Preparation method and application of silk fibroin hydrogel

Abstract

The invention provides a silk fibroin hydrogel preparation method, which comprises: S1, dissolving degummed silk in a neutral salt solution to obtain a silk fibroin dissolving solution; s2, diluting the silk fibroin dissolving solution prepared in the step S1 with a diluent to obtain a silk fibroin dissolving diluent; s3, carrying out physical cross-linking treatment on the silk fibroin dissolving diluent prepared in the step S2 or regulating the pH value for physical cross-linking or adding a surfactant for physical cross-linking, and incubating to obtain the silk fibroin hydrogel. The silk fibroin hydrogel is simple in preparation process, high in gelling rate, low in loss and high in raw material utilization rate.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and particularly relates to a preparation method and application of a silk fibroin hydrogel. Background Art

[0002] Silk is a natural high-quality fiber mainly composed of fibroin and sericin. Among them, fibroin, as the main component of silk, accounts for about 75%. The silk fibroin obtained by degumming and dissolving has excellent mechanical properties, good biocompatibility and easy processing characteristics, and can be made into various biomaterials, including scaffolds, microspheres, films, nanofibers and hydrogels, etc., and has received extensive attention and applications in many fields such as biomedicine, tissue engineering, food, cosmetics and optoelectronics in recent years.

[0003] Hydrogels are considered to be functional polymer materials formed by cross-linking hydrophilic polymer through physical or chemical methods, with a three-dimensional network structure and high water content, and have good permeability to many chemical and biological molecules. Hydrogels can reduce the friction force on the surrounding tissues due to their soft and moist characteristics. According to the material source, hydrogels can be divided into natural and synthetic polymer hydrogels. Natural polymer hydrogel materials also include natural polymer proteins such as gelatin, collagen, silk, and natural polymer polysaccharides such as hyaluronic acid, sodium alginate, chitosan, and agarose. Compared with synthetic polymer material hydrogels, natural polymer material raw materials have rich yields, wide sources, good biocompatibility and degradability, and have become the focus of attention of researchers. Silk fibroin has rich functional groups, mechanical properties and degradability, and has been deeply studied in many fields such as tissue engineering scaffolds, tissue adhesives, drug delivery carriers, and flexible wearable electronic devices.

[0004] Hydrogels are one of the main forms of silk fibroin materials and have shown broad application prospects in many fields such as biomedicine, tissue engineering, drug delivery, biomimetic mineralization, and medical aesthetic products, becoming products in the field of biomedical materials in recent years. Currently, the preparation of silk fibroin hydrogels mainly includes degumming, dissolving, and dialysis to obtain a pure silk fibroin aqueous solution, and then hydrogels are prepared by the following methods: (1) Physical cross-linking: Through physical methods such as freeze-thaw cycles, heating-cooling treatments, or ultraviolet irradiation, cross-linking structures are formed between silk fibroin molecules, thereby solidifying into hydrogels. (2) Chemical cross-linking: Cross-linking agents (such as glutaraldehyde, genipin, etc.) are added to the silk fibroin solution, and covalent bonds are formed between silk fibroin molecules through chemical reactions to prepare hydrogels. (3) Self-assembly: Under certain conditions, silk fibroin molecules can spontaneously assemble into hydrogels with specific structures. This method usually requires controlling conditions such as the concentration, pH value, and temperature of the solution. The long technological processes of these preparation methods, the instability of silk protein solutions, and the low yield of silk protein production limit the batch preparation and application of silk fibroin gels. Summary of the Invention

[0005] Technical problems to be solved: The purpose of the present invention is to provide a preparation method for silk fibroin hydrogels, to solve problems such as high loss of silk fibroin, long gel-forming process, and poor controllability in the previous preparation process, and to achieve the batch preparation and application of silk fibroin hydrogels.

