Methoxypolyethylene glycol epoxy chemically modified silk fibroin and method of making same

Abstract

The present application relates to methoxy polyethylene glycol epoxy chemical modified silk fibroin and its preparation method, belong to the field of biological extraction technology. The present application adds low concentration hydrogen bond inhibitor and pH regulator in regenerated silk fibroin solution, then adds trypsin for incubation, inactivation, solid-liquid separation to obtain silk fibroin solid; then mixed with inorganic salt solution for activation, after activation, co-incubation with methoxy polyethylene glycol epoxy, purification treatment, to obtain chemically grafted silk fibroin solution. The present application first proposes the technical scheme of low concentration hydrogen bond inhibition-pH regulation combined enzymolysis technology, dissolution modification technology of the precipitate layer after enzymolysis and inorganic salt solution activation / methoxy polyethylene glycol epoxy synergistic chemical modification technology, which not only solves the problems of low proteolysis effect and long-term maintenance of enzyme activity, but also successfully prepares the precipitate layer into a stable solution, greatly improves the success rate of silk fibroin chemical modification, and is beneficial to further development and use.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological extraction, in particular to methoxypolyethylene glycol chemically modified silk fibroin and a preparation method thereof. BACKGROUND

[0002] Silkworm silk is mainly composed of silk fibroin with a mass ratio of about 70% and sericin with a mass ratio of about 25%. Natural silkworm silk forms a regular, hydrophobic and high content of beta sheet region in the silkworm gland due to a large number of intermolecular hydrogen bonds, so that it can only swell in water and most organic solvents but not dissolve. In order to prepare spinning dope and other various properties of silk fibroin-based materials, it is necessary to dissolve the silk to obtain a large amount of silk fibroin solution. The conventional silk fibroin raw material is obtained by generating a regenerated silk fibroin solution, that is, through a plurality of processes such as degumming, dissolving, dialysis, purification and concentration. At present, the silkworm fiber dissolving system mainly includes inorganic salt system, organic solvent system and organic salt system, which is used to prepare a regenerated silk fibroin solution, and its essence is to destroy the existing hydrogen bond or inhibit the formation of intermolecular hydrogen bond, to break the beta sheet structure of silk fibroin and make the molecular chain exist in a random coil conformation and in a dissolved state.

[0003] There are mainly two ways to enzymatically hydrolyze silk fibroin solution in the prior art, way one: completely hydrolyze silk fibroin to obtain small molecular weight polypeptides contained in the supernatant, which is used to prepare small molecular weight polypeptide silk fibroin products or to study the molecular weight, amino acid type and content, and amino acid sequence. The primary structure of the silk fibroin after enzymatic hydrolysis is a small molecular weight polypeptide fragment, which almost does not contain the sequence of GAGAGS, and its secondary structure does not contain beta sheet conformation and crystallinity. No matter how modified, it is impossible to talk about the excellent mechanical properties of large molecular weight and containing beta sheet structure. Way two: partially hydrolyze silk fibroin to form supernatant layer (mainly amorphous region) and precipitate layer (mainly crystalline region), and study the changes of amino acid type and content, beta sheet amount, etc. of different layers. The emphasis of this way is chemical analysis test, which is irrelevant to the preparation of silk fibroin raw material.

[0004] Traditional methods of dissolving silk fibroin fiber, such as lithium bromide inorganic salt system, can dissolve the precipitated layer, but the solution after dialysis of lithium bromide will continue to agglomerate to form a precipitate, thereby limiting its application, not to mention subsequent purification and modification steps. Currently, the method of using polyethylene glycol to increase water solubility is only limited to regenerated silk fibroin solution, and the modification efficiency is low, and the modification success rate is not high. Lu Shenzhou et al. modified the silk fibroin film with polyethylene glycol glycidyl ether (Lu Shenzhou, Li Mingzhong, Liu Yang, et al. Modification of silk fibroin film with polyethylene glycol glycidyl ether [J]. Polymer Materials Science and Engineering, 2003, 19(1): 104-107): polyethylene glycol was first reacted with epichlorohydrin to form polyethylene glycol glycidyl ether, and then the silk fibroin solution and polyethylene glycol glycidyl ether were blended and dried into a film. This method changes the structure of the silk fibroin film, reduces its dissolution rate, and improves the tensile strength and elongation at break. Yang Hua et al. further characterized the structure and properties of the blended film after modification of silk fibroin on the basis of the above research (Yang Hua, Nie Tao, Cai Hui, et al. Study on polyethylene glycol glycidyl ether modified silk fibroin [J]. Chemical Industry and Science & Technology, 2013, 21(5): 4-8). However, both of these two papers are from the perspective of blending polyethylene glycol glycidyl ether with regenerated silk fibroin solution for modification, which not only has low modification efficiency and low modification success rate, but also polyethylene glycol glycidyl ether is synthesized, which increases the complexity of the process and the uncertainty of reagent residues. The patent with application number CN202410550980 discloses a preparation method of silk fibroin microspheres, one of the steps is: adding methoxy polyethylene glycol active ester to the regenerated silk fibroin solution to introduce polyethylene glycol to the amino residue of the silk fibroin molecule, obtaining methoxy polyethylene glycol active ester modified silk fibroin, thereby obtaining better water solubility than pure regenerated silk fibroin, reducing the agglomeration and precipitation of the prepared microspheres. However, this method is a modification of regenerated silk fibroin, and the modification efficiency is low and the chemical modification success rate is not high.

