Silk fiber material, preparation method and application

Abstract

The invention relates to the technical field of silk fiber materials, and discloses a silk fiber material and a preparation method and application thereof.The preparation method comprises the steps that degummed fibroin fiber serves as a matrix, and the degummed fibroin fiber is mildly modified through genipin-mediated chemical crosslinking or riboflavin-mediated photo-crosslinking or the combination of genipin-mediated chemical crosslinking and riboflavin-mediated photo-crosslinking; the modified degummed fibroin fiber shows excellent cell adhesion and proliferation performance, the biocompatibility is remarkably improved, meanwhile, the excellent mechanical property of natural silk is reserved, and the degummed fibroin fiber is suitable for various biomedical application scene requirements; and the modified degummed fibroin fiber also has fluorescence luminescence characteristics obviously different from those before modification, and is beneficial to visual positioning and monitoring in specific application scenes. The method can be compatible with an existing textile process, and has popularization and application values.

Description

Technical Field

[0001] This invention relates to the field of silk fiber material technology, specifically to a silk fiber material, its preparation method, and its application. Background Technology

[0002] my country is a major silkworm-producing country, with its annual silkworm cocoon production accounting for about 75% of the global total. After silk is degummed to remove sericin, degummed fibroin fiber is obtained. Since the degumming process does not damage the β-fold crystal structure inside the fiber, degummed fibroin fiber perfectly retains the excellent mechanical properties (high strength, high toughness) and luster of natural silk, and is currently widely used in the textile industry.

[0003] Degummed silk fibroin, as a natural protein fiber, possesses excellent biocompatibility and mechanical properties, making it a promising candidate for applications in biomedical materials. Currently, research on the application of degummed silk fibroin in biomedical materials is primarily based on silk fibroin protein. Silk fibroin is obtained by dissolving degummed silk fibroin in a specific salt solution (such as lithium bromide) or neutral salt, thereby disrupting the fiber's β-sheet crystal structure. During this dissolution and regeneration process, the stable β-sheet crystal structure of silk fibers is destroyed. Although silk fibroin can currently be processed into various forms such as microspheres, microcapsules, fibers, dressings, and hydrogels using different processing techniques (such as microfluidics) for application in various medical scenarios, the complexity of silk fibroin preparation and purification, as well as the cumbersome nature of subsequent processing, necessitates exploring and developing simpler and more efficient methods to expand the application range of silk fibroin fibers. In this regard, directly using degummed silk fibroin fibers as raw materials and undergoing specific treatments to prepare medical fiber materials has significant advantages. Summary of the Invention

[0004] In view of the problems mentioned in the background art, the purpose of this invention is to provide a silk fiber material, a preparation method and an application, which aims to prepare silk fiber material using degummed fibroin fiber as raw material, reduce the preparation cost and production process requirements of silk fiber material, and at the same time, the prepared silk fiber material has good biocompatibility and is suitable for biomedical application needs.

[0005] To achieve the above-mentioned technical effects, the present invention provides the following technical solution.

[0006] In a first aspect, the present invention provides a method for preparing silk fiber material, which includes the following steps: Step 1: Prepare degummed fibroin fibers: The degummed fibroin fibers are obtained by degumming raw silk from silkworm cocoons to remove sericin. Step 2: Modify the degummed silk fibroin fiber: The modification method of the degummed silk fibroin fiber is selected from any one of chemical crosslinking modification, photocrosslinking modification, and chemical-photocrosslinking combined modification; The chemical crosslinking modification includes the step of immersing the degummed silk fibroin fiber in crosslinking solution A in the dark for 0.5-3 hours for a chemical crosslinking reaction. The crosslinking solution A includes a buffer solution and 0.05-2.0 wt% genipin. The photocrosslinking modification includes the step of immersing the degummed silk fibroin fiber in crosslinking solution B and performing a photocrosslinking reaction for 1-3 minutes under irradiation with a 365-405nm light source. The crosslinking solution B includes a buffer solution, 0.2wt% riboflavin and 0.1wt% sodium persulfate. The chemical-photocrosslinking combined modification includes the steps of immersing the degummed silk fibroin fiber in crosslinking solution C, first performing a photocrosslinking reaction under 365-405nm light source irradiation for 1-3 min, and then performing a chemical crosslinking reaction in the dark for 0.5-3 h. The crosslinking solution C includes buffer solution, 0.05-2.0wt% genipin, 0.2wt% riboflavin and 0.1wt% sodium persulfate. Step 3: Wash the modified degummed silk fibers with deionized water and then dry them for later use.

