Preparation method for silk fibroin hydrogel

Abstract

Provided in the present application is a preparation method for a silk fibroin hydrogel. The preparation method comprises the following steps: adding natural silk to a sodium carbonate solution, and heating the solution to obtain degummed natural silk; dissolving the degummed natural silk in a lithium bromide solution, and adding glycidyl methacrylate thereto to perform a reaction, so as to obtain a reaction solution; dialyzing the reaction solution to obtain a supernate; freeze-drying the supernate to obtain a freeze-dried protein; and mixing an antibacterial agent solution with a photoinitiator solution, adding the freeze-dried protein thereto, and irradiating same with blue-violet light to obtain a silk fibroin hydrogel. In the present application, glycidyl methacrylate is used to modify the degummed natural silk (fibroin); and an antibacterial agent and a photoinitiator are added in the hydrogel-forming step, and irradiation is performed with blue-violet light to subject the modified fibroin to curing crosslinking, thereby preparing a silk fibroin hydrogel having antibacterial performance, which hydrogel can be widely used in the fields of biomedical materials, etc. The preparation method of the present application is simple and fast, and has a high hydrogel-forming speed.

Description

Preparation method of silk fibroin hydrogel Technical Field

[0001] The present application relates to the technical field of gel materials, and in particular to a method for preparing a silk fibroin hydrogel. Background Art

[0002] Silk fibroin has good biocompatibility, can promote the growth of human cells, and has certain biodegradability. Therefore, silk fibroin has received widespread attention in the fields of textiles, biomedicine, etc.

[0003] Gel is a physical state between solid and liquid with unique properties, especially hydrogel, which retains a large amount of water and can maintain a certain shape. It is currently widely used in artificial skin, tissue engineering materials, drug release, artificial muscle, biosensors and other aspects.

[0004] Silk fibroin hydrogels can be prepared from silk fibroin solutions, which gradually gel upon standing. These solutions can be used in wound repair, surgical sutures, and tissue repair. However, existing silk fibroin hydrogels suffer from drawbacks such as long gelling times and limited functionality (for example, lack of antibacterial properties), which restrict their practical application.

[0005] Summary of the Invention

[0006] In view of this, the present application proposes a method for preparing a silk fibroin hydrogel, which has a short gelation time and the prepared silk fibroin hydrogel has antibacterial properties.

[0007] One embodiment of the present application provides a method for preparing a silk fibroin hydrogel, comprising the following steps:

[0008] Degumming: Add silk to sodium carbonate solution and heat to obtain degummed silk;

[0009] Modification: dissolving the degummed silk in a lithium bromide solution, and then adding glycidyl methacrylate to react to obtain a reaction solution;

[0010] Dialysis: dialyzing the reaction solution to obtain a supernatant;

[0011] Lyophilization: lyophilizing the supernatant to obtain lyophilized protein;

[0012] Gelation: mixing the antibacterial agent solution and the photoinitiator solution, then adding the freeze-dried protein to obtain a freeze-dried protein solution, and irradiating the freeze-dried protein solution with blue-violet light to obtain the silk fibroin hydrogel.

[0013] In one embodiment, the antibacterial solution includes nanosilver solution, nanocopper solution or nanozinc solution.

[0014] In one embodiment, the photoinitiator solution is a phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt solution, and the mass volume concentration of the photoinitiator solution is 0.25% to 5%.

[0015] In one embodiment, the wavelength of the blue-violet light is 365 nm to 405 nm.

[0016] In one embodiment, the blue-violet light irradiation time is 10 s to 20 s.

[0017] In one embodiment, before the dissolving step, the preparation method further comprises: drying the degummed silk.

[0018] In one embodiment, after the reaction solution is dialyzed, the preparation method further comprises: centrifuging the dialyzed reaction solution to obtain the supernatant.

[0019] In one embodiment, the molar ratio of the antibacterial agent solution to the photoinitiator solution is (1-2):1.

[0020] In one embodiment, the concentration of the lithium bromide solution is 9.3 mol / L~9.5 mol / L.

[0021] In one embodiment, the concentration of the sodium carbonate solution is 0.02 mol / L~0.05 mol / L.

