Preparation and application of oxidative stress microenvironment-responsive smart semi-switchable hydrogels

Abstract

The invention relates to a preparation method and application of an intelligent semi-conversion hydrogel that responds to an oxidative stress microenvironment, and belongs to the field of biomedicine technology. The preparation method described in the present invention can encapsulate therapeutic drugs therein and place them in the injured area. By responding to the large amount of ROS released by the early oxidative stress of spinal cord injury (SCI), the hydrogel can achieve a transition from a gel state to a semi-gel sol state, thereby consuming ROS while causing explosive release of drugs, and quickly reaching drug therapeutic concentrations in the acute phase of SCI. After the SCI oxidative stress period, ROS release decreases, and under the action of reducing substances in the body, the hydrogel can be restored from a semi-sol-gel state to a gel state, thereby achieving long-term slow release of the drug to be released and promoting neural repair in the later stage of SCI. Through this gel state-semi-sol-gel state-gel state transition, the present invention can achieve the controlled release of intelligent drugs that respond to oxidative stress, thereby achieving the best SCI treatment effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a preparation method and application of an oxidative stress microenvironment-responsive intelligent semi-conversion hydrogel. Background Art

[0002] Spinal cord injury (SCI) is a serious and common central nervous system disorder that can lead to impaired sensory and motor function, placing a significant psychological and economic burden on patients and their families. Therefore, developing effective and feasible treatments is crucial. Pathologically, SCI is divided into primary and secondary injuries. Primary injury is an initial contusion or transection caused by mechanical compression or traction on the spinal cord, which can occur immediately. Secondary injury, on top of the primary injury, involves complex pathophysiological changes in the spinal cord tissue, including neuronal death, ischemia-reperfusion, edema, inflammation, and oxidative stress, which involve the immune system, nervous system, and blood circulation. These changes are key factors contributing to SCI impairment and hindering recovery. Therefore, SCI treatment should prioritize improving the microenvironment of the spinal cord injury to minimize the progression of secondary injury. Regeneration of blood vessels and neural circuits in the injured area is particularly crucial during treatment. Vascular regeneration can deliver nutrients and oxygen to the injured area and prevent the influx of neutrophils, inflammatory cells, and even toxins from the bloodstream, effectively improving the microenvironment for neural regeneration. Neural circuit regeneration is the basis for motor and sensory recovery after SCI. As the basic building block of neural circuits, the induced regeneration of neurons is the key to functional recovery after SCI. Currently, surgical decompression is often used in clinical practice to eliminate spinal cord compression, restore mechanical stability, and thus reduce the damage caused by primary injury. At the same time, drugs such as glucocorticoids and neurotrophic factors are also used to inhibit the progression of inflammation, oxidative stress, etc. and promote neurological function recovery. However, traditional drug delivery methods still have certain limitations.

[0003] Current drug delivery methods are generally divided into systemic intravenous injection, local injection at the injury site, or local administration by encapsulating the drug in a carrier. First, traditional intravenous systemic drug delivery methods generally select higher drug concentrations to achieve effective treatment of the target, which can easily lead to systemic drug toxicity. Lower drug concentrations often result in insufficient local drug concentration, making it impossible to achieve the ideal drug enrichment concentration, resulting in a significant reduction in therapeutic efficacy. Second, while local injection solves the problem of poor local drug enrichment, the single-injection mode of drug administration has the disadvantage that the drug is absorbed and cleared in a short period of time and cannot have long-term effects. While fractional injections can continuously replenish local drug concentrations, they can cause pain to the patient. Third, while encapsulating the drug in a traditional hydrogel drug delivery system and placing it at the injury site can meet the local drug concentration and maintain release for a long time, traditional hydrogel drug delivery systems cannot intelligently control the drug release rate, resulting in drug release only at a predetermined rate and failing to meet the pathophysiological and spatiotemporal characteristics of spinal cord injury, resulting in ultimately low treatment efficiency.

