A hydrogel and its application in ischemic heart disease

Abstract

The present invention discloses a hydrogel and its application in ischemic heart disease, relating to the technical field of myocardial infarction repair. The present invention provides a multifunctional hydrogel with injectability, enabling in-situ injection. This hydrogel combines the comprehensive characteristics of improving the conductivity of the infarcted area, generating oxygen, scavenging ROS, promoting M2 polarization of macrophages, and promoting angiogenesis. Its excellent performance is outstanding in alleviating MI / RI, improving the local microenvironment, and enhancing myocardial repair function, providing a new direction for the treatment of heart injury. At the same time, the CSA-δ hydrogel formed after loading drugs has the ability of drug sustained release, which enables the drugs to be released continuously and stably, thereby prolonging the drug action time.

Description

Technical Field

[0001] The present invention relates to the technical field of myocardial infarction repair, and in particular, to a hydrogel and its application in ischemic heart disease. Background Art

[0002] Myocardial infarction (MI) is a major disabling and lethal disease globally. Its pathogenesis is mainly related to the rupture of coronary atherosclerotic plaques or endothelial erosion, especially with a higher incidence in elderly patients. Although the current treatment strategy focuses on myocardial reperfusion, the reperfusion process itself can trigger further death of myocardial cells (i.e., reperfusion injury), leading to progressive loss of cardiac function and heart failure. Due to the extremely low proliferative capacity of adult mammalian myocardial cells, existing treatment methods are difficult to effectively repair the large number of missing myocardial cells after myocardial infarction reperfusion (MI / R).

[0003] The occurrence of myocardial reperfusion injury involves various complex mechanisms, including calcium overload, inflammatory response, mitochondrial dysfunction, and oxidative stress. These mechanisms work together to further exacerbate myocardial cell loss, expand the necrotic area, and significantly increase the risk of myocardial fibrosis. As the fibrosis process intensifies, the structure and function of the heart gradually deteriorate, which may lead to left ventricular remodeling, decline in cardiac function, and ultimately develop into chronic heart failure. In addition, reperfusion injury may also trigger severe arrhythmias, further endangering the life safety of patients. These concurrent problems significantly affect the overall effect of reperfusion therapy and become an important obstacle restricting the improvement of the long-term prognosis of patients.

[0004] Recent studies have shown that by gene editing to target and inhibit the key factor CBX7, myocardial cells can be induced to re-enter the proliferative state, thereby reducing the degree of injury in young mice during ischemic reperfusion injury. However, for the disease of MI, more attention should be paid to the biological characteristics of the elderly population. In addition, gene therapy methods are limited by the long clinical translation cycle and high technical threshold. In contrast, developing small molecule inhibitors targeting CBX7 has significant advantages such as strong specificity and clear action mechanism, and is more easily clinically translated.

[0005] However, traditional oral or injection administration methods have problems such as poor targeting and low utilization rate, and there is an urgent need for innovative drug delivery systems. The in-situ sustained-release strategy based on biomaterials provides a new direction to break through this bottleneck. Gel materials have been widely used in the field of tissue engineering due to their soft and moist texture and the characteristics of mimicking the human ECM. Research reveals that this biomaterial can not only provide the necessary mechanical support for myocardial repair but also act as a transmission medium for cells or drugs, playing a key role. With the in-depth understanding of the changes in the myocardial microenvironment after myocardial infarction, intelligent and functional hydrogels have been developed for cardiac repair. Zou et al. designed a Gel@Exo conductive hydrogel to enhance the electrical coupling between cardiomyocytes. The application of this hydrogel effectively improved cardiac function, including increasing the ejection fraction and fractional shortening rate, and reducing the fibrotic area. Li et al. developed a microRNA-21-5p delivery system based on functionalized mesoporous silica nanoparticles, which can achieve controllable delivery through the injection of hydrogel and promote angiogenesis, significantly reducing the infarct area. However, problems such as hypoxia, oxidative stress, insufficient angiogenesis, and insufficient electrical coupling still exist, and a good environment for myocardial regeneration cannot be provided.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The purpose of the present invention is to provide a hydrogel and its application in ischemic heart disease to improve the myocardial microenvironment (problems such as hypoxia, oxidative stress, insufficient angiogenesis, and insufficient electrical coupling) from the perspective of gel materials, and provide a good environment for myocardial regeneration; in addition, by loading small molecule inhibitors with the hydrogel material, the disadvantages of traditional administration methods are avoided, the residence time of small molecules in the heart is increased, thereby increasing the utilization rate, and providing a new solution for the treatment after myocardial reperfusion injury.

[0008] The present invention is implemented as follows:

[0009] In the first aspect, the present invention provides a hydrogel, which includes: an aqueous sodium alginate solution containing melanin nanoparticles, a reducing agent, and an aqueous calcium gluconate solution containing calcium peroxide nanoparticles. The volume ratio of the aqueous sodium alginate solution containing melanin nanoparticles to the aqueous calcium gluconate solution containing calcium peroxide nanoparticles is 2.5:1 - 3.5:1. The final concentration of melanin nanoparticles in the hydrogel is 0.8 - 1.5 mg / ml. The mass fraction of sodium alginate in the aqueous sodium alginate solution is 2.5%–3.5%. The concentration of calcium peroxide nanoparticles in the aqueous calcium gluconate solution is 0.8–1.5 mg / ml. The mass fraction of calcium gluconate in the aqueous calcium gluconate solution is 2.5%–3.5%. The ratio of the aqueous sodium alginate solution to the reducing agent is 1 mL:0.4–0.6 mg, and the reducing agent is selected from ascorbic acid, glutathione, or cysteine.

[0010] In a second aspect, the present invention also provides a method for preparing a hydrogel, which comprises the following steps: adding a reducing agent to an aqueous sodium alginate solution containing melanin nanoparticles, and then mixing with an aqueous calcium gluconate solution containing calcium peroxide nanoparticles to obtain a mixed solution.

[0011] In a third aspect, the present invention also provides a hydrogel containing a small molecule inhibitor, which comprises the above-mentioned hydrogel or a hydrogel prepared by the method for preparing the above-mentioned hydrogel and a small molecule inhibitor, and the small molecule inhibitor is δ-Amyrenone.

[0012] In a fourth aspect, the present invention also provides a method for preparing a hydrogel containing a small molecule inhibitor, which comprises: first adding δ-Amyrenone to an aqueous calcium gluconate solution containing calcium peroxide nanoparticles, mixing to obtain an aqueous calcium gluconate solution containing δ-Amyrenone and calcium peroxide nanoparticles, and then mixing the aqueous sodium alginate solution containing melanin nanoparticles, the reducing agent with the aqueous calcium gluconate solution containing δ-Amyrenone and calcium peroxide nanoparticles.

[0013] In a fifth aspect, the present invention also provides the use of the hydrogel or the hydrogel containing a small molecule inhibitor in the preparation of a drug for preventing and / or treating ischemic heart disease.

[0014] The present invention has the following beneficial effects:

[0015] The present invention provides a hydrogel (CSA), which has injectability and can achieve in-situ injection. In addition, the CSA hydrogel exhibits strong long-term oxygen-producing ability and has significant potential to alleviate the hypoxic environment in the myocardial infarction area.

[0016] The CSA hydrogel exhibits good electrical conductivity and can provide an electrical environment similar to myocardial tissue. By introducing melanin and calcium peroxide, the electrical conductivity of the CSA hydrogel has been significantly improved, which is consistent with the electrical signal conduction level of natural myocardial tissue, showing great application potential in cardiac tissue engineering repair.

[0017] The CSA hydrogel exhibits good biocompatibility in cell experiments. Even at a concentration of 10%, no obvious cytotoxicity was observed, and the cell viability remained good, providing strong biocompatibility support for its further application in animal experiments.

[0018] The CSA hydrogel can effectively scavenge ROS, reduce oxidative stress, and decrease the damage of cardiomyocytes and human umbilical vein endothelial cells in a ROS environment. In addition, the CSA hydrogel can promote the proliferation of vascular endothelial cells and the M2 polarization of macrophages, showing a certain cardioprotective effect. Specifically, it can reduce the infarct area and alleviate the degree of cardiac fibrosis, increase the ventricular wall thickness, and thus have a positive impact on cardiac function.

