Fibroin-based hydrogel optical fiber as well as preparation method and application thereof

Abstract

The invention discloses a fibroin-based hydrogel optical fiber as well as a preparation method and application thereof, and belongs to the technical field of hydrogel optical fibers. According to the preparation method, the fibroin-based hydrogel optical fiber with a chemical-physical double-crosslinking structure is constructed through a synergistic strategy of enzymatic crosslinking and ethanol solution post-treatment; the preparation method comprises the following steps: performing degumming treatment by using a sodium carbonate solution to prepare a silk protein solution, respectively preparing a core solution and a coating solution by using the silk protein solution, a horseradish peroxidase solution and an H2O2 solution, injecting the core solution into gel, then using an ethanol solution to reinforce the mechanical properties of the core solution, and then coating the coating solution, thereby obtaining the high-strength and high-toughness fiber. Finally, the fibroin-based hydrogel optical fiber with excellent mechanical properties, low optical loss and good biocompatibility is obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of hydrogel optical fibers, and in particular relates to a silk protein-based hydrogel optical fiber and a preparation method and application thereof. Background Art

[0002] In recent years, brain-computer interface (BCI) technology has developed rapidly. While the medical field has become a core application area for BCI technology, its application has expanded to diverse scenarios such as education, entertainment, competition, and the military, presenting a multi-dimensional commercial prospect. BCIs establish a bidirectional interactive channel between bioelectric signals and electronic devices, enabling motor control decoding and sensory information encoding, providing a technical foundation for functional reconstruction. Neural signals from BCI systems can be collected through two approaches: non-invasive scalp electrode recording and direct acquisition of cortical signals using implanted electrodes. Conventional metal microelectrodes, due to their rigid material, are susceptible to physical damage to delicate brain regions during implantation. Long-term use can also lead to electrochemical reactions, biotoxicity, chronic inflammation, neurodegeneration, and signal transduction barriers caused by glial cell encapsulation, seriously impacting their stability and lifespan. Against this backdrop, the development of BCI technology that does not require implanted electrodes has become a critical and pressing issue.

[0003] Fiber optic photometric recording technology can achieve parallel detection of neural activities in multiple brain regions by transmitting and collecting neuronal fluorescence signals through optical fibers. It provides important support for the integration of optogenetics and brain-computer interface systems, and promotes the construction of a bidirectional neural information transmission system based on optical principles. Currently, 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, etc. According to the materials, traditional silica-based optical fibers have excellent optical transparency and extremely low propagation loss, but their biocompatibility is insufficient, they are brittle and have high rigidity. As a result, these optical fibers can only be used temporarily for treatment or diagnosis, and have application limitations. Hydrogel optical fibers can well solve the above problems.

[0004] Recently, the preparation technology and functional properties of hydrogel optical fibers have received continuous attention from the academic community, and various research institutions have made breakthroughs. Thanks to the excellent tissue compatibility of hydrogels and the rapid response and accurate detection characteristics of optical fiber sensing systems, this type of new composite material has shown great application potential in pathological screening and real-time monitoring of physiological parameters, bringing more reliable and safe innovative solutions to the field of modern medical diagnosis. In 2017, the Yetisen team developed a hydrogel optical fiber whose core material is poly(acrylamide-co-polyethylene 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 change reversibly, thereby affecting the propagation characteristics of light. Using this characteristic, the optical fiber can quantitatively detect glucose within a certain range ("Yetisen AK, 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's team developed a quantum dot-doped hydrogel fiber ratio fluorescence sensor with a core-package structure, which can detect Fe 3+ions to achieve real-time in situ selective detection (“Zhou M, Guo J, Yang C. Ratiometric fluorescence sensor for Fe3+ ions detection based on quantumdot-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 the aptamer in a mesoporous chitosan hydrogel waveguide membrane, providing a more reliable, low-cost and suitable thrombin detection solution for complex biological samples (such as whole blood) (“Alamrani NA, Greenway GM, Pamme N, et al. Afeasibility study of a leaky waveguide aptasensor for thrombin[J]. Analyst, 2019, 144(20): 6048-6054.”), which provides new possibilities for the clinical transformation of optogenetic therapy technology. In 2015, Applegate's team reported a biocompatible optical fiber made entirely of silk fibroin (SF). By encapsulating a silk membrane (n=1.54) in a silk hydrogel (n=1.34), they created a flexible optical fiber with both mechanical strength and biocompatibility ("Applegate MB, Perotto G, Kaplan DL, et al. Biocompatible silk step-index optical waveguides [J]. Biomedical Optics Express, 2015, 6(11): 4221."). This fiber is made of environmentally friendly materials, avoiding the use of irritating solvents. Light is directly coupled into the fiber through a glass optical fiber, with a propagation loss of approximately 2 dB / cm. It can withstand manipulation and achieve light transmission within biological tissues, providing a functional prototype for in vivo photomedicine applications.In 2018, Zhong Cheng 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 between 48-91 kPa, which is 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 an alternative to silica fibers and can reduce the adverse reactions of tissues to implants. In 2022, Feng's team used polyethylene glycol (PEG) and PEG / Pluronic prepolymers to prepare core-type and core-cladding hydrogel optical fiber waveguides by 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 have shown that the waveguide can activate photochemical and optogenetic reactions, achieve light conduction in muscle tissue over 5 cm, and can trigger cell migration in 3D hydrogels and drug release of optogenetic engineering materials through light (“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 testing and optogenetics, they still face several challenges in practical application scenarios, such as insufficient compatibility between mechanical properties and biological environment, optical properties that still need to be optimized, and biocompatibility that needs to be further evaluated. 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 optogenetics and brain-computer interfaces. Summary of the Invention