[0007] Furthermore, in a preferred embodiment of the present invention, the modification method for the degummed silk fibroin fiber in step 2 is a chemical-photocrosslinking combined modification method.

[0008] Furthermore, the buffer solution is selected from deionized water, PBS buffer, and Tris buffer with a pH of 7.0-8.0.

[0009] Furthermore, in a preferred embodiment of the present invention, the degumming method in step 1 is an alkaline degumming process. The alkaline degumming process includes placing the raw silkworm cocoons in an alkaline solution and boiling for 30-60 minutes, followed by washing.

[0010] Furthermore, the strong alkaline solution is selected from a 0.05-0.5 wt% sodium carbonate solution.

[0011] Furthermore, in step 3, the drying method can be either freeze drying or low-temperature drying.

[0012] Furthermore, the temperature of the chemical crosslinking reaction in the chemical crosslinking modification and the chemical-photocrosslinking combined modification is 25-45℃.

[0013] This invention uses degummed silk fibroin fiber as a matrix and modifies it mildly through genipin-mediated chemical crosslinking, riboflavin-mediated photocrosslinking, or a combination of both. The modified degummed silk fibroin fiber exhibits excellent cell adhesion and proliferation properties, significantly improved biocompatibility, and also has fluorescent luminescence properties that are significantly different from those before modification.

[0014] Secondly, the present invention provides silk fiber materials prepared by the above-described preparation method.

[0015] Thirdly, the present invention provides the application of the above-mentioned silk fiber material in biomedical fiber materials.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The method for preparing silk fiber materials provided by the present invention modifies degummed silk fibroin fibers by genipin-mediated chemical crosslinking or riboflavin-mediated photocrosslinking or a combination of both, thereby giving the silk fiber materials better biocompatibility and making them suitable for various biomedical application scenarios. 2. The silk fiber material prepared by the preparation method provided by the present invention not only has excellent biocompatibility and is suitable for biomedical applications, but also has special fluorescent luminescence characteristics, which is beneficial for visual positioning and monitoring in specific application scenarios. 3. The modification methods used in this invention are all non-toxic or low-toxic systems, and the residues are easy to remove; 4. The preparation method provided by the present invention can use silk fibroin fibers degummed by alkaline method as raw materials. Compared with enzymatic degumming, the production process requirements are low and the cost is low. Moreover, the chemical crosslinking and photocrosslinking conditions involved in the modification method of the present invention are suitable for continuous yarn / fabric roll-to-roll processing, and can be compatible with existing textile processes, thus having promotion and application value. Attached Figure Description

[0017] Figure 1 This is a SEM image of the degummed silk fibroin fiber prepared in Example 1 of the present invention; Figure 2 These are microscopic images of degummed silk fibroin fibers prepared in Example 1 of the present invention under different light sources. Figure 3 This is a SEM image of the silk fiber material prepared in Example 2 of the present invention; Figure 4 These are microscopic images of the silk fiber material prepared in Example 2 of the present invention under different light sources. Figure 5 This is a SEM image of the silk fiber material prepared in Example 3 of the present invention; Figure 6 These are microscopic images of the silk fiber material prepared in Example 3 of the present invention under different light sources. Figure 7 This is a SEM image of the silk fiber material prepared in Example 4 of the present invention; Figure 8 These are microscopic images of the silk fiber material prepared in Example 4 of the present invention under different light sources. Figure 9 This is a cell viability detection diagram from the cell proliferation experiment of Example 5 of the present invention; Figure 10 This is a dual-fluorescence staining detection image of live / dead cells in the cell staining experiment of Example 5 of the present invention; Figure 11 This is a flowchart of the process for preparing silk fiber materials according to the present invention. Detailed Implementation

[0018] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative of the present invention and should not be considered as the entirety of the invention or as a limitation or restriction of the technical solution of the present invention. Unless otherwise specified, the materials and reagents used in the following embodiments can be obtained through conventional commercial channels.