[0019] Meanwhile, the CSA-δ hydrogel (CSA containing δ-Amyrenone) formed after loading drugs has the ability of drug sustained release, which enables the drugs to be continuously and stably released, thereby prolonging the drug action time; the CSA-δ hydrogel not only significantly promotes the regeneration and repair of cardiomyocytes, but also further enhances its repair effect by constructing a microenvironment conducive to myocardial recovery. The experimental results of the present invention indicate that the CSA-δ hydrogel has significant potential in the field of myocardial infarction repair, provides a new treatment strategy for the recovery of cardiac function after MI / R, and has important clinical application prospects. Brief Description of the Drawings

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as a limitation of the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0021] Figure 1 For the preparation and characterization of CaO2 NPs ((a) XRD pattern of CaO2; (b) FTIR spectrum of CaO2; (c) SEM image of CaO2, scale bar = 500 nm);

[0022] Figure 2 For the preparation and characterization result images of MNPs ((a) FTIR image of MNPs; (b) SEM image of MNPs, scale bar = 500 nm);

[0023] Figure 3 For the verification of the fluidity and injectability of SA and CSA hydrogels (the fluidity display of the two hydrogels in an inverted container and the experimental results of syringe writing);

[0024] Figure 4 For the rheological and FT-IR characterization of the hydrogels ((a) Rheological analysis of SA and CSA hydrogels; (b) Infrared spectra of calcium peroxide, ascorbic acid, melanin, SA, and CSA hydrogels);

[0025] Figure 5Conductivity results of SA and CSA hydrogels ((a) Brightness changes of LED bulbs in SA and CSA hydrogels. (b) Conductivity analysis of SA and CSA hydrogels. Data in the figure represent Mean±SD. Significant difference: **p<0.01);

[0026] Figure 6 Drug release curve of CSA-δ hydrogel;

[0027] Figure 7 Determination results of various parameters of hydrogel degradation ((a) Oxygen production curve of CSA hydrogel; (b) Degradation curve of CSA hydrogel; (c) Statistical results of pH value of CSA hydrogel products);

[0028] Figure 8 Biocompatibility test results of hydrogels (The green line in the figure represents 80% cell viability);

[0029] Figure 9 ROS scavenging level test results of hydrogels ((a) Effects of SA and CSA hydrogels on scavenging DPPH free radicals; (b) Representative fluorescence images of the activity of total intracellular reactive oxygen species (DCFH-DA) after treatment with SA and CSA hydrogels; Scale bar = 100um);

[0030] Figure 10 Angiogenesis promotion ability test results of hydrogels ((a) CCK-8 assay results of HUVECs treated with SA or CSA hydrogels for 3 days in a ROS environment; (b) Western-blot detection results of angiogenesis-related proteins (VWF, Vcam1, and CD31) in HUVECs treated with SA or CSA hydrogels for 3 days in a ROS environment. Data in the figure represent Mean±SD. Significant difference: ****p<0.0001);

[0031] Figure 11 Macrophage polarization promotion ability test results of hydrogels (Representative immunofluorescence images showing the expression of CD86 and CD206 in THP-1-derived macrophages under different treatments; Scale bar = 100 um);

[0032] Figure 12 In vivo ROS scavenging ability test of hydrogels (Representative fluorescence images of superoxide anion radical activity (DHE, red) in heart sections 1 day after treatment under different conditions; Scale bar = 100 um);

[0033] Figure 13 In vivo angiogenesis promotion ability test results of hydrogels (Representative fluorescence images of α-smooth muscle actin (α-SMA, red) in heart sections 28 days after treatment under different conditions; Scale bar = 100 um);

[0034] Figure 14 Figure showing the results of detecting the ability of promoting macrophage polarization in hydrogels ((a) Immunofluorescence images of CD86 and CD206 in myocardial tissue on the 1st day after treatment. (b) Immunofluorescence images of CD86 and CD206 in myocardial tissue on the 3rd day after treatment; scale bar = 50 μm);

[0035] Figure 15 Results of analyzing cardiac function indexes of rats' hearts by echocardiography 28 days after treatment ((a) M-mode echocardiogram images of rats in each group 28 days after treatment, which intuitively reflect the structure and motion state of the heart; (b) Doppler echocardiogram images, which further reveal the characteristics of cardiac hemodynamics; (c - e) Quantitative comparison of left ventricular function of rats in different groups 28 days after treatment. Data in the figure represent Mean±SD. Significant differences: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0036] Figure 16 Figure showing the results of Masson staining of hearts in each group 28 days after MI / R ((a) The figure shows the results of Masson trichrome staining of cardiac tissue sections in each group. The scale bar for the whole section is 1 mm, and the scale bar for the regional screenshot is 100 μm; (b) Based on these Masson trichrome staining images, we quantitatively analyzed the thickness of the anterior wall of the left ventricle. Data in the figure represent Mean±SD. Significant differences: *p<0.05, ***p<0.001, ****p<0.0001);

[0037] Figure 17 HE staining images of various organs of rats in each group 28 days after MI / R, scale bar = 200 μm. Detailed implementation manners

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0039] CSA-δ hydrogel: It is a hydrogel containing δ-Amyrenone.

[0040] MI / RI: Myocardial infarction reperfusion injury.

[0041] CSA: Injectable composite sodium alginate hydrogel.

[0042] SA: Injectable sodium alginate hydrogel.

[0043] MNPs: Melanin nanoparticles.

[0044] In a first aspect, the present invention provides a hydrogel, which comprises: an aqueous sodium alginate solution containing melanin nanoparticles, a reducing agent, and an aqueous calcium gluconate solution containing calcium peroxide nanoparticles. The volume ratio of the aqueous sodium alginate solution containing melanin nanoparticles to the aqueous calcium gluconate solution containing calcium peroxide nanoparticles is 2.5:1 - 3.5:1. The final concentration of melanin nanoparticles in the hydrogel is 0.8 - 1.5 mg / ml. The mass fraction of sodium alginate in the aqueous sodium alginate solution is 2.5% - 3.5%. The concentration of calcium peroxide nanoparticles in the aqueous calcium gluconate solution is 0.8 - 1.5 mg / ml. The mass fraction of calcium gluconate in the aqueous calcium gluconate solution is 2.5% - 3.5%. The ratio of the aqueous sodium alginate solution to the reducing agent is 1 mL:0.4 - 0.6 mg, and the reducing agent is selected from ascorbic acid, glutathione, or cysteine.

[0045] CaO2 (calcium peroxide), as a widely used solid oxygen-releasing agent, exhibits a more gentle and persistent oxygen-releasing characteristic compared to H2O2, which can effectively prevent the rapid accumulation of local reactive oxygen species and thus maintain the redox homeostasis. However, due to their large volume, conventional-sized CaO2 particles are prone to agglomeration when exposed to water, significantly slowing down the oxygen release rate. In contrast, nano-scale CaO2 particles can not only greatly accelerate the reaction kinetics but also effectively overcome the aggregation phenomenon between particles, optimizing their performance. The preparation method of calcium peroxide nanoparticles refers to the methods reported in the prior art such as (HUANG Y, FU Z, WANG H, etc. Calcium Peroxide-Based Hydrogels Enable Biphasic Release of Hydrogen Peroxide for Infected Wound Healing [J]. Advanced science (Weinheim, Baden-Wurttemberg, Germany), 2024, 11(40): e2404813). Calcium peroxide nanoparticles can slowly release oxygen to help supplement the oxygen in the infarcted area.

[0046] Ascorbic acid can regulate the acid-base balance and prevent the adverse effects of the alkaline environment generated by calcium peroxide on tissues.

[0047] The natural MNPs contained in squid ink are regarded as an important bioactive substance, possessing excellent protective functions, capable of effectively shielding ultraviolet radiation, resisting reactive oxygen species attacks, neutralizing toxins, and chelating metal ions. These naturally occurring melanins not only have good biocompatibility and biodegradability but also exhibit excellent redox activity and semi-conductive properties, thus showing great potential in biomedical applications. In this invention, melanin nanoparticles are added to the hydrogel, which has the effect of scavenging excessive ROS and can promote the polarization of macrophages into the M2 type, further contributing to the inhibition of the inflammatory response.

[0048] By introducing melanin and calcium peroxide, the conductivity of the CSA hydrogel has been significantly improved, which is consistent with the electrical signal conduction level of natural cardiac tissue, showing great application potential in cardiac tissue engineering repair.