[0022] This application uses glycidyl methacrylate to modify degummed silk (fibroin). An antimicrobial agent and a photoinitiator are added during the gelation step. The modified silk fibroin is then cured and cross-linked using violet light irradiation, thereby producing a silk fibroin hydrogel with antimicrobial properties. This method is simple and rapid, with a high gelation rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1 is a UV-visible spectrum of the silk fibroin hydrogels prepared in Example 1 and Comparative Example 1 of the present application.

[0024] FIG2 is the antibacterial ring test results of the silk fibroin hydrogels prepared in Example 1 and Comparative Example 1 of the present application.

[0025] FIG3 shows the antibacterial rate test results of the silk fibroin hydrogels prepared in Example 1 and Comparative Example 1 of the present application.

[0026] The following specific implementation methods will further illustrate the embodiments of the present application in conjunction with the above-mentioned drawings.

[0027] DETAILED DESCRIPTION

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the art of the present application. The terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present application.

[0029] The present application proposes a method for preparing a silk fibroin hydrogel, which includes the steps of degumming, modification, dialysis, freeze-drying and gelation.

[0030] The main components of silk are sericin and silk fibroin (SF, a protein also known as fibroin or silk). Sericin accounts for approximately 20%-30% of the silk's weight, while fibroin accounts for approximately 70%-80%. Degumming involves removing the sericin from the silk. Specifically, the silk is placed in a sodium carbonate (Na2CO3) solution and heated until no yellow colloid precipitate (the yellow colloid precipitate is sericin) is produced, yielding degummed silk (i.e., silk fibroin). The concentration of the Na2CO3 solution can range from, but is not limited to, 0.02 mol / L to 0.05 mol / L. For example, the concentration of the Na2CO3 solution can be 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, and so on. A Na2CO3 solution can be heated to boiling. Shredded silk from mulberry silkworms (silk cocoons) is then added to the boiling Na2CO3 solution and heated continuously to degummed silk. The degummed silk can then be washed with copious amounts of deionized water to remove sericin and excess sodium carbonate.

[0031] In some embodiments, after degumming, the degummed silk may be dried. For example, the degummed silk may be dried in a vacuum drying oven to completely dehydrate the degummed silk and obtain dry silk fibroin.

[0032] The modification step specifically involves dissolving the dried degummed silk (dried silk fibroin) in a lithium bromide solution, then adding glycidyl methacrylate (GMA) to react and produce a reaction solution. The reaction can be carried out under heating and stirring conditions. The reaction is complete when the solid material dissolves completely and the solution turns yellow. GMA should be added in the dark to prevent oxidative decomposition of the raw material. Due to the unique spatial structure of silk fibroin molecules, they are highly susceptible to crystallization and poorly soluble in water before modification. Methacrylation of silk fibroin with GMA introduces double bonds into the silk fibroin molecules. Due to the introduction of additional chemical groups, the modified silk fibroin molecules (SF-GMA) are less susceptible to crystallization and are soluble in water, allowing the SF-GMA to be photocured in the subsequent step to form a hydrogel. The use of GMA stabilizes the reaction, primarily through epoxide ring opening, without the production of acidic byproducts. Furthermore, GMA promotes reactions with amino groups. Theoretically, two GMA molecules can react with one free amino group, thus maximizing the number of vinyl groups on the SF. GMA also reacts with the hydroxyl and carboxyl groups in SF molecules. These functional groups can promote further methacrylation of SF through an ester exchange mechanism (as a secondary mechanism), increasing the amount of vinyl methacrylate, thereby maximizing the crosslinking density of the hydrogel and significantly shortening the gelation time of the hydrogel. In addition, the SF-GMA has a block structure that can load and encapsulate antimicrobial agents, making the formed silk fibroin hydrogel antimicrobial.

[0033] In some embodiments, the concentration of the lithium bromide solution is 9.3 mol / L to 9.5 mol / L. For example, the concentration of the lithium bromide solution can be 9.3 mol / L, 9.4 mol / L, 9.5 mol / L, and so on.

