Methoxy polyethylene glycol epoxidation modified silk fibroin and preparation method thereof

Abstract

The invention relates to methoxy polyethylene glycol epoxidation modified silk fibroin and a preparation method thereof, and belongs to the technical field of biological extraction. The method comprises the following steps: adding a low-concentration hydrogen bond inhibitor and a pH regulator into a regenerated silk fibroin solution, mixing, adding trypsin, incubating, inactivating, carrying out solid-liquid separation, and taking a solid phase to obtain a silk fibroin solid; then mixing and activating with an inorganic salt solution, performing epoxy co-incubation with methoxy polyethylene glycol after activation, and performing purification treatment to obtain a chemically grafted silk fibroin solution. The invention provides a low-concentration hydrogen bond inhibition-pH regulation combined enzymolysis technology, a dissolution modification technology of a precipitation layer after enzymolysis and a technical scheme of inorganic salt solution activation / methoxy polyethylene glycol epoxy synergistic chemical modification for the first time, so that the problems that the proteolysis effect is low and the enzymatic activity cannot be maintained for a long time are solved; and the precipitation layer is successfully prepared into a stable solution, so that the success rate of chemical modification of the silk fibroin is greatly improved, and subsequent further development and use are facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of biological extraction, in particular to methoxypolyethylene glycol epoxide chemically modified silk fibroin and a preparation method thereof. Background Art

[0002] Mulberry silk is primarily composed of fibroin (approximately 70% by weight) and sericin (approximately 25%). Natural mulberry silk, due to numerous hydrogen bonds between molecular chains within the silkworm's glands, forms regular, hydrophobic, and high-content β-pleated regions, resulting in the silk being swellable but not dissolving in water and most organic solvents. To prepare spinning solutions and other silk-based materials with various properties, the silk must be dissolved to produce a large amount of silk fibroin solution. Conventional silk fibroin raw materials are obtained by generating regenerated silk fibroin solutions through a multi-step process, including silk degumming, dissolution, dialysis, purification, and concentration. Currently, the main silk fiber dissolution systems used to prepare regenerated silk fibroin solutions include inorganic salt systems, organic solvent systems, and organic salt systems. Their essence lies in disrupting existing hydrogen bonds or inhibiting the formation of hydrogen bonds between molecular chains, thereby unraveling the β-pleated structure of silk fibroin, leaving the molecular chains in a random coiled conformation and in solution.

[0003] There are two main methods for enzymatically hydrolyzing silk fibroin solutions. Method 1: Completely hydrolyze the silk fibroin to obtain low-molecular-weight polypeptides in the supernatant, which are then used to prepare small-molecule polypeptide silk fibroin products or to study their molecular weight, amino acid species and content, and amino acid sequence. The primary structure of the enzymatically hydrolyzed silk fibroin is a low-molecular-weight polypeptide fragment, containing almost no GAGAGS sequences. Its secondary structure also lacks β-sheet conformation and crystallinity. Regardless of subsequent modification, the high molecular weight and excellent mechanical properties of β-sheet structure are out of the question. Method 2: Partially hydrolyze the silk fibroin to form a supernatant layer (primarily an amorphous region) and a precipitate layer (primarily a crystalline region). The amino acid species and content, as well as changes in β-sheet content, in the different layers are then studied. This method focuses on chemical analysis and testing and is not relevant to the preparation of silk fibroin raw materials.

[0004] Traditional methods for dissolving silk fibers, such as lithium bromide inorganic salt systems, can dissolve the precipitate layer, but after dialysis of lithium bromide, the solution will continue to aggregate to form a precipitate, which limits its application, not to mention the subsequent purification and modification steps. At present, the method of using polyethylene glycol to increase water solubility is limited to regenerated silk fibroin solution, with low modification efficiency and low modification success rate. Lu Shenzhou et al. used polyethylene glycol glycidyl ether to modify silk fibroin membrane (Lu Shenzhou, Li Mingzhong, Liu Yang, et al. Modification of silk fibroin membrane with polyethylene glycol glycidyl ether [J]. Polymer Materials Science and Engineering, 2003, 19(1): 104-107): first, polyethylene glycol was reacted with epichlorohydrin to form polyethylene glycol glycidyl ether, and then the silk fibroin solution and polyethylene glycol glycidyl ether were mixed and dried to form a membrane. This method changed the structure of the silk fibroin membrane, reduced its dissolution rate, and improved both tensile strength and elongation at break. Based on the above research, Yang Hua et al. further characterized the structure and properties of the blended membrane after modification of silk fibroin (Yang Hua, Nie Tao, Cai Hui, et al. Study on modified silk fibroin with polyethylene glycol glycidyl ether [J]. Chemical Technology, 2013, 21(5):4-8). However, both of these papers modified the membrane from the perspective of blending polyethylene glycol glycidyl ether with regenerated silk fibroin solution. Not only was the modification efficiency low, the modification success rate was also low, and polyethylene glycol glycidyl ether was synthesized, which increased the complexity of the process and the uncertainty of reagent residues. Patent application number CN202410550980 discloses a method for preparing silk fibroin microspheres. One step involves adding methoxypolyethylene glycol active ester to a regenerated silk fibroin solution and introducing polyethylene glycol onto amino residues in the silk fibroin molecules to obtain methoxypolyethylene glycol active ester-modified silk fibroin. This method achieves better water solubility than pure regenerated silk fibroin and reduces aggregation and precipitation of the prepared microspheres. However, this method, which modifies regenerated silk fibroin, suffers from low modification efficiency and a low success rate for chemical modification.

