Traditional Chinese medicine small molecule conductive hydrogel for repairing nerves and preparation method and application thereof

Abstract

The application discloses a traditional Chinese medicine small-molecule conductive hydrogel for repairing nerves and a preparation method and application thereof, and belongs to the technical field of biomedical materials.The preparation method is as follows: firstly, mix tannic acid and 5-formyl-2-furan boronic acid in a buffer solution and adjust the pH value to neutral or weak alkaline; then introduce carboxymethyl chitosan and poly(3,4-ethylenedioxythiophene) / polystyrene sulfonate solution; and construct a double dynamic crosslinking network by using borate ester bonds formed by the tannic acid and the 5-formyl-2-furan boronic acid and Schiff base bonds formed by the carboxymethyl chitosan and the 5-formyl-2-furan boronic acid.The hydrogel has excellent injectability and self-healing property, can realize antioxidation and anti-inflammation of a damaged microenvironment and reconstruction of a bioelectric pathway in cooperation, and thus promotes nerve regeneration and conduction recovery.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a small-molecule conductive hydrogel of traditional Chinese medicine, its preparation method and application. Background Technology

[0002] Nervous system injuries, including peripheral nerve injury (PNI) and central nervous system injury (SCI), are common and serious clinical conditions, usually caused by traumatic events such as traffic accidents or natural disasters, leading to long-term loss of motor and sensory functions. Disruption of the neural signaling network and the complex pathological microenvironment after injury are key factors restricting nerve repair. Currently, although autologous nerve transplantation is considered the "gold standard" of clinical treatment, it faces limitations such as limited donor availability, complications from donor site denervation, and size and shape mismatch. With the development of regenerative medicine, bridging nerve defects using biomaterial tissue engineering scaffolds has become an important alternative. Hydrogels, due to their ability to fill irregular injury cavities and provide physical support for nerve stumps, are considered ideal candidate materials for neural tissue engineering. However, traditional biomaterial scaffolds primarily focus on providing passive physical support and are insufficient to actively address the multiple pathological challenges following nerve injury.

[0003] Microenvironmental regulation following nerve injury is the primary barrier to tissue repair. During the acute phase of injury, the local microenvironment undergoes dramatic pathological changes, generating large amounts of reactive oxygen species (ROS) and triggering severe oxidative stress, directly damaging nerve cells and leading to a cascade of secondary damage. Simultaneously, intense inflammatory responses and the release of pro-inflammatory factors exacerbate tissue damage and inhibit axonal growth. Therefore, endowing hydrogels with highly efficient antioxidant and anti-inflammatory functions to scavenge ROS and regulate inflammatory phenotypes is crucial for improving the environment for nerve regeneration.

[0004] Furthermore, the nervous system possesses a highly specialized bioelectrical network, and its functional recovery depends not only on the connection of physical structures but also on the reconstruction of the continuity of electrical signal transmission. Traditional hydrogels with poor conductivity cannot reconstruct the bioelectrical pathways at both ends of damaged nerves, and the glial scars formed after injury further block nerve communication, making it difficult for regenerated axons to cross the lesion area. Although conductive hydrogels can simulate the endogenous electrical microenvironment and promote axonal extension, conventional conductive materials often lack the ability to actively regulate inflammation and oxidative stress in the early stages of injury.

[0005] To overcome these shortcomings, current conventional strategies typically involve physically encapsulating exogenous drugs or bioactive molecules to endow scaffolds with anti-inflammatory or antioxidant functions. However, such exogenous delivery systems often face limitations such as sudden drug release, short half-lives of active molecules, complex fabrication processes, and difficulties in quality control. Therefore, there is an urgent need to develop an integrated material that is easy to prepare and can effectively combine microenvironment regulation capabilities with efficient electrical conduction to achieve spatiotemporal synergistic repair after nerve injury. Summary of the Invention

[0006] To overcome the shortcomings and deficiencies of the existing technology, the purpose of this invention is to provide a small molecule conductive hydrogel of traditional Chinese medicine for nerve repair, its preparation method and application.

[0007] The technical solution provided by this invention is as follows: A small-molecule conductive hydrogel of traditional Chinese medicine, the hydrogel is composed of carboxymethyl chitosan (CMCh), ellagic acid (EA), 5-formyl-2-furanboronic acid (FFBA), and conductive polymer PEDOT:PSS; wherein, in the hydrogel, ellagic acid and 5-formyl-2-furanboronic acid form borate ester bonds, and carboxymethyl chitosan and 5-formyl-2-furanboronic acid form Schiff base bonds to construct a double dynamic crosslinking network.

[0008] Preferably, the concentrations of each component in the obtained hydrogel are: carboxymethyl chitosan 2~10.0% (w / v), ellagic acid 0.5~5.0% (w / v), 5-formyl-2-furanboronic acid 0.5~5.0% (w / v), and PEDOT:PSS 0.1~1.0% (w / v).