[0004] On October 25, 2011, the journal Nature Communications published a paper titled "Redox-responsive self-healing materials formed from host–guest polymers." The paper disclosed a stimulus-responsive network construction. However, based on the data provided in the paper, it was not possible to prepare a gel containing a stimulus-responsive network. Figure 7 According to the preparation process and feed ratio of this paper, no white solid substance can be obtained after precipitation. Figure 8 After synthesizing the materials according to the paper, PAA-Fc and PAA-CD could not form a host-guest structure and were in a liquid state. Summary of the Invention

[0005] To address the above-mentioned issues, the present invention provides a method for preparing and applying a smart semi-switchable hydrogel that responds to an oxidative stress microenvironment. The oxidative stress microenvironment-responsive smart semi-switchable hydrogel prepared by the present invention can encapsulate therapeutic drugs and deliver them to the injured area. By responding to the large amounts of ROS (reactive oxygen species) released by oxidative stress in the early stages of spinal cord injury, the hydrogel transforms from a gel state to a semi-gel sol state, consuming ROS while simultaneously releasing the drug in a burst, rapidly reaching therapeutic drug concentrations during the acute phase of SCI. After the oxidative stress phase of SCI, ROS release decreases. Under the action of reducing substances (such as glutathione and glucose) in the body, the hydrogel can revert from a semi-sol-gel state to a gel state, enabling long-term, slow release of the drug to be released, promoting neural repair in the later stages of SCI. Through this gel-semi-sol-gel-gel transition, the present invention enables controlled release of smart drugs responsive to oxidative stress, thereby achieving optimal SCI therapeutic efficacy. Furthermore, encapsulating the drug within the semi-switchable hydrogel allows for simultaneous drug release, achieving optimal SCI therapeutic results.

[0006] The molecular formulas or English abbreviations mentioned in the present invention correspond to the following:

[0007] PAA is polyacrylic acid; DMF is N,N-dimethylformamide; PyBOP is benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate; ET3N is triethylamine; CD is cyclodextrin; MWCO is the molecular weight cut-off of the dialysis bag; FcCONH2 is ferrocenylcarboxyethyldiamine; GelMA is methacrylated gelatin; ROS is reactive oxygen species.

[0008] A method for preparing an oxidative stress microenvironment-responsive intelligent semi-switchable hydrogel comprises the following steps:

[0009] Step 1: Preparation of PAA-CD

[0010] The dried PAA was dissolved in DMF, and then PyBOP and ET3N were added and stirred continuously at room temperature. CD was then added and stirred to react to obtain a modified polymer. The modified polymer was precipitated with acetone and centrifuged to collect a white solid modified polymer. The collected white solid modified polymer was dissolved in ultrapure water and then dialyzed against water to obtain a polymer solution, designated as polymer solution A. The polymer solution A was freeze-dried to obtain PAA-CD.

[0011] Step 2: Preparation of PAA-Fc

[0012] The dried PAA was dissolved in DMF, and then PyBOP and ET3N were added and stirred at room temperature to obtain solution B. FcCONH2 was dissolved in DMF and stirred at room temperature to obtain solution C. Solution C was added dropwise to solution B with sufficient stirring, and then diluted with ultrapure water. The diluted mixture was dialyzed against water to obtain a polymer solution, designated polymer solution D. Polymer solution D was freeze-dried to obtain PAA-Fc, which was stored under argon.

[0013] Step 3: Prepare semi-converted hydrogel and load drugs

[0014] The PAA-CD prepared in step 1 was dissolved in water to prepare a 4% by mass PAA-CD solution, the PAA-Fc prepared in step 2 was dissolved in water to prepare a 4% by mass PAA-Fc solution, and GelMA was dissolved in water to prepare a 30% by mass GelMA solution. The above three solutions were taken and mixed with the loaded drug in a volume ratio of 2:1:1 to finally obtain a semi-switchable hydrogel for oxidative stress microenvironment-responsive smart drug delivery and controlled release, and the loaded drug accounted for 10% of the total volume of the semi-switchable hydrogel.

