Antibacterial silk fibroin hydrogel as well as preparation method and application thereof

Abstract

The invention provides antibacterial silk fibroin hydrogel as well as a preparation method and application thereof, and relates to the technical field of biomedical materials. The antibacterial silk fibroin hydrogel provided by the invention is prepared from the following components: modified silk fibroin and copper montmorillonite, the modified silk fibroin is at least one of glycidyl methacrylate modified silk fibroin, methacrylic anhydride modified silk fibroin, isocyanate ethyl methacrylate modified silk fibroin and norbornene anhydride modified silk fibroin, and chemical crosslinking is formed among molecules of the modified silk fibroin. The copper montmorillonite and the modified silk fibroin form physical crosslinking through hydrogen bonds, and the mass ratio of the modified silk fibroin to the copper montmorillonite is 100: (1-10). The antibacterial silk fibroin hydrogel provided by the invention has good biological safety, long-acting antibacterial property, electrical conductivity and good mechanical property.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to an antibacterial silk fibroin hydrogel and a preparation method and application thereof. Background Art

[0002] Hydrogel has a cross-linked hydrophilic polymer network. It is soft and can absorb a large amount of water while maintaining a certain shape. It is widely used in the biomedical field. Such as drug sustained-release carriers, tissue engineering materials, artificial skin, artificial muscles, biosensors, wound dressings, etc. Silk fibroin is a natural protein with excellent properties such as good biodegradability, biocompatibility, good air permeability and moisture permeability, and immunogenicity. The silk fibroin molecular structure contains a large number of active groups such as hydroxyl, carboxyl and amino groups, which can react with various cross-linking agents to form a cross-linked network hydrogel. It is also possible to chemically modify the silk fibroin molecular chain using active groups such as hydroxyl, amino, carboxyl and thiol, introduce vinyl, and form a hydrogel through photocuring.

[0003] Silk fibroin hydrogel is widely used in the biomedical field. In order to give silk fibroin hydrogel broad-spectrum antibacterial properties, silver antibacterial agents are loaded into the hydrogel. However, the aggregation of nanosilver and the uncontrolled release of silver ions will produce certain cytotoxicity and silver is expensive. Some researchers have also loaded quaternary ammonium cationic surfactants into silk fibroin hydrogels, but small molecule surfactants are easily dissolved, making it difficult to achieve long-term antibacterial effects. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides an antimicrobial silk fibroin hydrogel and a preparation method and application thereof. The antimicrobial silk fibroin hydrogel has good biosafety, long-lasting antibacterial properties, electrical conductivity and good mechanical properties.

[0005] The specific technical solutions of the present invention are as follows: In a first aspect, the present invention provides an antibacterial silk fibroin hydrogel, the components of which include modified silk fibroin and copper montmorillonite, the modified silk fibroin is at least one of glycidyl methacrylate modified silk fibroin, methacrylic anhydride modified silk fibroin, ethyl methacrylate isocyanate modified silk fibroin and nadic anhydride modified silk fibroin, chemical crosslinks are formed between molecules of the modified silk fibroin, the copper montmorillonite and the modified silk fibroin are physically crosslinked through hydrogen bonds, and the mass ratio of the modified silk fibroin to the copper montmorillonite is 100:(1-10).

[0006] The antibacterial silk fibroin hydrogel provided by the present invention adopts copper montmorillonite as an antibacterial agent, copper ions in the copper montmorillonite can be slowly released from the interlayer of the montmorillonite into the antibacterial silk fibroin hydrogel, and by controlling the relative amounts of the copper montmorillonite and the modified silk fibroin, the antibacterial silk fibroin hydrogel forms a porous structure that is easy to exert antibacterial properties, so that the antibacterial silk fibroin hydrogel has long-lasting antibacterial properties and electrical conductivity; the copper montmorillonite and the modified silk fibroin form physical crosslinks through hydrogen bonds, so that the copper montmorillonite is uniformly dispersed in the antibacterial silk fibroin hydrogel, thereby improving the mechanical properties of the antibacterial silk fibroin hydrogel; and copper is an essential element for the human body, and its toxicity is far less than that of silver-based antibacterial agents, so it is safer and cheaper, and the copper montmorillonite antibacterial agent has higher biological safety and lower cost.