[0006] Technical solution: A preparation method for silk fibroin hydrogels, the preparation method includes the following steps: S1. Dissolve the degummed silk in a neutral salt solution to obtain a silk fibroin solution; S2. Dilute the silk fibroin solution prepared in step S1 with a diluent to obtain a diluted silk fibroin solution; S3. Perform physical cross-linking treatment on the diluted silk fibroin solution prepared in step S2, or adjust the pH for physical cross-linking, or add a surfactant and then perform physical cross-linking, and incubate to obtain a silk fibroin hydrogel.

[0007] Preferably, the preparation method further includes the following steps: Desalt the silk fibroin hydrogel prepared in step S3 by dialysis or ultrafiltration to obtain a salt-free silk fibroin hydrogel; Or, perform mineralization treatment on the silk fibroin hydrogel prepared in step S3 to obtain a mineralized silk fibroin composite hydrogel.

[0008] Preferably, the neutral salt solution in step S1 includes an aqueous lithium bromide solution and a ternary solution of calcium chloride / ethanol / water. The concentration of the lithium bromide solution is 8-10 mol / L, and the molar ratio of calcium chloride, ethanol, and water in the ternary solution is 1:2:8; the dissolution temperature is 50-80 °C, and the dissolution time is 1-8 h; the concentration of the silk fibroin solution is 12-25 wt%.

[0009] Preferably, the diluent in step S2 includes any one or more of deionized water, sodium chloride, or phosphate buffer solution.

[0010] Preferably, the concentration of silk fibroin in the silk fibroin dissolution diluent in step S2 is 0.05-7 wt%.

[0011] Preferably, the physical cross-linking method in step S3 includes any one or more of ultrasonic cross-linking, vortex cross-linking, or repeated freeze-thaw cross-linking.

[0012] Preferably, the incubation temperature in step S3 is 25-70 °C, and the time is 6-160 h.

[0013] Preferably, the surfactant in step S3 includes any one or two of Span 80 or Tween 20; the pH range is 5-7.

[0014] Preferably, the power of the ultrasonic cross-linking is 10-50 W, the ultrasonic time is 2-6 min, the vortex speed is 1000-3000 rpm, and the vortex time is 1-7 min.

[0015] Application of the silk fibroin hydrogel prepared by the above preparation method in biomedicine, tissue engineering, aesthetic medicine injection, food, and cosmetics.

[0016] Beneficial effects: The preparation method of the present invention has the following advantages: The preparation process of the silk fibroin hydrogel in the present invention is stable: After the silk is dissolved in a high-concentration neutral salt, the stable β-sheet structure is broken into an amorphous structure. In the presence of the neutral salt, the amorphous silk fibroin can be maintained stably, but the aqueous silk fibroin solution after dialysis is extremely unstable. The present invention reduces the concentration of the neutral salt in the solution by dilution without dialysis. Due to the presence of the neutral salt, the silk fibroin solution is extremely stable. At this time, physical or chemical actions are used to promote the folding of silk protein molecules to form a hydrogel. This process is based on the stable characteristics of the low-concentration neutral salt silk fibroin solution; In the present invention, the preparation rate of the silk fibroin hydrogel is high: during the process of dissolving silk in medium-concentration neutral salt, the molecular chains of silk fibroin are damaged, and the high-molecular silk fibroin molecules are cut into silk fibroin with different molecular weights. At this time, a large amount of small-molecule silk fibroin will be lost through the dialysis process, resulting in a low preparation rate of silk fibroin. In the present invention, the diluted silk fibroin solution is directly converted into a gel before dialysis. Since the silk fibroin undergoes intermolecular folding and aggregation, the loss of small-molecule silk fibroin is avoided, and the preparation rate of the silk fibroin gel is close to 100%; In the present invention, the preparation process of the silk fibroin hydrogel is short and efficient: dialysis takes the longest time in the preparation process of the silk fibroin solution, and ultrafiltration also takes a long time due to the instability of the solution. In the present invention, the diluted silk fibroin solution is directly converted into a gel. Although the gel contains neutral salt, the low concentration of neutral salt does not affect the application of the silk fibroin gel, and it can form a functional gel through mineralization and has new uses; The silk fibroin hydrogel of the present invention has a wide range of uses: due to the good biocompatibility of silk fibroin, the silk fibroin hydrogel composed of it can be applied in the fields of biomedicine, tissue engineering, aesthetic injection, food, cosmetics, etc.; The process for preparing the silk fibroin hydrogel of the present invention is short, efficient, high-yield, and low-cost, so it is very suitable for the batch preparation and wide application of the silk fibroin hydrogel. Brief Description of the Drawings

