Astragaloside-loaded conductive hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to astragaloside-loaded conductive hydrogel as well as a preparation method and application thereof. The preparation method comprises the following steps: uniformly mixing a methylacrylic anhydride gelatin aqueous solution with an organic solvent solution of astragaloside, and carrying out ultraviolet light cross-linking curing to form methylacrylic anhydride gelatin / astragaloside hydrogel; soaking the obtained hydrogel in hydrochloric acid containing a free radical initiator; then soaking and polymerizing in an aniline ethanol solution; and soaking and purifying to obtain the methylacrylic anhydride gelatin-polyaniline conductive hydrogel loaded with astragaloside. In-vitro experiments prove that the compound has a certain anti-inflammatory effect, can induce neural stem cells to be directionally differentiated into neurons and oligodendrocytes, and is beneficial to promoting nerve regeneration and myelin sheath formation; finally, in-vivo experiments prove that the composition can improve the athletic ability and tissue function recovery of SCI rats, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to a conductive hydrogel loaded with astragaloside and a preparation method and application thereof. BACKGROUND

[0002] Spinal cord injury (SCI) is a severe and incurable central nervous system disease that can cause partial or complete sensory and motor dysfunction below the injury plane, and is closely related to long-term disability, shortened life span, decreased quality of life, and increased economic burden. After SCI, a series of complex pathological processes occur, such as apoptosis, oxidative stress, axonal demyelination, and glial scar evolution. Currently, the treatment of SCI in the clinic is mainly based on drugs and surgery. Many macromolecular drugs are difficult to cross the blood-spinal cord barrier, and if they are to achieve a therapeutic dose at the injury site, they need to be taken in large amounts, which can easily cause systemic toxicity, lead to harmful off-target effects, and also cause complications such as pneumonia, urinary tract infection, and gastrointestinal bleeding. Surgical treatment cannot achieve axonal regeneration and connection, and cannot restore damaged nerve function.

[0003] With the rapid development of tissue engineering technology and material science, the use of biomaterials to load drugs to achieve sustained release and remodeling of the microenvironment of SCI can well solve the above problems. The development of hydrogels, especially conductive hydrogels, provides more possibilities for the treatment of SCI. They not only can be used as carriers for cell growth and adhesion, but also can deliver drugs to the local injured spinal cord in sufficient dose and duration, while avoiding harmful side effects associated with systemic administration. More importantly, the electrical conductivity of conductive hydrogels matches the physiological characteristics of neural tissue, which is conducive to the exchange of electrical signals between cells and the conduction of nerve impulses. Astragaloside (AS) is the main monomer active component of Chinese medicine Astragalus, which has been proven to have a wide range of pharmacological effects, including anti-inflammatory, anti-oxidative stress, neuroprotection, promotion of myelin regeneration, and immunomodulation, which are consistent with some pathological processes involved in SCI. However, there are few studies on its use in SCI, and currently there is no related research on the combination of conductive hydrogels to synergistically promote SCI repair. SUMMARY

[0004] In order to solve the problems in the prior art, the present application firstly constructs a gelatin methacryloyl-polyaniline@astragaloside (GMP@AS) conductive hydrogel loaded with astragaloside by an interface in-situ polymerization method; secondly, it is proved by in-vitro experiments that the conductive hydrogel has certain anti-inflammatory effect and can induce neural stem cells to differentiate into neurons and oligodendrocytes, which is beneficial to promoting nerve regeneration and myelination; finally, in-vivo experiments prove that the conductive hydrogel can improve the motor ability and tissue function recovery of SCI rats and has good application prospect.

[0005] In order to achieve the above-mentioned object, the present application provides the following technical solutions: The application provides a preparation method of a gelatin methacryloyl-polyaniline conductive hydrogel loaded with astragaloside, which comprises the following steps: (1) uniformly mixing a gelatin methacryloyl aqueous solution and an organic solvent solution of astragaloside to obtain a pre-polymerization liquid, then uniformly mixing the pre-polymerization liquid with a photoinitiator, and using ultraviolet light to cross-link and solidify the pre-polymerization liquid to form a gelatin methacryloyl / astragaloside hydrogel, and soaking the gelatin methacryloyl / astragaloside hydrogel in deionized water to remove ions that have not been gelled; (2) soaking the gelatin methacryloyl / astragaloside hydrogel in hydrochloric acid (HCL) containing a free radical initiator, then soaking the gelatin methacryloyl / astragaloside hydrogel in an aniline ethanol solution to polymerize, and sequentially soaking the obtained hydrogel in ultrapure water and an HCl solution to purify the hydrogel, so as to obtain a gelatin methacryloyl-polyaniline conductive hydrogel loaded with astragaloside (GMP@AS).