[0009] This invention further discloses a method for preparing the above-mentioned hydrogel, the method comprising the following steps: (1) Dissolve 5-formyl-2-furanboronic acid in PBS buffer, add alkaline solution to adjust the pH value, add ellagic acid at room temperature and react for 1 to 4 hours to obtain ellagic acid-5-formyl-2-furanboronic acid precursor solution; wherein the concentration range of ellagic acid is 1 to 10.0% (w / v) and the concentration range of 5-formyl-2-furanboronic acid is 1 to 10.0% (w / v). (2) Prepare carboxymethyl chitosan aqueous solution and PEDOT:PSS aqueous solution; wherein the concentration range of carboxymethyl chitosan is 8~40.0% (w / v) and the concentration range of PEDOT:PSS is 0.4~4% (w / v). (3) The ellagic acid-5-formyl-2-furanboronic acid precursor solution, carboxymethyl chitosan aqueous solution and PEDOT:PSS aqueous solution are mixed and stirred to obtain a small molecule conductive hydrogel of traditional Chinese medicine; wherein, the concentration of each component in the hydrogel is: carboxymethyl chitosan 2~10.0% (w / v), ellagic acid 0.5~5.0% (w / v), 5-formyl-2-furanboronic acid 0.5~5.0% (w / v), PEDOT:PSS 0.1~1.0% (w / v).

[0010] Preferably, in step (1), the pH value of the ellagic acid-5-formyl-2-furanboronic acid precursor solution is 7.0~7.4.

[0011] Preferably, in step (3), the volume ratio of the carboxymethyl chitosan aqueous solution, the PEDOT:PSS aqueous solution and the ellagic acid-5-formyl-2-furanboronic acid precursor solution is (1~2):(1~2):(1~4).

[0012] This invention further discloses the application of the above-mentioned small molecule conductive hydrogel of traditional Chinese medicine in the preparation of nerve injury repair materials.

[0013] Preferably, the nerve injury includes peripheral nerve injury and spinal cord injury.

[0014] This invention overcomes the shortcomings of existing technologies by providing a small-molecule conductive hydrogel made from traditional Chinese medicine for nerve repair, along with its preparation method and applications. The invention involves mixing ellagic acid and 5-formyl-2-furanboronic acid in a buffer solution and adjusting the pH to neutral or weakly alkaline. Subsequently, carboxymethyl chitosan and a poly(3,4-ethylenedioxythiophene) / polystyrene sulfonate solution are introduced. A dual dynamic cross-linking network is constructed using the borate ester bonds formed between ellagic acid and 5-formyl-2-furanboronic acid, and the Schiff base bonds formed between carboxymethyl chitosan and 5-formyl-2-furanboronic acid. This hydrogel exerts antioxidant and anti-inflammatory effects by releasing ellagic acid to regulate the damaged microenvironment. It also utilizes PEDOT:PSS to reconstruct the bioelectrical conduction pathways in the damaged area, promoting axonal regeneration and functional recovery. The hydrogel possesses excellent injectability and self-healing properties. Its core function lies in synergistically achieving antioxidant and anti-inflammatory effects on the damaged microenvironment and reconstructing bioelectrical pathways, thereby promoting nerve regeneration and conduction recovery. In vivo experiments have confirmed that this material can significantly improve motor and sensory function after peripheral nerve and spinal cord injury and promote nerve repair.

[0015] The beneficial effects of this invention after adopting the above technical solution are as follows: 1. Excellent dynamic properties: Thanks to the dual dynamic cross-linking network of borate ester bonds and Schiff base bonds, the hydrogel of this invention exhibits a significant shear-thinning effect and excellent self-healing ability. This allows the material to be injected via a minimally invasive method, precisely adapting to and filling irregular nerve injury cavities, while rapidly restoring structural integrity after implantation; 2. Significant Microenvironment Regulation Function: This invention utilizes the polyphenolic structure of ellagic acid, a small molecule from traditional Chinese medicine, to endow the hydrogel with intrinsic bioactivity. During the acute phase of injury, the material can efficiently scavenge reactive oxygen species (ROS) at the injury site, alleviating oxidative stress. Simultaneously, it possesses excellent anti-inflammatory capabilities, downregulating pro-inflammatory factor levels, thereby reshaping the biological microenvironment required for nerve repair. 3. Superior Bioelectric Reconstruction Capability: By introducing PEDOT:PSS to construct a stable conductive network, the hydrogel of this invention can simulate the endogenous electrical microenvironment of nerve tissue, establishing a "bioelectronic bridge" for electrical signal conduction at both ends of the damaged nerve. Experiments have shown that the presence of the conductive interface significantly promotes the directional growth of axons, remyelination, and electrophysiological recovery of nerve function. 4. Excellent biocompatibility and biocompatibility: All components of the hydrogel of this invention exhibit good biocompatibility, and their degradation kinetics are highly matched with the timescale of nerve regeneration. In vivo experiments have confirmed that this material can significantly improve motor and sensory functions and reduce muscle atrophy after denervation in peripheral nerve and spinal cord injury models, demonstrating extremely high potential for clinical translation. Attached Figure Description

[0016] Figure 1 This document describes the formation mechanism, microstructure, and conductive activity characterization of the conductive hydrogel of this invention. A shows a photograph of the hydrogel formation; B shows the injectability of the hydrogel; C shows a scanning electron microscope (SEM) image of the hydrogel, revealing its three-dimensional porous network structure; D shows the self-healing property of the hydrogel; E shows the electrochemical impedance spectroscopy (EIS) test results; F shows the conductivity of the hydrogel; and G shows a circuit diagram demonstrating its conductive activity under physiological conditions, illustrating the connection of the hydrogel to an isolated spinal cord to illuminate an LED.

[0017] Figure 2 The rheological properties of the conductive hydrogel are characterized; where A is the strain scan curve; B is the frequency scan curve; C is the viscosity-shear rate curve, showing the shear thinning behavior; D is the high and low strain alternating cycle test curve, showing the self-healing performance of the material; in the figure, EFC refers to the hydrogel prepared in Comparative Example 1 (EFC), and EFC-P refers to the hydrogel prepared in Example 1 (EFC-P0.3).

