Silk fibroin-based hydrogel optical fiber and preparation method and application thereof

Abstract

The application discloses a silk protein-based hydrogel optical fiber and a preparation method and application thereof, and belongs to the technical field of hydrogel optical fibers.The silk protein-based hydrogel optical fiber with a chemical-physical double crosslinking structure is constructed through a synergistic strategy of enzymatic crosslinking and ethanol solution post-processing; firstly, a degumming treatment is performed by using a sodium carbonate solution to prepare a silk protein solution; then, a core liquid and a coating liquid are respectively prepared by using the silk protein solution, a horseradish peroxidase solution and an H2O2 solution; the core liquid is injected into a gel, and then, an ethanol solution is used to strengthen the mechanical property of the core liquid; finally, the coating liquid is coated, and the silk protein-based hydrogel optical fiber with excellent mechanical property, low optical loss and good biocompatibility is obtained.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of hydrogel optical fiber, and particularly relates to a silk fibroin-based hydrogel optical fiber and a preparation method and application thereof. BACKGROUND

[0002] In recent years, the brain-computer interface technology has developed rapidly, and the medical field has become the core landing direction of the brain-computer interface technology, and at the same time, its application territory has extended to education, entertainment, competition and military and other multi-scene, showing a multi-dimensional commercialization prospect. The brain-computer interface realizes motion control decoding and perception information coding by constructing a bidirectional interaction channel between bioelectric signals and electronic devices, and provides a technical basis for functional reconstruction. The neural electrical signals of the brain-computer interface system can be collected through two ways: one is to use non-invasive scalp electrodes to record, and the other is to directly obtain the cortical signals by means of implanted electrodes. The conventional metal microelectrode is easy to cause physical damage to the fragile brain area in the implantation process due to the rigid material, and long-term use may also cause problems such as electrochemical reaction, biological toxicity, chronic inflammation, neurodegenerative disease, and signal transmission barrier caused by glial cell wrapping, which seriously affect its stability and service life. Under this background, the development of brain-computer interface technology without implanted electrodes has become an important issue to be solved.

[0003] The optical fiber photometry recording technology can realize the parallel detection of neural activity in multiple brain regions by conducting and collecting neuron fluorescence signals through optical fibers, and provides important support for the integration of optogenetics and brain-computer interface system, and promotes the construction of bidirectional neural information transmission system based on optical principles. The existing optical fibers are mainly divided into glass optical fibers, polymer optical fibers (including traditional plastic optical fibers and hydrogel optical fibers, etc.), special glass optical fibers and crystal optical fibers. Although the traditional silica-based optical fiber has excellent optical transparency and extremely low propagation loss, its insufficient biocompatibility, brittleness and high rigidity characteristics lead to the application limitation of this kind of optical fiber which can only be used temporarily for treatment or diagnosis, and the hydrogel optical fiber can well solve the above problems.

[0004] Recently, the preparation technology and functional characteristics of hydrogel optical fibers have attracted continuous attention from the academic community, and various research institutions have made breakthroughs. Due to the excellent tissue compatibility of hydrogel and the rapid response and accurate detection characteristics of optical fiber sensing system, this new type of composite material shows great application potential in pathological screening and physiological parameter real-time monitoring, and brings more reliable and safe innovation solutions to the field of modern medical diagnosis. In 2017, Yetisen team developed a hydrogel optical fiber, whose core material is poly(acrylamide-co-ethylene glycol diacrylate), and the outer layer is coated with calcium alginate. When the glucose concentration in the surrounding environment changes, the diameter of this hydrogel optical fiber will reversibly change, thereby affecting the propagation characteristics of light. Using this characteristic, the optical fiber can quantitatively detect glucose within a certain range (“Yetisen A K, Jiang N, Fallahi A, et al. Glucose-sensitive hydrogel optical fibers functionalized with phenylboronic acid [J]. Advanced Materials, 2017, 29(15): 1606380.”). In 2018, Yang Changxi team developed a quantum dot doped hydrogel optical fiber ratio fluorescence sensor with a core-cladding structure, which can detect Fe 3+Ion Real-time in-situ selective detection (“Zhou M, Guo J, Yang C. Ratiometric fluorescence sensor for Fe3+ ions detection based on quantum dot-doped hydrogel optical fiber[J]. Sensors and Actuators, B: Chemical, 2018, 264: 52-58.”). In 2019, Alamrani et al. developed an aptamer sensor based on leaky waveguide (LW) technology, immobilizing aptamers in mesoporous chitosan hydrogel waveguide films, providing a more reliable, low-cost and suitable thrombin detection scheme for complex biological samples such as whole blood (“Alamrani NA, Greenway GM, Pamme N, et al. A feasibility study of a leaky waveguide aptasensor for thrombin[J]. Analyst, 2019, 144(20): 6048-6054.”), providing new possibilities for the clinical translation of optogenetic therapy technology. In 2015, the Applegate team reported a biocompatible optical fiber composed entirely of silk fibroin (SF), which formed a flexible optical fiber with mechanical strength and biocompatibility by encapsulating silk films (n = 1.54) in silk hydrogels (n = 1.34) (“Applegate M B, Perotto G, Kaplan D L, et al. Biocompatible silk step-index optical waveguides[J]. Biomedical Optics Express, 2015, 6(11): 4221.”). The optical fiber is prepared using environmentally friendly materials, avoiding the use of irritating solvents, and the light is directly coupled into the glass optical fiber, with a propagation loss of about 2 dB / cm, which can withstand handling and achieve light transmission in biological tissues, providing a functional prototype for in vivo photomedicine applications.In 2018, Zhong et al. developed an alginate-polyacrylamide (alg-PAAm) hydrogel optical fiber suitable for in vivo optogenetics, with a propagation loss of 0.25 dB / cm and a Young's modulus of 48-91 kPa, similar to the mechanical properties of some human tissues ("Wang L, Zhong C, Ke D, et al. Ultrasoft and highly stretchable hydrogel optical fibers for In vivo optogenetic modulations [J]. Advanced Optical Materials, 2018, 6(16): 1800427."). This optical fiber can be used as a replacement for silica fibers, reducing the adverse reactions of tissues to implants. In 2022, Feng's team used polyethylene glycol (PEG)-based and PEG / Pluronic prepolymers to prepare core-type and core-cladding-type hydrogel optical fiber waveguides through room temperature extrusion printing combined with in-situ photopolymerization curing technology, with an optical loss as low as 0.1 dB / cm. In vitro experiments showed that this waveguide can activate photochemical and optogenetic reactions, achieve light transmission in muscle tissue over 5 cm, and enable light-triggered cell migration in 3D hydrogels and drug release from optogenetically engineered materials ("Feng J, Zheng Y, Bhusari S, et al. Printed degradable optical waveguides for guiding light into tissue [J]. Advanced Functional Materials, 2020, 30(45): 2004327."). Although the above studies show that hydrogel optical fibers have great potential in medical detection and optogenetics, they still face challenges in practical application scenarios, such as insufficient mechanical properties and biological environment adaptability, the need for optimization of optical properties, and the need for further evaluation of biocompatibility. Therefore, the development of hydrogel optical fibers with excellent mechanical properties, low optical loss, and good biocompatibility is of great significance for promoting the practical application of hydrogel materials in optogenetic technology and brain-computer interface fields. SUMMARY