[0015] In the step 1, the molecular weight of PAA is 450,000, the mass is 500 mg, the volume of DMF is 20 mL, the mass of the added PyBOP is 0.4 g, the volume of the added ET3N is 80 μL, and the mixture of DMF dissolved with PAA and the added PyBOP and ET3N is continuously stirred for 2 hours; the mass of the added CD is 0.79 g, and the stirring reaction time after the addition of CD is 24 hours; 200 mL of acetone is used, and the centrifugation conditions are centrifugal rotation at room temperature and a speed of 8900 rpm; the white solid modified polymer is dissolved in ultrapure water for 12 hours, and the water dialysis uses a dialysis tube with a MWCO of 14 kDa for 144 hours; the temperature for freeze-drying the polymer A solution is -80°C and the pressure is 1.3-13 Pa.

[0016] In the step 2, the molecular weight of PAA is 450,000, the mass is 500 mg, the volume of DMF in which PAA is dissolved is 40 mL, the mass of the added PyBOP is 0.4 g, the volume of the added ET3N is 80 μL, and the mixture of DMF in which PAA is dissolved and the added PyBOP and ET3N is continuously stirred for 2 hours; the mass of the added FcCONH2 is 0.19 g, the volume of DMF in which FcCONH2 is dissolved is 10 mL, and the mixture is stirred for 2 hours; solution C is added dropwise to solution B with stirring for 24 hours; the diluted mixture is dialyzed against water using a dialysis tube with a MWCO of 14 kDa for 144 hours; the temperature for freeze-drying the polymer solution D is -80°C and the pressure is 1.3-13 Pa.

[0017] The oxidative stress microenvironment-responsive smart semi-switchable hydrogel obtained according to the above preparation method can be applied in the following aspects:

[0018] preparing products for treating spinal cord injuries;

[0019] preparing products that promote nerve regeneration;

[0020] Preparation of products that promote recovery of motor function after spinal cord injury;

[0021] Prepare products that improve the microenvironment of spinal cord injury;

[0022] Prepare products that improve the repair effect of spinal cord injuries.

[0023] The working process and working principle of the present invention:

[0024] The present invention constructs a stimuli-responsive network with polyacrylic acid (PAA) as the backbone, onto which cyclodextrin (CD) and ferrocene (Fc) are grafted, respectively, to form PAA-CD and PAA-Fc networks. Under normal conditions, PAA-CD and PAA-Fc interact through host-guest interactions, forming a gel state with a pressure of approximately 100 Pa. The preparation method described herein uses methacrylated gelatin (GelMA) as the backbone network and the PAA-CD and PAA-Fc host-guest system as the stimuli-responsive network to construct a semi-switchable hydrogel system.

[0025] After adding H2O2 (the main component of ROS) into the semi-conversion hydrogel system, PAA-Fc was oxidized to PAA-Fc + , resulting in the disassembly of PAA-CD and PAA-Fc. During this process, GelMA is not affected and always maintains the skeleton state. Therefore, the semi-conversion hydrogel system realizes the transformation from gel state to semi-sol-gel state under the action of H2O2. After further adding reducing substances, Fc +It is reduced to Fc, resulting in the host-guest reassembly of PAA-CD and PAA-Fc to form a gel.

[0026] The semi-switchable hydrogel responds to the oxidative stress phase following spinal cord injury (during which a large amount of ROS is generated), transforming from a gel state to a semi-gel sol state, explosively releasing the encapsulated drug while simultaneously consuming ROS and mitigating the damage to spinal cord tissue caused by secondary injury. After the oxidative stress phase ends, due to the reduction in ROS levels, the semi-sol-gel hydrogel can transition back to a sol state under the action of reducing substances such as glutathione and glucose in the body, achieving long-term and slow drug release.

