CN120795112B

CN120795112B - A medical silk fibroin solution grafted with polysaccharides, its preparation method and application

Info

Publication number: CN120795112B
Application number: CN202511282837.4A
Authority: CN
Inventors: 刘也卓; 杨文华
Original assignee: Fuxiang Sitai Medical Technology Suzhou Co ltd
Current assignee: Fuxiang Sitai Medical Technology Suzhou Co ltd
Priority date: 2025-09-09
Filing date: 2025-09-09
Publication date: 2025-11-21
Anticipated expiration: 2045-09-09
Also published as: CN120795112A

Abstract

The invention relates to a medical silk fibroin solution grafted with polysaccharide, and a preparation method and application thereof, and belongs to the technical field of medical materials. The invention firstly carries out enzymolysis on silk by protease, obtains high beta-folded silk fibroin precipitate while simplifying the process, then treats the silk fibroin precipitate by a second organic solvent with low concentration, keeps the space shielding of internal amino while the terminal amino is exposed, simultaneously activates carboxyl of polysaccharide by triazolin diketone to obtain polysaccharide containing high-activity anhydride, and reacts the silk fibroin with the polysaccharide containing high-activity anhydride to obtain the medical silk fibroin solution of the grafted polysaccharide. The medical silk fibroin solution can be stably stored for a long time, keeps a solution state, has good performance, and has wide application in preparation of tissue engineering materials.

Description

Medical silk fibroin solution grafted with polysaccharide and preparation method and application thereof

Technical Field

The invention relates to the technical field of medical materials, in particular to a medical silk fibroin solution grafted with polysaccharide, and a preparation method and application thereof.

Background

The silk is composed of about 70% silk fibroin and about 25% sericin. Silk fibroin is a natural polymer material extracted from silk, and the molecular structure of the silk fibroin is composed of highly ordered beta-sheet crystalline regions and loosely disordered amorphous regions alternately. The crystallization area is mainly formed by repeatedly arranging small molecular amino acid residues such as glycine, alanine, serine and the like to form a tightly-stacked antiparallel beta-sheet structure, so that excellent mechanical strength and stability are provided for the silk fibroin, and the non-crystallization area is rich in large side chain amino acids such as phenylalanine, tyrosine, tryptophan and the like, so that the material has certain flexibility and functional modifier.

The conventional enzymolysis of silk fibroin is started from regenerated silk fibroin solution, silk is required to be degummed and dried to form degummed silk, protease is added into the regenerated silk fibroin solution formed by degummed silk dissolution and purification for enzymolysis, so that silk fibroin can be obtained.

After cleavage of the silk fibroin, the junction site between the amorphous and crystalline regions is excised, and silk fibroin with high beta-sheet content having repetition GAGAGS (G represents glycine, a represents alanine, S represents serine) precipitates due to large molecular weight, which have unique application advantages in the fields of materials and biomedicine. However, due to the exposure of a large number of hydrophobic regions caused by cleavage, the surface hydrophilicity of the silk fibroin is significantly reduced, thereby affecting the solubility thereof. In order to improve the solubility of silk fibroin, polysaccharide is often used for grafting modification of silk fibroin to improve the hydrophilicity of the silk fibroin, and in the process, the silk fibroin and the polysaccharide form gel through crosslinking or electrostatic action, so that the solubility of the silk fibroin is improved. However, these modification techniques all use gel form as the target product, the stability is poor, and the beta-sheet is largely lost in the preparation process, so that the solid material prepared from the silk fibroin product in gel state has insufficient rigidity and limited application.

Therefore, a new preparation method is needed to obtain the silk fibroin solution with good solution stability and mechanical property while simplifying the process flow.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to solve the problem that a silk fibroin solution with good stability, simple preparation process and good mechanical property is lacked in the prior art.

