Microenvironment responsive spinal cord repair hydrogel as well as preparation method and application thereof

Abstract

The invention relates to microenvironment responsive spinal cord repair hydrogel as well as a preparation method and application thereof, and belongs to the technical field of hydrogel. The hydrogel is prepared from the following raw materials in parts by mass: 0.8 to 1.5 parts of oxidized xyloglucan, 0.8 to 1.5 parts of methacrylic acid chitosan, 0.1 to 0.5 part of nano silicon particles and 0.7 to 2.0 parts of borax. A borate bond and a Schiff base bond are adopted as ROS response main bodies to form a continuous response dynamic network, intelligent response is realized through the dynamic reversible characteristic of the ROS, and the interference of various factors in a complex physiological environment is resisted through the dual dynamic network, so that the stable response capability of the material in a spinal cord injury microenvironment is kept for more than 15 days.

Description

Technical Field

[0001] The present invention belongs to the technical field of hydrogels, and particularly relates to a microenvironment-responsive spinal cord repair hydrogel and its preparation method and application. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and it is not necessarily regarded as an admission or an indication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Currently, the research on spinal cord injury (SCI) repair materials focuses on constructing three-dimensional structures with bioactivity, mechanical adaptability, and microenvironment responsiveness. The current mainstream technologies include synthetic polymer hydrogels, natural biomaterials, and composite hydrogel systems. Hydrogels have become a research hotspot due to their characteristics of biomimetic extracellular matrix, which can not only act as a stem cell / drug carrier for precise delivery but also form a physical barrier in situ through injectability to inhibit scar formation. However, existing repair materials have significant defects in terms of microenvironment responsiveness, anti-swelling property, self-healing property, bioactivity, antibacterial property, mechanical strength, drug loading capacity, etc., especially the following problems exist.

[0004] Insufficient anti-swelling property, including: (1) Swelling leads to a decrease in mechanical properties. After traditional hydrogels absorb water and swell, the three-dimensional network structure is prone to expand and deform, resulting in a significant reduction in mechanical strength. (2) The risk of compression on surrounding tissues. Traditional hydrogels (such as PVA or PEG-based) have too many hydrophilic groups and the water absorption swelling rate in the physiological environment exceeds 150%. The excessive swelling of the hydrogel can compress normal tissues, leading to the risk of increased intracranial pressure after implantation and causing secondary injury. (3) Insufficient stability and durability. Traditional chemically cross-linked hydrogels (such as single covalent bond cross-linking) are difficult to restore their original shape after swelling and have poor dynamic responsiveness.

[0005] Insufficient antibacterial property. The drug release of traditional hydrogels is mostly passive diffusion and cannot be dynamically regulated according to the infected microenvironment (such as pH, ROS level), resulting in premature drug release or insufficient local concentration. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a microenvironment-responsive spinal cord repair hydrogel and its preparation method and application. By introducing Schiff base bonds and borate ester bonds into the hydrogel system, the dynamic balance of the two bonds under local pH and ROS fluctuations is achieved, and further the self-healing rate is adapted to the microenvironment; the nano-silicon particles have a rigid support effect and can also achieve an anti-inflammatory effect.

[0007] To achieve the above purpose, the technical solution of the present invention is as follows:

[0008] In a first aspect, a microenvironment-responsive spinal cord repair hydrogel is prepared from the following raw materials in parts by mass: 0.8 to 1.5 parts of oxidized xyloglucan, 0.8 to 1.5 parts of methacrylated chitosan, 0.1 to 0.5 parts of nano-silicon particles, and 0.7 to 2.0 parts of borax.

[0009] Optionally, the preparation raw materials further include 95.5 to 97.2 parts of PBS buffer solution.

[0010] Optionally, the preparation raw materials include anti-inflammatory drugs and / or neurotrophic factors.

[0011] Optionally, the anti-inflammatory drugs include one or more of methylprednisolone, dexamethasone, and prednisolone.

[0012] Optionally, the concentration of methylprednisolone in the microenvironment-responsive spinal cord repair hydrogel is 0.5 to 5.0 mg / mL; or, the concentration of dexamethasone in the microenvironment-responsive spinal cord repair hydrogel is 0.1 to 2.0 mg / mL; or, the concentration of prednisolone in the microenvironment-responsive spinal cord repair hydrogel is 0.5 to 5 mg / mL.

[0013] Optionally, the neurotrophic factors include one or more of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophic factor-3 (NT-3).

[0014] Optionally, the concentration of nerve growth factor (NGF) in the microenvironment-responsive spinal cord repair hydrogel is 0.01 to 0.5 μg / mL; or, the concentration of brain-derived neurotrophic factor (BDNF) in the microenvironment-responsive spinal cord repair hydrogel is 0.05 to 1.0 μg / mL; or, the concentration of neurotrophic factor-3 (NT-3) in the microenvironment-responsive spinal cord repair hydrogel is 0.02 to 0.8 μg / mL.

[0015] In a second aspect, a method for preparing the above-mentioned microenvironment-responsive spinal cord repair hydrogel includes the following steps:

[0016] Add oxidized xyloglucan and methacrylated chitosan into the buffer solution respectively, and stir and react at room temperature for 4 to 6 h; then add nano-silicon particles and borax in sequence, and stir and react at 40 to 60 °C for 2 to 3 h.

[0017] Optionally, the loading methods of anti-inflammatory drugs and / or neurotrophic factors include one or more of physical adsorption method, chemical bond wrapping method, and in-situ mixing loading method.

[0018] Optionally, the physical adsorption method includes: using the hydroxyl or amino groups on the surface of nano-silicon particles to combine with drug molecules through hydrogen bonds or electrostatic interactions to achieve drug adsorption and loading, inhibit burst release, and extend the sustained release period.

[0019] Optionally, the chemical bond conjugation encapsulation method includes: constructing a double network structure through dynamic borate ester bonds and Schiff base bonds, encapsulating the drug in three-dimensional pores, and regulating the reversible dissociation of chemical bonds by means of a pH / ROS response mechanism to achieve intelligent drug release.

[0020] Optionally, the in-situ mixing and loading method includes: directly blending with a drug solution during the preparation of the hydrogel, embedding drug molecules into the network interior through a crosslinking reaction, and fixing the drug distribution by combining with the rigid support of nano-silicon particles.

[0021] Optionally, the buffer solution is PBS buffer solution.

[0022] Optionally, the preparation method of oxidized xyloglucan includes: mixing sodium periodate and xyloglucan in a mass ratio of 1:(2 - 5) to prepare a solution, stirring and reacting at room temperature for 4 - 6 h, adding ethylene glycol to terminate the reaction, and dialyzing to obtain oxidized xyloglucan.

[0023] Optionally, dialyze substances with a molecular weight cut-off of ≥3500 Da and freeze-dry to obtain oxidized xyloglucan.