[0005] From the above, it can be seen that there is no report in the prior art on the feasibility of increasing the water solubility and high chemical modification rate of the precipitation layer after silk fibroin enzymatic hydrolysis. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides methoxy polyethylene glycol epoxide chemically modified silk fibroin and its preparation method. Traditional silk fiber dissolution technology is limited to conventional silk fibroin solution. Whether it is lithium bromide system or polyethylene glycol derivative modification, it has only been practiced separately on silk fibroin solution. Different from conventional regenerated silk fibroin solution, the precipitate layer structure after enzymatic hydrolysis of the present invention is more regular, with high crystallinity, high β-folding amount, and a higher relative proportion of GAGAGS in the primary structure, but its water solubility is worse, and the active sites cannot be fully exposed, thereby limiting its subsequent use. Conventional single dissolution system cannot solve the problem of agglomeration and re-precipitation caused by dialysis after the precipitation layer is dissolved, and the modification success rate of a single modification system is not high. The present invention proposes for the first time a technical solution combining low-concentration hydrogen bond inhibition-pH regulation enzymatic hydrolysis technology, a dissolution modification technology of the precipitate layer after enzymatic hydrolysis, and an inorganic salt solution activation / methoxypolyethylene glycol epoxy synergistic modification technology. It not only solves the problems of low proteolysis effect and inability to maintain enzyme activity for a long time, but also successfully prepares the precipitate layer into a stable solution, greatly improving the success rate of silk fibroin modification, broadening its application range, and facilitating subsequent further development and use.

[0007] The present invention is achieved through the following technical solutions:

[0008] The first object of the present invention is to provide a method for preparing methoxypolyethylene glycol epoxide chemically modified silk fibroin, comprising the following steps:

[0009] S1. Adding a hydrogen bond inhibitor and a pH regulator with a final concentration of 0.01 M to 3 M to the regenerated silk fibroin solution, mixing, then adding trypsin to incubate and inactivate, and performing solid-liquid separation to obtain a solid phase to obtain a silk fibroin solid; the obtained silk fibroin solid has high crystallinity and high rigidity;

[0010] S2. The silk fibroin solid obtained in step S1 is mixed with an inorganic salt solution for activation, and after activation, it is co-incubated with methoxy polyethylene glycol epoxide, and purified to obtain a methoxy polyethylene glycol epoxide chemically modified silk fibroin solution.

[0011] Furthermore, 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] Furthermore, in step S1, the hydrogen bond inhibitor is one or more of CaCl2 / ethanol / water, LiBr, NaSCN and ZnCl2.

[0014] Furthermore, 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] And / or, the pH regulator adjusts the pH value of the regenerated silk fibroin solution to 6-9.

[0016] Furthermore, in step S1, the final concentration of trypsin added is 0.01 wt%-0.3 wt%.

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

[0018] And / or, the incubation condition is: incubation at 25° C.-37° C. for 24 h-48 h.

[0019] Furthermore, in step S1, the solid-liquid separation is performed by centrifugation; after the solid-liquid separation, a washing step is further included; the washing includes water washing and alcohol washing, and the alcohol includes methanol, ethanol, aromatic alcohol, etc.

[0020] Furthermore, in step S2, the silk fibroin solid is first activated with an inorganic salt solution to destroy the β-pleated hydrogen bond network to promote dissolution and fully expose the amino acid sites. The anions / cations combine with the functional groups on the silk fibroin molecular chain to form positive and negative charge micro-regions; the methoxy polyethylene glycol epoxy added subsequently reacts nucleophilically with the anions and cations under the activation of the anions and cations, thereby efficiently completing the chemical modification.

[0021] Furthermore, 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 anion in the inorganic salt solution is Cl - 、SO4 2- Br - 、SCN - 、CO3 2- and OH - One or more of;

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

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

[0024] Furthermore, the molecular weight of the methoxy polyethylene glycol epoxy is 1 kDa-100 kDa.