[0018] Figure 3This study characterizes the biocompatibility, swelling, and in vitro and in vivo degradation properties of the conductive hydrogel. A shows the results of the in vitro hemolysis experiment; B shows the cytotoxicity of the hydrogel to rat Schwann cells (RSCs); C shows the live / dead fluorescence staining of RSCs co-incubated with the hydrogel; D shows the swelling kinetics curve of the hydrogel; E shows the in vitro degradation curve; F shows ultrasound imaging monitoring after subcutaneous implantation; G shows the complete blood count of the animals after degradation; H shows the biochemical indicators of liver and kidney function; and I shows HE-stained sections of major organs.

[0019] Figure 4 The antioxidant and anti-inflammatory activities of the conductive hydrogel were characterized. A represents the statistical scavenging rates of DPPH, ABTS, PTIO radicals, and hydrogen peroxide; B represents the mRNA expression levels of pro-inflammatory factors (IL-1β, IL-6, TNF-α) in macrophages under LPS-induced activity, detected by qPCR.

[0020] Figure 5 This study demonstrates the effect of conductive hydrogel on promoting PC12 cell differentiation and axon growth. In the image, A shows a photograph of PC12 cells after co-culturing with the hydrogel; B shows quantitative statistics on the number of neurites and the average length of neurites.

[0021] Figure 6 This image shows the repair effect of conductive hydrogel in a sciatic nerve injury (PNI) model. A shows the gait footprint on postoperative day 28; B shows the Sciatic Nerve Function Index (SFI) score; C shows the recovery of hyperalgesia as assessed by a bipedal balance analgesia meter; D shows the electrophysiological assessment (CMAP) results at postoperative week 4; E shows the wet weight ratio of the gastrocnemius muscle; F shows an image of the gastrocnemius muscle; G shows Masson staining analysis of the gastrocnemius muscle; and H shows an HE-stained section of the injured sciatic nerve, demonstrating the recovery of the nerve bundle structure.

[0022] Figure 7 The study demonstrates the repair effect of conductive hydrogels in a spinal cord injury (SCI) model. A shows the reactive oxygen species (ROS) level at the site of spinal cord injury on postoperative day 3; BD shows the mRNA expression levels of inflammatory factors (IL-1β, TNF-α) at ​​the site of spinal cord injury on postoperative day 3; E shows the body motor function (BMS) score of mice within 6 weeks postoperatively; F shows the gait analysis of mice at week 6 after treatment; G shows the results of the inclined plane test; H shows the motor evoked potential (MEP) detection at week 6 postoperatively; and I shows the histological evaluation of the longitudinal section of the spinal cord, including HE staining and Masson staining, demonstrating the cavity and tissue continuity.

[0023] Figure 8To evaluate the molecular-level effect of conductive hydrogels on spinal cord injury repair, the expression levels of genes related to nerve regeneration and myelination (NF200, Tuj-1, GFAP, Syn, MBP, etc.) in the spinal cord injury area were detected by qPCR to evaluate the material's promoting effect on axonal extension and remyelination. Detailed Implementation

[0024] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but this does not constitute any limitation on the present invention.

[0025] Example 1 A method for preparing a small-molecule conductive hydrogel of traditional Chinese medicine (EFC-P0.3) includes the following steps: (1) Preparation of ellagic acid-5-formyl-2-furanboronic acid precursor solution: Dissolve 5-formyl-2-furanboronic acid in 10mM PBS buffer to prepare a 1.6% (w / v) solution. Add an appropriate amount of sodium hydroxide solution to adjust the pH value until the 5-formyl-2-furanboronic acid is completely dissolved. Then add ellagic acid to make its concentration reach 1.6% (w / v). Stir and react at room temperature in the dark for 2 hours to obtain a homogeneous precursor solution with a pH value of about 7.4. (2) Preparation of polymer solution: Dissolve carboxymethyl chitosan in deionized water to prepare an 8% (w / v) solution; dissolve PEDOT:PSS in water to prepare a 1.2% (w / v) solution; (3) Mixing and gelling: The above 8% carboxymethyl chitosan solution, 1.2% PEDOT:PSS solution, and ellagic acid-5-formyl-2-furanboronic acid precursor solution were mixed and stirred at a volume ratio of 1:1:2. The mixture was gelled in situ within 60 seconds; the final concentrations of each component of the resulting hydrogel were: ellagic acid 0.8% (w / v), 5-formyl-2-furanboronic acid 0.8% (w / v), carboxymethyl chitosan 2% (w / v), and PEDOT:PSS 0.3% (w / v).

[0026] Example 2 A method for preparing a low-concentration conductive polymer hydrogel (EFC-P0.1) is basically the same as that in Example 1, except that the concentration of the PEDOT:PSS solution in step (2) of this example is 0.4% (w / v). An 8% (w / v) carboxymethyl chitosan solution, a 0.4% (w / v) PEDOT:PSS solution, and an ellagic acid-5-formyl-2-furanboronic acid precursor solution are mixed and stirred at a volume ratio of 1:1:2. The final concentrations of the components of the resulting hydrogel are: ellagic acid 0.8% (w / v), 5-formyl-2-furanboronic acid 0.8% (w / v), carboxymethyl chitosan 2% (w / v), and PEDOT:PSS 0.1% (w / v).

