Grafting polysaccharide medical silk fibroin solution and its preparation method and application

Abstract

The present application relates to a kind of medical silk fibroin solution of grafted polysaccharide and its preparation method and application, belong to medical material technical field.The present application is first to silkworm silk protease enzymolysis, while obtaining the silk fibroin precipitate of high beta-fold in the process of simplification, then through the low concentration of second organic solvent treatment silk fibroin precipitate, while maintaining the spatial shielding of internal amino group in the terminal amino exposure;Carboxyl of polysaccharide is activated using triazoline diketone simultaneously, and polysaccharide containing high active anhydride is obtained;Silk fibroin is reacted with polysaccharide containing high active anhydride, and the medical silk fibroin solution of grafted polysaccharide of the present application can be obtained.The medical silk fibroin solution of the present application can be stored for a long time and keep solution state, and has good performance, and has wide application in the preparation of tissue engineering material.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and in particular to a medical silk fibroin solution grafted with polysaccharides, its preparation method, and its application. Background Technology

[0002] Silk is composed of approximately 70% fibroin and 25% sericin. Fibroin is a natural polymer extracted from silk, and its molecular structure consists of alternating highly ordered β-sheet crystalline regions and loosely ordered amorphous regions. The crystalline regions are mainly composed of repeating small-molecule amino acid residues such as glycine, alanine, and serine, forming a tightly packed antiparallel β-sheet structure, which endows fibroin with excellent mechanical strength and stability. The amorphous regions are rich in large side-chain amino acids such as phenylalanine, tyrosine, and tryptophan, giving the material a certain degree of flexibility and functional modifiability.

[0003] Conventional enzymatic hydrolysis of silk fibroin begins with a regenerated silk fibroin solution. To obtain this solution, silkworm silk must first be degummed and dried to form degummed silk. Then, protease is added to the regenerated silk fibroin solution obtained by dissolving and purifying the degummed silk to carry out enzymatic hydrolysis, thus yielding silk fibroin. This process involves multiple steps, is not only complex but also consumes a large amount of chemical reagents and generates a large amount of industrial wastewater.

[0004] After enzymatic cleavage, the connection sites between the amorphous and crystalline regions of silk fibroin are removed. Silk fibroin with a high β-sheet content and repeating GAGAGS (G for glycine, A for alanine, S for serine) tends to precipitate due to its large molecular weight. These precipitates offer unique advantages in materials science and biomedicine. However, enzymatic cleavage exposes a large number of hydrophobic regions, significantly reducing the hydrophilicity of the silk fibroin surface and thus affecting its solubility. To improve the solubility of silk fibroin, polysaccharides are often used to graft it onto the fibroin to enhance its hydrophilicity. In this process, silk fibroin and polysaccharides form a gel through cross-linking or electrostatic interactions, thereby improving the solubility of silk fibroin. However, these modification techniques all target gel-like products, resulting in poor stability and significant loss of β-sheets during preparation. Therefore, solid materials prepared from gel-state silk fibroin products lack rigidity, limiting their applications.

[0005] Therefore, there is an urgent need to find a new preparation method that can simplify the process while obtaining silk fibroin solutions with good solution stability and mechanical properties. Summary of the Invention

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

[0007] To address the aforementioned technical problems, this invention provides a medical-grade silk fibroin solution grafted with polysaccharides, its preparation method, and its applications. In the preparation of silk fibroin, it is commonly separated by enzymatic hydrolysis of silk. Traditional enzymatic hydrolysis techniques start from regenerated silk fibroin solution (i.e., silk fibroin solution after degumming), consuming large amounts of chemical reagents and generating significant amounts of industrial wastewater. This invention optimizes the process by directly dissolving the silk before enzymatic hydrolysis, improving process efficiency and reducing reagent and wastewater pollution. Furthermore, enzymatic hydrolysis of the undegummed silk yields a silk fibroin precipitate with a high β-sheet structure. Additionally, in existing technologies, the hydrophobic regions of the high β-sheet structure of silk fibroin are exposed, resulting in poor solubility of the precipitate in water, making it difficult to use directly as a raw material. This invention directionally grafts polysaccharides onto the terminal amino groups of silk fibroin while leaving the internal amino groups unaffected. This increases the water solubility of the silk fibroin and avoids excessive cross-linking between the silk fibroin and polysaccharides, preventing gel formation. Specifically, this invention treats silk fibroin precipitate with a low-concentration solvent, ensuring the exposure of terminal amino groups while maintaining the spatial shielding of internal amino groups. Simultaneously, triazolinide is used to activate the carboxyl groups of the polysaccharide, converting them into highly reactive acid anhydrides. This intermediate readily reacts with silk fibroin containing active amino groups, forming amide bonds between the terminal amino groups of the silk fibroin and the acid anhydride of the polysaccharide. This enhances the hydrophilicity of the silk fibroin and effectively inhibits self-crosslinking, thereby achieving long-term stable storage of the solution. Furthermore, the medical-grade silk fibroin solution grafted with polysaccharides of this invention promotes cell proliferation and possesses excellent mechanical properties, showing potential application value in the preparation of tissue engineering materials.