[0025] Furthermore, in step S2, the mass ratio of the methoxy polyethylene glycol epoxy to the silk fibroin solid is 1:200-1:1;

[0026] And / or, the incubation conditions are: incubation at 4°C-80°C for 0.5 h-72 h, preferably incubation at 25°C-40°C for 4 h-48 h;

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

[0028] The second object of the present invention is to provide methoxy polyethylene glycol epoxide chemically modified silk fibroin obtained by the preparation method.

[0029] The mechanism of the present invention is:

[0030] Low-concentration hydrogen bond inhibition-pH adjustment combined enzymatic hydrolysis: first add a low-concentration hydrogen bond inhibitor and a pH adjuster to adjust an environment that is conducive to the long-term maintenance of trypsin activity, and then add trypsin for incubation to obtain a precipitate with high crystallinity and high rigidity, which is essentially different from traditional silk fibroin in primary and secondary structures.

[0031] Inorganic salt solution activation-methoxy polyethylene glycol epoxy modification synergistic chemical modification: Because the precipitated part has worse water solubility, the silk fibroin precipitate is first activated and dissolved with an inorganic salt solution to expose the active sites and form charge micro-regions. Then methoxy polyethylene glycol epoxy is added for nucleophilic reaction, thereby forming a methoxy polyethylene glycol epoxy chemically modified silk fibroin solution with a high chemical modification success rate and more stability.

[0032] The above technical solution of the present invention has the following advantages over the prior art:

[0033] (1) Better structure. There is an essential difference between the traditional regenerated silk fibroin solution and the precipitate formed after trypsin hydrolysis of the present invention. The primary structure of the protein has changed. The G / A / S ratio of the precipitate after enzymatic hydrolysis is high, the crystallinity and rigidity are large, and the amino acid sequence is more regular and orderly. Therefore, the mechanical properties are better than those of the traditional regenerated silk fibroin solution.

[0034] (2) Maintaining enzymatic hydrolysis efficiency and enzyme activity for a long time. The enzymatic hydrolysis is carried out by low-concentration hydrogen bond inhibition-pH adjustment, which allows the silk protein chain to stretch more and expose the enzymatic hydrolysis site. At the same time, it can activate the enzyme itself, improve the enzymatic hydrolysis effect, and enable the enzyme to maintain its own activity for a long time.

[0035] (3) Higher success rate of chemical modification. The present invention adopts the method of inorganic salt solution activation-methoxy polyethylene glycol epoxy synergistic chemical modification. First, the silk fibroin is dissolved and activated with an inorganic salt solution, which destroys the hydrogen bonds to promote dissolution and fully unfolds the molecular chain to fully expose the amino acid sites. At the same time, it combines with the functional groups on the chain to form charge micro-regions, which is beneficial to the nucleophilic reaction of methoxy polyethylene glycol epoxy, greatly improving the success rate of chemical modification.

[0036] (4) The solution is more stable. The precipitate after silk fibroin enzymatic hydrolysis has a regular structure and poor water solubility. If only inorganic salt solution is added without methoxy polyethylene glycol epoxy, the solution will precipitate again after purification; if only methoxy polyethylene glycol epoxy is added without inorganic salt solution, the solution will be basically insoluble; under the synergistic effect of inorganic salt solution activation and 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 methoxy polyethylene glycol epoxy on traditional regenerated silk fibroin. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0038] Figure 1 The highly stable methoxy polyethylene glycol epoxy chemically modified silk fibroin solution formed in Example 1 of the present invention;

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

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

[0041] Figure 4 This is the infrared spectrum of silk fibroin before chemical modification with methoxy polyethylene glycol epoxide in Example 1;

[0042] Figure 5 This is the infrared spectrum of silk fibroin chemically modified with methoxy polyethylene glycol epoxide in Example 1;

[0043] Figure 6 This is a diagram showing the application of the methoxypolyethylene glycol epoxide chemically modified silk fibroin solution obtained in Example 1 to prepare a liquid wound dressing;

[0044] Figure 7 This is a stress-strain diagram of the methoxy polyethylene glycol epoxide chemically modified silk fibroin solution obtained in Example 1 after being allowed to stand to form a film. DETAILED DESCRIPTION

[0045] 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 the present invention and implement it. However, the embodiments are not intended to limit the present invention.