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

[0006] To achieve the above objectives, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing a silk protein-based hydrogel optical fiber, comprising the following steps:

[0008] (1) placing silk cocoons in a sodium carbonate solution for degumming, soaking, washing, and drying to obtain silk; mixing the obtained silk with a LiBr solution for sealing and dissolving, dialyzing, centrifuging, and concentrating to obtain a silk protein solution (SF solution);

[0009] (2) mixing the silk protein solution obtained in step (1) with a horseradish peroxidase solution and a H2O2 solution to obtain a core liquid; injecting the obtained core liquid into a gel, and then soaking it in an ethanol solution and water in sequence to obtain a silk protein-based hydrogel fiber core;

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

[0011] Technical principle: The present invention constructs a silk protein-based hydrogel optical fiber with a chemical-physical double cross-linking structure through a synergistic strategy of enzymatic cross-linking and ethanol solution post-treatment; the silk protein solution is first degummed using a sodium carbonate solution to prepare a silk protein solution, and then the core liquid and the coating liquid are prepared using the silk protein solution, horseradish peroxidase solution, and H2O2 solution respectively. After the core liquid is injected into a gel, an ethanol solution is used to enhance its mechanical properties, and then the coating liquid is coated, finally obtaining a silk protein-based hydrogel optical fiber with excellent mechanical properties, low optical loss and good biocompatibility.

[0012] Furthermore, in step (1), the ratio of the silk cocoons to the sodium carbonate solution is (2-5):500 (g / mL), and the concentration of the sodium carbonate solution is 0.02 mol / L.

[0013] Furthermore, in step (1), the degumming treatment is carried out at a temperature of 100-120° C. and for a time of 0.5-1 h.

[0014] Furthermore, in step (1), the degumming treatment is carried out at a temperature of 120° C. and for a time of 1 hour.

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

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

[0017] Furthermore, in step (1), the dialysate for the dialysis is deionized water, the dialysis time is two days, and the water is changed every 1 hour.

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

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

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

[0021] Furthermore, in step (2), the obtained core liquid is injected into a gel as follows: the obtained core liquid is injected into a mold tube, allowed to stand at 37°C for 40 minutes to complete cross-linking, and then immersed in an ethanol solution for curing, and then the mold tube is peeled off to obtain a silk protein-based hydrogel fiber core; the concentration of the ethanol solution is 80v / v%.

[0022] Furthermore, in step (2), the concentration of the ethanol solution is 80 v / v% and the soaking time in the ethanol solution is 2 hours. The present invention utilizes ethanol solution soaking to enhance the mechanical properties of the silk protein-based hydrogel fiber core, thereby improving the mechanical properties of the optical fiber.

[0023] Furthermore, in step (3), the concentration of the silk protein solution is 2 wt%; the volume ratio of the silk protein solution to the horseradish peroxidase solution and the H2O2 solution is 1 mL: (20-60) μL: (20-80) μL, 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 present invention uses a low-concentration SF hydrogel as the cladding, forming an internal and external concentration difference with the core, thereby obtaining an SF hydrogel optical fiber.

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

[0025] The present invention provides a silk protein-based hydrogel optical fiber prepared by the preparation method described in the above technical solution.

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

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

[0028] The present invention successfully cross-links an SF hydrogel core by optimizing parameters such as the degumming process, the concentration of the SF solution during cross-linking, and the usage ratio of the HRP solution and the H2O2 solution. Low-concentration SF hydrogel is then used as the cladding to form an internal and external concentration difference, ultimately yielding an SF hydrogel optical fiber.