[0008] The first objective of this invention is to provide a method for preparing a medical silk fibroin solution grafted with polysaccharides, comprising the following steps:

[0009] S1. Dissolve silk in a first organic solvent with a concentration of 6-10M, then add protease solution for incubation. After incubation, inactivate the protease. After centrifugation, obtain silk fibroin precipitate. Dissolve the silk fibroin precipitate in a second organic solvent with a concentration of 4-6M to obtain an enzymatically digested silk fibroin solution.

[0010] S2. Under the condition of a third organic solvent, a polysaccharide containing at least one carboxyl group is mixed and reacted with a triazolinone to obtain an activated polysaccharide.

[0011] S3. Mix the enzyme-digested silk fibroin solution from S1 with the activated polysaccharide from S2, and then dialyze to obtain a medical silk fibroin solution with grafted polysaccharide.

[0012] Further, 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. The concentration of the first organic solvent is greater than the concentration of the second organic solvent.

[0013] Further, the protease mentioned in step S1 is selected from one or more of α-chymotrypsin, pepsin, trypsin, proteinase K, papain, and alkaline protease.

[0014] Further, the mass ratio of the protease to silk in step S1 is 1:(5-500).

[0015] Further, the polysaccharide mentioned in step S2 is selected from one or more of sodium alginate, hyaluronic acid, carboxymethyl chitosan, agarose, chitosan, and carboxymethyl cellulose.

[0016] Further, the third organic solvent mentioned in step S2 is selected from... N , N -Dimethylformamide, dimethyl sulfoxide, N , N -Dimethylacetamide, N One or more of methylpyrrolidone.

[0017] Further, the mass ratio of triazolinedione to polysaccharide in step S2 is (10-1):1.

[0018] Furthermore, in step S2, the reaction temperature is 4-40℃ and the reaction time is 10-180 min.

[0019] Further, in step S2, after the polysaccharide and triazoline dione are mixed and reacted, a reaction regulator is added to terminate the reaction. The reaction regulator is selected from one or more of β-mercaptoethanol, dithiothreitol, cysteine, and glycine. The reaction regulator can react rapidly with triazoline dione to form a stable adduct (such as a thioester or disulfide bond), preventing the triazoline dione from further activating the carboxyl group.

[0020] Further, in step S3, the mass ratio of the silk fibroin precipitate dissolved in the enzymatically digested silk fibroin to the polysaccharide is 1:(1-10).

[0021] Furthermore, the mass ratio of the triazolinidone to the volume ratio of the third organic solvent is 1 g: (5-10) mL.

[0022] Furthermore, the reaction temperature in step S3 is 2-8℃, and the reaction time is 5-60 min.

[0023] The second objective of this invention is to provide a medical silk fibroin solution of grafted polysaccharides prepared by the above-described preparation method.

[0024] A third objective of this invention is to provide an application of the above-mentioned medical silk fibroin solution in the preparation of tissue engineering materials.

[0025] Furthermore, the tissue engineering materials include bone tissue materials and cell proliferation materials.

[0026] The mechanism of this invention is as follows:

[0027] One-step enzymatic hydrolysis of silk: Silk is directly dissolved in a first organic solvent, and then incubated with a protease for enzymatic hydrolysis. Silk is mainly composed of sericin and fibroin. The first organic solvent can dissolve sericin and fibroin and cause their molecular chains to unfold, while the protease simultaneously cleaves both. Due to the structural differences between sericin and fibroin, only the highly β-sheeted crystalline regions of fibroin containing repeating GAGAGS fragments precipitate, forming fibroin precipitate, while the amorphous regions of fibroin and sericin remain in the supernatant.