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

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

[0048] NaSCN was purchased from Jinan Huifengda with the product number HFD-248;

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

[0050] Ethanol was purchased from Bio-Lab with the catalog number L11024-VZU; ZnCl2 was purchased from Sinopharm with the catalog number H1L0491; PBS buffer was purchased from Bianzhen Biological with the catalog number bzw2106f; Tris-HCl buffer was purchased from Solebao with the catalog number T1160; HBSS buffer was purchased from Kanglang Biological with the catalog number KL-13487; MOPS buffer was purchased from Yuduo Biological with the catalog number LG-NA0016; acetate buffer was purchased from Xiens with the catalog number T-317015; trypsin was purchased from Aladdin with the catalog number T274333; KSCN inorganic salt was purchased from Naphthalene Biochemical with the catalog number NCS1453117; Fe Cl3 inorganic salt was purchased from Yuanmu Biological, product number is YM-0181; Zn SO4 inorganic salt was purchased from Naphthalene Biochemical with the product number NCS187378; NaCO3 inorganic salt was purchased from Baishengyue Biological with the product number S291934; LiCl inorganic salt was purchased from Yubo Biological with the product number YBP123954;

[0051] CaBr2 was purchased from Solebro, product number CB14717714;

[0052] mPEG-EPO was purchased from Carbon Technology with the catalog number 80010121.

[0053] Example 1:

[0054] This embodiment provides a method for preparing methoxy polyethylene glycol epoxide chemically modified silk fibroin, the specific steps are as follows:

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

[0056] Mulberry silk was boiled in a Na2CO3 aqueous solution for 1 h and dried to obtain degummed silk. The degummed silk was then dissolved in a 9.3 M lithium bromide (LiBr) solution and purified to obtain a regenerated silk fibroin solution.

[0057] The silk fibroin solution was diluted to 0.5 wt %, lithium bromide was added to a final concentration of 0.8 M, PBS buffer was added to adjust the pH to 8.0, and then trypsin was added to a final concentration of 0.1 wt %. The mixture was incubated at 37°C for 24 h; then the enzyme was inactivated by heating in a boiling water bath for 10 min.

[0058] S2. Collection of precipitate: centrifuge the inactivated enzymatic hydrolysate at 5000 rpm for 5 min, discard the supernatant, collect the precipitate, and wash it 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 was first activated with KSCN inorganic salt solution, the final concentration of the inorganic salt solution was 10 M, the mass ratio of the inorganic salt solution to the silk fibroin was 1:1, mPEG-EPO was added to the activated solution, the average molecular weight of mPEG-EPO was 4 kDa, the mass ratio of mPEG-EPO to silk fibroin was 3:100, and the solution was incubated at 25°C for 12 h; the incubated solution was placed in a dialysis bag with a molecular weight cutoff of 14 kDa and purified in pure water for 24 h to obtain a highly stable mPEG-EPO chemically modified silk fibroin solution (such as Figure 1 shown).

[0060] Example 2:

[0061] This embodiment provides a method for preparing methoxy polyethylene glycol epoxide chemically modified silk fibroin, the specific steps are as follows:

[0062] S1. Low concentration hydrogen bond inhibition-pH adjustment combined with enzymatic hydrolysis:

[0063] Mulberry silk was boiled in a Na2CO3 aqueous solution for 1 h and dried to obtain degummed silk. The degummed silk was then dissolved in a 9.3 M lithium bromide (LiBr) solution and purified to obtain a regenerated silk fibroin solution.

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

[0065] S2. Collection of precipitate: The inactivated enzymatic hydrolysate was centrifuged at 3000 rpm for 10 min, the supernatant was discarded, the precipitate was collected, and washed with water and methanol to complete the collection of the silk fibroin solid.

[0066] S3, inorganic salt solution activation - methoxy polyethylene glycol epoxy synergistic chemical modification of silk fibroin: the silk fibroin solid collected in S2 was first The activated solution was activated with Cl3 inorganic salt solution, the final concentration of the inorganic salt solution was 4 M, the mass ratio of the inorganic salt solution to the silk fibroin was 5:1, mPEG-EPO was added to the activated solution, the average molecular weight of mPEG-EPO was 1 kDa, and the mass ratio of mPEG-EPO to the silk fibroin was 1:200. The solution was incubated at 40°C for 4 h. The incubated solution was placed in a dialysis bag with a molecular weight cutoff of 10 kDa and purified in pure water for 48 h to obtain a highly stable mPEG-EPO chemically modified silk fibroin solution.

[0067] Example 3:

[0068] This embodiment provides a method for preparing methoxy polyethylene glycol epoxide chemically modified silk fibroin, the specific steps are as follows:

[0069] S1. Low concentration hydrogen bond inhibition-pH adjustment combined with enzymatic hydrolysis:

[0070] Mulberry silk was boiled in a Na2CO3 aqueous solution for 1 h and dried to obtain degummed silk. The degummed silk was then dissolved in a 9.3 M lithium bromide (LiBr) solution and purified to obtain a regenerated silk fibroin solution.