[0017] Figure 1 is the effect diagram of the silk fibroin hydrogel prepared by the present invention; Figure 2 is the scanning electron micrograph of the silk fibroin hydrogel prepared by the present invention; Figure 3 is the infrared spectrum of the silk fibroin hydrogel prepared in Example 1; Figure 4 is the rheological diagram of the silk fibroin hydrogel prepared in Example 1; Figure 5 is the effect diagram of the silk fibroin hydrogel prepared in Comparative Example 1; Figure 6 is the effect diagram of the silk fibroin hydrogel prepared in Comparative Example 2; Figure 7 is the effect diagram of the silk fibroin hydrogel prepared in Comparative Example 3; Figure 8 is the effect diagram of the silk fibroin hydrogel prepared in Comparative Example 4; Figure 9 is the effect diagram of the silk fibroin hydrogel prepared in Comparative Example 5. Detailed Description of the Invention

[0018] The present invention will be further described below in conjunction with embodiments. The following embodiments are explanations of the present invention, and the present invention is not limited to the following embodiments: Example 1

[0019] A method for preparing a silk fibroin hydrogel, the preparation method comprising the following steps: S1. Dissolve the degummed silk in a ternary solution of calcium chloride / ethanol / water. The molar ratio of calcium chloride, ethanol and water in the ternary solution is 1:2:8. The dissolution temperature is 50 °C and the dissolution time is 4 h to obtain a silk fibroin solution with a concentration of 12 wt%. S2. Dilute the silk fibroin solution prepared in step S1 with deionized water to obtain a diluted silk fibroin solution with a concentration of 2.2 wt%. S3. Perform ultrasonic cross-linking treatment on the diluted silk fibroin solution prepared in step S2. The power of ultrasonic cross-linking is 50 W, the ultrasonic time is 2 min, and incubate at 35 °C for 48 h to obtain a silk fibroin hydrogel.

[0020] Example 2

[0021] A method for preparing a silk fibroin hydrogel, the preparation method comprising the following steps: S1. Dissolve the degummed silk in a ternary solution of calcium chloride / ethanol / water. The molar ratio of calcium chloride, ethanol and water in the ternary solution is 1:2:8. The dissolution temperature is 60 °C and the dissolution time is 2 h to obtain a silk fibroin solution with a concentration of 22 wt%. S2. Dilute the silk fibroin solution prepared in step S1 with deionized water to obtain a diluted silk fibroin solution with a concentration of 6 wt%. S3. Perform ultrasonic cross-linking treatment on the diluted silk fibroin solution prepared in step S2. The power of ultrasonic cross-linking is 40 W, the ultrasonic time is 4 min, and incubate at 55 °C for 24 h to obtain a silk fibroin hydrogel.

[0022] Example 3

[0023] A method for preparing a silk fibroin hydrogel, the preparation method comprising the following steps: S1. Dissolve the degummed silk in an 8 mol / L lithium bromide solution to obtain a silk fibroin solution with a concentration of 18 wt%. S2. Dilute the silk fibroin solution prepared in step S1 with deionized water to obtain a diluted silk fibroin solution with a concentration of 2 wt%. S3. Vortex crosslink the fibroin protein dissolution diluent prepared in step S2 at a vortex speed of 1000 rpm for 5 min, and incubate it at 55 °C for 24 h to obtain a fibroin hydrogel.

