Nano-enzyme drug-loaded hydrogel compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials. The invention provides a nano-enzyme-loaded hydrogel compound as well as a preparation method and application thereof.The nano-enzyme-loaded hydrogel compound is prepared by taking thermo-sensitive chitosan / sodium glycerophosphate hydrogel as a material substrate, jointly loading Mn-POM nano-enzyme and a TRPA1 channel antagonist HC-030031 on the material substrate, and stirring under an ice bath condition. The nano-enzyme drug-loaded hydrogel compound prepared by the preparation method disclosed by the invention integrates in-situ forming, ROS (reactive oxygen species) removal, microenvironment response drug release and synergistic anti-inflammation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a nanoenzyme-loaded drug-coated hydrogel composite, its preparation method, and its application. Background Technology

[0002] Chronic inflammation is a common pathological basis for the development of many major diseases, such as arthritis, atherosclerosis, neurodegenerative diseases, and chronic enteritis. During this process, reactive oxygen species (ROS), such as superoxide anions (O2·kJ / L), play a crucial role. - Excessive production of ROS (hydrogen peroxide, H2O2) and hydroxyl radicals (·OH) leads to oxidative stress, which in turn activates key inflammatory signaling pathways such as NF-κB, forming a vicious cycle of mutually reinforcing oxidative stress and inflammation, thus exacerbating tissue damage. Therefore, developing local drug delivery systems that can precisely and efficiently remove ROS and synergistically reduce inflammation is of great significance for the treatment of inflammatory diseases.

[0003] Locally injectable hydrogels have become ideal carriers for local drug delivery due to their good biocompatibility and sustained retention at the lesion site. Among them, the chitosan / β-glycerophosphate (CS / GP) thermosensitive hydrogel system has been extensively studied due to its good biocompatibility, biodegradability, and sol-gel transition at body temperature. However, existing CS / GP drug-loaded hydrogels used for anti-inflammatory purposes are mostly passive drug release carriers, lacking the ability to regulate the inflammatory microenvironment (such as high ROS levels), resulting in limited functionality and therapeutic efficacy.

[0004] Polyoxometalates (POMs) are a class of well-defined metal-oxygen clusters with abundant redox properties, and their enzymatic activity can be modulated by the central metal atom. Currently, there are no reports on the use of POM-based nanozymes with well-defined ROS-scavenging activities (such as superoxide dismutase (SOD) and catalase (CAT)) co-loaded with anti-inflammatory drugs in smart hydrogels for synergistic anti-inflammatory therapy. Constructing an integrated hydrogel system that responds to the inflammatory microenvironment, possesses both highly efficient ROS scavenging capabilities and controllable drug release is a key technical challenge for achieving synergistic anti-inflammatory therapy. Summary of the Invention

[0005] To address the problems existing in the background technology, the present invention provides a nanoenzyme drug-loaded hydrogel composite, its preparation method and application, which integrates in-situ molding, ROS removal, microenvironment-responsive drug release and synergistic anti-inflammatory effects.

[0006] To achieve the above objectives, in a first aspect, the present invention provides a method for preparing a nanoenzyme-loaded hydrogel composite, wherein a thermosensitive chitosan / sodium glycerophosphate hydrogel is used as a substrate, and Mn-POM nanoenzyme and TRPA1 channel antagonist HC-030031 are co-loaded on the substrate and stirred under ice bath conditions to obtain the composite.

[0007] Furthermore, the specific steps include the following:

[0008] S1. Preparation of Mn-POM nanozymes;

[0009] S2. Dissolve TRPA1 channel antagonist HC-030031 in DMSO and sonicate under ice bath conditions to obtain solution A;

[0010] S3. Dissolve chitosan in glacial acetic acid and stir to dissolve, to obtain solution B;

[0011] S4. Dissolve sodium β-glycerophosphate in ultrapure water, stir at 37°C, and treat with an ice bath to obtain solution C;

[0012] S5. Stir under ice bath conditions, and after thoroughly mixing solution B and solution C, add solution A and Mn-POM nanozyme to form a uniform composite sol, thus obtaining the nanozyme-loaded hydrogel composite.