[0027] Beneficial effects of the present invention:

[0028] Compared to existing technologies, the preparation method described in this invention can produce an intelligent semi-convertible hydrogel that responds to an oxidative stress microenvironment. Compared to traditional hydrogels, this device exhibits ROS-responsive activity, enabling burst release of drugs while simultaneously scavenging ROS during oxidative stress. Once ROS concentration decreases, the drug can be released slowly and over a long period of time, achieving intelligent controlled drug release based on an oxidative stress microenvironment. This method avoids the systemic drug toxicity that can occur with intravenous systemic administration, addressing the inherent limitation of achieving high local concentrations of drugs. It also eliminates the short half-life and rapid clearance of localized drugs after injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the mutual conversion process of stimulus-responsive networks in the semi-switch hydrogel prepared by the method described in Example 1 of the present invention.

[0030] Figure 2 This is the in vitro drug release curve of the semi-switching hydrogel prepared by the method described in Example 1 of the present invention.

[0031] Figure 3 This is a fluorescence microscope image of the in vitro ROS scavenging ability of the semi-converted hydrogel prepared by the method described in Example 1 of the present invention, wherein Hoechst is nicotinate hexamethylenetetramine and ROS is reactive oxygen species.

[0032] Figure 4 This is a fluorescence microscope image of the in vitro angiogenesis-promoting ability of the semi-converted hydrogel prepared by the method described in Example 1 of the present invention.

[0033] Figure 5 This is a fluorescence microscope image of the ability of the semi-converted hydrogel prepared by the method described in Example 1 of the present invention to promote the differentiation of neural stem cells in vitro, wherein Dapi is 4',6-diamidino-2-phenylindole and MAP2 is microtubule-associated protein 2.

[0034] Figure 6 This is a rat footprint analysis image showing the ability of the semi-conversion hydrogel prepared by the method described in Example 1 of the present invention to promote motor function recovery in rats after spinal cord injury.

[0035] Figure 7 This is a photograph of the material state after precipitation during the preparation of PAA-CD in the background technology of the present invention.

[0036] Figure 8 This is a photograph of the material state after the blend of PAA-CD and PAA-Fc prepared in the background technology of the present invention. DETAILED DESCRIPTION

[0037] Embodiment 1 of the present invention.

[0038] A method for preparing an oxidative stress microenvironment-responsive intelligent semi-switchable hydrogel comprises the following steps:

[0039] Step 1: Preparation of PAA-CD

[0040] 500 mg of dried PAA with a molecular weight of 450,000 was dissolved in 20 mL of DMF, and then 0.4 g of PyBOP and 80 μL of ET3N were added and stirred continuously at room temperature for 2 hours. Then, 0.79 g of CD was added and stirred for 24 hours to obtain a modified polymer. The modified polymer was precipitated with 200 mL of acetone and centrifuged at 8900 rpm at room temperature to collect a white solid modified polymer. The collected white solid modified polymer was dissolved in ultrapure water for 12 hours and then dialyzed with water using a dialysis tubing with a MWCO of 14 kDa for 144 hours to obtain a polymer solution, named polymer solution A. The polymer solution A was freeze-dried at -80°C and a pressure of 1.3-13 Pa to obtain PAA-CD.

[0041] Step 2: Preparation of PAA-Fc

[0042] 500 mg of dried PAA with a molecular weight of 450,000 was dissolved in 40 mL of DMF, and then 0.4 g of PyBOP and 80 μL of ET3N were added and stirred for 2 hours to obtain solution B. 0.19 g of FcCONH2 was dissolved in 10 mL of DMF and stirred for 2 hours to obtain solution C. Solution C was added dropwise to solution B at room temperature, and the reaction mixture was stirred for 24 hours and then diluted with ultrapure water. The mixture was dialyzed against water using dialysis tubing with a MWCO of 14 kDa for 144 hours to obtain a polymer solution, designated polymer solution D. Polymer solution D was freeze-dried at -80°C and a pressure of 1.3-13 Pa to obtain PAA-Fc, which was stored under argon.