[0005] To solve the above technical problems, the present application provides a silk protein-based hydrogel optical fiber and its preparation method and application. The silk protein-based hydrogel optical fiber prepared by the present application has excellent mechanical properties, low optical loss, and good biocompatibility.

[0006] To achieve the above purpose, the present application provides the following technical solutions:

[0007] The application provides a preparation method of a silk fibroin-based hydrogel optical fiber, comprising the following steps:

[0008] (1) degumming treatment is performed on the cocoon in a sodium carbonate solution, soaking, cleaning and drying are performed, and silk is obtained; the obtained silk and a LiBr solution are mixed for sealed dissolution, dialysis, centrifugation and concentration are performed, and a silk fibroin solution (SF solution) is obtained;

[0009] (2) the silk fibroin solution obtained in step (1) is mixed with a horseradish peroxidase solution and an H2O2 solution to obtain a core liquid; injection gelation is performed on the obtained core liquid, and then the silk fibroin-based hydrogel fiber core is obtained by sequentially immersing in an ethanol solution and water;

[0010] (3) the silk fibroin solution obtained in step (1) is mixed with a horseradish peroxidase solution and an H2O2 solution to obtain a coating liquid; the silk fibroin-based hydrogel fiber core obtained in step (2) is immersed in the coating liquid for coating treatment, and the silk fibroin-based hydrogel optical fiber is obtained.

[0011] Technical principle: the silk fibroin-based hydrogel optical fiber with a chemical-physical double crosslinking structure is constructed through the synergistic strategy of enzymatic crosslinking and ethanol solution post-treatment; the silk fibroin solution is prepared by using the sodium carbonate solution for degumming treatment, then the core liquid and the coating liquid are prepared by using the silk fibroin solution, the horseradish peroxidase solution and the H2O2 solution respectively, the core liquid is subjected to injection gelation and then treated with the ethanol solution to enhance the mechanical property, and then the coating liquid is coated, and finally the silk fibroin-based hydrogel optical fiber with excellent mechanical property, low optical loss and good biocompatibility is obtained.

[0012] Further, in step (1), the cocoon and the sodium carbonate solution are used in a ratio of (2-5) : 500 (g / mL), and the concentration of the sodium carbonate solution is 0.02 mol / L.

[0013] Further, in step (1), the degumming treatment is performed at a temperature of 100-120 DEG C for 0.5-1 h.

[0014] Further, in step (1), the degumming treatment is performed at a temperature of 120 DEG C for 1 h.

[0015] Further, in step (1), the soaking reagent is warm water, the temperature of the warm water is 40 DEG C, and the soaking time is 20 min; the cleaning reagent is deionized water, and the cleaning is performed 4-5 times; and the drying temperature is 60 DEG C.

[0016] Further, in step (1), the ratio of the amount of silk and LiBr solution is 1:15 (g / mL); the concentration of the LiBr solution is 9.3 mol / L; the temperature of the sealed dissolving is 60℃, and the time is 6-7 h.

[0017] Further, in step (1), the dialysis liquid is deionized water, and the dialysis time is two days, and the water is changed every 1 h.

[0018] Further, in step (1), the centrifugation is performed twice; the centrifugation conditions are 9000 r / min, 4℃, and 20 min.

[0019] Further, in step (1), the concentration is performed in a 10 wt% polyethylene glycol solution.

[0020] Further, in step (2), the concentration of the silk fibroin solution is 3-5 wt%, and further preferably 4-5 wt%; the volume ratio of the silk fibroin solution, the horseradish peroxidase solution, and the H2O2 solution is 1 mL:(20-60) μL:(20-80) μL, and more preferably 1 mL:(40-60) μL:(60-80) μL; the concentration of the horseradish peroxidase solution is 250 U / mL, and the concentration of the H2O2 solution is 40 mM.