[0005] As can be seen from the above, there is no report in the prior art on the feasibility of increasing the water solubility of the precipitated layer after enzymatic hydrolysis of silk fibroin and high chemical modification rate. SUMMARY

[0006] To solve the above technical problems, the application provides a methoxypolyethylene glycol epoxy chemically modified silk fibroin and a preparation method thereof. Traditional silk fiber dissolution technology is limited to conventional silk fibroin solution, and whether lithium bromide system or polyethylene glycol derivative modification is only practiced on the silk fibroin solution. Unlike conventional regenerated silk fibroin solution, the precipitated layer after enzymatic hydrolysis has a more regular structure, high crystallinity, high beta folding amount, and a higher relative proportion of GAGAGS in the primary structure, but has poorer water solubility and cannot fully expose active sites, thereby limiting its subsequent use. The conventional single dissolution system cannot solve the problem of reprecipitation of the precipitated layer after dialysis, and the success rate of the single modification system is not high. The application first proposes a low-concentration hydrogen bond inhibition-pH adjustment combined enzymatic hydrolysis technology, a precipitated layer dissolution modification technology after enzymatic hydrolysis, and an inorganic salt solution activation / methoxypolyethylene glycol epoxy synergistic modification technical solution, which not only solves the problems of low enzymatic hydrolysis effect and long-term maintenance of enzyme activity, but also successfully prepares the precipitated layer into a stable solution, greatly improves the success rate of silk fibroin modification, widens the application range, and is beneficial to further development and use.

[0007] The application is implemented by the following technical solutions.

[0008] The first object of the application is to provide a preparation method of methoxypolyethylene glycol epoxy chemically modified silk fibroin, comprising the following steps:

[0009] S1, adding a hydrogen bond inhibitor and a pH adjuster to a regenerated silk fibroin solution to obtain a mixture with a final concentration of 0.01 M-3 M, then adding trypsin for incubation, inactivation, solid-liquid separation to obtain a silk fibroin solid, and the obtained silk fibroin solid is a silk fibroin solid with high crystallinity and high rigidity;

[0010] S2, mixing the silk fibroin solid obtained in step S1 with an inorganic salt solution for activation, then co-incubating with methoxypolyethylene glycol epoxy, and purifying to obtain a methoxypolyethylene glycol epoxy chemically modified silk fibroin solution.

[0011] Further, in step S1, the regenerated silk fibroin is obtained by degumming, dissolving and purifying silk.

[0012] And / or, the concentration of the regenerated silk fibroin in the regenerated silk fibroin solution is 0.5 wt%-40 wt%.

[0013] Further, in step S1, the hydrogen bond inhibitor is one or more of CaCl2 / ethanol / water, LiBr, NaSCN and ZnCl2.

[0014] Further, in step S1, the pH regulator is one or more of Tris-HCl buffer, HEPES buffer, PBS buffer, HBSS buffer, MOPS buffer and acetate buffer.

[0015] Further, the pH regulator adjusts the pH value of the regenerated silk fibroin solution to 6-9.

[0016] Further, in step S1, the added trypsin has a final concentration of 0.01 wt%-0.3 wt%.

[0017] Further, in step S1, the mass ratio of trypsin to regenerated silk fibroin in the regenerated silk fibroin solution is 1:5-1:500.

[0018] Further, the incubation condition is 25°C-37°C for 24 h-48 h.

[0019] Further, in step S1, the solid-liquid separation method is centrifugation; the solid-liquid separation further includes a washing step; the washing includes water washing and alcohol washing, and the alcohol includes methanol, ethanol, aromatic alcohol, etc.

[0020] Further, in step S2, the fibroin solid is first activated with an inorganic salt solution to destroy the hydrogen bond network of β-sheet, promote dissolution and fully expose the amino acid sites, and then the methoxypolyethylene glycol epoxy is added to react with the fibroin under the activation of the anion and cation, thereby efficiently completing the chemical modification.

[0021] Further, in step S2, the cation in the inorganic salt solution is one or more of Na + , Mg 2+ , Cu 2+ , Zn 2+ , Li + , K + , Fe 3+ and Ca 2+ ; and the anion in the inorganic salt solution is one or more of Cl - , SO4 2- , Br - , SCN - , CO3 2- and OH - .

[0022] Further, the concentration of the inorganic salt solution is 4 M-15 M.

[0023] Further, in step S2, the mass ratio of the inorganic salt solution to the fibroin solid is 40:1-1:1.

[0024] Further, the methoxypolyethylene glycol epoxy has a molecular weight of 1 kDa-100 kDa.

[0025] Further, in the step S2, the mass ratio of the methoxypolyethylene glycol epoxy to the silk fibroin solid is 1:200-1:1.

[0026] And / or, the incubation condition is: 4℃-80℃ incubation for 0.5 h-72 h, preferably 25℃-40℃ incubation for 4 h-48 h.

[0027] And / or, the molecular weight cut-off of the purification is 10 kDa-100 kDa.

[0028] The second object of the present application is to provide the methoxypolyethylene glycol epoxy chemically modified silk fibroin prepared by the preparation method.