[0027] Example 3 A method for preparing a high-concentration conductive hydrogel includes the following steps: (1) Preparation of ellagic acid-5-formyl-2-furanboronic acid precursor solution: Prepare 5-formyl-2-furanboronic acid to 3% (w / v), add ellagic acid to 3% (w / v), adjust the pH to 7.2, and react for 3 hours; (2) Prepare a 12% (w / v) carboxymethyl chitosan solution and a 1.6% (w / v) PEDOT:PSS solution; (3) Mix carboxymethyl chitosan, PEDOT:PSS and precursor solution in a volume ratio of 2:1:1; the final concentrations of each component of the resulting hydrogel are: 1.5% (w / v) ellagic acid, 1.5% (w / v) 5-formyl-2-furanboronic acid, 3% (w / v) carboxymethyl chitosan and 0.4% (w / v) PEDOT:PSS.

[0028] Comparative Example 1 The method for preparing a hydrogel (EFC) without conductive components is basically the same as that in Example 1, except that an equal volume of deionized water is used instead of the PEDOT:PSS solution. The resulting hydrogel consists of 0.8% (w / v) ellagic acid, 0.8% (w / v) 5-formyl-2-furanboronic acid, and 2% (w / v) carboxymethyl chitosan, and does not possess conductive activity.

[0029] Example of effect I. Implementation Content 1. Preparation of hydrogels and characterization of their physicochemical properties and conductive activity (1) Preparation and microstructure observation of hydrogels Each precursor solution (including EA+FFBA mixture, CMCh solution, and PEDOT:PSS dispersion) was placed in a sample vial, and its state was observed and photographed. Simultaneously, the prepared hydrogel samples (including Comparative Example 1 (EFC), Example 1 (EFC-P0.3), and Example 2 (EFC-P0.1)) were placed in a sample vial, and their macroscopic gelation state was observed and photographed using methods such as inversion. The prepared hydrogel samples (including Comparative Example 1 (EFC) and Example 1 (EFC-P0.3), note: in the following text, mainly Comparative Example 1 hydrogel (EFC), Example 1 hydrogel (EFC-P0.3), and Example 2 hydrogel (EFC-P0.1) were selected for experiments. Unless otherwise specified, EFC refers to the hydrogel in Comparative Example 1 (EFC), and EFC-P refers to the hydrogel in Example 1 (EFC-P0.3)) were rapidly frozen in liquid nitrogen and then transferred to a freeze dryer for complete freeze-drying. The sample was fractured using liquid nitrogen, fixed on a sample stage, and sputtered with gold. Its three-dimensional porous network structure was then observed using a scanning electron microscope (SEM).

[0030] (2) Conductivity test Measured using an electrochemical workstation via electrochemical impedance spectroscopy (EIS). The hydrogel was sandwiched between two parallel stainless steel electrodes at 10... -1 Up to 10 5 Impedance is measured within the Hz frequency range. According to the formula ( =d / (R×A))Calculate the conductivity, where d is the thickness, A is the contact area, and R is the resistance value.

[0031] (3) Experiment on conduction in detached bodies A series circuit consisting of a 3V DC power supply, an LED indicator, and fresh isolated mouse spinal cord tissue was constructed. After creating a 2 mm defect in the middle of the spinal cord to disconnect the circuit, conductive hydrogel was filled in situ into the defect gap, and the on / off state of the LED was observed to evaluate the ability of the electrical pathway to be reconstructed.

[0032] (4) Rheological test The following tests were conducted using a rotational rheometer at 25°C: a) Strain scan: a fixed angular frequency of 1 Hz, strain range of 0.1% to 1000%, to determine the linear viscoelastic region. b) Frequency scan: a fixed strain of 1%, frequency range of 0.1 to 100 rad / s. c) Shear thinning test: assess viscosity change over a shear rate range of 0.1 to 100 s⁻¹. d) Self-healing test: perform alternating high and low strain cycles, i.e., instantaneously switch between 1% low strain and 1000% high strain, and observe the recovery of the storage modulus.

[0033] 2. Evaluation of the biocompatibility and in vitro / in vivo degradation of the hydrogel (1) Hemolysis test An 8% red blood cell suspension was mixed with an equal volume of hydrogel and incubated at 37°C for 1 hour. After centrifugation, the absorbance of the supernatant at 490 nm was measured, and the hemolysis rate was calculated.

[0034] (2) Cytotoxicity assay (MTT method) Rat Schwann cells (RSCs) and mouse macrophages (RAW264.7) were co-cultured with hydrogel extract for 24, 48, and 72 hours, respectively. After incubation with MTT solution, formazan crystals were dissolved with dimethyl sulfoxide, and absorbance was measured at 570 nm to calculate cell viability. Cell viability was further assessed using a live / dead staining assay: the co-cultured cells were discarded from the culture medium and washed with PBS, then incubated with a staining working solution containing calcein (live cell staining, green fluorescence) and propidium iodide (dead cell staining, red fluorescence) at 37°C in the dark for 20-30 minutes. Subsequently, fluorescence microscopy was used to observe and image the cells. By observing the distribution and ratio of green and red fluorescence, the effect of the hydrogel extract on cell viability and cell morphology was visually evaluated.

[0035] (3) Swelling and in vitro degradation The hydrogel was immersed in PBS solution at 37°C, and the weight was measured periodically to calculate the swelling ratio. In vitro degradation was evaluated by monitoring the percentage of mass loss of the hydrogel in PBS.

