After filtration, it was mixed with equal volumes of 3 mg / g pepsin solution (the mass ratio of pepsin to silk was 1:100). The reaction was carried out at 37°C for 18 h. The enzyme was inactivated by heating at 100°C for 20 min. The solution was centrifuged at 8000 rpm for 5 min, and the lower layer was removed and washed to obtain the silk fibroin precipitate.

[0052] The silk fibroin precipitate was dissolved in 30 mL of 5.0 M zinc chloride solution to obtain the enzymatically cleaved silk fibroin solution.

[0053] S2. Activate polysaccharide carboxyl groups with triazolinedione: Dissolve 5 g of triazolinedione in 30 mL N , N -dimethylacetamide, add 1 g of carboxymethyl chitosan and mix and dissolve it. The mass ratio of triazolinedione to carboxymethyl chitosan is 5:1. React at 40 ° C for 10 min, and then add cysteine ​​to terminate the reaction.

[0054] S3. Directed grafting of polysaccharide onto silk fibroin: The enzymatically cleaved silk fibroin solution in S1 and the activated polysaccharide solution in S2 were mixed and reacted. The mass ratio of silk fibroin precipitate to polysaccharide was 1:5, and the reaction was carried out at 8°C for 5 min.

[0055] The reaction mixture was placed in a dialysis bag with a molecular weight cutoff of 20 kDa and dialyzed with water for 36 h to obtain a medical silk fibroin solution grafted with polysaccharide.

[0056] Example 4

[0057] S1. One-step enzymatic hydrolysis of silk: 15 g of silk was dissolved in 100 mL of 10.0 M calcium chloride-ethanol-water ternary solvent (molar ratio of 1:2:8). After filtration, the solution was mixed with equal volumes of 3 mg / g proteinase K solution (the mass ratio of proteinase K to silk was 1:50). The reaction was incubated at 37°C for 16 h, heated at 95°C for 15 min to inactivate the enzyme, and centrifuged at 4000 rpm for 10 min. The lower layer was removed and washed to obtain the silk fibroin precipitate.

[0058] The silk fibroin precipitate was dissolved in 15 mL of 6.0 M calcium chloride-ethanol-water ternary solvent (molar ratio 1:2:8) to obtain the enzyme-cut silk fibroin solution.

[0059] S2, activation of the carboxyl group of polysaccharide by triazolin dione: 4 g of triazolin dione was dissolved in 24 mL of dimethylformamide, 2 g of chitosan was added and dissolved, the mass ratio of triazolin dione to chitosan was 2:1, and the reaction was carried out at 4°C for 180 min, and then β-mercaptoethanol was added to terminate the reaction. N - methylpyrrolidone, 2 g of agarose was added and dissolved, the mass ratio of triazolin dione to agarose was 2:1, and the reaction was carried out at 25°C for 60 min, and then glycine was added to terminate the reaction.

[0060] S3, polysaccharide-directed grafting of silk fibroin: the enzyme-cut silk fibroin solution in S1 and the activated polysaccharide solution in S2 were mixed, the mass ratio of silk fibroin precipitate to polysaccharide was 1:3, and the reaction was carried out at 4°C for 10 min.

[0061] The reaction mixture was placed in a dialysis bag with a molecular weight cut-off of 10 kDa, and dialyzed in PBS buffer for 20 h to obtain a medical silk fibroin solution grafted with polysaccharide.

[0062] Example 5

[0063] S1, one-step enzymatic hydrolysis of silk: 15 g of silk was dissolved in 100 mL of 9.0 M lithium bromide solution, filtered, and mixed with an equal volume of 3 mg / g papain solution (mass ratio of papain to silk 1:50), reacted at 37°C for 24 h, inactivated the enzyme by heating at 100°C for 15 min, centrifuged at 5000 rpm for 10 min, and then the lower layer was taken out and washed to obtain a silk fibroin precipitate.

[0064] The silk fibroin precipitate was dissolved in 15 mL of 5.5 M lithium bromide solution to obtain an enzyme-cut silk fibroin solution.

[0065] S2, activation of the carboxyl group of polysaccharide by triazolin dione: 4 g of triazolin dione was dissolved in 24 mL of dimethylformamide, 2 g of chitosan was added and dissolved, the mass ratio of triazolin dione to chitosan was 2:1, and the reaction was carried out at 4°C for 180 min, and then β-mercaptoethanol was added to terminate the reaction. N , N - dimethylformamide, 2 g of chitosan was added and dissolved, the mass ratio of triazolin dione to chitosan was 2:1, and the reaction was carried out at 4°C for 180 min, and then β-mercaptoethanol was added to terminate the reaction.

