High-strength, antibacterial regenerated silk fibroin hydrogel fiber and preparation method thereof

Abstract

The application discloses a kind of high-strength, antibacterial regenerated silk fibroin hydrogel fibers and preparation method thereof, belong to the field of biomedical materials.The preparation method of the application includes: extracting regenerated silk fibroin, preparing spinning solution to carry out wet spinning, orientation traction, antibacterial zinc ion adsorption.The fiber formed by orientation traction coagulation bath in the application, and then it is recrystallized and solidified, which significantly increases the mechanical properties of hydrogel fiber, the breaking strength can reach 726-804MPa, the crystallinity is 44.64%-47.98%, the beta-fold content is 68.45%-85.12%, and it has significant antibacterial effect.The preparation process is simple, there is no organic solvent residue, convenient operation, stable method and easy to realize.Through wet spinning technology, zinc ion crosslinking coordination and loading are combined, the synergistic improvement of high strength and antibacterial performance of the fiber is realized, and the high requirements of biomedical materials on mechanical properties and biocompatibility are met.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, and specifically relates to a high-strength, antibacterial regenerated silk fibroin hydrogel fiber and its preparation method. Background Technology

[0002] The main components of silkworm cocoons include sericin and fibroin. After degumming, fibroin exhibits superior mechanical properties, good biocompatibility, and high tunability, and has been recognized as a safe and reliable biomaterial. The secondary structure of fibroin is mainly composed of β-sheets and amorphous domains (such as α-helices and random coils). The β-sheet structure endows silk fibers with high tensile strength and stiffness, while the random coils and α-helices enhance their extensibility and flexibility. Based on these properties, fibroin fibers prepared from fibroin show broad application prospects in the biomedical field. Their potential applications are not limited to wearable intelligent bandages, artificial heart valves, artificial muscles / ligaments, bone screws, artificial cartilage, and medical sutures. The development of these materials not only meets the modern medical demand for high-performance biomaterials but also provides important support for innovation in tissue engineering, regenerative medicine, and medical devices.

[0003] Hydrogels are hydrophilic polymers with a three-dimensional network structure formed through physical or chemical cross-linking. Hydrogel fibers have wide applications in the field of biomedical materials due to their good biocompatibility, excellent flexibility, deformation adaptability, high water content, and bioactivity. However, due to the special structure of hydrogel fibers, the following disadvantages exist when applied to biomedical materials: (1) Low mechanical strength: the polymer network structure breaks under external force; (2) Insufficient toughness: the fibers are prone to fatigue and damage after repeated stretching; (3) Poor structural stability: in dry or extreme environments, hydrogels are prone to dehydration and brittleness, resulting in insufficient mechanical properties. The defects in mechanical properties and structural stability of hydrogel fibers severely hinder their practical application. Although macromolecular crosslinking (CN 116716731 ​​A), double-network crosslinking (such as patents CN 116163027 A and CN 118029004A), and interpenetrating crosslinking (CN 115537957 B) can effectively improve the mechanical properties of hydrogel fibers, they are still insufficient to meet the mechanical performance requirements of high-strength and high-toughness biomedical materials. Therefore, how to overcome the conflict between strength and toughness and obtain hydrogels that are structurally stable and possess both high strength and high toughness remains an urgent problem to be solved. Summary of the Invention

[0004] To address the shortcomings and deficiencies of the existing technology, the primary objective of this invention is to provide a method for preparing high-strength, antibacterial regenerated silk fibroin hydrogel fibers. This method involves dissolving, dialyzing, and freeze-drying degummed silkworm cocoons to obtain silk fibroin, followed by wet spinning, orientation drawing, and recrystallization to produce regenerated silk fibroin hydrogel fibers with both strength and toughness. Finally, the hydrogel fibers are impregnated in an antibacterial solution to obtain antibacterial regenerated silk fibroin hydrogel fibers. This invention significantly improves the mechanical properties of the hydrogel fibers by orientation drawing of the hydrogel fibers obtained in the coagulation bath, followed by recrystallization and solidification. Zinc ion crosslinking further enhances the mechanical properties of the fibers, while also imparting certain antibacterial properties.

[0005] This invention first provides a method for preparing high-strength regenerated silk fibroin hydrogel fibers, comprising the following steps:

[0006] (1) Extraction and preparation of regenerated silk fibroin: Natural silkworm cocoons are washed with deionized water, dried, degummed, dried, dissolved with lithium bromide, dialyzed, filtered, centrifuged and freeze-dried to obtain regenerated silk fibroin;

[0007] (2) Wet spinning to prepare regenerated hydrogel fibers: The silk fibroin obtained in step (1) is dissolved in formic acid to prepare spinning solution. The spinning solution is pumped into methanol coagulation bath through a hose by a microfluidic pump to obtain regenerated silk fibroin hydrogel fibers.

[0008] (3) Orientation traction and recrystallization solidification: The regenerated silk fibroin hydrogel fiber obtained in step (2) is oriented and tractioned using a multi-functional mechanical testing instrument. The traction fiber is then immersed in a recrystallization bath for recrystallization and solidification to obtain high-strength regenerated silk fibroin hydrogel fiber.

