Method for preparing an initial equivalent, conformational transition rate different sf hydrogel

Abstract

The application belongs to the technical field of biomedicine and relates to a preparation method of SF hydrogel with equivalent initial characteristics and different conformation transition rates. Two SF hydrogel precursor solutions alpha and beta are mixed in different proportions, crosslinked under certain conditions for a certain time, and thus SF hydrogel with equivalent initial characteristics and different conformation transition rates is obtained. The SF in the SF hydrogel precursor solutions alpha and beta is the same, and the concentration of the SF is the same. The stress-strain curves of the two SF hydrogels formed after the crosslinking reaction of the SF hydrogel precursor solutions alpha and beta are equivalent, the average pore diameters are equivalent, and the uniformity of crosslinking points is different. The SF hydrogel with equivalent initial characteristics and different conformation transition rates is successfully prepared.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedicine, and relates to a preparation method of SF hydrogel with initial equivalence and different conformation transition rates. BACKGROUND

[0002] As a kind of natural protein material, SF (silk fibroin) has been widely studied and concerned due to its excellent biocompatibility, degradability and processing convenience. Among various SF materials, chemically cross-linked SF hydrogel not only inherits the natural advantages of SF, but also exhibits unique extracellular matrix biomimetic characteristics and highly adjustable mechanical properties. In recent years, researchers have attempted to use SF hydrogel in the fields of in vitro cell culture, corneal materials, in vivo tissue repair, etc., and have confirmed that it has good application prospects in related fields.

[0003] However, in the in vivo implantation or in vitro cell culture environment, the free movement and self-assembly of the hydrophobic segment of the SF molecule in the chemically cross-linked SF hydrogel will cause the conformation of the SF molecule in the hydrogel to transform from the random coil conformation to the β-sheet conformation with a lower energy level. This will inevitably lead to a dynamic conformational transition of the SF molecule in the hydrogel, and further cause dynamic changes in the pore size, softness and light transmittance and other physical characteristics of the SF hydrogel material. A large number of research reports have confirmed that the pore size, softness and other characteristic parameters of the material can have a profound impact on the adhesion, proliferation and differentiation behaviors of cells. The conformational transition generally occurs obviously within 1-3 weeks in the initial use period, and the relevant period is also generally considered as the key period in which the characteristics of the material affect the behaviors of cells. Based on this, the protein conformational transition microenvironment in the SF hydrogel is also likely to effectively regulate the adhesion and differentiation behaviors of cells, thereby bringing an important unknown influence on the application of such important biological materials in the fields of living cell encapsulation culture, in vitro tissue construction and in vivo tissue repair, etc.

[0004] Up to now, there is no report on the research and development of in-depth and effective regulation and utilization strategies for the similar conformation transition microenvironment in the above-mentioned interdisciplinary field, especially the development of SF hydrogel construction strategies with equivalent initial characteristics (initial characteristics refer to the compression mechanical properties and average pore size characteristics of SF hydrogel at the initial stage. Studies have shown that the compression mechanical properties and pore size characteristics of biomaterials have a profound impact on the behavior of cell proliferation and differentiation. In the process of studying the influence of the protein conformation transition microenvironment in SF hydrogel on cell behavior, if the compression mechanical properties and average pore size of the hydrogel at the initial stage are not equivalent, they will be mixed with protein conformation transition to affect the relevant cell behavior, so that the influence of protein conformation transition on cell behavior cannot be accurately obtained) and different protein conformation transition rates. The construction of related material platform is expected to study and reveal the influence of protein conformation transition microenvironment on cell behavior under the premise of excluding various interference factors, and finally provide important guidance for the effective regulation of protein conformation transition in SF hydrogel and its reasonable use in the biomedical field.

[0005] At present, a small number of researchers have begun to try to construct SF hydrogels with "different conformation transition rates" and use them to study the influence of related factors on cell behavior. For example, the literature (ACS Applied Materials & Interfaces 2022, 14, 7531-7550) uses SF combined with carboxymethyl cellulose and gelatin to prepare several composite hydrogels with "different conformation transition rates" and investigates their influence on stem cell differentiation. However, this scheme is to change the protein conformation transition rate in the composite hydrogel by regulating the ratio of SF component to the other two components, so the initial chemical components, mechanical properties and crosslinking density of the hydrogel materials in different groups are greatly different, which cannot meet the requirement of equivalent initial characteristics. The literature (Functional Polymers 2024, 37, 312-321) uses a single SF material component to construct SF hydrogels with different protein conformation transition rates based on the strategy of regulating the chemical crosslinking density and investigates their influence on the proliferation of cells adhered to the surface of the hydrogel. Although this scheme excludes the interference of different chemical compositions to a certain extent, the initial average pore size and mechanical properties of the hydrogels in different groups are still significantly different (due to different chemical crosslinking densities).

[0006] In summary, the existing strategies cannot realize the preparation of SF hydrogels with equivalent initial characteristics and different protein conformation transition rates. SUMMARY

[0007] The purpose of the present application is to solve the problems existing in the prior art and provide a preparation method of SF hydrogel with equivalent initial characteristics and different conformation transition rates.

[0008] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0009] The preparation method of the SF hydrogel with different initial equivalent and conformation transition rates comprises the following steps:

[0010] The SF in the SF hydrogel precursor solution α and the SF hydrogel precursor solution β is the same, and the concentration of the SF is the same.

[0011] The SF hydrogel precursor solution α, the SF hydrogel precursor solution β, the certain condition and the certain time meet the following requirements: the stress-strain curve of the SF hydrogel formed by the cross-linking reaction of the SF hydrogel precursor solution α under the certain condition for the certain time is equivalent (i.e. almost completely the same) to the stress-strain curve of the SF hydrogel formed by the cross-linking reaction of the SF hydrogel precursor solution β under the certain condition for the certain time, the average pore size is equivalent, and the uniformity of the cross-linking points is different.