[0034] The dialysis step is specifically as follows: the reaction solution is placed in a dialysis bag and dialyzed for three days, with the water changed three times a day. The dialysis can remove lithium bromide and impurities such as unreacted silk fibroin and GMA. When the liquid in the dialysis bag becomes turbid from clear and the dialysis bag expands, it indicates that the dialysis is complete. In some embodiments, the dialyzed SF-GMA can also be centrifuged to remove the supernatant. The speed of the centrifuge can be 5000 rpm, and the time can be 1 h. The speed and time of the centrifugation can be adjusted according to actual needs.

[0035] The freeze-drying step specifically includes: placing the supernatant in a -20°C refrigerator and freezing overnight, and then placing it in a freeze dryer and freeze-drying overnight to obtain a sponge-like (porous) freeze-dried protein.

[0036] The gelation step is specifically as follows: mixing an antibacterial agent solution with a photoinitiator solution, then adding the freeze-dried protein, stirring and ultrasonic treatment are performed to promote the dissolution of the freeze-dried protein to obtain a freeze-dried protein solution, and irradiating the freeze-dried protein solution with blue-violet light to obtain the silk fibroin hydrogel with antibacterial properties.

[0037] In some embodiments, the antibacterial solution includes a nano-silver solution, a nano-copper solution, or a nano-zinc solution.

[0038] In some embodiments, the photoinitiator solution is a lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP, CAS No. 85073-19-4) solution, and the mass volume concentration (w / v) of the photoinitiator solution is 0.25% to 5%. For example, the mass volume concentration of the photoinitiator solution can be 0.25%, 0.30%, 0.40%, 0.50%, 1%, 2%, 3%, 4%, 5%, etc.

[0039] In some embodiments, the molar ratio of the antimicrobial solution to the photoinitiator solution is (1-2):1. For example, when the antimicrobial solution is a nanosilver solution and the photoinitiator solution is a LAP solution, when the nanosilver solution is added dropwise to the LAP solution, the LAP content in the mixture of the two is a maximum of 0.1 mol (in which case the molar ratio of nanosilver to LAP is 1:1) and a minimum of 0.05 mol (in which case the molar ratio of nanosilver to LAP is 2:1) when the nanosilver content is 0.1 mol.

[0040] In some embodiments, the wavelength of the blue-violet light is 365 nm to 405 nm. For example, the wavelength of the blue-violet light is 365 nm, 370 nm, 375 nm, 380 nm, 390 nm, 400 nm, 405 nm, etc. The photoinitiator LAP can cause the dissolved freeze-dried protein to undergo a cross-linking reaction under irradiation with the blue-violet light having a wavelength of 365 nm to 405 nm, thereby forming a silk fibroin hydrogel.

[0041] In some embodiments, the blue-violet light irradiation time is 10 s to 20 s.

[0042] The present application will be further described below with reference to specific embodiments.

[0043] Example 1

[0044] (1) Degumming: Add 3.18 g of sodium carbonate solid to 1.5 L of ultrapure water to prepare a sodium carbonate solution with a concentration of 0.02 M (mol / L), and heat the sodium carbonate solution until it boils; weigh 20 g of mulberry silk (silk cocoon), cut it into pieces, and then put the cut mulberry silk into the boiling sodium carbonate solution. Continue heating for about 1 h until no yellow gelatinous precipitate is produced, indicating that all the sericin has been removed. Stop heating, pour off the sodium carbonate solution, take out the degummed silk, and wash the degummed silk with a large amount of deionized water.

[0045] (2) Drying: Place the cleaned degummed silk in a vacuum drying oven for 12 hours to completely dehydrate it. Weigh the dehydrated degummed silk on a precision balance to obtain about 15 g of dry silk fibroin.

[0046] (3) Modification: Prepare 40 ml of 9.3 M lithium bromide solution, take 4 g of dried degummed silk (dried silk fibroin) and dissolve it in the lithium bromide solution, place the beaker containing lithium bromide and silk fibroin solution on a heating stirrer, and add GMA to the beaker. Set the heating stirrer to 60°C and continue heating and stirring for 4 hours until the solid matter is completely dissolved and the solution turns yellow, to obtain a reaction solution, the main component of which is the modified silk fibroin molecule (SF-GMA).

[0047] (4) Dialysis: Place the reaction solution in the beaker into a dialysis tape and dialyze for three days, changing the water three times a day. Pour the dialyzed liquid into a centrifuge tube and centrifuge it at 5000 rpm for 1 hour. Remove the supernatant.