[0009] In one embodiment of the present invention, high-strength regenerated silk fibroin hydrogel fibers are immersed in an antibacterial solution. After the solution becomes clear, the fibers are removed and sterilized to obtain high-strength, antibacterial regenerated silk fibroin hydrogel fibers.

[0010] In one embodiment of the present invention, in step (1), the specific process of washing, drying, degumming and drying is as follows: cut the natural silkworm cocoons into pieces, wash them with deionized water 4-5 times and dry them, take the washed and dried silkworm cocoons and place them in Na2CO3 solution, boil them for 1-2 hours to degumme them, wash them with deionized water 4-5 times after degumming, and dry them.

[0011] In one embodiment of the present invention, in step (1), the mass-to-volume ratio of silkworm cocoon to Na2CO3 solution is 25-35g:10-14L, and the concentration of Na2CO3 solution is 0.02-0.025mol / L.

[0012] In one embodiment of the present invention, in step (1), the process of dissolving lithium bromide specifically involves taking degummed and dried silkworm cocoons and dissolving them in a lithium bromide solution, stirring and dissolving them in a water bath at 60°C for 4-6 hours.

[0013] In one embodiment of the present invention, in step (1), the mass-to-volume ratio of the degummed and dried silkworm cocoons to the lithium bromide solution is 25-27g:90-100mL, and the concentration of the lithium bromide solution is 9-9.4mol / L.

[0014] In one embodiment of the present invention, in step (1), the dialysis process specifically involves cooling to room temperature after the silkworm cocoons have dissolved, and then performing dialysis for 48-72 hours, changing the deionized water at least 3 times every 24 hours; the molecular weight cutoff during dialysis is 3500 Da.

[0015] In one embodiment of the present invention, in step (1), the filtration, centrifugation, and freeze-drying process specifically involves filtering the initial extract obtained by dialysis using a nylon filter cloth (500 mesh), then centrifuging it at a speed of 5000 r / min, and taking the supernatant for freeze-drying. The freeze-dried product needs to be stored at -80°C.

[0016] In one embodiment of the present invention, in step (2), the concentration of the spinning solution is 12-15%.

[0017] In one embodiment of the present invention, in step (2), the spinning solution is pumped into the methanol coagulation bath at a speed of 0.5 to 1 mL / min, and the diameter of the hose is 200 μm.

[0018] In one embodiment of the present invention, in step (2), the spinning solution is soaked in a 95% methanol coagulation bath for 10 min to 30 min.

[0019] In one embodiment of the present invention, in step (2), the methanol coagulation bath further contains zinc ions, the concentration of which is 0.5-5%, preferably 0.5-4.5%, more preferably 1-4.5%, and even more preferably 1.5-3%.

[0020] In one embodiment of the present invention, in step (2), the methanol coagulation bath contains methanol and zinc chloride, and the concentration of zinc ions in the methanol coagulation bath is 0.5-5%.

[0021] In one embodiment of the present invention, in step (3), the ambient temperature during orientation traction is 23-25°C, the air humidity is 50%, the traction speed is v = 200 mm / min, and after orientation traction, tension is fixed with a force of 1 N for 2 min-3 min.

[0022] In one embodiment of the present invention, in step (3), the traction multiple of the orientation traction is 4 to 8 times, preferably 5 to 7 times, and more preferably 6 times.

[0023] In one embodiment of the present invention, in step (3), the solution of the recrystallization bath is a 75% ethanol solution, the fiber is in the recrystallization bath for 10 min-30 min, the curing temperature is 25-37℃, and the curing time is 10-30 min.

[0024] In one embodiment of the present invention, in step (3), the solution of the recrystallization bath further contains zinc ions, the concentration of which is 0.5-5%, preferably 0.5-4.5%, more preferably 1-4.5%, and even more preferably 1.5-3%.

[0025] In one embodiment of the present invention, in step (3), the recrystallization bath contains ethanol and zinc chloride, and the concentration of zinc ions in the recrystallization bath is 0.5-5%.

[0026] In one embodiment of the present invention, the antibacterial solution is an aqueous solution containing zinc ions, the soaking time is 4-6 hours, and the concentration of zinc ions in the solution is 0.1-5%, preferably 0.5-5%, more preferably 1-4%, and even more preferably 1.5-3.5%. The aqueous solution containing zinc ions is preferably an aqueous solution of zinc chloride.

[0027] In one embodiment of the present invention, the sterilization process is high-temperature and high-pressure sterilization, wherein the sterilization temperature is 121℃-134℃ and the pressure is 1.02-2.03 kg / cm². 2 The sterilization time is 30-60 minutes.

[0028] The present invention also discloses a high-strength, antibacterial regenerated silk fibroin hydrogel fiber prepared by the above method.

[0029] In one embodiment of the present invention, the high-strength, antibacterial regenerated silk fibroin hydrogel fiber has a diameter of 35-45 μm, a fracture stress of 724-804 MPa, and a fracture strain of 23.7-36.38%.

[0030] This invention also discloses the application of the above-mentioned high-strength, antibacterial regenerated silk fibroin hydrogel fiber in the fields of high-performance biomedical materials such as wearable intelligent bandages, artificial heart valves, artificial muscles / ligaments, bone screws, artificial cartilage, and medical sutures.