[0006] Preferably, in step (1), the grafting rate of the gelatin methacryloyl is 55-65%.

[0007] Preferably, in step (1), the concentration of the gelatin methacryloyl aqueous solution is 10-15%; the volume ratio of the gelatin methacryloyl astragaloside solution to the organic solvent solution of astragaloside is 1:0.0025-1:0.025; and more preferably, the volume ratio of the gelatin methacryloyl astragaloside solution to the organic solvent solution of astragaloside is 1:0.015.

[0008] Preferably, in step (1), the organic solvent is DMSO.

[0009] Preferably, in step (1), the mass ratio of the gelatin methacryloyl to the photoinitiator is (9-10):(1-1.2).

[0010] Preferably, in step (1), the volume ratio of the organic solvent solution of astragaloside IV to the prepolymer solution is (0.0025-0.025):1.

[0011] Preferably, in step (2), the free radical initiator is 0.05-0.1 M ammonium persulfate.

[0012] Preferably, in step (2), the soaking is performed in 0.8-1 M hydrochloric acid for 2-4 hours.

[0013] Preferably, in step (2), the soaking is performed in 0.1-0.2 M aniline ethanol solution for 4-6 hours.

[0014] Preferably, in step (2), the hydrogel is soaked in ultrapure water for 2-3 days and in 0.8-1 M HCl solution for 1 day.

[0015] The astragaloside IV-loaded methacrylic anhydride gelatin-polyaniline conductive hydrogel prepared by the above preparation method.

[0016] Preferably, in the astragaloside IV-loaded methacrylic anhydride gelatin-polyaniline conductive hydrogel, the concentration of astragaloside IV is 12.5 µg / ml-125 µg / ml; further preferably, the concentration of astragaloside IV is 75 µg / ml.

[0017] The astragaloside IV-loaded methacrylic anhydride gelatin-polyaniline conductive hydrogel is used for preparing a spinal cord injury repair material.

[0018] Compared with the prior art, the present application has the following beneficial effects: The astragaloside IV-loaded conductive hydrogel has good biocompatibility and drug release performance, and the porous three-dimensional structure is conducive to the growth of neural cells. In vitro, it can induce primary neural stem cells to differentiate into neurons and oligodendrocytes, and promote the transformation of BV2 cells from pro-inflammatory M1 type to anti-inflammatory M2 type. In vivo experiments show that the astragaloside IV-loaded conductive hydrogel can significantly reduce the volume of the cyst cavity at the lesion site, effectively reduce scar formation, and promote myelin formation and neuron connection, thereby restoring interrupted nerve function and improving rat motor function, and has good application prospects. Compared with the prior art, it has the effects of anti-inflammatory, promoting nerve regeneration, promoting myelin formation and improving motor function. Compared with the clinical treatment, the astragaloside IV-loaded conductive hydrogel developed in the present application can avoid repeated drug use after one-time implantation, and has better nerve repair effect. BRIEF DESCRIPTION OF DRAWINGS

[0019] The drawings accompanying the specification of this application form a part thereof, serve to provide further understanding of the application, and together with the description of the exemplary embodiments of the application and explanations thereof, make an explanation of the application, and do not constitute an improper limitation of the application. In the drawings: Figure 1 Chemical structure of methyl methacrylate gelatin, polyaniline, astragaloside IV.

[0020] Figure 2 Figure 1 is a picture of the appearance and internal morphology of GMP conductive hydrogel; A) picture of the appearance of GMP conductive hydrogel; B) picture of the internal morphology of GMP conductive hydrogel observed by scanning electron microscopy.

[0021] Figure 3 Figure 3 is a picture of the proliferation activity of cells loaded with different concentrations of astragaloside IV in conductive hydrogel.

[0022] Figure 4 Figure 4 is a picture of the cumulative release of astragaloside IV of GMP@AS at different time points.