[0029] The silk protein-based hydrogel optical fiber prepared by the present invention exhibits excellent comprehensive performance: the breaking stress reaches 616.4 kPa, and the structural stability of 63% swelling degree is maintained after swelling equilibrium; the light transmission loss is as low as 2.2 dB / cm, which can effectively transmit signals; there is no obvious cytotoxicity and good biocompatibility, and it is expected to be used as an implantable biomaterial in the fields of brain-computer interfaces and tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0031] Figure 1 The UV spectra of SF hydrogels prepared using the SF solutions obtained under conditions ③, ④, and ⑤ in Example 1;

[0032] Figure 2 The tensile fracture stress of the silk protein-based hydrogel fiber core prepared using SF solutions of different concentrations in Example 2;

[0033] Figure 3 The tensile breaking stress of the silk protein-based hydrogel fiber core at different HRP solution dosages in Example 3;

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

[0035] Figure 5 This is a curve showing the change in swelling degree over time of the silk protein-based hydrogel optical fiber prepared in Example 5 in PBS buffer solution;

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

[0037] Figure 7 The results of the correlation between the length and optical power of the silk protein-based hydrogel optical fiber prepared in Example 5 are as follows;

[0038] Figure 8 Figure 5 shows the toxic effect of the SF solution prepared in step (1) of Example 5 on 4T1 cells and LO2 hepatocytes (a), the cell proliferation rate data of the SF hydrogel core prepared in step (2) of Example 5 co-cultured with 4T1 cells for different lengths of time (b), and the merged image 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 following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0040] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0041] Unless otherwise specified, the raw materials in the examples of the present invention were purchased from commercial sources.

[0042] Example 1

[0043] A method for preparing a silk protein solution, comprising the following specific steps:

[0044] (1) 2 g of chopped silk cocoons were added to 500 mL of 0.02 mol / L sodium carbonate solution and degummed at ①80°C for 30 min; ②80°C for 1 h; ③100°C for 1 h; ④120°C for 30 min; and ⑤120°C for 1 h. The degummed silk was washed with deionized water and soaked in 40°C warm water for 20 min. The degummed silk was then washed with deionized water 4-5 times. The resulting silk was loosened and dried in an oven at 60°C overnight.

[0045] (2) Take 1g of treated silk and add 15mL of 9.3mol / L LiBr solution, seal it, place it in a 60℃ oven to dissolve for 6-7h, then put it in a dialysis bag (molecular weight cutoff 3500Da), and dialyze it in deionized water for two days, changing the water once every 1h to remove LiBr. After the dialysis is completed, centrifuge the dialyzed silk protein solution twice, and the centrifugation conditions are 9000r / min, 4℃, and 20min. Put the centrifuged silk protein solution in a dialysis bag, and then place it in a 10wt% polyethylene glycol solution and concentrate it to 2wt%, 3wt%, 4wt%, and 5wt% respectively to obtain a silk protein solution (SF solution), which is sealed and stored in a 4℃ refrigerator for use.

[0046] During the experiment, it was found that when degumming was performed under conditions ① and ②, SF easily turned into a white physical gel after 2-3 days of dialysis and when concentrated, and it was impossible to reach the target concentration for preparing a hydrogel with excellent mechanical properties; therefore, the following characterization and experiments only focus on the SF solutions obtained under conditions ③, ④, and ⑤.

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

[0048] 1. Molecular weight of silk

[0049] The SF solution with a concentration of 2 wt% obtained under conditions ③, ④, and ⑤ in Example 1 was diluted to 0.1 wt% as the starting concentration. The intrinsic viscosity [η] of the SF solution was measured using an Ubbelohde viscometer using the capillary elution method. Since [η] is closely related to the average molecular weight of the polymer, There is a semi-empirical relationship, and the molecular weight of silk can be calculated according to formula 1

[0050]

[0051] In Formula 1, K is the proportionality coefficient and α is the shape parameter. In the present invention, K≈0.01 dL / g and α≈0.5.