[0024] Example 4

[0025] A method for preparing a fibroin hydrogel, the preparation method comprising the following steps: S1. Dissolve the degummed silk in a lithium bromide solution with a concentration of 10 mol / L to obtain a fibroin protein dissolution solution with a concentration of 24 wt%; S2. Dilute the fibroin protein dissolution solution prepared in step S1 with deionized water to obtain a fibroin protein dissolution diluent with a concentration of 6.5 wt%; S3. Vortex crosslink the fibroin protein dissolution diluent prepared in step S2 at a vortex speed of 3000 rpm for 7 min, and incubate it at 55 °C for 24 h to obtain a fibroin hydrogel.

[0026] Example 5

[0027] A method for preparing a fibroin hydrogel, the preparation method comprising the following steps: S1. Dissolve the degummed silk in a lithium bromide solution with a concentration of 8.2 mol / L to obtain a fibroin protein dissolution solution with a concentration of 15 wt%; S2. Dilute the fibroin protein dissolution solution prepared in step S1 with deionized water to obtain a fibroin protein dissolution diluent with a concentration of 6.5 wt%; S3. Vortex crosslink the fibroin protein dissolution diluent prepared in step S2 at a vortex speed of 2000 rpm for 6 min, and incubate it at 55 °C for 24 h to obtain a fibroin hydrogel; S4. Dialyze the fibroin hydrogel prepared in step S3 under running water for 72 h to remove salt, and obtain a salt-free fibroin hydrogel.

[0028] Example 6

[0029] A method for preparing a fibroin hydrogel, the preparation method comprising the following steps: S1. Dissolve the degummed silk in a lithium bromide solution with a concentration of 9 mol / L to obtain a fibroin protein dissolution solution with a concentration of 15 wt%; S2. Dilute the fibroin protein dissolution solution prepared in step S1 with deionized water to obtain a fibroin protein dissolution diluent with a concentration of 5.2 wt%; S3. Vortex crosslink the fibroin protein dissolution diluent prepared in step S2 at a vortex speed of 3000 rpm for 5 min, and incubate it at 55 °C for 24 h to obtain a fibroin hydrogel; S4. Dialyze the fibroin hydrogel prepared in step S3 under running water for 72 h to remove salt, obtaining a salt-free fibroin hydrogel.

[0030] Comparative Example 1 A preparation method of a fibroin hydrogel, the preparation method comprising the following steps: S1. Dissolve the degummed silk in a lithium bromide solution with a concentration of 10 mol / L to obtain a fibroin protein dissolution solution with a concentration of 24 wt%; S2. Dilute the fibroin protein dissolution solution prepared in step S1 with deionized water to obtain a fibroin protein dissolution diluent with a concentration of 12 wt%; S3. Vortex crosslink the fibroin protein dissolution diluent prepared in step S2 at a vortex speed of 3000 rpm for 7 min, and incubate it at 55 °C for 24 h to obtain a fibroin hydrogel.

[0031] Comparative Example 2 A preparation method of a fibroin hydrogel, the preparation method comprising the following steps: S1. Dissolve the degummed silk in a lithium bromide solution with a concentration of 10 mol / L to obtain a fibroin protein dissolution solution with a concentration of 24 wt%; S2. Dilute the fibroin protein dissolution solution prepared in step S1 with deionized water to obtain a fibroin protein dissolution diluent with a concentration of 0.01 wt%; S3. Vortex crosslink the fibroin protein dissolution diluent prepared in step S2 at a vortex speed of 3000 rpm for 7 min, and incubate it at 55 °C for 24 h to obtain a fibroin hydrogel.

[0032] Comparative Example 3 A preparation method of a fibroin hydrogel, the preparation method comprising the following steps: S1. Dissolve the degummed silk in a magnesium chloride solution with a concentration of 10 mol / L, filter the insoluble silk, and obtain a fibroin protein dissolution solution with a concentration of 24 wt%; S2. Dilute the fibroin protein dissolution solution prepared in step S1 with deionized water to obtain a fibroin protein dissolution diluent with a concentration of 6.5 wt%; S3. Vortex crosslink the fibroin protein dissolution diluent prepared in step S2 at a vortex speed of 3000 rpm for 7 min, and incubate it at 55 °C for 24 h to obtain a fibroin hydrogel.