[0043] Step 3: Prepare semi-converted hydrogel and load drugs

[0044] Platelet-rich plasma lysate (PL) + 500 ng / ml nerve growth factor (NGF) was selected as the drug to be loaded in this example.

[0045] The PAA-CD prepared in step 1 was dissolved in water to prepare a 4% by mass PAA-CD solution, the PAA-Fc prepared in step 2 was dissolved in water to prepare a 4% by mass PAA-Fc solution, and GelMA was dissolved in water to prepare a 30% by mass GelMA solution. 20 ml, 10 ml, and 10 ml of the above three solutions were taken in sequence and mixed with platelet-rich plasma lysate (PL) + 500 ng / ml nerve growth factor (NGF) to finally obtain a semi-switchable hydrogel for oxidative stress microenvironment-responsive smart drug delivery and controlled release, and the platelet-rich plasma lysate (PL) + 500 ng / ml nerve growth factor (NGF) accounted for 10% of the total volume of the semi-switchable hydrogel.

[0046] See also Figure 1 and Figure 2 , which is Example 2 of the present invention, is an in vitro drug release experiment of the semi-switch hydrogel prepared according to Example 1, as a verification of the release ability of the hydrogel-loaded drug.

[0047] Following the preparation method described in Example 1, platelet-rich plasma lysate (PL) plus 500 ng / ml nerve growth factor (NGF) was selected as the loading agent to prepare a drug release model to investigate drug loading and drug release behavior in cell culture medium. NGF protein concentration was measured using an enzyme-linked immunosorbent assay (ELISA) kit to assess overall drug release.

[0048] a. Drug release in reduced state

[0049] According to the preparation method described in the present invention, a semi-switchable hydrogel for oxidative stress microenvironment-responsive smart drug delivery and controlled release was prepared. 400 µL of the semi-switchable hydrogel was placed in a test tube and 2.5 mL of DMEM medium was added. The DMEM medium was replaced every 10 hours, and 20 µL of the replaced DMEM medium was used to measure the NGF concentration using an ELISA kit and calculate the NGF release.

[0050] b. Drug release in oxidative state

[0051] A semi-switchable hydrogel for oxidative stress microenvironment-responsive smart drug delivery and controlled release was prepared according to the preparation method described in Example 1. 400 µL of the semi-switchable hydrogel was placed in a test tube, and 2.5 mL of DMEM medium was added. The oxidation reactant, H₂O₂, was also added to the test tube (preparing multiple sets of identical test tubes). NGF protein concentration was measured using an ELISA kit from one test tube every 10 hours to assess protein release at different time points and under different oxidative states.

[0052] See also Figure 1 and Figure 3 As shown in Example 3 of the present invention, the in vitro ROS scavenging ability of the semi-converted hydrogel prepared according to Example 1 is determined, which is a product used in the preparation of a product for improving the microenvironment of spinal cord injury.

[0053] To establish an in vitro oxygen-glucose deprivation / reoxygenation (OGD / R) model: After cells reached satisfactory confluency, the culture medium was aspirated, the cells were washed with PBS, and sugar-free DMEM medium was added. The culture plate was then placed in a gas box and aerated with anaerobic air (95% N₂, 5% CO₂) and placed in a 37°C incubator for 12 hours. The culture medium was then replaced with normal medium and incubated in a 37°C incubator with 5% CO₂ for another 12 hours, thus establishing an in vitro oxygen-glucose deprivation / reoxygenation (OGD / R) model.

[0054] Cell Grouping and Treatment: Rat brain microvascular endothelial cells (RBMVEC) were used as an in vitro model cell line. The cells were divided into a blank group, an OGD / R group, and a hydrogel group. The blank group received no treatment; the OGD / R group received no treatment except for the OGD / R treatment; and the hydrogel group received the drug-free semi-converted hydrogel prepared according to the present invention in the culture medium simultaneously with the OGD / R treatment. After the OGD / R treatment, the cells were loaded with a DCFH-DA fluorescent probe and ROS clearance was observed using fluorescence microscopy.