[0052] Experimental results: The intrinsic viscosity of the SF solution obtained under condition ③ was 2.0805 dL / g. Substituting this into Equation 1, the average molecular weight of the silk was calculated to be 43,300 Da. The intrinsic viscosity of the SF solution obtained under condition ④ was 1.8755 dL / g. Substituting this into Equation 1, the average molecular weight of the silk was calculated to be 35,200 Da. The intrinsic viscosity of the SF solution obtained under condition ⑤ was 1.408 dL / g. Substituting this into Equation 1, the average molecular weight of the silk was calculated to be 19,800 Da. It can be seen that the higher the degumming temperature and the longer the degumming time, the lower the average molecular weight of the silk obtained. This is because high temperature and long degumming time cause the peptide bonds of the silk fibroin to break through thermal degradation and hydrolysis, shortening the molecular chain and thus reducing the average molecular weight. Furthermore, because high-molecular-weight SF has longer peptide chains, they are more likely to entangle with each other, have abundant interaction sites, and are more likely to form β-folds to form physical crosslinks. During the subsequent concentration process, it is very easy to turn into a white crosslinked substance, making it impossible to obtain an SF solution of the ideal concentration. Furthermore, the high-molecular-weight SF solution has a high viscosity, which is not conducive to experimental operation. This also confirms that SF degummed under conditions ① and ② easily turns into a white physical gel after dialysis for 2-3 days and when concentrated. On the contrary, if the SF molecular weight is too low, crosslinking may be difficult and the material performance may be reduced.

[0053] 2. Transmittance of SF hydrogel

[0054] To prepare a hydrogel with excellent optical properties, the transmittance of the SF hydrogel was measured in the wavelength range of 200-800 nm using an ultraviolet-near-infrared-visible spectrophotometer. The preparation process of the SF hydrogel is as follows: first, the 5 wt% SF solution obtained under conditions ③, ④, and ⑤ in Example 1 was taken out of the refrigerator, 2 mL of the solution was pipetted 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, the mixture was evenly mixed, and then placed in a 37°C oven for cross-linking. After the cross-linking reaction lasted for 1 hour, the SF hydrogel was obtained. Figure 1 The UV spectrum of SF hydrogel prepared by using SF solution obtained under conditions ③, ④, and ⑤ in Example 1. Figure 1 As can be seen, the transmittance of SF hydrogels in the 300-800nm ​​range increases with increasing debonding temperature and time. The SF hydrogel prepared using the SF solution obtained under condition ⑤ has the highest transmittance, while the SF hydrogel prepared using the SF solution obtained under condition ③ has a relatively low transmittance. The SF hydrogel prepared using the SF solution obtained under condition ④ has a transmittance between the two. As a precursor material for hydrogel optical fibers, high transmittance is essential.

[0055] Example 2

[0056] A method for preparing a silk protein-based hydrogel fiber core, comprising the following specific steps:

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

[0058] (2) The core liquid obtained in step (1) was quickly injected into a polytetrafluoroethylene (PTFE) tubular mold with an inner diameter of 1.8 mm using a syringe with a needle, and allowed to stand at 37°C for 40 minutes to complete cross-linking. The mold was then immersed in an 80 v / v% ethanol solution for 48 hours. The PTFE tube was cut open along the end, the PTFE tube was peeled off, and the hydrogel fiber was completely removed. The fiber was immersed in an 80 v / v% ethanol solution again for 2 hours, and then immersed in deionized water for 2 hours to obtain a silk protein-based hydrogel fiber core.

[0059] Since the cross-linking speed of the SF solution with a concentration of 5wt% is too fast to be injected into the PTFE tube, the following only conducts quasi-static tensile fracture load tests on the silk protein-based hydrogel fiber core prepared using SF solutions with concentrations of 2wt%, 3wt% and 4wt%.

[0060] Example 3

[0061] A method for preparing a silk protein-based hydrogel fiber core, comprising the following specific steps:

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

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

[0064] Example 4

[0065] A method for preparing a silk protein-based hydrogel fiber core, comprising the following specific steps:

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

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

[0068] The mechanical properties of the silk protein-based hydrogel core prepared in Example 2-4 were tested by quasi-static tensile breaking load. Figure 2-4 The test process is as follows: the upper end of the sample is fixed to the magnetic clamp system, suspended vertically with a clamping length of 5mm, and the lower end is connected to a detachable weight loading device. Using the progressive loading method, the axial tensile load is gradually applied in increments of Δm=0.5g (at intervals of 10s) until the sample breaks, and the critical fracture mass m is recorded. critical The tensile fracture stress is calculated according to formula 2:

[0069]

[0070] In formula 2, r is the core radius of the fiber. After demolding, r = 0.45 mm = 4.5 × 10 -4 m; g = 9.8 m / s 2 .