In order to solve the technical problems, the invention provides a medical silk fibroin solution grafted with polysaccharide, and a preparation method and application thereof. In the process of preparing silk fibroin, silk fibroin is often separated by enzymolysis of silk, and the traditional enzymolysis technology begins with regenerated silk fibroin solution (namely silk fibroin solution after silk degumming), consumes a large amount of chemical reagents, and generates a large amount of industrial wastewater. The invention optimizes the process, and the silk is directly dissolved and then subjected to enzymolysis, so that the process efficiency is improved, and the pollution of reagents and wastewater is reduced. Meanwhile, silk is subjected to enzymolysis under the condition of not degumming, so that silk fibroin sediment with a high beta-sheet structure can be obtained. In addition, in the prior art, the silk fibroin hydrophobic region with a high beta-sheet structure is exposed, so that the solubility of silk fibroin sediment in water is poor, and the silk fibroin is difficult to directly use as a raw material. According to the invention, the polysaccharide is directionally grafted on the terminal amino group of the silk fibroin without the influence of the internal amino group, so that the water solubility of the silk fibroin can be increased, and the excessive crosslinking of the silk fibroin and the polysaccharide to form gel can be avoided. Specifically, the invention treats the silk fibroin precipitate through a low concentration solvent, ensures the exposure of the terminal amino group and simultaneously keeps the space shielding of the internal amino group, simultaneously activates the carboxyl group of the polysaccharide by triazolin dione, and converts the carboxyl group into highly reactive anhydride, the intermediate is extremely easy to react with the silk fibroin containing active amino groups, and the terminal amino group of the silk fibroin and the anhydride of the polysaccharide form an amide bond so as to enhance the hydrophilicity of the silk fibroin and effectively inhibit the occurrence of self-crosslinking phenomenon, thereby realizing the long-term stable storage of the solution. In addition, the medical silk fibroin solution grafted with polysaccharide can promote cell proliferation, has good mechanical properties, and has potential application value in preparation of tissue engineering materials.

The first object of the invention is to provide a method for preparing medical silk fibroin solution grafted with polysaccharide, which comprises the following steps:

S1, dissolving silk in a first organic solvent with the concentration of 6-10M, adding a protease solution for incubation, inactivating protease after incubation, centrifuging to obtain a silk fibroin precipitate, and dissolving the silk fibroin precipitate in a second organic solvent with the concentration of 4-6M to obtain an enzyme silk fibroin solution;

S2, mixing and reacting polysaccharide containing at least one carboxyl group with triazolin diketone in the presence of a third organic solvent to obtain activated polysaccharide;

S3, mixing the enzyme silk fibroin solution in the S1 with the polysaccharide activated in the S2 for reaction, and dialyzing to obtain the medical silk fibroin solution grafted with the polysaccharide.

Further, the first organic solvent and the second organic solvent in the step S1 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 solution is greater than the concentration of the second organic solvent.

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

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

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

Further, the third organic solvent in the step S2 is selected from one or more of N, N-dimethylformamide, dimethyl sulfoxide, N-dimethylacetamide, and N-methylpyrrolidone.

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

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

Further, in the step S2, after the polysaccharide and the triazolin diketone are mixed and reacted, a reaction mediator is added to terminate the reaction, wherein the reaction mediator is one or more selected from beta-mercaptoethanol, dithiothreitol, cysteine and glycine. The reaction mediator is capable of reacting rapidly with the triazolinedione to form a stable adduct (e.g., a thioester or disulfide bond) that prevents the triazolinedione from continuing to activate the carboxyl group.

Further, the mass ratio of the silk fibroin precipitate dissolved in the enzyme silk fibroin in the step S3 to the polysaccharide is 1 (1-10).

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

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

The second object of the invention is to provide a medical silk fibroin solution of grafted polysaccharide prepared by the preparation method.

The third object of the invention is to provide an application of the medical silk fibroin solution in preparing tissue engineering materials.

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

The mechanism of the invention is as follows:

And (3) carrying out enzymolysis on silk by a one-step method, namely directly dissolving the silk in a first organic solvent, and adding protease for incubation and enzymolysis. The silk mainly comprises sericin and silk fibroin, and the first organic solvent can dissolve the sericin and the silk fibroin and enable molecular chains of the sericin and the silk fibroin to be spread, and protease enzyme cuts the sericin and the silk fibroin simultaneously. Due to the structural differences between sericin and silk fibroin, only the crystalline region of the high beta-sheet with the repeat GAGAGS fragment of silk fibroin precipitates to form silk fibroin precipitate, and the amorphous region of silk fibroin and silk fibroin remain in the supernatant.