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

[0072] S2. Collection of precipitate: centrifuge the inactivated enzymatic hydrolysate at 6000 rpm for 5 min, discard the supernatant, collect the precipitate, and wash it with water and ethanol to complete the collection of silk fibroin solid.

[0073] S3, inorganic salt solution activation - methoxy polyethylene glycol epoxy synergistic chemical modification of silk fibroin: the silk fibroin solid collected in S2 was first treated with Zn The activated solution was activated with SO4 inorganic salt solution, the final concentration of the inorganic salt solution was 6 M, the mass ratio of the inorganic salt solution to the silk fibroin was 10:1, mPEG-EPO was added to the activated solution, the average molecular weight of mPEG-EPO was 10 kDa, the mass ratio of mPEG-EPO to the silk fibroin was 1:1, and the solution was incubated at 37°C for 48 h. The incubated solution was placed in a dialysis bag with a molecular weight cutoff of 80 kDa and purified in pure water for 36 h to obtain a highly stable mPEG-EPO chemically modified silk fibroin solution.

[0074] Example 4:

[0075] This embodiment provides a method for preparing methoxy polyethylene glycol epoxide chemically modified silk fibroin, the specific steps are as follows:

[0076] S1. Low concentration hydrogen bond inhibition-pH adjustment combined with enzymatic hydrolysis:

[0077] Mulberry silk was boiled in a Na2CO3 aqueous solution for 1 h and dried to obtain degummed silk. The degummed silk was then dissolved in a 9.3 M lithium bromide (LiBr) solution and purified to obtain a regenerated silk fibroin solution.

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

[0079] S2. Collection of precipitate: The inactivated enzymatic hydrolysate was centrifuged at 5000 rpm for 5 min, the supernatant was discarded, the precipitate was collected, and washed with water and benzyl alcohol to complete the collection of the silk fibroin solid.

[0080] S3. Inorganic salt solution activation - methoxy polyethylene glycol epoxy synergistic chemical modification of silk fibroin: The silk fibroin solid collected in S2 above was first activated with Na2CO3 inorganic salt solution. The final concentration of the inorganic salt solution was 10 M, and the mass ratio of the inorganic salt solution to the silk fibroin was 20:1. mPEG-EPO was added to the activated solution. The average molecular weight of mPEG-EPO was 20 kDa, and the mass ratio of mPEG-EPO to silk fibroin was 1:100. The solution was incubated at 37°C for 12 h. The incubated solution was placed in a dialysis bag with a molecular weight cutoff of 50 kDa and purified in pure water for 18 h to obtain a highly stable mPEG-EPO chemically modified silk fibroin solution.

[0081] Example 5:

[0082] This embodiment provides a method for preparing methoxy polyethylene glycol epoxide chemically modified silk fibroin, the specific steps are as follows:

[0083] S1. Low concentration hydrogen bond inhibition-pH adjustment combined with enzymatic hydrolysis:

[0084] Mulberry silk was boiled in a Na2CO3 aqueous solution for 1 h and dried to obtain degummed silk. The degummed silk was then dissolved in a 9.3 M lithium bromide (LiBr) solution and purified to obtain a regenerated silk fibroin solution.

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

[0086] S2. Collection of precipitate: centrifuge the inactivated enzymatic hydrolysate at 4000 rpm for 10 min, discard the supernatant, collect the precipitate, and wash it with water and ethanol to complete the collection of silk fibroin solid.

[0087] S3. Inorganic salt solution activation - methoxy polyethylene glycol epoxy synergistic chemical modification of silk fibroin: The silk fibroin solid collected in S2 above was first activated with LiCl inorganic salt solution. The final concentration of the inorganic salt solution was 9.3 M, and the mass ratio of the inorganic salt solution to the silk fibroin was 40:1. mPEG-EPO was added to the activated solution. The average molecular weight of mPEG-EPO was 6 kDa, and the mass ratio of mPEG-EPO to silk fibroin was 1:150. The solution was incubated at 40°C for 8 h. The incubated solution was placed in a dialysis bag with a molecular weight cutoff of 50 kDa, first purified in LiCl inorganic salt solution for 8 h, and then purified in pure water for 12 h to obtain a highly stable mPEG-EPO chemically modified silk fibroin solution.

[0088] Example 6:

[0089] This embodiment provides a method for preparing methoxy polyethylene glycol epoxide chemically modified silk fibroin, the specific steps are as follows:

[0090] S1. Low concentration hydrogen bond inhibition-pH adjustment combined with enzymatic hydrolysis:

[0091] Mulberry silk was boiled in a Na2CO3 aqueous solution for 1 h and dried to obtain degummed silk. The degummed silk was then dissolved in a 9.3 M lithium bromide (LiBr) solution and purified to obtain a regenerated silk fibroin solution.