[0071] Figure 2 The tensile fracture stress of the silk protein-based hydrogel fiber core prepared using SF solutions of different concentrations in Example 2. Figure 2 It can be seen that with the increase of SF solution concentration, the breaking stress of the silk protein-based hydrogel fiber core gradually increases. When the SF solution concentration is 4wt%, the breaking strength of the silk protein-based hydrogel fiber core reaches the maximum, which is 354.4kPa. Therefore, a SF solution with a concentration of 4wt% is selected to prepare the silk protein-based hydrogel fiber core.

[0072] Figure 3 The tensile fracture stress of the silk protein-based hydrogel fiber core under different HRP solution dosages in Example 3. Figure 3 It can be seen that when the dosage of HRP solution is 20-60 μL, the breaking stress of the silk protein-based hydrogel fiber core gradually increases and reaches a maximum of 477.7 kPa at 60 μL; the breaking strength of the silk protein-based hydrogel fiber core with the addition of 80 μL HRP solution drops sharply to 130.9 kPa. The following reasons may be the following: the cross-linking speed is too fast, resulting in excessive local cross-linking density, forming stress concentration points, and reducing the overall mechanical properties. Therefore, it is most appropriate to choose 1 mL SF solution to add 60 μL HRP solution (250 U / mL).

[0073] Figure 4 The tensile breaking stress of the silk protein-based hydrogel fiber core under different H2O2 solution dosages in Example 4 is shown in FIG. Figure 4It can be seen that when the amount of H2O2 solution is within the range of 20-80μL, the fracture stress of the silk fibroin-based hydrogel core gradually increases with the increase in H2O2 solution dosage, reaching a peak of 616.4kPa at 80μL. However, when the amount of H2O2 solution is further increased to 100μL, the fracture strength drops sharply to 439.1kPa. This may be due to two reasons: first, excessive H2O2 causes irreversible inactivation of HRP, directly interrupting the cross-linking reaction; second, excessive H2O2 may oxidize the silk fibroin peptide chains, destroying the molecular chain integrity and thus reducing the stability of the cross-linked network. Based on the experimental results, the optimal cross-linking condition is determined to be 80μL of H2O2 solution (40mM) per 1mL of SF solution.

[0074] Based on the experimental results of Examples 2-4, the optimal cross-linking conditions were determined as follows: the concentration of SF solution was 4 wt %, and 60 μL HRP solution (250 U / mL) and 80 μL H 2 O 2 solution (40 mM) were added to every 1 mL SF solution.

[0075] Example 5

[0076] A method for preparing a silk protein-based hydrogel optical fiber, comprising the following specific steps:

[0077] (1) Preparation of SF solution: 3 g of chopped silk cocoons were placed in 500 mL of 0.02 mol / L sodium carbonate solution and degummed in an oil bath at 120°C for 30 min. The degummed silk was washed with deionized water and soaked in 40°C warm water for 20 min. The degummed silk was then washed with deionized water for 4-5 times. The resulting silk was loosened and dried in an oven at 60°C overnight. 1 g of treated silk was added to 15 mL of 9.3 mol / L LiBr solution, sealed, and placed in an oven at 60°C to dissolve for 6-7 h. The solution was then packed in a dialysis bag and dialyzed in deionized water for two days, with the water changed every 1 h to remove LiBr. After dialysis, the resulting silk protein solution was centrifuged twice at 9000 r / min, 4°C, and 20 min. The centrifuged silk protein solution was packed in a dialysis bag and then placed in a 10 wt% polyethylene glycol solution for concentration. The mass fraction of the SF solution was determined by the evaporation weighing method to obtain silk protein solutions (SF solutions) with concentrations of 2 wt% and 4 wt%, respectively. The solutions were sealed and stored in a refrigerator at 4°C until use.

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

[0079] (3) Preparation of SF hydrogel optical fiber: 1 mL of the 2 wt% SF solution obtained in step (1) was placed in a culture dish, and 60 μL of HRP solution (250 U / mL) and 80 μL of H₂O₂ solution (40 mM) were added to obtain a uniform coating solution. The silk protein-based hydrogel fiber core obtained in step (2) was immersed in the coating solution for 3 min, then removed and suspended vertically at room temperature for 0.5 h to obtain a silk protein-based hydrogel optical fiber (SF hydrogel optical fiber).

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

[0081] 1. Determination of swelling characteristics

[0082] Three SF hydrogel optical fibers prepared in Example 5 with similar specifications were taken, the initial mass w0 was accurately weighed, and then placed in a PBS buffer solution at 37°C. At the set time points (10, 20, 30, 40, 50, 60, and 120 minutes), samples were taken out in sequence, and the mass w1 was measured after the surface liquid was removed by filter paper. Based on the initial mass w0, the swelling rate (SR) was calculated using Equation 3, and the results are shown in Figure 3. Figure 5 .