[0028] The enzymatically hydrolyzed silk fibroin precipitate exhibits a highly repetitive GAGAGS secondary structure, with some amino groups located at the terminal ends and others on internal side chains, particularly lysine side chains. Conventional methods for dissolving silk fibroin precipitates involve using high-concentration secondary organic solvents, which exposes the ε-amino groups of lysine residues within the silk fibroin, leading to excessive cross-linking and gelation of the polysaccharide. This invention, however, uses a low-concentration secondary organic solvent to expose only the terminal amino groups of the silk fibroin, preventing excessive exposure of internal amino groups. Simultaneously, triazolinone is used to convert the carboxyl groups of the polysaccharide into highly reactive anhydrides. The terminal amino groups of the silk fibroin form amide bonds with the anhydrides of the polysaccharide, enhancing the hydrophilicity of the silk fibroin and effectively inhibiting self-cross-linking, thereby achieving long-term stable storage of the solution.

[0029] The beneficial effects of this invention are:

[0030] (1) The process is more simplified. Traditionally, polysaccharides react with the terminal amino groups of silk fibroin to form amide bonds, thereby obtaining a silk fibroin solution with directionally grafted polysaccharides. Silk fibroin enzymatic hydrolysis requires silk degumming, dissolution, and purification steps to obtain a regenerated silk fibroin solution, and then adding protease to the regenerated silk fibroin solution for enzymatic hydrolysis. This invention adopts a one-step enzymatic hydrolysis method, directly dissolving the silk and adding protease for enzymatic hydrolysis, omitting the degumming, dissolution, and purification steps, greatly reducing chemical reagents and industrial pollutants, simplifying the process, increasing production efficiency, and lowering costs.

[0031] (2) High solution stability. The medical silk fibroin solution with grafted polysaccharides prepared in this invention has good stability. No precipitation was observed within 90 days. This not only exceeds the stability of traditional regenerated silk fibroin solutions and EDC / NHS modified silk fibroin solutions, but also avoids excessive cross-linking of polysaccharides to form gels, which is beneficial for further development and use.

[0032] (3) Directional grafting of polysaccharides endows the silk fibroin solution with good properties. The present invention uses a low concentration of a second organic solvent to expose only part of the amino groups of the silk fibroin, especially the terminal amino groups, without exposing the amino groups of the internal side chains, thereby directionally grafting polysaccharides onto the terminal amino groups of the silk fibroin and avoiding byproducts and gelation caused by excessive cross-linking.

[0033] (4) Excellent mechanical properties and broad application prospects. The film prepared from the medical silk fibroin solution of the grafted polysaccharide of the present invention has good mechanical properties and cell proliferation promotion effect, and has broad application prospects in the preparation of tissue engineering materials, especially bone tissue engineering materials and cell proliferation materials. Attached Figure Description

[0034] Figure 1 These are process flow diagrams of the present invention and Comparative Example 1. The left diagram is the process flow diagram of the medical silk fibroin solution grafted with polysaccharides of the present invention, and the right diagram is the process flow diagram of the silk fibroin product of Comparative Example 1.

[0035] Figure 2 These are graphs showing the results of sericin residue determination in Examples 1-6 and Comparative Example 1 of the present invention;

[0036] Figure 3 This is the infrared spectrum of polysaccharide grafted with silk fibroin in Example 1 of the present invention. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0039] The source and catalog number of the reagents are shown below.

