Preparation method and application of oxidative stress microenvironment response intelligent semi-conversion hydrogel

Abstract

The invention discloses a preparation method and application of oxidative stress microenvironment response intelligent semi-conversion hydrogel, and belongs to the technical field of biological medicine. According to the preparation method disclosed by the invention, a therapeutic drug can be wrapped in the drug and placed in a local injury part, and a large amount of ROS released by oxidative stress in the early stage of spinal cord injury (SCI) is responded, so that conversion from a gel state to a semi-gel sol state is realized, and explosive release of the drug is caused while the ROS is consumed; the medicine treatment concentration is quickly reached in the acute stage of SCI; after the SCI oxidative stress period, ROS release is reduced, and under the action of in-vivo reducing substances, the hydrogel can be recovered from a semi-sol-gel state to a gel state, so that long-term slow release of to-be-released drugs is realized, and later-stage neural restoration of SCI is promoted; through the conversion from the gel state to the semi-sol gel state to the gel state, intelligent drug controlled release responding to oxidative stress can be achieved, and therefore the optimal SCI treatment effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a preparation method and application of an intelligent semi-convertible hydrogel responsive to an oxidative stress microenvironment. Background Art

[0002] Spinal cord injury (SCI) is a serious and common central nervous system disease, which can lead to impaired sensory and motor functions, bringing huge psychological and economic burdens to patients and their families. Therefore, it is very necessary to develop effective and feasible treatment measures. Pathologically, SCI is divided into primary injury and secondary injury. The primary injury is the initial contusion or transection caused by mechanical compression or traction of the spinal cord, which can occur instantly at the moment of injury. The secondary injury is based on the primary injury, and complex pathophysiological changes such as neuronal death, ischemia-reperfusion, edema, inflammation and oxidative stress occur in the spinal cord tissue, which involve the immune system, nervous system and blood circulation, and are the key factors leading to SCI disorders and hindering recovery. Therefore, the treatment of SCI should focus on improving the microenvironment of spinal cord injury to minimize the progression of secondary injury. During the treatment process, the regeneration of blood vessels and nerve circuits in the injured area is particularly crucial. Blood vessel regeneration can transport nutrients and oxygen to the injured local area, and prevent neutrophils, inflammatory cells and even toxins in the blood from entering the injured site, which can effectively improve the nerve regeneration microenvironment. The regeneration of nerve circuits is the basis for the recovery of motor and sensory functions after SCI. As the basic constituent unit of nerve circuits, the induced regeneration of neurons is the key to functional recovery after SCI. Currently, surgical decompression is often used clinically to relieve spinal cord compression and restore mechanical stability, thereby reducing 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 nerve function recovery. However, the traditional drug delivery methods still have certain limitations.

[0003] Current administration methods are generally divided into systemic intravenous injection, local injection at the injury site, or local administration by encapsulating the drug in a certain carrier. First, in the traditional systemic intravenous administration method, a relatively high drug concentration is usually selected to achieve effective treatment of the target, which is likely to cause systemic drug toxicity; when the drug concentration is low, there is usually insufficient local drug concentration, and the ideal drug enrichment concentration cannot be achieved, resulting in a significant reduction in the treatment effect. Second, in the local injection method, although the defect of poor local enrichment of the drug is solved, the one-time injection mode of the drug has the disadvantage that the drug is absorbed and cleared in the short term and cannot act for a long time; although multiple injections can continuously supplement the local drug concentration, it will cause pain to the patient. Third, although 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 a long-term release, the traditional hydrogel drug delivery system cannot intelligently regulate the drug release rate, resulting in the drug being released only at a fixed rate and not conforming to the pathophysiological spatio-temporal characteristics after spinal cord injury, leading to low final 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", which disclosed a construction of a stimulus-responsive network. However, based on the data provided in this paper, a gel containing a stimulus-responsive network cannot be prepared. For example, Figure 7 , according to the preparation process and feed ratio in this paper, no white solid substance can be obtained after precipitation. For example, Figure 8 , after synthesizing the material according to this paper, PAA-Fc and PAA-CD cannot form a host-guest structure and present a liquid state. Summary of the Invention