[0007] Furthermore, the mass ratio of the modified silk fibroin to the copper montmorillonite is 100:(1-5). By further optimizing the relative amounts of copper montmorillonite and modified silk fibroin, the copper montmorillonite in the antibacterial silk fibroin hydrogel can be dispersed more evenly, avoiding the occurrence of agglomeration and precipitation, thereby making the antibacterial silk fibroin hydrogel have better comprehensive properties.

[0008] In a second aspect, the present invention further provides a method for preparing the above antimicrobial silk fibroin hydrogel, comprising the following steps: S1. Dissolving modified silk fibroin in water to form a modified silk fibroin aqueous solution, wherein the mass concentration of the modified silk fibroin in the modified silk fibroin aqueous solution is 20-35%; the modified silk fibroin is at least one of glycidyl methacrylate modified silk fibroin, methacrylic anhydride modified silk fibroin, ethyl methacrylate isocyanate modified silk fibroin and nadic anhydride modified silk fibroin; S2, adding copper montmorillonite to the modified silk fibroin aqueous solution in step S1, stirring evenly to obtain a mixed solution, wherein the mass ratio of the copper montmorillonite to the modified silk fibroin is (1-10):100; S3. Add a photoinitiator to the mixed solution described in step S2, stir evenly and then irradiate with blue light to obtain an antibacterial silk fibroin hydrogel.

[0009] The method for preparing the antibacterial silk fibroin hydrogel provided by the present invention comprises the following steps: firstly forming a modified silk fibroin aqueous solution with a mass concentration of 20-35%, then adding copper montmorillonite with a mass ratio of (1-10):100 to the modified silk fibroin aqueous solution, stirring to uniformly disperse the copper montmorillonite in the modified silk fibroin aqueous solution, and subsequently, under blue light irradiation, a photoinitiator generates free radicals, thereby photo-crosslinking the modified silk fibroin and the copper montmorillonite to form the antibacterial silk fibroin hydrogel. The modified silk fibroin used is a natural polymer with the advantages of abundant resources and good biocompatibility; the entire preparation process uses water as a solvent, which is green, low-carbon and environmentally friendly.

[0010] In a possible embodiment, the modified silk fibroin in step S1 is prepared by the following steps: D1, cutting the silk cocoons into pieces to remove the pupae, adding them into a 0.5% Na2CO3 aqueous solution and boiling them for 30 min, wherein the bath ratio of the silk cocoons to the Na2CO3 aqueous solution is 1:100, then washing the silk cocoons with deionized water for 8-10 times, and then drying them to obtain degummed silk fibroin; D2, weighing 10 g of the degummed silk fibroin described in step D1, adding it to 50 mL of 9.3 M LiBr aqueous solution, and dissolving it in a 60 °C water bath to obtain a degummed silk fibroin mixed solution; D3. Slowly add at least one of glycidyl methacrylate, methacrylic anhydride, ethyl isocyanate methacrylate and nadic anhydride to the degummed silk fibroin mixed solution described in step D2, and then dialyze and freeze-dry after the reaction to obtain modified silk fibroin.

[0011] The preparation method of the modified silk fibroin has the advantages of readily available raw materials, simple steps and high production efficiency.

[0012] In a possible implementation manner, the copper montmorillonite in step S2 is prepared by the following steps: M1. Copper sulfate pentahydrate is dispersed in water to form a copper sulfate pentahydrate aqueous solution, sodium montmorillonite is added to the copper sulfate pentahydrate aqueous solution, and then stirred in a water bath at 55-65° C. to react to obtain a copper montmorillonite solution; M2. The copper montmorillonite solution described in step M1 is centrifuged in a centrifuge to obtain a solid-liquid separation product, and then the supernatant of the solid-liquid separation product is poured out, and the remaining part is washed with an ethanol solution, and the washing is repeated multiple times until there is no Cu in the upper clear liquid. 2+ ions, and a blue precipitate was obtained; M3. Put the blue precipitate described in step M2 into an oven to dry, and then grind and sieve to obtain copper montmorillonite powder.

[0013] The copper montmorillonite preparation method uses copper sulfate pentahydrate and sodium montmorillonite as raw materials, reacts at a temperature of 55-65°C to obtain a copper montmorillonite solution, and obtains copper montmorillonite powder through centrifugation and drying. The method has the advantages of simple process and easy control.