[0036] (4) In vivo evaluation Hydrogel was subcutaneously injected into the back of SD rats, and the residual volume was monitored weekly using a high-frequency small animal ultrasound imaging system. At the end of the experiment, whole blood was collected for complete blood count and liver and kidney function biochemical analysis. Simultaneously, major organs were removed, fixed in 4% paraformaldehyde, and stained with hematoxylin and eosin (HE) to observe tissue morphological changes.

[0037] 3. Evaluation of the antioxidant and anti-inflammatory activities of the hydrogel (1) Free radical scavenging experiment The scavenging ability of the hydrogel for different free radicals was evaluated using the DPPH, ABTS, and PTIO methods, respectively, and the scavenging effect on hydrogen peroxide was evaluated using the titanium sulfate method. The absorbance changes were recorded by spectrophotometer and the scavenging rate was calculated.

[0038] (2) In vitro anti-inflammatory experiment An inflammation model was established in RAW264.7 macrophages by inducing them with 100 ng / mL LPS, followed by co-culturing with hydrogel extract for 24 hours. Total RNA was extracted from the cells using the TRIzol method and reverse transcribed. The mRNA expression levels of pro-inflammatory factors such as IL-1β, IL-6, and TNF-α were detected by real-time quantitative PCR (qPCR). GADPH was used as an internal control, and the primer sequences are as follows: GADPH-F: 5'-TGTGTCCGTCGTGGATCTGA-3'; GADPH-R: 5'-TTGCTGTTGAAGTCGCAGGAG-3'; IL-1β-F: 5'-TGGTGTGTGACGTTCCCATT-3'; IL-1β-R: 5'-TGTCGTTGCTTGGTTCTCCT-3'; IL-6-F: 5'-CGGCCTTCCCTACTTCACAA-3'; IL-6-R: 5'-GCAAGTGCATCATCGTTGTTC-3'; TNF-α-F: 5'-ACTCCAGGCGGTGCCTATGT-3'; TNF-α-R: 5'-GTGAG GGTCTGGGCCATAGAA-3'.

[0039] 4. Evaluation of the effects of hydrogels on neuronal differentiation and axonal growth Undifferentiated PC12 cells were cultured in NGF-containing induction medium for 24 hours, then replaced with hydrogel extraction medium. Calcein staining was performed on days 4 and 6, and neurite growth was observed using laser confocal microscopy. Axon number and length were counted using ImageJ software.

[0040] 5. Therapeutic effect of hydrogel in rat sciatic nerve injury model (PNI) (1) Model establishment and drug administration Adult SD rats (weighing 200-250 grams) were anesthetized via intraperitoneal injection of sodium pentobarbital solution. The rats were immobilized in a prone position, and the hair on the back of the right hind limb was clipped and the area disinfected. The skin was incised along the femur, and the muscles were bluntly dissected to expose the right sciatic nerve. A constant pressure was applied to the sciatic nerve notch using non-toothed hemostatic forceps for 2 minutes. A translucent, flat lesion approximately 2 mm wide was observed at the compression site, without nerve rupture, confirming successful model establishment. Subsequently, 50 μL of conductive hydrogel or a control sample was injected subependymally at the nerve injury site using a micropipette. After the material gelled, the muscles and skin were sutured layer by layer, and the rat was placed on a heating pad until it awoke.

[0041] (2) Functional assessment A. Gait Analysis and Sciatic Nerve Function Index (SFI) Calculation: Gait testing was performed on day 28 post-surgery. Black ink was applied to the hind paws of rats, and they were allowed to autonomously traverse a 50 cm long, darkened walking tunnel with white paper laid on the bottom for footprint recording. Samples were selected from continuous and clear footprints, and the following measurements were recorded: experimental side footprint length (EPL), normal side footprint length (NPL); experimental side toe spread width (ETS), normal side toe spread width (NTS); experimental side inter-toe distance (EIT), normal side inter-toe distance (NIT). SFI values ​​were calculated using the Bain formula, with approximately 0 points representing normal function and approximately -100 points representing complete functional loss.

[0042] B. Assessment of Hyperalgesia: The weight-bearing difference between the two hind limbs was measured using a bipedal balance analgesia meter. The weight distribution between the affected (right) and normal (left) hind limbs was recorded when the rat was standing at rest, and the bilateral weight-bearing difference was calculated. The smaller the difference, the lower the animal's pain sensitivity to mechanical stimuli, indicating better recovery of sensory function.

[0043] (3) Electrophysiological assessment Electrophysiological monitoring was performed 4 weeks post-surgery. Rats were anesthetized and the bilateral sciatic nerves were re-exposed. Compound muscle action potentials (CMAPs) were recorded using an electrochemical workstation. The stimulating electrode was placed proximal to the lesion, the recording electrode was placed on the gastrocnemius muscle belly, and the grounding electrode was placed on the tail. Constant pulse stimulation was administered, and the peak amplitude and latency of the CMAPs were recorded and analyzed. The ratio of the experimental to the control side was calculated to assess the transduction capacity of the damaged nerve for motor commands and the quality of axonal regeneration.

[0044] (4) Histological and muscle evaluation Evaluation of target muscle wet weight and fibrosis At the end of the experiment, the bilateral gastrocnemius and soleus muscles were completely dissected and weighed immediately. The weight ratio of the affected side to the healthy side was calculated to assess the degree of muscle atrophy caused by denervation. Subsequently, Masson staining was performed on the muscle tissue to observe collagen deposition between muscle fibers, and the degree of muscle fibrosis was assessed by calculating the proportion of collagen fiber area.