[0092] The silk fibroin solution was diluted to 6 wt %, and a NaSCN solution with a final concentration of 0.1 M was added. The pH was adjusted to 8.0 by adding acetate buffer, and then trypsin was added with a final concentration of 0.05 wt %. The mixture 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 precipitate: The inactivated enzymatic hydrolysate was centrifuged at 7000 rpm for 5 min, the supernatant was discarded, the precipitate was collected, and washed with water and methanol to complete the collection of the silk fibroin solid.

[0094] S3. Inorganic salt solution activation - methoxy polyethylene glycol epoxy synergistic chemical modification of silk fibroin: The silk fibroin solid collected in the above S2 was first activated with CaBr2 inorganic salt solution. The final concentration of the inorganic salt solution was 15 M, and the mass ratio of the inorganic salt solution to the silk fibroin was 20:1. mPEG-EPO was added to the activated solution. The average molecular weight of mPEG-EPO was 8 kDa, and the mass ratio of mPEG-EPO to silk fibroin was 1:100. The solution was incubated at 30°C for 24 h. The incubated solution was placed in a dialysis bag with a molecular weight cutoff of 20 kDa, first purified in CaBr2 inorganic salt solution for 6 h, and then purified 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 methoxypolyethylene glycol epoxide chemically modified silk fibroin, which is similar to Example 1, except that: in step S1, PBS buffer is not added, and the remaining steps are consistent with Example 1, and the pH value of the resulting solution is >9.

[0097] Comparative Example 2:

[0098] This comparative example provides a method for preparing methoxypolyethylene glycol epoxide 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 remaining steps are consistent with Example 1.

[0099] Comparative Example 3:

[0100] This comparative example provides a method for preparing methoxypolyethylene 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 remaining steps are consistent with Example 1.

[0101] Comparative Example 4:

[0102] This comparative example provides a method for preparing methoxypolyethylene glycol epoxide chemically modified silk fibroin, which is similar to Example 1, except that: in step S3, pure water is used to replace the KSCN inorganic salt solution, and the remaining steps are consistent with Example 1.

[0103] Comparative Example 5:

[0104] This comparative example provides a method for preparing silk fibroin, which is similar to Example 1, except that mPEG-EPO is not added in step S3, and the remaining steps are the same as Example 1. The obtained silk fibroin solution is a flocculent precipitate, such as Figure 2 shown.

[0105] Comparative Example 6:

[0106] This comparative example provides a method for preparing a traditional regenerated silk fibroin chemically modified with methoxy polyethylene glycol epoxide, and the specific steps are as follows:

[0107] Mulberry silk was boiled in a Na2CO3 aqueous solution for 1 h and dried to obtain degummed silk. The degummed silk was then dissolved in a 9.3 M lithium bromide (LiBr) solution and purified to obtain a regenerated silk fibroin solution.

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

[0109] Comparative Example 7:

[0110] This 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 from the above examples and comparative examples were subjected to relevant tests, and the test items were as follows:

[0113] (1) Enzyme activity test: The enzyme catalytic substrate method was used to test the percentage of residual enzyme activity: similar to step S1, without adding silk fibroin, hydrogen bond inhibitor, pH regulator and protease were added for incubation, samples were taken at different time points, substrate was added for catalytic reaction, and enzyme activity was quantified by ultraviolet spectroscopy. The method was based on the current Chinese Pharmacopoeia - Trypsin <Potency Determination>.

[0114] (2) Enzymatic hydrolysis effect test: The ratio of the sum of the specific arginine (R) and lysine (K) amino acid contents in the precipitate after enzymatic hydrolysis was tested to verify the effect of enzymatic hydrolysis. The amino acid content was measured using an amino acid analyzer. The calculation formula is:

[0115] Completeness of enzymatic hydrolysis = 1-(R+K) ratio of total amino acids / theoretical value of 5.3‰

[0116] Note: Silk fibroin contains 5263 amino acids, of which R and K have 28 and are only located on both sides of the chain segment. Therefore, the theoretical ratio of R+K amino acids to all amino acids before enzymatic hydrolysis is 28 / 5263=5.3‰; trypsin is a specific protease, and its cleavage sites are R and K; after trypsin cleavage of silk fibroin, the precipitated silk fibroin segments collected only have R and K at the end (the ratio is almost zero), and the rest of the segments do not contain R and K. Therefore, the enzymatic hydrolysis effect can be verified based on the ratio of the content of R+K amino acids in the silk fibroin precipitate to the total amino acid content.