The enzymatic silk fibroin precipitate has a secondary structure with high repetition GAGAGS, and part of amino groups in a molecular chain are positioned at the tail end and part of amino groups in an internal side chain, especially a lysine side chain. The conventional way to solubilize the silk fibroin precipitate is to use a high concentration of a second organic solvent for solubilization, which results in exposure of epsilon-amino groups of the internal lysines of the silk fibroin, which in turn results in excessive crosslinking and gelation of the polysaccharide. The invention uses the second organic solvent with low concentration to make the silk fibroin only expose the terminal amino group, but not the internal amino group, and simultaneously uses the triazolin diketone to convert the carboxyl group of the polysaccharide into the high-reactivity anhydride, and the terminal amino group of the silk fibroin and the anhydride of the polysaccharide form an amide bond so as to enhance the hydrophilicity of the silk fibroin and effectively inhibit the occurrence of self-crosslinking phenomenon, thereby realizing the long-term stable storage of the solution.

The invention has the beneficial effects that:

(1) The process is simplified. The traditional polysaccharide reacts with the terminal amino group of the silk fibroin to form an amide bond, so that the silk fibroin solution of the oriented graft polysaccharide is obtained. Silk fibroin enzymolysis is carried out by degumming silk, dissolving and purifying to obtain regenerated silk fibroin solution, and then adding protease into the regenerated silk fibroin solution for enzymolysis. The invention adopts one-step enzymolysis, directly dissolves silk and then adds protease for enzymolysis, omits the process steps of degumming, dissolving and purifying, greatly reduces chemical reagents and industrial pollutants, simplifies the process, has higher production efficiency and lower cost.

(2) The solution stability is high. The medical silk fibroin solution of the grafted polysaccharide prepared by the invention has good stability, no precipitation is observed within 90 days, the stability of the traditional regenerated silk fibroin solution and EDC/NHS modified silk fibroin solution is exceeded, excessive crosslinking of the polysaccharide is avoided to form gel, and the subsequent further development and use are facilitated.

(3) The polysaccharide directional grafting imparts good properties to the silk fibroin solution. The invention adopts the second organic solvent with low concentration, only part of silk fibroin amino groups, especially terminal amino groups, are exposed, and amino groups of internal side chains are not exposed, so that polysaccharide is directionally grafted to the terminal amino groups of the silk fibroin, and byproducts and gelation caused by excessive crosslinking are avoided.

(4) Excellent mechanical property and wide application prospect. The film prepared from the medical silk fibroin solution grafted with polysaccharide has good mechanical property and effect of promoting cell proliferation, and has wide application prospect in preparation of tissue engineering materials, in particular bone tissue engineering materials and cell proliferation materials.

Drawings

FIG. 1 is a process flow diagram of the present invention and comparative example 1, the left hand drawing is a process flow diagram of a medical silk fibroin solution of the present invention grafted with polysaccharide, and the right hand drawing is a process flow diagram of a silk fibroin product of comparative example 1;

FIG. 2 is a graph showing the results of the sericin residue measurement of examples 1 to 6 of the present invention and comparative example 1;

FIG. 3 is an infrared spectrum of the polysaccharide grafted silk fibroin of example 1 of the present invention.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

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

The sources and numbers of reagents are shown below.

Lithium bromide (LiBr) was purchased from aladine under the trade designation L108934;

sodium thiocyanate (NaSCN) was purchased from Jinan Toyoda under the designation HFD-248;

Zinc chloride (ZnCl ₂) was purchased from chinese medicine under the product number H1L0491;

Calcium chloride (CaCl ₂) -ethanol-water ternary solvent, wherein CaCl ₂ is purchased from Ji Yesheng chemical industry and has the product number JYS1545;

ethanol is purchased from Beaulaibo under the product number L11024-VZU;

Trypsin was purchased from aledine under the designation T274333;

Alpha-chymotrypsin was purchased from micllin under the trade designation C804761;

Pepsin was purchased from alatin under the trade designation P110928;

Proteinase K was purchased from Shanghai Chemie Co., ltd, cat# JK-038;