[0014] In a possible implementation, the mass concentration of the copper sulfate pentahydrate aqueous solution in step M1 is 10-20 g / L. By controlling the mass concentration of the copper sulfate pentahydrate aqueous solution to 10-20 g / L, it is beneficial to obtain a higher reaction rate.

[0015] In a possible implementation, the mass ratio of the sodium montmorillonite to the copper sulfate pentahydrate in step M1 is 2: 1. By controlling the mass ratio of the sodium montmorillonite to the copper sulfate pentahydrate to be 2: 1, the purity and yield of the copper montmorillonite can be improved.

[0016] In a possible implementation, the photoinitiator in step S3 is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

[0017] In a possible implementation, the mass ratio of the photoinitiator to the modified silk fibroin in step S3 is (0.1-1): 100. By controlling the relative amounts of the photoinitiator and the modified silk fibroin, while the amount of the photoinitiator is sufficient to cause the cross-linking reaction of the modified silk fibroin, the waste of resources caused by excessive use of the photoinitiator and the reduction of the performance of the antibacterial silk fibroin hydrogel are avoided.

[0018] In a possible implementation, the wavelength of the blue light in step S3 is 365-405 nm, and the irradiation time is 1-5 min. By controlling the wavelength of the blue light used for irradiation and the irradiation time, the irradiation light intensity and light quantity are moderate, while ensuring that the modified silk fibroin can be induced to undergo a cross-linking reaction by the photoinitiator, it is also avoided that the modified silk fibroin is destroyed due to excessive energy of the irradiation light wave.

[0019] In a third aspect, the present invention also provides the application of the antimicrobial silk fibroin hydrogel in the biomedical field.

[0020] Based on the common knowledge in this field, the above-mentioned implementation modes can be combined arbitrarily.

[0021] The reagents and raw materials used in the present invention are commercially available.

[0022] The positive and progressive effects of the present invention are: The antibacterial silk fibroin hydrogel provided by the present invention uses copper montmorillonite formed by copper intercalation between montmorillonite layers as an antibacterial agent, and is compounded with modified silk fibroin to form an antibacterial silk fibroin hydrogel. The antibacterial silk fibroin hydrogel not only has long-lasting bactericidal properties and biosafety, but also has good mechanical properties and conductive properties. The antibacterial rate of the antibacterial silk fibroin hydrogel can be as high as 99.9%, the compression strain can be as high as 70%, and the stress can be as high as 0.325 Mpa. The preparation method of the antibacterial silk fibroin hydrogel provided by the present invention has the advantages of low cost and green environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a cross-sectional scanning electron micrograph of the antibacterial silk fibroin hydrogel prepared in Example 2.

[0024] Figure 2This is the element EDS mapping diagram of the antibacterial silk fibroin hydrogel prepared in Example 2.

[0025] Figure 3 This is a graph showing the test results of the antibacterial performance of the sustained-release solution of the antibacterial silk fibroin hydrogel prepared in Examples 1-3 against Staphylococcus aureus.

[0026] Figure 4 This is a graph showing the test results of the antibacterial performance of the sustained-release solution of the antibacterial silk fibroin hydrogel prepared in Examples 1-3 against Escherichia coli.

[0027] Figure 5 This is a compression performance test data chart of the hydrogels prepared in Examples 1-3 and Comparative Example 1.

[0028] Figure 6 The conductive properties test data of the hydrogels prepared in Examples 1-3 and Comparative Example 1 are shown in FIG. DETAILED DESCRIPTION

[0029] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation method and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable changes derived therefrom are still within the scope of protection of the claims of the present invention.

[0030] It should be noted that the endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0031] Unless otherwise defined, all terms, symbols and other scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with conventionally understood meanings are defined herein for the purpose of clarification or ease of reference, and such definitions herein should not be construed as indicating significant differences from conventional understandings in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents and instruments is carried out in accordance with the protocols and parameters given by the manufacturer.