[0023] Figure 5 Figure 5 is a picture of the in vitro anti-inflammatory effect of GMP@AS conductive hydrogel; A) immunofluorescence picture of Arg-1 after co-culturing with BV-2 cells in each group; B) statistical chart of fluorescence intensity of Arg-1; C) immunofluorescence picture of iNOS after co-culturing with BV-2 cells in each group; D) statistical chart of fluorescence intensity of iNOS.

[0024] Figure 6 Figure 6 is a picture of the directional induction of neural stem cell differentiation by GMP@AS conductive hydrogel. A) fluorescence staining chart of neuron marker Tuj-1 in each group; B) statistical analysis of the percentage of Tuj-1+ cells in the total cell number in each group; C) fluorescence staining chart of oligodendrocyte marker MBP in each group; D) statistical analysis of the percentage of MBP+ cells in the total cell number in each group.

[0025] Figure 7 Figure 7 is a picture that GMP@AS conductive hydrogel can promote the recovery of motor function of SCI rats. A) morphology of hind limbs of rats in each group at 6 weeks after operation; B) BBB score of motor function of rats in each group at 6 weeks after operation; C) footprint test chart of rats in each group at 6 weeks after operation (the footprints of forelimbs are represented by blue color and the footprints of hind limbs are represented by red color).

[0026] Figure 8 Figure 8 is a picture that GMP@AS conductive hydrogel can reduce the volume of cyst cavity in the injury area. A) HE staining chart of horizontal section of injury area in each group at 6 weeks after operation; B) statistical chart of cyst cavity area.

[0027] Figure 9 Figure 9 is a picture of the promoting effect of GMP@AS conductive hydrogel on the regeneration of neurons of SCI rats. A) NeuN immunofluorescence chart of spinal cord tissue in each group; B) statistical chart of the number of neurons in each group.

[0028] Figure 10 Figure 10 is a chart of LFB staining of spinal cord tissue in each group. DETAILED DESCRIPTION

[0029] The advantages and features of the present application will become more apparent with the following detailed description of specific embodiments. The embodiments are exemplary only, and do not limit the scope of the present application. Those skilled in the art will understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present application without departing from the spirit and scope of the present application, and such modifications and substitutions fall within the scope of the present application.

[0030] Example 1 The preparation method of the methacrylated gelatin-polyaniline (GMP@AS) conductive hydrogel loaded with different concentrations of astragaloside A includes the following steps: First, 500 mg of methacrylated gelatin (chemical structural formula as shown in Figure 1 , with a grafting rate of 55%) freeze-dried powder was added to 4 ml of phosphate buffer solution (PBS), and the solution was completely dissolved in a 70°C water bath to prepare a 12.5% methacrylated gelatin solution (GM solution).

[0031] Secondly, prepare the astragaloside A (AS) solution. Weigh 5 mg of astragaloside A powder and dissolve it in 1 ml of dimethyl sulfoxide (DMSO) solvent to prepare a 5 mg / ml astragaloside A solution stock solution. Then, add the AS solution to the GM solution at a volume ratio of GM@AS solution:AS solution of 1:0.0025, stir uniformly, and eliminate bubbles to prepare a methacrylated gelatin@astragaloside A solution (GM@AS solution) with an AS concentration of 12.5 µg / ml.

[0032] Finally, mix the GM@AS solution and the photoinitiator at a dose of 9:1. Use 405 nm wavelength ultraviolet light to irradiate the above solution to crosslink and solidify to form a GM@AS hydrogel. Soak it in deionized water for 1 day to remove unglued ions. Then, soak it in a 1M hydrochloric acid (HCl) solution containing 0.08M ammonium persulfate (APS) for 4 hours. Next, soak it in a 0.16M aniline ethanol solution for 4 hours, and polymerize it into a GMP@AS hydrogel with an AS concentration of 12.5 µg / ml. Finally, sequentially soak it in ultrapure water and 1M HCl solution for 2 days and 1 day, respectively, to remove unglued ions. Scanning electron microscopy is used to observe the morphology of the freeze-dried GMP@AS conductive hydrogel.

[0033] Figure 2 It is shown that the prepared conductive hydrogel is doped with polyaniline (polyaniline structural formula as shown in Figure 1(As shown) After polymerization, it is a dark green semi-solid with a loose, porous internal microstructure, with pore sizes mostly around 100µm. This three-dimensional network structure creates a large specific surface area, which can serve as a drug carrier and facilitates cell adhesion and penetration as well as rapid electron transport, laying the structural foundation for the subsequent climbing of nerve cells after implantation.