[0012] The SF hydrogel with different initial equivalent and conformation transition rates refers to the SF hydrogel with equivalent initial characteristics and different protein conformation transition rates.

[0013] As a preferred technical scheme:

[0014] The preparation method of the SF hydrogel with different initial equivalent and conformation transition rates as described above, the SF hydrogel precursor solution α is an SF / HRP / H2O2 system, the SF hydrogel precursor solution β is an SF / X / SPS system, and the core gel-forming principles of the two are the same, both of which are to form a chemical cross-linking type SF hydrogel by relying on the generation of an appropriate di-tyrosine bond, HRP is horseradish peroxidase, H2O2 is hydrogen peroxide, X is Ru or Rf, Ru is a ruthenium compound, Rf is riboflavin, and SPS is sodium persulfate.

[0015] The preparation method of the SF hydrogel with different initial equivalent and conformation transition rates as described above, the specific steps are as follows:

[0016] (1) HRP solution is added to the SF solution, and then H2O2 solution is added to obtain the SF hydrogel precursor solution α, which is cross-linked under the temperature T for the time t to obtain the SF hydrogel A;

[0017] (2) The SF hydrogel A is subjected to compression test by using an electronic universal testing machine to obtain the stress-strain curve of the SF hydrogel A, and the cross section of the SF hydrogel A after freeze-drying is photographed by using a scanning electron microscope to obtain the average pore size of the SF hydrogel A.

[0018] (3) adding the X solution into the SF solution, then adding the SPS solution to obtain a plurality of SF hydrogel precursor solutions B with different amounts of X, and crosslinking under visible light irradiation at light intensity L for a time t to obtain a plurality of SF hydrogels B;

[0019] (4) performing compression test on the SF hydrogel B by using an electronic universal testing machine to obtain the stress-strain curve of the SF hydrogel B, and performing section shooting on the SF hydrogel B after freeze-drying by using a scanning electron microscope to obtain the average pore size of the SF hydrogel B;

[0020] (5) finding the SF hydrogel B equivalent to the SF hydrogel A in stress-strain curve and average pore size, and recording the corresponding SF hydrogel precursor solution B as SF hydrogel precursor solution β;

[0021] (6) mixing the SF hydrogel precursor solution α and the SF hydrogel precursor solution β in different proportions, and crosslinking under visible light irradiation at light intensity L for a time t at temperature T to obtain the SF hydrogel with initial equivalence and different conformation transition rates.

[0022] The preparation method of the SF hydrogel with initial equivalence and different conformation transition rates as described above, in step (1), the volume ratio of the SF solution to the HRP solution is 1 mL: 15-50 μL, and the volume ratio of the HRP solution to the H2O2 solution is 1:0.3-1:0.7.

[0023] The preparation method of the SF hydrogel with initial equivalence and different conformation transition rates as described above, in step (1), the concentration of the SF solution is 2-10 wt%, the concentration of the HRP solution is 800-1200 U / mL, the concentration of the H2O2 solution is 0.3-0.7 mol / L, T is 35-39℃, and t is 30 min or more.

[0024] The preparation method of the SF hydrogel with initial equivalence and different conformation transition rates as described above, in step (3), the volume ratio of the SF solution to the X solution is 1 mL: 10-40 μL, and the volume ratio of the X solution to the SPS solution is 1:0.8-1:1.2.

[0025] The preparation method of the SF hydrogel with initial equivalence and different conformation transition rates as described above, in step (3), the concentration of the SF solution is 2-10 wt%, the concentration of the SPS solution is 0.3-0.7 mol / L, L is 30-80 mW cm -2 ; when X is Ru, the concentration of the X solution is 3-7 mol / L; and when X is Rf, the concentration of the X solution is 0.03-0.07 mol / L.

[0026] In the step (6) of the preparation method of the SF hydrogel with different initial equivalent, conformation transition rate as described above, the mixing of the SF hydrogel precursor solution α and the SF hydrogel precursor solution β in different proportions means mixing the SF hydrogel precursor solution α and the SF hydrogel precursor solution β in different volume ratios, for example, in the volume ratios of 1:0, 3:1, 1:1, 1:3, and 0:1.

[0027] In the preparation method of the SF hydrogel with different initial equivalent, conformation transition rate as described above, the preparation steps of SF in all SF solutions are as follows:

[0028] (a) degumming;

[0029] The silk is placed in a sodium carbonate solution with a concentration of 2-5 wt% and a temperature of 95-100°C for 20-60 min, washed with deionized water for at least 3 times, and dried to obtain SF fibers;

[0030] (b) dissolving;

[0031] The SF fibers are dissolved in a lithium bromide solution with a concentration of 9-9.3 mol / L and a temperature of 40-60°C for 2-4 h to obtain an SF solution;

[0032] (c) dialysis;

[0033] The SF solution obtained in step (b) is filtered and then placed in a dialysis bag, and dialyzed with deionized water, which is changed three times a day, until the conductivity of the SF solution is ≤4.0 μS / cm;

[0034] (d) freeze-drying;

[0035] The filtered dialysis solution is injected into a mold and subjected to freezing treatment in an environment of -20 to -80°C, and then the frozen body is placed in a freeze dryer to obtain SF by freeze-drying.