[0019] Example 1: Preparation of degummed silk fibroin fibers Weigh 1g of silkworm cocoon using a balance and add it to 100mL of Na2CO3 solution (0.5wt%). Heat and boil for 0.5h, then wash with deionized water to remove sericin from the surface of the silk. Repeat the degumming process several times. After degumming, place the silk in an oven to obtain dried degummed fibroin fibers.

[0020] The degummed silk fibroin fibers prepared above were characterized using scanning electron microscopy, stereomicroscopy, and laser confocal microscopy. (See also...) Figure 1 As shown, the degummed silk fibroin fibers prepared in this embodiment have no obvious color ( Figure 1 a) The fiber surface has a slightly rough structure ( Figure 1 (b and c). See also Figure 2 As shown, in the dry state, the degummed silk fibroin fibers prepared in this embodiment exhibit fluorescence under 405 nm and 488 nm laser light. Figure 2 a); Under wet conditions, the degummed silk fibroin fibers prepared in this embodiment exhibited obvious swelling ( Figure 2 b1), and exhibited fluorescence attenuation under 405 nm and 488 nm laser excitation. Figure 2 b).

[0021] Example 2: Preparation of chemically cross-linked modified silk fiber materials Using the degummed silk fibroin fiber from Example 1 as the substrate, the following steps were then performed: (1) Prepare cross-linking solution: 0.5 wt% genipin in PBS buffer (pH 7.0).

[0022] (2) Immerse the degummed silk fiber in the above crosslinking solution and react at 35°C in the dark for 3 hours. It can be gently left to stand or shaken / circulated. After the reaction is completed, wash repeatedly with deionized water until the liquid is colorless. Dry at low temperature or freeze dry for later use.

[0023] The prepared silk fiber material was characterized using scanning electron microscopy, stereomicroscopy, and laser confocal microscopy. (See also...) Figure 3 As shown, the silk fiber material prepared in this embodiment has a brighter luster than that in Example 1. Figure 3 a) The fiber surface is smoother than that in Example 1. Figure 3 (b and c); see also Figure 4 As shown, in the wet state, the silk fiber material prepared in this embodiment exhibited bright fluorescence under laser excitation at 405 nm, 488 nm, 561 nm and 640 nm. Figure 4 ).

[0024] Example 3: Preparation of photocrosslinked modified silk fiber materials Using the degummed silk fibroin fiber from Example 1 as the substrate, the following steps were then performed: (1) Prepare cross-linking solution: 0.2 wt% riboflavin and 0.1 wt% sodium persulfate in PBS buffer (pH 7.0).

[0025] (2) Immerse the degummed silk fiber in the above crosslinking solution and carry out a photocrosslinking reaction for 1-3 minutes under 365nm ultraviolet light irradiation; after the reaction, wash repeatedly with deionized water until the liquid is colorless; dry at low temperature or freeze dry for later use.

[0026] The prepared silk fiber material was characterized using scanning electron microscopy, stereomicroscopy, and laser confocal microscopy. (See also...) Figure 5 As shown, the silk fiber material prepared by 2 minutes of ultraviolet light irradiation in this embodiment exhibits a colorful hue. Figure 5 a) The fiber surface is smoother than that in Example 1. Figure 5 (b and c); see also Figure 6 As shown, in the wet sample state, after 1 min ( Figure 6 a) 2 min ( Figure 6 b) and 3 min ( Figure 6 c) The silk fiber material obtained by ultraviolet light irradiation showed fluorescence under both 405 nm and 488 nm laser excitation, and showed stronger fluorescence under 488 nm laser than in Example 1.

[0027] Example 4: Preparation of chemically-photocrosslinked modified silk fiber materials Using the degummed silk fibroin fiber from Example 1 as the substrate, the following steps were then performed: (1) Prepare cross-linking solution: 0.5 wt% genipin, 0.2 wt% riboflavin, and 0.1 wt% sodium persulfate in PBS buffer (pH 7.0).