[0048] (5) Freeze-drying: Freeze the supernatant in a -20°C refrigerator overnight, and then freeze-dry it in a freeze dryer overnight to obtain a sponge-like (porous) freeze-dried protein.

[0049] (6) Gel formation: The silver nanoparticles solution was mixed with a 0.25% (w / v) LAP solution using a magnetic stirrer and stirred for 5 to 6 hours. The resulting mixture had a molar ratio of silver nanoparticles to LAP of 1:1. Lyophilized protein was added to the mixture of silver nanoparticles and LAP, stirred, and ultrasonically treated to promote the dissolution of the lyophilized protein. After the lyophilized protein was completely dissolved, the lyophilized protein solution was irradiated with blue-violet light at a wavelength of 365 nm. The transition from liquid to hydrogel state was observed. After irradiation with blue-violet light for more than ten seconds (10 s to 20 s), the silk fibroin hydrogel AgNPs-GSF carrying silver nanoparticles was generated.

[0050] Nanosilver is not prone to developing drug resistance and has a strong inhibitory and killing effect on dozens of pathogenic microorganisms, including Escherichia coli, Staphylococcus aureus, Salmonella, and Streptococcus pneumoniae. Silver nanoparticles-loaded silk fibroin hydrogel, AgNPs-GSF, exhibits excellent antibacterial properties. It can release silver nanoparticles into the environment, inhibiting bacterial growth. Its unique bactericidal mechanism also prevents bacteria from developing drug resistance.

[0051] Comparative Example 1

[0052] In Comparative Example 1, the degumming, drying, modification, dialysis, and lyophilization steps were identical to those in Example 1 and are not further described here. The only difference between Comparative Example 1 and Example 1 is the gelation step. In Comparative Example 1, the gelation step involves adding the lyophilized protein to the LAP solution, stirring, and ultrasonicating to promote its dissolution. After complete dissolution, the lyophilized protein solution is irradiated with 365 nm violet light to produce the silk fibroin hydrogel, GMA-SF.

[0053] Test Example 1: UV-Vis Spectroscopy

[0054] The AgNPs-GSF aqueous solution before irradiation in the gelation step of Example 1 (i.e., the solution after the freeze-dried protein is completely dissolved but before light curing) is added to a 96-well plate, and the GMA-SF aqueous solution before irradiation in the gelation step of Comparative Example 1 (i.e., the solution after the freeze-dried protein is completely dissolved but before light curing) is added to a 96-well plate. The nanosilver solution is added to the 96-well plate, and then the aqueous solution is irradiated with blue-violet light (365 nm) in the 96-well plate to cause photocrosslinking to form a hydrogel. The 96-well plate is then scanned using a microplate reader in the range of 260 nm-700 nm to obtain the UV-visible spectrum shown in Figure 1.

[0055] As shown in Figure 1, the nanosilver solution exhibits a distinct absorption peak at 415 nm, attributed to surface plasmon resonance. The silk fibroin hydrogel of Example 1 exhibits a distinct absorption peak at 415 nm, demonstrating successful loading of the nanosilver into the silk fibroin hydrogel (resulting in AgNPs-GSF) while maintaining its original particle size. The silk fibroin hydrogel of Comparative Example 1 (GMA-SF) exhibits no absorption peak at 415 nm.

[0056] Test Example 2: Inhibition Ring Test

[0057] (1) Preparation of hydrogel samples: Prepare several metal sheets with a diameter of 10 mm. Use a pipette to apply the AgNPs-GSF aqueous solution (i.e., the solution after the freeze-dried protein is completely dissolved but before light curing) in the gelation step of Example 1 before irradiation on both sides of the metal sheet, with a smear amount of 50 μL on each side. After smearing, irradiate with blue-violet light (365 nm) to cure it. Repeat the above operation using the GMA-SF aqueous solution (i.e., the solution after the freeze-dried protein is completely dissolved but before light curing) in the gelation step of Comparative Example 1 before irradiation. Take three samples from each group, and after curing, sterilize them by ultraviolet irradiation and seal them for future use.