[0033] Comparative Example 4 A method for preparing a fibroin hydrogel, the preparation method comprising the following steps: S1. Dissolve the degummed silk in a ternary solution of calcium chloride / ethanol / water, where the molar ratio of calcium chloride, ethanol, and water in the ternary solution is 1:2:8, the dissolution temperature is 60 °C, and the dissolution time is 2 h to obtain a fibroin protein dissolution solution with a concentration of 22 wt%; S2. Dialyze the fibroin protein dissolution solution prepared in step S1 to remove calcium ions to obtain a fibroin protein aqueous solution, and concentrate the fibroin protein aqueous solution to obtain a fibroin protein solution with a concentration of 6 wt%; S3. Add calcium chloride to the fibroin protein solution prepared in step S2, and perform ultrasonic crosslinking treatment. The power of ultrasonic crosslinking is 40 W, the ultrasonic time is 4 min, and incubate it at 55 °C to obtain a fibroin hydrogel.

[0034] Comparative Example 5 A method for preparing a fibroin hydrogel, the preparation method comprising the following steps: S1. Dissolve the degummed silk in a ternary solution of calcium chloride / ethanol / water, where the molar ratio of calcium chloride, ethanol, and water in the ternary solution is 1:2:8, the dissolution temperature is 60 °C, and the dissolution time is 2 h to obtain a fibroin protein dissolution solution with a concentration of 22 wt%; S2. Dialyze the fibroin protein dissolution solution prepared in step S1 to remove calcium ions to obtain a fibroin protein aqueous solution, and concentrate the fibroin protein aqueous solution to obtain a fibroin protein solution with a concentration of 6 wt%; S3. Perform ultrasonic crosslinking treatment on the fibroin protein solution prepared in step S2. The power of ultrasonic crosslinking is 40 W, the ultrasonic time is 4 min, and incubate it at 55 °C for 360 h to obtain a fibroin hydrogel.

[0035] Performance test: After freeze-drying the hydrogels prepared in the examples and comparative examples, soak them in water to remove metal ions, then dry and weigh them, and calculate the loss rate; the specific calculation method is as follows: The degummed silk is denoted as G1, and the dried and weighed silk is denoted as G2: Loss rate = (the degummed silk denoted as G1 - the dried and weighed silk denoted as G2) / the degummed silk denoted as G1 * 100% As can be seen from the above table, the hydrogel obtained by the preparation method of the present invention has a lower loss rate compared with the hydrogel materials obtained by the previous preparation methods, and can save the usage amount of raw materials to the greatest extent.

[0036] The following further explains the experimental phenomena in conjunction with the accompanying drawings of the specification: From Figure 1 , 2 it can be seen that the silk fibroin hydrogel has a certain fluidity, but it is not in a solution state and has injectability. The scanning electron microscope image shows that the microstructure of the silk fibroin hydrogel is sheet-like, loose and fragile; as Figure 3 shown, the secondary structures of the silk fibroin solution and the silk fibroin hydrogel prepared in Example 1 were detected. Figure 1 It shows that the silk fibroin solution exhibits an amide I structure at 1640 cm -1 -1, and the amide I of the silk fibroin hydrogel is 1620 cm -1 -1, and the peak shifts, indicating the transformation of silk fibroin from a random coil to a β-sheet structure. Figure 4 From the rheological properties, it can be known that the storage modulus of the silk fibroin hydrogel is about 269 Pa, and the loss modulus (39.1 Pa) is lower than the storage modulus, further confirming the formation of the gel.

[0037] As Figure 5 shown, the appearance of the hydrogel prepared in Comparative Example 1 has no obvious change, the solution is light yellow, is a flowing liquid, and has not formed a gel.

[0038] As Figure 6 shown, flocculates precipitate in the hydrogel prepared in Comparative Example 2, the protein is separated from the liquid, and no gel is formed; therefore, it can be seen from the above two figures that neither too high nor too low concentration of the silk fibroin solution diluent can form a gel, and controlling the concentration of the diluent within a reasonable range is beneficial to the formation of the hydrogel.