[0013] Further, the specific operation of S1 is as follows: Dissolve 0.4414g of ammonium molybdate in 10mL of ultrapure water, sonicate for 10min, then add 5mL of 0.108mM manganese chloride solution and 4mL of saturated vitamin C solution at 37℃ and 500~700r / min, continue stirring for 2h, dialyze for 6-24h, and then freeze dry to obtain the product.

[0014] Furthermore, in S3, the concentration of glacial acetic acid is 0.1M.

[0015] Further, the specific operation of S3 is as follows: dissolve chitosan in glacial acetic acid, and stir magnetically for 10 minutes at a temperature of 37°C and a rotation speed of 500~700 r / min to obtain a solution B with a concentration of 2% (w / v).

[0016] Further, the specific operation of S4 is as follows: dissolve sodium β-glycerophosphate in ultrapure water to prepare a 56% (w / v) sodium β-glycerophosphate solution; stir magnetically for 10 min at a temperature of 37℃ and a rotation speed of 500~700 r / min, and then place in an ice bath for 5 min to obtain a solution C with a concentration of 56% (w / v).

[0017] Furthermore, in the nanoenzyme-loaded hydrogel composite, the final concentration of chitosan is 1.5% (w / v); the final concentration of sodium β-glycerophosphate is 14% (w / v); the final concentration of Mn-POM nanoenzyme is 75 μg / mL; and the final concentration of HC030031 is 10 μM.

[0018] Secondly, the present invention provides a nanoenzyme-loaded hydrogel composite, which is prepared by the above-described preparation method.

[0019] Thirdly, the present invention provides an application of a nanoenzyme-loaded drug-eluting hydrogel composite, which is prepared by the above-described preparation method and is used in the preparation of drugs for treating inflammatory diseases.

[0020] This application has the following beneficial effects:

[0021] 1. The nanoenzyme-loaded hydrogel complex of the present invention (CS / GP-HC@Mn-POM thermosensitive injectable hydrogel) has excellent injectability, in-situ gelation and long-lasting sustained-release properties. Its preparation method is simple and has good biocompatibility. Utilizing the highly efficient peroxidase / catalase-like catalytic activity of manganese-based polyoxometalate nanoclusters (Mn-POM nanoenzymes), it can continuously scavenge overexpressed reactive oxygen free radicals at the lesion site, significantly reduce cellular oxidative stress damage, and create a favorable microenvironment for tissue repair.

[0022] 2. The nanoenzyme-loaded hydrogel complex of the present invention (CS / GP-HC@Mn-POM thermosensitive injectable hydrogel) targets the pathological microenvironment such as neuroinflammatory diseases. By simultaneously exerting the specific antagonistic effect of HC030031 on the TRPA1 pain-sensing channel and the catalytic scavenging function of Mn-POM, it effectively inhibits the ROS-related inflammatory cascade while blocking pain signal transduction. This system can also further regulate key inflammatory signaling pathways, downregulate the expression of pro-inflammatory factors such as TNF-α and IL-1β, and promote the polarization of macrophages towards the repair phenotype, thereby achieving multiple synergistic effects of antioxidation, anti-inflammation and neuroprotection, providing a new strategy for local targeted treatment of chronic pain and inflammatory diseases. Attached Figure Description

[0023] Figure 1 The image shows a transmission electron microscope (TEM) image of the Mn-POM nanozyme in Example 2; where A is the TEM image of the Mn-POM nanozyme and B is the mapping image of the Mn-POM nanozyme.

[0024] Figure 2The image shows a scanning electron microscope (SEM) image of the nanoenzyme-loaded hydrogel complex (i.e., CS / GP-HC@Mn-POM thermosensitive injectable hydrogel) in Example 2; where A is the SEM image of the nanoenzyme-loaded hydrogel complex and B is the mapping image of the nanoenzyme-loaded hydrogel complex.

[0025] Figure 3 The thermo-sensitive properties of the nanoenzyme-loaded hydrogel complex (i.e., CS / GP-HC@Mn-POM thermo-sensitive injectable hydrogel) in Example 2 are shown in Figure 2. Among them, A is the inverted test diagram of the nanoenzyme-loaded hydrogel complex, and B is the thermo-sensitive rheological diagram of the nanoenzyme-loaded hydrogel complex.