[0066] S3, polysaccharide-directed grafting of silk fibroin: the enzyme-cut silk fibroin solution in S1 and the activated polysaccharide solution in S2 were mixed, the mass ratio of silk fibroin precipitate to polysaccharide was 1:4, and the reaction was carried out at 4°C for 20 min.

[0067] The reaction mixture was placed in a dialysis bag with a molecular weight cut-off of 10 kDa, and dialyzed in water medium for 24 h to obtain a medical silk fibroin solution grafted with polysaccharide.

[0068] Example 6

[0069] S1, one-step enzymatic hydrolysis of silk: 20 g of silk was dissolved in 100 mL of 9.3 M lithium bromide solution, filtered, and mixed with an equal volume of 3 mg / g alkaline protease solution (mass ratio of alkaline protease to silk was 3:200), and reacted at 37°C for 18 h. The enzyme was inactivated by heating at 100°C for 10 min, and centrifuged at 4000 rpm for 10 min. The lower layer was taken out and washed to obtain a silk fibroin precipitate.

[0070] The silk fibroin precipitate was dissolved in 20 mL of 4.5 M lithium bromide solution to obtain an enzyme-cut silk fibroin solution.

[0071] S2, activation of polysaccharide carboxyl group by triazolinone: 5 g of triazolinone was dissolved in 50 mL of dimethyl sulfoxide, and 1 g of hydroxymethyl cellulose was added and dissolved. The mass ratio of triazolinone to hydroxymethyl cellulose was 5:1, and the reaction was carried out at 4°C for 150 min. Glycine was then added to terminate the reaction.

[0072] S3, silk fibroin directed grafting of polysaccharide: the enzyme-cut silk fibroin solution in S1 and the activated polysaccharide solution in S2 were mixed, and the mass ratio of silk fibroin precipitate to polysaccharide was 1:5. The reaction was carried out at 4°C for 15 min.

[0073] The reaction mixture was placed in a dialysis bag with a molecular weight cut-off of 20 kDa, and dialyzed in PBS buffer for 18 h to obtain a grafted polysaccharide medical silk fibroin solution.

[0074] Comparative Example 1

[0075] This comparative example provides a method for preparing a silk fibroin product. Similar to Example 1, the only difference is that in step S1, the silk is first added to a 0.5 g / L Na2CO3 solution and boiled for 1 h. The degummed silk is then taken out, washed with water for 3 times, and dried. Then it is dissolved in a 9.3 M lithium bromide solution, dialyzed to obtain a regenerated silk fibroin solution, and then incubated with protease. The remaining steps are consistent with Example 1. The comparison of the process flow is shown in Figure 1 .

[0076] Comparative Example 2

[0077] This comparative example provides a method for preparing a silk fibroin product. Similar to Example 1, the only difference is that in step S1, the silk fibroin precipitate is dissolved in water instead of lithium bromide solution. The remaining steps are consistent with Example 1.

[0078] Comparative Example 3

[0079] The comparative example provides a preparation method of a silk fibroin product, which is similar to Example 1, except that in step S1, the silk fibroin precipitate is dissolved in a 9.3 M lithium bromide solution, and the remaining steps are consistent with Example 1.

[0080] Comparative Example 4

[0081] The comparative example provides a preparation method of a silk fibroin product, which is similar to Example 1, except that in step S2, the sodium alginate is directly dissolved in water, and then mixed and reacted with the enzyme-cut silk fibroin solution in S3, and the remaining steps are consistent with Example 1.

[0082] Comparative Example 5

[0083] The comparative example provides a preparation method of a silk fibroin product, which is similar to Example 1, except that in step S2, 0.6 g of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 1.2 g of N-hydroxysuccinimide (NHS) are used to activate the polysaccharide carboxyl group, and the remaining steps are consistent with Example 1.

[0084] Test Example

[0085] The samples obtained in the above examples and comparative examples were subjected to relevant tests, and the test items are as follows.

[0086] (1) Silk sericin residual amount determination: The method mentioned in the patent with the patent number CN20201056391.7 entitled "Polypeptide antibody for directly detecting silk sericin and preparation method and application" was used to determine the residual amount of silk sericin in the silk fibroin precipitate in step S1 of Examples 1-6 and the silk fibroin precipitate prepared in step S1 of Comparative Example 1, and the results are shown in Table 1. Figure 2

[0087] (2) Property observation: The medical silk fibroin solution of grafted polysaccharide prepared in Examples 1-6 and the silk fibroin products prepared in Comparative Examples 1-5 were observed by naked eye under colorless cool white light without background interference to observe the properties of the silk fibroin solution.