[0032] Example 1

[0033] This embodiment provides an antimicrobial silk fibroin hydrogel, which is prepared by the following steps: (1) Preparation of glycidyl methacrylate modified silk fibroin: The silk cocoons were removed from the pupa, added to a 0.5% Na2CO3 aqueous solution and boiled for 30 min, with a bath ratio of 1:100 between the silk cocoons and the aqueous solution, and the process was repeated three times, followed by washing and drying to obtain degummed silk fibroin; 10 g of degummed silk fibroin was weighed, added to 50 mL of a 9.3 M LiBr aqueous solution, dissolved in a 60 °C water bath, and then 571 mmol of glycidyl methacrylate (GMA) was slowly added dropwise, reacted for 3 h, dialyzed for 3 days, and freeze-dried for 48 h to obtain glycidyl methacrylate-modified silk fibroin; (2) Preparation of copper montmorillonite: Weigh 7.5 g of copper sulfate pentahydrate and dissolve it in 500 mL of water. Add 15 g of sodium montmorillonite and stir in a 60 °C water bath for 6 h. Centrifuge at 8000 r / min for 3 minutes. Pour out the supernatant and wash with 50% ethanol solution. Repeat several times until there is no Cu in the supernatant. 2+ , drying, grinding and passing through a 200-mesh sieve, and sealing and storing to obtain copper montmorillonite powder; (3) Preparation of antibacterial silk fibroin hydrogel: 3 g of glycidyl methacrylate modified silk fibroin prepared in step (1) and 0.03 g of copper montmorillonite powder prepared in step (2) were weighed and dissolved in 10 mL of deionized water. The mixture was stirred at a constant speed for 30 min. 300 μL of 10% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate photoinitiator (LAP) was added under light-proof conditions. The mixture was stirred for another 10 min. The mixture was irradiated with a blue light flashlight with a wavelength of 405 nm for 5 min to obtain an antibacterial silk fibroin hydrogel.

[0034] Example 2

[0035] This embodiment provides an antimicrobial silk fibroin hydrogel, which is prepared by the following steps: (1) Preparation of glycidyl methacrylate modified silk fibroin: The silk cocoons were removed from the pupa, added to a 0.5% Na2CO3 aqueous solution and boiled for 30 min, with a bath ratio of 1:100 between the silk cocoons and the aqueous solution, and the process was repeated three times, followed by washing and drying to obtain degummed silk fibroin; 10 g of degummed silk fibroin was weighed, added to 50 mL of a 9.3 M LiBr aqueous solution, dissolved in a 60 °C water bath, and then 571 mmol of glycidyl methacrylate (GMA) was slowly added dropwise, reacted for 3 h, dialyzed for 3 days, and freeze-dried for 48 h to obtain glycidyl methacrylate-modified silk fibroin; (2) Preparation of copper montmorillonite: Weigh 7.5 g of copper sulfate pentahydrate and dissolve it in 500 mL of water. Add 15 g of sodium montmorillonite and stir in a 60 °C water bath for 6 h. Centrifuge at 8000 r / min for 3 minutes. Pour out the supernatant and wash with 50% ethanol solution. Repeat several times until there is no Cu in the supernatant. 2+ , drying, grinding and passing through a 200-mesh sieve, and sealing and storing to obtain copper montmorillonite powder; (3) Preparation of antibacterial silk fibroin hydrogel: 3 g of glycidyl methacrylate modified silk fibroin prepared in step (1) and 0.06 g of copper montmorillonite powder prepared in step (2) were weighed and dissolved in 10 mL of deionized water. The mixture was stirred at a constant speed for 30 min. 300 μL of 10% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate photoinitiator (LAP) was added under light-proof conditions. The mixture was stirred for another 10 min. The mixture was irradiated with a blue light flashlight with a wavelength of 405 nm for 5 min to obtain an antibacterial silk fibroin hydrogel.