[0021] Further, in step (2), the obtained core liquid is injected into a mold tube to form a gel, specifically, the obtained core liquid is injected into a mold tube, and is left to stand at 37℃ for 40 min to complete crosslinking, and then is soaked in an ethanol solution to solidify, and then the mold tube is peeled off to obtain a silk fibroin-based hydrogel fiber core; the concentration of the ethanol solution is 80 v / v%.

[0022] Further, in step (2), the concentration of the ethanol solution is 80 v / v%; the soaking time in the ethanol solution is 2 h. The ethanol solution soaking is used to strengthen the mechanical properties of the silk fibroin-based hydrogel fiber core, and further improve the mechanical properties of the optical fiber.

[0023] Further, in step (3), the concentration of the silk fibroin solution is 2 wt%; the volume ratio of the silk fibroin solution, the horseradish peroxidase solution, and the H2O2 solution is 1 mL:(20-60) μL:(20-80) μL, and more preferably 1 mL:(40-60) μL:(60-80) μL; the concentration of the horseradish peroxidase solution is 250 U / mL, and the concentration of the H2O2 solution is 40 mM. The SF hydrogel with a low concentration is used as a cladding layer, a concentration difference is formed between the core and the cladding, and thus the SF hydrogel optical fiber is obtained.

[0024] Further, in step (3), the temperature of the coating treatment is room temperature, and the time is 0.5 h.

[0025] The application provides a silk fibroin protein-based hydrogel optical fiber prepared by the preparation method.

[0026] The application also provides application of the silk fibroin protein-based hydrogel optical fiber in optogenetic technology and brain-computer interface for non-therapeutic purposes.

[0027] Compared with the prior art, the application has the following advantages and technical effects:

[0028] The application successfully crosslinks the SF hydrogel fiber core by optimizing the degumming process, the concentration of the SF solution during crosslinking, the proportion of the HRP solution and the H2O2 solution, and the like, uses the SF hydrogel with low concentration as the cladding layer, forms a concentration difference between the inside and the outside, and finally obtains the SF hydrogel optical fiber.

[0029] The silk fibroin protein-based hydrogel optical fiber prepared by the application has excellent comprehensive performance: the breaking stress is 616.4 kPa, the structural stability is maintained at 63% swelling degree after swelling balance; the optical transmission loss is as low as 2.2 dB / cm, and the signal transmission can be effectively performed; the obvious cytotoxicity is not present, and the good biocompatibility is present, so that the silk fibroin protein-based hydrogel optical fiber is expected to be used as an implanted biomaterial in the field of brain-computer interface and tissue engineering. BRIEF DESCRIPTION OF DRAWINGS

[0030] The accompanying drawings, which form a part of the present application, are used to provide further understanding of the present application, and the illustrative embodiments of the present application and their description are used to explain the present application, and do not constitute improper limitations on the present application. In the drawings:

[0031] Figure 1 The ultraviolet spectrum of the SF hydrogel prepared by using the SF solution obtained under conditions ③, ④ and ⑤ in embodiment 1 is shown in the figure;

[0032] Figure 2 The tensile breaking stress of the silk fibroin protein-based hydrogel fiber core prepared by using different concentrations of SF solution in embodiment 2 is shown in the figure;

[0033] Figure 3 The tensile breaking stress of the silk fibroin protein-based hydrogel fiber core under different HRP solution dosages in embodiment 3 is shown in the figure;

[0034] Figure 4 The tensile breaking stress of the silk fibroin protein-based hydrogel fiber core under different H2O2 solution dosages in embodiment 4 is shown in the figure;

[0035] Figure 5 The curve of the swelling degree of the silk fibroin protein-based hydrogel optical fiber prepared in embodiment 5 in the PBS buffer solution with time is shown in the figure.

[0036] Figure 6 Scanning electron microscope images of the silk fibroin-based hydrogel optical fiber prepared in Example 5, wherein the upper image is a cross-sectional view and the lower image is a partial enlarged view of the cross-section;

[0037] Figure 7 Results of the correlation between the length and the optical power of the silk fibroin-based hydrogel optical fiber prepared in Example 5;

[0038] Figure 8 Toxicity of the SF solution prepared in step (1) of Example 5 to 4T1 cells and L02 liver cells (a), cell proliferation rate data of the SF hydrogel fiber core prepared in step (2) of Example 5 co-cultured with 4T1 cells for different lengths of time (b), and merged images of live cell (Live, green) and dead cell (Dead, red) fluorescence staining and bright field (BF) images of the control group and the hydrogel treatment group (c). DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0040] In order to make the above objectives, features and advantages of the present application more apparent, the present application will be further described in detail below with reference to the drawings and specific embodiments.

[0041] Unless otherwise specified, the raw materials in the embodiments of the present application are obtained by commercial purchase.

[0042] Example 1

[0043] A preparation method of a silk fibroin solution, and the specific steps are as follows:

[0044] (1) 2 g of cut silkworm cocoons were added to 500 mL of a sodium carbonate solution with a concentration of 0.02 mol / L, and degumming treatment was performed under the following conditions: ① 80℃ for 30 min; ② 80℃ for 1 h; ③ 100℃ for 1 h; ④ 120℃ for 30 min; and ⑤ 120℃ for 1 h. The degummed silk was washed with deionized water and soaked in warm water at 40℃ for 20 min, and then washed with deionized water for 4-5 times. The obtained silk was pulled apart and dried in an oven at 60℃ overnight.