[0049] To achieve in-situ drug release and optimize the microenvironment after myocardial infarction reperfusion, it is particularly suitable to use a flowable hydrogel that can be directly injected into the myocardium as a drug delivery carrier. In this invention, by adjusting the ratio of sodium alginate to calcium ions, an injectable hydrogel with fluidity is prepared. Further, using this hydrogel as the substrate, ascorbic acid, melanin nanoparticles, and calcium peroxide nanoparticles are incorporated during the preparation process to successfully synthesize the CSA hydrogel.

[0050] The CSA hydrogel also exhibits strong long-term oxygen production ability, with significant potential to alleviate the hypoxic environment in the myocardial infarction area.

[0051] The CSA hydrogel shows good biocompatibility in cell experiments. Even at a concentration of 10%, no obvious cytotoxicity is observed, and the cell viability remains good, providing strong biocompatibility support for its further application in animal experiments.

[0052] In addition, the CSA hydrogel can effectively scavenge ROS, reduce oxidative stress, and reduce the damage of cardiomyocytes and human umbilical vein endothelial cells in the ROS environment. In addition, the CSA hydrogel can promote the proliferation of vascular endothelial cells and the M2 type polarization of macrophages, showing a certain cardioprotective effect. Specifically, it can reduce the infarct area and the degree of cardiac fibrosis, increase the ventricular wall thickness, and thus have a positive impact on cardiac function.

[0053] In an alternative embodiment, the volume ratio of the sodium alginate aqueous solution containing melanin nanoparticles to the calcium gluconate aqueous solution containing calcium peroxide nanoparticles is 2.5:1, 2.8:1, 3:1 or 3.5:1. The final concentration of melanin nanoparticles in the hydrogel is 0.8 mg / ml, 1 mg / ml, 1.2 mg / ml, 1.4 mg / ml or 1.5 mg / ml. The mass fraction of sodium alginate in the sodium alginate aqueous solution is 2.5%, 2.8%, 3% or 3.5%. The concentration of calcium peroxide nanoparticles in the calcium gluconate aqueous solution is 0.8 mg / ml, 1 mg / ml, 1.2 mg / ml, 1.4 mg / ml or 1.5 mg / ml. The mass fraction of calcium gluconate in the calcium gluconate aqueous solution is 2.5%, 2.6%, 3%, 3.2% or 3.5%. The ratio of the sodium alginate aqueous solution to ascorbic acid is 1 mL: 0.4 mg, 1 mL: 0.5 mg or 1 mL: 0.6 mg.

[0054] In a preferred embodiment of the application of the present invention, the melanin nanoparticles are natural melanin nanoparticles or modified melanin nanoparticles.

[0055] In a preferred embodiment of the application of the present invention, the natural melanin nanoparticles are prepared from the raw material of cuttlefish ink sac. In one embodiment, the cuttlefish ink sac is mixed with water, impurities are removed, and then the precipitate is taken for freeze-drying to obtain natural melanin nanoparticles. In addition, the powder in the cuttlefish ink sac can also be directly ultrasonically washed in deionized water, and the melanin nanoparticles are separated by centrifugation, washed and dried.

[0056] Modification is carried out, for example, by an organic solvent such as APTES (3-aminopropyltriethoxysilane), and then dried and ultrasonically dispersed. The modification is used to improve mechanical properties, UV shielding efficiency, etc.

[0057] In a second aspect, the present invention also provides a method for preparing a hydrogel, which includes the following steps: adding ascorbic acid to the sodium alginate aqueous solution containing melanin nanoparticles, and then mixing it with the calcium gluconate aqueous solution containing calcium peroxide nanoparticles to obtain a mixed solution.

[0058] In a preferred embodiment of the application of the present invention, the preparation method further includes: centrifuging the mixed solution.

[0059] In a third aspect, the present invention also provides a hydrogel containing a small molecule inhibitor, which includes the above-mentioned hydrogel or the hydrogel prepared by the preparation method of the above-mentioned hydrogel and a small molecule inhibitor, and the small molecule inhibitor is δ-Amyrenone.

[0060] δ-Amyrenone is a small molecule inhibitor of CBX7 identified by the inventors through screening. It can regulate the cell cycle, inhibit apoptosis, and promote the proliferation of cardiomyocytes. When loaded into the hydrogel, it exhibits certain cardioprotective effects, specifically reducing the infarct area, alleviating the degree of cardiac fibrosis, increasing the ventricular wall thickness, and thus having a positive impact on cardiac function. The CSA-δ hydrogel not only significantly promotes the regeneration and repair of cardiomyocytes but also further enhances its repair effect by constructing a microenvironment conducive to myocardial recovery. The experimental results of this invention indicate that the CSA-δ hydrogel has significant potential in the field of myocardial infarction repair, providing a new treatment strategy for the recovery of cardiac function after MI / R and potentially having important clinical application prospects.

[0061] In a preferred embodiment of the application of the present invention, each milliliter of the hydrogel contains 10 mg of the small molecule inhibitor.

[0062] Fourthly, the present invention also provides a preparation method of the hydrogel containing the small molecule inhibitor, which includes: first, mixing δ-Amyrenone with an aqueous calcium gluconate solution containing calcium peroxide nanoparticles to obtain an aqueous calcium gluconate solution containing δ-Amyrenone and calcium peroxide nanoparticles, and then mixing an aqueous sodium alginate solution containing melanin nanoparticles, ascorbic acid with the aqueous calcium gluconate solution containing δ-Amyrenone and calcium peroxide nanoparticles.

[0063] Fifthly, the present invention also provides the application of the hydrogel, or the hydrogel containing the small molecule inhibitor, in the preparation of drugs for preventing and / or treating ischemic heart disease.

[0064] In a preferred embodiment of the application of the present invention, the above-mentioned ischemic heart disease is selected from any one of myocardial infarction, myocardial infarction reperfusion injury, ventricular fibrosis, myocarditis, arrhythmia, angina pectoris, heart failure, coronary heart disease, and myocardial ischemia, and the hydrogel is administered by injection.

[0065] In this embodiment, a multifunctional hydrogel drug delivery system is developed, aiming to achieve the precise in-situ release of δ-Amyrenone at the damaged heart site and improve the microenvironment. This hydrogel drug delivery system consists of sodium alginate and calcium gluconate as the skeleton, which can be in-situ injected into the cardiac infarction area and has electrical conductivity similar to myocardial tissue. In addition, calcium peroxide nanoparticles are filled in the hydrogel, which can slowly release oxygen to help supplement oxygen in the infarcted area, and ascorbic acid is added to regulate the acid-base balance, prevent the alkaline environment generated by calcium peroxide from having an adverse effect on tissues, and scavenge ROS at the same time. Meanwhile, melanin nanoparticles are added to the hydrogel, which can scavenge excessive ROS and promote the polarization of macrophages into the M2 type, further contributing to the inhibition of the inflammatory response. That is to say, the core of this invention focuses on constructing a stable carrier platform to ensure the efficient release of δ-Amyrenone in the specific cardiac environment and simultaneously create a microscopic environment conducive to cardiac tissue repair, opening up a novel and effective strategic approach for the treatment of ischemic heart disease.

[0066] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0067] Example 1

[0068] Screening of the compound δ-Amyrenone.

[0069] The inventors obtained the information of CBX7 protein from the Uniport database and a small molecule compound library from TargetMol, and screened out the compound with the most potential specific binding ability through the Discovery Studio software. Based on the LibdockScore, we screened out the most promising small molecule δ-Amyrenone for subsequent verification. Table 1 lists some information of the top 10 small molecule compounds in terms of docking scores, and the binding mode and energy stability of the candidate compound δ-Amyrenone were verified by the AutoDock software. Among them, δ-Amyrnone and CBX7 have a binding energy of -11.61 kcal / mol, and the ligand efficiency is -0.37 kcal / mol, indicating very stable binding.

[0070] Table 1 Top 10 small molecule compounds in terms of docking scores after high-throughput screening

[0071]

[0072] Example 2

[0073] In this example, a multifunctional hydrogel drug delivery system is developed to achieve the precise in-situ release of δ-Amyrenone at the damaged heart site and improve the microenvironment. This example provides a preparation method for an injectable composite hydrogel drug delivery system (CSA hydrogel).

[0074] 1. Experimental materials

[0075] 1.1 Experimental cell lines

[0076] The human cardiomyocyte line AC16, the human monocyte line THP-1, and the human umbilical vein endothelial cell HUVEC are all derived from ATCC.