[0026] Figure 4 The drug release rate diagram is shown for the nanozyme drug-loaded hydrogel complex (i.e., CS / GP-HC@Mn-POM thermosensitive injectable hydrogel) in Example 2.

[0027] Figure 5 This is a graph showing the superoxide dismutase activity evaluation results of the Mn-POM nanozyme in Example 3;

[0028] Figure 6 This is a graph showing the catalase activity evaluation results of the Mn-POM nanozyme in Example 3;

[0029] Figure 7 The image shows the results of the Mn-POM nanozyme's cytotoxicity verification against RAW 264.7 cells in Example 4.

[0030] Figure 8 This is a diagram showing the results of the repair verification of damaged RAW 264.7 cells by Mn-POM nanozyme in Example 4;

[0031] Figure 9 This is a graph showing the evaluation results of the hemolytic effect of the Mn-POM nanozyme in Example 4;

[0032] Figure 10 The figure shows the cytotoxicity verification results of the nanozyme drug-loaded hydrogel complex (i.e., CS / GP-HC@Mn-POM thermosensitive injectable hydrogel) in Example 4.

[0033] Figure 11 This is a graph showing the effect of CS / GP-HC@Mn-POM in Example 5 on the ability to scavenge reactive oxygen species.

[0034] Figure 12 The graph shows the evaluation results of CS / GP-HC@Mn-POM inhibiting the TRPA1 pathway in Example 5;

[0035] Figure 13 This is a graph showing the in vivo therapeutic effect of CS / GP-HC@Mn-POM in Example 5. Detailed Implementation

[0036] The present application will be further described in detail below with reference to the embodiments.

[0037] Unless otherwise specified, the raw materials used in the embodiments and comparative examples of this application are all commercially available.

[0038] Example 1: This example describes the preparation of a nano-enzyme-loaded hydrogel composite (i.e., CS / GP-HC@Mn-POM thermosensitive injectable hydrogel):

[0039] The components of this nanoenzyme-loaded hydrogel complex include: Mn-POM nanoenzyme, TRPA1 channel antagonist HC-030031, and thermosensitive chitosan / sodium glycerophosphate hydrogel.

[0040] This nanoenzyme-loaded hydrogel composite uses a thermosensitive chitosan / sodium glycerophosphate hydrogel as the substrate. Mn-POM nanoenzymes and TRPA1 channel antagonist HC-030031 are co-loaded onto the thermosensitive chitosan / sodium glycerophosphate hydrogel and prepared by thorough stirring under ice bath conditions.

[0041] Specifically, the preparation steps of this nano-enzyme drug-loaded hydrogel complex are as follows:

[0042] S1. Weigh 0.4414 g of ammonium molybdate and dissolve it in 10 mL of ultrapure water. After ultrasonic treatment (power: 220 W, frequency: 40 kHz) for 10 min, add 5 mL of 0.108 mM manganese chloride solution and 4 mL of saturated vitamin C solution at 37 °C and continue stirring for 2 hours. Dialyze the solution in a dialysis bag (source leaf regenerated cellulose dialysis bag (1000) 45 mm, 6.4 mL / cm) for 6 hours and then freeze-dry to obtain Mn-POM nanozyme.

[0043] S2, S2, Dissolve TRPA1 channel antagonist HC-030031 in 1 mL of DMSO and sonicate under ice bath conditions to obtain a solution concentration of 10 mM, thus obtaining solution A;

[0044] S3. Dissolve chitosan (CS) in glacial acetic acid (0.1M) to prepare a 2% (w / v) CS solution. Stir magnetically at 500 r / min and 37℃ for 10 min to obtain solution B.

[0045] S4. Dissolve sodium β-glycerophosphate (GP) in ultrapure water to prepare a 56% (w / v) sodium β-glycerophosphate solution; specifically, stir magnetically at 500 r / min and 37℃ for 10 min, and then place in an ice bath for 5 min to obtain solution C.

[0046] S5. Under ice bath conditions and at a rotation speed of 500 r / min, take 2.5 mL of solution C and add it dropwise to 7.5 mL of solution B at a ratio of 1:3 while stirring. Add 10 μL of solution A and 0.75 mg of Mn-POM nanozyme while stirring until a uniform composite sol is formed, thus obtaining the nanozyme-loaded hydrogel complex.