[0045] B. Neurological Histological Analysis Damaged nerve tissue was collected, fixed in 4% paraformaldehyde, dehydrated, and embedded to prepare transverse and longitudinal sections. HE staining was performed, and the integrity of nerve bundles, axonal alignment, and inflammatory cell infiltration were observed under a microscope to evaluate the promoting effect of hydrogel materials on nerve structure reconstruction.

[0046] 6. Therapeutic effects of hydrogels in a mouse model of spinal cord injury (SCI) (1) Establishment of spinal cord hemisection model and drug treatment Adult Kunming mice (weighing 25-30 grams) were used in the experiment. After general anesthesia via intraperitoneal injection of sodium pentobarbital solution, the mice were fixed on the operating table, their backs were shaved and disinfected. A midline incision was made centered on the tenth thoracic vertebra (T10), and the muscles were dissected layer by layer to expose the spine. A laminectomy was carefully performed using bone forceps to expose the T10 segment of the spinal cord. A transverse hemisection was performed on the left side of the spinal cord using a microscalpel, reaching the midline. After confirming the injury, 10 μL of conductive hydrogel or a control sample was immediately injected in situ at the spinal cord defect. After the material gelled, the muscles and skin were sutured layer by layer. Postoperatively, the mice were assisted with artificial urination daily until their spontaneous urination function was restored.

[0047] (2) Detection of the pathological microenvironment in the acute phase A. Detection of reactive oxygen species (ROS) levels On the third day after surgery, spinal cord tissue from the injured segment and adjacent areas of mice in each group was collected and frozen sectioned. The sections were incubated with a dihydroethidium (DHE) fluorescent probe, and the intensity of red fluorescence in the spinal cord tissue of each group was observed by fluorescence microscopy to quantitatively assess the ability of the hydrogel to clear superoxide anions from the injured area.

[0048] B. Quantitative analysis of inflammatory factors Total RNA was extracted from the injured spinal cord segment and purified using the TRIzol method. The mRNA expression levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were detected using real-time quantitative PCR (qPCR). The fold change of each factor relative to the internal reference gene was calculated to evaluate the regulatory effect of the hydrogel on the inflammatory microenvironment during the acute phase of injury.

[0049] (3) Assessment of motor function recovery A behavioral score From week 1 to week 6 post-surgery, mice were assessed for motor function using the Basso Mouse Scale (BMS) at fixed times each week. Hock joint movement, foot placement, gait coordination, and tail position were observed in an open field and recorded using a scoring system from 0 (complete paralysis) to 9 (complete normal).

[0050] B. Inclined Plate Experiment The inclined plane test was performed weekly to assess the grip strength and balance of the mice's hind limbs. The mice were placed on a rough inclined plane with an adjustable angle, and the maximum tilt angle that the mice could withstand while maintaining their balance and not slipping was recorded.

[0051] C. Electrophysiological assessment At week 6 post-surgery, the continuity of neural conduction pathways was assessed using motor evoked potentials (MEP). Stimulating electrodes were placed in the motor cortex of the brain, while recording electrodes were placed in the belly of the tibialis anterior muscle in the contralateral hind limb. The peak amplitude of the electrical signals was recorded to evaluate the efficiency of regenerated axons in traversing the lesion area and transmitting nerve impulses.

[0052] (4) Histological evaluation and molecular mechanism analysis A. Spinal cord structure and scar characteristics At the end of the experiment (6 weeks post-operation), spinal cord tissue was collected, fixed in 4% paraformaldehyde, and embedded in paraffin. Longitudinal sections of the spinal cord were prepared and stained with hematoxylin and eosin (HE) to observe the area of ​​the spinal cord injury cavity and the continuity of the tissue; Masson staining was performed to observe the deposition of collagen fibers in the injured area and to assess the extent of collagen scar formation.

[0053] B. Expression of genes related to neural regeneration The expression of key genes in spinal cord tissue was quantitatively detected using qPCR. The detection indicators included: neuronal and axonal markers (NF200, Tuj-1), synaptic-associated protein (Syn-1), myelin-associated protein (MBP), and astrocyte activation markers (GFAP). By analyzing the expression differences of these genes, the molecular-level effects of conductive hydrogel on axonal regeneration, remyelination, and inhibition of glial scar formation were revealed. GAPDH was used as an internal reference gene, and the primer sequences are as follows: GAPDH F: 5'-CCTCGTCCCGTAGACAAAATG-3'; GAPDH R: 5'-TGAGGTCAATGAAGGGGTCGT-3'; GFAP F: 5'-ACCCGTTCCTCCATAAAGGC-3'; GFAP R: 5'-CCAGTTGTCGACTAGGACCG-3'; NF200 F: 5'-GCAGACATTGCCTCCTACCA-3'; NF200 R: 5'-GAGAAGGGACTCGGACCAAA-3'; MBP F: 5'-GGACAGTGATGTGTTTGGGGAG-3'; MBP R: 5'-TCTCGGGAAAAGAGGCGGA-3'; Syn-1 F: 5'-ACCTCGGTGGTGTTTGGCTT-3'; Syn-1 R: 5'-TGCCCGTAATCGGGTTGA-3'; Tuj-1 F: 5'-CGATGAGCACGGCATAGACC-3'; Tuj-1 R: 5'-CAGCACCACTCTGACCAAAGATAA-3'.

[0054] (5) Statistical analysis All experimental data in this invention are expressed as mean ± standard deviation (mean ± SD). Statistical significance was determined using one-way ANOVA combined with Tukey's post-hoc test. A p-value < 0.05 was considered statistically significant.