[0029] The mechanism of the present application is:

[0030] Low-concentration hydrogen bond inhibition-pH adjustment combined with enzymatic hydrolysis: first, low-concentration hydrogen bond inhibitors and pH regulators are added to adjust the environment conducive to the long-term maintenance of trypsin activity, and then trypsin is added for incubation, thereby obtaining a precipitated part with high crystallinity and large rigidity, which is fundamentally different from traditional silk fibroin in primary and secondary structure.

[0031] Inorganic salt solution activation-methoxypolyethylene glycol epoxy modification synergistic chemical modification: because the precipitated part has poorer water solubility, the silk fibroin precipitate is first activated and dissolved with an inorganic salt solution to expose active sites and form a charge microregion, and then methoxypolyethylene glycol epoxy is added for nucleophilic reaction, thereby forming a methoxypolyethylene glycol epoxy chemically modified silk fibroin solution with high success rate and better stability.

[0032] The above technical solutions of the present application have the following advantages compared with the prior art:

[0033] (1) The structure is more optimal. The precipitate formed after trypsin enzymolysis of the traditional regenerated silk fibroin solution is fundamentally different from the present application, the primary structure of the protein has changed, the G / A / S ratio in the enzymolysis precipitate is high, the crystallinity and rigidity are large, and the amino acid sequence is more regular and orderly, so the mechanical properties are also more optimal compared with the traditional regenerated silk fibroin solution.

[0034] (2) Long-term maintenance of enzymolysis efficiency and enzyme activity. Low-concentration hydrogen bond inhibition-pH adjustment is used for enzymolysis, which can make the silk fibroin chain more relaxed and expose the enzymolysis sites, and at the same time activate the enzyme itself, improve the enzymolysis effect, and make the enzyme maintain its activity for a long time.

[0035] (3) The success rate of chemical modification is higher. The inorganic salt solution activation-methoxy polyethylene glycol epoxy synergistic chemical modification method is adopted, the silk fibroin is dissolved and activated by the inorganic salt solution, the hydrogen bond is destroyed to promote dissolution, the molecular chain is fully unfolded to fully expose the amino acid sites, the functional groups on the chain are combined to form a charge micro area, and then the nucleophilic reaction of methoxy polyethylene glycol epoxy is facilitated, so that the success rate of chemical modification is greatly improved.

[0036] (4) The solution is more stable. The precipitate after enzymatic hydrolysis of silk fibroin has a regular structure, and its water solubility is also poorer. Only the inorganic salt solution is added, and the methoxy polyethylene glycol epoxy is not added, and the solution is precipitated again after purification; only the inorganic salt solution is not added, and the methoxy polyethylene glycol epoxy is added, and the solution is basically insoluble; under the synergistic effect of inorganic salt solution activation-methoxy polyethylene glycol epoxy chemical modification, the stability of the modified silk fibroin solution is greater than 30 days, far exceeding the modified solution of traditional regenerated silk fibroin by methoxy polyethylene glycol epoxy. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, in which:

[0038] Figure 1 is the high-stability methoxy polyethylene glycol epoxy chemically modified silk fibroin solution formed in Example 1 of the present application;

[0039] Figure 2 is the flocculent precipitate formed in Comparative Example 5 of the present application;

[0040] Figure 3 is the XRD graph of the silk fibroin precipitate crystallinity test of Comparative Example 1, Comparative Example 2 and Example 1;

[0041] Figure 4 is the infrared spectrum of the methoxy polyethylene glycol epoxy chemically modified silk fibroin before Example 1;

[0042] Figure 5 is the infrared spectrum of the methoxy polyethylene glycol epoxy chemically modified silk fibroin after Example 1;

[0043] Figure 6 is the application diagram of the methoxy polyethylene glycol epoxy chemically modified silk fibroin solution obtained in Example 1 prepared into a liquid adhesive plaster;

[0044] Figure 7 is the stress-strain diagram of the methoxy polyethylene glycol epoxy chemically modified silk fibroin solution obtained in Example 1 after standing into a film. DETAILED DESCRIPTION

[0045] The application will be further described in connection with the drawings and specific embodiments so that those skilled in the art can better understand and implement the application, but the embodiments are not limiting to the application.

[0046] The experimental methods used in the following examples are conventional methods, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0047] LiBr was purchased from Aladdin with the product code L108934;

[0048] NaSCN was purchased from Jinan Hui Fengda with the product code HFD-248;

[0049] CaCl2 / ethanol / water solution, wherein CaCl2 was purchased from Jiyesheng Chemical with the product code JYS1545;

[0050] Ethanol was purchased from Bao'elai Bo with the product code L11024-VZU; ZnCl2 was purchased from National Medicine with the product code H1L0491; PBS buffer was purchased from Bianzhi Biology with the product code bzwl206f; Tris-HCl buffer was purchased from Solay Bo with the product code T1160; HBSS buffer was purchased from Kanglang Biology with the product code KL-13487; MOPS buffer was purchased from Yudi Biology with the product code LG-NA0016; acetate buffer was purchased from Xinesi with the product code T-317015; trypsin was purchased from Aladdin with the product code T274333; KSCN inorganic salt was purchased from Naisi Biochemical with the product code NCS1453117; Fe Cl3 inorganic salt was purchased from Yuanmu Biology with the product code YM-0181; Zn SO4 inorganic salt was purchased from Naisi Biochemical with the product code NCS187378; NaCO3 inorganic salt was purchased from Baishengyue Biology with the product code S291934; LiCl inorganic salt was purchased from Yubo Biology with the product code YBP123954;

[0051] CaBr2 was purchased from Solay Bo with the product code CB14717714;

[0052] mPEG-EPO was purchased from Carbon Technology with the product code 80010121.