[0031] In this invention, lithium bromide disrupts the natural hydrogen bond network structure of silk fibroin, enabling the successful preparation of regenerated silk fibroin through dialysis and freeze-drying. Formic acid dissolves the regenerated silk fibroin and promotes its self-assembly by shielding the electrostatic repulsion effect in concentrated solutions. Furthermore, methanol is considered the optimal cross-linking agent for silk fibroin recrystallization because it rapidly promotes the formation of hydrophobic crystalline regions (such as β-sheet structures) in the regenerated silk fibroin. Zinc ions introduced into methanol can cross-link and coordinate with active serine residues in silk fibroin, forming a stable ionic bond network and improving the fiber's mechanical properties. Orientation traction treatment alters the protein chain arrangement, making it more orderly and increasing the number of interchain hydrogen bonds, significantly improving the fiber's mechanical properties. Zinc ion loading imparts excellent antibacterial properties to the fiber. These treatment methods provide important support for the preparation of high-strength, antibacterial regenerated silk fibroin hydrogel fibers for application in the biomedical field.

[0032] Beneficial effects:

[0033] (1) This invention prepares silk fibroin by dissolving, dialysis, and freeze-drying degummed silkworm cocoons. After wet spinning, the fibers are then oriented and recrystallized to produce regenerated silk fibroin hydrogel fibers with both strength and toughness. Finally, the hydrogel fibers are impregnated in an antibacterial solution to obtain antibacterial regenerated silk fibroin hydrogel fibers. This invention involves vertically oriented and drawn fibers formed after coagulation in a coagulation bath. On the one hand, directional oriented drawing can induce the molecular chains and other structures in the fiber to align along the axial direction through physical action, resulting in a dense internal structure, increased fiber crystallinity, and the induction of β-sheet formation, thereby greatly improving the mechanical properties of the fiber. On the other hand, the method of first oriented drawing and then recrystallizing in a recrystallization bath can prolong the recrystallization and solidification time, further contributing to the improvement of the fiber's mechanical properties.

[0034] (2) In this invention, a certain amount of zinc chloride is added to both the coagulation bath and the recrystallization bath. During the coagulation and recrystallization processes, zinc ions can coordinate and crosslink with serine in silk fibroin to form ionic covalent bonds. The crosslinking of zinc ions can further improve the mechanical properties of the fiber. At the same time, the embedding of zinc ions also endows the fiber with good antibacterial properties.

[0035] (3) After the high-strength regenerated silk fibroin hydrogel fiber after recrystallization and solidification is immersed in an antibacterial solution for a period of time, antibacterial ions can be adsorbed on the surface of the fiber, realizing the synergistic improvement of the fiber's high strength and antibacterial performance, while meeting the high requirements of biomedical materials for mechanical properties and biocompatibility.

[0036] (4) The high-strength regenerated silk fibroin hydrogel fiber prepared by this invention has greatly improved strength and toughness compared with other high-strength hydrogel fibers, and the experimental method can be extended to other polymer hydrogel fibers such as PVA and polyacrylamide.

[0037] (5) The hydrogel fibers prepared by this invention are transparent, have a smooth surface, no obvious defects, and good appearance. The fiber diameter is 35-45μm, the tensile strength can reach 726-804MPa, and the elongation at break is 26.02-36.62%, with excellent mechanical properties. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the preparation method of the high-strength, antibacterial regenerated silk fibroin fiber of the present invention.

[0039] Figure 2 These are schematic diagrams of the traction method in Examples 1-10(A) and the wet spinning method in Comparative Examples 2-9(B) of the present invention.

[0040] Figure 3 The mechanical property test results of regenerated silk fibroin hydrogel fibers with different traction ratios prepared in Examples 1 to 10 of the present invention are shown. A and B are the stress-strain curves of fibers in Examples 6 to 10 and Examples 1 to 5, respectively. C and D are the fracture stress and fracture strain data of fibers in Examples 1 to 10, respectively.

[0041] Figure 4 The stress-strain curves (A), fracture stress (B), and fracture strain (C) of regenerated silk fibroin hydrogel fibers with different spinning solution concentrations prepared in Examples 1, 14-16 of this invention are shown.

[0042] Figure 5 The stress-strain curves (A), fracture stress (B), and fracture strain (C) of regenerated silk fibroin hydrogel fibers with different zinc ion crosslinking concentrations prepared in Examples 1, 11-13 of this invention are shown.

[0043] Figure 6 These are physical images of the fibers used in Embodiments 1, 2, 6, and 7 of the present invention.

[0044] Figure 7 The images shown are scanning electron microscope (SEM) images of Embodiments 1, 2, 6, 7 and Comparative Example 1 of the present invention.

[0045] Figure 8 The Fourier transform infrared spectra are those of Embodiments 1, 2, 6, 7 and Comparative Example 1 of the present invention.

[0046] Figure 9The XRD patterns are those of Embodiment 1, Embodiment 2, Embodiment 6, Embodiment 7 and Comparative Example 1 of the present invention.

[0047] Figure 10 The secondary structure and crystallinity analysis of Examples 1, 2, 6, 7 and Comparative Example 1 of the present invention are presented.