[0039] As Figure 7 shown, the appearance of the hydrogel prepared in Comparative Example 3 shows milky white, but the solution has strong fluidity after inversion and no gel is formed. Although the same neutral salt is used for dissolution, the magnesium ions in the neutral salt may inhibit the formation of the gel, while the metal ions in the present invention do not inhibit the formation of the gel.

[0040] As Figure 8 shown, after introducing calcium chloride and ultrasonic treatment in the hydrogel prepared in Comparative Example 4, the solution presents a flowing milky white liquid state and still cannot incubate to form a hydrogel, while in Example 2, calcium ions combine with silk fibroin during the dissolution process and can promote the formation of the gel.

[0041] As Figure 9As shown, for the hydrogel prepared in Comparative Example 5, the formation of the hydrogel can be observed using the inversion method, but the incubation time is as long as 15 days and the loss rate is also higher. For the hydrogel prepared in Example 3, the incubation time only needs 24 hours, and the gel time is greatly shortened.

[0042] Obviously, the above examples are only for illustration purposes and are not intended to limit the implementation methods. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation methods here. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for preparing a silk fibroin hydrogel, characterized in that, The preparation method comprises the following steps: S1. Dissolve the degummed silk in a neutral salt solution to obtain a silk fibroin solution; S2. Dilute the silk fibroin solution prepared in step S1 with a diluent to obtain a diluted silk fibroin solution; S3. Perform physical crosslinking treatment on the diluted silk fibroin solution prepared in step S2, or adjust the pH for physical crosslinking, or add a surfactant and then perform physical crosslinking, and incubate to obtain a silk fibroin hydrogel.

2. The preparation method of the silk fibroin hydrogel according to claim 1, characterized in that, The preparation method further comprises the following steps: Desalt the silk fibroin hydrogel prepared in step S3 by dialysis or ultrafiltration to obtain a salt-free silk fibroin hydrogel; Or, perform mineralization treatment on the silk fibroin hydrogel prepared in step S3 to obtain a mineralized silk fibroin composite hydrogel.

3. The preparation method of the silk fibroin hydrogel according to claim 1, characterized in that: The neutral salt solution in step S1 includes an aqueous lithium bromide solution and a ternary solution of calcium chloride / ethanol / water. The concentration of the lithium bromide solution is 8-10 mol / L, and the molar ratio of calcium chloride, ethanol and water in the ternary solution is 1:2:8; the dissolution temperature is 50-80 °C, and the dissolution time is 1-8 h; the concentration of the silk fibroin solution is 12-25 wt%.

4. The preparation method of the silk fibroin hydrogel according to claim 1, characterized in that: The diluent in step S2 includes any one or more of deionized water, sodium chloride or phosphate buffer solution.

5. The preparation method of the silk fibroin hydrogel according to claim 1, characterized in that: The concentration of silk fibroin in the diluted silk fibroin solution in step S2 is 0.05-7 wt%.

6. The preparation method of the silk fibroin hydrogel according to claim 1, characterized in that: The physical crosslinking method in step S3 includes any one or more of ultrasonic crosslinking, vortex crosslinking or repeated freeze-thaw crosslinking.

7. The preparation method of the silk fibroin hydrogel according to claim 1, characterized in that: The incubation temperature in step S3 is 25-70 °C, and the time is 6-160 h.

8. The preparation method of the silk fibroin hydrogel according to claim 1, characterized in that: The surfactant in step S3 includes any one or both of Span 80 or Tween 20; the pH range is 5-7.

9. The preparation method of the silk fibroin hydrogel according to claim 6, characterized in that: The power of the ultrasonic crosslinking is 10-50 W, the ultrasonic time is 2-6 min, the vortex speed is 1000-3000 rpm, and the vortex time is 1-7 min.

10. Application of the silk fibroin hydrogel prepared by the preparation method according to claim 1 in biomedicine, tissue engineering, aesthetic injection, food and cosmetics.

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