[0077] 1.2 Experimental reagents

[0078] Table 2 Main experimental materials and reagents

[0079]

[0080] 1.3 Reagent preparation

[0081] Cell culture medium: For every 500 ml of basal medium (DMEM / F-12 can be selected for AC16, RPMI 1640 medium for THP-1, and DMEM medium for HUVEC), add 5.6 ml of double antibody and 56 ml of FBS.

[0082] 2. Experimental methods

[0083] 2.1 Preparation of calcium peroxide nanoparticles (CaO2NPs)

[0084] (1) Preparation of the reaction solution: First, completely dissolve 3 grams of calcium chloride in 30 ml of distilled water, then add 15 ml of 1 molar ammonia solution and 120 ml of polyethylene glycol 200 (PEG200), and continuously stir at room temperature until evenly mixed.

[0085] (2) Slow addition of hydrogen peroxide: Slowly add 30 ml of 30% hydrogen peroxide solution to the above stirred mixture at a rate not exceeding 20 - 30 drops / second.

[0086] (3) Adjust the pH value and observe the precipitation: After the addition of hydrogen peroxide is complete, adjust the pH value of the mixed solution to 10 by continuously adding ammonia solution. During this process, white or light yellow precipitates will gradually form in the solution. Continue to stir for 2 hours to ensure full reaction.

[0087] (4)Extraction and purification of the product: The mixture was centrifuged at 12,000 g for 5 minutes using a centrifuge to separate the precipitate. After discarding the supernatant, the precipitate was washed three times with a sodium hydroxide solution with a pH of 13, and then washed twice with distilled water. Finally, the washed precipitate was dried in an oven at 80 °C for 2 hours to obtain the final product.

[0088] 2.2 Preparation of natural melanin nanoparticles (MNPs)

[0089] (1)Sample preparation

[0090] The ink sacs of cuttlefish were purchased from the Taobao store "Qingdao Xinjie Aquaculture" and taken out from a -20 °C refrigerator.

[0091] (2)Dissolution and filtration

[0092] A certain amount of ink sacs was added to distilled water, kneaded and stirred until dissolved, and then stirred evenly with a magnetic stirrer for 12 h to dissolve impurities such as lipids. After precipitation, the supernatant was discarded, and distilled water was added again. This step was repeated 6 times.

[0093] (3)Freezing and lyophilization

[0094] The final precipitate was frozen at -80 °C and then lyophilized.

[0095] 2.3 Scanning electron microscope characterization of MNPs and CaO2 NPs

[0096] To maintain the original morphology of MNPs and CaO2 NPs, we took out the samples from the lyophilized state. Subsequently, scanning electron microscopy was used to conduct a detailed characterization of the microstructure of the lyophilized samples.

[0097] 2.4 X-ray diffraction characterization of CaO2 NPs

[0098] The lyophilized CaO2 NPs were taken for X-ray diffraction (XRD) analysis. First, the sample was ground and evenly spread to ensure a flat surface, and then scanned in an X-ray diffractometer. The scanning range was set from 2θ 20° to 60°, using Cu Kα radiation (wavelength 1.5406 Å), and the test was carried out at a scanning rate of 0.5° / min.

[0099] 2.5 Preparation of injectable composite hydrogel drug delivery system (CSA hydrogel).

[0100] Preparation of CSA hydrogel: First, prepare 3 ml of an aqueous solution of melanin nanoparticles with a concentration of 1.34 mg / ml (the concentration after final gelation is 1.34 mg / ml), and based on this, prepare a 3% (by mass) aqueous solution of sodium alginate containing melanin nanoparticles. At the same time, add 1.5 mg of ascorbic acid during the stirring process. Then, prepare a 3% (by mass) aqueous solution of calcium gluconate (the reaction temperature needs to be appropriately increased), and add 1 mg of calcium peroxide nanoparticles, and stir well to make it evenly dispersed. Subsequently, mix the two solutions of the aqueous solution of sodium alginate and the aqueous solution of calcium gluconate in a volume ratio of 3:1 (in this example, 3 ml of the aqueous solution of melanin nanoparticles and 1 ml of the aqueous solution of calcium gluconate), ensuring that the solution does not form lumps and is evenly fused during the process. After completion, continue to stir for 10 minutes to ensure full mixing. Finally, remove the bubbles by centrifugation to obtain an injectable composite hydrogel.

[0101] Example 3

[0102] In this example, the preparation of CSA-δ (CSA-δ-Amyrenone) hydrogel is carried out.

[0103] On the basis of the CSA hydrogel, add 40 mg of the small molecule inhibitor δ-Amyrenone to the system. After adding calcium peroxide to the aqueous solution of calcium gluconate, then add δ-Amyrenone, and finally synthesize the CSA-δ hydrogel with the sodium alginate solution with melanin.

[0104] Comparative Example 1

[0105] Preparation of injectable sodium alginate hydrogel (SA hydrogel)

[0106] First, prepare a 3% (by mass) aqueous solution of sodium alginate and a 3% (by mass) aqueous solution of calcium gluconate. It should be noted that the operating temperature needs to be appropriately increased when preparing the calcium gluconate solution. While continuously stirring 3 ml of the aqueous solution of sodium alginate, gradually add 1 ml of the aqueous solution of calcium gluconate, ensuring that the solution does not form lumps and is evenly mixed during the dropping process. After the dropping is completed, continue to stir for 10 minutes for full mixing. Then, remove the bubbles in the solution by high-speed centrifugation to finally obtain an injectable sodium alginate hydrogel.

[0107] Experimental Example 1

[0108] In this experimental example, the hydrogels prepared in Examples 2 - 3 and Comparative Example 1 are respectively experimented.

[0109] I. Experimental method

[0110] 1. Fourier transform infrared spectroscopy (FT-IR) characterization.

[0111] To improve the stability of the samples, the hydrogels of CaO2, MNPs, ascorbic acid, and SA (Comparative Example 1) and CSA (Example 2) were first freeze-dried. Subsequently, these samples were placed in the sample cell of a Fourier transform infrared spectrometer, and the spectral scanning range was set. By executing the spectral scanning program, the FT-IR spectra of each sample were obtained.

[0112] 2. Conductive characterization was performed on the hydrogels prepared from SA (Comparative Example 1) and CSA (Example 2).

[0113] A constant voltage circuit system was designed, and this system used a light-emitting diode (LED) as an indicating element. In this circuit, components such as the hydrogel and the light-emitting diode were connected in sequence. By directly observing the change in the light-emitting brightness of the light-emitting diode, we could indirectly evaluate the conductive efficiency of the hydrogel. To further quantitatively evaluate the conductivity of the hydrogel, a resistivity tester was used for systematic experiments. First, hydrogel samples were prepared. Then, the electrodes of the resistivity tester were accurately placed on both sides of the hydrogel sample to ensure full contact between the electrodes and the sample, so as to accurately measure the resistivity.

[0114] 3. The oxygen production capacity of the CSA hydrogel prepared in Example 2 was tested.

[0115] CSA with a volume of 20 mL was prepared and added to 40 mL of water. After vacuum treatment to remove air, the system was connected to a gas chromatograph to regularly (once every two days) monitor and determine the amount of oxygen generated.

[0116] 4. The pH change during the degradation process of the CSA hydrogel prepared in Example 2 was tested.

[0117] 2 mL of the composite hydrogel CSA was prepared. The hydrogel was placed in neutral water with several times its volume, and its degradation time was observed. The pH value of the degraded liquid was detected every day, and the undegraded part was fished out and weighed on the 1st, 2nd, 4th, 7th, and 15th days respectively.

[0118] 5. Rheological property tests were performed on the hydrogels prepared from SA (Comparative Example 1) and CSA (Example 2).

[0119] To verify the rheological properties of the two hydrogels, systematic experiments were carried out using a rheometer. First, hydrogel samples were prepared, and the storage modulus (G') and loss modulus (G'') of the samples were recorded by the rheometer in the frequency range from 0.1 Hz to 100 Hz.

[0120] 6. The drug release curve of the CSA-δ hydrogel prepared in Example 3 was determined.

[0121] Synthesize a hydrogel containing 1 mg of the small molecule inhibitor δ-Amyrenone and add it to a brown bottle containing 2 mL of PBS buffer at pH 7.4. Place the bottle in a constant temperature shaking incubator at 37°C. In the initial stage of the experiment (0 - 2 days), take out 200 μL of the sample from the bottle every 12 hours and supplement an equal volume of PBS to keep the total volume unchanged; in the next five days (2 - 7 days), change to sampling once every 24 hours; and in the following eight days (7 - 15 days), adjust to sampling once every 48 hours. Use a microplate reader equipped with a UV spectrophotometer function to measure the absorbance of each sample at a wavelength of 230 nm and record the obtained data in detail. By converting these absorbance values into the corresponding drug concentrations, we further plot the drug release curve to evaluate the slow drug release performance of the CSA-δ hydrogel.