[0048] Figure 11 The results of the evaluation of the zinc ion adsorption capacity and antibacterial performance of zinc ion solutions of different concentrations in Example 1 of the present invention are shown. Detailed Implementation

[0049] To ensure that the objectives, features, and advantages of the present invention are clearer and easier to understand, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0050] The present invention can be implemented in various ways, and may be carried out in addition to the specific details described herein. Those skilled in the art can extend its application without departing from the core ideas of the invention. Therefore, the present invention should not be limited to the specific embodiments disclosed.

[0051] Unless otherwise specified, all raw materials used in this invention are commercially available.

[0052] The materials obtained in the embodiments of the present invention were subjected to performance testing according to the following method:

[0053] The microstructure of hydrogel fibers was observed using scanning electron microscopy (SEM, Hitachi SU1510), Fourier transform infrared spectroscopy (FTIR, Nicoletis 10), and X-ray diffraction (XRD, D2 PHASERA26-X1-A2E0B2A0); traction and mechanical tests were performed using a microcomputer-controlled electronic universal testing machine (WDW-1).

[0054] The mechanical property testing method for hydrogel fibers is as follows: a hydrogel fiber sample with a length of 50 mm is taken and subjected to uniaxial tensile testing at a tensile speed of 20 mm / min until the fiber breaks. The stress and strain at the time of breakage are recorded.

[0055] Explanation of the English abbreviations for fibers in the examples and comparative examples:

[0056] RSF-Zn 2+ crosslinked (drawn): Zinc chloride is added to both the coagulation bath and the recrystallization bath when preparing hydrogel fibers, and orientation traction is required after the coagulation bath.

[0057] RSF-Zn 2+ crosslinked: Zinc chloride is added to both the coagulation bath and the recrystallization bath when preparing hydrogel fibers, but no orientation traction is performed after the coagulation bath.

[0058] RSF (drawn): When preparing hydrogel fibers, zinc chloride is not added to either the coagulation bath or the recrystallization bath, but orientation traction is required after the coagulation bath.

[0059] RSF: When preparing hydrogel fibers, no zinc chloride is added to the coagulation bath or recrystallization bath, and no orientation traction is performed after the coagulation bath.

[0060] Example 1

[0061] A method for preparing high-strength, antibacterial regenerated silk fibroin hydrogel fiber, comprising the following steps:

[0062] (1) Degumming of silkworm cocoons: Cut natural silkworm cocoons into pieces, wash them 4-5 times with deionized water and dry them. Take 35 grams of washed and dried silkworm cocoons and place them in 0.02M Na2CO3 solution, boil for 1 hour to degumme them, wash them 4-5 times with deionized water and dry them.

[0063] (2) Extraction of regenerated silk fibroin: Weigh 27g of degummed and dried silkworm cocoons and dissolve them in 100mL of lithium bromide solution with a concentration of 9.3mol / L. Stir and dissolve in a water bath at 60℃ for 6 hours. After the silkworm cocoons are dissolved, cool to room temperature and treat by dialysis (MWCO = 3500Da) for 72h, changing the deionized water at least 3 times every 24h. After dialysis, filter the initial extract using a nylon filter membrane (500 mesh), and then centrifuge at a speed of 5000r / min. Take the supernatant and freeze-dry it to obtain regenerated silk fibroin.

[0064] (3) Preparation of regenerated silk fibroin hydrogel by wet spinning: Weigh 1.5g of the regenerated silk fibroin obtained in step (2) and dissolve it in 10mL of formic acid to prepare a spinning solution with a concentration of 15%. The spinning solution is squeezed into a zinc chloride methanol coagulation bath solution with a mass concentration of 2.5% by passing it through a 200μm tube at a speed of V=0.5mL / min, and soaked for 10min to obtain regenerated silk fibroin hydrogel fibers. The zinc chloride methanol coagulation bath solution is composed of 2.5g of zinc chloride and 100mL of 95% methanol.

[0065] (4) Orientation Traction and Recrystallization: The regenerated silk fibroin hydrogel fiber obtained in step (3) was taken out and subjected to orientation traction at a speed of v = 200 mm / min on a multifunctional mechanical testing instrument at an ambient temperature of 23℃ and an air humidity of 50%. The traction process is as follows: Figure 2As shown in Figure A, the hydrogel fiber was fixed under tension with a force of 1 N for 2 minutes. After traction, the hydrogel fiber was immersed in a 2.5% zinc chloride ethanol recrystallization bath for 30 minutes for recrystallization and solidification, followed by drying at 37°C for 5 minutes to obtain high-strength regenerated silk fibroin hydrogel fiber RSF-Zn. 2+ The zinc chloride ethanol coagulation bath solution is composed of 2.5g zinc chloride and 100mL 75% ethanol.

[0066] Example 2

[0067] Example 2 differs from Example 1 in that the orientation traction process in step (4) is omitted, resulting in hydrogel fiber RSF-Zn. 2+ crosslinked.

[0068] Example 3

[0069] The difference between Example 3 and Example 1 is that the directional traction multiple in step (4) is 2 times.

[0070] Example 4

[0071] The difference between Example 4 and Example 1 is that the directional traction multiple in step (4) is 4 times.

[0072] Example 5

[0073] The difference between Example 5 and Example 1 is that the directional traction multiple in step (4) is 8 times.