[0034] Examples 2-6 The difference from Example 1 is that the volume ratio of GM@AS solution to AS mother liquor is 1:0.005 (Example 2), 1:0.01 (Example 3), 1:0.015 (Example 4), 1:0.02 (Example 5), and 1:0.025 (Example 6), respectively; the AS concentration in the GM@AS solution is 25µg / ml (Example 2), 50µg / ml (Example 3), 75µg / ml (Example 4), 100µg / ml (Example 5), and 125µg / ml (Example 6), respectively; and correspondingly, the AS concentration in the GMP@AS conductive hydrogel is 25µg / ml (Example 2), 50µg / ml (Example 3), 75µg / ml (Example 4), 100µg / ml (Example 5), and 125µg / ml (Example 6), respectively.

[0035] The optimal loading concentration of astragaloside IV-loaded GMP conductive hydrogels: The conductive hydrogels loaded with different concentrations of astragaloside A obtained in Examples 1-6 were co-cultured with PC12 cells for 48 hours. Then, the proliferation ability of the conductive hydrogels loaded with different concentrations of astragaloside A was determined by the CCK-8 kit, so as to evaluate the optimal loading concentration of astragaloside A on the GMP conductive hydrogel for subsequent experiments.

[0036] Figure 3 The results showed that after 48 hours of culture, the astragaloside A concentration of 75 µg / mL (Example 4) had the strongest promoting effect on cell proliferation, at 87.7 ± 2.5%. The conductive hydrogel exhibited good in vitro biocompatibility at this drug concentration. 75 µg / mL is likely the optimal loading concentration for GMP@AS to exert its effective effect.

[0037] Sustained-release properties of conductive hydrogel for astragaloside A: Dynamic release experiment was used to evaluate the in vitro drug release property of GMP@AS. 1 g of GMP@AS prepared in Example 4 was placed in 50 mL of PBS solution with pH = 7.4 and incubated at 37°C with constant temperature oscillation. During the experiment, samples were taken every 24 hours and the supernatant was collected. Then, the release amount of astragaloside was determined by ultraviolet spectrophotometry: first, the characteristic absorption peak of astragaloside was determined, and then a standard solution with gradient concentration (5-50 μg / mL) was prepared to establish a linear regression equation between standard concentration and absorbance value. The absorbance value of the sample to be tested was determined at the same wavelength, and the astragaloside concentration was calculated by substituting the standard curve equation. Finally, the drug release rate was determined by the ratio of the cumulative release amount to the initial drug loading amount.

[0038] Figure 4 It is shown that GMP@AS presents a fast-slow trend in the release of astragaloside, and half (50.3 ± 4.6%) of the drug is released in the first 4 days; more than 85% of AS is released on the 12th day. It is illustrated that GMP@AS has drug release performance and prolongs the action time of the drug.

[0039] GMP@AS conductive hydrogel has in vitro anti-inflammatory effect: To evaluate the in vitro anti-inflammatory effect of GMP@AS, BV-2 cells were first stimulated with 1 μg / ml lipopolysaccharide (LPS) for 24 hours to polarize them to M1 type and release inflammatory factors, and a cell inflammation model was established. Then, different interventions were applied to the induced BV-2 cells. After 24 hours, the effects of different treatment groups on the polarization of BV-2 cells to M1 / M2 phenotype were observed by cell immunofluorescence staining. The steps of immunofluorescence staining are as follows: the tissue or cells are fixed in 4% paraformaldehyde for 30 minutes and washed with PBS. Then, the samples are soaked in PBS containing 1% and 0.3% Triton-100X for 10-20 min. 5% horse serum albumin is used for blocking for 1 hour. After washing, the corresponding first antibody is incubated overnight at 4°C. Then, the corresponding second antibody is incubated at room temperature for 1 hour. DAPI is used to stain the cell nucleus, and the slice is sealed. Finally, the fluorescence image is obtained by confocal fluorescence microscope, and analyzed by ImageJ software.