[0036] Example 3

[0037] This embodiment provides an antimicrobial silk fibroin hydrogel, which is prepared by the following steps: (1) Preparation of glycidyl methacrylate modified silk fibroin: The silk cocoons were removed from the pupa, added to a 0.5% Na2CO3 aqueous solution and boiled for 30 min, with a bath ratio of 1:100 between the silk cocoons and the aqueous solution, and the process was repeated three times, followed by washing and drying to obtain degummed silk fibroin; 10 g of degummed silk fibroin was weighed, added to 50 mL of a 9.3 M LiBr aqueous solution, dissolved in a 60 °C water bath, and then 571 mmol of glycidyl methacrylate (GMA) was slowly added dropwise, reacted for 3 h, dialyzed for 3 days, and freeze-dried for 48 h to obtain glycidyl methacrylate-modified silk fibroin; (2) Preparation of copper montmorillonite: Weigh 7.5 g of copper sulfate pentahydrate and dissolve it in 500 mL of water. Add 15 g of sodium montmorillonite and stir in a 60 °C water bath for 6 h. Centrifuge at 8000 r / min for 3 minutes. Pour out the supernatant and wash with 50% ethanol solution. Repeat several times until there is no Cu in the supernatant. 2+ , drying, grinding and passing through a 200-mesh sieve, and sealing and storing to obtain copper montmorillonite powder; (3) Preparation of antibacterial silk fibroin hydrogel: 3 g of glycidyl methacrylate modified silk fibroin prepared in step (1) and 0.15 g of copper montmorillonite powder prepared in step (2) were weighed and dissolved in 10 mL of deionized water. The mixture was stirred at a constant speed for 30 min. 300 μL of 10% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate photoinitiator (LAP) was added under light-proof conditions. The mixture was stirred for another 10 min. The mixture was irradiated with a blue light flashlight with a wavelength of 405 nm for 5 min to obtain an antibacterial silk fibroin hydrogel.

[0038] Comparative Example 1 This comparative example provides a silk fibroin hydrogel, which is prepared by the following steps: (1) Preparation of glycidyl methacrylate modified silk fibroin: The silk cocoons were removed from the pupa, added to a 0.5% Na2CO3 aqueous solution and boiled for 30 min, with a bath ratio of 1:100 between the silk cocoons and the aqueous solution, and the process was repeated three times, followed by washing and drying to obtain degummed silk fibroin; 10 g of degummed silk fibroin was weighed, added to 50 mL of a 9.3 M LiBr aqueous solution, dissolved in a 60 °C water bath, and then 571 mmol of glycidyl methacrylate (GMA) was slowly added dropwise, reacted for 3 h, dialyzed for 3 days, and freeze-dried for 48 h to obtain glycidyl methacrylate-modified silk fibroin; (2) Preparation of silk fibroin hydrogel: Weigh 3.0 g of the glycidyl methacrylate-modified silk fibroin prepared in step (1) and dissolve it in 10 mL of deionized water. Stir at a constant speed for 30 minutes. Add 300 μL of 10% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate photoinitiator (LAP) in a dark environment. Stir for another 10 minutes. Irradiate with a blue light flashlight with a wavelength of 405 nm for 5 minutes to obtain a silk fibroin hydrogel.

[0039] The hydrogels in the embodiments and comparative examples were tested for performance, and the test methods and results are as follows: 1. Characterization of morphology and element distribution The cross-sectional morphology of the antimicrobial silk fibroin hydrogel prepared in Example 2 was observed by SEM scanning electron microscopy. Figure 1 As shown, it can be seen that the antimicrobial silk fibroin hydrogel prepared in Example 2 has a porous structure. The antimicrobial silk fibroin hydrogel in the corresponding area was subjected to elemental analysis, and the EDS Mapping diagram of each element is shown in FIG. Figure 2 As shown, it can be seen that copper montmorillonite is uniformly dispersed in the modified silk fibroin matrix.

[0040] 2. Antibacterial performance test 1) Antibacterial rate test Test method: The antibacterial performance was measured by the oscillation method: 10 mg of the dried gel sample was added to a 10 mL centrifuge tube. After UV sterilization for 2 hours, 3 mL of PBS was added, and then 2 mL of 10 5 CFU of bacterial solution (10 8 CFU bacterial solution was diluted with PBS, 10 μL 10 8 CFU of bacterial solution was added to 10 mL PBS), shaken, sealed and placed in a 37 ℃ constant temperature shaker for shaking culture for 16-24 h; 100 μL of the culture was added to 10 mL PBS to make 10 3 Then take 100 μL of bacterial solution and add 900 μL of PBS to dilute to 10 2 100 μL of the bacterial solution with a concentration of 1.5 μg / mL was taken and placed in the middle of the culture plate with agar medium, spread evenly, and cultured at 37 °C overnight, and then the number of colonies was calculated. The calculation formula of the inhibition rate is: inhibition rate (%) = (1-sample colony number / control colony number) * 100%.