Papain was purchased from Sigma under the accession number P3250;

alkaline protease was purchased from beijing bai-oibo under the product number QN0397;

Sodium alginate is purchased from the company of pharmaceutical excipients, inc. of West An Jin Hunan, with the product number 0045;

hyaluronic acid was purchased from Solarbio under the accession number S7020;

Carboxymethyl chitosan was purchased from Sigma under the accession number 926043;

Agarose was purchased from Sigma under the accession number a9045;

the chitosans were purchased from beijing bai-oibo under the product number QN0147;

carboxymethyl cellulose is available from Alatin under the trade designation C104977;

N, N-dimethylformamide was purchased from microphone under the trade designation N807509;

dimethyl sulfoxide was purchased from michelin under the designation D806645;

n, N-dimethylacetamide was purchased from Melin under the trade designation N807171;

n-methylpyrrolidone was purchased from Melin under the trade designation M814045;

beta-mercaptoethanol was purchased from mikrin under the trade designation M828395;

dithiothreitol is available from macril under the designation D806827;

Cysteine was purchased from microphone under the product number D831312;

glycine was purchased from Sigma under the accession number G8790;

phosphate buffered saline (PBS buffer) was purchased from a convenient organism under the accession number bzw2106f;

triazoline dione was purchased from STANDARDS under the trade designation ZC68005;

sodium carbonate (Na ₂CO₃) was purchased from chinese medicine under the trade designation 10019260.

Example 1

S1, carrying out enzymolysis on silk by a one-step method, namely dissolving 10 g silk in 100 mL of 9.3M lithium bromide solution, filtering, mixing with 3 mg/g trypsin solution in an equal volume (the mass ratio of trypsin to silk is 3:100), reacting for 20 h at 37 ℃, heating for 10 min at 100 ℃, inactivating enzyme, centrifuging for 10 min at 5000 rpm, taking the lower layer, and cleaning to obtain the silk fibroin precipitate.

The silk fibroin precipitate was dissolved in 10 mL of 4.0M lithium bromide solution to obtain an enzyme silk fibroin solution.

S2, triazolin diketone activated polysaccharide carboxyl, namely dissolving 2g triazolin diketone in 10 mL of N, N-dimethylformamide, adding 2g sodium alginate for mixed dissolution, reacting 180 min at 4 ℃ with the mass ratio of the triazolin diketone to the sodium alginate being 1:1, and then adding beta-mercaptoethanol for terminating the reaction.

S3, polysaccharide directional grafting silk fibroin, namely mixing the enzyme silk fibroin solution in S1 and the activated polysaccharide solution in S2, wherein the mass ratio of silk fibroin sediment to polysaccharide is 1:1, and reacting at 2 ℃ for 60 min.

The reaction mixture is placed in a dialysis bag with the molecular weight cut-off of 10 kDa, and is dialyzed for 24 h by aqueous medium to obtain medical silk fibroin solution of grafted polysaccharide.

Example 2

S1, carrying out enzymolysis on silk by a one-step method, namely dissolving 50 g silk in 100 mL of 6.0M sodium thiocyanate solution, filtering, mixing with 1:1 mg/g alpha-chymotrypsin solution in equal volume (the mass ratio of the alpha-chymotrypsin to the silk is 1:500), reacting for 24 hours at 37 ℃, heating for 15min at 100 ℃, inactivating enzyme, centrifuging for 5min at 6000 rpm, taking the lower layer, and cleaning to obtain silk fibroin precipitate.

The silk fibroin precipitate was dissolved in 50 mL of 4.5M sodium thiocyanate solution to obtain enzyme silk fibroin solution.

S2, triazolin dione activated polysaccharide carboxyl is prepared by dissolving 10 g of triazolin dione in 50 mL of dimethyl sulfoxide, adding 1 g of hyaluronic acid for mixed dissolution, mixing the triazolin dione and the hyaluronic acid for reaction at 15 ℃ for 120 min, and adding dithiothreitol for terminating reaction.

S3, polysaccharide directional grafting silk fibroin, namely mixing the enzyme silk fibroin solution in the S1 and the activated polysaccharide solution in the S2, wherein the mass ratio of silk fibroin sediment to polysaccharide is 1:10, and reacting at 4 ℃ for 15 min.