[0036] Principle of the invention:

[0037] The application innovatively proposes to construct SF hydrogels with equivalent initial characteristics and different protein conformation transition rates based on the regulation of chemical crosslinking point uniformity. The core principles involved are as follows: ① In the case of equivalent crosslinking point density and different crosslinking point uniformity, the overall compressive mechanical properties and average pore size of SF hydrogels are basically equivalent; ② In the case of equivalent crosslinking point density and low crosslinking point uniformity, the length of the free segment of SF molecules in the hydrogel is also more uneven. In this case, there are both "very short" free segments and "longer" free segments. As a result, those "longer" free segments are relatively easy to move freely and trigger the transition of SF molecules from random coil conformation to a lower energy state of β-sheet conformation, thereby accelerating the conformation transition process of the hydrogel; ③ In the case of equivalent crosslinking point density and high crosslinking point uniformity, the length of the free segment of SF molecules in the hydrogel is also more uniform. The free segment is of intermediate value and is relatively "short". The slow free movement of the "short" segment will significantly slow down the conformation transition process of the corresponding hydrogel.

[0038] In summary, it is theoretically feasible to regulate the conformation transition rate of SF hydrogels based on the chemical crosslinking point uniformity while ensuring that the initial characteristics of the hydrogels are equivalent. However, there are still some challenges in preparing SF hydrogels with equivalent crosslinking point density but different crosslinking point uniformity.

[0039] To solve this problem, the application selects the SF / HRP / H2O2 system and the SF / X / SPS system (specifically the SF / Ru / SPS system or the SF / Rf / SPS system) with similar crosslinking principles to prepare the SF hydrogel material. Specifically, both systems form chemical crosslinking type SF hydrogels by relying on the formation of appropriate di-tyrosine bonds, and the reaction conditions are very mild, which is beneficial for the encapsulation and culture of living cells in the hydrogel. The SF / HRP / H2O2 system only needs to be incubated at 37°C (body temperature) to complete crosslinking and gelation, and the SF / X / SPS system only needs to be irradiated under visible light to complete crosslinking and gelation. The gelation principle of the SF / HRP / H2O2 and SF / Ru / SPS systems for forming SF hydrogels is shown in Figure 1 .

[0040] The reasons that the SF / HRP / H2O2 system and the SF / X / SPS system can be used to successfully construct SF hydrogels with equivalent crosslinking point density but different crosslinking point uniformity are as follows. Both of the two types of crosslinking reaction systems are dependent on the generation of appropriate di-tyrosine bonds to form chemical crosslinking type hydrogels, and the biggest difference between them is that the molecular weight (Mw=44000 Da) of HRP is much larger than that of X. According to molecular dynamics and diffusion theory, small molecular substances have higher diffusion coefficients and weaker intermolecular forces than large molecular substances in the same medium, so they can be dispersed more quickly and uniformly in unit time. Under other similar conditions, compared with HRP with an "abnormally large" molecular weight, small molecular substances Ru or Rf can easily and uniformly disperse into the raw material system to form a hydrogel crosslinking network with excellent uniformity. The crosslinking points in the hydrogel formed by the SF / X / SPS system are "more uniform", and relatively speaking, the crosslinking points in the hydrogel formed by the SF / HRP / H2O2 system are "less uniform". Therefore, it can be inferred that under the same crosslinking density, compared with the SF / HRP / H2O2 system, the crosslinking density of the SF / X / SPS system is more uniform, and the conformational transition speed is slower.

[0041] Beneficial effects:

[0042] The application innovatively proposes a regulation strategy based on crosslinking point uniformity to construct SF hydrogels with equivalent initial conditions and different conformational transition rates. In addition, the synthesis of the hydrogels is also based on mild crosslinking reactions, which greatly guarantees that the hydrogel system is non-toxic and harmless to cells, thereby facilitating the packaging and culture observation of living cells. This strategy not only provides an important material platform for precisely studying the influence of the protein conformational transition microenvironment in SF hydrogels on cell behavior, but also provides valuable references and important guidance for the effective regulation of protein conformational transition in hydrogels and the development of high-efficiency tissue repair hydrogel scaffolds. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 A schematic diagram of the gel formation principle of the SF / HRP / H2O2 and SF / Ru / SPS systems for forming SF hydrogels;

[0044] Figure 2 A stress-strain curve of the alpha (SF / HRP / H2O2), beta (SF / Ru / SPS) and alpha:beta=1:1 hydrogels of Example 1;

[0045] Figure 3 The average pore size of the alpha (SF / HRP / H2O2), beta (SF / Ru / SPS) and alpha:beta=1:1 hydrogels of Example 1;

[0046] Figure 4Conformational transition of the α (SF / HRP / H2O2), β (SF / Ru / SPS) and α:β = 1:1 hydrogels of Example 1 incubated in a simulated cell culture environment for different days;

[0047] Figure 5 Proliferation of the stem cells in the α (SF / HRP / H2O2) hydrogel encapsulating the stem cells for different days of culture;

[0048] Figure 6 Proliferation of the stem cells in the α:β = 1:1 hydrogel encapsulating the stem cells for different days of culture;

[0049] Figure 7 Proliferation of the stem cells in the β (SF / Ru / SPS) hydrogel encapsulating the stem cells for different days of culture. DETAILED DESCRIPTION

[0050] The application will be further described below in connection with specific embodiments. It should be understood that these embodiments are only used to illustrate the application and not used to limit the scope of the application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the application after reading the content of the application, and these equivalent forms also fall within the scope of the appended claims.

[0051] The following are the test methods of the relevant performance indicators in each example:

[0052] Conductivity: tested by FG3 conductivity meter.