[0122] 7. Perform cell biocompatibility testing on the CSA hydrogel prepared in Example 2.

[0123] Seed human cardiomyocytes onto a 96-well plate at different densities and wait for the cells to stabilize. Prepare the hydrogel according to the protocol in Example 2, replacing the aqueous solution during the production process with cell culture medium. Mix the prepared hydrogel with the complete medium at different ratios (0%, 1%, 5%, 8%, 10%, 20%). Replace the cell culture medium with the previously prepared hydrogel-medium mixture, add 200 μL to each well, and continue culturing for 24 h, 72 h, 168 h (the mixture prepared in advance needs to be replaced midway for the longer time group). After the end of the culture period, remove the culture solution from each well, and then add CCK-8 mixed solution to each well. Place the 96-well plate containing the CCK-8 mixed solution in an incubator at 37°C and let it stand for 2 hours. Then, transfer the 96-well plate to a microplate reader and measure the absorbance value at 450 nm. Use these absorbance data to analyze cell viability and evaluate the biocompatibility performance of the prepared hydrogel on human cardiomyocytes.

[0124] 8. Perform the performance test of scavenging DPPH on the hydrogels prepared from SA (Comparative Example 1) and CSA (Example 2).

[0125] Weigh an appropriate amount of DPPH powder, dissolve it in absolute ethanol, mix and vortex it thoroughly to prepare a DPPH stock solution with a concentration of 1 mM. Dilute the stock solution to 0.1 mM with absolute ethanol to obtain a DPPH sub-solution. Prepare 1 ml of SA hydrogel and CSA hydrogel respectively according to the schemes of Comparative Example 1 and Example 2. Take 300 μl of PBS (as the control group), 300 μl of SA hydrogel and CSA hydrogel (as the experimental groups) and add them to 800 μl of the DPPH sub-solution. Incubate in the dark for 6 hours, and then measure the absorbance of the three groups of mixed liquids at 517 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the scavenging rate using Formula 1. Set 3 parallel samples for each group.

[0126] (Formula 1)

[0127] 9. Perform ROS performance tests on the hydrogels prepared from SA (Comparative Example 1) and CSA (Example 2).

[0128] Evaluate the levels of various reactive oxygen species (ROS) in cells by staining with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Culture cardiomyocytes in a normal environment or in a ROS microenvironment induced by 200 μM H2O2, and add or not add a certain concentration of SA and CSA hydrogels respectively. After 72 h, incubate with 10 μM DCFH-DA in the dark for 15 - 25 minutes. After washing with PBS, stain with 4',6-diamidino-2-phenylindole (DAPI). Observe and record the samples under a fluorescence microscope.

[0129] 10. Perform angiogenesis-promoting performance tests on the hydrogels prepared from SA (Comparative Example 1) and CSA (Example 2).

[0130] Culture HUVEC cells in a special medium, inoculate them in a 96-well plate at a certain density, and incubate them in an incubator for 8 - 12 hours to adhere. Subsequently, divide the HUVEC cells into two groups: the normal culture environment group and the ROS microenvironment group induced by 200 μM H2O2. Under the two environments, add or not add a certain concentration of SA and CSA hydrogels respectively, and set a total of four experimental conditions. After culturing for 72 hours, remove the medium, and add CCK-8 working solution (prepared according to the reagent instruction manual) to each well. Place the 96-well plate containing the CCK-8 working solution in the incubator and incubate for 2 hours to allow the reaction to occur fully. After the incubation ends, transfer the 96-well plate to an ELISA reader and measure the absorbance at 450 nm. Analyze the absorbance values and calculate the cell survival rate to judge the promoting effect of the hydrogel on the proliferation of HUVEC cells under ROS microenvironment conditions.

[0131] Meanwhile, culture HUVEC cells under the above four conditions. After the culture is completed, collect cell samples, perform protein extraction, and verify the expression of angiogenesis-related indicators in HUVEC cells through Western blot experiments to further evaluate the functional role of the hydrogel.

[0132] 11. Test the performance of promoting macrophage polarization of the hydrogels prepared by SA (Comparative Example 1) and CSA (Example 2).

[0133] (1) Cell culture: Culture THP-1 cells in a medium. After induction with PMA for 24 hours, make them adhere to the wall. Subsequently, inoculate the cells into a 6-well plate at a certain density. After the cells are stable, divide them into two groups: a normal culture environment group and a ROS microenvironment group induced by 200 μM H2O2. Under these two environments, add or not add a certain concentration of SA and CSA hydrogels respectively, and set a total of four experimental conditions for culturing for 24 hours.

[0134] (2) Cell fixation: Gently rinse the cells with pre-cooled PBS to remove the medium. Add 4% paraformaldehyde to fix the cells for 10 - 15 minutes at room temperature. Wash the cells 3 times with PBS, 5 minutes each time, to remove the excess fixative.

[0135] (3) Cell permeabilization: Use 0.1% Triton X-100 permeabilization solution to treat for 5 - 10 minutes to help antibodies penetrate into the cells. Wash the cells 3 times with PBS, 5 minutes each time.

[0136] (4) Blocking: Use 5% goat serum as a blocking solution and perform blocking treatment at room temperature for 30 to 60 minutes to prevent non-specific binding reactions from occurring.

[0137] (5) Primary antibody incubation: Add the primary antibody (such as the main antibody against the target protein), dilute it according to the manufacturer's instructions, and incubate at 4°C for 10 - 12 hours. Then wash 3 times with PBS, 5 minutes each time.

[0138] (6) Secondary antibody incubation: Add the secondary antibody of the same species as the primary antibody to the sample and incubate at room temperature for 1 to 2 hours. Then wash 3 times with PBS, 5 minutes each time.

[0139] (7) Nuclear staining: Add DAPI for nuclear staining and incubate for 5 - 10 minutes. Wash the cells 3 times with PBS, 5 minutes each time.

[0140] (8) Microscopic observation: Observe and record the fluorescence signal using a fluorescence microscope.

[0141] 12. Statistical analysis methods

[0142] The data obtained from the experiments were expressed in the form of mean ± standard deviation and evaluated by statistical methods such as T - test and one - way analysis of variance (ANOVA). In the statistical analysis results, a P - value less than 0.05 was set as the criterion for significant difference. Among them, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001, all of which are used to indicate significant differences in statistical significance.

[0143] II. Experimental results

[0144] 1. Preparation and characterization of CaO2 NPs

[0145] As a widely used solid oxygen - releasing agent, compared with H2O2, CaO2 exhibits a more gentle and persistent oxygen - releasing characteristic, which can effectively prevent the rapid accumulation of local reactive oxygen species and thus maintain the redox homeostasis. However, due to their large volume, conventional - sized CaO2 particles tend to agglomerate when encountering water, significantly slowing down the oxygen - release rate. In contrast, nano - sized CaO2 particles can not only greatly accelerate the reaction kinetics but also effectively overcome the aggregation between particles, optimizing their performance.

[0146] We prepared calcium peroxide nanoparticles using the method reported previously. The X - ray diffraction (XRD) pattern of the obtained sample is as shown in Figure 1 a. Its main diffraction peaks (2θ ≈ 31.7°, 36.2°, 47.3°, etc.) are completely consistent with the standard diffraction pattern of calcium peroxide (PDF#03 - 0865), confirming the phase purity of the sample. At the same time, we carried out FTIR tests on the prepared sample, and the results are as shown in Figure 1 b. In the infrared spectrum, the positions of the characteristic peaks are consistent with those reported in the literature, further proving the chemical composition and structure of the sample. In addition, we used scanning electron microscopy (SEM) to observe the morphology of the sample, and the results are as shown in Figure 1 c. The SEM image clearly shows that most of the prepared calcium peroxide particles are distributed in the nanoscale, further verifying the successful preparation of nanoparticle formation. In summary, through the comprehensive characterization of XRD, FTIR, and SEM, we successfully prepared calcium peroxide nanoparticles with pure crystal phase, clear chemical structure, and uniform particle size, laying a solid foundation for further research on their oxygen - releasing performance and applications.