[0055] See also Figure 1 and Figure 4As shown in Example 4 of the present invention, the semi-converted hydrogel prepared according to Example 1 is used to verify the ability of promoting angiogenesis in vitro. It is used in the preparation of products for treating spinal cord injury and the preparation of products for improving the repair effect of spinal cord injury.

[0056] Cells were divided into the following groups: blank group, hydrogel group, and hydrogel + drug treatment group. The blank group received no treatment. The hydrogel group received the drug-free semi-converted hydrogel prepared by the present invention after cell inoculation. The hydrogel group received the drug-added semi-converted hydrogel prepared by the present invention after cell inoculation.

[0057] Use a 4°C pre-cooled pipette tip to absorb Matrigel gel (matrix gel), evenly spread it in a 4°C pre-cooled 96-well plate, and incubate it in a 37°C incubator for 1 hour to allow the Matrigel gel to solidify. Select RBMVEC in the logarithmic phase and in good growth condition, digest and resuspend them, and prepare 2*10 4 100 μL of cell suspension was evenly inoculated into each well of a 96-well plate covered with Matrigel and incubated in a 37°C, 5% CO2 incubator. After 6 hours, tubule formation on the surface of the Matrigel was observed under a microscope and photographed.

[0058] See also Figure 1 and Figure 5 As shown in Example 5 of the present invention, the semi-converted hydrogel prepared according to Example 1 is used to verify the ability of promoting neural stem cell differentiation in vitro. It is applicable to the preparation of products for treating spinal cord injury, promoting nerve regeneration, and improving the repair effect of spinal cord injury.

[0059] Cell Grouping and Treatment: Primary rat hippocampal neural stem cells were used. Cells were divided into a blank group, a hydrogel group, and a hydrogel + drug treatment group. The blank group received no treatment. The hydrogel group received the drug-free semi-converted hydrogel prepared by the present invention after cell inoculation. The hydrogel group received the drug-added semi-converted hydrogel prepared by the present invention after cell inoculation. Each group was cultured for 5 days.

[0060] Cell immunofluorescence:

[0061] (1) Cell fixation: Remove the culture medium, wash with PBS (phosphate buffer solution) three times, and add 500 μL of 4% paraformaldehyde to fix the cells at room temperature for 20 minutes.

[0062] (2) Cell permeabilization: After the cells are fixed, they are washed three times with PBS, each time for 5 minutes, and then 500 μL of 0.3% Triton X-100 is added and incubated at room temperature for 15 minutes to permeabilize the membrane.

[0063] (3) Blocking: Remove the permeabilization solution, wash with PBS three times, 5 minutes each time, and add 500 μL of 1% BSA solution to block at room temperature for 1 hour.

[0064] (4) Primary antibody incubation: After removing the blocking solution, wash with PBS three times, 5 minutes each time, add 500 μL of 1:500 diluted MAP2, and incubate at 4°C overnight.

[0065] (5) Secondary antibody incubation: Remove the primary antibody, wash with PBS three times, 5 minutes each time, add 500 μL of 1:500 diluted fluorescent-labeled secondary antibody, and incubate at room temperature in the dark for 2 hours.

[0066] (6) Nuclear staining: Remove the secondary antibody, wash with PBS three times, 5 minutes each time, add 100 μL of 1 μmol / L DAPI staining solution, and incubate in the dark for 5 minutes.

[0067] (7) Sealing: Aspirate the DAPI staining solution, wash with PBS three times, 5 minutes each time, pick up the cell slide with tweezers, and place it upside down on a slide coated with anti-fluorescence quenching mounting medium.