[0028] (2) The degummed silk fiber is immersed in the above crosslinking solution and first subjected to photocrosslinking reaction under 365nm ultraviolet light for 2 min, and then reacted at 35℃ for 3 h in the dark. After the reaction is completed, it is repeatedly washed with deionized water until the liquid is colorless. It is then dried at low temperature or freeze-dried for later use.

[0029] The prepared silk fiber material was characterized using scanning electron microscopy, stereomicroscopy, and laser confocal microscopy. (See also...) Figure 7 As shown, the silk fiber material prepared in this embodiment exhibits a colorful hue. Figure 7 a) The fiber surface is smoother than that in Example 1. Figure 7 (b and c); see also Figure 8 As shown, in the wet state, the silk fiber material prepared in this embodiment exhibited bright fluorescence under laser excitation at 488 nm and 561 nm. Figure 8 ).

[0030] Example 5: Biocompatibility Evaluation 1. Evaluation was conducted using cell proliferation experiments. The biocompatibility of the fiber materials prepared in Examples 1 to 4 was assessed using a mouse fibroblast (NIH3T3 cell) proliferation assay. 1 × 10⁻⁶ cells were used per well. 4 NIH3T3 cell suspension was seeded into 96-well plates and cultured at 37°C for 24 h. Cells adhered to the plates. The plates were then treated with solutions containing 10% fetal bovine serum (FBS), 1 U / mL penicillin, and 1×10⁻⁶ ppm. -3 1 mg / mL streptomycin was prepared in DMEM medium for the following fiber material sample solutions: Example 1 (unmodified / SF), Example 2 (chemically crosslinked / GS), Example 3 (photocrosslinked / RS), and Example 4 (chemical-photocrosslinked / RGS). The control group was prepared with an equal volume of 10% (FBS), 1 U / mL penicillin, and 1×10⁻⁶ oz. -3 The cells were cultured in DMEM medium containing mg / mL streptomycin, with three replicates per group. After 1 and 3 days of culture, the absorbance of each group at 450 nm was measured using a microplate reader with a Cell Counting Kit-8 (CCK8) to plot the change in cell viability over time.

[0031] See Figure 9 As shown, after co-incubating the fiber materials prepared in Examples 2 to 4 with NIH3T3 cells for 1 day and 3 days, their cell viability was higher than that of the control group and Example 1. The cell viability increased significantly on the 3rd day, with the fiber material in Example 4 showing the most significant increase.

[0032] 2. Evaluation was performed using a live / dead cell double staining assay. To more directly observe the effect of silk fiber materials on cell viability, the "live / dead cell dual fluorescence staining assay" was used to assess the viability of cells after co-culturing with silk fiber materials.

[0033] The experimental method is as follows: 1. Cell culture and treatment: 5 × 10⁵ cells per well 4 NIH3T3 cell suspension was seeded into 12-well plates and cultured at 37°C for 24 h to allow adherence. Then, the fiber material to be tested was irradiated with UV for 1 h and added to DMEM medium (containing 10% fetal bovine serum (FBS), 1 U / mL penicillin, and 1×10⁻⁶ mg / mL cellulose). -3 A 1 mg / mL solution of streptomycin was prepared; then 100 μL of the solution was added to wells containing cells as the experimental group; the control group was prepared by adding DMEM medium without fibrous material to wells containing cells.

[0034] Based on the type of fiber material to be tested, there were four experimental groups: Experimental group 1 used the fiber material of Example 1, Experimental group 2 used the fiber material of Example 2, Experimental group 3 used the fiber material of Example 3, and Experimental group 4 used the fiber material of Example 4.

[0035] 2. Staining: Remove the mixed solution (experimental group) or DMEM medium (control group) from the wells, add staining working solution to the wells, repeat 2 wells for each group, incubate for 72 h, and observe the stained cells using a fluorescence microscope.