[0147] 2. Preparation and characterization of MNPs

[0148] The natural MNPs contained in squid ink are regarded as an important bioactive substance, possessing excellent protective functions, capable of effectively shielding ultraviolet radiation, resisting reactive oxygen species attacks, neutralizing toxins, and chelating metal ions. These naturally occurring melanins not only have good biocompatibility and biodegradability but also exhibit excellent redox activity and semi-conductive properties, thus showing great potential in biomedical applications.

[0149] We successfully extracted melanin nanoparticles by simple centrifugation and stirring methods. According to the results of scanning electron microscopy (SEM) ( Figure 2 shown in b), the obtained MNPs are spherical, with a uniform size distribution, and the particle size is less than 500 nm. In addition, we carried out Fourier transform infrared spectroscopy (FTIR) tests on the prepared samples, and the results are as Figure 2 shown in a. The characteristic peaks of the FTIR spectrum are completely consistent with the literature reports, further confirming the accuracy of the chemical composition and structure of the samples.

[0150] 3. Characterization of SA hydrogel and CSA hydrogel.

[0151] To achieve in-situ drug release and optimize the microenvironment after myocardial infarction reperfusion, it is particularly suitable to use a flowable hydrogel that can be directly injected into the myocardium as a drug delivery carrier. In this experimental section, we prepared an injectable hydrogel with fluidity by adjusting the ratio of sodium alginate to calcium ions. Further, based on this hydrogel as the substrate, ascorbic acid, MNPs, and CaO2 NPs were incorporated during the preparation process to successfully synthesize CSA hydrogel. As Figure 3 shown, both of these hydrogels exhibit moderate fluidity. Although not as fluid as pure water, slight flow can be observed when the container is inverted. Through syringe loading and writing tests, we verified that these hydrogels have excellent injectability.

[0152] Figure 4 Shown in a, the storage modulus (G') of SA and CSA hydrogels is higher than the loss modulus (G''), which reveals that the prepared materials possess the typical characteristics of hydrogels. However, G' is relatively low, indicating that the hydrogel is in a weak gel state. To confirm that CaO2, ascorbic acid, and MNPs are successfully loaded in the composite hydrogel, we used FT-IR analysis to detect each component and CSA. As Figure 4 shown in b, the corresponding relationship between the characteristic peaks of CSA and each component not only confirmed the presence of CaO2 and MNPs but also effectively verified the loading of ascorbic acid.

[0153] 4. Characterization of the electrical conductivity of injectable hydrogels and injectable composite hydrogels.

[0154] To quantify the conductivity properties of SA and CSA, we constructed a circuit system integrated with light-emitting diodes to visualize their electrical properties. As Figure 5 shown in a of Figure 5 , although the SA hydrogel can drive the light-emitting diode to emit light, its brightness is relatively low. In contrast, the CSA hydrogel significantly enhances the brightness of the LED, which may be attributed to the introduction of melanin. Melanin is a natural conductive material, and its conjugated molecular structure can effectively promote electron transfer. When melanin nanoparticles are dispersed in the hydrogel, they form conductive pathways in the gel network, significantly improving the conductivity of the hydrogel. In addition, the high specific surface area of melanin nanoparticles enables them to be evenly distributed in the hydrogel and interact with the matrix. This uniform distribution helps to form a continuous conductive network, further enhancing the conductive performance. Comprehensive analysis shows that the CSA hydrogel exhibits good application potential in regulating cardiac tissue electrophysiology.

[0155] The conductivity range of natural myocardium is 10 -4 ~10 S / cm, which provides an important reference for the design of conductive hydrogels for myocardial infarction repair. To further verify the conductive properties of the two hydrogels, we used a resistivity tester to quantitatively analyze their conductivity. As Figure 5 shown in b of Figure 5 , the conductivity of the SA hydrogel is 0.084 ± 0.007 S / m, showing relatively low conductivity; while the conductivity of the CSA hydrogel is increased to 0.115 ± 0.006 S / m, which is consistent with the electrical signal conduction level of natural myocardial tissue.

[0156] 5. Determination of the drug release curve of CSA-δ hydrogel.

[0157] In the in vitro drug release test, we used the CSA-δ hydrogel loaded with 1 mg of δ-Amyrenone. During the experiment, samples were continuously collected and analyzed to plot the drug release curve (see Figure 6 ). The experimental results show that CSA exhibits obvious slow-release characteristics under neutral conditions: on the second day, the drug release rate exceeds 50%, and by the 15th day, the release rate exceeds 85%. These data indicate that the hydrogel has excellent drug slow-release performance, providing strong support for its application in long-acting drug delivery.

[0158] 6. Detection of the oxygen production capacity, degradation and pH change of CSA hydrogel

[0159] When CaO2 comes into contact with water, it decomposes into H2O2 and Ca(OH)2, and then H2O2 further decomposes into oxygen and water. To control the accumulation of H2O2, ascorbic acid was introduced into the hydrogel system. Ascorbic acid plays a dual role: First, as an antioxidant, it can effectively catalyze the decomposition of H2O2 and accelerate this process; Second, as an acidic component, it helps to neutralize the local alkaline environment caused by Ca(OH)2. Moreover, the antioxidant property of ascorbic acid can prevent the calcium overload problem caused by damaged cellular calcium channels, thus maintaining the normal function of cells.

[0160] As Figure 7 shown in a, CSA exhibits stable oxygen release performance, with a continuous release time of approximately 8 to 10 days. Subsequently, at around day 10, the oxygen generation significantly slows down to nearly stop. To monitor the degradation process of the hydrogel, hydrogel samples were taken out and weighed on the 1st, 2nd, 4th, 7th, and 15th days of the experiment, and a degradation curve was plotted based on this (see Figure 7 b). The results show that the hydrogel achieved nearly complete degradation within approximately 15 days. In addition, the pH value of the degradation product solution was detected, and its value was always maintained within the range of 7.1 to 7.4, which is close to the physiological pH (refer to Figure 7 c). This finding indicates that the composite hydrogel can effectively maintain the physiological stability of the environment during degradation, laying a solid foundation for its potential applications in the biomedical field.

[0161] 7. Detection of the cytocompatibility of the CSA hydrogel.

[0162] Biocompatibility is a key indicator to measure the suitability of hydrogels as biomedical materials. To comprehensively evaluate the potential toxic effects of hydrogels on cells, we adopted the CCK-8 detection technique to conduct relevant experiments, and the obtained results are as Figure 8 shown. For the four groups with a CSA hydrogel proportion of 1% to 10%, compared with the control group, the cell viability exceeded 80% during both short-term culture (24 hours) and long-term culture (72 hours and 168 hours), that is, no obvious cytotoxicity was exhibited. However, the 20% CSA hydrogel group may have certain toxicity or inhibitory effects on cells, especially during long-term culture, and this effect may gradually become apparent. The research results show that the CSA hydrogel has good biocompatibility with cells within a certain concentration range, demonstrating its application potential in the biomedical field. This finding also provides a reference concentration for subsequent research.

[0163] 8. Detection of the ROS scavenging level of the CSA hydrogel.

[0164] The DPPH method is one of the main methods for colorimetric determination of antioxidant capacity. To verify the ability of MNPs and ascorbic acid to endow the hydrogel with the ability to scavenge reactive oxygen species in highly oxidative stress myocardial infarction tissues, we evaluated the antioxidant potential of SA and CSA hydrogels. As Figure 9 shown in a of Figure 9 , the DPPH scavenging ability of the CSA hydrogel was significantly higher than that of the SA hydrogel, indicating the important role of MNPs and ascorbic acid in free radical scavenging. The abundant antioxidant groups (such as phenolic hydroxyl groups) in MNPs are the key factors for scavenging ROS. The excellent antioxidant activity exhibited by the CSA hydrogel helps to effectively scavenge harmful ROS in the myocardial infarction area, thereby improving and remodeling the infarct microenvironment.

[0165] To further verify the ability of the CSA hydrogel to scavenge ROS, we used the DCFH-DA staining method to evaluate the intracellular ROS level. The results showed that after 3 days of culture, in the SA hydrogel ( Figure 9 shown in b of Figure 9 ), the green intensity of DCFH-DA was almost the same as that of the hydrogen peroxide culture group, indicating a relatively high ROS level. However, the fluorescence intensity of DCFH-DA in the CSA hydrogel group was significantly reduced and was close to the level of normally cultured cells.