[0024] Optionally, the preparation method of methacrylated chitosan includes: dissolving chitosan in an acetic acid solution, then adding methacrylic anhydride in an amount 0.36 - 1.92 times the mass of chitosan, stirring and reacting at 60 - 80 °C for 6 - 10 h, adding an alkaline substance to neutralize to pH = 6.5 - 7, diluting, and dialyzing to obtain methacrylated chitosan.

[0025] Optionally, dialyze substances with a molecular weight cut-off of 8000 - 14000 Da and freeze-dry to obtain methacrylated chitosan.

[0026] Optionally, the alkaline substance includes an aqueous solution of sodium bicarbonate.

[0027] Optionally, the preparation method of nano-silicon particles includes: dispersing 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane in 5 - 6 times the volume of ultrapure water, adding 2-aminophenol in an amount 0.24 - 0.30 times the mass of 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane, stirring and reacting at 70 - 80 °C for 100 - 150 min, and dialyzing to obtain nano-silicon particles.

[0028] Optionally, dialyze substances with a molecular weight cut-off of 400 - 600 Da and freeze-dry to obtain nano-silicon particles.

[0029] In a third aspect, the above microenvironment-responsive spinal cord repair hydrogel is used in at least one of the following a1)-a5), including:

[0030] a1) Preparing a product for promoting spinal cord repair;

[0031] a2) Preparing a product for continuously scavenging ROS in the microenvironment during spinal cord repair;

[0032] a3) Preparing a product for continuously regulating the pH of the microenvironment during spinal cord repair;

[0033] a4) Preparing a product for antibacterial during spinal cord repair;

[0034] a5) Preparing a product for sustained release of drugs during spinal cord repair.

[0035] The beneficial effects of the present invention are as follows:

[0036] 1. The present invention uses borate ester bonds and Schiff base bonds as ROS-responsive entities to form a continuously responsive dynamic network, which achieves intelligent response through its dynamic reversible characteristics and can effectively trigger bond cleavage even in a low-concentration ROS environment. This dynamic bond has pH responsiveness, is stable at physiological pH (7.4), and dissociates reversibly in the local acidic / alkaline microenvironment of the injury area, dynamically regulating the network density and balancing water absorption swelling and mechanical strength: in an acidic environment (pH < 6.5), the imine bond (-C=N-) undergoes reversible cleavage to neutralize hydrogen ions in the environment and increase the pH; when the microenvironment returns to neutral, the network self-repairs through the re-bonding of amino groups and aldehyde groups, enabling the material to quickly self-repair after ROS response and avoiding the problem of structural collapse caused by bond cleavage in traditional materials (the mechanical strength loss of traditional materials after response reaches more than 50%), maintaining the integrity of the scaffold to continuously regulate the microenvironment; in an alkaline condition (pH > 7.4), the borate ester bond tends to be stable and complements the Schiff base bond to prevent excessive degradation of the material in the late stage of inflammation. The material adjusts the crosslinking density through the concentration of tetraborate ions (0.02 - 0.05 mol / L) during pH fluctuations, and then dynamically adjusts the network porosity to achieve an intelligent match of the drug release curve; by means of a dual dynamic network to resist interference from various factors in the complex physiological environment, the material maintains a stable response ability in the spinal cord injury microenvironment for more than 15 days.

[0037] 2. The added nano-silicon particles in the present invention strengthen the three-dimensional network through physical filling and interfacial interaction, significantly improving the elastic modulus of the material and enabling it to match the mechanical property range of spinal cord tissue, which is 0.2 - 1.0 kPa. The rigid support can prevent the structure from collapsing, providing physical anchor points for dynamic bond recombination and avoiding structural misalignment during the self-healing process of traditional soft gels. The hydrophobic properties of the nano-silicon particles and borate ester bonds can reduce water molecule penetration and inhibit excessive swelling. At the same time, their dynamic properties allow the network to restore structural integrity through bond recombination after swelling, avoiding irreversible swelling deformation of traditional hydrogels. The surface of the nano-silicon particles (SiNPs) can bind amino groups, which can quickly adhere to the negatively charged bacterial cell membrane surface through electrostatic interaction, destroying the membrane integrity, enabling the SiNPs to achieve broad-spectrum antibacterial activity without relying on antibiotics, and significantly enhancing the inhibitory effects on Gram-positive bacteria (such as Staphylococcus aureus) and Gram-negative bacteria (such as Escherichia coli). The anti-inflammatory property of the silicon particles combined with the ROS responsiveness of the dynamic borate ester bonds and Schiff base bonds endows the composite hydrogel with strong anti-inflammatory properties.

[0038] 3. Aiming at the problems of mismatched mechanical strength and weak sustained-release function in traditional drug delivery systems, the present invention forms a stable three-dimensional pore structure through the OXG-CSMA-borax dynamic borate ester bond / Schiff base bond double-network structure. This network can not only adapt to the drug diffusion rate through the breakage-recombination of dynamic bonds, but also load drug molecules on the surface of nano-silicon particles to provide multiple drug-loading sites. Compared with the collagen system that relies on single physical adsorption in traditional repair material technologies, this design significantly improves the drug-loading capacity and achieves sustained release, avoiding secondary damage to nerve tissue caused by sudden drug release. In the acidic microenvironment of the injury area, the Schiff base bond selectively breaks to release neurotrophic factors; when the ROS concentration increases, the Schiff base bond further dissociates to accelerate the release of antioxidant drugs. This sequential control characteristic overcomes the defect of "insufficient release timing regulation" in the background technology, realizing multi-stage precise drug delivery of early anti-inflammatory - mid-term promoting regeneration - late-stage barrier repair, which highly matches the spinal cord injury repair process. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0040] Figure 1 It is the scanning electron microscope detection result of the microenvironment-responsive spinal cord repair hydrogel in Example 1; among them, (a) schematic diagram of the pore structure Figure 1 ; (b) distribution diagram of four elements, C (red), N (purple), Si (blue), O (green).

[0041] Figure 2It is the staining result diagram of cell live / dead staining after co-culturing the present invention with mesenchymal stem cells in Example 3.

[0042] Figure 3 It is the staining result diagram of nerve tissue regeneration and repair in spinal cord injured mice in Example 4; among them, (a) Immunofluorescence staining of typical markers of neurons and astrocyte markers 4 weeks after establishing a semi-transverse spinal cord injury model in mice; (b) Statistical analysis of the fluorescence intensity of astrocyte markers; (c) Statistical analysis of the fluorescence intensity of neuron cell markers.

[0043] Figure 4 It is the evaluation result diagram of neuroinflammation in Example 4; Immunofluorescence staining of neuroinflammatory indexes 4 weeks after establishing a semi-transverse spinal cord injury model in mice.

[0044] Figure 5 It is the evaluation result diagram of the functional analysis of spinal cord injured mice in Example 4, (a) BMS score result diagram.