[0074] Example 6

[0075] The difference between Example 6 and Example 1 is that the coagulation bath solution in step (3) is a methanol solution, and the ethanol in step (4) does not contain zinc chloride. The hydrogel fiber obtained is named RSF (drawn).

[0076] Example 7

[0077] The difference between Example 7 and Example 6 is that the orientation traction process in step (4) is omitted, and hydrogel fiber RSF is obtained.

[0078] Example 8

[0079] The difference between Example 8 and Example 6 is that the directional traction multiple in step (4) is 2 times.

[0080] Example 9

[0081] The difference between Example 9 and Example 6 is that the directional traction multiple in step (4) is 4 times.

[0082] Example 10

[0083] The difference between Example 10 and Example 6 is that the directional traction multiple in step (4) is 8 times.

[0084] Example 11

[0085] The difference between Example 11 and Example 2 is that the mass concentration of zinc chloride in the coagulation bath solution in step (3) is 0.5%, and the concentration of zinc chloride in the recrystallization bath in step (4) is 0.5%.

[0086] Example 12

[0087] The difference between Example 12 and Example 2 is that the mass concentration of zinc chloride in the coagulation bath solution in step (3) is 1.0%, and the concentration of zinc chloride in the recrystallization bath in step (4) is 1.0%.

[0088] Example 13

[0089] The difference between Example 13 and Example 2 is that the mass concentration of zinc chloride in the coagulation bath solution in step (3) is 5%, and the concentration of zinc chloride in the recrystallization bath in step (4) is 5%.

[0090] Example 14

[0091] The difference between Example 14 and Example 7 is that the concentration of the spinning solution in step (3) is 8%.

[0092] Example 15

[0093] The difference between Example 15 and Example 7 is that the concentration of the spinning solution in step (3) is 12%.

[0094] Example 16

[0095] The difference between Example 16 and Example 7 is that the concentration of the spinning solution in step (3) is 18%.

[0096] Example 17

[0097] The difference between Example 17 and Example 1 is that Example 17 also includes a step of antibacterial ion adsorption on the high-strength regenerated silk fibroin hydrogel fiber obtained in step (4): 1g of recrystallized and solidified hydrogel fiber is weighed and placed in a 2.5% zinc chloride aqueous solution. The solution is stirred thoroughly for 6 hours. After the solution becomes clear, the fiber is removed and subjected to high temperature and high pressure (121℃) at a pressure of 1.02kg / cm. 2 Sterilization treatment for 30 min yielded hydrogel fibers RSF-Zn. 2+ crosslinked(drawn). Where drawn represents oriented traction.

[0098] Comparative Example 1

[0099] The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 only includes step (1).

[0100] Comparative Example 2

[0101] The difference between Comparative Example 2 and Example 1 is that the processes of steps (3) and (4) are as follows:

[0102] Preparation of regenerated silk fibroin hydrogel by wet spinning: The preparation process is as follows Figure 2 B. Weigh 1.5g of the regenerated silk fibroin obtained in step (2) and dissolve it in 10mL of formic acid to prepare a spinning solution with a concentration of 15%. The spinning solution is squeezed into a zinc chloride methanol coagulation bath solution with a mass concentration of 2.5% through a 200μm tube at a speed of V = 0.5mL / min. After passing through the traction winding wheel 1 (ω1 = 20r / min), it is immersed in a zinc chloride ethanol recrystallization bath solution with a mass concentration of 2. Under the action of the traction winding wheel 2 (ω2 = 20r / min), it is stretched (traction ratio is 2 times). The fibers collected by the winding wheel 2 are soaked in ethanol for 30min, taken out and dried to obtain regenerated silk fibroin hydrogel fibers.

[0103] Comparative Example 3

[0104] The difference between Comparative Example 3 and Comparative Example 2 is that ω2 and ω2 / ω1 are changed so that their ratio is 1.25, which is equivalent to a traction of 4 times.

[0105] Comparative Example 4

[0106] The difference between Comparative Example 4 and Comparative Example 2 is that ω2 and ω2 / ω1 are changed so that their ratio is 1.5, which is equivalent to a traction of 6 times.

[0107] Comparative Example 5

[0108] The difference between Comparative Example 5 and Comparative Example 2 is that ω2 and ω2 / ω1 are changed so that their ratio is 2, which is equivalent to a traction of 8 times.

[0109] Comparative Example 6

[0110] The difference between Comparative Example 6 and Example 6 is that the processes of steps (3) and (4) are as follows:

[0111] Preparation of regenerated silk fibroin hydrogel by wet spinning: Weigh 1.5g of the regenerated silk fibroin obtained in step (2) and dissolve it in 10mL of formic acid to prepare a spinning solution with a concentration of 15%. The spinning solution is squeezed into a 95% methanol coagulation bath at a speed of V = 0.5mL / min through a 200μm flexible tube. After passing through traction winding wheel 1 (ω1 = 20r / min), it is immersed in a 75% ethanol recrystallization bath. Under the action of traction winding wheel 2 (ω2 = 20r / min), it is drawn (drawing ratio is 2 times). The drawing process is as follows... Figure 2 As shown in B, the fibers collected by the winding wheel 2 are soaked in ethanol for 30 minutes, then removed and dried to obtain regenerated silk fibroin hydrogel fibers.