[0045] (2) Take 1 g of the treated silk and add 15 mL of a LiBr solution with a concentration of 9.3 mol / L, seal, and place in an oven at 60°C to dissolve for 6-7 h, then place in a dialysis bag (molecular weight cut-off 3500 Da) and dialyze in deionized water for two days, changing the water every 1 h to remove the LiBr. After dialysis, centrifuge the silk fibroin solution twice at 9000 r / min, 4°C, and 20 min. Then place the centrifuged silk fibroin solution in a dialysis bag, and then place in a 10 wt% polyethylene glycol solution to concentrate to 2 wt%, 3 wt%, 4 wt%, and 5 wt% respectively, to obtain a silk fibroin solution (SF solution), and store in a 4°C refrigerator.

[0046] During the experiment, it was found that when degumming was performed under conditions ① and ②, the SF was extremely easy to become a white physical gel after dialysis for 2-3 days and during concentration, and could not reach the target concentration for preparing a hydrogel with excellent mechanical properties; therefore, the following characterization and experiments were only for the SF solution obtained under conditions ③, ④, and ⑤.

[0047] Performance test of the silk fibroin solution prepared in Example 1

[0048] 1. Molecular weight of silk

[0049] Take the 2 wt% SF solution obtained under conditions ③, ④, and ⑤ in Example 1, dilute to 0.1 wt% as the starting concentration, and use a capillary efflux method to measure the intrinsic viscosity [η] of the SF solution using an Ubbelohde viscometer. Since [η] and the average relative molecular mass of the polymer There is a semi-empirical relationship, and the molecular weight of the silk can be calculated according to formula 1

[0050]

[0051] In formula 1, K is a proportionality coefficient, and a is a shape parameter. In the present application, K ≈ 0.01 dL / g, and a ≈ 0.5.

[0052] The experimental results are as follows: the intrinsic viscosity [η] of the SF solution obtained under condition ③ is 2.0805 dL / g, and the average relative molecular mass of the silk is calculated to be 43300 Da by substituting formula 1; the intrinsic viscosity [η] of the SF solution obtained under condition ④ is 1.8755 dL / g, and the average relative molecular mass of the silk is calculated to be 35200 Da by substituting formula 1; the intrinsic viscosity [η] of the SF solution obtained under condition ⑤ is 1.408 dL / g, and the average relative molecular mass of the silk is calculated to be 19800 Da by substituting formula 1. It can be seen that the higher the degumming temperature and the longer the time, the smaller the average relative molecular mass of the silk obtained; this is because high temperature and long time degumming cause the silk fibroin peptide bond to break and the molecular chain to shorten through thermal degradation and hydrolysis reaction, thereby reducing the average relative molecular mass. Moreover, because the SF with high molecular weight has longer peptide chains, it is easy to entangle with each other, has abundant interaction sites and is more prone to form β-pleated sheet to form physical crosslinking, and is extremely easy to become white crosslinked material during subsequent concentration process, and it is difficult to obtain SF solution with ideal concentration, and the SF solution with high molecular weight has high viscosity, which is not conducive to experimental operation. This also proves that the SF degummed under conditions ① and ② is extremely easy to become white physical gel after dialysis for 2-3 days and during concentration.

[0053] 2. Light transmittance of SF hydrogel

[0054] To prepare a hydrogel with excellent optical properties, the light transmittance of the SF hydrogel was measured in the wavelength range of 200-800 nm by using a UV-visible near-infrared spectrophotometer. The preparation process of the SF hydrogel is as follows: first, the SF solution with a concentration of 5 wt% obtained under conditions ③, ④ and ⑤ in Example 1 was taken out of the refrigerator, 2 mL of the solution was taken by using a pipette into a 1 cm cuvette, 50 μL of horseradish peroxidase (HRP, 250 U / mL) solution and 60 μL of hydrogen peroxide (H2O2, 40 mM) solution were added and mixed uniformly, and then the mixture was placed in a 37℃ oven for crosslinking. After 1 h of crosslinking reaction, the SF hydrogel was obtained. Figure 1 The UV spectrum of the SF hydrogel prepared by using the SF solution obtained under conditions ③, ④ and ⑤ in Example 1 is shown in FIG. 2. It can be seen that the transmittance of the SF hydrogel increases continuously in the range of 300-800 nm as the degumming temperature increases and the degumming time extends. Figure 1 It can be seen that the transmittance of the SF hydrogel prepared by using the SF solution obtained under condition ⑤ is the highest, the transmittance of the SF hydrogel prepared by using the SF solution obtained under condition ③ is relatively low, and the transmittance of the SF hydrogel prepared by using the SF solution obtained under condition ④ is between the two. As a precursor material of hydrogel optical fiber, it is necessary to ensure a relatively high transmittance.

[0055] Example 2

[0056] A preparation method of a silk fibroin-based hydrogel fiber core, the specific steps are as follows:

[0057] (1) Take 1 mL of the SF solution with a concentration of 2 wt%, 3 wt%, 4 wt%, and 5 wt% obtained under condition IV of Example 1 into a sample bottle, respectively, and add 40 μL of HRP solution (250 U / mL) and 40 μL of H2O2 solution (40 mM) into each sample bottle using a syringe, and mix well to obtain a core solution.