[0045] (b) Catwalk gait analysis result diagram. Detailed implementation manners

[0046] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0047] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0048] For the experimental methods without specific conditions noted in the following examples, they are generally in accordance with conventional conditions. For the raw materials and reagents used in the following examples, unless otherwise specified, they can be obtained commercially.

[0049] Term explanation:

[0050] Burst release effect: It refers to the phenomenon that drugs are suddenly released under specific conditions in pharmaceutical preparations.

[0051] Reactive oxygen species (ROS): It refers to the general term of oxygen-containing free radicals and peroxides that are easy to form free radicals and are related to oxygen metabolism in living organisms.

[0052] Creep: The phenomenon that the strain of a solid material increases with the extension of time under the condition of constant stress.

[0053] Swelling ratio: It refers to the degree of water absorption and swelling of a material in a solution, which can be calculated by the following formula: Swelling ratio (%) = (W2 - W1) / W1 x 100%. Where W1 is the initial weight of the material, and W2 is the weight after soaking in the solution for a certain period of time.

[0054] Compression modulus: The ratio of compressive stress to compressive strain when an object is under triaxial compression.

[0055] Tensile strength: It refers to the stress at which a material produces the maximum uniform plastic deformation.

[0056] Enzyme-linked immunosorbent assay (ELISA): An immunoassay method that binds an antigen or antibody to the surface of a solid-phase carrier, and uses the specific binding of antigen and antibody and the enzyme-labeled on the antibody or antigen to catalyze a specific substrate to produce a color reaction to achieve the detection of the target substance.

[0057] BBB locomotor score: A standardized scoring system used to evaluate the recovery of hindlimb motor function in experimental animals after spinal cord injury, and is now widely used in neuroscience and spinal cord injury research.

[0058] Flexural strength: It refers to the maximum stress that a material can withstand when it ruptures or reaches a specified bending moment under the action of a bending load, and this stress is the maximum normal stress during bending.

[0059] BMS score: A tool used to evaluate the limb motor function of patients with spinal cord injury. It consists of two scores, namely "motor score" and "sensory score". The motor score determines the degree of limb movement of the patient, and the sensory score is used to evaluate the patient's ability to perceive tactile stimuli.

[0060] Catwalk gait analysis: A complete system and useful tool for quantitatively evaluating gait changes in rodents caused by pain or other movement defects. The system can be used to evaluate animal models of nerve trauma, nerve atrophy, nerve diseases, and pain symptom groups.

[0061] Example 1

[0062] (1) A microenvironment-responsive spinal cord repair hydrogel, and the preparation method includes:

[0063] S1. Mix an aqueous solution of sodium periodate (NaIO4) with a mass concentration of 10% and an aqueous solution of xyloglucan (commercially available, purity ≥ 98%, sugar components: xylose 34%, glucose 45%, galactose 17%, arabinose 2%, other sugars 2%) with a mass concentration of 10%, and control the mass ratio of sodium periodate / xyloglucan solutes to be 1:2; stir magnetically in the dark at room temperature (500 rpm, 25 °C, 4 h), then add ethylene glycol (purity ≥ 99%) with a mass ratio of 5.5 to potassium periodate and stir for 1 - 2 minutes under the same conditions to terminate the reaction to obtain a first mixed solution; subsequently, dialyze the first mixed solution in deionized water for 72 h to retain macromolecular substances with a molecular weight cut-off ≥ 3500 Da, and freeze-dry the retained substances to obtain oxidized xyloglucan (OXG).

[0064] S2. Prepare an acetic acid solution with a mass concentration of 1% using glacial acetic acid, dissolve chitosan (commercially available, purity ≥ 98%, deacetylation degree ≥ 75%, molecular weight 100 - 350 KDa) in the 1% acetic acid solution so that the mass ratio of chitosan to glacial acetic acid is 1:1 to form a second mixed solution; drop methacrylic anhydride (MA, purity ≥ 95%, containing about 0.2% of a hindered phenol antioxidant as a stabilizer) into the second mixed solution, control the mass ratio of chitosan to methacrylic anhydride to be 1:0.36 (the specific ratio depends on the molecular weight, deacetylation degree of chitosan, and the purity of methacrylic anhydride), and stir magnetically in the dark at 60 °C (500 rpm, 6 h); then neutralize with an aqueous solution of sodium bicarbonate with a mass fraction of 10% to pH = 6.5 and dilute 5 times in volume to end the reaction to obtain a crude CSMA solution; dialyze the crude CSMA solution in deionized water for 3 - 4 days to retain macromolecular substances with a molecular weight cut-off of 8000 - 14000 Da to remove unreacted reagents; freeze-dry the retained substances to obtain a white sponge-like product, which is methacrylated chitosan (CSMA).

[0065] S3. Use 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane as the silicon source, stir ultrapure water in a 70 °C constant temperature water bath (300 - 500 rpm), and add a silicon source with a volume of 1 / 5 of the ultrapure water volume (the purity of 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane ≥ 99%); then add 2-aminophenol (purity

[0066] Using hydrazine hydrate (≥98.5%) as a reducing agent, 4.0 mg of 2-aminophenol was added per milliliter of the silicon source, and the reaction was continuously stirred at a constant temperature of 70 °C for 150 min, gradually generating a third dispersion liquid in which silicon nanoparticles were dispersed. The third dispersion liquid was dialyzed with deionized water to retain substances with a molecular weight cut-off of 400 - 600 Da. The dialysis continued for 8 hours to remove unreacted small molecule impurities. The retained substances were freeze-dried to obtain the required nano-silicon particles (SiNPs), and their particle size was detected to be 50 - 100 nm.

[0067] S4. Mix 1 part of the freeze-dried powder of oxidized xyloglucan (OXG) and 1 part of methacrylated chitosan (CSMA) with 98 parts of PBS buffer solution, and stir magnetically in the dark at room temperature (600 rpm, 25 °C, 4 h) to obtain a xyloglucan-chitosan hydrogel composite solution based on dynamic Schiff base bonds. Add nano-silicon particles (SiNPs) to the xyloglucan-chitosan hydrogel composite solution to make its mass fraction in the system 0.3%, and stir at a speed of 600 rpm at a temperature of 40 °C for 2 h to obtain a composite solution. Add commercially available high-purity borax (purity ≥99%) to the composite solution to dissolve it, so that the concentration in terms of tetraborate ions is 0.04 mol / L. Stir magnetically in the dark at room temperature (600 rpm, 40 °C, 2 h) to form a xyloglucan-chitosan-borax-silicon particle cross-linked network based on dynamic borate ester bonds, which is a microenvironment-responsive spinal cord repair hydrogel. In this step, the mass ratio of oxidized xyloglucan (OXG), methacrylated chitosan (CSMA), nano-silicon particles (SiNPs), borax and PBS buffer solution is 1:1:0.3:1.5:96.2.