[0040] The results are shown in Figure 5 The control group is a blank control group without LPS induction. Microglial cells with specific markers can be divided into pro-inflammatory M1 type and anti-inflammatory M2 type. Arg-1 is used to label M2 type cells (results are shown in Figure 5 A, 5B), and iNOS is used to label M1 type cells (results are shown in Figure 5C, 5D). After LPS treatment, the LPS group showed low expression of Arg-1 and high expression of iNOS, indicating that LPS induced BV-2 cells to polarize to the pro-inflammatory M1 type. After GMP@AS intervention treatment, the fluorescence intensity of Arg-1 was significantly increased, and the fluorescence intensity of iNOS was significantly decreased (P<0.001), indicating that GMP@AS can induce microglial cells to polarize to M2 type and play an anti-inflammatory role.

[0041] GMP@AS conductive hydrogel can induce neural stem cells to differentiate into neurons and oligodendrocytes: Neural stem cells were extracted from the cerebral cortex of E17-18 rat embryos and digested into single cells using Accutase enzyme. The cells were seeded in DMEM / F12 growth medium containing 2% B27 neuronal supplement, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and 1% penicillin / streptomycin. When the diameter of the neural sphere exceeded 200 µm, it was passaged. When studying the differentiation of neural stem cells, the culture medium was removed EGF and bFGF growth factors. The cells were changed every 2-3 days. The above cells were cultured in a 37°C incubator with 5% CO2. After 5 days, Tuj-1 primary antibody was used to label and stain neurons (results as shown in Figure 6 A, 6B), and MBP was used to label and stain oligodendrocytes (results as shown in Figure 6 C, 6D). Methylprednisolone (MP) is a commonly used drug for the clinical treatment of SCI and is used as a positive control.

[0042] The differentiation of neural stem cells into neurons and oligodendrocytes helps to regenerate neural tissue and myelin. Figure 6 It was shown that the growth of neural stem cells on GMP@AS conductive hydrogel was easy to differentiate into neurons, with a significant difference (P<0.001), and the proportion of Tuj-1+ cells was 47.6±4.5%. In addition, compared with the Control and MP groups, the proportion of neural stem cells differentiated into oligodendrocytes was higher in the GMP@AS environment, with a significant difference compared with the other two groups. The above results showed that GMP@AS has the effect of directional induction of neural stem cell differentiation, which may be one of the reasons for its neuroprotective effect.

[0043] Construction of rat SCI model: SD rats were randomly divided into 4 groups: sham operation group (Sham) group, spinal cord injury (SCI) group (n=12), positive drug methylprednisolone (MP) group and methyl methacrylate gelatin-polyaniline conductive hydrogel loaded with astragaloside (GMP@AS) group. The SCI model of SD rats was established. The rats were anesthetized with 2% sodium pentobarbital, fixed in a prone position on the operating table, and the back skin was exposed. A 3-4 cm longitudinal incision was made along the spinous process, and the subcutaneous fascia and muscle tissue were separated layer by layer to expose the vertebrae. Under a microscope, the drill was used to open the lamina at T9-T10 to expose the spinal cord. The Allen's impactor was used to hit the spinal cord with parameters of depth 1.0 mm, speed 2.0 m / s, and duration 150 ms. After injury, the rat tail and hindlimb appeared tremor. The dura mater was incised under a microscope, and the hydrogel material in different groups was placed, and finally the incision was sutured layer by layer. The rats in the sham operation group were only exposed to the spinal cord without hitting. The MP group was injected with positive control drug methylprednisolone, 30 mg / kg, 1 time / day, for 14 days after modeling. All model animals were isolated according to the conventional standard, and the ventilation and bedding were changed regularly. Artificial urination was performed twice a day until the urination function recovered.

[0044] GMP@AS conductive hydrogel can promote the recovery of motor function of SCI rats: To observe the recovery process of motor function of rats in each group after injury, BBB score was performed at 1, 4, 7, 14, 21, 28, 35, 42 d after modeling operation. The BBB score was scored in the range of 0-21, reflecting the joint movement, stability, paw position, gait, forelimb and hindlimb coordination, and trunk position of the hindlimb. 0 points indicate complete paralysis, no movement of the hindlimb, and 21 points indicate free movement of the hindlimb, normal walking posture. In addition, footprint test was performed on rats in each group at 42 d after operation, which can directly reflect the movement coordination of rat limbs. In this test, the forelimbs of the rats were stained with blue ink, and the hindlimbs were stained with red ink. Make it walk on white paper to leave a footprint, through the analysis of the footprint, the gait parameters such as the rotation angle of the forepaw and hindpaw, the stride, the support point, and the vertical distance between the forelimb and the hindlimb can be obtained.