[0166] In summary, through the synergistic action of MNPs and ascorbic acid, the CSA hydrogel exhibits excellent free radical scavenging ability. This property not only effectively alleviates the oxidative damage caused by ROS, but also provides strong support for the optimization and functional recovery of the myocardial infarction microenvironment, highlighting the potential application value of the CSA hydrogel in cardiac tissue engineering.

[0167] 9. Detection of the angiogenesis-promoting ability of the CSA hydrogel.

[0168] The viability of human umbilical vein endothelial cells (HUVECs) in the ROS microenvironment was evaluated by the CCK-8 assay. After 3 days of continuous culture, oxidative stress injury led to a significant decrease in the viability of HUVECs, while the application of the CSA hydrogel enhanced the viability of these endothelial cells to a certain extent ( Figure 10 shown in a of Figure 10 ). Subsequently, Western-blot experiments were used to detect the expression of angiogenesis-related proteins, including von Willebrand factor (VWF), vascular cell adhesion molecule 1 (Vcam1), and CD31. As Figure 10 shown in b of Figure 10 , compared with the SA hydrogel, the CSA hydrogel significantly upregulated the expression of angiogenesis-related genes. That is to say, the CSA hydrogel improved the viability of HUVECs by scavenging ROS, thus having the potential to promote angiogenesis in the MI region.

[0169] 10. Detection of the macrophage polarization-promoting ability of the CSA hydrogel.

[0170] The effects of SA or CSA hydrogels on the polarization of THP-1-derived macrophages in the ROS microenvironment were investigated by immunofluorescence experiments. After continuous culture for 1 day, immunofluorescence staining was then performed to show the expression of CD86 (a surface marker of M1 macrophages) and CD206 (a surface marker of M2 macrophages) in THP-1-derived macrophages after different treatments ( Figure 11 ). After adding hydrogen peroxide, the number of CD86-positive THP-1-derived macrophages increased significantly, indicating their polarization into the M1 phenotype. At the same time, the number of CD206-positive cells did not change significantly. Apparently, the CSA hydrogel reduced the signal of CD86 and increased the signal of CD206, suggesting that the CSA hydrogel induced macrophage polarization from M1 to M2.

[0171] The experimental results of this example show that:

[0172] (1) The CSA hydrogel has excellent injectability and can achieve in-situ injection. At the same time, the CSA-δ hydrogel formed after loading drugs has the ability of drug sustained release, which enables the drug to be continuously and stably released, thereby prolonging the drug action time. In addition, the CSA hydrogel exhibits strong long-term oxygen production ability and has significant potential to relieve the hypoxic environment in the myocardial infarction area.

[0173] (2) The CSA hydrogel shows good conductivity and can provide an electrical environment similar to that of myocardial tissue. By introducing melanin and calcium peroxide, the conductivity of the CSA hydrogel has been significantly improved, and its conductivity is 0.115 ± 0.006 S / m, which is consistent with the electrical signal conduction level of natural myocardial tissue, showing great application potential in cardiac tissue engineering repair.

[0174] (3) The CSA hydrogel shows good biocompatibility in cell experiments. Even at a concentration of 10%, no obvious cytotoxicity was observed, and the cell viability remained good, providing strong biocompatibility support for its further application in animal experiments.

[0175] (4) The CSA hydrogel effectively scavenges ROS, reduces oxidative stress, and reduces the damage of cardiomyocytes and human umbilical vein endothelial cells in the ROS environment. In addition, the CSA hydrogel can promote the proliferation of vascular endothelial cells and the M2 polarization of macrophages, and is expected to promote angiogenesis and tissue repair in the myocardial infarction area.

[0176] Experimental Example 2

[0177] The therapeutic effect of the CSA-δ hydrogel on elderly rats with MI / R was evaluated.

[0178] In this experimental example, an MI / R model of aged rats was established, and a sham operation (Sham) group was set as a control to systematically verify the comprehensive effects of CSA-δ hydrogel in scavenging ROS, promoting angiogenesis, regulating macrophage polarization, and restoring cardiac function, thereby evaluating the therapeutic effect of this hydrogel. We will adopt a variety of evaluation methods, including DHE staining, immunofluorescence analysis, echocardiography, determination of cardiac function parameters, Masson's trichrome staining, and HE staining, to comprehensively and deeply explore the performance of CSA-δ hydrogel in therapeutic applications.

[0179] 1. Experimental materials

[0180] 1.1 Experimental animals

[0181] SPF-grade SD rats provided by Dashuo in Chengdu were used as experimental subjects, regardless of gender, and continued to be cultured until 2 years old. To ensure the hygienic conditions of the animals, the experimental rats were housed in an environment with a 12-hour light-dark cycle control and had free access to clean drinking water and high-quality feed. All animal experiments were strictly conducted in accordance with the approval and guiding principles of the Experimental Animal Ethics Review Committee of Sichuan Provincial People's Hospital.

[0182] 2.2 Experimental materials and reagents

[0183] Table 3 Main experimental materials and reagents

[0184]

[0185] 2. Experimental methods

[0186] 2.1 Prepare SA hydrogel, CSA hydrogel, and CSA-δ hydrogel according to the methods of Examples 2-3 and Comparative Example 1.

[0187] 2.2 Construction of MI / R model and administration

[0188] An MI / R model was constructed for aged SD rats: First, expose the pleura and open the chest between the second and third intercostal spaces on the left side of the chest. Then, open the chest between the second and third left costal spaces to expose the heart. Use 7-0 silk thread to tie a slipknot at the origin of the left anterior descending branch of the coronary artery and occlude the left anterior descending branch. Ensure that there is no obvious bleeding in the heart cavity during the operation and keep the operation area clean. Finally, suture the operation area layer by layer to ensure full adhesion of the tissue layers. After 40 minutes of ischemia treatment, remove the coronary artery ligation thread. At the end of the experimental period, euthanize the animals with an overdose of isoflurane and remove the main organ tissues for further analysis.

[0189] After that, multiple-point myocardial injections of SA, CSA hydrogel, and CSA-δ hydrogel were performed around the cardiac ischemic area, with care taken to avoid spillage. In the sham operation group, only thoracotomy was performed without ligation of the left anterior descending branch. In the PBS group and the drug administration group, PBS and δ-Amyrenone were injected via the caudal vein, respectively.

[0190] 2.3 DHE staining of frozen sections.

[0191] (1)Tissue collection: The rat hearts were collected on the first day after infarction. Immediately after removal, the hearts were placed in a -80°C refrigerator to ensure tissue viability and integrity.

[0192] (2)Freezing embedding and sectioning: The preserved heart tissue was placed in a freezing section embedding agent. After complete freezing, the tissue was sectioned into 5-μm-thick slices using a freezing microtome, and the slices were transferred onto glass slides for subsequent experiments.

[0193] (3)DHE staining: The tissue sections were washed three times with PBS for 3 minutes each to thoroughly remove the residual embedding agent. According to the DHE kit instructions, the mother liquor was diluted to 10 μM and stained at 37°C for 30 minutes in the dark. Subsequently, the sections were washed three times again with PBS, stained with DAPI for 10 minutes, and then mounted after three washes with PBS. Finally, the sections were observed under a fluorescence microscope and the results were recorded.

[0194] 2.4 Collection of organ tissues, pathological HE staining, and Masson staining of cardiac tissues.

[0195] (1)Collection of tissue samples: The rats were divided into three groups according to different experimental purposes: the group on the first day after drug administration, from which cardiac tissues were taken; the group on the third day after drug administration, from which cardiac tissues were taken; and the group on the 28th day after drug administration, from which, in addition to cardiac tissues, liver, spleen, lung, and kidney organs were also collected. Among them, the cardiac tissues of the first-day group were frozen at -80°C for later use, and the organs of the remaining groups were fixed with 4% paraformaldehyde for more than 24 hours for later use.

[0196] (2)Embedding and sectioning of tissues: The samples in (1) were dehydrated in a dehydrator and then embedded in paraffin in a paraffin embedding machine. The embedded and frozen tissues were sectioned to a thickness of 5 microns. The sections were picked up with glass slides in warm water for subsequent processing.

[0197] (3)HE staining procedure: The samples were placed in an automatic staining machine, and the program of dewaxing + HE staining was selected. After staining, a neutral resin coverslip was applied. The images were observed and recorded using a microscope.

[0198] (4)Masson trichrome staining: The staining operation was performed according to Section 2.3.8.