[0047] Example 2: Morphological and structural characterization and evaluation of the nanoenzyme-loaded hydrogel complex prepared in Example 1:

[0048] (1) The Mn-POM nanozyme prepared in Example 1 was characterized by transmission electron microscopy.

[0049] The results are as follows Figure 1 As shown, Figure 1 -A indicates that the morphology of this Mn-POM nanozyme is a regular spherical or near-spherical aggregate of particles, with relatively uniform particle size. Figure 1 The mapping analysis in -B shows that the element distribution is relatively uniform and verifies the successful incorporation of Mn.

[0050] (2) The nanoenzyme-loaded hydrogel complex prepared in Example 1 was characterized by scanning electron microscopy.

[0051] The results are as follows Figure 2 As shown, Figure 2 -A shows that the nanozyme-loaded hydrogel composite exhibits a porous network structure, which is beneficial for the more uniform dispersion of the Mn-POM nanozyme and the desired drug within the hydrogel matrix. Figure 2 The mapping analysis in -B provides a visual representation of the successful incorporation of Mn-POM nanozymes.

[0052] (3) The nanoenzyme-loaded hydrogel complex prepared in Example 1 was characterized using a rheological analyzer.

[0053] The results are as follows Figure 3 As shown, Figure 3 -A shows that the nanoenzyme-loaded hydrogel composite remains liquid at room temperature, exhibiting free-flowing properties. The sample rapidly (within 2 minutes) transitions to a gel state at 37°C using the "inverted method," demonstrating its thermally responsive gel properties. Figure 3 In section B, the thermosensitive properties of the hydrogel were further characterized by rheological testing. The results showed that as the temperature increased, the storage modulus (G') and loss modulus (G'') of the hydrogel gradually intersected. When the temperature rose to 37℃, G' exceeded G'', indicating that the system had completed the sol-gel transition.

[0054] (4) The drug release and degradation behavior of the nanoenzyme-loaded hydrogel complex prepared in Example 1 was studied.

[0055] The results are as follows Figure 4 As shown, the cumulative drug release was measured on days 5, 10, and 15. The results showed that the nanozyme-loaded hydrogel composite could slowly and stably release the loaded nanozyme and drug over time. On day 15, the cumulative release rates of both the Mn-POM nanozyme and the TRPA1 channel antagonist (HC-030031) were greater than 60%. This demonstrates that the nanozyme-loaded hydrogel composite exhibits an appropriate drug release rate.

[0056] Example 3: Evaluation of the activity of the Mn-POM nanozyme prepared in Example 1:

[0057] (1) Determination of ·O2 using a superoxide anion assay kit - Clearance capability.

[0058] Mn-POM nanozymes of different concentrations (25, 50, 75, 100, 125, 150 μg / mL) were mixed with superoxide anion exchange kit reagents, and their UV absorbance at 550 nm was measured. The superoxide dismutase (SOD) activity was calculated according to the kit instructions.

[0059] The results are as follows Figure 5 As shown, the characteristic absorption peak at 550 nm decreases with increasing concentration of Mn-POM nanozyme, indicating that Mn-POM nanozyme has a positive effect on ·O2. - Its scavenging ability is concentration-dependent and it exhibits good superoxide dismutase-like activity.

[0060] (2) The activity of catalase was verified using a portable dissolved oxygen meter.

[0061] Mn-POM nanozymes of different concentrations (25, 50, 75, 100 μg / mL) were added to 2 mM H2O2 solution, and the change in oxygen content in the solution was measured to evaluate catalase activity.

[0062] The results are as follows Figure 6 As shown, the oxygen production rate gradually increases with the increase of Mn-POM nanozyme concentration, indicating that Mn-POM nanozyme has good catalase-like activity and promotes the decomposition of H2O2 into O2.

[0063] Example 4: Biosafety evaluation of the Mn-POM nanozyme prepared in Example 1:

[0064] (1) The biosafety of Mn-POM nanozymes was evaluated using the CCK-8 assay for cell proliferation and toxicity.