[0068] (2) Microscopic observation results

[0069] The prepared microenvironment-responsive spinal cord repair hydrogel was detected by scanning electron microscopy, and the results are as Figure 1 shown. Among them, Figure 1 (a) in is a schematic diagram of the pore structure under scanning electron microscopy Figure 1 , Figure 1 (b) in is a schematic diagram of the pore structure under scanning electron microscopy Figure 2 . Specifically, by adjusting the concentration of tetraborate ions (controlling the cross-linking density of borate ester bonds) and the formation ratio of Schiff base bonds (affecting the dynamic network reorganization ability), the gradient control of the three-dimensional network cross-linking degree is realized. Figure 1 (c) in is the distribution of four elements C, N, Si, and O. It can be seen that the distribution of these elements in the material is relatively uniform, indicating that each element has been successfully introduced and has good dispersion in the material matrix. This shows that the components are mixed evenly during the material preparation process, which may contribute to their subsequent synergistic effects in biological applications or functional performances.

[0070] (3) Mechanical performance detection

[0071] Its initial compressive strength was detected to be 1.0 kPa; the composite hydrogel was immersed in physiological saline for 72 hours to become a swollen hydrogel and then detected. Its volume swelling ratio was 8% (swelling ratio < 10%), the compressive modulus was 87% of the initial value, and 93% of the compressive strength was maintained. The low swelling ratio also reduced the risk of compression on surrounding tissues.

[0072] A universal material testing machine (INSTRON 5944) was used to conduct cyclic compression tests on the swollen hydrogel: the compression strain was set to 50% and the frequency was 1 Hz, and 30 consecutive cycles of loading-unloading were carried out. The results showed that the initial cyclic compression stress was 0.85 kPa, and the stress decreased to 0.81 kPa after 30 cycles, with an attenuation rate of 4.7%. The stress-strain curves of each cycle were highly overlapped, and the attenuation rate of the hysteresis loop area was only 3.2%, indicating that the dynamic bond network could effectively dissipate energy through the real-time recombination of Schiff base bonds and borate ester bonds; compared with traditional single-network hydrogels, the synergistic effect of dual dynamic bonds significantly improved the anti-fatigue performance. The stress attenuation rate was ≤ 5% after 30 cycles of compression.

[0073] Scanning electron microscopy was synchronously used to observe the pore structure changes, and the chemical bond recombination rates of borate ester bonds and Schiff base bonds were analyzed by FT-IR (experimental data showed that 98% of bond recombination was completed within 1 hour); at the same time, the real-time recovery data of the mechanical properties of the material under different pH / ROS conditions were monitored, indicating that the microenvironment-responsive spinal cord repair hydrogel prepared in this example was based on the pH-responsive characteristics of borate ester bonds (stable at physiological pH 7.4 and reversibly dissociated in an acidic microenvironment) and the ROS sensitivity of Schiff base bonds, and could achieve dynamic network recombination after swelling; experiments showed that there was no spinal cord compression phenomenon after implantation in the animal model, and it could adapt to the dynamic water changes in the injury area, avoiding secondary injuries caused by mechanical mismatch.

[0074] Its initial tensile strength was detected as follows: standard dumbbell-shaped specimens were prepared according to ASTM D412 standard. After the samples were equilibrated in the test environment (25 °C, 90% humidity) for 24 hours to eliminate residual stress, the tensile strength was measured to be 1.27 mpa. Then, the samples were cut perpendicular to the tensile direction, and the end faces were joined and placed. The macroscopic incision was closed within 0.5 hour at 25 °C, and the tensile strength was measured to be 1.10 mpa after 2 hours, recovering to more than 85% of the original value; after 16 h of healing, after 2 more damage-healing tests (3 times in total), the tensile strength was measured to be 1.16 mpa, still maintaining more than 90% of the mechanical properties (the traditional single dynamic bond system only recovered < 50% and failed after repeated repairs); experiments showed that the hydrogel implanted in the animal model maintained structural integrity in the spinal cord dynamic contraction / expansion environment without crack generation.

[0075] The flexural strength of standard dumbbell-shaped specimens was detected using the three-point bending test method, and the result was an average flexural strength of 2.7 mpa.

[0076] Soak with H2O2 solutions of different concentration gradients in combination with the placed end face positions, and control the local ROS level by adjusting the time; or treat the cut interface with different pH buffers (such as PBS with pH 5.0 - 8.0) to simulate microenvironment fluctuations; during the process, use fluorescently labeled dynamic Schiff base bond precursors (higher fluorescence intensity at the fracture and weaker at the uniform distribution), and use a confocal microscope to detect the change in fluorescence intensity at the incision in real time, calculate the self-healing rate = fluorescence intensity at the incision / original fluorescence intensity to determine the self-healing rate; it was found that an increase in the level of reactive oxygen species (ROS) in the damaged area triggers the preferential recombination of Schiff base bonds, and local pH fluctuations regulate the dynamic balance of borate ester bonds, achieving the adaptation of the self-healing rate to the microenvironment. In the experiment, when the ROS concentration ≥ 5 μM, the self-healing rate increased by 2.3 times.

[0077] During the damage-healing test, use in-situ spectroscopy to analyze the infrared spectrum of the incision area and observe the change in the characteristic peak intensity of Schiff base bonds (C=N,

[0078] ~1640 cm-1) and borate ester bonds (B-O, ~1380 cm-1), calculate the bond recombination rate. The ratio of the C=N peak intensity after 1 hour to the initial C=N peak intensity is about 99.1% based on multiple experimental data, and the ratio of the B-O peak intensity after 1 hour to the initial B-O peak intensity is about 98.4%; it shows that 98% of the bond recombination is completed within 1 hour after the borate ester bond and Schiff base bond are triggered to break by ROS, and the compressive strength of the material is restored to 95% of the initial value (the strength of traditional materials loses 50% and is irreversible after fracture), maintaining the integrity of the scaffold to continuously regulate the microenvironment.

[0079] (4) Antibacterial performance detection

[0080] Use the agar diffusion method to measure its antibacterial performance: First, culture the bacteria to the logarithmic growth phase (OD600 ≈ 0.5), dilute to 10 6 CFU / mL, take 100 μL of the bacterial suspension and spread it evenly on the LB agar plate; gently press a sterile hydrogel disc (diameter 6 mm, thickness 2 mm) on the agar surface, and culture at 37 °C for 18 - 24 hours, measure the diameter of the inhibition zone (including the hydrogel contact area and the surrounding transparent ring). The inhibition zone areas of the composite hydrogel loaded with SiNPs against Staphylococcus aureus and Escherichia coli reach 196.5 ± 10.3 mm 2 and 61.3 ± 1.1 mm 2 , indicating its significant ability to inhibit bacterial proliferation; compared with the traditional antibiotic loading system, it avoids the risk of bacterial drug resistance.

[0081] In the experiment against methicillin-resistant Staphylococcus aureus (MRSA), the experimental group used the microenvironment-responsive spinal cord repair hydrogel prepared in this example, and the control group used methicillin antibiotics. The inhibition rate of biofilm formation in the experimental group reached over 82% (less than 30% in the control group), and the area of the inhibition zone reached 167.9 ± 13.1 mm 2 , indicating that it can break through the traditional antibiotic resistance barrier and achieve efficient synergistic antibacterial effects.