[0055] II. Research Results 1. Physicochemical properties and conductive activity of hydrogels like Figure 1 As shown, Figure 1 A demonstrates the hydrogel formation process, confirming through a vial inversion experiment that ellagic acid, 5-formyl-2-furanboronic acid, carboxymethyl chitosan, and PEDOT:PSS solution can rapidly form a gel within 60 seconds after mixing. Figure 1 B and 1D show that the hydrogel has good injectability and self-healing properties. Figure 1 Scanning electron microscopy (SEM) images of C show that the hydrogel has a typical three-dimensional porous network structure, which is conducive to material exchange and cell migration. Figure 1 E and 1F indicate that as the concentration of the conductive component PEDOT:PSS increases, the impedance of the hydrogel decreases significantly, demonstrating good conductivity. Figure 1 The isolated spinal cord-LED series circuit model constructed by G shows that after implanting conductive hydrogel in the spinal cord defect gap, the LED light was lit again, proving that the hydrogel can establish a stable electrical connection at the interface of moist physiological tissue and reconstruct the bioelectrical conduction pathway.

[0056] 2. Rheological properties of hydrogels The results are as follows Figure 2 As shown. Figure 2 A is the strain scan curve, which shows that the storage modulus (G') is higher than the loss modulus (G") in the low strain range, and a gel-sol transition occurs when the strain exceeds a certain threshold. Figure 2 B is the frequency scan curve, which shows that G' and G" exhibit a weak frequency dependence in the range of 0.1 to 100 rad / s, confirming the stable three-dimensional cross-linked network. Figure 2 C shows that as the shear rate increases, the viscosity of the hydrogel decreases by orders of magnitude, exhibiting significant shear-thinning behavior and giving it excellent injection properties. Figure 2The high and low strain alternating cycle test of D showed that the modulus of the hydrogel could be rapidly restored after the high strain was removed, demonstrating its excellent self-healing ability based on dynamic covalent bonds.

[0057] 3. Biocompatibility, swelling and degradation properties of hydrogels The results are as follows Figure 3 As shown. Figure 3 The hemolysis test results of A showed that the hemolysis rate of the hydrogel group was far below the 5% biosafety threshold. Figure 3 MTT assays and live / dead staining assays (B and 3C) confirmed that rat Schwann cells (RSC) and mouse macrophages (RAW264.7) maintained high survival rates after 72 hours of co-culturing in hydrogel extract, demonstrating good cell compatibility. Figure 3 D and Figure 3 E demonstrates the excellent swelling properties of the hydrogel and its slow and controllable in vitro degradation mode. Figure 3 F shows that ultrasound monitoring images recorded a gradual decrease in the volume of the hydrogel within 35 days after subcutaneous implantation in rats; Figure 3 Hematological and biochemical analyses following the G and 3H experiments showed that liver and kidney function indicators were within the normal physiological range. Figure I shows that HE-stained sections of major organs (heart, liver, spleen, lung, and kidney) and the skin at the implantation site showed no pathological changes, confirming the excellent in vivo safety of the material.

[0058] 4. In vitro antioxidant and anti-inflammatory activities of hydrogels The results are as follows Figure 4 As shown. Figure 4 A shows that the hydrogel exhibits significant scavenging effects on DPPH, ABTS, PTIO free radicals, and hydrogen peroxide (H2O2), confirming its broad-spectrum reactive oxygen species (ROS) scavenging ability. Figure 4 The qPCR results of B showed that in the LPS-induced macrophage inflammation model, hydrogel treatment significantly reduced the mRNA expression levels of pro-inflammatory factors IL-1β, IL-6 and TNF-α, demonstrating that the ellagic acid component endowed the material with significant anti-inflammatory activity.

[0059] 5. Hydrogels promote neuronal differentiation and axonal growth. The results are as follows Figure 5 As shown. Figure 5 A shows the differentiation morphology of PC12 cells in hydrogel culture, with obvious neural synaptic extensions visible. Figure 5 Quantitative analysis of B showed that, compared with the control group, the number and average length of neurites in the hydrogel group were significantly increased, proving that the electroactive microenvironment constructed by the material is beneficial to promoting the occurrence and growth of neuronal axons.

[0060] 6. The repair effect of hydrogel on sciatic nerve injury (PNI) The results are as follows Figure 6 As shown. Figure 6 Figures A and 6B show that on postoperative day 28, the conductive hydrogel group showed the most significant gait recovery, with its sciatic nerve function index (SFI) score being significantly better than that of other treatment groups. Figure 6 The results of the bipedal balance test in group C showed that the weight-bearing difference in the hind limbs of the rats in this group was significantly reduced, which effectively alleviated the pain hypersensitivity induced by nerve damage. Figure 6 Electrophysiological assessment of D showed that the composite muscle action potential (CMAP) amplitude was highest in the conductive hydrogel group at week 4 post-surgery, indicating good recovery of nerve conduction function. Figure 6 E and 6F showed that the gastrocnemius muscle atrophy was reduced and the wet weight ratio was increased in this group. Figure 6 G showed a significant reduction in muscle fibrosis after treatment with conductive hydrogel. Figure 6 HE staining of H nerve tissue showed that the nerve bundles were neatly arranged and the inflammatory infiltration was reduced after treatment with conductive hydrogel.