[0040] Lithium bromide (LiBr) was purchased from Aladdin, catalog number L108934;

[0041] Sodium thiocyanate (NaSCN) was purchased from Jinan Huifengda, product number HFD-248;

[0042] Zinc chloride (ZnCl2) was purchased from Sinopharm, catalog number H1L0491;

[0043] The ternary solvent of calcium chloride (CaCl2)-ethanol-water, wherein CaCl2 was purchased from Jiyesheng Chemical Co., Ltd., with product number JYS1545;

[0044] Ethanol was purchased from Bio-Raybo, product number L11024-VZU;

[0045] Trypsin was purchased from Aladdin, catalog number T274333;

[0046] α-chymotrypsin was purchased from Maclean's, catalog number C804761;

[0047] The pepsin was purchased from Aladdin, product number P110928;

[0048] Proteinase K was purchased from Shanghai Jingke Chemical Technology Co., Ltd., product number JK-038;

[0049] Papain was purchased from Sigma, product number P3250;

[0050] The alkaline protease was purchased from Beijing Bio-Lab, product number QN0397;

[0051] Sodium alginate was purchased from Xi'an Jinxiang Pharmaceutical Excipients Co., Ltd., item number 0045;

[0052] Hyaluronic acid was purchased from Solarbio, product number S7020;

[0053] Carboxymethyl chitosan was purchased from Sigma, product number 926043;

[0054] The agarose was purchased from Sigma, product number A9045;

[0055] Chitosan was purchased from Beijing Bio-Lab, product number QN0147;

[0056] Carboxymethyl cellulose was purchased from Aladdin, product number C104977;

[0057] N , N - Dimethylformamide was purchased from Maclean's, product number N807509;

[0058] Dimethyl sulfoxide was purchased from Maclean, product number D806645;

[0059] N , N - Dimethylacetamide was purchased from Maclean's, product number N807171;

[0060] N 2-Methylpyrrolidone was purchased from Maclean's, catalog number M814045;

[0061] β-mercaptoethanol was purchased from Maclean's, catalog number M828395;

[0062] Dithiothreitol was purchased from Maclean's, product number D806827;

[0063] Cysteine ​​was purchased from Maclean's, catalog number D831312;

[0064] Glycine was purchased from Sigma, product number G8790;

[0065] Phosphate buffer solution (PBS buffer solution) was purchased from Bianzhen Biotechnology, catalog number bzw2106f;

[0066] Triazoline dione was purchased from STANDARDS, catalog number ZC68005;

[0067] Sodium carbonate (Na2CO3) was purchased from Sinopharm, product number 10019260.

[0068] Example 1

[0069] 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 an equal volume of 3 mg / g trypsin solution (the mass ratio of trypsin to silk is 3:100). React at 37℃ for 20 h, heat at 100℃ for 10 min to inactivate the enzyme, centrifuge at 5000 rpm for 10 min, take the lower layer, wash, and obtain silk fibroin precipitate.

[0070] The silk fibroin precipitate was dissolved in 10 mL of 4.0 M lithium bromide solution to obtain an enzymatically digested silk fibroin solution.

[0071] S2, Triazoline-activated polysaccharide carboxyl groups: Dissolve 2 g of triazoline in 10 mL of... N , N 2 g of sodium alginate was added to dimethylformamide and dissolved. The mass ratio of triazoline dione to sodium alginate was 1:1. The mixture was reacted at 4°C for 180 min, and then β-mercaptoethanol was added to terminate the reaction.

[0072] S3, Polysaccharide-directed grafting of silk fibroin: The enzymatically digested silk fibroin solution in S1 and the activated polysaccharide solution in S2 are mixed, with the mass ratio of silk fibroin precipitate to polysaccharide being 1:1, and the mixture is reacted at 2℃ for 60 min.

[0073] The reaction mixture was placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed with water for 24 h to obtain a medical silk fibroin solution of grafted polysaccharide.

[0074] Example 2

[0075] S1. One-step enzymatic hydrolysis of silk: Dissolve 50 g of silk in 100 mL of 6.0 M sodium thiocyanate solution, filter, and mix with an equal volume of 1 mg / g α-chymotrypsin solution (the mass ratio of α-chymotrypsin to silk is 1:500). React at 37℃ for 24 h, heat at 100℃ for 15 min to inactivate the enzyme, centrifuge at 6000 rpm for 5 min, take the lower layer, wash, and obtain silk fibroin precipitate.

[0076] The silk fibroin precipitate was dissolved in 50 mL of 4.5 M sodium thiocyanate solution to obtain an enzymatically digested silk fibroin solution.

[0077] S2. Triazoline-dione activates the carboxyl group of polysaccharides: Dissolve 10 g of triazoline-dione in 50 mL of dimethyl sulfoxide, add 1 g of hyaluronic acid and mix to dissolve. The mass ratio of triazoline-dione to hyaluronic acid is 10:1. Mix the two and react at 15℃ for 120 min. Then add dithiothreitol to terminate the reaction.