[0005] To solve the above problems, the present invention provides a method for preparing an oxidation stress microenvironment-responsive intelligent semi-convertible hydrogel and its application. The oxidation stress microenvironment-responsive intelligent semi-convertible hydrogel prepared by the present invention can encapsulate therapeutic drugs therein and place them at the local injury site. By responding to a large amount of ROS (reactive oxygen species) released during the early stage of spinal cord injury due to oxidation stress, a transformation from the gel state to the semi-gel sol state is achieved, thereby consuming ROS while causing explosive drug release and rapidly reaching the drug treatment concentration during the acute phase of SCI. After the oxidation stress period of SCI, the release of ROS decreases. Under the action of reducing substances in the body (such as glutathione, glucose, etc.), the hydrogel can return from the semi-sol gel state to the gel state, thereby realizing the long-term slow release of the drug to be released and promoting nerve repair in the later stage of SCI. Through this transformation of the gel state - semi-sol gel state - gel state, the present invention can achieve intelligent drug controlled release in response to oxidation stress, thereby achieving the best treatment effect for SCI and the best drug release effect. At the same time, by encapsulating drugs in the semi-convertible hydrogel, the effects of the drugs can be exerted simultaneously, thereby achieving the best treatment effect for SCI.

[0006] The molecular formulas or English abbreviations mentioned in the present invention are corresponding as follows: PAA is polyacrylic acid; DMF is N,N-dimethylformamide; PyBOP is benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; ET3N is triethylamine; CD is cyclodextrin; MWCO is the molecular weight cut-off of the dialysis bag; FcCONH2 is ferrocene carboxyethyl diamine; GelMA is methacrylated gelatin; ROS is reactive oxygen species.

[0007] A method for preparing an oxidation stress microenvironment-responsive intelligent semi-convertible hydrogel, comprising the following steps: Step 1. Prepare PAA-CD Dissolve dry PAA in DMF, then add PyBOP and ET3N, and continuously stir at room temperature, then add CD, and stir for reaction to obtain a modified polymer; precipitate the modified polymer with acetone, centrifuge, and collect the white solid modified polymer; dissolve the collected white solid modified polymer in ultrapure water, then perform water dialysis to obtain a polymer solution, named A polymer solution, and freeze-dry the A polymer solution to obtain PAA-CD; Step 2. Prepare PAA-Fc Dissolve the dried PAA in DMF, then add PyBOP and ET3N, and stir well at room temperature to obtain solution B; dissolve FcCONH2 in DMF and stir well at room temperature to obtain solution C; add solution C dropwise to solution B, stir well, and then dilute with ultrapure water; perform water dialysis on the diluted mixture to obtain a polymer solution, named D polymer solution. Freeze-dry the D polymer solution to obtain PAA-Fc, and store it in argon. Step 3: Prepare a semi-converted hydrogel and load the drug Dissolve the PAA-CD prepared in Step 1 in water to obtain a 4% (mass percentage) PAA-CD solution, dissolve the PAA-Fc prepared in Step 2 in water to obtain a 4% (mass percentage) PAA-Fc solution, dissolve GelMA in water to obtain a 30% (mass percentage) GelMA solution. Take the above three solutions and mix them with the loaded drug in a volume ratio of 2:1:1 to finally obtain a semi-converted hydrogel for intelligent drug delivery and controlled release in response to the oxidative stress microenvironment, and make the loaded drug account for 10% of the total volume of the semi-converted hydrogel.

[0008] In the above 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 added PyBOP is 0.4 g, the volume of added ET3N is 80 μL, and the continuous stirring time of the mixture of DMF dissolved with PAA and added PyBOP and ET3N is 2 hours; the mass of added CD is 0.79 g, and the stirring reaction time after adding CD is 24 hours; the amount of acetone used is 200 mL, and the centrifugation condition is centrifugal rotation at room temperature with a rotation speed of 8900 rpm; the time for dissolving the white solid modified polymer in ultrapure water is 12 h, the water dialysis uses a dialysis tube with a MWCO of 14 kDa, and the dialysis time is 144 hours; the temperature for freeze-drying the A polymer solution is -80 °C, and the pressure is 1.3 - 13 Pa.