[0112] Comparative Example 7

[0113] The difference between Comparative Example 7 and Comparative Example 6 is that ω2 is changed so that the ratio of ω2 / ω1 is 1.25, which is equivalent to a traction of 4 times.

[0114] Comparative Example 8

[0115] The difference between Comparative Example 8 and Comparative Example 6 is that ω2 is changed so that the ratio of ω2 / ω1 is 1.5, which is equivalent to a traction of 6 times.

[0116] Comparative Example 9

[0117] The difference between Comparative Example 9 and Comparative Example 6 is that ω2 is changed so that the ratio of ω2 / ω1 is 2, which is equivalent to 8 times the traction.

[0118] Tables 1 and 2 show the diameter and mechanical properties of the hydrogel fibers under different traction ratios in Examples 1-5 and Examples 6-10. The stress-strain curves of the hydrogel fibers under different traction ratios are shown below. Figure 3 As shown in A and 3B, from Table 1, Table 2 and Figure 3 The experimental results show that as the traction strength increases, its mechanical properties become superior, but an 8-fold traction leads to a decrease in mechanical properties. Excessive traction causes the internal hydrogen bonds to break, negatively impacting mechanical properties. From... Figure 3 As can be seen from C and 3D, compared with RSF, the zinc ion cross-linked fiber exhibits superior mechanical properties after traction. Its optimal traction ratio is 6 times.

[0119] Table 1. Diameter and mechanical properties of hydrogel fibers in Examples 1-5 (Mean ± SD, N = 3)

[0120]

[0121] Table 2. Diameter and mechanical properties of hydrogel fibers in Examples 6-10 (Mean ± SD, N = 3)

[0122]

[0123] Comparative Examples 2-5 and 6-9 were prepared using conventional traction processes to produce zinc ion crosslinked and uncrosslinked hydrogel fibers, respectively. Their diameter and mechanical properties are shown in Tables 3 and 4. Comparing the data in Tables 1 and 2, it can be seen that the orientation traction combined with recrystallization method of this invention is more beneficial for improving the mechanical properties of hydrogel fibers compared to conventional traction processes. This is mainly because conventional traction utilizes guide rollers and winding rollers, but requires controlling the speed and tension of the rollers to traction the fibers. Although this method can achieve integrated spinning, the solidification and recrystallization time of the hydrogel fibers is not long enough, and the traction is mainly achieved through the centrifugal speed of the rollers, making it impossible to perform orientation traction along the axial direction of the spinning process. Therefore, the improvement effect on the mechanical properties of the fibers is relatively limited. Furthermore, during the spinning process, fiber breakage and slippage are very likely to occur, easily leading to uneven fiber traction. The orientation-traction method employed in this invention, through step-by-step processing, effectively avoids insufficient fiber coagulation and recrystallization time. Directional orientation-traction induces the molecular chains and other structures within the fiber to align axially through physical action, resulting in a denser internal structure, increased fiber crystallinity, and the induction of β-sheet formation, thereby significantly improving the fiber's mechanical properties. The coordination crosslinking of zinc ions with serine in silk fibroin forms ionic covalent bonds, which, to a certain extent, enhances the mechanical properties of wet-spun fibers.

[0124] Table 3. Diameter and mechanical properties of hydrogel fibers in Comparative Examples 2–5 (Mean ± SD, N = 3)

[0125]

[0126] Table 4. Diameter and mechanical properties of hydrogel fibers in Comparative Examples 6–9 (Mean ± SD, N = 3)

[0127]

[0128] The concentration of the spinning solution was adjusted without traction to verify the effect of the spinning solution concentration on the mechanical properties of hydrogel fibers. The stress-strain curves and mechanical properties of fibers prepared with spinning solutions of different concentrations are shown below. Figure 4 As shown in the figure, SF represents silk fibroin, and 8% (SF) represents a spinning solution concentration of 8%. It can be seen from the figure that the mechanical properties of the fiber are best when the spinning solution concentration is 15%. When the spinning solution concentration increases to 18%, its viscosity is too high, which can easily cause clogging of the spinning head, resulting in uneven fiber and thus affecting its mechanical properties.

[0129] The zinc ion concentration was adjusted without traction to verify its effect on fiber mechanical properties. The stress-strain curves and mechanical properties of fibers prepared with different zinc ion crosslinking concentrations are shown below. Figure 5 As shown, from Figure 5 It can be seen that 2.5% zinc ion crosslinking concentration is optimal. Too low a concentration (such as 1%) leads to incomplete crosslinking, while too high a concentration (5%) leads to increased fiber brittleness, which affects mechanical properties.