[0036] 3. Microscope settings: Green Channel: Excitation light at 490nm, observing live cells; Red channel: Laser light at 545nm, used to observe dead cells; Overlay images: Visually display the distribution and proportion of live and dead cells in the same field of view.

[0037] See the experimental results. Figure 10 As shown, no obvious cell death was found in experimental groups 2 to 4, while obvious cell death was found in the control group and experimental group 1, indicating that the silk fiber materials provided in Examples 2 to 4 have good biocompatibility.

[0038] In summary, the silk fiber materials provided in Examples 2 to 4 exhibit excellent cell adhesion and proliferation properties, good biocompatibility, and, because the β-sheet crystal structure of the fiber is not significantly damaged, it essentially retains the excellent mechanical properties of natural silk, effectively avoiding the cumbersome processing steps involved in silk fibroin purification. This provides a new method for utilizing silk materials. Therefore, the silk fiber materials provided by this invention have enormous application potential in various fields, including cell culture, facial masks, sutures, patches, vascular / nerve conduits, absorbable wraps, tissue engineering scaffolds, analytical testing materials, medical aesthetic materials, and implantable / interventional devices.

[0039] Since this invention can use alkali-degummed silk fibroin fibers as raw materials, and the chemical and photocrosslinking conditions involved in the modification method are suitable for continuous yarn / fabric roll-to-roll processing, this invention is compatible with existing textile processes. In practical applications, the production process for preparing silk fiber materials can be referenced. Figure 11 conduct.

[0040] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing silk fiber material, characterized in that: Includes the following steps: Step 1: Prepare degummed fibroin fibers: Silkworm cocoons are degummed to remove sericin and obtain the degummed fibroin fibers. Step 2: Modify the degummed silk fibroin fiber: The modification method of the degummed silk fibroin fiber is selected from any one of chemical crosslinking modification, photocrosslinking modification, and chemical-photocrosslinking combined modification; The chemical crosslinking modification includes the step of immersing the degummed silk fibroin fiber in crosslinking solution A in the dark for 0.5-3 hours for a chemical crosslinking reaction. The crosslinking solution A includes a buffer solution and 0.05-2.0 wt% genipin. The photocrosslinking modification includes the step of immersing the degummed silk fibroin fiber in crosslinking solution B and performing a photocrosslinking reaction for 1-3 minutes under irradiation with a 365-405nm light source. The crosslinking solution B includes a buffer solution, 0.2wt% riboflavin and 0.1wt% sodium persulfate. The chemical-photocrosslinking combined modification includes the steps of immersing the degummed silk fibroin fiber in crosslinking solution C, first performing a photocrosslinking reaction under 365-405nm light source irradiation for 1-3 min, and then performing a chemical crosslinking reaction in the dark for 0.5-3 h. The crosslinking solution C includes buffer solution, 0.05-2.0wt% genipin, 0.2wt% riboflavin and 0.1wt% sodium persulfate. Step 3: Wash the modified degummed silk fibers with deionized water and then dry them for later use.

2. The preparation method according to claim 1, characterized in that: In step 2, the modification method for degummed fibroin fibers is a combined chemical-photocrosslinking modification method.

3. The preparation method according to claim 1, characterized in that: The buffer solution is selected from deionized water, PBS buffer, and Tris buffer with a pH of 7.0-8.

0.

4. The preparation method according to claim 1, characterized in that: In step 1, the degumming treatment method selected is the alkaline degumming process.

5. The preparation method according to claim 4, characterized in that: The alkaline degumming process includes the steps of boiling silkworm cocoons in an alkaline solution for 30-60 minutes and then washing them.

6. The preparation method according to claim 5, characterized in that: The strong alkaline solution is selected from a 0.05-0.5 wt% sodium carbonate solution.

7. The preparation method according to claim 1, characterized in that: In step 3, the drying method can be either freeze drying or low-temperature drying.

8. The preparation method according to claim 1, characterized in that: The temperature of the chemical crosslinking reaction in the chemical crosslinking modification and the chemical-photocrosslinking combined modification is 25-45℃.

9. The silk fiber material prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the silk fiber material according to claim 9 in biomedical fiber materials.

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