[0061] 7. The repair effect of hydrogels on spinal cord injury (SCI) The results are as follows Figure 7 As shown. Figure 7 As shown in Figures A through D, on postoperative day 3, the conductive hydrogel group significantly reduced the level of reactive oxygen species (ROS) in the spinal cord injury site and downregulated the expression of inflammatory factors IL-1β, IL-6, and TNF-α, effectively improving the early injury microenvironment. Figure 7 E showed that during the 6-week observation period after surgery, the BMS scores of the mice in this group steadily recovered. Figure 7 F shows that the gait recovery was most significant in the conductive hydrogel group. Figure 7 G shows that the angle of the inclined plane test steadily increased after treatment with conductive hydrogel, and the motor coordination was significantly improved. Figure 7 Motor evoked potential (MEP) assays at H showed that the conductive hydrogel group recorded electrical signals with increased amplitude, demonstrating the effective restoration of nerve impulse conduction across the injury area. Figure 7 Histological analysis of group I showed that the volume of the cavity in the spinal cord injury center was reduced, collagen fiber scar formation was suppressed, and tissue continuity was well maintained.

[0062] 8. Molecular-level evaluation of hydrogel therapy for spinal cord injury The results are as follows Figure 8 As shown. Figure 8The results of qPCR analysis of spinal cord tissue at week 6 post-surgery are presented. The mRNA expression levels of neural regeneration-related genes (NF200, Tuj-1, S100, Syn) and remyelination-related genes (MBP) were significantly upregulated in the conductive hydrogel group, while the expression of astrocyte activation marker (GFAP) was significantly suppressed. These results confirm that this material promotes axonal extension and functional regeneration at the molecular level by synergistically mimicking the bioelectrical microenvironment and regulating the metabolic microenvironment.

[0063] III. Conclusion This invention's hydrogel achieves a deep integration of physical properties and biological functions through the synergistic effect of borate ester bonds and Schiff base bonds. Its excellent injectability and self-healing ability provide a reliable mechanical basis for adapting to complex nerve injury cavities. Experimental results show that this hydrogel system can achieve spatiotemporal synergistic nerve repair through a staged intervention mechanism: in the early stage of injury, ellagic acid efficiently scavenges reactive oxygen species and regulates the inflammatory response, effectively optimizing the metabolic microenvironment of the damaged area; during the repair period, a stable conductive network reconstructs bioelectrical signal transduction pathways, significantly promoting axonal regeneration and remyelination. In vivo experiments in sciatic nerve injury and spinal cord injury models showed significant recovery of motor and sensory functions, confirming the material's significant efficacy in reducing muscle atrophy and promoting tissue reconstruction.

[0064] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A small-molecule conductive hydrogel made from traditional Chinese medicine, characterized in that, The hydrogel is composed of carboxymethyl chitosan (CMCh), ellagic acid (EA), 5-formyl-2-furanboronic acid (FFBA), and conductive polymer PEDOT:PSS. In the hydrogel, ellagic acid and 5-formyl-2-furanboronic acid form borate ester bonds, and carboxymethyl chitosan and 5-formyl-2-furanboronic acid form Schiff base bonds, constructing a dual dynamic crosslinking network.

2. The hydrogel as described in claim 1, characterized in that, The concentrations of each component in the resulting hydrogel are as follows: carboxymethyl chitosan 2~10.0% (w / v), ellagic acid 0.5~5.0% (w / v), 5-formyl-2-furanboronic acid 0.5~5.0% (w / v), and PEDOT:PSS 0.1~1.0% (w / v).

3. The method for preparing the hydrogel according to claim 1, characterized in that, The method includes the following steps: (1) Dissolve 5-formyl-2-furanboronic acid in PBS buffer, add alkaline solution to adjust the pH value, add ellagic acid at room temperature and react for 1 to 4 hours to obtain ellagic acid-5-formyl-2-furanboronic acid precursor solution; wherein the concentration range of ellagic acid is 1 to 10.0% (w / v) and the concentration range of 5-formyl-2-furanboronic acid is 1 to 10.0% (w / v). (2) Prepare carboxymethyl chitosan aqueous solution and PEDOT:PSS aqueous solution; wherein the concentration range of carboxymethyl chitosan is 8~40.0% (w / v) and the concentration range of PEDOT:PSS is 0.4~4% (w / v). (3) The ellagic acid-5-formyl-2-furanboronic acid precursor solution, carboxymethyl chitosan aqueous solution and PEDOT:PSS aqueous solution are mixed and stirred to obtain a small molecule conductive hydrogel of traditional Chinese medicine; wherein, the concentration of each component in the hydrogel is: carboxymethyl chitosan 2~10.0% (w / v), ellagic acid 0.5~5.0% (w / v), 5-formyl-2-furanboronic acid 0.5~5.0% (w / v), PEDOT:PSS 0.1~1.0% (w / v).

4. The preparation method according to claim 3, characterized in that, In step (1), the pH value of the ellagic acid-5-formyl-2-furanboronic acid precursor solution is 7.0~7.

4.

5. The preparation method according to claim 3, characterized in that, In step (3), the volume ratio of the carboxymethyl chitosan aqueous solution, the PEDOT:PSS aqueous solution and the ellagic acid-5-formyl-2-furanboronic acid precursor solution is (1~2):(1~2):(1~4).

6. The application of the small molecule conductive hydrogel of traditional Chinese medicine as described in claim 1 or 2 in the preparation of nerve injury repair materials.

7. The application as described in claim 6, characterized in that, The nerve injuries include peripheral nerve injuries and spinal cord injuries.

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