[0009] In the above Step 2, the molecular weight of PAA is 450,000, the mass is 500 mg, the volume of DMF for dissolving PAA is 40 mL, the mass of added PyBOP is 0.4 g, the volume of added ET3N is 80 μL, and the continuous stirring time of the mixture of DMF dissolved with PAA and added PyBOP and ET3N is 2 hours; the mass of added FcCONH2 is 0.19 g, the volume of DMF for dissolving FcCONH2 is 10 mL, and the stirring time of the mixture of the two is 2 hours; add solution C dropwise to solution B, and the stirring time is 24 hours; for water dialysis of the diluted mixture, use a dialysis tube with a MWCO of 14 kDa, and the dialysis time is 144 hours; the temperature for freeze-drying the D polymer solution is -80 °C, and the pressure is 1.3 - 13 Pa.

[0010] The intelligent semi-convertible hydrogel responsive to the oxidative stress microenvironment obtained according to the above preparation method can be applied in the following aspects: Preparing products for treating spinal cord injury; Preparing products for promoting nerve regeneration; Preparing products for promoting the recovery of motor function after spinal cord injury; Preparing products for improving the microenvironment of spinal cord injury; Preparing products for enhancing the repair effect of spinal cord injury.

[0011] The working process and principle of the present invention: The present invention constructs a stimulus-responsive network, which uses polyacrylic acid (PAA) as the main chain, and grafts cyclodextrin (CD) and ferrocene (Fc) onto it respectively to construct the PAA-CD and PAA-Fc networks. In the normal state, PAA-CD and PAA-Fc combine in the form of host-guest interaction to form a gel state of about 100 Pa. The preparation method of the present invention uses methacrylated gelatin (GelMA) as the backbone network and the above-mentioned PAA-CD and PAA-Fc host-guest system as the stimulus-responsive network to construct a semi-convertible hydrogel system.

[0012] After adding H2O2 (the main component of ROS) to the semi-convertible hydrogel system, PAA-Fc is 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 backbone state. Therefore, the semi-convertible hydrogel system realizes the transition from the gel state to the semi-sol-gel state under the action of H2O2. After further adding reducing substances, Fc + is reduced to Fc, resulting in the re-host-guest assembly of PAA-CD and PAA-Fc to form a gel.

[0013] The above semi-convertible hydrogel can respond to the oxidative stress stage after spinal cord injury (a large amount of ROS is generated in this stage), change from the gel state to the semi-gel-sol state to explosively release the drugs encapsulated therein, and at the same time consume ROS to reduce the harm of secondary injury to spinal cord tissue. After the oxidative stress stage ends, due to the decrease in the content of ROS, the semi-convertible hydrogel in the semi-sol-gel state can change to the sol state under the action of reducing substances such as glutathione and glucose in the body, realizing the long-term and slow release of drugs.

[0014] The beneficial effects of the present invention: Compared with the prior art, the oxidative stress microenvironment-responsive intelligent semi-switchable hydrogel can be prepared by the preparation method described in the present invention. Compared with traditional hydrogels, this device has ROS-responsive activity, can scavenge ROS while triggering the explosive release of drugs during the oxidative stress stage, and realizes the slow and long-term release of drugs after the ROS concentration decreases, that is, realizes the intelligent drug controlled release based on the oxidative stress microenvironment. It avoids the systemic drug toxicity that may be caused by the intravenous systemic administration method, solves the defect that it cannot achieve high local drug enrichment, and also eliminates the short half-life and easy rapid clearance after local drug injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the mutual conversion process of the stimulus-responsive network in the semi-switchable hydrogel prepared by the method described in Example 1 of the present invention.

[0016] Figure 2 In vitro drug release curve of the semi-switchable hydrogel prepared by the method described in Example 1 of the present invention.

[0017] Figure 3 Fluorescence microscopy image of the in vitro ROS scavenging ability of the semi-switchable hydrogel prepared by the method described in Example 1 of the present invention, where Hoechst is hexidium nicotinate and ROS is reactive oxygen species.

[0018] Figure 4 Fluorescence microscopy image of the in vitro angiogenesis-promoting ability of the semi-switchable hydrogel prepared by the method described in Example 1 of the present invention.

[0019] Figure 5 Fluorescence microscopy image of the in vitro ability of the semi-switchable hydrogel prepared by the method described in Example 1 of the present invention to promote the differentiation of neural stem cells, where Dapi is 4',6-diamidino-2-phenylindole and MAP2 is microtubule-associated protein 2.

[0020] Figure 6 Rat footprint imprint analysis image of the ability of the semi-switchable hydrogel prepared by the method described in Example 1 of the present invention to promote the recovery of motor function after spinal cord injury in rats.