[0078] S3, Polysaccharide-directed grafting of silk fibroin: The enzymatically digested silk fibroin solution in S1 and the activated polysaccharide solution in S2 are mixed, with the mass ratio of silk fibroin precipitate to polysaccharide being 1:10, and the mixture is reacted at 4℃ for 15 min.

[0079] 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 of grafted polysaccharide.

[0080] Example 3

[0081] S1. One-step enzymatic hydrolysis of silk: Dissolve 30 g of silk in 100 mL of 8 M zinc chloride solution, filter, and mix with an equal volume of 3 mg / g pepsin solution (mass ratio of pepsin to silk is 1:100). React at 37℃ for 18 h, heat at 100℃ for 20 min to inactivate the enzyme, centrifuge at 8000 rpm for 5 min, take the lower layer, wash, and obtain silk fibroin precipitate.

[0082] The silk fibroin precipitate was dissolved in 30 mL of 5.0 M zinc chloride solution to obtain an enzymatically digested silk fibroin solution.

[0083] S2, Triazoline-dione activates the carboxyl group of polysaccharides: Dissolve 5 g of triazoline-dione in 30 mL of... N , N Add 1 g of carboxymethyl chitosan to dimethylacetamide and mix and dissolve. The mass ratio of triazolinone to carboxymethyl chitosan is 5:1. React at 40℃ for 10 min, and then add cysteine ​​to terminate the reaction.

[0084] S3, Polysaccharide-directed grafting of silk fibroin: The enzymatically digested silk fibroin solution in S1 and the activated polysaccharide solution in S2 are mixed and reacted. The mass ratio of silk fibroin precipitate to polysaccharide is 1:5. The reaction is carried out at 8℃ for 5 min.

[0085] 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 of grafted polysaccharide.

[0086] Example 4

[0087] S1. One-step enzymatic hydrolysis of silk: Dissolve 15 g of silk in 100 mL of 10.0 M calcium chloride-ethanol-water ternary solvent (molar ratio 1:2:8), filter, and mix with an equal volume of 3 mg / g proteinase K solution (mass ratio of proteinase K to silk is 1:50). React at 37℃ for 16 h, heat at 95℃ for 15 min to inactivate the enzyme, centrifuge at 4000 rpm for 10 min, take the lower layer, wash, and obtain silk fibroin precipitate.

[0088] The silk fibroin precipitate was dissolved in 15 mL of a 6.0 M calcium chloride-ethanol-water ternary solvent (molar ratio 1:2:8) to obtain an enzymatically digested silk fibroin solution.

[0089] S2, Triazoline-dione activates the carboxyl group of polysaccharides: Dissolve 4 g of triazoline-dione in 24 mL of... N Add 2 g of agarose to triazolinone and mix to dissolve. The mass ratio of triazolinone to agarose is 2:1. React at 25°C for 60 min, and then add glycine to terminate the reaction.

[0090] S3, Polysaccharide-directed grafting of silk fibroin: The enzymatically digested silk fibroin solution from S1 and the activated polysaccharide solution from S2 are mixed. The mass ratio of silk fibroin precipitate to polysaccharide is 1:3, and the reaction is carried out at 4℃ for 10 min.

[0091] The reaction mixture was placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed with PBS buffer for 20 h to obtain a medical silk fibroin solution of grafted polysaccharide.

[0092] Example 5

[0093] S1. One-step enzymatic hydrolysis of silk: Dissolve 15 g of silk in 100 mL of 9.0 M lithium bromide solution, filter, and mix with an equal volume of 3 mg / g papain solution (mass ratio of papain to silk is 1:50). React at 37℃ for 24 h, heat at 100℃ for 15 min to inactivate the enzyme, centrifuge at 5000 rpm for 10 min, take the lower layer, wash, and obtain silk fibroin precipitate.

[0094] The silk fibroin precipitate was dissolved in 15 mL of 5.5 M lithium bromide solution to obtain an enzymatically digested silk fibroin solution.