The reaction mixture is placed in a dialysis bag with the molecular weight cut-off of 14 kDa, and is dialyzed for 18 h by taking PBS buffer solution as a medium, so as to obtain the medical silk fibroin solution of the grafted polysaccharide.

Example 3

S1, carrying out enzymolysis on silk by a one-step method, namely dissolving 30 g silk in 100 mL of 8M zinc chloride solution, filtering, mixing with 3 mg/g pepsin solution in an equal volume (the mass ratio of pepsin to silk is 1:100), reacting for 18 h at 37 ℃, heating for 20 min at 100 ℃, inactivating enzyme, centrifuging for 5min at 8000 rpm, taking out the lower layer, and cleaning to obtain the silk fibroin precipitate.

The silk fibroin precipitate was dissolved in 30 mL of 5.0M zinc chloride solution to obtain enzyme silk fibroin solution.

S2, triazolin dione activated polysaccharide carboxyl, namely dissolving 5g triazolin dione in 30 mL of N, N-dimethylacetamide, adding 1g carboxymethyl chitosan for mixed dissolution, reacting at 40 ℃ for 10min, and adding cysteine for terminating the reaction, wherein the mass ratio of the triazolin dione to the carboxymethyl chitosan is 5:1.

S3, polysaccharide directional grafting silk fibroin, namely mixing and reacting the enzyme silk fibroin solution in S1 and the activated polysaccharide solution in S2, wherein the mass ratio of silk fibroin sediment to polysaccharide is 1:5, and reacting at 8 ℃ for 5 min.

The reaction mixture is placed in a dialysis bag with the molecular weight cut-off of 20 kDa, and is dialyzed for 36 h by taking water as a medium, so as to obtain the medical silk fibroin solution of the grafted polysaccharide.

Example 4

S1, carrying out enzymolysis on silk by a one-step method, namely dissolving 15g silk in 100 mL of 10.0M calcium chloride-ethanol-water ternary solvent (the molar ratio is 1:2:8), filtering, mixing with 3 mg/g proteinase K solution in equal volume (the mass ratio of proteinase K to silk is 1:50), reacting for 16 h at 37 ℃, heating for 15 min at 95 ℃, inactivating enzyme, centrifuging for 10 min at 4000 rpm, taking the lower layer, and cleaning to obtain the silk fibroin precipitate.

The silk fibroin precipitate is taken and dissolved in 15 mL of 6.0M calcium chloride-ethanol-water ternary solvent (molar ratio 1:2:8) to obtain enzyme silk fibroin solution.

S2, triazolin dione activated polysaccharide carboxyl, namely dissolving 4g triazolin dione in 24 mL N-methyl pyrrolidone, adding 2g agarose for mixing and dissolving, reacting at 25 ℃ for 60 min when the mass ratio of the triazolin dione to the agarose is 2:1, and then adding glycine to terminate the reaction.

S3, polysaccharide directional grafting silk fibroin, namely mixing the enzyme silk fibroin solution in S1 and the activated polysaccharide solution in S2. The mass ratio of silk fibroin precipitate to polysaccharide was 1:3, and reacted at 4 ℃ for 10 min.

The reaction mixture is placed in a dialysis bag with the molecular weight cut-off of 10 kDa, and is dialyzed for 20 h by taking PBS buffer solution as a medium, so as to obtain the medical silk fibroin solution of the grafted polysaccharide.

Example 5

S1, carrying out enzymolysis on silk by a one-step method, namely dissolving 15 g silk in 100 mL of 9.0M lithium bromide solution, filtering, mixing with 3 mg/g papain solution in equal volume (the mass ratio of papain to silk is 1:50), reacting for 24 hours at 37 ℃, heating 15 min at 100 ℃, centrifuging 10 min at 5000 rpm, taking the lower layer, and cleaning to obtain the silk fibroin precipitate.

The silk fibroin precipitate was dissolved in 15 mL of 5.5M lithium bromide solution to obtain enzyme silk fibroin solution.