[0053] Example 1:

[0054] The embodiment provides a preparation method of methoxypolyethylene glycol epoxy chemically modified silk fibroin, and the specific steps are as follows:

[0055] S1, low concentration hydrogen bond inhibition-pH adjustment combined with enzymatic hydrolysis:

[0056] Boil the mulberry silk in Na2CO3 aqueous solution for 1 h, dry to obtain degummed silk, dissolve the degummed silk in 9.3 M lithium bromide (LiBr) solution, and purify to obtain regenerated silk fibroin solution;

[0057] Dilute the regenerated silk fibroin solution to 0.5 wt%, add lithium bromide to a final concentration of 0.8 M, add PBS buffer to adjust the pH to 8.0, then add trypsin to a final concentration of 0.1 wt%, and incubate at 37°C for 24 h; then inactivate the enzyme by heating in a boiling water bath for 10 min.

[0058] S2, collection of precipitate: centrifuge the inactivated enzyme solution at 5000 rpm for 5 min, discard the supernatant, collect the precipitate layer, and wash with water and ethanol to complete the collection of silk fibroin solid.

[0059] S3, inorganic salt solution activation-methoxy polyethylene glycol epoxy synergistic chemical modification of silk fibroin: the silk fibroin solid collected in S2 above is first activated with a KSCN inorganic salt solution, the final concentration of the inorganic salt solution is 10 M, the mass ratio of inorganic salt solution to silk fibroin is 1:1, mPEG-EPO is added to the activated and dissolved solution, the average molecular weight of mPEG-EPO is 4 kDa, the mass ratio of mPEG-EPO to silk fibroin is 3:100, and the solution is incubated at 25°C for 12 h; the incubated solution is loaded into a dialysis bag with a molecular weight cut-off of 14 kDa, and purified in pure water for 24 h to obtain a high-stability mPEG-EPO chemically modified silk fibroin solution (as shown in Figure 1

[0060] Example 2:

[0061] The present embodiment provides a preparation method of methoxy polyethylene glycol epoxy chemically modified silk fibroin, and the specific steps are as follows:

[0062] S1, low concentration hydrogen bond inhibition-pH adjustment combined with enzymolysis:

[0063] Boil the mulberry silk in Na2CO3 aqueous solution for 1 h, dry to obtain degummed silk, dissolve the degummed silk in 9.3 M lithium bromide (LiBr) solution, and purify to obtain regenerated silk fibroin solution;

[0064] Dilute the regenerated silk fibroin solution to 10 wt%, add NaSCN to a final concentration of 0.3 M, add Tris-HCl buffer to adjust the pH to 6.0, then add trypsin to a final concentration of 0.1 wt%, and incubate at 25°C for 48 h; then inactivate the enzyme by heating in a boiling water bath for 15 min.

[0065] ​S2, collection of precipitate: centrifuge the inactivated enzymatic solution at 3000 rpm for 10 min, discard the supernatant, collect the precipitate layer, and wash with water and methanol to complete the collection of fibroin solid.

[0066] S3, inorganic salt solution activation-methoxy polyethylene glycol epoxy synergistic chemical modification of fibroin: the fibroin solid collected in S2 above is first activated with Fe Cl3 inorganic salt solution activation, the final concentration of inorganic salt solution is 4 M, the mass ratio of inorganic salt solution to fibroin is 5:1, mPEG-EPO is added to the activated solution, the average molecular weight of mPEG-EPO is 1 kDa, the mass ratio of mPEG-EPO to fibroin is 1:200, and the solution is incubated at 40°C for 4 h; the incubated solution is loaded into a dialysis bag with a molecular weight cut-off of 10 kDa, and purified in pure water for 48 h to obtain a high-stability mPEG-EPO chemically modified fibroin solution.

[0067] Example 3:

[0068] The present embodiment provides a preparation method of methoxy polyethylene glycol epoxy chemically modified fibroin, and the specific steps are as follows:

[0069] S1, low concentration hydrogen bond inhibition-pH adjustment combined enzymolysis:

[0070] The mulberry silk is boiled in Na2CO3 aqueous solution for 1 h, and dried to obtain degummed silk. The degummed silk is dissolved in a 9.3 M lithium bromide (LiBr) solution, and a regenerated fibroin solution is obtained by purification.

[0071] The regenerated fibroin solution is diluted to 5 wt%, 0.01 M CaCl2 / ethanol / water solution (wherein the molar ratio of CaCl2, ethanol and water is 1:2:8) is added, HEPES buffer is added to adjust the pH to 7.4, then trypsin is added, the final concentration of trypsin is 0.01 wt%, and the solution is incubated at 37°C for 48 h; and then heated in a boiling water bath for 20 min to inactivate the enzyme.

[0072] S2, collection of precipitate: centrifuge the inactivated enzymatic solution at 3000 rpm for 10 min, discard the supernatant, collect the precipitate layer, and wash with water and methanol to complete the collection of fibroin solid.