[0082] After injecting the microenvironment-responsive spinal cord repair hydrogel obtained in this example into the injury defect area of the mouse spinal cord hemisection model, its inflammatory response in vivo was systematically evaluated. Fluorescent staining was performed through relevant inflammatory indicators. The results showed that compared with the simple injury group, the fluorescence intensity of the nerve inflammation indicator in the injury site of the treatment group was significantly reduced, and the range was significantly reduced, indicating that the nerve inflammation in the treatment group was significantly reduced. This material can significantly inhibit the growth of pathogenic microorganisms and reduce the inflammation level, demonstrating excellent antibacterial and anti-inflammatory properties, as Figure 4 shown.

[0083] (5) Detection of the effect of clearing microenvironment ROS

[0084] A ROS-simulated microenvironment was constructed in an incubator at 37°C and 5% CO2; the ROS scavenging performance was detected using the DCFH-DA fluorescent probe method, including: immersing 20 μL of the hydrogel sample (5 mm in diameter and 2 mm in thickness) in 1 mL of serum-free medium containing 10 μM DCFH-DA, incubating at 37°C for 30 minutes, washing and transferring to a new medium; adding H2O2 to 0.5 mM to trigger ROS in the medium, and immediately starting timing and recording the initial fluorescence intensity (excitation wavelength 488 nm, emission wavelength 525 nm); at time points of 0, 6, 12, 24, 48, 72, 120, and 180 hours, the DCF fluorescence intensity in the medium was detected using a fluorescence microplate reader. The DCF fluorescence intensity reflects the residual amount of ROS, and the scavenging rate (ROS scavenging rate) and half-life were calculated accordingly:

[0085] Among them, F0 is the initial fluorescence intensity after adding H2O2, and Ft is the fluorescence intensity at each time point.

[0086] By fitting the curve of the ROS scavenging rate changing with time, the time required for the fluorescence intensity to drop to 50% of the initial value was calculated, that is, the ROS scavenging half-life.

[0087] The hydrogel samples were respectively the composite hydrogel loaded with SiNPs prepared in this example (denoted as NP-Gel), the traditional hydrogel modified with MnO2 nanoparticles (denoted as MnO2-Gel), and the single-network hydrogel without dynamic bonds (selecting polyethylene glycol hydrogel, the internal chemical bonds of which are mainly ether bonds and carbon-carbon single bonds, which are non-dynamic bonds, denoted as Control-Gel, and its preparation raw materials mainly include polyethylene glycol, polyethylene glycol diacrylate, and PBS buffer solution, which has good biocompatibility, but has a high swelling rate, low mechanical strength and lacks dynamic response ability). The test results are shown in Table 1.

[0088] Table 1

[0089]

[0090] It shows that the hydrogel (NP-Gel) prepared in this example can continuously scavenge ROS in the oxidative stress microenvironment after spinal cord injury simulated by H2O2 solution: the scavenging rate is stably maintained above 85% within 72 hours, significantly higher than that of the control group (MnO2-Gel is only 45%, Control-Gel is only 25%); the scavenging rate of the traditional material (MnO2-Gel) drops sharply after 24 hours, while NP-Gel realizes continuous scavenging due to the dynamic recombination of Schiff base bonds.

[0091] Through exponential decay fitting, the half-life of NP-Gel is 182 ± 5 hours (close to 180 hours). The half-life of MnO2-Gel is only 24 ± 3 hours, which is consistent with traditional materials; Control-Gel: has no significant scavenging ability, and the half-life cannot be calculated.

[0092] The above specific test processes and data show that the hydrogel prepared in this example has the function of continuously scavenging ROS in the microenvironment during spinal cord repair, and products that can continuously scavenge ROS in the microenvironment during spinal cord repair can be prepared, proving the breakthrough advantage of the hydrogel of the present invention in ROS scavenging performance.

[0093] (6) Detection of the effect of mediating microenvironment pH

[0094] Construct a pH simulation microenvironment in a 37°C, 5% CO2 incubator: use 5 mL of PBS buffer solution with a pH of 6.0 (containing 10 mM lactic acid, simulating the local acidic inflammatory environment after spinal cord injury) to construct a common acidic injury environment, and use 5 mL of Tris-HCl buffer solution with a pH of 8.0 to construct an occasional alkaline inflammatory environment. Immerse the hydrogel to be tested (diameter 5 mm × thickness 2 mm) in the acidic and alkaline buffer solutions simulating different environments and oscillate at 37°C (50 rpm). Samples are taken at time points of 0, 2, 4, 6, 12, 24, 48, and 72 h to measure the real-time pH value (pH t) Calculate the time to reach physiological pH (i.e., the time required to adjust the pH to 7.4 ± 0.05); the hydrogels to be tested include: the hydrogel prepared in this example, traditional pH-responsive hydrogels (polyacrylic acid-based, PAA gels), and non-responsive hydrogels (PEG gels).

[0095] The results in the acidic injury environment are shown in Table 2; the results in the alkaline inflammatory environment are shown in Table 3.

[0096] Table 2

[0097]

[0098] Table 3

[0099]

[0100] Whether in an acidic (pH 6.0) or alkaline (pH 8.0) microenvironment, the composite hydrogel loaded with SiNPs of the present invention can synergistically use Schiff base bonds and borate ester bonds to accurately adjust the pH to 7.4 ± 0.05 within 26 h and 24 h respectively, and maintain this pH range until the end of 72 h. Compared with traditional non-responsive (PEG) and single-responsive (PAA) hydrogels, its pH adjustment is more efficient and accurate, showing excellent dynamic balance ability in a complex pH fluctuation environment.

[0101] Through the above specific test process and data, it shows that the hydrogel prepared in this example has the ability to continuously adjust the pH of the microenvironment, and products that can continuously adjust the pH of the microenvironment can be prepared, proving the breakthrough advantage of the hydrogel of the present invention in microenvironment pH adjustment.

[0102] Comparative Example 1

[0103] The difference between this comparative example and Example 1 is that in the preparation method, step S3 for preparing nano-silicon particles (SiNPs) is not adopted, and nano-silicon particles (SiNPs) are not added in step S4. In the mechanical property test, its compressive strength is only 0.15 kPa and its flexural strength is 0.2 kPa.

[0104] Comparing Comparative Example 1 with Example 1 shows that introducing nano-silicon particles (SiNPs) with a particle size of 50 - 100 nm in Example 1 can provide physical anchoring sites to avoid structural misalignment during the self-healing process; the flexural strength of the healed material is increased by 15% - 25% compared with that without introducing nano-silicon (the collapse rate of the traditional soft gel structure > 40%); it is close to the elastic modulus range of spinal cord tissue (0.2 - 1 kPa), confirming that its mechanical properties are compatible with the spinal cord and meet the requirements for spinal cord repair.