[0021] Figure 7 Photo of the substance state after precipitation during the preparation of PAA-CD in the background technology of the present invention.

[0022] Figure 8 Photo of the substance state after blending PAA-CD and PAA-Fc in the background technology of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0023] Example 1 of the present invention.

[0024] A method for preparing an intelligent semi-conversion hydrogel responsive to an oxidative stress microenvironment, comprising the following steps: Step 1: Prepare PAA-CD Dissolve 500 mg of dry PAA with a molecular weight of 450,000 in 20 mL of DMF, then add 0.4 g of PyBOP and 80 μL of ET3N, and continuously stir at room temperature for 2 hours. Then add 0.79 g of CD and stir and react for 24 hours to obtain a modified polymer. Precipitate the modified polymer with 200 mL of acetone, and after centrifugal rotation at 8900 rpm at room temperature, collect the white solid modified polymer. Dissolve the collected white solid modified polymer in ultrapure water for 12 h, and then perform water dialysis for 144 hours using a dialysis tube with an MWCO of 14 kDa to obtain a polymer solution, named A polymer solution. Freeze-dry the A polymer solution at -80°C under a pressure of 1.3 - 13 Pa to obtain PAA-CD; Step 2: Prepare PAA-Fc Dissolve 500 mg of dry PAA with a molecular weight of 450,000 in 40 mL of DMF, then add 0.4 g of PyBOP and 80 μL of ET3N, and stir for 2 hours to obtain solution B. Dissolve 0.19 g of FcCONH2 in 10 mL of DMF and stir for 2 hours to obtain solution C. Dropwise add solution C to solution B at room temperature, stir the reaction mixture for 24 hours, and then dilute with ultrapure water. Perform water dialysis on the mixture for 144 hours using a dialysis tube with an MWCO of 14 kDa to obtain a polymer solution, named D polymer solution. Freeze-dry the D polymer solution at -80°C under a pressure of 1.3 - 13 Pa to obtain PAA-Fc, and store it in argon; Step 3: Prepare a semi-conversion hydrogel and load drugs Select platelet-rich plasma lysate (PL) + 500 ng / ml nerve growth factor (NGF) as the drug to be loaded in this example.

[0025] Dissolve the PAA-CD prepared in Step 1 in water to obtain a 4% (by mass) PAA-CD solution, dissolve the PAA-Fc prepared in Step 2 in water to obtain a 4% (by mass) PAA-Fc solution, dissolve GelMA in water to obtain a 30% (by mass) GelMA solution. Sequentially take 20 ml, 10 ml, and 10 ml of the above three solutions, and mix them together with platelet-rich plasma lysate (PL) + nerve growth factor (NGF) at 500 ng / ml to finally obtain a semi-convertible hydrogel with intelligent drug delivery and controlled release in response to the oxidative stress microenvironment, and make the platelet-rich plasma lysate (PL) + nerve growth factor (NGF) at 500 ng / ml account for 10% of the total volume of the semi-convertible hydrogel.

[0026] Please refer to Figure 1 and Figure 2 , which is Example 2 of the present invention. For the in vitro drug release experiment of the semi-convertible hydrogel prepared according to Example 1, it is used to verify the drug release ability of the hydrogel loaded with drugs.

[0027] According to the preparation method described in Example 1, select platelet-rich plasma lysate (PL) + nerve growth factor (NGF) at 500 ng / ml as the drug-loaded preparation to prepare a drug release model to study the drug loading of the semi-convertible hydrogel and its drug release behavior in cell culture medium. The NGF protein concentration is measured by an enzyme-linked immunosorbent assay (ELISA) kit to evaluate the release of the total drug.

[0028] a. Drug release under reducing conditions Prepare a semi-convertible hydrogel with intelligent drug delivery and controlled release in response to the oxidative stress microenvironment according to the preparation method described in the present invention. Take 400 μL of the semi-convertible hydrogel and place it in a test tube, and add 2.5 mL of DMEM medium. Replace the DMEM medium every 10 h, and take 20 μL of the replaced DMEM medium for measuring the NGF concentration by an ELISA kit to calculate the NGF release.