[0073] S3, inorganic salt solution activation-methoxy polyethylene glycol epoxy synergistic chemical modification of fibroin: the fibroin solid collected in S2 above is first activated with Fe SO4 inorganic salt solution activation, the final concentration of inorganic salt solution is 6 M, the mass ratio of inorganic salt solution to silk fibroin is 10:1, mPEG-EPO is added in the activated solution, the average molecular weight of mPEG-EPO is 10 kDa, the mass ratio of mPEG-EPO to silk fibroin is 1:1, and the solution is incubated at 37°C for 48 h; the incubated solution is loaded into a dialysis bag with a molecular weight cut-off of 80 kDa, and purified in pure water for 36 h to obtain a high-stability mPEG-EPO chemically modified silk fibroin solution.

[0074] Example 4:

[0075] The embodiment provides a preparation method of methoxypolyethylene glycol epoxy chemically modified silk fibroin, and the specific steps are as follows:

[0076] S1, low-concentration hydrogen bond inhibition-pH adjustment combined with enzymolysis:

[0077] The silk of Bombyx mori is boiled in a Na2CO3 aqueous solution for 1 h, and dried to obtain degummed silk. The degummed silk is dissolved in a 9.3 M lithium bromide (LiBr) solution, and a regenerated silk fibroin solution is obtained by purification.

[0078] The regenerated silk fibroin solution is diluted to 15 wt%, a ZnCl2 solution with a final concentration of 3 M is added, an HBSS buffer is added to adjust the pH to 9.0, and then trypsin is added, the final concentration of trypsin is 0.2 wt%, and the solution is incubated at 37°C for 30 h. Then, the enzyme is inactivated by heating in a boiling water bath for 10 min.

[0079] S2, collection of precipitates: the inactivated enzymolysis solution is centrifuged at 5000 rpm for 5 min, the supernatant is discarded, and the precipitate layer is collected and washed with water and benzyl alcohol to complete the collection of silk fibroin solids.

[0080] S3, inorganic salt solution activation-methoxypolyethylene glycol epoxy cooperative chemical modification of silk fibroin: the silk fibroin solids collected in S2 are first activated with a Na2CO3 inorganic salt solution, the final concentration of the inorganic salt solution is 10 M, the mass ratio of the inorganic salt solution to silk fibroin is 20:1, mPEG-EPO is added in the activated solution, the average molecular weight of mPEG-EPO is 20 kDa, the mass ratio of mPEG-EPO to silk fibroin is 1:100, and the solution is incubated at 37°C for 12 h; the incubated solution is loaded into a dialysis bag with a molecular weight cut-off of 50 kDa, and purified in pure water for 18 h to obtain a high-stability mPEG-EPO chemically modified silk fibroin solution.

[0081] Example 5:

[0082] The embodiment provides a preparation method of methoxypolyethylene glycol epoxy chemically modified silk fibroin, and specific steps are as follows:

[0083] S1, low concentration hydrogen bond inhibition-pH adjustment combined enzymolysis:

[0084] Boil the mulberry silk in a Na2CO3 aqueous solution for 1 h, and dry to obtain degummed silk; the degummed silk is put into a 9.3 M lithium bromide (LiBr) solution to be dissolved, and a regenerated silk fibroin solution is obtained through purification;

[0085] The regenerated silk fibroin solution is diluted to 3 wt%, 1 M lithium bromide is added at a final concentration, a MOPS buffer is added to adjust the pH to 7.4, then trypsin is added, the final concentration of trypsin is 0.3 wt%, and the mixture is incubated at 37°C for 22 h; and then the enzyme is inactivated by heating in a boiling water bath for 15 min.

[0086] S2, collection of the precipitate: the inactivated enzymolysis solution is centrifuged at 4000 rpm for 10 min, the supernatant is discarded, and the precipitate is collected and washed with water and ethanol to complete the collection of the silk fibroin solid.

[0087] S3, inorganic salt solution activation-methoxypolyethylene glycol epoxy synergistic chemical modification of silk fibroin: the silk fibroin solid collected in the above S2 is activated with a LiCl inorganic salt solution, the final concentration of the inorganic salt solution is 9.3 M, the mass ratio of the inorganic salt solution to the silk fibroin is 40:1, mPEG-EPO with an average molecular weight of 6kDa is added to the activated and dissolved solution, the mass ratio of mPEG-EPO to the silk fibroin is 1:150, and the mixture is incubated at 40°C for 8 h; the incubated solution is loaded into a dialysis bag with a molecular weight cut-off of 50kDa, and is purified in the LiCl inorganic salt solution for 8 h and then in pure water for 12 h, to obtain a high-stability mPEG-EPO chemically modified silk fibroin solution.

[0088] Embodiment 6:

[0089] The embodiment provides a preparation method of methoxypolyethylene glycol epoxy chemically modified silk fibroin, and specific steps are as follows:

[0090] S1, low concentration hydrogen bond inhibition-pH adjustment combined enzymolysis:

[0091] Boil the mulberry silk in a Na2CO3 aqueous solution for 1 h, and dry to obtain degummed silk; the degummed silk is put into a 9.3 M lithium bromide (LiBr) solution to be dissolved, and a regenerated silk fibroin solution is obtained through purification;

[0092] The solution of the regenerated silk fibroin was diluted to 6 wt%, a NaSCN solution with a final concentration of 0.1 M was added, the pH was adjusted to 8.0 by adding acetate buffer, then trypsin was added, the final concentration of trypsin was 0.05 wt%, and the solution was incubated at 37°C for 48 h; then the enzyme was inactivated by heating in a boiling water bath for 10 min.