[0117] (3) Precipitation crystallinity test: XRD was used to test the crystallinity of the sample. The test results were fitted with Peakfit software for the peaks of the crystalline and amorphous regions. The crystallinity was calculated using the normalized area method. Figure 3 By performing peak fitting on 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, observe the properties of the silk fibroin / solution with the naked eye or under colorless cold white light without background interference.

[0119] (5) Solution stability: After sterilizing the prepared silk fibroin solution sample, place it in a refrigerator at 4°C, observe the time it takes for the silk fibroin solution to form micelles / produce precipitation, and record the solution stability period.

[0120] (6) Success rate of chemical modification of methoxy polyethylene glycol epoxide: The methoxy polyethylene glycol epoxide content in the purified solution was determined by high-speed gel chromatography, and the success rate of chemical modification was calculated based on the added amount.

[0121] (7) Infrared spectroscopy was used to determine the chemical grafting success of silk fibroin after chemical modification with methoxy polyethylene glycol epoxide. Figures 4 and 5 It can be seen that after the methoxy polyethylene glycol epoxide chemically modified silk fibroin, the characteristic peak of silk fibroin β folding at 1620 cm -1 and 1520cm -1 Significantly weakened, 2887cm -1 The peak at 1109 cm is the methyl vibration peak in the methoxy polyethylene glycol epoxy group. -1The peaks are characteristic skeleton vibration peaks of COC ethers in methoxy polyethylene glycol epoxy, indicating that methoxy polyethylene glycol epoxy was successfully chemically grafted onto silk fibroin.

[0122] Table 1 shows the test results of low-concentration hydrogen bond inhibition-pH adjustment combined enzymatic hydrolysis, as follows:

[0123] Table 1

[0124]

[0125] In Comparative Example 1, no pH regulator was used during the enzymatic hydrolysis process. Although the low concentration of hydrogen bond inhibitor added can hinder the formation of β-sheets, allowing the silk fibroin chains to stretch more, thereby fully exposing the enzymatic hydrolysis sites and activating the enzyme itself, which improves the enzymatic hydrolysis efficiency to a certain extent, the pH environment of the incubation solution (pH>9) is not conducive to the long-term maintenance of enzyme activity. Therefore, after 2 hours of incubation, the residual activity percentage of the enzyme is only about 50%, which is far lower than the approximately 90% in the examples. As the enzyme activity gradually decreases, the crystallinity of the silk fibroin finally obtained is lower than that of the examples. At the same time, the enzymatic hydrolysis completeness of 75% is lower than the nearly complete enzymatic hydrolysis in the examples, further illustrating the importance of the pH environment for long-term maintenance of enzyme activity. Due to the low crystallinity and incomplete enzymatic hydrolysis caused by enzyme inactivation, the target precipitate structure was not obtained, so no further testing was performed.

[0126] Comparative Example 2 did not add a hydrogen bond inhibitor during the enzymatic hydrolysis process. Although the addition of a pH adjuster can maintain enzyme activity, and the enzyme activity after 2 hours of incubation is about 90%, which is the same as that of the comparative example, the lack of a hydrogen bond inhibitor cannot effectively prevent the formation of intermolecular hydrogen bonds to a certain extent. Firstly, the silk fibroin chains cannot stretch out in a single chain form, thereby not fully exposing the enzymatic hydrolysis sites; secondly, it cannot effectively activate the enzyme itself and improve the enzymatic hydrolysis efficiency. As a result, the enzymatic hydrolysis completeness is only 79%, which is lower than the nearly complete enzymatic hydrolysis of the example. As a result, the crystallinity of the precipitate is lower than that of the example, and does not meet the target requirements.

[0127] Comparative Example 3 added a high concentration of a hydrogen bond inhibitor during the enzymatic hydrolysis process. While the enzyme activity and hydrolysis completeness were comparable to those in Example 1, the high concentration of the hydrogen bond inhibitor prevented the target fragment from forming a β-sheet after enzymatic hydrolysis, causing it to precipitate. Therefore, the high concentration of the hydrogen bond inhibitor prevented the target precipitate from forming.

[0128] Table 2 shows the test results of inorganic salt solution activation-methoxy polyethylene glycol epoxy synergistic chemical modification, as follows:

[0129] Table 2

[0130]

[0131] No inorganic salt solution was added in Comparative Example 4. The absence of inorganic salt resulted in the silk fibroin precipitate existing as an unstable suspension in pure water. This suspension was not effectively dissolved when methoxypolyethylene glycol epoxy was added, and it was even less likely to expose active sites to form charged microdomains. This resulted in a very low success rate for chemical modification and the inability to form a solution, making it unsuitable for subsequent use.