[0053] Example 1

[0054] A preparation method of an initial equivalent SF hydrogel with different conformational transition rates, the specific steps are as follows:

[0055] (1) Preparation of raw materials;

[0056] Silk: extracted from "Qiufeng Baiyu" variety of cocoon, produced in Jiangsu;

[0057] Sodium carbonate solution: solvent is deionized water, concentration is 2wt%, temperature is 95℃;

[0058] Deionized water;

[0059] Lithium bromide solution: solvent is deionized water, concentration is 9mol / L, temperature is 40℃;

[0060] HRP solution: HRP is horseradish peroxidase (CAS number is 9003-99-0), solvent is deionized water, concentration is 800U / mL;

[0061] H2O2 solution: H2O2 is hydrogen peroxide, solvent is deionized water, concentration is 0.3 mol / L;

[0062] Ru solution: Ru is tris(2,2'-bipyridyl) dichloro ruthenium(II) hexahydrate, solvent is deionized water, concentration is 3 mol / L;

[0063] SPS solution: SPS is sodium persulfate, solvent is deionized water, concentration is 0.3 mol / L;

[0064] (2) Preparation of SF;

[0065] (2.1) Put the silk into the sodium carbonate solution and cook for 20 min, wash with deionized water for 3 times, and dry to obtain SF fiber;

[0066] (2.2) Dissolve the SF fiber in lithium bromide solution for 2 h to obtain SF solution;

[0067] (2.3) The SF solution obtained in step (2.2) is filtered and loaded into a dialysis bag, and dialyzed with deionized water, which is replaced three times a day, until the conductivity of the SF solution is 4 μS / cm;

[0068] (2.4) The dialyzed filtrate is injected into a mold and subjected to freezing treatment in an environment of -20℃, and then the frozen body is placed in a freeze dryer to obtain SF by freeze drying;

[0069] (3) Preparation of SF hydrogel with different initial equivalent and conformational transition rate;

[0070] (3.1) Prepare a SF solution with a concentration of 2wt% (solvent is deionized water), add HRP solution to the SF solution, then add H2O2 solution to obtain SF hydrogel precursor solution α, and crosslink at a temperature of 35℃ for 35 min to obtain SF hydrogel A; wherein the volume ratio of SF solution to HRP solution is 1 mL:15 μL, and the volume ratio of HRP solution to H2O2 solution is 1:0.3;

[0071] (3.2) The stress-strain curve of SF hydrogel A is obtained by compression test using an electronic universal testing machine, and the average pore size of SF hydrogel A is obtained by statistical analysis of the cross-section of freeze-dried SF hydrogel A photographed by scanning electron microscope;

[0072] (3.3) Prepare a SF solution with a concentration of 2wt% (solvent is deionized water), add Ru solution to the SF solution, then add SPS solution to obtain a plurality of SF hydrogel precursor solutions B with different amounts of Ru, and crosslink under light intensity of 30 mW cm -2under visible light irradiation for 35 min to obtain a plurality of SF hydrogels B; wherein the volume ratio of the SF solution to the Ru solution is 1 mL:10 μL, and the volume ratio of the Ru solution to the SPS solution is 1:0.8;

[0073] (3.4) The stress-strain curve of the SF hydrogel B was obtained by compression test using an electronic universal testing machine, and the average pore size of the SF hydrogel B was obtained by taking a section of the freeze-dried SF hydrogel B and then counting using a scanning electron microscope;

[0074] (3.5) The SF hydrogel B equivalent to the stress-strain curve and the average pore size of the SF hydrogel A was found, and the corresponding SF hydrogel precursor solution B was recorded as SF hydrogel precursor solution β;

[0075] (3.6) The SF hydrogel precursor solution α and the SF hydrogel precursor solution β were mixed in a volume ratio of 1:0, 3:1, 1:1, 1:3, and 0:1, and then cross-linked under visible light irradiation at a temperature of 35℃ and an illumination intensity of 30 mW cm -2 for 35 min to obtain initial equivalent SF hydrogels with different protein conformation transition rates, which were recorded as α(SF / HRP / H2O2) hydrogel, α:β=3:1 hydrogel, α:β=1:1 hydrogel, α:β=1:3 hydrogel, and β(SF / Ru / SPS) hydrogel. The α(SF / HRP / H2O2) hydrogel, the α:β=1:1 hydrogel, and the β(SF / Ru / SPS) hydrogel were tested to verify that they have equivalent initial characteristics and different protein conformation transition rates, as follows:

[0076] The α(SF / HRP / H2O2) hydrogel, the α:β=1:1 hydrogel, and the β(SF / Ru / SPS) hydrogel were tested, and the initial characteristics of the three hydrogels were as shown in Figure 2 , Figure 3 Thus, the three hydrogels have equivalent initial characteristics.

[0077] The three hydrogels were immersed in DMEM complete medium and then incubated in a cell incubator at a temperature of 37℃, a relative humidity of 95%, and a CO2 volume concentration of 5% for 10 days, with the medium being replaced every two days. The β-sheet conformation content of the three hydrogels at different days was as shown in Figure 4 From the graph, it can be seen that the α(SF / HRP / H2O2) hydrogel has the fastest conformation transition in the simulated cell culture environment, the β(SF / Ru / SPS) hydrogel has the slowest conformation transition, and the conformation transition speed of the α:β=1:1 hydrogel is between the two. Thus, the three hydrogels have different protein conformation transition rates.

[0078] In summary, the hydrogel with the fastest conformation transition can be constructed by the SF / HRP / H2O2 system alone, the hydrogel with the slowest conformation transition can be constructed by the SF / Ru / SPS system alone, and the SF hydrogel with a conformation transition at an intermediate speed can be constructed by adjusting the volume ratio of the two similar gelation systems.