[0068] (8) Fluorescence observation: Store the slides at 4°C away from light, observe under a fluorescence microscope or confocal fluorescence microscope, and take pictures.

[0069] Please refer to the figure Figure 1 and Figure 6 The figure shows Example 6 of the present invention, which is an experiment on promoting motor function recovery after spinal cord injury in rats using the semi-converted hydrogel prepared according to Example 1. The invention is used in preparing a product for promoting motor function recovery after spinal cord injury.

[0070] A rat spinal cord clamp injury model was established. All rats were acclimated to a constant temperature and humidity environment with free access to food and water for three days before modeling. Body weight was measured and anesthesia was administered with an intraperitoneal injection of 1% sodium pentobarbital (40 mg / kg). Modeling was initiated after the rats were deeply anesthetized. After skin preparation on the back, the rats were immobilized in the prone position. The skin was disinfected with iodine tincture and covered with a sterile drape, centered at the highest point of the dorsal spine (the T10 spinous process). A longitudinal incision of approximately 3.0 cm was made along the dorsal midline, centered at the highest point of the dorsal spine. Subcutaneous tissue, fascia, and musculature flanking the spinous processes were gradually dissected to expose the T9-T10 spinous processes. Distraction of the accessory tissues was performed with a spreader to fully expose the dorsal spinal structures. After the spinous processes were removed with rongeurs, the posterior lamina of T9 and T10 was carefully removed along the interlaminar space, extending bilaterally to the facet joints, to fully expose the spinal dura mater. If bleeding occurred, gauze was applied to stop bleeding. The incision was then rinsed with saline to ensure a clean surgical field. Insert the two blades of a 50g arterial clamp into the space between the spinal cord and the vertebral canal on both sides. Slowly loosen the clamp and continue compression for 60 seconds. At this point, the rat's hind limbs and tail will twitch rapidly, followed by loss of motor function in both lower limbs, indicating that the model is successful. Slowly withdraw the clamp, and dark red congestion will be seen under the dura mater at the site of spinal cord compression. Press again to stop bleeding, gently wash the wound with normal saline, suture it, and disinfect the sutured incision again with iodine. After surgery, 2ml of normal saline is injected intraperitoneally to replenish body fluids, and post-anesthesia resuscitation is performed in a warming blanket. After waking up, it can be seen that the rat has lost motor function in both lower limbs. After surgery, continuous intramuscular injection of penicillin is given for 5 days to prevent postoperative infection. Massage the bladder twice a day to assist urination (1 week). Ensure sufficient feed and water, change the bedding frequently, and try to avoid unnecessary rat deaths.

[0071] Animals were divided into two groups and treated: a spinal cord injury group and a treatment group. The spinal cord injury group received no treatment after the clamp injury, and the wound was sutured normally. The treatment group received a drug-loaded hydrogel prepared by the present invention applied to the spinal cord surface after the clamp injury. Both groups of rats were housed in a clean, ventilated, constant temperature, and humidity environment in a dedicated experimental rat breeding room for four weeks.

[0072] To observe the coordination of hindlimb movements in rats after spinal cord injury, we conducted a footprint imprinting experiment on three groups of rats. Before the experiment, a narrow, transparent tunnel approximately 80 cm long and 8 cm wide was prepared. A long strip of white paper was laid at the bottom of the tunnel, and a small amount of animal feed was placed at one end. Rats were captured and their hind limbs were painted with non-toxic red ink. After the rats calmed down, they were placed at the other end of the tunnel and allowed to walk freely along the tunnel. The footprints of the rats' forelimbs and hind limbs were then imprinted on the pre-laid white paper. If the rats stopped in the tunnel, the paper was replaced and the experiment was repeated to ensure that the rats continued to walk continuously within the tunnel. After the walk was completed, the paper with the footprints was collected, and the rats' footprints were observed and analyzed to evaluate the recovery of their limb walking function. The upper image shows the footprints of rats in the spinal cord injury group, and the lower image shows the footprints of rats in the treatment group. Clear paw prints were observed in the treatment group, indicating significant recovery of hindlimb motor function.