[0088] (3) Solution stability: The medical silk fibroin solution of grafted polysaccharide prepared in Examples 1-6 and the silk fibroin products prepared in Comparative Examples 1-5 were respectively placed in a 4°C refrigerator for cold storage, and the state of the solution was observed and recorded within 90 days.

[0089] (4) Infrared spectroscopy was used to determine the grafting state of silk fibroin in the medical silk fibroin solution of grafted polysaccharide of Example 1, and qualitative judgment was made on whether the chemical grafting was successful. As can be seen from Table 2, Figure 3 after the polysaccharide was directionally grafted with silk fibroin, the characteristic peak of silk fibroin β-fold was 1620 cm -1 and 1520 cm​-1 obvious weakening, 3296 cm -1 obvious weakening, 3296 cm -1 obvious weakening, 3296 cm

[0090] (5) Mechanical property test: The grafted polysaccharide medical silk fibroin solution prepared in Examples 1-6 was placed in a petri dish and naturally placed into a film, and the silk fibroin products of Comparative Examples 1-5 were also placed in a petri dish and naturally placed into a film. A TST-01M intelligent electronic tensile tester was used to test the tensile breaking strength and elongation at break, with a tensile speed of 10 mm / min, a sample width of 10 mm, and a clamping length of 10 mm. The results are shown in Table 1.

[0091] Table 1 Stability and mechanical property test results

[0092] Comparative Example 1 starts with silk, and after degumming and drying treatment, degummed silk is obtained, which is then dissolved in a first organic solvent, and then purified to obtain a traditional regenerated silk fibroin solution, which is then subjected to enzymatic hydrolysis. The final silk fibroin product is the same as that of Examples 1-6, and the polysaccharide is successfully chemically grafted onto the enzyme-cut silk fibroin to form a clear and transparent solution with a stability of more than 90 days, and the mechanical properties are also similar. However, the traditional enzymatic hydrolysis method represented by Comparative Example 1 starts with regenerated silk fibroin solution for enzymatic hydrolysis, and from silk to regenerated silk fibroin solution, multiple processes are required, which consumes a large amount of chemical reagents and industrial wastewater, especially in the degumming process. Whether it is alkaline solution boiling or acid solution boiling, it will cause non-specific hydrolysis of silk (different from the specific point hydrolysis of enzymes). From the degumming effect of Figure 2 there is no significant difference in the amount of silk glue residue between Examples 1-6 and Comparative Example 1, which is less than 20 ng / mg, i.e. 0.002%.

[0093] Comparative Example 2 uses pure water to dissolve the silk fibroin precipitate. The absence of a second organic solvent causes the silk fibroin precipitate to exist in an unstable suspended state, and the activated polysaccharide added subsequently cannot be effectively chemically grafted and modified, so that the final product does not form a solution, but exists in the form of a precipitate, and cannot be used subsequently.

[0094] Comparative Example 3 uses a high concentration of the second organic solvent to dissolve the silk fibroin precipitate. The high concentration of the second organic solvent causes the secondary structure of the silk fibroin precipitate to be completely opened, the silk fibroin chain is completely stretched out, the silk fibroin terminal and internal amino groups are fully exposed, and the subsequently added activated polysaccharide is chemically modified and cross-linked at multiple sites, forming a "molecular brush" structure, resulting in the modified solution being excessively cross-linked and ultimately forming a gel, which is not conducive to further use. At the same time, due to excessive grafting modification, the silk fibroin maintains a random coil conformation, resulting in a decrease in film rigidity (breaking strength) and an increase in flexibility (breaking elongation). The grafted polysaccharide medical silk fibroin solution of Examples 1-6 is prepared by dissolving the silk fibroin precipitate in a low concentration of the second organic solvent in step S1, which causes the terminal amino groups of the silk fibroin to react with the activated polysaccharide to form an anhydride, reducing the formation of endogenous cross-linking sites, thereby effectively inhibiting the occurrence of self-crosslinking.

[0095] Comparative Example 4 uses unactivated polysaccharide to blend with the enzyme-cut silk fibroin solution. The unactivated polysaccharide cannot be chemically grafted and modified with the silk fibroin, and the essence is the physical blending of the two, and the subsequent dialysis step can completely remove the polysaccharide, so that the hydrogen bonds between the silk fibroin molecules again dominate, and then the flocculent precipitate is produced, which cannot form a stable solution for further use.