[0105] Example 2

[0106] The difference between Example 2 and Example 1 lies in: adding a drug loading process. Specifically, in S4, methylprednisolone is dissolved in PBS buffer (pH 7.4), mixed with oxidized xyloglucan and methacrylated chitosan, and the final concentration of methylprednisolone is controlled to be 0.3 mg / mL. Subsequently, nano-silicon particles and borax are added in sequence, and stirred and reacted at 40 °C for 3 hours. The loading of anti-inflammatory drugs is achieved by the formation of hydrogen bonds between the hydroxyl groups on the surface of nano-silicon and the ester groups of methylprednisolone.

[0107] Based on the synergistic effect of borate ester bonds and Schiff base bonds, a dual-responsive network is constructed to achieve precise drug release triggered by dual factors of pH / ROS. Verified by the rat spinal cord contusion model, after implantation, the hydrogel group loaded with methylprednisolone: ① ELISA detection shows that the drug concentration in the injury area reaches the peak value (12.3 ± 1.2 μg / g) at 72 h, which is significantly higher than that of the intravenous injection group (4.1 ± 0.8 μg / g); ② The time to maintain the effective concentration (>5 μg / g) within 28 days reaches 19.5 ± 2.3 days, which is 2.3 times longer than that of the gelatin microsphere carrier; ③

[0108] The BBB motor score reaches 14.2 ± 1.5 points in the 4th week, which is 58% higher than that of the blank polyethylene glycol hydrogel group, proving that its sustained-release system effectively promotes functional recovery.

[0109] By binding the hydroxyl groups on the surface of the added nano-silicon particles to the drug molecules, the drug loading amount is significantly increased and the burst release of the drug is inhibited: the drug release rate in the initial 12 hours is reduced from 48% ± 5% of the traditional carrier to 12% ± 2%; in artificial cerebrospinal fluid (aCSF), the drug sustained-release period is extended to more than 14 days (the traditional system ≤ 7 days).

[0110] By regulating the concentration of tetraborate ions to adjust the cross-linking density of the composite hydrogel, a gradient response of the material to pH 4-9 is achieved, enabling the material to dynamically adjust the network porosity when the pH fluctuates, realizing an intelligent matching of the drug release curve. In the acute phase, anti-inflammatory drugs (the composite anti-inflammatory drug is methylprednisolone) can be rapidly released, and in the repair phase, neurotrophic factors (the composite neurotrophic factor is NGF) are slowly released. The release efficiency is increased by more than 35% compared with the single-responsive system.

[0111] Example 3

[0112] The microenvironment-responsive spinal cord repair hydrogel prepared in Example 1 was co-cultured with mesenchymal stem cells and then subjected to live-dead staining.

[0113] The steps include: After trypsin digestion, mesenchymal stem cells are resuspended in the hydrogel precursor solution, and the cell density is controlled at 1×10 6cells / mL; Subsequently, the cell-hydrogel mixture was evenly dispensed into 10-cm dishes and incubated in a constant temperature incubator at 37°C and 5% CO2 for 30 - 60 min. After the hydrogel was crosslinked and formed, complete medium was added for further culture; the medium was changed every 2 days during the culture process. On the 7th day of culture, the hydrogel samples containing cells were taken out for Live / Dead staining: A mixed working solution of Calcein-AM (2 μM) and propidium iodide PI (4 μM) was added dropwise to the surface of the samples and incubated at 37°C for 30 minutes, and then slowly rinsed 2 - 3 times with PBS to remove excess dye; the stained hydrogels were observed under a confocal laser scanning microscope.

[0114] In the observation results, Calcein-AM can be cleaved by live cell esterase into green fluorescent products to label live cells; PI can penetrate dead cells with damaged cell membranes and bind to DNA, showing red fluorescence; the activity, distribution, and biocompatibility of cells in the hydrogel can be evaluated through image analysis, and the results are as Figure 2 shown; it can be seen that the present invention has good biocompatibility.

[0115] Example 4

[0116] For experimental mice, the T10 spinal cord segment was exposed through laminectomy, and a 2-mm full-thickness transverse defect model was created with micro scissors. Guided by a stereotaxic apparatus, a mesenchymal stem cell suspension (concentration 1×10 6 cells / μL) was precisely injected into the central area of the semi-transverse spinal cord injury of the mice using a micro syringe (specification 33G), and the single-point injection volume was 2 μL; after the cells adhered for 1 hour, the hydrogel prepared in Example 1 was slowly injected through the same path to cover the stem cell transplantation area, and the injection volume was 5 μL. The mice were anesthetized at the 4th week after injury, and spinal cord tissue blocks (including the injury area and about 5 mm of tissue before and after) were taken out and fixed in 4% paraformaldehyde (PFA) for 24 hours, and then dehydrated in 30% sucrose solution until the tissue sank to the bottom. The dehydrated tissue was embedded in OCT embedding medium, and transverse or sagittal sections with a thickness of 10 - 20 μm were prepared on a cryostat and attached to glass slides for standby. The sections were rinsed 3 times with PBS (5 minutes each time), and then permeabilized with 0.3% Triton X-100 for 10 - 15 minutes; then blocked with 5% BSA or normal goat serum at room temperature for 1 hour to block non-specific binding; after blocking, the excess liquid was aspirated, the primary antibody mixture was added, and incubated overnight at 4°C; the next day, washed 3 times with PBS, added the fluorescently labeled secondary antibody, and incubated in the dark for 1 hour; after the incubation was completed, washed 3 times with PBS again, dropped the mounting medium containing DAPI for mounting; finally, observed and photographed under a confocal microscope to record the distribution of neuronal axons and astrocytes and the situation of glial scars.

[0117] The results of confocal microscopy are as follows Figure 4 shown: It can be seen that the present invention significantly promotes the differentiation of stem cells into neurons, as well as promotes the regeneration and repair of nerve tissue. The hydrogel rapidly gels to form a three-dimensional encapsulation structure, and its dynamic network pores can provide anchoring sites for stem cells. At the same time, the microenvironment is continuously regulated through the pH / ROS response mechanism; this operation mode avoids mechanical stress damage caused by the pre-mixing of stem cells and the hydrogel through physical separation, and the barrier formed after gelation can effectively inhibit the infiltration of inflammatory cells and reduce the mechanical compression of the graft on the surrounding tissues.

[0118] Figure 3 (a) shows the immunofluorescence staining of typical markers of neurons and astrocyte markers 4 weeks after establishing a semi-transverse spinal cord injury model in mice. Figure 3 (b) shows the statistical analysis of the fluorescence intensity of astrocyte markers. Figure 3 (c) shows the statistical analysis of the fluorescence intensity of neuron cell markers; it shows that this material can effectively improve the survival rate of stem cells in the in vivo microenvironment, and at the same time reduce the secondary injury of the graft to the spinal cord tissue; compared with the simple injury group, the present invention significantly promotes the regeneration and tissue repair of nerve tissue at the injury site, promotes the extension and remyelination of nerve axons, and provides a better biological scaffold solution for the cell therapy of spinal cord injury.