[0045] Results are shown in Figure 7 A, the hindlimbs of rats in each group after 6 weeks of intervention were observed, and the hindlimbs of rats in the Sham group were not different from normal rats; the hindlimbs of rats in the SCI group were paralyzed, the muscle atrophy was serious, the foot was downward, and no active movement was produced; the rats in the positive control (MP) group were slightly stronger than the SCI group, and one hindlimb appeared foot dorsum on the ground, but still could not support the body weight; the hindlimb shape of the GMP@AS group was obviously improved, the foot claw could support the ground, the foot ankle was eversion, there was no obvious spasm, and it could support most of the body weight, the tail was slightly raised, and the function recovered best. The BBB score showed that Figure 7B), in the first 2 weeks, the motor function of each group of rats was in the rapid recovery stage, and the scores from high to low were GMP@AS group (8.3±0.7), MP group (5.1±1.1), and SCI group (2.6±0.4) except for the sham group. After 2 weeks, the recovery rate of each group of rats slowed down, and the final scores from high to low were GMP@AS group (12.1±0.7), MP group (6.8±1.2), and SCI group (3.8±0.7). At the end of the evaluation, the rats in the GMP@AS group basically reached the palm weight-bearing movement, and could walk with the hind limbs and occasionally coordinate the movement of the fore and hind limbs, with a score significantly higher than that of the other groups (P<0.001). Footprint experiment showed that Figure 7 C), 6 weeks later, the rats in the SCI group showed obvious double hind limb dragging gait, without active support and step generation, and only front limb crawling was observed. The MP group showed unilateral limb movement, with intermittent footprints, short support time, and rhythm disorder of fore and hind limb movement, which could not fully support the body weight. The GMP@AS group had continuous footprints, could continuously walk with foot weight-bearing, and the fore and hind limbs moved alternately, with significant improvement in average stride and rotation angle.

[0046] GMP@AS conductive hydrogel can reduce the local cyst cavity area of SCI: To observe the growth of the damaged local spinal cord tissue and the formation of the cyst cavity, HE staining was performed on the spinal cord tissue sections. The specific steps were as follows: after perfusing the rats, the spinal cord tissue to be stained was taken out, fixed with 4% paraformaldehyde overnight, and then immersed in PBS. Finally, the tissue sample was embedded and cut into 20 μm thick sections, and the frozen sections were sequentially stained, differentiated, returned to blue, and transparented according to the instructions. Finally, the images were observed under a confocal microscope.

[0047] Figure 8 As shown in the figure, the Sham group showed a clear normal layered structure of the spinal cord tissue, and no cavity was formed. The neuron cell volume was large and the structure was complete. The remaining injury groups all had cysts of different sizes and irregular shapes. The SCI group had the largest cavity formation area (33.6±3.4%), the internal structure of the spinal cord tissue was chaotic, and chronic inflammatory reaction was accompanied. The neuron cell nucleus was pyknosis and deep staining, and it was reduced, indicating that a large number of neurons were destroyed. The cyst cavity area of the MP group was significantly smaller than that of the SCI group (P<0.001), and the cyst cavity area of the two groups accounted for (14.1±1.6%) and (9±0.6%), respectively, and the difference between the two groups was statistically significant. Except for the Sham group, the cyst cavity area of the GMP@AS group was the smallest (3.9±1.5%), which may be related to the growth and infiltration of nerve fibers, indicating that the conductive hydrogel GMP@AS loaded with astragaloside A can effectively reduce the cavity area and promote tissue regeneration and repair.

[0048] GMP@AS conductive hydrogel can promote nerve regeneration in SCI rats: To observe the promoting effect of GMP@AS on the peripheral nerve regeneration of SCI rats, the neuron marker NeuN was stained by immunofluorescence. As shown in Figure 9 Figure 6, the neuron cell body of the Sham group was clear and complete, and the number of neurons was rich. The number of neurons around the injury of the SCI group decreased sharply, and the regeneration ability was very limited under the condition of no intervention. The number of neurons of the MP group increased to a certain extent, but the cells were shrunk or deformed, which was significantly less than that of the GMP@AS group (P<0.001). The neurons around the injury of the GMP@AS group were dense, which was significantly more than that of the MP and SCI groups (P<0.001), indicating that the implantation of GMP@AS conductive hydrogel had obvious promoting effect on the regeneration of neurons.