[0029] b. Drug release under oxidative conditions Prepare a semi-convertible hydrogel with intelligent drug delivery and controlled release in response to the oxidative stress microenvironment according to the preparation method described in Example 1. Take 400 μL of the semi-convertible hydrogel and place it in a test tube, add 2.5 mL of DMEM medium, and at the same time add the oxidative reactant H2O2 to the test tube (prepare multiple identical test tubes). Take 1 test tube to be tested every 10 h and use an ELISA kit to detect the NGF protein concentration to evaluate the protein release at different time points and under different oxidative conditions.

[0030] Please refer to Figure 1 and Figure 3As shown, this is Example 3 of the present invention. The determination of the in vitro ROS scavenging ability of the semi-converted hydrogel prepared according to Example 1 belongs to the application in the preparation of products for improving the microenvironment of spinal cord injury.

[0031] Construction of in vitro oxygen-glucose deprivation / reoxygenation model (OGD / R): After the cell confluence was satisfactory, the original culture medium was aspirated completely, and after washing with PBS, sugar-free DMEM medium was added. Then the culture plate was placed in an airtight box, and anaerobic gas with a volume fraction of 95% N2 and 5% CO2 was introduced and placed in an incubator at 37°C for 12 h. After that, the culture medium was changed to normal medium and continued to be incubated in an incubator at 37°C with a volume fraction of 5% CO2 for 12 h, thereby constructing the in vitro oxygen-glucose deprivation / reoxygenation model (OGD / R).

[0032] Cell grouping and treatment: Rat brain microvascular endothelial cells (RBMVEC) were selected as the in vitro model cells. The cells were divided into a blank group, an OGD / R group, and a hydrogel group. The blank group was not treated with anything. The OGD / R group was not treated with anything except for OGD / R. The hydrogel group was added with the semi-converted hydrogel without drugs prepared according to the present invention in the culture medium while undergoing OGD / R treatment. After the OGD / R treatment was completed, DCFH-DA fluorescent probe was loaded and the ROS scavenging situation was observed under a fluorescence microscope.

[0033] Please refer to Figure 1 and Figure 4 As shown, this is Example 4 of the present invention. The verification of the in vitro angiogenesis-promoting ability of the semi-converted hydrogel prepared according to Example 1 belongs to the application in the preparation of products for treating spinal cord injury and the preparation of products for improving the repair effect of spinal cord injury.

[0034] Cell grouping: Blank group, hydrogel group, hydrogel + drug treatment group. The blank group was not treated with anything. The hydrogel group was added with the semi-converted hydrogel without drugs prepared according to the present invention after inoculating the cells. The hydrogel + drug treatment group was added with the semi-converted hydrogel with drugs prepared according to the present invention after inoculating the cells.

[0035] Use a pre-cooled pipette tip at 4°C to aspirate Matrigel (matrix gel), spread it evenly in a pre-cooled 96-well plate at 4°C, and place it in an incubator at 37°C for 1 h to solidify the Matrigel. Select the logarithmic phase and RBMVEC with good growth status, digest and resuspend them to prepare a cell suspension of 2*10 4 / mL. 100 μL of the cell suspension was evenly inoculated into each well of the 96-well plate coated with Matrigel and placed in an incubator at 37°C with 5% CO2 for incubation. After 6 h, the formation of tubules on the surface of Matrigel was observed under a microscope, photographed, and pictures were collected.

[0036] Please refer to Figure 1 andFigure 5 As shown in the figure, this is Example 5 of the present invention, which verifies the ability of the semi-converted hydrogel prepared according to Example 1 to promote the differentiation of neural stem cells in vitro. It belongs to the application in preparing products for treating spinal cord injury, preparing products for promoting nerve regeneration, and preparing products for improving the repair effect of spinal cord injury.

[0037] Cell grouping and treatment: Primary rat hippocampal neural stem cells were selected. They were divided into a blank group, a hydrogel group, and a hydrogel + drug treatment group. The blank group was not treated with anything. After inoculating the cells, the hydrogel group was added with the semi-converted hydrogel without drugs prepared by the present invention, and the hydrogel + drug treatment group was added with the semi-converted hydrogel with drugs prepared by the present invention. They were cultured for 5 days respectively.

[0038] Cell immunofluorescence: (1) Cell fixation: Remove the culture medium, wash 3 times with PBS (phosphate buffer solution), add 500 μL of 4% paraformaldehyde to fix the cells at room temperature for 20 min.

[0039] (2) Cell permeabilization: After cell fixation, wash 3 times with PBS, 5 min for each wash, and then add 500 μL of 0.3% Triton X-100 to incubate at room temperature for 15 min for permeabilization.