[0083]

[0084] The mechanical properties and optical transmission performance of hydrogel optical fiber are restricted by the swelling characteristics. When the swelling degree is high, the material is prone to deformation due to water absorption, resulting in a decrease in mechanical properties. At the same time, the loss during optical signal transmission may increase. Figure 5 This is a curve showing the change in swelling degree of the silk protein-based hydrogel optical fiber prepared in Example 5 in PBS buffer solution over time. Figure 5It shows that the peak swelling rate of the SF hydrogel optical fiber prepared in Example 5 is only 63%. The low swelling rate enables the SF hydrogel optical fiber to maintain good mechanical strength and stable light transmission efficiency, effectively avoiding problems such as nerve tissue damage, signal transmission distortion, and equipment operation failure caused by water absorption and expansion of traditional materials, providing important technical guarantees for the practical application of long-term implantable neural interfaces in clinical scenarios.

[0085] 2. Mechanical Properties Evaluation

[0086] The testing method is the same as the method for testing the mechanical properties of the silk protein-based hydrogel fiber core prepared in Examples 2-4.

[0087] The SF hydrogel fiber prepared in Example 5 achieved a measured breaking stress of 616.4 kPa, significantly higher than that of a single physically cross-linked SF hydrogel fiber (10-100 kPa). This strength surpasses the upper limit of a single chemically cross-linked SF hydrogel fiber (100-500 kPa), reaching the mid-to-high-end strength of dual-crosslinked hydrogels. This demonstrates that the synergistic effect of physical and enzymatic chemical cross-linking significantly enhances the hydrogel's network density and load transfer efficiency. For an implantable medical optical fiber, a strength of 616.4 kPa is sufficient to withstand the daily creeping deformation of muscle tissue.

[0088] 3. Morphology and structure characterization

[0089] The SF hydrogel optical fiber prepared in Example 5 was frozen with liquid nitrogen, and after being brittle, it was freeze-dried by vacuum freeze drying equipment. The microscopic morphology and structure of the cross section were then observed using a transmission scanning electron microscope. The results are shown in FIG. Figure 6 .

[0090] Figure 6 This is a scanning electron microscope image of the silk protein-based hydrogel optical fiber prepared in Example 5, wherein the upper image is a cross-sectional view and the lower image is a partial magnified view of the cross section. Figure 6 It can be seen that the silk protein-based hydrogel optical fiber prepared in Example 5 has a uniform profile, with slight differences in the inner and outer microscopic networks, but no obvious core-sheath stratification. This may be because the synergistic effect of physical-chemical double cross-linking blurs the boundary, and the outer layer of low-concentration hydrogel shrinks and weakens the interface during dehydration. 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 inner layer has a high-concentration network with dense cross-linking, corresponding to its fracture stress of 616.4 kPa, which ensures structural strength; the outer layer has a low concentration that matches the 63% swelling rate, balancing hydrophilicity and stability. The core layer has a high molecular chain density and a higher refractive index than the sheath layer, which meets the requirements of total reflection. The core-sheath function of the hydrogel optical fiber of the present invention lies in the synergistic composition-performance gradient, rather than relying on morphological stratification. The fuzzy interface and concentration gradient jointly provide support for the light-guiding performance and mechanical stability of the hydrogel optical fiber.

[0091] 4. Optical loss

[0092] Optical transmission loss is measured using an intrusive test setup, with the primary equipment consisting of an optical power meter and a light-emitting device. The experimental process consists of two phases: the initial phase records the baseline value (P1), which is the light intensity reading when the light source is directly connected to the power meter; the subsequent phase obtains the measured value (P2), measuring the change in signal intensity after transmission through the fiber under test. The optical loss per unit length (α) is calculated using Equation 4.

[0093]

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

[0095] The optical loss per unit length of the SF hydrogel fiber prepared in Example 5 was measured to be 2.2 dB / cm, which is comparable to other polymer optical fibers (0.02-5 dB / cm) and much lower than that of natural silk materials (28 dB / cm), demonstrating a significant transmission performance advantage. The hydrogel optical fiber prepared in this invention, with its low optical loss characteristics, can effectively reduce signal attenuation during transmission, and therefore can generally better meet the practical needs of brain-computer interfaces in terms of optical fiber signal transmission.