[0049] GMP@AS conductive hydrogel can promote the regeneration of myelin of SCI rats: To observe the regeneration of myelin around the injury, the spinal cord tissue sections were stained by LFB. First, the sections were immersed in LFB staining solution overnight at room temperature. Then, the sections were immersed in differentiation solution, ethanol and tar violet staining solution in turn. Then, the sections were transparentized by xylene, and finally, the sections were mounted with neutral balsam, and observed under a microscope.

[0050] As shown in Figure 10 Figure 7, the myelin density of the Sham group was the highest, and the staining was the deepest. The myelin sheath was arranged in order, and was dense and uniform, which was in sharp contrast with the gray matter. The myelin sheath in the center of the injury of the SCI group almost completely disappeared, and presented a large area of light blue. The positive control MP group showed extensive demyelination, and the boundary between white matter and gray matter was relatively blurred, and the fiber arrangement was chaotic. The GMP@AS group was obviously improved, and the axon was wrapped by a large amount of myelin sheath, and the myelinated nerve fiber was significantly higher than that of other groups, but the fiber direction of the myelin sheath in the center of the injury was still relatively irregular.

Claims

1. A method for preparing a conductive hydrogel loaded with astragaloside A, characterized in that, Includes the following steps: (1) Mix the methacrylic anhydride gelatin solution with the organic solvent solution of astragaloside A to obtain a prepolymer solution; then mix it with a photoinitiator and use ultraviolet light to crosslink and cure to form methacrylic anhydride gelatin / astragaloside A hydrogel. Soak the gelatin in deionized water to remove the ungelled components. (2) The methacrylic anhydride gelatin / astragaloside hydrogel was soaked in hydrochloric acid containing a free radical initiator; then soaked in aniline ethanol solution and polymerized; the resulting hydrogel was then soaked in ultrapure water and HCl solution in sequence and purified to obtain methacrylic anhydride gelatin-polyaniline conductive hydrogel loaded with astragaloside.

2. The method for preparing the conductive hydrogel loaded with astragaloside A according to claim 1, characterized in that, In step (1), the grafting rate of the methacrylic anhydride gelatin is 55-65%.

3. The method for preparing the conductive hydrogel loaded with astragaloside A according to claim 1, characterized in that, In step (1), the methacrylic anhydride gelatin solution is a PBS solution of methacrylic anhydride gelatin; the concentration of methacrylic anhydride gelatin in the prepolymer solution is 10-15%.

4. The method for preparing the conductive hydrogel loaded with astragaloside A according to claim 1, characterized in that, Step (1), the organic solvent is DMSO.

5. The method for preparing the conductive hydrogel loaded with astragaloside A according to claim 1, characterized in that, In step (1), the mass ratio of methacrylic anhydride gelatin solution to photoinitiator is (9~10):(1~1.2); preferably, the volume ratio of astragaloside A organic solvent solution to prepolymer solution is (0.0025~0.025):

1.

6. The method for preparing the conductive hydrogel loaded with astragaloside A according to claim 1, characterized in that, In step (2), the free radical initiator is 0.05~0.1M ammonium persulfate.

7. The method for preparing the conductive hydrogel loaded with astragaloside A according to claim 1, characterized in that, Step (2): Soak in 0.8~1M hydrochloric acid for 2~4 hours.

8. The method for preparing the conductive hydrogel loaded with astragaloside A according to claim 1, characterized in that, Step (2): Soak in 0.1-0.2M aniline ethanol solution for 4-6 hours; preferably, soak the hydrogel in ultrapure water for 2-3 days and in 0.8-1M HCl solution for 1 day.

9. The methacrylic anhydride-modified gelatin-polyaniline conductive hydrogel loaded with astragaloside A prepared by any one of claims 1-8, wherein the concentration of astragaloside A in the hydrogel is 12.5~125µg / ml.

10. The application of the methacrylic anhydride-modified gelatin-polyaniline conductive hydrogel loaded with astragaloside A according to claim 9 in the preparation of spinal cord injury repair materials.

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