[0093] S2, collection of the precipitate: the inactivated enzyme solution was centrifuged at 7000 rpm for 5 min, the supernatant was discarded, and the precipitate was collected and washed with water and methanol to complete the collection of the silk fibroin solid.

[0094] S3, activation of the inorganic salt solution-methoxy polyethylene glycol epoxy cooperative chemical modification of silk fibroin: the silk fibroin solid collected in S2 was first activated with a CaBr2 inorganic salt solution, the final concentration of the inorganic salt solution was 15 M, the mass ratio of the inorganic salt solution to the silk fibroin was 20:1, mPEG-EPO with an average molecular weight of 8 kDa was added to the solution after activation, the mass ratio of mPEG-EPO to silk fibroin was 1:100, and the solution was incubated at 30°C for 24 h; the incubated solution was loaded into a dialysis bag with a molecular weight cut-off of 20 kDa, and was first purified in a CaBr2 inorganic salt solution for 6 h and then in pure water for 12 h to obtain a stable mPEG-EPO chemically modified silk fibroin solution.

[0095] Comparative Example 1:

[0096] This comparative example provides a preparation method of methoxy polyethylene glycol epoxy chemically modified silk fibroin, which is similar to Example 1, except that in step S1, no PBS buffer is added, and the rest of the steps are consistent with Example 1, and the pH of the obtained solution is > 9.

[0097] Comparative Example 2:

[0098] This comparative example provides a preparation method of methoxy polyethylene glycol epoxy chemically modified silk fibroin, which is similar to Example 1, except that in step S1, no hydrogen bond inhibitor (0.8 M lithium bromide) is added, and the rest of the steps are consistent with Example 1.

[0099] Comparative Example 3:

[0100] This comparative example provides a preparation method of methoxy polyethylene glycol epoxy chemically modified silk fibroin, which is similar to Example 1, except that in step S1, a high concentration of hydrogen bond inhibitor (9.3 M lithium bromide) is added, and the rest of the steps are consistent with Example 1.

[0101] Comparative Example 4:

[0102] The comparative example provides a preparation method of methoxypolyethylene glycol epoxy chemically modified silk fibroin, which is similar to example 1, except that in step S3, the KSCN inorganic salt solution is replaced with pure water, and the remaining steps are consistent with example 1.

[0103] Comparative example 5:

[0104] The comparative example provides a preparation method of silk fibroin, which is similar to example 1, except that in step S3, no mPEG-EPO is added, and the remaining steps are consistent with example 1. The obtained silk fibroin solution is flocculent precipitate, as shown in Figure 2

[0105] Comparative example 6:

[0106] The comparative example provides a preparation method of methoxypolyethylene glycol epoxy chemically modified traditional regenerated silk fibroin, and the specific steps are as follows:

[0107] The silk of Bombyx mori is boiled in a Na2CO3 aqueous solution for 1 h, and dried to obtain degummed silk. The degummed silk is dissolved in a 9.3 M lithium bromide (LiBr) solution, and a regenerated silk fibroin solution is obtained by purification;

[0108] The mPEG-EPO is added to the above-mentioned regenerated silk fibroin solution, the average molecular weight of the mPEG-EPO is 4 kDa, and the mass ratio of the mPEG-EPO to the silk fibroin is 3:100. The solution is incubated at 25°C for 12 h. The incubated solution is loaded into a dialysis bag with a molecular weight cut-off of 14 kDa, and purified in pure water for 24 h to obtain a polyethylene glycol chemically modified traditional regenerated silk fibroin solution.

[0109] Comparative example 7:

[0110] The comparative example provides a preparation method of polyethylene glycol 300 modified silk fibroin, which is similar to example 1, except that in step S3, polyethylene glycol 300 (PEG300) is used instead of mPEG-EPO, and the remaining steps are consistent with example 1.

[0111] Test example:

[0112] The samples obtained in the above examples and comparative examples are subjected to relevant tests, and the test items are as follows:

[0113] (1) Enzyme activity test: the residual activity percentage of the enzyme is tested by the enzyme catalytic substrate method: similar to step S1, no silk fibroin is added, and a hydrogen bond inhibitor, a pH adjuster and a protease are added for incubation. Samples are taken at different time points, and a substrate is added for catalytic reaction. The enzyme activity is quantified by ultraviolet spectroscopy method. The method is referred to the current Chinese Pharmacopoeia-Trypsin <Purity Determination>.

[0114] ​(2) Enzymolysis effect test: test the ratio of the sum of the contents of specific arginine (R) and lysine (K) in the precipitate after enzymolysis, and verify the effect of enzymolysis. The content of amino acid is determined by an amino acid analyzer. The calculation formula is:

[0115] Enzymolysis completeness = 1 - (R+K) ratio of total amino acid / 5.3‰ theoretical value

[0116] Note: silk fibroin contains 5263 amino acids, of which R and K are 28, and only at the two sides of the chain segment, so the theoretical ratio of R+K two kinds of amino acids to all amino acids before enzymolysis is 28 / 5263=5.3‰; trypsin is a specific protease, and its enzyme cutting point is R and K; after trypsin enzymolysis of silk fibroin, the collected silk fibroin precipitate has R and K only at the end (the ratio is almost zero), and the rest of the chain segment does not contain R and K, so the enzymolysis effect can be verified according to the ratio of the content of R+K two kinds of amino acids to the total amino acid content in the silk fibroin precipitate.