[0096] 5. Length and optical power correlation test

[0097] Three groups of 10 cm long SF hydrogel optical fiber samples prepared in Example 5 were used as test objects. During the experiment, a step-by-step shortening method was adopted, reducing the sample length by 1 cm each time. The insertion-type light intensity detection technology was used to accurately measure the light transmission efficiency at each shortening stage. Through a systematic analysis of the collected data, the relationship between the change in optical fiber length and its optical performance was explored. The results are shown in Figure 2. Figure 7 Fiber length is one of the key parameters that restrict optical power. Exploring the relationship between hydrogel fiber length and optical power will help clarify its effectiveness and stability in optical signal transmission.

[0098] Figure 7 The results of the correlation between the length and optical power of the silk protein-based hydrogel optical fiber prepared in Example 5 are shown in Figure 5. The experimental data were fitted with a function to obtain R 2=0.997, indicating a good fitting effect. The data shows that as the fiber length shortens, the output optical power gradually increases. When the fiber length is about 3 cm, the output optical power can reach about 5500 nW. From a practical application perspective, researchers can reasonably select the appropriate fiber length based on the fitting curve and the target optical power value, thereby achieving precise control of the optical signal quality. In addition, by conducting length test experiments, the optimal fiber length for implantation can be further screened, ensuring a higher signal power while providing key parameter support for the development of adapted implantable brain-computer interface devices.

[0099] 6. Cytocompatibility assessment

[0100] 6.1 Cytotoxicity of SF Solution

[0101] The CCK-8 method was used to evaluate the in vitro cytotoxicity of the 4% SF solution prepared in step (1) of Example 5. The test subjects were the relative survival rates of 4T1 cells and LO2 cells. The test process was as follows: 4T1 cells and LO2 cells were seeded at a density of 8000 cells per well in a 96-well cell culture plate, 100 μL of culture medium was added to each well, and the cells were cultured overnight in an incubator to allow the cells to adhere to the bottom of the plate. The SF solution was dissolved in sterile PBS to prepare a 20% aqueous solution by mass. After sterilization by filtration through a 0.22 μm sterile filter membrane, it was diluted with fresh culture medium to form a gradient concentration of SF solution (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 hours. After the culture was completed, the supernatant was removed and 100 μL of fresh DMEM medium containing 10% CCK-8 was added to each well. After incubation for 2 hours, the absorbance (OD value) at 450 nm was measured using a microplate reader. The experiment set up cells without SF solution as the control group (control) and SF solution without cells as the blank group (blank). Both groups were treated according to the same process. The relative cell viability was calculated according to formula 5, and the results are shown in Figure 8 Part a in .

[0102]

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

[0104] 6.2 Cytotoxicity of SF Hydrogel Core (CCK-8 Assay)

[0105] 4T1 cells were used as a model to evaluate the in vitro cytotoxicity of the SF hydrogel core prepared in step (2) of Example 5. 4T1 cells were cultured at a rate of 1×10 4 Cells were seeded in a 24-well plate at a density of cells / well, 500 μL of culture medium was added to each well, and cultured overnight until the cells adhered. The SF hydrogel core prepared in step (2) of Example 5 was cut into hydrogel blocks with a diameter of 2 mm and a thickness of 2 mm. The original culture medium of the adhered cells was discarded, 500 μL of fresh DMEM was added to each well, and the above-mentioned hydrogel blocks were placed in the wells and cultured for 6 h, 24 h and 48 h, respectively. After the culture was completed, the hydrogel blocks were removed, the culture medium was discarded, 400 μL of culture medium containing 10% CCK-8 was added to each well, and the incubation continued for 2 h. 100 μL was transferred to a 96-well plate, and the absorbance (OD value) at 450 nm was detected by an enzyme marker. In the experiment, cells without hydrogel blocks were set as the control group and hydrogel blocks without cells were set as the blank group. They were treated according to the same process. The relative cell survival rate of the cell group without hydrogel blocks after 6 h of culture was taken as the 100% benchmark value. The relative cell survival rate was calculated according to formula 5, and the results are shown in Table 5. Figure 8 Part b of .

[0106] 6.3 Cytotoxicity of SF Hydrogel (Live-Dead Staining Method)

[0107] During the experiment, 4T1 cells were suspended at a concentration of 1×10 4 The cells were inoculated into a 35mm confocal culture dish (1mL per well) at a concentration of cells / mL and placed in a constant temperature incubator for adherent culture for 24 hours. After the cells have completed adherence, the original culture medium was discarded and 1mL of fresh 1640 culture medium solution was added. After the cut hydrogel blocks (i.e., the hydrogel blocks with a diameter of 2mm and a thickness of 2mm cut from the SF hydrogel core prepared in step (2) of Example 5 in 6.2) were immersed in the culture system, another culture dish without hydrogel blocks was used as a control, and the standard culture conditions were maintained for co-culture for 24 hours. After the culture was terminated, the hydrogel blocks were taken out, the residual culture medium was removed, and they were gently washed three times with sterile PBS buffer to remove impurity interference. Subsequently, 1640 culture medium containing Live-Dead dye was added, and finally an inverted fluorescence microscope was used for cell viability detection and image acquisition. The results are shown in FIG. Figure 8 Part c in .