Claims

1. A method for preparing an oxidative stress microenvironment-responsive smart semi-switch hydrogel, characterized in that: The steps include: Step 1: Preparation of PAA-CD Dried PAA was dissolved in DMF, and then PyBOP and ET3N were added and stirred continuously at room temperature. CD was then added and stirred to react to obtain the modified polymer. The modified polymer was precipitated with acetone and centrifuged to collect a white solid modified polymer. The collected white solid modified polymer was dissolved in ultrapure water and then dialyzed against water to obtain a polymer solution, designated as polymer solution A. The polymer solution A was freeze-dried to obtain PAA-CD. Step 2: Preparation of PAA-Fc Dissolve dry PAA in DMF, then add PyBOP and ET3N, and stir thoroughly at room temperature to obtain solution B; dissolve FcCONH2 in DMF, and stir thoroughly at room temperature to obtain solution C; Solution C was added dropwise to Solution B with sufficient stirring, and then diluted with ultrapure water. The diluted mixture was dialyzed against water to obtain a polymer solution, designated as Polymer Solution D. Polymer Solution D was freeze-dried to obtain PAA-Fc, which was stored under argon. Step 3: Prepare semi-converted hydrogel and load drugs The PAA-CD prepared in step 1 was dissolved in water to prepare a 4% by mass PAA-CD solution, the PAA-Fc prepared in step 2 was dissolved in water to prepare a 4% by mass PAA-Fc solution, and GelMA was dissolved in water to prepare a 30% by mass GelMA solution. The above three solutions were taken and mixed with the loaded drug in a volume ratio of 2:1:1 to finally obtain a semi-switchable hydrogel for oxidative stress microenvironment-responsive smart drug delivery and controlled release, and the loaded drug accounted for 10% of the total volume of the semi-switchable hydrogel.

2. The method for preparing an oxidative stress microenvironment-responsive smart semi-switch hydrogel according to claim 1, characterized in that: In the step 1, the molecular weight of PAA is 450,000, the mass is 500 mg, the volume of DMF is 20 mL, the mass of the added PyBOP is 0.4 g, the volume of the added ET3N is 80 μL, and the mixture of DMF dissolved with PAA and the added PyBOP and ET3N is continuously stirred for 2 hours; the mass of the added CD is 0.79 g, and the stirring reaction time after the addition of CD is 24 hours; 200 mL of acetone is used, and the centrifugation conditions are centrifugal rotation at room temperature and a speed of 8900 rpm; the white solid modified polymer is dissolved in ultrapure water for 12 hours, and the water dialysis uses a dialysis tube with a MWCO of 14 kDa for 144 hours; the temperature for freeze-drying the polymer A solution is -80°C and the pressure is 1.3-13 Pa.

3. The method for preparing an oxidative stress microenvironment-responsive smart semi-switch hydrogel according to claim 1, characterized in that: In the step 2, the molecular weight of PAA is 450,000, the mass is 500 mg, the volume of DMF in which PAA is dissolved is 40 mL, the mass of the added PyBOP is 0.4 g, the volume of the added ET3N is 80 μL, and the mixture of DMF in which PAA is dissolved and the added PyBOP and ET3N is continuously stirred for 2 hours; the mass of the added FcCONH2 is 0.19 g, the volume of DMF in which FcCONH2 is dissolved is 10 mL, and the mixture is stirred for 2 hours; solution C is added dropwise to solution B with stirring for 24 hours; the diluted mixture is dialyzed against water using a dialysis tube with a MWCO of 14 kDa for 144 hours; the temperature for freeze-drying the polymer solution D is -80°C and the pressure is 1.3-13 Pa.

4. Use of the oxidative stress microenvironment-responsive smart semi-switch hydrogel obtained by the preparation method according to claim 1 in the preparation of spinal cord injury drugs.

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