[0117] (3) Precipitate crystallinity test: XRD is used to test the crystallinity of the sample, and the test results are subjected to peak fitting of the crystalline and amorphous regions by Peakfit software, and the crystallinity is calculated by the normalized area method. By Figure 3 peak fitting of the XRD spectrum, it can be calculated that the crystallinity of Example 1 is higher than that of Comparative Examples 1 and 2.

[0118] (4) Property observation: during the preparation of the silk fibroin solution, the properties of the silk fibroin / solution are observed by naked eye or under colorless cool white light without background interference.

[0119] (5) Solution stability: after sterilization, the prepared silk fibroin solution sample is placed in a 4℃ refrigerator for cold storage, the time for the silk fibroin solution to form micelles / precipitate is observed, and the solution stable period is recorded.

[0120] (6) Success rate of methoxy polyethylene glycol epoxy chemical modification: high-speed gel chromatography is used to determine the content of methoxy polyethylene glycol epoxy in the purified liquid, and the success rate of chemical modification is calculated according to the amount of addition.

[0121] (7) Infrared spectroscopy is used to determine the silk fibroin after methoxy polyethylene glycol epoxy chemical modification, and qualitative judgment is made on whether the chemical grafting is successful. From Figure 4~5 it can be seen that after the silk fibroin is chemically modified by methoxy polyethylene glycol epoxy, the characteristic peaks of the silk fibroin β sheet at 1620 cm -1 and 1520 cm -1 are significantly weakened, and 2887 cm -1 is the methyl vibration peak in methoxy polyethylene glycol epoxy, and 1109 cm -1The peak at 1100 cm-1 is the characteristic peak of C-O-C ether skeleton vibration of methoxyl polyethylene glycol epoxy, which indicates that methoxyl polyethylene glycol epoxy is successfully grafted onto silk fibroin.

[0122] Table 1 is a test result of low concentration hydrogen bond inhibitor-pH adjustment combined enzymatic hydrolysis, as follows:

[0123] Table 1

[0124]

[0125] In Comparative Example 1, there is no pH adjuster in the enzymatic hydrolysis process. Although the low concentration of hydrogen bond inhibitor added can hinder the formation of β-pleated sheet, so that the silk fibroin chain can be more relaxed, thereby fully exposing the enzymatic hydrolysis site, and activating the enzyme itself, to a certain extent, the enzymatic hydrolysis efficiency is improved, but the pH environment (pH>9) of the incubation solution is not conducive to the long-term maintenance of enzyme activity, so after 2 hours of incubation, the percentage of residual enzyme activity is only about 50%, which is much lower than the about 90% in the examples. Under the condition of gradually decreasing enzyme activity, the final silk fibroin crystallinity obtained is lower than that in the examples, and the enzymatic hydrolysis completeness of 75% is lower than the nearly complete enzymatic hydrolysis in the examples, which further illustrates the importance of the pH environment to the long-term maintenance of enzyme activity. Due to the low crystallinity and incomplete enzymatic hydrolysis caused by enzyme inactivation, the target precipitated structure is not obtained, so no further test is carried out.

[0126] In Comparative Example 2, no hydrogen bond inhibitor is added in the enzymatic hydrolysis process. Although the pH adjuster added can maintain the activity of the enzyme, about 90% of the enzyme activity after 2 hours of incubation has no difference from that in the comparative example, but the lack of hydrogen bond inhibitor cannot effectively prevent the formation of intermolecular hydrogen bonds to a certain extent, which can not effectively relax the silk fibroin chain in a single chain form, thereby not fully exposing the enzymatic hydrolysis site; secondly, it cannot effectively activate the enzyme itself to improve the enzymatic hydrolysis efficiency, so the enzymatic hydrolysis completeness is only 79%, which is lower than the nearly complete enzymatic hydrolysis in the examples, and thus the crystallinity of the precipitate is lower than that in the examples, which cannot meet the target requirements.

[0127] In Comparative Example 3, a high concentration of hydrogen bond inhibitor is added in the enzymatic hydrolysis process. Although the enzyme activity and enzymatic hydrolysis completeness have no difference from those in the examples, the high concentration of hydrogen bond inhibitor causes the target fragment after enzymatic hydrolysis to be unable to form β-pleated sheet and precipitate. Therefore, the high concentration of hydrogen bond inhibitor cannot obtain the target precipitated layer.

[0128] Table 2 is a test result of inorganic salt solution activation-methoxyl polyethylene glycol epoxy synergistic chemical modification, as follows:

[0129] Table 2

[0130]

[0131] Comparative Example 4 does not add inorganic salt solution. The absence of inorganic salt results in the fibroin precipitated in pure water to exist in an unstable suspension, which cannot be effectively dissolved when adding methoxypolyethylene glycol epoxy, and it is impossible to expose active sites to form charge microzones. Not only is the success rate of chemical modification very low, but also the solution cannot be formed, which is not suitable for further use.

[0132] Comparative Example 5 does not add methoxypolyethylene glycol epoxy. The anions and cations in the inorganic salt solution can not only effectively destroy the intermolecular hydrogen bonds to form β-pleated sheets, and then the fibroin precipitate can be destroyed to form a solution by hydrogen bonds, but also the anions and cations can combine with the functional groups on the fibroin molecular chain to form active sites. However, since methoxypolyethylene glycol epoxy is not added, the active sites gradually disappear due to the dialysis of anions and cations during purification, and intermolecular hydrogen bonds again dominate, thereby producing flocculent precipitates and failing to form a stable solution for further use.