[0058] (2) Prepare LB medium: Prepare the medium solution using ultrapure water, tryptone, yeast extract, and NaCl. Sterilize the solution in a steam autoclave at high temperature and high pressure. Once the medium solution cools to approximately 40-50°C, use an electric pipette to transfer the medium into a sterile culture dish (plate) and allow to cool for later use.

[0059] (3) Bacteria coating: Use a pipette to take an appropriate amount of bacterial solution (E. coli) and inoculate it on the cooled solid culture medium. Then use a sterile spreading rod to evenly spread the bacterial solution on the surface of the culture medium to obtain a plate inoculated with E. coli. Follow the same steps to obtain a plate inoculated with Staphylococcus aureus.

[0060] (4) Placing materials: Use tweezers to place the metal sheet coated with evenly coated hydrogel in the middle of the plate and gently press it to make it adhere. Then, turn the plate upside down and place it in a 37°C constant temperature incubator.

[0061] (5) Observation results: After 12 h of incubation, the samples were taken out of the incubator and the growth of the colonies and the formation of inhibition rings were observed and recorded. The results are shown in Figure 2.

[0062] The inhibition zone is a relatively intuitive and convenient method for qualitatively determining whether a material possesses antibacterial properties. If bacteria can grow on the agar plate surrounding the material, it indicates that the material has no killing or inhibitory effect on bacteria. If one or more sterile areas appear around the material, these areas are called inhibition zones. Generally speaking, the larger the inhibition zone, the greater the antibacterial effect of the material.

[0063] Referring to Figure 2, the GMA-SF hydrogel prepared in Comparative Example 1 did not produce an inhibition zone on a plate inoculated with Staphylococcus aureus and Escherichia coli, indicating that the GMA-SF hydrogel has no antibacterial activity against these two bacteria. In contrast, the AgNPs-GSF hydrogel in Example 1 produced relatively clear inhibition zones on plates inoculated with both Staphylococcus aureus and Escherichia coli, demonstrating that the AgNPs-GSF hydrogel prepared in Example 1 of the present application exhibits significant inhibitory and killing effects against both Staphylococcus aureus and Escherichia coli.

[0064] Test Example 3: Antibacterial Rate Test

[0065] (1) Sample preparation: The AgNPs-GSF aqueous solution before irradiation in the gelation step of Example 1 (i.e., the solution after the freeze-dried protein is completely dissolved but before light curing) and the GMA-SF aqueous solution before irradiation in the gelation step of Comparative Example 1 were placed in a 48-well plate and irradiated with blue-violet light (365 nm) in the 48-well plate to solidify into a hydrogel at the bottom of the well plate.

[0066] (2) Preparation of test samples: Add alcohol to the 48-well plate with solidified hydrogel to allow the alcohol to soak the hydrogel, then replace the alcohol with PBS (phosphate buffered saline), repeat several times to remove the alcohol to prevent the alcohol from affecting the antibacterial test results, and place the sample under ultraviolet light for 30 minutes, dry it, and set aside.

[0067] (3) LB medium preparation: Prepare a solution using ultrapure water, tryptone, yeast extract, and NaCl. Sterilize the solution in a steam autoclave at high temperature and high pressure. Once the culture medium cools to approximately 40-50°C, use an electric pipette to transfer the culture medium into a sterile culture dish (plate) and allow to cool for later use.

[0068] (4) Co-culture + dilution coating counting test: Dilute the bacterial solution (Escherichia coli, Staphylococcus aureus) to 10 6 CFU / mL, 400 μL of bacterial solution was evenly added to the test samples of Example 1 and Comparative Example 1, and then placed in a 37°C constant temperature shaker for 12 h. After the shaking culture was completed, the bacterial solution was diluted 10 4 , 10 5 , 10 6 After multiples, take 100 μL of the diluted bacterial solution and spread it on the culture medium of the plate. Place the inoculated plate in a constant temperature incubator and culture it for 18 hours. Take out the plate, take a picture of it, and count the number of colonies. Each material can be tested in triplicate.

[0069] The oscillating co-culture-dilution plate method is a common method for testing the sensitivity of bacteria to antimicrobial substances. The material and bacterial solution are co-cultured and the bacterial solution after co-culture is used for coating. It can more objectively evaluate the antibacterial rate of the material, and can also quantitatively measure it and calculate the antibacterial rate.