[0095] S2, Triazoline-dione activates the carboxyl group of polysaccharides: Dissolve 4 g of triazoline-dione in 24 mL of... N , N 2 g of chitosan was added to dimethylformamide and dissolved. The mass ratio of triazolinone to chitosan was 2:1. The mixture was reacted at 4°C for 180 min, and then β-mercaptoethanol was added to terminate the reaction.

[0096] S3, Polysaccharide-directed grafting of silk fibroin: The enzymatically digested silk fibroin solution in S1 and the activated polysaccharide solution in S2 are mixed, with the mass ratio of silk fibroin precipitate to polysaccharide being 1:4, and the mixture is reacted at 4℃ for 20 min.

[0097] The reaction mixture was placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed with water for 24 h to obtain a medical silk fibroin solution of grafted polysaccharide.

[0098] Example 6

[0099] S1. One-step enzymatic hydrolysis of silk: Dissolve 20 g of silk in 100 mL of 9.3 M lithium bromide solution, filter, and mix with an equal volume of 3 mg / g alkaline protease solution (mass ratio of alkaline protease to silk is 3:200). React at 37℃ for 18 h, heat at 100℃ for 10 min to inactivate the enzyme, centrifuge at 4000 rpm for 10 min, take the lower layer, wash, and obtain silk fibroin precipitate.

[0100] The silk fibroin precipitate was dissolved in 20 mL of 4.5 M lithium bromide solution to obtain an enzymatically digested silk fibroin solution.

[0101] S2. Triazoline-dione activates the carboxyl group of polysaccharides: Dissolve 5 g of triazoline-dione in 50 mL of dimethyl sulfoxide, add 1 g of hydroxymethyl cellulose and mix to dissolve. The mass ratio of triazoline-dione to hydroxymethyl cellulose is 5:1. React at 4℃ for 150 min, and then add glycine to terminate the reaction.

[0102] S3, Polysaccharide-directed grafting of silk fibroin: The enzymatically digested silk fibroin solution in S1 and the activated polysaccharide solution in S2 are mixed, with the mass ratio of silk fibroin precipitate to polysaccharide being 1:5, and the mixture is reacted at 4℃ for 15 min.

[0103] The reaction mixture was placed in a dialysis bag with a molecular weight cutoff of 20 kDa and dialyzed with PBS buffer for 18 h to obtain a medical silk fibroin solution of grafted polysaccharide.

[0104] Comparative Example 1

[0105] This comparative example provides a method for preparing a silk fibroin product, similar to Example 1, except that in step S1, silk is first added to a 0.5 g / L Na2CO3 solution and boiled for 1 h. The degummed silk is then removed, washed three times with water, dried, and dissolved in a 9.3 M lithium bromide solution. The solution is then purified by dialysis to obtain a regenerated silk fibroin solution, which is then incubated with protease. The remaining steps are consistent with Example 1. (Comparison of process flow is provided.) Figure 1 As shown.

[0106] Comparative Example 2

[0107] This comparative example provides a method for preparing a silk fibroin product, which is similar to Example 1, except that in step S1, the silk fibroin precipitate is dissolved in water but not in lithium bromide solution, and the remaining steps are the same as in Example 1.

[0108] Comparative Example 3

[0109] This comparative example provides a method for preparing 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 the same as in Example 1.

[0110] Comparative Example 4

[0111] This comparative example provides a method for preparing a silk fibroin product, which is similar to Example 1, except that in step S2, sodium alginate is directly dissolved in water, and then mixed and reacted with the enzyme-digested silk fibroin solution in S3. The remaining steps are the same as in Example 1.

[0112] Comparative Example 5

[0113] This comparative example provides a method for preparing a silk fibroin product, 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 carboxyl group of the polysaccharide, and the remaining steps are the same as in Example 1.

[0114] Test case

[0115] The samples obtained from the above embodiments and comparative examples were subjected to relevant tests, and the test items are as follows.

[0116] (1) Determination of residual sericin: The residual 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 were determined using the method mentioned in the patent CN20201056391.7 entitled "Polypeptide Antibody for Direct Detection of Silk Sericin and its Preparation Method and Application". The results are as follows: Figure 2 As shown.

[0117] (2) Observation of properties: The medical silk fibroin solutions of grafted polysaccharides prepared in Examples 1-6 and the silk fibroin products prepared in Comparative Examples 1-5 were observed by the naked eye under colorless cold white light without background interference.