[0041] Test results: The antibacterial rate data of the antibacterial silk fibroin hydrogels prepared in Examples 1-3 and the silk fibroin hydrogel prepared in Comparative Example 1 are shown in Table 1: It can be seen from the data in Table 1 that the antibacterial silk fibroin hydrogels prepared in Examples 1-3 have a very high antibacterial rate, which is significantly better than the antibacterial performance of the silk fibroin hydrogel prepared in Comparative Example 1. In Examples 1-3, the antibacterial silk fibroin hydrogels prepared in Examples 2 and 3 have a higher antibacterial rate, indicating that with the increase of montmorillonite content, the antibacterial performance is significantly improved, and the antibacterial rate of the antibacterial silk fibroin hydrogel in Example 3 is as high as 99.9% or more.

[0042] 2) Sustained-release antibacterial performance test Test method: Weigh 300 mg of each of the antibacterial silk fibroin hydrogels prepared in Example 1 to Example 3, sterilize them in a 75% ethanol glass container, add the samples to EP tubes in a sterile operating table, seal the tubes after adding 2 mL of PBS to each tube, and place them in a 37°C constant temperature shaker for sustained release for 1, 3, 5 and 7 days. After absorbing 500 µL of supernatant on the sterile operating table, add 500 µL of PBS, seal the extracted supernatant and store it in a 4°C refrigerator to obtain 1, 3, 5 and 7 days of sustained release solution.

[0043] 100 µL of slow-release solution was added to a 96-well plate, PBS was used as a blank control, and 100 µL of 10 7 -10 8The bacterial solution containing CFU was cultured in a 37 °C incubator for 16-24 h. The absorbance was measured using an ELISA reader (wavelength 620 nm). Inhibition rate (%) = (1- sample absorbance / control absorbance) * 100%.

[0044] Test results: Test results such as Figure 3 and Figure 4 As shown in the figure, it can be seen that the antibacterial silk fibroin protein hydrogels prepared in Examples 1-3 all show a sustained release effect, and as the sustained release time increases, the antibacterial rate of the sustained release liquid of the antibacterial silk fibroin protein hydrogel prepared in Examples 1-3 against Staphylococcus aureus and Escherichia coli not only does not decrease, but gradually increases, indicating that copper ions can be slowly released from the hydrogel, indicating that the antibacterial silk fibroin protein hydrogel prepared in Examples 1-3 has excellent long-term antibacterial properties.

[0045] 3. Compression performance test The hydrogels prepared in Examples 1-3 and Comparative Example 1 were respectively made into cylindrical samples of the same size and subjected to compression performance tests using a universal material testing machine. Six parallel tests were performed for each sample. The results are shown in FIG. Figure 5 As shown in the figure, the compressive strain and stress of the antibacterial silk fibroin hydrogel prepared in Examples 1-3 are better than those of the silk fibroin hydrogel prepared in Comparative Example 1, and with the increase of the copper montmorillonite content, the compressive strain and stress are significantly increased, indicating that the uniform dispersion of copper montmorillonite in the modified silk fibroin matrix is ​​conducive to the large amount of silanol in montmorillonite to form physical crosslinks with silk fibroin through hydrogen bonding, thereby improving the mechanical properties of the hydrogel.

[0046] 4. Conductivity test The conductivity was measured using a ST512-S2T-2A four-probe tester (Beijing Zhongxi Yuanda Co., Ltd.) with a test current of 1 mA and a voltage of 2 V. The results are as follows: Figure 6 As can be seen from the figure, the conductivity of the antibacterial silk fibroin hydrogels prepared in Examples 1-3 is higher than that of the silk fibroin hydrogel prepared in Comparative Example 1, and as the content of copper montmorillonite increases, the conductivity of the antibacterial silk fibroin hydrogel is enhanced, and it is expected to be used in biosensors.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibacterial silk fibroin hydrogel, characterized in that: The components of the antibacterial silk fibroin hydrogel include modified silk fibroin and copper montmorillonite, wherein the modified silk fibroin is at least one of glycidyl methacrylate modified silk fibroin, methacrylic anhydride modified silk fibroin, ethyl methacrylate isocyanate modified silk fibroin and nadic anhydride modified silk fibroin, chemical crosslinks are formed between molecules of the modified silk fibroin, the copper montmorillonite and the modified silk fibroin are physically crosslinked through hydrogen bonds, and the mass ratio of the modified silk fibroin to the copper montmorillonite is 100:(1-10).