[0119] The mice were scored by the BMS at each time point and the Catwalk gait analysis of the mice was performed at the end stage. The system quantitatively evaluated the recovery of the hind limb motor function and gait characteristics of the mice. The results are as follows Figure 5 shown, where Figure 5 (a) in is the BMS score result graph, Figure 5 (b) in is the Catwalk gait analysis result graph. Compared with the injury group, the treatment group of the present invention showed significantly higher BMS scores at each time point after surgery (P<0.05), indicating that it can effectively promote the recovery of motor function after spinal cord injury; at the same time, the gait parameters of the mice (including stride length, stance phase duration, footprint symmetry, etc.) were significantly improved, and the gait pattern was closer to that of the normal control group (P<0.05), further verifying the function improvement effect of the present invention in spinal cord injury repair.

[0120] Example 5

[0121] The difference between this embodiment and Embodiment 1 lies in: adding a drug loading process. Specifically, in S4, the anti-inflammatory drug dexamethasone and the neurotrophic factor NGF are respectively dissolved in PBS buffer, pre-mixed with nano-silicon particles (SiNPs) for 30 minutes, and preliminary loading is achieved through hydrogen bond binding between the hydroxyl groups on the silicon particle surface and the drug molecules. Subsequently, the drug-loaded nano-silicon particles are added to the premixed solution of oxidized xyloglucan (OXG) and chitosan methacrylate (CSMA), where the concentration of dexamethasone in the system is 0.5 mg / mL, and the concentration of the neurotrophic factor NGF in the system is 50 ng / mL. A double-network hydrogel is formed through dynamic borate ester bond / Schiff base bond cross-linking, enabling the drugs to be further embedded in the three-dimensional pore structure. Finally, double drug loading is completed by magnetic stirring at 40 °C (600 rpm, 2 hours).

[0122] An in vitro simulated spinal cord injury microenvironment (alternating cycles of pH 6.5 + ROS 0.2 mM acidic oxidation conditions and pH 7.4 + ROS

[0123] 0.05 mM neutral recovery conditions) was adopted, and the cumulative release rates of dexamethasone and NGF were quantitatively analyzed by high-performance liquid chromatography (HPLC).

[0124] The test results were as follows: In the acute phase, flow cytometry was used to monitor the average amount of sudden drug release in the first 6 hours after ROS triggering, which was 84%. In the repair phase, the sustained release curve of NGF within 28 days was measured by ELISA, and the daily average release amount was 4%. It shows that the double dynamic network constructed by borate ester bond and Schiff base bond, combined with the PH / ROS dual-responsive mechanism, realizes the burst release of anti-inflammatory drugs (such as dexamethasone) in the acute phase (PH ≤ 6.5 + ROS ≥ 0.2 mM), and the sustained release of neurotrophic factor (NGF) in the repair phase (PH 7.4 + ROS ≤ 0.05 mM), enabling the material to maintain a stable response in a complex physiological environment (such as intermittent ROS fluctuations).

[0125] The xyloglucan used in the present invention is a mixed sugar, and the component ratios of xyloglucans from different sources are different, and their molecular sizes are also different. According to different parameters, it is necessary to explore the hydrogel construction conditions to achieve the best performance. Tamarind xyloglucan has a multi-branched sugar structure and a high content of active hydroxyl groups, which significantly optimizes the biocompatibility of the dynamic network, while other sources of xyloglucan do not have advantages in terms of multi-branched sugar structure and high content of active hydroxyl groups, and cannot achieve the same level of cross-linking density, biological activity, and response sensitivity as tamarind-derived xyloglucan.

[0126] Xyloglucan has significant advantages in the repair of the central nervous system such as the spinal cord. Its natural polysaccharide structure endows excellent biocompatibility and biodegradability, and can mimic the extracellular matrix environment of nerve cells, promoting neuron adhesion and axonal directional growth. As a natural polymer derived from tamarind, after xyloglucan forms aldehyde groups through oxidation, it can form dynamic Schiff base bonds with the amino groups of chitosan to construct a pH / ROS dual-responsive network, precisely matching the acid-base fluctuations and oxidative stress changes in the spinal cord injury microenvironment. Compared with synthetic materials, the hydroxyl groups on its polysaccharide chain can enhance the water retention and lubricity of the hydrogel, reducing tissue friction damage after implantation. In addition, the three-dimensional pore structure of xyloglucan is more conducive to nutrient delivery and metabolic waste clearance. When combined with nanosilicon particles synergistically, it can form a support network that mechanically adapts to the spinal cord tissue, effectively maintaining the morphological stability of the damaged site.

[0127] Compared with the prior art, the present invention also has the following outstanding advantages:

[0128] 1. Although there are single dynamic network designs containing borate ester bonds or Schiff base bonds in the prior art, their pH response range is limited (e.g., only effective in neutral to alkaline environments), and they cannot achieve dynamic self-regulation under local acid / base fluctuations. The dual dynamic bonds of the present invention, through their synergistic effect, enable the hydrogel to preferentially trigger the reversible dissociation of Schiff base bonds under acidic (pH ≤ 6.5) conditions, while maintaining the network integrity through the stability of borate ester bonds at physiological pH (7.4), solving the problems of mechanical strength collapse or excessive swelling caused by the single bond response of traditional materials. In addition, the rigid support of nanosilicon particles further enhances the structural stability of the network under pH fluctuations, breaking through the contradiction between the stability and response sensitivity of traditional systems.

[0129] 2. Although there are solutions that use borate ester bonds or Schiff base bonds alone in the prior art, the core innovation of the present invention lies in the optimization and upgrade of the ROS response mechanism through the composite action of the dynamic synergistic network of borate ester bonds and Schiff base bonds and nanosilicon particles. Traditional single dynamic bond systems have defects such as fixed response thresholds and low self-healing efficiency. However, the present invention, through the pH / ROS dual response coupling of dual dynamic bonds and the rigid support of nanosilicon, can still maintain structural stability through bond recombination in a low-concentration ROS environment, while achieving improved anti-swelling properties and antibacterial function integration. This multi-component synergistic effect enables the system to exhibit a continuous response ability and mechanical adaptability in complex physiological environments that cannot be achieved by traditional single dynamic bond systems, constituting a substantial innovation different from the prior art.