[0118] (3) Solution stability: The medical silk fibroin solutions of grafted polysaccharides prepared in Examples 1-6 and the silk fibroin products prepared in Comparative Examples 1-5 were placed in a refrigerator at 4°C and the state of the solutions was observed and recorded within 90 days.

[0119] (4) The grafting status of the silk fibroin in the medical silk fibroin solution of the grafted polysaccharide in Example 1 was determined by infrared spectroscopy to qualitatively determine whether the chemical grafting was successful. Figure 3 It can be seen that after polysaccharide is directionally grafted onto silk fibroin, the characteristic peak of silk fibroin β-sheet is 1620 cm⁻¹. -1 and 1520cm -1 Significantly weakened, 3296cm -1 The broad peak at 1031 cm⁻¹ is due to the superposition of hydrogen bond vibrations of hydroxyl groups in carbohydrate molecules. -1 The strong peak is the stretching vibration peak of the glycosidic bond (COC) in the polysaccharide, indicating that the polysaccharide was successfully chemically grafted onto silk fibroin.

[0120] (5) Mechanical property testing: The medical silk fibroin solutions of grafted polysaccharides prepared in Examples 1-6 were placed in petri dishes and allowed to stand naturally to form films. The silk fibroin products of Comparative Examples 1-5 were also placed in petri dishes and allowed to stand to form films. The tensile strength and elongation at break were tested using a TST-01M intelligent electronic tensile testing instrument. The tensile speed was 10 mm / min, the sample width was 10 mm, and the clamping length was 10 mm. The results are shown in Table 1.

[0121] Table 1. Results of stability and mechanical property tests

[0122]

[0123] Comparative Example 1 started with silkworm silk, which underwent degumming and drying to obtain degummed silk. This degummed silk was then dissolved in a first organic solvent and purified to obtain a traditional regenerated silk fibroin solution. This solution was then enzymatically hydrolyzed, resulting in a silk fibroin product similar to that of Examples 1-6. Polysaccharides were successfully chemically grafted onto the enzymatically hydrolyzed silk fibroin, forming a clear, transparent solution with a stability greater than 90 days and similar mechanical properties. However, the traditional enzymatic hydrolysis method represented by Comparative Example 1 all started from the regenerated silk fibroin solution. The process from silkworm silk to regenerated silk fibroin solution requires multiple additional steps, consuming large quantities of chemical reagents and industrial wastewater. Especially during the degumming process, whether boiling in an alkaline or acidic solution, non-specific hydrolysis of the silk occurs (unlike the specific site hydrolysis of enzymes). Figure 2 Regarding the degumming effect, there was no significant difference in the sericin residue of Examples 1-6 and Comparative Example 1, all of which were below 20 ng / mg, i.e. 0.002%.

[0124] Comparative Example 2 used pure water to dissolve the silk fibroin precipitate. The lack of a second organic solvent caused the silk fibroin precipitate to exist in an unstable suspension state. The subsequently added activated polysaccharide could not be effectively chemically grafted and modified with it. Therefore, it did not form a solution but existed in the form of a precipitate and could not be used for further processing.

[0125] Comparative Example 3 used a high-concentration second organic solvent to dissolve the silk fibroin precipitate. The high concentration of the second organic solvent completely opened the β-sheet of the secondary structure of the silk fibroin precipitate, fully unfolding the silk fibroin chains and exposing the terminal and internal amino groups. The subsequently added activated polysaccharide underwent multi-site chemical modification and cross-linking, forming a "molecular brush" structure. This resulted in over-cross-linking of the modified solution, ultimately forming a gel, which is detrimental to further use. Simultaneously, due to over-grafting modification, the silk fibroin maintained a random coil conformation, leading to a decrease in film rigidity (tensile strength) and an increase in flexibility (elongation at break). In contrast, the medical silk fibroin solutions with grafted polysaccharides in Examples 1-6, during preparation, resulted in the silk fibroin precipitate in step S1 being dissolved in a low-concentration second organic solvent. This exposed the terminal amino groups of the silk fibroin. The terminal amino groups reacted with the activated polysaccharide to form anhydrides, reducing the formation of endogenous cross-linking sites and effectively inhibiting self-cross-linking.