[0065] Mouse mononuclear macrophage leukemia cells (RAW 264.7) were selected as the research subject, and approximately 5 × 10⁶ cells were used. 3 RAW264.7 cells were seeded in 96-well plates and treated with DMEM medium containing different concentrations of Mn-POM nanozyme (12.5, 25, 50, 75, 100, 150 μg / mL) for 24 hours. Then, 10 μL of CCK-8 assay solution was added to each well, and the cells were incubated together for 1 hour. Cell viability was calculated by measuring absorbance at 450 nm using a microplate reader.

[0066] The results are as follows Figure 7 As shown, when the concentration of Mn-POM nanozyme was below 75 μg / mL, the survival rate of RAW 264.7 cells was above 70%; when the concentration of nanozyme exceeded 75 μg / mL, the survival rate of RAW 264.7 cells began to decrease significantly and fell below 70%, indicating that at low concentrations (75 μg / mL), the nanozyme maintained relative safety for RAW 264.7 cells.

[0067] Following the same experimental steps as described above, after RAW 264.7 cells were plated for 24 hours, the old culture medium was removed. First, 400 μM H2O2 culture medium was added for stimulation for 4 hours, and then fresh Mn-POM culture medium of different concentrations was added for co-incubation. Subsequent experimental steps were the same as described above.

[0068] The results are as follows Figure 8 As shown, H2O2 stimulation significantly reduced the survival rate of RAW 264.7 cells, indicating that H2O2 can damage RAW 264.7 cells and inhibit their growth and proliferation. However, with the addition of Mn-POM nanozyme, the survival rate of RAW 264.7 cells increased to varying degrees with the increase of Mn-POM nanozyme concentration, indicating that Mn-POM nanozyme has a certain degree of repair ability for damaged RAW 264.7 cells.

[0069] (2) The biocompatibility of Mn-POM nanozymes was evaluated using a hemolysis experiment.

[0070] Whole blood was collected from the orbital cavity of C57 mice and placed in anticoagulant tubes containing EDTA. After thorough shaking, the cells were centrifuged at 3000 rpm for 15 minutes to obtain blood cells. Mn-POM nanozyme was diluted with PBS to obtain solutions of different concentrations. 1 mL of nanomaterial solution (25, 50, 75, 100, 125, 150 μg / mL), 1 mL of ultrapure water (ddH2O), and 1 mL of normal saline (NS) were mixed with 20 μL of blood cells respectively. The mixtures were incubated at 37°C for 4 hours, then centrifuged at 3000 rpm for 15 minutes. The supernatant was pipetted into 96-well plates, and the absorbance at 542 nm was measured using a microplate reader. Three replicates were used for each group.

[0071] The results are as follows Figure 9 As shown, in the measured Mn-POM nanozyme solutions of different concentrations, the hemolysis rate of cells was far below the international safety standard of 5%, indicating that Mn-POM nanozyme has excellent blood compatibility and biosafety.

[0072] (3) The biosafety of the nanozyme-loaded hydrogel complex was tested using a live-dead assay.

[0073] Mouse mononuclear macrophage leukemia cells (RAW 264.7) were selected as the research subject. RAW 264.7 cells were seeded in six-well plates (10 cells per well). 5 In this study, cells were incubated at 37°C for 24 hours with extracts of NS (i.e., physiological saline group), Mn-POM (i.e., nanozyme group), HC@Mn-POM (i.e., nanozyme + drug complex group), and CS / GP-HC@Mn-POM (i.e., nanozyme drug-loaded hydrogel complex group, which is simplified as CS / GP-HC@Mn in the following text and corresponding figures). Then, the cells were stained with a live and dead cell staining kit and photographed under a confocal microscope.

[0074] The results are as follows Figure 10 As shown, all cells were in a live state and exhibited green fluorescence in the image. No obvious dead cells (red fluorescence) were observed, indicating that the nanoenzyme-loaded hydrogel composite has good biocompatibility.

[0075] Example 5: Evaluation of the cell assay results of the product obtained in Example 1:

[0076] (1) Evaluation of the ability to scavenge reactive oxygen species.