[0079] In addition, since both systems are gelled under extremely mild (cell-friendly) conditions, the combination of the two systems is also gelled under mild conditions. Thus, the hydrogel formation process is non-toxic to cells, facilitating the encapsulation of living cells and subsequent three-dimensional cell culture and comprehensive investigation of cell behavior. To confirm this point, the following experiments were conducted:

[0080] An α(SF / HRP / H2O2) hydrogel encapsulating stem cells, an α:β = 1:1 hydrogel encapsulating stem cells, and a β(SF / Ru / SPS) hydrogel encapsulating stem cells were prepared, wherein:

[0081] The preparation process of the α(SF / HRP / H2O2) hydrogel encapsulating stem cells is basically the same as that of the α(SF / HRP / H2O2) hydrogel, except that before the HRP solution is added to the SF solution, an equal volume of bone marrow mesenchymal stem cell suspension is also added to the SF solution, and the number of stem cells in the suspension is 10 × 10 5 cells / mL;

[0082] The preparation process of the α:β = 1:1 hydrogel encapsulating stem cells is basically the same as that of the α:β = 1:1 hydrogel, except that when the SF hydrogel precursor solution α and the SF hydrogel precursor solution β are mixed, an equal volume of bone marrow mesenchymal stem cell suspension is also added, and the number of stem cells in the suspension is 10 × 10 5 cells / mL;

[0083] The preparation process of the β(SF / Ru / SPS) hydrogel encapsulating stem cells is basically the same as that of the β(SF / Ru / SPS) hydrogel, except that before the Ru solution is added to the SF solution, an equal volume of bone marrow mesenchymal stem cell suspension is also added to the SF solution, and the number of stem cells in the suspension is 10 × 10 5 cells / mL;

[0084] After the α(SF / HRP / H2O2) hydrogel encapsulating stem cells, the α:β = 1:1 hydrogel encapsulating stem cells, and the β(SF / Ru / SPS) hydrogel encapsulating stem cells were immersed in DMEM complete medium, they were incubated in a cell incubator at a temperature of 37°C, a relative humidity of 95%, and a CO2 volume concentration of 5% for 10 days, with the medium being changed every two days, and the cell viability was observed under a microscope.Figures 5 to 7 As can be seen from the above, the cell viability of the cells in the three hydrogels gradually increased after long-term culture (indirectly indicating that the number of cells gradually increased), and obvious cell proliferation occurred.

[0085] Therefore, the SF / HRP / H2O2 and SF / Ru / SPS systems alone or the composite based on the two systems can successfully construct SF hydrogels with equivalent compressive mechanical properties and average pore sizes, and these hydrogels exhibit good biocompatibility in cell culture, providing a powerful tool for exploring the influence of protein conformational transition on cell behavior.

[0086] Example 2

[0087] A method for preparing an initial equivalent SF hydrogel with different conformational transition rates, the specific steps are as follows:

[0088] (1) Preparation of raw materials;

[0089] Silk: extracted from "Qiufeng Baiyu" variety of cocoon, produced in Jiangsu;

[0090] Sodium carbonate solution: solvent is deionized water, concentration is 2wt%, temperature is 95℃;

[0091] Deionized water;

[0092] Lithium bromide solution: solvent is deionized water, concentration is 9mol / L, temperature is 40℃;

[0093] HRP solution: HRP is horseradish peroxidase (CAS number is 9003-99-0), solvent is deionized water, concentration is 900U / mL;

[0094] H2O2 solution: H2O2 is hydrogen peroxide, solvent is deionized water, concentration is 0.4mol / L;

[0095] Ru solution: Ru is tris(2,2'-bipyridine) dichloro ruthenium (II) hexahydrate, solvent is deionized water, concentration is 5mol / L;

[0096] SPS solution: SPS is sodium persulfate, solvent is deionized water, concentration is 0.4mol / L;

[0097] (2) Preparation of SF;

[0098] (2.1) Put the silk into the sodium carbonate solution and cook for 30min, wash with deionized water for 4 times, and dry to obtain SF fibers;

[0099] (2.2) Dissolve the SF fibers in the lithium bromide solution for 2h to obtain an SF solution;

[0100] (2.3) The SF solution obtained in step (2.2) is filtered and then put into a dialysis bag, and dialyzed with deionized water, which is changed three times a day, until the conductivity of the SF solution is 3 μS / cm;

[0101] (2.4) The dialyzed filtrate is injected into a mold, and then subjected to freezing treatment in an environment at -30°C, and then the frozen body is placed in a freeze dryer to obtain SF by freeze drying;

[0102] (3) Preparation of SF hydrogel with initial equivalent and different conformational transition rates;

[0103] (3.1) An SF solution with a concentration of 4wt% (deionized water as solvent) is prepared, and then HRP solution is added, followed by the addition of H2O2 solution, to obtain an SF hydrogel precursor solution α, which is subjected to crosslinking reaction at a temperature of 36°C for 40 min to obtain SF hydrogel A; wherein the volume ratio of the SF solution to the HRP solution is 1 mL: 15 μL, and the volume ratio of the HRP solution to the H2O2 solution is 1:0.3;

[0104] (3.2) The SF hydrogel A is subjected to compression test by using an electronic universal testing machine to obtain the stress-strain curve of the SF hydrogel A, and at the same time, the cross-section of the freeze-dried SF hydrogel A is photographed by using a scanning electron microscope, and then statistics are obtained to obtain the average pore size of the SF hydrogel A;