[0058] (2) The core solution obtained in step (1) is quickly injected into a polytetrafluoroethylene (PTFE) tubular mold with an inner diameter of 1.8 mm using a syringe with a needle, and is left to stand at 37°C for 40 min to complete crosslinking, then is soaked in an ethanol solution with a concentration of 80 v / v% for 48 h, is cut along the port of the PTFE tube, the PTFE tube is peeled off, the hydrogel fiber is completely taken out, is soaked in an ethanol solution with a concentration of 80 v / v% again for 2 h, and then is taken out after being soaked in deionized water for 2 h, to obtain a silk fibroin-based hydrogel fiber core.

[0059] Since the crosslinking speed of the SF solution with a concentration of 5 wt% is too fast, it cannot be injected into the PTFE tube, so only the silk fibroin-based hydrogel fiber cores prepared using SF solutions with concentrations of 2 wt%, 3 wt%, and 4 wt% are tested for quasi-static tensile breaking load.

[0060] Example 3

[0061] A preparation method of a silk fibroin-based hydrogel fiber core, the specific steps are as follows:

[0062] (1) Take 1 mL of the SF solution with a concentration of 4 wt% obtained under condition IV of Example 1 into a sample bottle, and add 20 μL, 40 μL, 60 μL, and 80 μL of HRP solution (250 U / mL) and 40 μL of H2O2 solution (40 mM) into the sample bottle using a syringe, respectively, and mix well to obtain a core solution.

[0063] Step (2) is the same as that in Example 2.

[0064] Example 4

[0065] A preparation method of a silk fibroin-based hydrogel fiber core, the specific steps are as follows:

[0066] (1) Take 1 mL of the SF solution with a concentration of 4 wt% obtained under condition IV of Example 1 into a sample bottle, and add 20 μL, 40 μL, 60 μL, and 80 μL of H2O2 solution (40 mM) and 60 μL of HRP solution (250 U / mL) into the sample bottle using a syringe, respectively, and mix well to obtain a core solution.

[0067] Step (2) is the same as that in Example 2.

[0068] The mechanical properties of the silk fibroin-based hydrogel fiber cores prepared in Example 2-4 were tested by quasi-static tensile breaking load, and the results are shown in Table 1. Figures 2-4 The test process is as follows: the upper end of the sample is fixed to a magnetic clamp system, vertically suspended, the clamping length is 5 mm, the lower end is connected to a detachable weight loading device, a gradual load method is used, the axial tensile load is gradually applied in increments of Δm = 0.5 g (interval 10 s), until the sample breaks, and the critical breaking mass m critical The tensile breaking stress is calculated according to formula 2:

[0069]

[0070] In formula 2, r is the radius of the fiber core, which is measured to be r = 0.45 mm = 4.5 x 10 -4 m after demolding; g = 9.8 m / s 2 .

[0071] Figure 2 The tensile breaking stress of the silk fibroin-based hydrogel fiber cores prepared in Example 2 using different concentrations of SF solution. As can be seen from Table 2, Figure 2 With the increase of the concentration of SF solution, the breaking stress of the silk fibroin-based hydrogel fiber core gradually increases, and when the concentration of SF solution is 4wt%, the breaking strength of the silk fibroin-based hydrogel fiber core reaches the maximum, which is 354.4kPa, so the concentration of 4wt% SF solution is selected to prepare the silk fibroin-based hydrogel fiber core.

[0072] Figure 3 The tensile breaking stress of the silk fibroin-based hydrogel fiber cores in Example 3 under different amounts of HRP solution. As can be seen from Table 3, Figure 3 When the amount of HRP solution is 20-60 μL, the breaking stress of the silk fibroin-based hydrogel fiber core gradually increases, and reaches the maximum of 477.7 kPa at 60 μL; the breaking strength of the silk fibroin-based hydrogel fiber core added with 80 μL of HRP solution decreases sharply to 130.9 kPa, which may be due to the following reasons: the crosslinking speed is too fast, causing the local crosslinking density to be too high, forming a stress concentration point, and reducing the overall mechanical properties, so it is most appropriate to select 1 mL of SF solution added with 60 μL of HRP solution (250 U / mL).

[0073] Figure 4 The tensile breaking stress of the silk fibroin-based hydrogel fiber cores in Example 4 under different amounts of H2O2 solution. As can be seen from Table 4, Figure 4It can be seen that when the amount of H2O2 solution is in the range of 20-80 μL, the breaking stress of the silk fibroin-based hydrogel core gradually increases with the increase of the amount of H2O2 solution, and reaches a peak value of 616.4 kPa at 80 μL; while the amount of H2O2 solution continues to increase to 100 μL, the breaking strength drops to 439.1 kPa. This may be due to two reasons: one is that excessive H2O2 leads to irreversible inactivation of HRP, directly interrupting the cross-linking reaction; the other is that excessive H2O2 may oxidize silk fibroin peptide chains, destroy the integrity of molecular chains, and thus reduce the stability of the cross-linked network. According to the experimental results, it is determined that the optimal cross-linking condition is to add 80 μL of H2O2 solution (40 mM) to 1 mL of SF solution.

[0074] According to the experimental results of Examples 2-4, the optimal cross-linking condition is determined as follows: the concentration of SF solution is 4 wt%, 60 μL of HRP solution (250 U / mL) and 80 μL of H2O2 solution (40 mM) are added to 1 mL of SF solution.