[0077] RAW 264.7 macrophages were induced at a rate of 2 × 10⁻⁶. 5Cells were cultured at a density of 1000 cells / mL, 2 mL per well, evenly distributed in 6-well cell culture plates and incubated until complete cell adhesion. The old culture medium was then removed, and the cells were stimulated with 400 mM H2O2 for 2 h, followed by the addition of 75 μg / mL Mn-POM. The cells were incubated at 37°C with 5% CO2 for 24 h. The DCFH-DA fluorescent probe was diluted 1:1000 with serum-free medium to prepare a 10 μM working solution. The cell supernatant was removed, and the cells were washed three times with PBS. 1 mL of diluted DCFH-DA was added, and the cells were co-incubated for 20 min. The cells were then washed three times with serum-free cell culture medium to thoroughly remove any unadapted DCFH-DA, and the results were analyzed using confocal microscopy.

[0078] The results are as follows Figure 11 As shown, no obvious ROS (green fluorescence) was observed in the cells of the NS group and the CS / GP-HC@Mn group; a large amount of ROS was observed in the cells of the H2O2 group after stimulation with H2O2; while the green fluorescence around the damaged cells of the H2O2+CS / GP-HC@Mn group was significantly reduced after treatment with Mn-POM nanozyme, indicating that Mn-POM nanozyme has a significant promoting effect on the recovery of RAW 264.7 cells damaged by oxidative stimulation.

[0079] (2) Evaluation of inhibition of the TRPA1 pathway in cells.

[0080] Using trigeminal neuron cells CP-R314 as the research object, the experiment was divided into a blank control group, a nanozyme-loaded hydrogel complex group (CS / GP-HC@Mn), a hydrogen peroxide (H2O2) stimulation group, and a treatment group (H2O2+CS / GP-HC@Mn). After co-incubation of cells, TRPA1 antibody was added, and the expression of TRPA1 channels in trigeminal neuron cells was detected by immunofluorescence.

[0081] The results are as follows Figure 12 As shown, in the blank group and the nanozyme-loaded hydrogel complex group, TRPA1 channel was not significantly expressed (no obvious red fluorescence was observed); while in the H2O2-stimulated group, the expression level of TRPA1 channel in cells was significantly increased, and obvious red fluorescence could be observed; while in the treatment group (H2O2+CS / GP-HC@Mn), the expression level of TRPA1 gradually decreased, and the intensity of red fluorescence decreased, indicating that the treatment system can inhibit the expression of this channel and block the cellular pain pathway.

[0082] (3) Evaluation of the effects of animal experiments.

[0083] SPF-grade SD rats were used as experimental animals and were divided into 5 groups: sham-operated group—no modeling, pain-free group;

[0084] TN + saline group – model established, no treatment group;

[0085] TN+CS / GP@Mn-POM—Modeling, nanozyme therapy group only;

[0086] TN+CS / GP-HC—Modeling, HC treatment group only;

[0087] TN+CS / GP-HC@Mn—Modeling, Nanoenzyme-loaded hydrogel complex therapy

[0088] All rats underwent surgical model establishment. Except for the sham-operated group, the other experimental animals were used to establish a rat model of trigeminal neuralgia using the CT-guided sliding litmus injection method. After model establishment, the rats in each group were given corresponding drug intervention treatment. The experimental feeding period was 7 days. On the 7th day, dissection and paraffin embedding sections were performed, and immunofluorescence staining was used for treatment.

[0089] The results are as follows Figure 13 As shown, in the sham-operated group (Sham), no significant expression of neurotransmitters and cellular components (CD86, CD206, IL-1β, SP) was observed. In the TN + saline group, staining of neurotransmitters and intercellular components was significant after modeling. In the treatment groups (TN + CS / GP@Mn-POM group, TN + CS / GP-HC group, TN + CS / GP-HC@Mn group), the corresponding staining patterns changed significantly, with CD206 expression gradually increasing, while the expression of CD86, IL-1β, SP, and other substances gradually decreased. These results indicate that the CS / GP-HC@Mn treatment group can improve the pain microenvironment of trigeminal neuralgia and regulate the pain perception-inhibition pathway.