[0132] Methoxy polyethylene glycol epoxy was not added in Comparative Example 5. The anions and cations in the inorganic salt solution can not only effectively destroy the β-folding formed by intermolecular hydrogen bonds, thereby breaking the hydrogen bonds of the silk fibroin precipitate to form a solution, but also the anions and cations can combine with the functional groups on the silk fibroin molecular chain to form active sites. However, due to the lack of methoxy polyethylene glycol epoxy, the active sites gradually disappeared during the purification process due to the cations and anions being dialyzed out, and the intermolecular hydrogen bonds once again became dominant, resulting in the formation of flocculent precipitation, and the inability to form a stable solution for further use.

[0133] Comparative Example 6 uses a traditional regenerated silk fibroin solution to add methoxy polyethylene glycol epoxy for chemical modification. Since no enzymatic hydrolysis is performed, its structure is different from the precipitate after enzymatic cleavage, and thus the crystallinity is low. Furthermore, since no inorganic salt solution is added, the silk fibroin peptide chain cannot fully expose the amino acid sites, no anion / cation binds to the functional groups on the silk fibroin molecular chain, and no positive and negative charge micro-regions can be formed. Therefore, the added methoxy polyethylene glycol epoxy lacks sufficient sites for nucleophilic reaction, and the success rate of chemical modification is not high. When the success rate of chemical modification is not high, firstly, the methoxy polyethylene glycol epoxy that has not been successfully chemically grafted is dialyzed and purified, lacking sufficient steric hindrance formed by polyethylene glycol, and secondly, the unmodified amino acid correlation ratio is higher. During the solution placement process, the intermolecular hydrogen bonds gradually take the dominant position 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 methoxy polyethylene glycol epoxy. It does not have active sites for chemical modification with silk fibroin. Its essence is physical blending with silk fibroin and is completely dialyzed out in the subsequent purification step. Therefore, the success rate of chemical modification is almost zero, and intermolecular hydrogen bonds dominate again, resulting in flocculent precipitation, which cannot form a stable solution for further use.

[0135] Application Example 1:

[0136] The stable mPEG-EPO chemically modified silk fibroin solution obtained in Example 1 was sterilized and placed in a spray bottle. The antibacterial and film-forming properties of silk fibroin were used to prepare a liquid bandage. The liquid bandage was sprayed on the wound. After about 60 seconds, a dense film quickly formed to repair and protect the wound. Figure 6 The stress-strain diagram of the obtained dense film is shown in FIG. Figure 7 shown.

[0137] Application Example 2:

[0138] The stable mPEG-EPO chemically modified silk fibroin solution obtained in Examples 1-6 was allowed to stand naturally in a watch glass to form a film. The tensile 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 embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for preparing methoxy polyethylene glycol epoxide chemically modified silk fibroin, characterized in that: The following steps are involved: S1. Adding a hydrogen bond inhibitor and a pH regulator with a final concentration of 0.01 M to 3 M to the regenerated silk fibroin solution, mixing, then adding trypsin to incubate and inactivate, and performing solid-liquid separation to obtain a solid phase to obtain a silk fibroin solid; S2. The silk fibroin solid obtained in step S1 is mixed with an inorganic salt solution for activation, and then incubated with methoxy polyethylene glycol epoxide after activation. After purification, a methoxy polyethylene glycol epoxide chemically modified silk fibroin solution is obtained.

2. The preparation method according to claim 1, characterized in that In step S1, the hydrogen bond inhibitor is one or more of CaCl2 / ethanol / water, LiBr, NaSCN and ZnCl2.

3. The preparation method according to claim 1, characterized in that 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; And / or, the pH regulator adjusts the pH value of the regenerated silk fibroin solution to 6-9.

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

5. The preparation method according to claim 1, characterized in that In step S1, the mass ratio of trypsin to regenerated silk fibroin in the regenerated silk fibroin solution is 1:5-1:500; And / or, the incubation condition is: incubation at 25° C.-37° C. for 24 h-48 h.

6. 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 anion in the inorganic salt solution is Cl - 、SO4 2- Br - 、SCN - 、CO3 2- and OH - One or more of; And / or, the concentration of the inorganic salt solution is 4 M-15 M.

7. 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 40:1-1:

1.

8. 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.

9. The preparation method according to claim 1, characterized in that In step S2, the mass ratio of the methoxy polyethylene glycol epoxy to the silk fibroin solid is 1:200-1:1; And / or, the incubation condition is: incubation at 4° C.-80° C. for 0.5 h-72 h.

10. The methoxypolyethylene glycol epoxide chemically modified silk fibroin obtained by the preparation method according to any one of claims 1 to 9.

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