[0133] Comparative Example 6 uses a traditional regenerated fibroin solution to add methoxypolyethylene glycol epoxy for chemical modification. Since no enzymatic hydrolysis is performed, the structure is different from that of the precipitate after enzymatic hydrolysis, and thus the crystallinity is low. Moreover, since no inorganic salt solution is added, the fibroin peptide chain cannot fully expose the amino acid sites, and no anions / cations combine with the functional groups on the fibroin molecular chain to form positive / negative charge microzones. Therefore, when the added methoxypolyethylene glycol epoxy performs nucleophilic reaction, there are not enough sites, and the success rate of chemical modification is not high. In the case of a low success rate of chemical modification, firstly, the methoxypolyethylene glycol epoxy that is not successfully grafted is purified by dialysis, and there is not enough steric hindrance formed by polyethylene glycol, and secondly, the proportion of unmodified amino acids is higher, and during the storage of the solution, intermolecular hydrogen bonds gradually dominate again, resulting in a solution stability of only 3 days, which is much lower than the more than 30 days of the present embodiment.

[0134] Comparative Example 7 uses PEG300 for modification. PEG300 is different from methoxypolyethylene glycol epoxy, and there is no active site for chemical modification with fibroin. Its essence is physical blending with fibroin, and it is completely dialyzed out in the subsequent purification step. Therefore, the success rate of chemical modification is almost zero, intermolecular hydrogen bonds again dominate, and flocculent precipitates are produced, which cannot form a stable solution for further use.

[0135] Application Example 1

[0136] The stable mPEG-EPO chemically modified fibroin solution obtained in Example 1 is sterilized and filled into a spray bottle. By using the antibacterial property and film-forming property of fibroin, a liquid bandage is prepared, which is sprayed on the wound. After about 60 s, a dense film is quickly formed to repair and protect the wound. Figure 6 The stress-strain diagram of the obtained dense film is as follows:Figure 7 as shown.

[0137] Example 2:

[0138] The mPEG-EPO chemically modified silk fibroin solution obtained in Examples 1-6 was left to stand naturally to form a film in a petri dish, and the tensile breaking strength and elongation at break were tested using a TST-01M intelligent electronic tensile tester at 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 3:

[0139] Table 3

[0140]

[0141] Obviously, the above examples are merely illustrative and are not intended to limit the embodiments. Based on the above description, other different forms of changes or variations can be made by those of ordinary skill in the art. It is not necessary or possible to exhaust all embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for preparing methoxylated polyethylene glycol epoxidized silk fibroin, characterized in that, Includes the following steps: S1. Add hydrogen bond inhibitors and pH adjusters to the regenerated silk fibroin solution to a final concentration of 0.01 M-3 M, mix, then add trypsin for incubation to inactivate, separate the solid phase and obtain silk fibroin solid. S2. The silk fibroin solid obtained in step S1 is mixed with an inorganic salt solution for activation. After activation, it is co-incubated with methoxy polyethylene glycol epoxy and purified to obtain a methoxy polyethylene glycol epoxy modified silk fibroin solution. In step S1, the regenerated silk fibroin is obtained by degumming, dissolving, and purifying silk. In step S1, the hydrogen bond inhibitor is one or more of CaCl2 / ethanol / water, LiBr, NaSCN, and ZnCl2; In step S1, the pH adjuster adjusts the pH of the regenerated silk fibroin solution to 6-9; In step S1, the mass ratio of trypsin to regenerated silk fibroin in the regenerated silk fibroin solution is 1:5-1:500; the incubation conditions are: 25℃-37℃ for 24 h-48 h. In step S2, the mass ratio of the methoxy polyethylene glycol epoxy to the silk fibroin solid is 1:200-1:1; the incubation conditions are: 4℃-80℃ for 0.5 h-72 h.

2. The preparation method according to claim 1, characterized in that, In step S1, the pH adjuster is one or more of Tris-HCl buffer, HEPES buffer, PBS buffer, HBSS buffer, MOPS buffer, and acetate buffer.

3. The preparation method according to claim 1, characterized in that, In step S1, the final concentration of the added trypsin is 0.01 wt%-0.3 wt%.

4. The preparation method according to claim 1, characterized in that, In step S2, the cation in the inorganic salt solution is Na. + Mg 2+ Cu 2+ Zn 2+ Li + K + Fe 3+ and Ca 2+ One or more of the following; the anion in the inorganic salt solution is Cl. - SO4 2- ,Br - SCN - CO3 2- and OH - One or more of them.

5. The preparation method according to claim 1, characterized in that, In step S2, the concentration of the inorganic salt solution is 4M-15M.

6. The preparation method according to claim 1, characterized in that, In step S2, the mass ratio of the inorganic salt solution to the silk fibroin solid is 40:1 to 1:

1.

7. The preparation method according to claim 1, characterized in that, In step S2, the molecular weight of the methoxy polyethylene glycol epoxy is 1 kDa-100 kDa.

8. The methoxylated polyethylene glycol epoxidized silk fibroin obtained by the preparation method of any one of claims 1-7.

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