[0096] Comparative Example 5 uses EDC / NHS to graft silk fibroin and polysaccharide. The silk fibroin and polysaccharide themselves have very low reaction efficiency under mild conditions. EDC and NHS are a pair of "partner" catalysts that first activate the carboxyl group on the polysaccharide chain into a highly reactive intermediate, which then reacts with the amino group on the silk fibroin chain to form a three-dimensional network structure, which easily forms a three-dimensional network structure, which encapsulates water molecules, and finally forms a hydrogel.

[0097] Example 7

[0098] The grafted polysaccharide medical silk fibroin solution of examples 1-6 was prepared into porous scaffolds by freeze-drying method, the specific steps are as follows: the grafted polysaccharide medical silk fibroin solution was placed in a-20℃ refrigerator, completely frozen and then placed in a freeze dryer, vacuumized to form a solid powder, the powder was dissolved in a serum-containing culture medium and inoculated with L929 fibroblasts, and the cell proliferation was detected by cell proliferation toxicity detection method (CCK-8 method) for 3 days. The cell proliferation rate of the grafted polysaccharide medical silk fibroin solution prepared in examples 1-6 reached 124.4% at most, showing excellent cell proliferation effect, the reason is that the silk fibroin porous scaffold prepared from the medical silk fibroin solution of examples 1-6 not only can increase the specific surface area to accommodate more cells, but also can provide contact guidance, promote cell stretching, and activate focal adhesion kinase (FAK) and extracellular regulated protein kinase / mitogen-activated protein kinase (ERK / MAPK) signaling pathway, promote cell spreading and proliferation, and polysaccharide further provides nutrients to provide energy for cells. The silk fibroin product of comparative example 3 was in gel state, L929 fibroblasts were inoculated by the same method and the cell proliferation was detected by cell proliferation toxicity detection method (CCK-8 method) for 3 days, the specific surface area of the gel state was low, and its proliferation effect was not as good as examples 1-6. The test results are shown in table 2.

[0099] Table 2 cell proliferation rate experimental results

[0100] Obviously, the above examples are only examples for clearly illustrating, not limiting the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments are not required to be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for preparing a medical silk fibroin solution grafted with polysaccharide, characterized in that: The following steps are involved: S1, dissolving silk in a first organic solvent at a concentration of 6-10M, then adding a protease solution for incubation, inactivating the protease after incubation, and centrifuging to obtain a silk fibroin precipitate, dissolving the silk fibroin precipitate in a second organic solvent at a concentration of 4-6M to obtain an enzymatically cut silk fibroin solution; S2. mixing a polysaccharide containing at least one carboxyl group with triazolinedione in the presence of a third organic solvent to react to obtain an activated polysaccharide; S3, mixing the enzyme-cut silk fibroin solution in S1 with the activated polysaccharide in S2 for reaction, dialyzing, and obtaining a medical silk fibroin solution grafted with polysaccharide.

2. The preparation method according to claim 1, characterized in that In step S1, the first organic solvent and the second organic solvent are independently selected from one or more of lithium bromide, sodium thiocyanate, zinc chloride, and calcium chloride-ethanol-water ternary solvents; or, in step S2, the third organic solvent is selected from N , N -dimethylformamide, dimethyl sulfoxide, N , N -dimethylacetamide, N - one or more of methylpyrrolidone.

3. The preparation method according to claim 1, characterized in that The protease in step S1 is selected from one or more of α-chymotrypsin, pepsin, trypsin, proteinase K, papain, and alkaline protease.

4. The preparation method according to claim 1, characterized in that The mass ratio of protease to silk in step S1 is 1:(5-500).

5. The preparation method according to claim 1, characterized in that The polysaccharide in step S2 is selected from one or more of sodium alginate, hyaluronic acid, carboxymethyl chitosan, agarose, chitosan, and carboxymethyl cellulose; or the mass ratio of the triazolinedione to the polysaccharide is (10-1):

1.

6. The preparation method according to claim 1, characterized in that The mass ratio of the silk fibroin precipitate dissolved in the enzymatically chopped silk fibroin solution in step S3 to the polysaccharide is 1:(1-10).

7. The preparation method according to claim 1, characterized in that The mass ratio of the triazolinedione to the volume of the third organic solvent is 1 g: (5-10) mL.

8. The preparation method according to claim 1, characterized in that The reaction temperature in step S3 is 2-8° C., and the reaction time is 5-60 min.

9. The preparation method according to any one of claims 1 to 8 is used to prepare a medical silk fibroin solution grafted with polysaccharide.

10. Use of the medical silk fibroin solution according to claim 9 in preparing tissue engineering materials.

Images