[0040] (3) Blocking: Remove the permeabilization solution, wash 3 times with PBS, 5 min for each wash, add 500 μL of 1% BSA solution to block at room temperature for 1 h.

[0041] (4) Primary antibody incubation: After removing the blocking solution, wash 3 times with PBS, 5 min for each wash, add 500 μL of MAP2 diluted 1:500, and incubate overnight at 4°C.

[0042] (5) Secondary antibody incubation: Remove the primary antibody, wash 3 times with PBS, 5 min for each wash, add 500 μL of the fluorescently labeled secondary antibody diluted 1:500, and incubate in the dark at room temperature for 2 h.

[0043] (6) Nuclear staining: Remove the secondary antibody, wash 3 times with PBS, 5 min for each wash, add 100 μL of 1 μmol / L DAPI staining solution, and incubate in the dark for 5 min.

[0044] (7) Mounting: Aspirate the DAPI staining solution, wash 3 times with PBS, 5 min for each wash, pick up the cell slide with forceps, and invert it onto a glass slide coated with anti-fluorescence quenching mounting medium.

[0045] (8) Fluorescence observation: The glass slide was stored in the dark at 4°C, observed under a fluorescence microscope or a confocal fluorescence microscope, and photographed.

[0046] Please refer to Figure Figure 1 and Figure 6As shown in Example 6 of the present invention, the semi-conversion hydrogel prepared according to Example 1 promotes the recovery of motor function after spinal cord injury in rats. The invention belongs to the product used in preparing a product for promoting the recovery of motor function after spinal cord injury.

[0047] Implementation of rat spinal cord clamp injury model: All rats were kept at constant temperature and humidity, and were allowed to feed and drink water freely. After 3 days of adaptive feeding, the model was established. The rats were weighed and anesthetized with 1% sodium pentobarbital (40 mg / kg) by intraperitoneal injection. The model was established after the rats entered deep anesthesia. After the skin of the back was prepared, the rats were fixed in a prone position. The highest protrusion of the rat's back spine (T10 spinous process) was disinfected with iodine tincture and a sterile medium sheet was laid. With the highest point of the back spine as the center, the skin was cut longitudinally along the midline of the back for about 3.0 cm, and the subcutaneous tissue, fascia and muscle tissue on both sides of the spinous process were separated layer by layer to expose the T9-T10 spinous process. The expander expanded the accessory tissue to fully expose the dorsal structure of the spine. After the spinous process was bitten off with a bone rongeur, the posterior lamina of T9 and T10 was carefully bitten off along the interlaminar space, and both sides reached the articular process joint to fully expose the spinal dura mater. If there is bleeding, press with gauze to stop bleeding, and wash the incision with saline to make the surgical field clean. Insert 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 to compress for 60s. At this time, the rat's hind limbs and tail twitch rapidly, and then the motor function of both lower limbs is lost, indicating that the model is successful. Slowly withdraw the clamp, and dark red congestion can be seen under the dura mater at the spinal cord compression site. Compression is performed again to stop bleeding, the wound is gently washed with saline, sutured, and the sutured incision is disinfected again with iodine. After surgery, 2ml of 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 motor function of both lower limbs is lost. Penicillin is continuously injected intramuscularly for 5 days after surgery to prevent postoperative infection, and the bladder is massaged twice a day to assist urination (1 week), ensuring sufficient feed and water, changing bedding frequently, and trying to avoid unnecessary rat deaths.

[0048] Animal grouping and treatment: spinal cord injury group and treatment group. The spinal cord injury group was not treated after clamp injury, and the wound was sutured normally; the treatment group was covered with the hydrogel containing therapeutic drugs prepared by the present invention on the surface of the spinal cord after clamp injury. The two groups of rats were raised in a special breeding room for experimental rats in a clean, ventilated, constant temperature and humidity environment for 4 weeks.