[0105] (3.3) An SF solution with a concentration of 4wt% (deionized water as solvent) is prepared, and then Ru solution is added, followed by the addition of SPS solution, to obtain a plurality of SF hydrogel precursor solutions B with different amounts of Ru, which are subjected to crosslinking reaction under visible light irradiation at an intensity of 40 mW cm -2 for 40 min to obtain a plurality of SF hydrogels B; wherein the volume ratio of the SF solution to the Ru solution is 1 mL: 10 μL, and the volume ratio of the Ru solution to the SPS solution is 1:0.8;

[0106] (3.4) The SF hydrogel B is subjected to compression test by using an electronic universal testing machine to obtain the stress-strain curve of the SF hydrogel B, and at the same time, the cross-section of the freeze-dried SF hydrogel B is photographed by using a scanning electron microscope, and then statistics are obtained to obtain the average pore size of the SF hydrogel B;

[0107] (3.5) The SF hydrogel B equivalent to the stress-strain curve and the average pore size of the SF hydrogel A is found, and the corresponding SF hydrogel precursor solution B is recorded as SF hydrogel precursor solution β;

[0108] (3.6) Mixing SF hydrogel precursor solution α and SF hydrogel precursor solution β in a volume ratio of 1:0, 3:1, 1:1, 1:3, 0:1, respectively, and then irradiating with visible light at a temperature of 36℃ and an illumination intensity of 40 mW cm -2 for 40 min to obtain initial equivalent SF hydrogels with different conformational transition rates.

[0109] Example 3

[0110] A method for preparing an initial equivalent SF hydrogel with different conformational transition rates, comprising the following steps:

[0111] (1) Preparation of raw materials;

[0112] Silk: extracted from "Qiufeng Baiyu" variety of cocoon, produced in Jiangsu;

[0113] Sodium carbonate solution: solvent is deionized water, concentration is 3wt%, and temperature is 98℃;

[0114] Deionized water;

[0115] Lithium bromide solution: solvent is deionized water, concentration is 9.3 mol / L, and temperature is 50℃;

[0116] HRP solution: HRP is horseradish peroxidase (CAS No. 9003-99-0), solvent is deionized water, and concentration is 1000 U / mL;

[0117] H2O2 solution: H2O2 is hydrogen peroxide, solvent is deionized water, and concentration is 0.5 mol / L;

[0118] Ru solution: Ru is tris(2,2'-bipyridine)dichlororuthenium(II) hexahydrate, solvent is deionized water, and concentration is 7 mol / L;

[0119] SPS solution: SPS is sodium persulfate, solvent is deionized water, and concentration is 0.5 mol / L;

[0120] (2) Preparation of SF;

[0121] (2.1) Putting silk into sodium carbonate solution and boiling for 40 min, washing with deionized water for 4 times, and drying to obtain SF fiber;

[0122] (2.2) Dissolving SF fiber in lithium bromide solution for 3 h to obtain SF solution;

[0123] (2.3) Filtering the SF solution obtained in step (2.2), and then loading into a dialysis bag and dialyzing with deionized water, changing deionized water three times a day until the conductivity of the SF solution is 3 μS / cm;

[0124] (2.4) The dialyzed filtrate is injected into a mold and subjected to freezing treatment in an environment of -50°C, and then the frozen body is placed in a freeze dryer to obtain SF by freeze-drying;

[0125] (3) Prepare SF hydrogel with initial equivalence and different conformational transition rates;

[0126] (3.1) Prepare an SF solution with a concentration of 6wt% (deionized water as solvent), add an HRP solution to the SF solution, then add an H2O2 solution to obtain an SF hydrogel precursor solution α, and cross-link at a temperature of 37°C for 45 min to obtain SF hydrogel A; wherein the volume ratio of the SF solution to the HRP solution is 1 mL:50 μL, and the volume ratio of the HRP solution to the H2O2 solution is 1:0.7;

[0127] (3.2) Perform compression testing on SF hydrogel A using an electronic universal testing machine to obtain the stress-strain curve of SF hydrogel A, and at the same time, perform cross-section photography on the freeze-dried SF hydrogel A using a scanning electron microscope to obtain the average pore size of SF hydrogel A;

[0128] (3.3) Prepare an SF solution with a concentration of 6wt% (deionized water as solvent), add a Ru solution to the SF solution, then add an SPS solution to obtain multiple SF hydrogel precursor solutions B with different amounts of Ru, and cross-link under visible light irradiation at an intensity of 60 mW cm -2

[0129] (3.4) Perform compression testing on SF hydrogel B using an electronic universal testing machine to obtain the stress-strain curve of SF hydrogel B, and at the same time, perform cross-section photography on the freeze-dried SF hydrogel B using a scanning electron microscope to obtain the average pore size of SF hydrogel B;

[0130] (3.5) Find SF hydrogel B that is equivalent in stress-strain curve and average pore size to SF hydrogel A, and record the corresponding SF hydrogel precursor solution B as SF hydrogel precursor solution β;

[0131] (3.6) Mix SF hydrogel precursor solution α and SF hydrogel precursor solution β at volume ratios of 1:0, 3:1, 1:1, 1:3, and 0:1, and cross-link under visible light irradiation at an intensity of 60 mW cm -2 ​​

[0132] Example 4

[0133] A method for preparing an initial equivalent, conformational transition rate different SF hydrogel, the specific steps are as follows:

[0134] (1) Preparation of raw materials;

[0135] Silk: extracted from "Qiufeng Baiyu" variety of cocoon, produced in Jiangsu;

[0136] Sodium carbonate solution: solvent is deionized water, concentration is 4wt%, temperature is 100℃;

[0137] Deionized water;

[0138] Lithium bromide solution: solvent is deionized water, concentration is 9mol / L, temperature is 60℃;

[0139] HRP solution: HRP is horseradish peroxidase (CAS number is 9003-99-0), solvent is deionized water, concentration is 1100U / mL;