[0075] Example 5

[0076] A preparation method of a silk fibroin-based hydrogel optical fiber, the specific steps are as follows:

[0077] (1) Preparation of SF solution: 3 g of cut silkworm cocoon was taken into 500 mL of 0.02 mol / L sodium carbonate solution, and degumming was carried out in 120°C oil bath for 30 min. The degummed silk was washed with deionized water and soaked in 40°C warm water for 20 min. The silk was washed with deionized water for 4-5 times, and then was placed in a 60°C oven for overnight drying. 1 g of treated silk was added into 15 mL of 9.3 mol / L LiBr solution, sealed and placed in a 60°C oven for 6-7 h, and then was packed with dialysis bag and dialyzed in deionized water for two days, with water changed every 1 h to remove LiBr. After dialysis, the obtained silk fibroin solution was centrifuged twice at 9000 r / min, 4°C and 20 min. The centrifuged silk fibroin solution was packed with dialysis bag, and then was concentrated in 10 wt% polyethylene glycol solution. The mass fraction of SF solution was measured by evaporation weighing method, and 2 wt% and 4 wt% silk fibroin solutions (SF solutions) were obtained, which were sealed and stored in a 4°C refrigerator for use.

[0078] (2) Preparation of SF hydrogel fiber core: Take 1 mL of the SF solution with a concentration of 4 wt% obtained in step (1) into a sample bottle, and add 60 μL of HRP solution (250 U / mL) and 80 μL of H2O2 solution (40 mM) into the sample bottle using a syringe, mix well to obtain the core solution. The obtained core solution is quickly injected into a PTFE tubular mold with an inner diameter of 1.8 mm using a syringe with a needle, and is left to stand in a 37°C oven for 40 min to complete the crosslinking, and then is soaked in an ethanol solution with a concentration of 80 v / v% for 48 h, cut along the PTFE tube port, peel off the PTFE tube, take out the hydrogel fiber completely, and then soak in an ethanol solution with a concentration of 80 v / v% for 2 h, and then soak in deionized water for 2 h to obtain the silk fibroin-based hydrogel fiber core.

[0079] (3) Preparation of SF hydrogel optical fiber: Take 1 mL of the SF solution with a concentration of 2 wt% obtained in step (1) into a culture dish, and add 60 μL of HRP solution (250 U / mL) and 80 μL of H2O2 solution (40 mM) into the culture dish to obtain a uniform coating solution. The silk fibroin-based hydrogel fiber core obtained in step (2) is immersed in the coating solution for 3 min and then taken out, and then is vertically hung at room temperature for 0.5 h to obtain the silk fibroin-based hydrogel optical fiber (SF hydrogel optical fiber).

[0080] Performance test of the silk fibroin-based hydrogel optical fiber prepared in Example 5

[0081] 1. Swelling property determination

[0082] Three SF hydrogel optical fibers prepared in Example 5 with similar specifications are taken, and the initial mass w0 is accurately weighed, and then is placed in a PBS buffer solution at 37°C. At the set time nodes (10, 20, 30, 40, 50, 60 and 120 min), the sample is taken out in turn, and the surface liquid is absorbed by filter paper to determine the mass w1. The swelling rate (SR) is calculated based on the initial mass w0 using formula 3, and the results are shown in Table 1. Figure 5 .

[0083]

[0084] The mechanical properties and optical transmission properties of the hydrogel optical fiber are restricted by the swelling properties. When the swelling degree is high, the material is easy to deform due to water absorption, which leads to a decrease in the mechanical properties, and the loss in the process of optical signal transmission may increase. Figure 5 The curve of the swelling degree of the silk fibroin-based hydrogel optical fiber prepared in Example 5 in the PBS buffer solution with time is shown in FIG. 1. Figure 5The results show that the peak swelling ratio of the SF hydrogel optical fiber prepared in Example 5 is only 63%. The low swelling ratio enables the SF hydrogel optical fiber to maintain good mechanical strength and stable optical transmission efficiency, effectively avoiding problems such as nerve tissue damage, signal transmission distortion and equipment failure caused by water absorption and swelling of traditional materials. This provides important technical support for the practical application of long-term implantable neural interfaces in clinical scenarios.

[0085] 2. Mechanical performance evaluation

[0086] The testing method is the same as the method used to test the mechanical properties of the silk protein-based hydrogel cores prepared in Examples 2-4 above.

[0087] The fracture stress of the SF hydrogel optical fiber prepared in Example 5 was measured to be 616.4 kPa. This strength is much higher than that of SF hydrogel fibers with single physical cross-linking (10-100 kPa) and has exceeded the strength limit of SF hydrogel fibers with single chemical cross-linking (100-500 kPa), reaching the mid-to-high level of double cross-linked hydrogels. This indicates that the synergistic effect of physical cross-linking and enzymatic chemical cross-linking significantly improves the network density and load transfer efficiency of the hydrogel. As an implantable medical optical fiber, a strength of 616.4 kPa is sufficient to resist the daily peristaltic deformation of muscle tissue.

[0088] 3. Morphological and structural characterization

[0089] The SF hydrogel optical fiber prepared in Example 5 was subjected to liquid nitrogen freezing treatment. After brittle fracture, it was freeze-dried using a vacuum freeze-drying device. Subsequently, the microstructure and cross-section were observed using emission scanning electron microscopy. The results are shown in […]. Figure 6 .