[0070] The experimental results of the antibacterial rate test are shown in Figure 3. As shown in Figure 3, even at the smallest dilution factor (10 4 ), in the experimental group of AgNPs-GSF hydrogel co-culture prepared in Example 1, no colonies were formed for either Escherichia coli or Staphylococcus aureus (the total bacterial colony count was 0). In the control group of GMA-SF hydrogel co-cultured prepared in Comparative Example 1, at 10 4 At a dilution multiple of , the colonies of the two bacteria almost covered the entire plate, indicating that the AgNPs-GSF hydrogel prepared in Example 1 of the present application had excellent antibacterial properties.

[0071] The antibacterial rate is calculated according to the following formula: Wherein, η represents the antibacterial rate of AgNPs-GSF prepared in Example 1, a represents the total bacterial colony count after oscillation co-culture of GMASF prepared in Comparative Example 1, and A represents the total bacterial colony count after oscillation co-culture of AgNPs-GSF prepared in Example 1. 6 At the dilution multiple of 10, the colony counts of Staphylococcus aureus in the three culture media after oscillation co-culture of GMASF prepared in Comparative Example 1 were 30, 50 and 87 respectively. 6 At a dilution factor of , the colony counts of E. coli in the three culture media after shaking co-culture of GMASF prepared in Comparative Example 1 were 56, 61, and 75, respectively. The antibacterial rate of the AgNPs-GSF hydrogel prepared in Example 1 of the present application was 100%.

[0072] This application uses glycidyl methacrylate to modify degummed silk (fibroin). An antimicrobial agent and a photoinitiator are added during the gelation step. The modified silk fibroin is then cured and cross-linked using violet light irradiation, thereby producing a silk fibroin hydrogel with antimicrobial properties. This method is simple and rapid, with a high gelation rate.

[0073] The above description is some specific implementation methods of the present application, but in actual application, it is not limited to these implementation methods. For ordinary technicians in this field, other variations and changes made according to the technical concept of the present application should fall within the scope of protection of the present application.

Claims

1. A method for preparing a silk fibroin hydrogel, characterized in that: The preparation method comprises the following steps: Degumming: Add silk into sodium carbonate solution and heat to obtain degummed silk; Modification: dissolving the degummed silk in a lithium bromide solution, and then adding glycidyl methacrylate to react to obtain a reaction solution; Dialysis: dialyzing the reaction solution to obtain a supernatant; Freeze-drying: freeze-drying the supernatant to obtain freeze-dried protein; Gelation: mixing the antibacterial agent solution with the photoinitiator solution, then adding the freeze-dried protein to obtain a freeze-dried protein solution, and irradiating the freeze-dried protein solution with blue-violet light to obtain the silk fibroin hydrogel.

2. The preparation method according to claim 1, characterized in that The antibacterial agent solution includes nano silver solution, nano copper solution or nano zinc solution.

3. The preparation method according to claim 1, characterized in that: The photoinitiator solution is a phenyl (2,4,6-trimethylbenzoyl) lithium phosphate solution, and the mass volume concentration of the photoinitiator solution is 0.25% to 5%.

4. The preparation method according to claim 1, characterized in that: The wavelength of the blue-violet light is 365 nm to 405 nm.

5. The preparation method according to claim 1, characterized in that: The blue-violet light irradiation time is 10 s to 20 s.

6. The preparation method according to claim 1, characterized in that: Before the dissolving step, the preparation method further comprises: drying the degummed silk.

7. The preparation method according to claim 1, characterized in that: After the reaction solution has completed dialysis, the preparation method further comprises: centrifuging the reaction solution after dialysis to obtain the supernatant.

8. The preparation method according to claim 1, characterized in that: The molar ratio of the antibacterial agent solution to the photoinitiator solution is (1-2):

1.

9. The preparation method according to claim 1, characterized in that: The concentration of the lithium bromide solution is 9.3 mol / L~9.5 mol / L.

10. The preparation method according to claim 1, characterized in that: The concentration of the sodium carbonate solution is 0.02 mol / L~0.05 mol / L.

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