[0140] H2O2 solution: H2O2 is hydrogen peroxide, solvent is deionized water, concentration is 0.6mol / L;

[0141] Rf solution: Rf is riboflavin, solvent is deionized water, concentration is 0.03mol / L;

[0142] SPS solution: SPS is sodium persulfate, solvent is deionized water, concentration is 0.6mol / L;

[0143] (2) Preparation of SF;

[0144] (2.1) Put the silk into the sodium carbonate solution and cook for 50min, wash with deionized water for 5 times, and dry to obtain SF fiber;

[0145] (2.2) Dissolve the SF fiber in the lithium bromide solution for 4h to obtain an SF solution;

[0146] (2.3) After filtering the SF solution obtained in step (2.2), it is loaded into a dialysis bag and dialyzed with deionized water, which is replaced three times a day, until the conductivity of the SF solution is 2μS / cm;

[0147] (2.4) Inject the dialyzed filtrate into a mold and freeze treat in an environment of-70℃, then place the frozen body in a freeze dryer to obtain SF by freeze drying;

[0148] (3) Preparation of an initial equivalent, conformational transition rate different SF hydrogel;

[0149] (3.1) Prepare a SF solution (solvent is deionized water) with a concentration of 8wt%, add HRP solution into the SF solution, then add H2O2 solution, to obtain SF hydrogel precursor solution alpha, crosslinking reaction at a temperature of 38℃ for 50min to obtain SF hydrogel A; wherein the volume ratio of SF solution to HRP solution is 1mL:50μL, and the volume ratio of HRP solution to H2O2 solution is 1:0.7;

[0150] (3.2) Perform compression test on SF hydrogel A by using electronic universal testing machine to obtain the stress-strain curve of SF hydrogel A, and at the same time, perform cross-section shooting on the freeze-dried SF hydrogel A by using scanning electron microscope to obtain the average pore size of SF hydrogel A;

[0151] (3.3) Prepare a SF solution (solvent is deionized water) with a concentration of 8wt%, add Rf solution into the SF solution, then add SPS solution, to obtain multiple SF hydrogel precursor solutions B with different amounts of Rf, crosslinking reaction under visible light irradiation with a light intensity of 70mW cm -2 for 50min to obtain multiple SF hydrogels B; wherein the volume ratio of SF solution to Rf solution is 1mL:40μL, and the volume ratio of Rf solution to SPS solution is 1:1.2;

[0152] (3.4) Perform compression test on SF hydrogel B by using electronic universal testing machine to obtain the stress-strain curve of SF hydrogel B, and at the same time, perform cross-section shooting on the freeze-dried SF hydrogel B by using scanning electron microscope to obtain the average pore size of SF hydrogel B;

[0153] (3.5) Find SF hydrogel B equivalent to SF hydrogel A in stress-strain curve and average pore size, and record the corresponding SF hydrogel precursor solution B of SF hydrogel B as SF hydrogel precursor solution beta;

[0154] (3.6) Mix SF hydrogel precursor solution alpha and SF hydrogel precursor solution beta according to the volume ratio of 1:0, 3:1, 1:1, 1:3 and 0:1, and crosslinking reaction under visible light irradiation with a light intensity of 70mW cm -2 for 50min at a temperature of 38℃, to obtain SF hydrogels with initial equivalence and different conformational transition rates.

[0155] Example 5

[0156] A preparation method of SF hydrogel with initial equivalence and different conformational transition rates, the specific steps are as follows:

[0157] (1) Preparation of raw materials;

[0158] Silk: extracted from "Qiufeng Baiyu" variety of cocoon, produced in Jiangsu;

[0159] Sodium carbonate solution: solvent is deionized water, concentration is 4wt%, temperature is 100℃;

[0160] Deionized water;

[0161] Lithium bromide solution: solvent is deionized water, concentration is 9.3mol / L, temperature is 60℃;

[0162] HRP solution: HRP is horseradish peroxidase (CAS No. 9003-99-0), solvent is deionized water, concentration is 1200U / mL;

[0163] H2O2 solution: H2O2 is hydrogen peroxide, solvent is deionized water, concentration is 0.7mol / L;

[0164] Rf solution: Rf is riboflavin, solvent is deionized water, concentration is 0.07mol / L;

[0165] SPS solution: SPS is sodium persulfate, solvent is deionized water, concentration is 0.7mol / L;

[0166] (2) Preparation of SF;

[0167] (2.1) Put the silk into the sodium carbonate solution and boil for 60min, wash with deionized water for 5 times, and dry to obtain SF fiber;

[0168] (2.2) Dissolve the SF fiber in the lithium bromide solution for 4h to obtain SF solution;

[0169] (2.3) After filtering the SF solution obtained in step (2.2), it is loaded into a dialysis bag and dialyzed with deionized water, which is replaced three times a day, until the conductivity of the SF solution is 2μS / cm;

[0170] (2.4) Inject the dialyzed filtrate into a mold and freeze at-80℃, then freeze-dry the frozen body to obtain SF;

[0171] (3) Preparation of SF hydrogel with different initial equivalent and conformational transition rate;

[0172] (3.1) Prepare a SF solution with a concentration of 10wt% (solvent is deionized water), add HRP solution to the SF solution, then add H2O2 solution to obtain SF hydrogel precursor solution α, crosslink at a temperature of 39℃ for 50min to obtain SF hydrogel A; wherein the volume ratio of SF solution to HRP solution is 1mL:50μL, and the volume ratio of HRP solution to H2O2 solution is 1:0.7;