S2, triazolin dione activated polysaccharide carboxyl, namely dissolving 4g triazolin dione in 24 mL of N, N-dimethylformamide, adding 2 g chitosan for mixing and dissolving, reacting 180 min at 4 ℃ in a mass ratio of 2:1 of the triazolin dione to the chitosan, and then adding beta-mercaptoethanol to terminate the reaction.

S3, polysaccharide directional grafting silk fibroin, namely mixing the enzyme silk fibroin solution in S1 and the activated polysaccharide solution in S2, wherein the mass ratio of silk fibroin sediment to polysaccharide is 1:4, and reacting at 4 ℃ for 20 min.

Example 6

S1, carrying out enzymolysis on silk by a one-step method, namely dissolving 20g silk in 100 mL of 9.3M lithium bromide solution, filtering, mixing with 3: 3 mg/g alkaline protease solution in equal volume (the mass ratio of alkaline protease to silk is 3:200), reacting for 18 h at 37 ℃, heating for 10min at 100 ℃, inactivating enzyme, centrifuging for 10min at 4000 rpm, taking out the lower layer, and cleaning to obtain the silk fibroin precipitate.

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

S2, triazolin dione activated polysaccharide carboxyl is prepared by dissolving 5 g triazolin dione in 50 mL dimethyl sulfoxide, adding 1g hydroxymethyl cellulose for mixed dissolution, reacting at 4 ℃ for 150 min in a mass ratio of the triazolin dione to the hydroxymethyl cellulose, and then adding glycine for terminating the reaction.

S3, polysaccharide directional grafting silk fibroin, namely mixing the enzyme silk fibroin solution in the S1 and the activated polysaccharide solution in the S2, wherein the mass ratio of silk fibroin sediment to polysaccharide is 1:5, and reacting at 4 ℃ for 15 min.

The reaction mixture is placed in a dialysis bag with the molecular weight cut-off of 20 kDa, and is dialyzed for 18 h by taking PBS buffer solution as a medium, so as to obtain the medical silk fibroin solution of the grafted polysaccharide.

Comparative example 1

The comparative example provides a process for the preparation of a silk fibroin product, similar to example 1, except that in step S1 silk is first added to a 0.5 g/L Na ₂CO₃ solution to boil for 1 h, the degummed silk is fished out, washed with water for 3 times, dried, redissolved in a 9.3M lithium bromide solution, dialyzed and purified to obtain a regenerated silk fibroin solution, then protease is added for incubation, and the rest steps are identical to those of example 1. A comparison of the process flows is shown in figure 1.

Comparative example 2

This comparative example provides a process for the preparation of a silk fibroin product, similar to example 1, except that in step S1 the silk fibroin precipitate was dissolved in water, but not in lithium bromide solution, and the remaining steps were identical to example 1.

Comparative example 3

This comparative example provides a process for the preparation of a silk fibroin product, similar to example 1, except that in step S1 the silk fibroin precipitate was dissolved in 9.3M lithium bromide solution, the remaining steps being identical to example 1.

Comparative example 4

This comparative example provides a process for the preparation of a silk fibroin product, similar to example 1, except that in step S2 sodium alginate is directly dissolved in water and then mixed with the enzyme silk fibroin solution in S3 for reaction, the remaining steps are identical to example 1.

Comparative example 5

This comparative example provides a process for the preparation of a silk fibroin product, similar to example 1, except that 0.6g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 1.2g N-hydroxysuccinimide (NHS) were used to activate the polysaccharide carboxyl groups in step S2, the remainder of the procedure being identical to that of example 1.

Test case

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

(1) Measurement of residual amount of sericin the residual amount of sericin in the sericin precipitate obtained in step S1 of examples 1 to 6 and the sericin precipitate obtained in step S1 of comparative example 1 was measured by the method mentioned in the patent No. CN20201056391.7 entitled polypeptide antibody for direct detection of sericin, preparation method and use, and the result is shown in FIG. 2.

(2) And (3) observing the properties of the medical silk fibroin solution of the grafted polysaccharide prepared in the examples 1-6 and the silk fibroin products prepared in the comparative examples 1-5 respectively under colorless and white light without background interference, and observing the properties of the silk fibroin solution with naked eyes.