[0090] In summary, the nanoenzyme-loaded drug-eluting hydrogel complex CS / GP-HC@Mn (i.e., CS / GP-HC@Mn-POM thermosensitive injectable hydrogel) provided by this invention has significant therapeutic advantages: it achieves minimally invasive and precise implantation and long-term sustained drug release through its simple thermosensitive in-situ molding characteristics. Its core component, manganese-based polyoxometalate (Mn-POM), with its highly efficient peroxidase / catalase-like activity, can continuously clear excess reactive oxygen free radicals accumulated at the lesion site, effectively alleviating oxidative stress damage. At the same time, the loaded TRPA1 specific antagonist HC030031 can directly block pain signal transmission and synergize with the catalytic function of Mn-POM to jointly inhibit the ROS-triggered inflammatory cascade response, downregulate the expression of key pro-inflammatory factors such as TNF-α and IL-1β, and thus regulate the immune microenvironment, creating favorable conditions for the repair of nerve tissue. Therefore, it achieves multiple synergistic effects of antioxidation, anti-inflammation, and analgesia in the treatment of neuroinflammatory diseases and chronic pain.

[0091] The applicant declares that the above embodiments illustrate the detailed method of the present invention. These methods are merely preferred embodiments, and the scope of protection of the present invention is not limited thereto. Any modifications to the present invention, equivalent substitutions or alterations to the technical solutions and inventive concepts made by those skilled in the art within the scope of the present invention's technology are all within the scope of protection and disclosure of the present invention.

Claims

1. A method for preparing a nanoenzyme-loaded drug-coated hydrogel composite, characterized in that, Using a thermosensitive chitosan / sodium glycerophosphate hydrogel as the substrate, Mn-POM nanozyme and TRPA1 channel antagonist HC-030031 were co-loaded onto the substrate and stirred under ice bath conditions to obtain the final product.

2. The method for preparing the nanoenzyme-loaded hydrogel composite according to claim 1, characterized in that, Specifically, the steps include the following: S1. Preparation of Mn-POM nanozymes; S2. Dissolve TRPA1 channel antagonist HC-030031 in DMSO and sonicate under ice bath conditions to obtain solution A; S3. Dissolve chitosan in glacial acetic acid and stir to dissolve, to obtain solution B; S4. Dissolve sodium β-glycerophosphate in ultrapure water, stir at 37°C, and treat with an ice bath to obtain solution C; S5. Stir under ice bath conditions, and after thoroughly mixing solution B and solution C, add solution A and Mn-POM nanozyme to form a uniform composite sol, thus obtaining the nanozyme-loaded hydrogel composite.

3. The method for preparing the nanoenzyme-loaded hydrogel composite according to claim 1 or 2, characterized in that, The specific operation of S1 is as follows: Dissolve 0.4414g of ammonium molybdate in 10mL of ultrapure water, sonicate for 10min, add 5mL of 0.108mM manganese chloride solution and 4mL of saturated vitamin C solution at 37℃ and 500~700r / min, continue stirring for 2h, dialyze for 6-24h, and then freeze dry to obtain the product.

4. The method for preparing the nanoenzyme-loaded hydrogel composite according to claim 1, characterized in that, In S3, the concentration of glacial acetic acid is 0.1M.

5. The method for preparing the nanoenzyme-loaded hydrogel composite according to claim 1 or 4, characterized in that, The specific operation of S3 is as follows: dissolve chitosan in glacial acetic acid, and stir magnetically for 10 minutes at a temperature of 37℃ and a speed of 500~700r / min to obtain a solution B with a concentration of 2% (w / v).

6. The method for preparing the nanoenzyme-loaded hydrogel composite according to claim 1, characterized in that, The specific operation of S4 is as follows: dissolve sodium β-glycerophosphate in ultrapure water to prepare a 56% (w / v) sodium β-glycerophosphate solution; stir magnetically for 10 min at a temperature of 37℃ and a rotation speed of 500~700 r / min, and then place in an ice bath for 5 min to obtain a solution C with a concentration of 56% (w / v).

7. The method for preparing the nanoenzyme-loaded hydrogel composite according to claim 1, characterized in that, In the nanoenzyme-loaded hydrogel composite, the final concentration of chitosan is 1.5% (w / v); the final concentration of sodium β-glycerophosphate is 14% (w / v); the final concentration of Mn-POM nanoenzyme is 75 μg / mL; and the final concentration of HC030031 is 10 μM.

8. A nanoenzyme-loaded drug-coated hydrogel composite, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 7.

9. An application of a nanoenzyme-loaded hydrogel composite, characterized in that, The nanoenzyme-loaded hydrogel composite is prepared by the preparation method according to any one of claims 1 to 7 and is used in the preparation of drugs for treating inflammatory diseases.

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