[0173] (3.2) The stress-strain curve of the SF hydrogel A was obtained by compression test using an electronic universal testing machine, and the average pore size of the SF hydrogel A was obtained by statistical analysis after taking the cross-section of the freeze-dried SF hydrogel A using a scanning electron microscope;

[0174] (3.3) An SF solution (solvent: deionized water) with a concentration of 10wt% was prepared, and Rf solution was added to the SF solution, followed by the addition of SPS solution, to obtain a plurality of SF hydrogel precursor solutions B with different amounts of Rf, which were crosslinked under irradiation of visible light with an intensity of 80mW cm -2 for 50min to obtain a plurality of SF hydrogels B; wherein the volume ratio of the SF solution to the Rf solution was 1mL:40μL, and the volume ratio of the Rf solution to the SPS solution was 1:1.2;

[0175] (3.4) The stress-strain curve of the SF hydrogel B was obtained by compression test using an electronic universal testing machine, and the average pore size of the SF hydrogel B was obtained by statistical analysis after taking the cross-section of the freeze-dried SF hydrogel B using a scanning electron microscope;

[0176] (3.5) The SF hydrogel B equivalent to the stress-strain curve and the average pore size of the SF hydrogel A was found, and the corresponding SF hydrogel precursor solution B was recorded as SF hydrogel precursor solution β;

[0177] (3.6) The SF hydrogel precursor solution α and the SF hydrogel precursor solution β were mixed in a volume ratio of 1:0, 3:1, 1:1, 1:3 and 0:1, and crosslinked under irradiation of visible light with an intensity of 80mW cm -2 for 50min at a temperature of 39℃, to obtain SF hydrogels with initial equivalence and different conformational transition rates.

Claims

1. A method for preparing SF hydrogels with initial equivalents and different conformational transition rates, characterized in that: After mixing SF hydrogel precursor solution α and SF hydrogel precursor solution β in different proportions and subjecting them to cross-linking reaction for a certain time under certain conditions, SF hydrogels with initial equivalents but different conformational transition rates were obtained. The SF in the SF hydrogel precursor solution α and the SF hydrogel precursor solution β are the same, and the concentration of SF is the same; The SF hydrogel precursor solution α, the SF hydrogel precursor solution β, the certain conditions, and the certain time satisfy: the SF hydrogel formed by the cross-linking reaction of the SF hydrogel precursor solution α under the certain conditions for the certain time and the SF hydrogel formed by the cross-linking reaction of the SF hydrogel precursor solution β under the certain conditions for the certain time have equivalent stress-strain curves, equivalent average pore sizes, and different cross-linking point uniformity; The SF hydrogel precursor solution α is a SF / HRP / H2O2 system, and the SF hydrogel precursor solution β is a SF / X / SPS system, HRP is horseradish peroxidase, H2O2 is hydrogen peroxide, X is Ru or Rf, Ru is ruthenium, Rf is riboflavin, and SPS is sodium persulfate; SF hydrogels with equivalent initial characteristics but different conformational transition rates refer to SF hydrogels with equivalent initial characteristics but different protein conformational transition rates.

2. The method for preparing SF hydrogels with initial equivalent and different conformational transition rates according to claim 1, characterized in that: The specific steps are as follows: (1) HRP solution was added to the SF solution, followed by H2O2 solution to obtain SF hydrogel precursor solution α. ​​The cross-linking reaction was carried out at temperature T for time t to obtain SF hydrogel A. (2) Obtain the stress-strain curve of SF hydrogel A and, at the same time, obtain the average pore size of SF hydrogel A; (3) Adding X solution to the SF solution, and then adding SPS solution, to obtain multiple SF hydrogel precursor solutions B with different X dosages, and subjecting them to crosslinking reaction for a time t under visible light irradiation with a light intensity L, to obtain multiple SF hydrogels B; (4) Obtaining the stress-strain curve of SF hydrogel B and, at the same time, obtaining the average pore size of SF hydrogel B; (5) Find SF hydrogel B with equivalent stress-strain curve and average pore size to SF hydrogel A, and record the corresponding SF hydrogel precursor solution B as SF hydrogel precursor solution β; (6) After mixing SF hydrogel precursor solution α and SF hydrogel precursor solution β in different volume ratios, the cross-linking reaction time t is carried out under visible light irradiation at temperature T and light intensity L to obtain SF hydrogels with initial equivalents and different conformational transition rates.

3. The method for preparing SF hydrogels with initial equivalent and different conformational transition rates according to claim 2, characterized in that: In step (1), the volume ratio of SF solution to HRP solution is 1 mL:15~50 μL, and the volume ratio of HRP solution to H2O2 solution is 1:0.3~0.

7.

4. The method for preparing SF hydrogels with initial equivalent and different conformational transition rates according to claim 2, characterized in that: In step (1), the concentration of the SF solution is 2-10 wt%, the concentration of the HRP solution is 800-1200 U / mL, the concentration of the H2O2 solution is 0.3-0.7 mol / L, T is 35-39°C, and t is more than 30 min.

5. The method for preparing SF hydrogels with initial equivalent and different conformational transition rates according to claim 2, characterized in that: In step (3), the volume ratio of SF solution to X solution is 1 mL:10~40 μL, and the volume ratio of X solution to SPS solution is 1:0.8~1.

2.

6. The method for preparing SF hydrogels with initial equivalent and different conformational transition rates according to claim 2, characterized in that: In step (3), the concentration of SF solution is 2~10wt%, the concentration of SPS solution is 0.3~0.7mol / L, and L is 30~80mWcm -2 When X is Ru, the concentration of the X solution is 3~7 mol / L; when X is Rf, the concentration of the X solution is 0.03~0.07 mol / L.

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