(3) Solution stability the medical silk fibroin solutions of the graft polysaccharides prepared in examples 1-6 and silk fibroin products prepared in comparative examples 1-5 were respectively put into a refrigerator at 4 ℃ for refrigeration, and the state of the solutions was observed and recorded over 90 days.

(4) And (3) determining grafting state of silk fibroin in the medical silk fibroin solution of the grafted polysaccharide in the embodiment 1 by an infrared spectrometry method, and qualitatively judging whether chemical grafting is successful. As can be seen from FIG. 3, after the polysaccharide is directionally grafted with the silk fibroin, characteristic peaks 1620cm ^-1 and 1520cm ^-1 of the silk fibroin are obviously weakened, hydrogen bond vibration of hydroxyl groups in saccharide molecules is overlapped at 3296cm ^-1 to form a broad peak ‌, and a strong peak at 1031cm ^-1 is an expansion vibration peak of glycosidic bonds (C-O-C) in the polysaccharide, which indicates that the polysaccharide is successfully chemically grafted to the silk fibroin.

(5) Mechanical property test, namely placing medical silk fibroin solution of the grafted polysaccharide prepared in the examples 1-6 in a surface dish for natural standing and film forming, placing silk fibroin products of the comparative examples 1-5 in the surface dish for standing and film forming, and testing tensile breaking strength and breaking elongation by using a TST-01M intelligent electronic tensile tester, wherein the tensile speed is 10 mm/min, the sample width is 10 mm and the clamping length is 10 mm. The results are shown in Table 1.

TABLE 1 stability and mechanical Property test results

Comparative example 1 degummed silk is obtained by degumming and drying silk, then is dissolved in a first organic solvent, is purified to obtain a traditional regenerated silk fibroin solution, is subjected to enzymolysis, and finally forms a silk fibroin product which is the same as that of examples 1-6, and polysaccharide is successfully chemically grafted to the silk fibroin after enzyme digestion to form a clear and transparent solution with stability of more than 90 days, and meanwhile, the mechanical properties are similar. However, the conventional enzymolysis method represented by comparative example 1 starts enzymolysis from regenerated silk fibroin solution, multiple processes are added from silk to regenerated silk fibroin solution, and a large amount of chemical reagents and industrial wastewater are consumed, especially the degumming process, and nonspecific hydrolysis (different from specific point hydrolysis of enzyme) is caused on silk in the alkaline solution boiling or the acid solution boiling. From the degumming effect of FIG. 2, the residual sericin amounts of examples 1 to 6 and comparative example 1 were not significantly different, and were each less than 20 ng/mg, i.e., 0.002%.

Comparative example 2 pure water was used to solubilize the silk fibroin precipitate. The absence of the second organic solvent, which results in the precipitation of the silk fibroin in an unstable suspension state, does not allow an effective chemical grafting modification of the subsequently added activated polysaccharide and therefore does not eventually form a solution, but exists in the form of a precipitate, which is not available for subsequent use.

Comparative example 3 the silk fibroin precipitate was dissolved using a high concentration of a second organic solvent. The second organic solvent with high concentration enables the beta-sheet of the secondary structure of the silk fibroin sediment to be completely unfolded, the silk fibroin chain is completely unfolded, the tail end and the internal amino groups of the silk fibroin are fully exposed, the activated polysaccharide added subsequently and the activated polysaccharide are subjected to multi-point chemical modification and cross-linking, and the grafted polysaccharide forms a molecular brush structure, so that the modified solution is excessively cross-linked to finally form gel, and the subsequent further use is not facilitated. Meanwhile, due to excessive grafting modification, the silk fibroin maintains a random coil conformation, so that the rigidity (breaking strength) of the film is reduced, and the flexibility (breaking elongation) is increased. In the preparation process of the medical silk fibroin solution of the grafted polysaccharide in examples 1-6, the silk fibroin precipitate in the step S1 is dissolved in the second organic solvent with low concentration, so that the amino groups at the exposed tail ends of the silk fibroin react with the activated polysaccharide to form anhydride, and the formation of endogenous crosslinking sites is reduced, thereby effectively inhibiting the occurrence of self-crosslinking phenomenon.