[0130] Fiber electron microscopy characterization

[0131] The high-strength regenerated silk fibroin hydrogel fibers prepared in Examples 1-2 and 6-7 were pre-frozen at -20°C and -80°C, respectively, and then placed in a freeze dryer. After the samples were completely freeze-dried at -70°C and 1 Pa, they were removed, and cross-sections were cut using a sharp scalpel blade and observed under a scanning electron microscope (SEM) to examine their microstructure. (Visual observation...) Figure 6 The results showed that the fiber surface exhibited a certain metallic luster after traction, indicating that the traction treatment had a significant impact on the fiber's appearance. SEM characterization results ( Figure 7 This further revealed the microstructural characteristics of the fibers. Undrawn fibers had a diameter of approximately 200 μm and exhibited a random network structure on their surface. In contrast, fibers treated with six times the draw weight had a diameter reduced to approximately 40 μm, and their surface structure transformed into an oriented arrangement along the draw direction. This oriented arrangement indicates that the internal crystals and chemical bonds of the fibers rearranged along a specific direction during the draw process, thereby enhancing the mechanical properties of the fibers to some extent. In summary, the draw treatment significantly affects the diameter and surface structure of the fibers, and this structural change is closely related to the improvement in their mechanical properties.

[0132] Fiber Fourier Transform Infrared Spectroscopy (FTIR) Analysis

[0133] The high-strength regenerated silk fibroin hydrogel fibers prepared in Examples 1-2 and 6-7 were freeze-dried. 2 mg of the freeze-dried fiber sample was weighed and 200 mg of potassium bromide was added to each sample. After thorough grinding, the samples were compressed into tablets using a tablet press at a pressure of 20 MPa. Subsequently, the samples were scanned using attenuated total reflectance (ATR) mode of a Fourier transform infrared spectroscopy (FTIR) instrument, with a scanning wavelength range of 500-4000 nm and a scanning frequency of 32 scans / second. The peak spectral data were recorded.

[0134] Sample Fourier results as follows Figure 8 As shown, traction can significantly increase the peak intensity of the amide II (Amide II) crystal domain, thereby improving its mechanical properties, 542 cm⁻¹. -1 The absorption peak is the Zn-O functional group absorption peak, indicating that zinc ions and serine hydroxyl groups of silk fibroin form a coordination bond structure.

[0135] Fiber X-ray diffraction (XRD) analysis

[0136] X-ray diffraction analysis of the freeze-dried samples of Examples 1-2 and 6-7 was performed under Cu-Kα monochromatic radiation using a Rigaku D / Max-2550PC instrument at 45 kV and 30 mA to assess the crystallinity of silk fibroin. The analysis was conducted using a transmission mode-dependent sensitive detector at 0.02° step intervals and wavelengths of [missing information]. Under these conditions, the sample was scanned in the 2θ range of 5° to 60°, and the scanned peak spectral data were recorded.

[0137] X-ray diffraction (XRD) pattern Figure 9 The results showed that characteristic peaks of β-sheets associated with amide II were observed at 9.1°, 18.9°, 20.7°, and 24°, indicating that traction treatment can promote the formation of β-sheet structures in fibers. Further quantitative analysis ( Figure 10 RSF and RSF-Zn were compared. 2+ crosslinked, RSF(drawn) and RSF-Zn 2+ The secondary structure and crystallinity of crosslinked (drawn) fibers and natural silk were analyzed. Results showed that zinc ion crosslinking and orientation drawing treatment significantly promoted the formation of β-sheet structures, especially in RSF-Zn fibers. 2+ Crosslinked (drawn) fibers, with β-sheet content ranging from 68.45% to 85.12% and crystallinity from 44.64% to 47.98%, are significantly higher than the β-sheet content (50.54% to 65.89%) and crystallinity (34.18% to 35.93%) of natural silk fibers. These results highlight the superiority of RSF-Zn. 2+ The significant advantages of crosslinked (drawn) fibers in terms of mechanical properties.

[0138] Test of zinc ion adsorption capacity and antibacterial properties of fiber

[0139] 0.5% Zn was prepared according to the method in Example 17. 2+ 1% Zn 2+ 2.5% Zn 2+ 5% Zn 2+ Antibacterial solution (0.5% Zn) 2+ For a 0.5% zinc chloride aqueous solution (corresponding to a mass concentration of 0.5%), weigh out 0.5g of dried zinc chloride solution. 2+ 1% Zn 2+ 2.5% Zn 2+ 5% Zn 2+ RSF-Zn sterilized by immersion in solution 2+The crosslinked (drawn) sample was placed in 2 mL of sterile PBS solution and incubated at 37°C with shaking for 24 h to obtain the extract.

[0140] Use a smear stick to apply a concentration of 1×10 5 CFU / mL of Staphylococcus aureus (S. aureus ATCC 25923) and Escherichia coli (E. coli ATCC 25922) were evenly inoculated onto LB agar plates. Ten μL of each sample extract was taken from 2 mL and added to sterile blank antimicrobial susceptibility testing discs (6 mm in diameter). The discs were placed on bacterial plates and then incubated at 37°C with 5% CO2 for 12 hours. After incubation, a vertical tangent was drawn from the edge of the inhibition zone to the edge of the antimicrobial susceptibility testing disc, and the distance was measured using ImageJ software to evaluate the antimicrobial effect of the fiber.

[0141] Figure 11 The antibacterial effects of hydrogel fibers impregnated with zinc ion solutions of different concentrations (0.5%, 1%, 2.5%, and 5%) are presented. Figure 11 It can be seen that the diameter of the antibacterial zone around the fiber is positively correlated with the zinc ion concentration, but the difference in antibacterial effect between 2.5% and 5% concentrations is not significant. This phenomenon reveals that the cross-linked hydrogel fiber has a saturation point for the adsorption of zinc ions, and concentrations exceeding 2.5% do not substantially help improve the antibacterial effect.