[0126] Comparative Example 4 used an unactivated polysaccharide mixed with an enzyme-digested silk fibroin solution. The unactivated polysaccharide and silk fibroin cannot be chemically grafted and modified; their relationship is essentially a physical blend. The subsequent dialysis step can completely remove the polysaccharide, so the hydrogen bonds between silk fibroin molecules become dominant again, resulting in flocculent precipitate that cannot form a stable solution for further use.

[0127] Comparative Example 5 used EDC / NHS to graft silk fibroin and polysaccharides. Silk fibroin and polysaccharides themselves have very low reaction efficiency under mild conditions. EDC and NHS act as a pair of catalysts, first activating the carboxyl groups on the polysaccharide chain into a highly reactive intermediate. This intermediate then undergoes an efficient amidation reaction with the amino groups on the silk fibroin chain, thereby connecting the two macromolecular chains through covalent bonds. This easily forms a three-dimensional network structure that encapsulates water molecules, ultimately forming a hydrogel.

[0128] Example 7

[0129] The medical silk fibroin solutions of grafted polysaccharides from Examples 1-6 were used to prepare porous scaffolds via freeze-drying. Specifically, the medical silk fibroin solutions of grafted polysaccharides were placed in a -20°C freezer, completely frozen, and then placed in a freeze dryer under negative pressure until a solid powder was formed. The powder was dissolved in serum-containing culture medium and inoculated into L929 fibroblasts. Cell proliferation was detected after 3 days using the Cell Proliferation-Toxicity Assay (CCK-8 assay). The medical silk fibroin solutions of grafted polysaccharides prepared in Examples 1-6 showed the highest cell proliferation rate of 124.4%, demonstrating excellent cell proliferation-promoting effects. This is because the porous scaffolds prepared from the medical silk fibroin solutions of Examples 1-6 not only increase the specific surface area to accommodate more cells but also provide contact guidance, promote cell extension, and activate focal adhesion kinase (FAK) and extracellular regulated protein kinase / mitogen-activated protein kinase (ERK / MAPK) signaling pathways, promoting cell spread and proliferation. Furthermore, the polysaccharides provide nutrients and energy to the cells. The silk fibroin product in Comparative Example 3 was in a gel state. L929 fibroblasts were inoculated using the same method, and cell proliferation was detected after 3 days using the cell proliferation toxicity assay (CCK-8 assay). The gel state had a lower specific surface area, and its proliferation effect was not as good as that of Examples 1-6. The test results are shown in Table 2.

[0130] Table 2 Results of cell proliferation rate experiment

[0131]

[0132] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a medical silk fibroin solution grafted with polysaccharides, characterized in that, Includes the following steps: S1. Dissolve silk in a first organic solvent with a concentration of 6-10M, then add protease solution for incubation. After incubation, inactivate the protease, centrifuge to obtain silk fibroin precipitate, and dissolve the silk fibroin precipitate in a second organic solvent with a concentration of 4-6M to obtain an enzymatically digested silk fibroin solution. S2. Under the condition of the presence of a third organic solvent, a polysaccharide containing at least one carboxyl group is mixed and reacted with a triazoline dione to obtain an activated polysaccharide, wherein the polysaccharide is selected from one or more of sodium alginate, hyaluronic acid, carboxymethyl chitosan, agarose, chitosan, and carboxymethyl cellulose. S3. Mix the enzyme-digested silk fibroin solution from S1 with the activated polysaccharide from S2, and then dialyze to obtain a medical silk fibroin solution with grafted polysaccharide. In step S1, the first and second organic solvents are independently selected from one or more of lithium bromide, sodium thiocyanate, zinc chloride, and calcium chloride-ethanol-water ternary solvents; in step S2, the third organic solvent is selected from... N , N -Dimethylformamide, dimethyl sulfoxide, N , N -Dimethylacetamide, N One or more of methylpyrrolidone.

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

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

4. The preparation method according to claim 1, characterized in that, The mass ratio of the triazoline dione to the polysaccharide is (10-1):

1.

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

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

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

8. The medical silk fibroin solution with grafted polysaccharides is prepared by the preparation method according to any one of claims 1-7.

9. The application of the medical silk fibroin solution according to claim 8 in the preparation of tissue engineering materials.

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