[0090] Figure 6 The images shown are scanning electron microscope (SEM) images of the silk fibroin-based hydrogel optical fiber prepared in Example 5. The top image is a cross-sectional view, and the bottom image is a magnified view of a portion of the cross-section. Figure 6 As can be seen, the silk fibroin-based hydrogel optical fiber prepared in Example 5 has a uniform profile and subtle differences in the micro-networks of the inner and outer layers, but no obvious core-sheath delamination. This may be due to the synergistic effect of physical-chemical double crosslinking blurring the boundaries, and the shrinkage of the low-concentration hydrogel in the outer layer during dehydration, which weakens the interface. Although there is a lack of clear layered boundaries in morphology, the difference in SF concentration between the inner and outer layers forms a functional gradient: the high-concentration network in the inner layer is densely crosslinked, corresponding to a fracture stress of 616.4 kPa, ensuring structural strength; the low-concentration outer layer matches a swelling ratio of 63%, balancing hydrophilicity and stability. The core layer has a high molecular chain density and a higher refractive index than the sheath layer, meeting the requirements for total internal reflection. The core-sheath function of the hydrogel optical fiber of this invention lies in the synergistic effect of the composition-performance gradient, rather than relying on morphological delamination. The blurred interface and concentration gradient together provide support for the light guiding performance and mechanical stability of the hydrogel optical fiber.

[0091] 4. Optical loss

[0092] Optical transmission loss measurement adopts an insertion detection scheme, and the main equipment includes an optical power meter and a light-emitting device. The experimental process is divided into two stages: the initial stage records the reference value (P1), that is, the light intensity reading when the light source is directly connected with the power meter; the subsequent stage obtains the measured value (P2), that is, the intensity change of the measured signal after transmission through the optical fiber. The unit length optical loss (a) is calculated by using formula 4.

[0093]

[0094] In formula 4, L represents the optical fiber length parameter in centimeters.

[0095] The unit length optical loss of the SF hydrogel optical fiber prepared in Example 5 is 2.2 dB / cm, which is comparable to other polymer optical fibers (0.02-5 dB / cm) and far lower than natural silk materials (28 dB / cm), showing obvious transmission performance advantages. The hydrogel optical fiber prepared by the present application can effectively reduce the attenuation of the signal in the transmission process due to its low optical loss characteristics, and therefore can better meet the actual needs of brain-computer interface in optical fiber transmission signal as a whole.

[0096] 5. Length and optical power correlation test

[0097] Three groups of SF hydrogel optical fiber samples with a length of 10 cm prepared in Example 5 were taken as test objects. During the experiment, the step-by-step shortening method was used, and the sample length was reduced by 1 cm each time. The insertion optical intensity detection technology was used to accurately measure the optical transmission efficiency at each shortening stage. Through systematic analysis of the collected data, the relationship between the length change of the optical fiber and its optical performance was explored, and the results are shown in Figure 7 The length of the optical fiber is one of the key parameters that restricts the optical power. Exploring the correlation between the length of the hydrogel optical fiber and the optical power helps to clarify its effectiveness and stability in optical signal transmission.

[0098] Figure 7 The length and optical power correlation results of the silk protein-based hydrogel optical fiber prepared in Example 5. The experimental data are fitted by a function, and the R 2= 0.997, indicating a good fitting effect. The data showed that as the length of the optical fiber was shortened, the output optical power showed a gradual upward trend, and when the length of the optical fiber was about 3 cm, the output optical power could reach about 5500 nW. From the perspective of practical application, researchers could reasonably select the appropriate fiber length according to the fitting curve and the target optical power value, so as to realize the precise control of the quality of the optical signal. In addition, by carrying out length test experiments, the optimal fiber length for implantation could be further screened, which could guarantee a higher signal power and provide key parameter support for the development of adaptive implantable brain-computer interface devices.

[0099] 6. Cell compatibility evaluation

[0100] 6.1 Cytotoxicity of SF solution

[0101] The CCK-8 method was used to evaluate the in vitro cytotoxicity of the SF solution prepared in step (1) of Example 5 with a concentration of 4%, and the relative survival rate of 4T1 cells and L02 cells was detected. The detection process was as follows: 4T1 cells and L02 cells were inoculated in a 96-well cell culture plate at a density of 8000 per well, 100 μL of culture medium was added to each well, and the cells were cultured in an incubator overnight to adhere to the bottom of the plate. The SF solution was dissolved in sterile PBS to prepare a 20% aqueous solution, filtered through a 0.22 μm sterile filter to remove bacteria, and then diluted with fresh culture medium to prepare SF solution with gradient concentrations (0, 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1.6 mg / mL, 3.2 mg / mL, 6.4 mg / mL, 12.8 mg / mL) to obtain SF culture medium solution. After the cells adhered, the original culture medium was discarded, and 100 μL of freshly prepared SF culture medium solution was added to each well, and the culture was continued for 48 h. After the culture ended, the supernatant was removed, 100 μL of fresh DMEM medium containing 10% CCK-8 was added to each well, and after 2 h of incubation, the absorbance (OD value) at 450 nm was measured using a microplate reader. The cells without the addition of SF solution were set as the control group (control), and the SF solution without cells was set as the blank group (blank), and both groups were treated according to the same process. The relative survival rate of the cells was calculated according to Formula 5, and the results are shown in part a of Table 1. Figure 8

[0102]

[0103] In Formula 5, OD sample is the absorbance value of the sample, OD control is the absorbance value of the control group, and OD blank is the absorbance value of the blank group.