2. The antibacterial silk fibroin hydrogel according to claim 1, characterized in that: The mass ratio of the modified silk fibroin to the copper montmorillonite is 100:(1-5).

3. A method for preparing the antibacterial silk fibroin hydrogel according to claim 1 or 2, characterized in that: The following steps are involved: S1. Dissolving modified silk fibroin in water to form a modified silk fibroin aqueous solution, wherein the mass concentration of the modified silk fibroin in the modified silk fibroin aqueous solution is 20-35%; the modified silk fibroin is at least one of glycidyl methacrylate modified silk fibroin, methacrylic anhydride modified silk fibroin, ethyl methacrylate isocyanate modified silk fibroin and nadic anhydride modified silk fibroin; S2, adding copper montmorillonite to the modified silk fibroin aqueous solution in step S1, stirring evenly to obtain a mixed solution, wherein the mass ratio of the copper montmorillonite to the modified silk fibroin is (1-10):100; S3. Add a photoinitiator to the mixed solution described in step S2, stir evenly and then irradiate with blue light to obtain an antibacterial silk fibroin hydrogel.

4. The method for preparing the antibacterial silk fibroin hydrogel according to claim 3, characterized in that: The modified silk fibroin described in step S1 is prepared by the following steps: D1, cutting the silk cocoons into pieces to remove the pupae, adding them into a 0.5% Na2CO3 aqueous solution and boiling them for 30 min, wherein the bath ratio of the silk cocoons to the Na2CO3 aqueous solution is 1:100, then washing the silk cocoons with deionized water for 8-10 times, and then drying them to obtain degummed silk fibroin; D2, weighing 10 g of the degummed silk fibroin described in step D1, adding it to 50 mL of 9.3 M LiBr aqueous solution, and dissolving it in a 60 °C water bath to obtain a degummed silk fibroin mixed solution; D3. Slowly add at least one of glycidyl methacrylate, methacrylic anhydride, ethyl isocyanate methacrylate and nadic anhydride to the degummed silk fibroin mixed solution described in step D2, and then dialyze and freeze-dry after the reaction to obtain modified silk fibroin.

5. The method for preparing the antibacterial silk fibroin hydrogel according to claim 3, characterized in that: The copper montmorillonite described in step S2 is prepared by the following steps: M1. Copper sulfate pentahydrate is dispersed in water to form a copper sulfate pentahydrate aqueous solution, sodium montmorillonite is added to the copper sulfate pentahydrate aqueous solution, and then stirred in a water bath at 55-65° C. to react to obtain a copper montmorillonite solution; M2. The copper montmorillonite solution described in step M1 is centrifuged in a centrifuge to obtain a solid-liquid separation product, and then the supernatant of the solid-liquid separation product is poured out, and the remaining part is washed with an ethanol solution, and the washing is repeated multiple times until there is no Cu in the upper clear liquid. 2+ ions, and a blue precipitate was obtained; M3. Put the blue precipitate described in step M2 into an oven to dry, and then grind and sieve to obtain copper montmorillonite powder.

6. The method for preparing the antibacterial silk fibroin hydrogel according to claim 5, characterized in that: The mass concentration of the copper sulfate pentahydrate aqueous solution in step M1 is 10-20 g / L; And / or, the mass ratio of the sodium montmorillonite to the copper sulfate pentahydrate in step M1 is 2:

1.

7. The method for preparing the antibacterial silk fibroin hydrogel according to claim 3, characterized in that: The photoinitiator in step S3 is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

8. The method for preparing the antibacterial silk fibroin hydrogel according to claim 3, characterized in that: The mass ratio of the photoinitiator to the modified silk fibroin in step S3 is (0.1-1):

100.

9. The method for preparing the antibacterial silk fibroin hydrogel according to claim 3, characterized in that: In step S3, the wavelength of the blue light is 365-405 nm, and the irradiation time is 1-5 min.

10. Use of the antimicrobial silk fibroin hydrogel according to claim 1 or 2 in the biomedical field.

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