[0142] In summary, this invention successfully developed an innovative high-strength, antibacterial regenerated silk fibroin hydrogel fiber. By optimizing the traditional wet spinning process and introducing zinc ion-silk fibroin coordination crosslinking and orientation traction technology, the precise preparation of high-strength regenerated silk fibroin hydrogel fibers of different diameters was achieved. Based on the negatively charged surface of silk fibroin, the fiber can achieve stable loading of zinc ions through electrostatic adsorption, thereby endowing the fiber with inherent antibacterial properties. This strategy not only significantly improves the mechanical properties of the fiber but also achieves long-lasting antibacterial function. This invention proposes a simple and promising preparation method, providing an innovative strategy for developing high-strength biomedical hydrogel sutures, intelligent wearable bandages, and other medical materials. It also opens up new research directions for the design and application of multifunctional materials in the field of regenerative medicine, and is expected to have wide applications in clinical medicine.

[0143] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing high-strength regenerated silk fibroin hydrogel fibers, characterized in that, Includes the following steps: (1) Extraction and preparation of regenerated silk fibroin: Natural silkworm cocoons were washed with deionized water, dried, degummed, dried, dissolved with lithium bromide, dialyzed in a 3500 Da dialysis bag, filtered, centrifuged and freeze-dried to obtain regenerated silk fibroin; (2) Preparation of regenerated hydrogel fibers by wet spinning: The silk fibroin obtained in step (1) is dissolved in formic acid to prepare a spinning solution. The spinning solution is pumped into a methanol coagulation bath through a hose using a microfluidic pump to obtain regenerated silk fibroin hydrogel fibers. The concentration of the spinning solution is 12-15%. The methanol coagulation bath contains zinc chloride, and the mass concentration of the zinc chloride is 0.5-5%. (3) Orientation traction and recrystallization solidification: The regenerated silk fibroin hydrogel fiber obtained in step (2) is vertically oriented and tractioned using a multifunctional mechanical testing instrument. The traction multiple is 5 to 7 times. The traction-obtained fiber is immersed in a recrystallization bath for recrystallization and solidification to obtain high-strength regenerated silk fibroin hydrogel fiber. The solution in the recrystallization bath contains zinc chloride, and the mass concentration of the zinc chloride is 0.5 to 5%.

2. The preparation method according to claim 1, characterized in that, In step (1), the degumming process involves placing the washed and dried silkworm cocoons in a Na2CO3 solution and boiling them for 1-2 hours to degumme them. During degumming, the mass-to-volume ratio of the silkworm cocoons to the Na2CO3 solution is 25-35g:10-14L, and the concentration of the Na2CO3 solution is 0.02-0.025mol / L.

3. The preparation method according to claim 1, characterized in that, In step (1), the process of dissolving lithium bromide specifically involves dissolving degummed and dried silkworm cocoons in a lithium bromide solution and stirring in a water bath at 60°C for 4-6 hours. The mass-to-volume ratio of the degummed and dried silkworm cocoons to the lithium bromide solution is 25-27g:90-100mL, and the concentration of the lithium bromide solution is 9-9.4mol / L.

4. The preparation method according to claim 1, characterized in that, In step (2), the spinning solution is pumped into the methanol coagulation bath at a speed of 0.5~1mL / min, the diameter of the hose is 200μm, and the spinning solution is soaked in the 95% methanol coagulation bath for 10min-30min.

5. The preparation method according to claim 1, characterized in that, In step (3), the ambient temperature during orientation traction is 23-25℃, the air humidity is 50%, the traction speed is v=200mm / min, and after orientation traction, the tension is fixed with a force of 1N for 2min-3min.

6. The preparation method according to claim 1, characterized in that, In step (3), the recrystallization bath solution is a 75% ethanol solution, the fiber is in the recrystallization bath for 10-30 minutes, the curing temperature is 25-37 ℃, and the curing time is 10-30 minutes.

7. The preparation method according to claim 1, characterized in that, The method also includes the steps of immersing high-strength regenerated silk fibroin hydrogel fibers in an antibacterial solution, waiting for the solution to become clear, removing the fibers and sterilizing them to obtain high-strength, antibacterial regenerated silk fibroin hydrogel fibers, wherein the antibacterial solution is an aqueous solution of zinc chloride, the immersion time is 4-6 hours, and the mass concentration of zinc chloride in the solution is 0.1-5%.

8. The preparation method according to claim 7, characterized in that, The sterilization process is high-temperature, high-pressure sterilization, with a temperature of 121℃-134℃ and a pressure of 1.02-2.03 kg / cm². 2 The sterilization time is 30-60 minutes.

9. The regenerated silk fibroin hydrogel fiber prepared by any one of claims 1 to 8, wherein the regenerated silk fibroin hydrogel fiber has a diameter of 35-45 μm, a fracture stress of 726-804 MPa, and a fracture strain of 26.02-36.62%.

10. The application of the regenerated silk fibroin hydrogel fiber as described in claim 9 in wearable intelligent bandages, artificial heart valves, artificial muscles / ligaments, bone screws, artificial cartilage, and medical sutures.

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