Comparative example 4 blend of unactivated polysaccharide with enzyme silk fibroin solution was used. The unactivated polysaccharide and the silk fibroin cannot be subjected to chemical grafting modification, the essence of the unactivated polysaccharide and the silk fibroin is physical blending of the two, and the polysaccharide can be completely removed through dialysis in the subsequent dialysis step, so that intermolecular hydrogen bonding of the silk fibroin is dominant again, floccule precipitation is generated, and a stable solution cannot be formed for further use.

Comparative example 5 silk fibroin and polysaccharide were grafted with EDC/NHS. The reaction efficiency of the silk fibroin and the polysaccharide is very low under mild conditions, EDC and NHS are used as a pair of partner catalysts, carboxyl on a polysaccharide chain is activated into a high-reactivity intermediate, and the high-reactivity intermediate and amino on the silk fibroin chain undergo high-efficiency amidation reaction, so that two macromolecular chains are connected through covalent bonds, a three-dimensional network structure is very easy to form, and water molecules are wrapped in the three-dimensional network structure, so that the hydrogel is finally formed.

Example 7

The medical silk fibroin solution of the grafted polysaccharide in the embodiment 1-6 is prepared into a porous bracket by a freeze drying method, and the specific steps are that the medical silk fibroin solution of the grafted polysaccharide is put into a refrigerator at-20 ℃, completely frozen and then put into a freeze dryer, vacuumized under negative pressure until solid powder is completely formed, the powder is dissolved in a serum-containing culture medium and inoculated with L929 fibroblasts, and cell proliferation is detected for 3 days by a cell proliferation toxicity detection method (CCK-8 method). The medical silk fibroin solutions of the grafted polysaccharides prepared in examples 1-6 showed excellent cell proliferation promoting effect with the cell proliferation rate up to 124.4%, because the silk fibroin porous scaffolds prepared from the medical silk fibroin solutions of examples 1-6 not only increased specific surface area to accommodate more cells, but also provided contact guidance, promoted cell expansion, and activated Focal Adhesion Kinase (FAK) and extracellular regulated protein kinase/mitogen activated protein kinase (ERK/MAPK) signaling pathways, promoted cell spreading and proliferation, and the polysaccharides further provided nutrients to provide energy to cells. The silk fibroin product of comparative example 3 was in a gel state, and the proliferation effect was not as good as examples 1 to 6 by inoculating L929 fibroblasts by the same method and detecting cell proliferation by a cell proliferation toxicity detection method (CCK-8 method) for 3 days, with a lower gel specific surface area. The test results are shown in Table 2.

TABLE 2 results of cell proliferation rate experiments

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims

1. The preparation method of the medical silk fibroin solution grafted with polysaccharide is characterized by comprising the following steps:

S2, mixing and reacting polysaccharide containing at least one carboxyl group with triazolin diketone in the presence of a third organic solvent to obtain activated polysaccharide, wherein the polysaccharide is selected from one or more of sodium alginate, hyaluronic acid, carboxymethyl chitosan, agarose, chitosan and carboxymethyl cellulose;

s3, mixing the enzyme silk fibroin solution in the S1 with the polysaccharide activated in the S2 for reaction, and dialyzing to obtain a medical silk fibroin solution grafted with the polysaccharide;

The first organic solvent and the second organic solvent in the step S1 are independently selected from one or more of lithium bromide, sodium thiocyanate, zinc chloride and calcium chloride-ethanol-water ternary solvents, and the third organic solvent in the step S2 is selected from one or more of N, N-dimethylformamide, dimethyl sulfoxide, N-dimethylacetamide and N-methylpyrrolidone.

2. The method according to claim 1, wherein the protease in step S1 is one or more selected from the group consisting of α -chymotrypsin, pepsin, trypsin, proteinase K, papain and alkaline protease.

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

4. The method according to claim 1, wherein the mass ratio of triazolinedione to polysaccharide is (10-1): 1.

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

6. The method according to claim 1, wherein the volume ratio of the mass of the triazolinedione to the third organic solvent is 1g (5-10) mL.

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

8. The medical silk fibroin solution of grafted polysaccharide prepared by the preparation method of any one of claims 1-7.

9. The use of the medical silk fibroin solution of claim 8 for preparing tissue engineering materials.