[0104] 6.2 Cytotoxicity of SF hydrogel core (CCK-8 method) ​

[0105] The in vitro cytotoxicity of the SF hydrogel core prepared in Example 5, step (2) was evaluated using 4T1 cells as a model. 4T1 cells were seeded in 24-well plates at a density of 1 x 10 4 The original culture medium of the adherent cells was discarded, and 500 μΐ of fresh DMEM was added to each well, followed by the addition of the SF hydrogel core prepared in Example 5, step (2) to each well. The cells were cultured for 6 h, 24 h, and 48 h, respectively. After the incubation, the SF hydrogel core was removed, and the culture medium was discarded. Then, 400 μΐ of culture medium containing 10% CCK-8 was added to each well, and the incubation was continued for 2 h. Then, 100 μΐ was transferred to a 96-well plate, and the optical density (OD) at 450 nm was measured using a microplate reader. The cells without the SF hydrogel core were used as a control group, and the cells without the cells were used as a blank group. The relative survival rate of the cells cultured for 6 h without the SF hydrogel core was used as a reference value of 100%. The relative survival rate of the cells was calculated according to Formula 5, and the results are shown in part b of Figure 8 .

[0106] 6.3 Cytotoxicity of SF hydrogel (Live-Dead staining method)

[0107] The 4T1 cell suspension was seeded in a 35-mm confocal special culture dish (1 mL per well) at a concentration of 1 x 10 4 After the cells were adhered, the original culture solution was removed, and 1 mL of fresh 1640 culture solution was added. The SF hydrogel core prepared in Example 5, step (2) was immersed in the culture system, and another culture dish without the SF hydrogel core was used as a control. The culture was continued for 24 h under standard culture conditions. After the culture was terminated, the SF hydrogel core was removed, and the residual culture solution was removed. The SF hydrogel core was gently washed three times with sterile PBS buffer to remove impurities. Then, 1640 culture medium containing Live-Dead staining agent was added, and the cell viability was detected and the images were collected using an inverted fluorescence microscope. The results are shown in part c of Figure 8 .

[0108] Figure 8 Toxicity of the SF solution prepared in Example 5, step (1) on 4T1 cells and L02 hepatocytes (a), cell proliferation rate data of the SF hydrogel core prepared in Example 5, step (2) and 4T1 cells cultured for different lengths of time (b), and merged images of live cells (Live, green), dead cells (Dead, red) fluorescent staining, and bright field (BF) images of the control group and the SF hydrogel treatment group (c). From Figure 8As shown in part a, when the SF solution concentration was 12.8 mg / mL, neither type of cell showed a significant toxic reaction, thus confirming that the SF solution has good biocompatibility. From Figure 8 As shown in section b, in the culture system containing SF hydrogel, the proliferation rate of 4T1 cells was basically consistent with that of the control group, and both groups of cells showed a continuous growth trend. Notably, after 24 h and 48 h of culture, the cell proliferation rate in the hydrogel group was slightly higher than that in the control group, indicating that SF hydrogel not only did not inhibit cell growth, but also, to some extent, promoted cell proliferation. Combined with... Figure 8 The fluorescence imaging results in section c show that after 48 hours of culture, the number of live cells in the hydrogel-treated group was significantly higher than that in the control group, the proportion of dead cells was lower, and the cell morphology remained normal. Based on the above experimental results, the SF hydrogel prepared in this invention exhibits extremely weak toxicity to 4T1 cells, demonstrating excellent cell compatibility and possessing potential application value in the field of biomedical materials.

[0109] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing a silk fibroin-based hydrogel optical fiber, characterized by, The method comprises the following steps: (1) degumming the cocoon in a sodium carbonate solution, soaking, cleaning and drying to obtain silk, mixing the obtained silk with a LiBr solution to seal and dissolve, dialyzing, centrifuging and concentrating to obtain a silk fibroin solution; the degumming temperature is 100-120℃, and the degumming time is 0.5-1h; (2) mixing the silk fibroin solution obtained in step (1) with a horseradish peroxidase solution and an H2O2 solution to obtain a core solution; injecting the obtained core solution into a PTFE tubular mold with an inner diameter of 1.8mm using a syringe with a needle, standing at 37℃ for 40min to complete crosslinking, then soaking in an ethanol solution with a concentration of 80v / v% for 48h, cutting along the PTFE tube port, peeling off the PTFE tube, taking out the hydrogel fiber completely, soaking in an ethanol solution with a concentration of 80v / v% again for 2h, then soaking in deionized water for 2h and taking out, to obtain a silk fibroin-based hydrogel core; the concentration of the silk fibroin solution is 3-4wt%; the volume ratio of the silk fibroin solution, the horseradish peroxidase solution and the H2O2 solution is 1mL:(20-60)μL:(20-80)μL; the concentration of the horseradish peroxidase solution is 250U / mL, and the concentration of the H2O2 solution is 40mM; (3) mixing the silk fibroin solution obtained in step (1) with a horseradish peroxidase solution and an H2O2 solution to obtain a coating solution; immersing the silk fibroin-based hydrogel core obtained in step (2) in the coating solution to perform coating treatment, to obtain the silk fibroin-based hydrogel optical fiber; the concentration of the silk fibroin solution is 2wt%; the volume ratio of the silk fibroin solution, the horseradish peroxidase solution and the H2O2 solution is 1mL:(20-60)μL:(20-80)μL; the concentration of the horseradish peroxidase solution is 250U / mL, and the concentration of the H2O2 solution is 40mM; the coating treatment temperature is room temperature, and the coating treatment time is 0.5h.

2. The production method according to claim 1, characterized by, In step (1), the cocoon and the sodium carbonate solution are used in a ratio of (2-5):500 (g / mL), and the concentration of the sodium carbonate solution is 0.02mol / L.

3. A silk fibroin-based hydrogel optical fiber prepared by the preparation method of any one of claims 1-2.

4. Use of the silk fibroin-based hydrogel optical fiber of claim 3 in optogenetic technology and brain-computer interface for non-therapeutic purposes.

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