[0108] Figure 8 The toxic effect of the SF solution prepared in step (1) of Example 5 on 4T1 cells and LO2 liver cells (a), the cell proliferation rate data of the SF hydrogel core prepared in step (2) of Example 5 co-cultured with 4T1 cells for different lengths of time (b), and the combined image of live cell (Live, green), dead cell (Dead, red) fluorescence staining and bright field (BF) images of the control group and the hydrogel treatment group (c). Figure 8As can be seen from part a of the figure, when the concentration of SF solution was 12.8 mg / mL, both cells showed no significant toxicity, which confirmed that SF solution has good biocompatibility. Figure 8 As can be seen from the data in part b, in the culture system with SF hydrogel, the proliferation rate of 4T1 cells was basically the same as that of the control group, and both groups of cells showed a continuous growth trend. It is worth noting that after 24h and 48h of culture, the cell proliferation rate of the hydrogel group was slightly higher than that of the control group, which indicates that SF hydrogel not only did not inhibit cell growth, but on the contrary showed a certain degree of promotion effect on cell proliferation. Figure 8 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 in the control group, the proportion of dead cells was lower, and the cell morphology remained normal. These experimental results demonstrate that the SF hydrogel prepared in this invention exhibits minimal cytotoxicity to 4T1 cells, demonstrating excellent cytocompatibility and potential application 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 changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for preparing a silk protein-based hydrogel optical fiber, characterized in that: The following steps are involved: (1) placing silk cocoons in a sodium carbonate solution for degumming, soaking, washing, and drying to obtain silk; mixing the obtained silk with a LiBr solution, sealing and dissolving the mixture, and dialyzing, centrifuging, and concentrating to obtain a silk protein solution; (2) mixing the silk protein solution obtained in step (1) with a horseradish peroxidase solution and a H2O2 solution to obtain a core liquid; injecting the obtained core liquid into a gel, and then soaking it in an ethanol solution and water in sequence to obtain a silk protein-based hydrogel fiber core; (3) The silk protein solution obtained in step (1) is mixed with the horseradish peroxidase solution and the H2O2 solution to obtain a coating liquid; the silk protein-based hydrogel fiber core obtained in step (2) is immersed in the coating liquid for coating treatment to obtain the silk protein-based hydrogel optical fiber.

2. The preparation method according to claim 1, characterized in that In step (1), the usage ratio of the silk cocoons and the sodium carbonate solution is (2-5):500 (g / mL), and the concentration of the sodium carbonate solution is 0.02 mol / L.

3. The preparation method according to claim 1, characterized in that In step (1), the degumming treatment is carried out at a temperature of 100-120° C. and for a time of 0.5-1 h.

4. The preparation method according to claim 1, characterized in that In step (2), the concentration of the silk protein solution is 3-5wt%; the volume ratio of the silk protein solution to 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.

5. The preparation method according to claim 1, characterized in that In step (2), the obtained core liquid is injected into a gel as follows: the obtained core liquid is injected into a mold tube, allowed to stand at 37°C for 40 minutes to complete cross-linking, and then immersed in an ethanol solution for solidification, and then the mold tube is peeled off to obtain a silk protein-based hydrogel fiber core.

6. The preparation method according to claim 1, characterized in that In step (2), the concentration of the ethanol solution is 80 v / v%; and the soaking time in the ethanol solution is 2 hours.

7. The preparation method according to claim 1, characterized in that In step (3), the concentration of the silk protein solution is 2 wt%; the volume ratio of the silk protein solution to the horseradish peroxidase solution and the H2O2 solution is 1 mL: (20-60) μL: (20-80) μL; the concentration of the horseradish peroxidase solution is 250 U / mL, and the concentration of the H2O2 solution is 40 mM.

8. The preparation method according to claim 1, characterized in that In step (3), the coating treatment is carried out at room temperature for 0.5 h.

9. A silk protein-based hydrogel optical fiber prepared by the preparation method according to any one of claims 1 to 8.

10. An application of the silk protein-based hydrogel optical fiber according to claim 9 in optogenetics and brain-computer interface for non-therapeutic purposes.

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