[0130] 3. Although there are existing solutions containing borate ester bonds and Schiff base bonds in the prior art, the self-healing ability of the present invention still has significant innovation. The key lies in the combination of the synergistic response mechanism of dual dynamic bonds (borate ester bonds / Schiff base bonds) and the physical support of nano-silicon particles, which solves the core problems of the lack of dynamic balance and mechanical mismatch in traditional double-bond systems. Specifically, existing double-bond systems often only achieve simple coexistence of bonds and fail to establish an adaptive balance mechanism for the two dynamic bonds in the pH / ROS microenvironment, resulting in a contradiction between healing efficiency and mechanical recovery (such as network collapse caused by excessive dissociation of a single bond under acidic conditions). In contrast, the present invention regulates the dissociation threshold and recombination rate of the two bonds, enabling the Schiff base bond to preferentially respond to ROS and trigger rapid recombination, while the borate ester bond maintains network stability through the regulation of the concentration of tetraborate ions, forming a staged self-healing mode. At the same time, the rigid support of nano-silicon particles provides physical anchor points for the recombination of dynamic bonds, overcoming the structural misalignment problem during the self-healing process of traditional soft gels and achieving simultaneous improvement in healing efficiency and mechanical properties. This combined design of double-bond synergistic response and physical strengthening exceeds the performance boundaries of existing single double-bond systems in terms of self-healing mechanism.

[0131] 4. The core inventive point of the present invention is to construct a dual dynamic network through the synergistic action of borate ester bonds and Schiff base bonds, combined with the rigid support of nano-silicon particles and multiple drug-loading sites, achieving a timing control function that cannot be achieved by traditional single dynamic bond systems. Although there are existing solutions containing dual dynamic bonds in the prior art, their synergistic response mechanism of dynamic bonds is insufficient, making it difficult to achieve precise temporal release of anti-inflammatory drugs and neurotrophic factors in complex physiological environments. The present invention regulates the network porosity through the dynamic balance of double bonds, enabling the drug release to highly match the spinal cord injury repair stage and breaking through the technical bottlenecks of traditional drug-loading systems in temporal regulation and spatial positioning, which is a substantial technical improvement not achieved by existing double-bond solutions.

[0132] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A microenvironment-responsive spinal cord repair hydrogel, characterized in that, It is made from the following raw materials in parts by mass: 0.8 - 1.5 parts of xyloglucan oxide, 0.8 - 1.5 parts of methacrylated chitosan, 0.1 - 0.5 parts of nano-silicon particles, and 0.7 - 2.0 parts of borax.

2. The microenvironment-responsive spinal cord repair hydrogel according to claim 1, wherein The preparation raw materials include 95.5 - 97.2 parts of PBS buffer solution.

3. The microenvironment-responsive spinal cord repair hydrogel according to claim 1, wherein, The preparation raw materials include anti-inflammatory drugs and / or neurotrophic factors; The anti-inflammatory drugs include one or more of methylprednisolone, dexamethasone, and prednisolone; or, the neurotrophic factors include one or more of NGF, BDNF, and NT-3.

4. The microenvironment-responsive spinal cord repair hydrogel according to claim 3, wherein The concentration of methylprednisolone in the microenvironment-responsive spinal cord repair hydrogel is 0.5 - 5.0 mg / mL; or, the concentration of dexamethasone in the microenvironment-responsive spinal cord repair hydrogel is 0.1 - 2.0 mg / mL; or, the concentration of prednisolone in the microenvironment-responsive spinal cord repair hydrogel is 0.5 - 5 mg / mL; Or, the concentration of NGF in the microenvironment-responsive spinal cord repair hydrogel is 0.01 - 0.5 μg / mL; or, the concentration of BDNF in the microenvironment-responsive spinal cord repair hydrogel is 0.05 - 1.0 μg / mL; or, the concentration of NT-3 in the microenvironment-responsive spinal cord repair hydrogel is 0.02 - 0.8 μg / mL.

5. A method for preparing a microenvironment-responsive spinal cord repair hydrogel according to any one of claims 1-4, characterized in that, It includes the following steps: Add xyloglucan oxide and methacrylated chitosan into the buffer solution respectively, and stir and react at room temperature for 4 - 6 h; then add nano-silicon particles and borax in sequence, and stir and react at 40 - 60 °C for 2 - 3 h.

6. The preparation method of the microenvironment-responsive spinal cord repair hydrogel according to claim 5, wherein The loading methods of anti-inflammatory drugs and / or neurotrophic factors include one or more of physical adsorption method, chemical bonding encapsulation method, and in-situ mixing loading method; Or, the physical adsorption method includes: using the hydroxyl or amino groups on the surface of nano-silicon particles to combine with drug molecules through hydrogen bonds or electrostatic interactions to achieve drug adsorption and loading; Or, the chemical bonding encapsulation method includes: constructing a double-network structure through dynamic borate ester bonds and Schiff base bonds to encapsulate the drug in three-dimensional pores; Or, the in-situ mixing loading method includes: directly blending with the drug solution during the preparation of the hydrogel, and embedding drug molecules into the network interior through cross-linking reaction; Or, the buffer solution is PBS buffer solution.

7. The preparation method of the microenvironment-responsive spinal cord repair hydrogel according to claim 5, characterized in that, The preparation method of xyloglucan oxide includes: mixing sodium periodate and xyloglucan in a mass ratio of 1:(2 - 5) to prepare a solution, stirring and reacting at room temperature for 4 - 6 h, then adding ethylene glycol to terminate the reaction, and dialyzing to obtain xyloglucan oxide; Or, dialyze the substances with a molecular weight cut-off ≥ 3500 Da, and freeze-dry to obtain xyloglucan oxide.

8. The preparation method of the microenvironment-responsive spinal cord repair hydrogel according to claim 5, wherein, The preparation method of methacrylated chitosan includes: dissolving chitosan in acetic acid solution, then adding methacrylic anhydride with a mass 0.36 - 1.92 times that of chitosan, stirring and reacting at 60 - 80 °C for 6 - 10 h, adding an alkaline substance to neutralize to pH = 6.5 - 7, diluting, and then dialyzing to obtain methacrylated chitosan; Or, dialyze the substances with a molecular weight cut-off of 8000 - 14000 Da, and freeze-dry to obtain methacrylated chitosan; Or, the alkaline substance includes an aqueous solution of sodium bicarbonate.

9. The preparation method of the microenvironment-responsive spinal cord repair hydrogel according to claim 5, wherein, The preparation method of the nano-silicon particles includes: dispersing 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane in 5 to 6 times the volume of ultrapure water, adding 2-aminophenol which is 0.24 to 0.30 times the mass of 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane thereto, stirring and reacting at 70 to 80 °C for 100 - 150 min, and dialyzing to obtain the nano-silicon particles; Or, dialyzing the substance with a dialysis cut-off molecular weight of 400 - 600 Da, and freeze-drying to obtain the nano-silicon particles.

10. Application of a microenvironment-responsive spinal cord repair hydrogel as described in any one of claims 1 - 4 in at least one of the following a1) - a5): a1) Preparing a product for promoting spinal cord repair; a2) Preparing a product for continuously scavenging microenvironment ROS during spinal cord repair; a3) Preparing a product for continuously adjusting the microenvironment pH during spinal cord repair; a4) Preparing a product for antibacterial during spinal cord repair; a5) Preparing a product for sustained drug release during spinal cord repair.

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