[0049] To observe the coordination of hindlimb movement in rats after spinal cord injury, we conducted footprint experiments on three groups of rats. Before the experiment, a long and narrow transparent channel about 80 cm long and about 8 cm wide was prepared. A long strip of white paper was pre-laid at the bottom of the channel, and a small amount of animal feed was placed at one end of the channel. The rat was picked up and non-toxic red ink was applied to its hindlimbs. After the rat calmed down, it was placed at the other end of the channel and allowed to move forward freely along the channel. At this time, the footprints of the rat's front and hind limbs would be printed on the pre-laid white paper. If the rat stopped in the channel, the white paper was replaced and the experiment was repeated to ensure that the rat walked continuously in the channel. After walking, the white paper with footprints was collected, and the footprints of the rat were observed and analyzed to evaluate the recovery of the walking function of the rat's limbs. The figure above shows the footprints of the rats in the spinal cord injury group, and the figure below shows the footprints of the rats in the treatment group. Clear paw prints can be seen in the rats in the treatment group, indicating that the recovery of hindlimb motor function is obvious.

Claims

1. A preparation method of an intelligent semi-convertible hydrogel responsive to oxidative stress microenvironment, characterized in that, It includes the following steps: Step 1: Prepare PAA-CD Dissolve the dried PAA in DMF, then add PyBOP and ET3N, and continuously stir at room temperature. Then add CD and stir for reaction to obtain a modified polymer; Precipitate the modified polymer with acetone, centrifuge, and collect the white solid modified polymer; dissolve the collected white solid modified polymer in ultrapure water, then perform water dialysis to obtain a polymer solution, named A polymer solution. Freeze-dry the A polymer solution to obtain PAA-CD; Step 2: Prepare PAA-Fc Dissolve the dried PAA in DMF, then add PyBOP and ET3N, and stir well at room temperature to obtain solution B; dissolve FcCONH2 in DMF and stir well at room temperature to obtain solution C; Dropwise add solution C to solution B and stir well, then dilute with ultrapure water; perform water dialysis on the diluted mixture to obtain a polymer solution, named D polymer solution. Freeze-dry the D polymer solution to obtain PAA-Fc and store it in argon; Step 3: Prepare a semi-converted hydrogel and load drugs Dissolve the PAA-CD prepared in Step 1 in water to obtain a 4% (mass percentage) PAA-CD solution, dissolve the PAA-Fc prepared in Step 2 in water to obtain a 4% (mass percentage) PAA-Fc solution, dissolve GelMA in water to obtain a 30% (mass percentage) GelMA solution. Take the above three solutions and mix them with the loaded drug in a volume ratio of 2:1:1 to finally obtain a semi-converted hydrogel with oxidative stress microenvironment-responsive intelligent drug delivery and controlled release, and make the loaded drug account for 10% of the total volume of the semi-converted hydrogel.

2. The preparation method of an intelligent semi-convertible hydrogel responsive to oxidative stress microenvironment according to claim 1, characterized in that: In the above 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 added PyBOP is 0.4 g, the volume of added ET3N is 80 μL, and the continuous stirring time of the mixture of DMF dissolved with PAA and added PyBOP and ET3N is 2 hours; the mass of added CD is 0.79 g, and the stirring reaction time after adding CD is 24 hours; the amount of acetone used is 200 mL, and the centrifugation condition is centrifugal rotation at room temperature with a rotation speed of 8900 rpm; the time for dissolving the white solid modified polymer in ultrapure water is 12 h, the water dialysis uses a dialysis tube with a MWCO of 14 kDa, and the dialysis time is 144 hours; the temperature for freeze-drying the A polymer solution is -80 °C, and the pressure is 1.3 - 13 Pa.

3. The preparation method of an intelligent semi-convertible hydrogel responsive to oxidative stress microenvironment according to claim 1, characterized in that: In the second step described above, the molecular weight of PAA is 450000, the mass is 500 mg, the volume of DMF for dissolving PAA is 40 mL, the mass of added PyBOP is 0.4 g, the volume of added ET3N is 80 μL, and the continuous stirring time of the mixture of DMF dissolving PAA and added PyBOP and ET3N is 2 hours; the mass of added FcCONH2 is 0.19 g, the volume of DMF for dissolving FcCONH2 is 10 mL, and the stirring time of the mixture of the two is 2 hours; the C solution is added dropwise to the B solution, and the stirring time is 24 hours; the diluted mixture is subjected to water dialysis using a dialysis tube with MWCO of 14 kDa, and the dialysis time is 144 hours; the temperature for freeze-drying the D polymer solution is -80 °C, and the pressure is 1.3 - 13 Pa.

4. Application of the oxidative stress microenvironment-responsive intelligent semi-conversion hydrogel obtained by the preparation method according to claim 1 in the treatment of spinal cord injury.

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