[0199] (5)Immunofluorescence staining analysis: After the paraffin removal pretreatment of the heart tissue samples by an automatic staining machine, the immunofluorescence staining operation is carried out according to the following steps:

[0200] a: Acquisition, fixation and dehydration process of heart samples: Construct an MI / R model of aged mice according to the steps shown in 2.2. After 24 hours, anesthetize and humanely sacrifice the mice, and then remove the hearts for experiments. The heart tissue needs to be immersed in a 4% paraformaldehyde solution for 24 hours to ensure the integrity of the tissue structure is maintained. Next, dehydrate the heart tissue using an automatic tissue dehydration device.

[0201] b: Paraffin embedding and thin section preparation of heart tissue: The dehydrated heart tissue needs to be further paraffin-embedded for subsequent sectioning. When sectioning, ensure that the thickness of the heart tissue thin sections is 5 μm to meet the specific requirements of the experiment.

[0202] c: Dewaxing and hydration: Place the paraffin sections in an automatic staining machine for baking and dewaxing, and complete hydration.

[0203] d: Antigen retrieval: Place the sections in a steam pressure cooker containing sodium citrate buffer (pH 6.0) for high-temperature and high-pressure retrieval for 5 - 10 minutes. After natural cooling to room temperature, rinse the sections with PBS 3 times, 5 minutes each time.

[0204] e: Permeabilization and blocking: Treat the sections with a 0.5% Triton X-100 solution to enhance the penetration ability of antibodies in the tissue. Subsequently, block the sections with goat serum at room temperature for 30 minutes to reduce the interference of non-specific binding and ensure the accuracy of the experimental results.

[0205] f: Primary antibody incubation: Add the primary antibody according to the proportion in the instruction manual, cover the section tissue, and incubate overnight at 4 °C in a wet box. Subsequently, wash the sections with PBS 3 times, 5 minutes each time.

[0206] g: Secondary antibody incubation: Then add the fluorescence-labeled secondary antibody (select the corresponding secondary antibody according to the host of the primary antibody, and the dilution ratio is shown in the instruction manual), incubate at 37 °C for 1 hour, and wash 3 times with PBS again, 5 minutes each time.

[0207] h: DAPI staining and mounting: Stain with DAPI at 37 °C for 20 minutes to label the cell nuclei. Wash with PBS 3 times, 5 minutes each time, to remove the excess stain. Drop an appropriate amount of mounting medium, cover the section with a coverslip, and gently flatten it to prevent air bubbles.

[0208] i: Microscopic Observation and Photography: The sections were observed using a fluorescence microscope, and the target fluorescence signal and nuclear staining signal were sequentially photographed using appropriate excitation wavelengths. The images were saved for subsequent analysis.

[0209] 2.5 Detection of Cardiac Function Indexes

[0210] To evaluate the effects of different treatment measures on cardiac function, echocardiography technology was used to detect relevant indexes. After successfully constructing the MI / R model and implementing different treatments, after 28 days, the mice in each group were anesthetized with isoflurane, and a small animal ultrasonic imaging system of the Fujifilm brand was used for cardiac function detection. Doppler ultrasound in C mode and M-mode ultrasound in B mode were mainly used to accurately measure the mitral valve blood flow and comprehensively evaluate cardiac function indexes, respectively.

[0211] 3. Experimental Results:

[0212] 3.1 ROS Scavenging Effect of CSA-δ Hydrogel in Vivo.

[0213] MI / R can trigger a large amount of oxidative stress. To verify the antioxidant efficacy of CSA-δ hydrogel, we used DHE staining technology to visually display the distribution of intracellular superoxide anions (a type of ROS). Specifically, as Figure 12 shown, on the first day after PBS injection in the myocardial infarction area, we clearly observed strong DHE signals, which directly indicated a large accumulation of ROS in this area. Further, compared with the SA hydrogel and δ-Amyrenone treatment groups, when we injected CSA and CSA-δ hydrogels on the first day, the DHE fluorescence was significantly weakened, indicating that the CSA hydrogel can effectively reduce the accumulation of ROS, and the addition of δ-Amyrenone does not affect its ability to scavenge ROS. The above results verified the antioxidant properties of CSA and CSA-δ hydrogels, indicating that it can protect cardiomyocytes from oxidative damage by scavenging ROS in the early stage of myocardial infarction reperfusion.

[0214] 3.2 Angiogenesis Promotion Effect of CSA-δ Hydrogel in Vivo.

[0215] After MI / R, angiogenesis plays a crucial role in the effective repair of the myocardium, which can promote the restoration of oxygen supply and rescue dying cardiomyocytes. In view of this, we deeply explored the effect of CSA-δ hydrogel on the vascular density in the myocardial infarction area, and the results are as Figure 13As shown. In the study, we used immunofluorescence staining to detect α-smooth muscle actin (α-SMA), a marker protein of mature neovascularization, as a basis for evaluating vascular density. Compared with the PBS group, more neovascularization was found in the SA hydrogel group and the δ-Amyrenone group. More importantly, the highest arterial density was observed in the CSA and CSA-δ hydrogel groups compared with other groups. Through visual observation, it was found that the number of blood vessels in the CSA and CSA-δ hydrogel groups was significantly higher than that in other groups. These results indicate that the CSA-δ hydrogel has a strong ability to promote angiogenesis in vivo, that is, the CSA-δ hydrogel may achieve the therapeutic effect of left ventricular reconstruction by increasing the vascular density in the infarcted area.

[0216] 3.3 Effect of CSA-δ hydrogel on promoting macrophage polarization in vivo.

[0217] To evaluate macrophage polarization, we performed immunofluorescence staining analysis on cardiac tissues on the 1st and 3rd days after myocardial infarction to detect surface markers for analyzing the pro-inflammatory and anti-inflammatory phases. In Figure 14 the immunofluorescence images shown in a, it was observed that on the first day of treatment, the marker of M1 macrophages, CD86, was significantly expressed in the myocardial infarction area in the PBS group, the δ-Amyrenone group, and the SA hydrogel group. Interestingly, compared with the PBS, δ-Amyrenone, and SA hydrogel groups, the CD86-positive M1 macrophages were relatively reduced in the CSA and CSA-δ hydrogel groups. In contrast, almost no CD206-positive M2 macrophages were observed in each group on the 1st day. These results indicate that in the early stage of myocardial infarction (the 1st day), the introduction of CSA and CSA-δ hydrogels can down-regulate the number of M1 macrophages, while there is no significant change in M2 macrophages. It is worth noting that on the third day after treatment, a significant difference was shown in the expression of CD206.

[0218] Figure 14 The immunofluorescence images in b show that compared with the PBS, δ-Amyrenone, and SA hydrogel groups, the expression of CD206 was significantly up-regulated in the CSA and CSA-δ hydrogel groups, indicating that the CSA hydrogel induced M2 macrophage polarization in the myocardial infarction area. At the same time, the CD86-positive cells in the CSA and CSA-δ hydrogel groups were significantly reduced, indicating that the CSA and CSA-δ hydrogels down-regulated M1 macrophages and inhibited the inflammatory response in the early stage of myocardial infarction. Overall, the CSA-δ hydrogel effectively reduced the number of pro-inflammatory M1 macrophages and promoted the polarization of macrophages into M2 macrophages with repair-promoting effects from 1 to 3 days after treatment, which is beneficial to the recovery process of myocardial tissue.

[0219] 3.4 Detection and verification of the repair effect of CSA-δ hydrogel on cardiac function in rats.

[0220] To explore the effect of CSA-δ hydrogel on the repair of cardiac function in elderly rats after MI / R, echocardiography was performed 28 days after the treatment in each group. The M-mode ultrasound in B-mode was used to evaluate the systolic and diastolic capabilities of the hearts in each group. As Figure 15 shown in a of Figure [X], among the 6 groups, the Sham group had the largest range of systolic activity, and the CSA-δ treatment group showed a similar systolic amplitude. The anterior ventricular walls of the PBS group and the SA group were nearly horizontal, showing the characteristics of fibrosis. The CSA group and the δ-Amyrenone group had a certain improvement compared with the PBS group, with a larger systolic amplitude. In addition, the cardiac function was further evaluated using Doppler ultrasound in C-mode. The results showed that the δ-Amyrenone group, the SA group, and the CSA group could increase the E / A peak ratio. However, the CSA-δ group had a higher E / A peak ratio, demonstrating a more prominent therapeutic effect (see Figure 15 b of Figure [X] and Figure 15 e of Figure [X]). At the same time, we also deeply analyzed key echocardiogram parameters such as ejection fraction (EF) and fractional shortening (FS) ( Figure 15 c of Figure [X], Figure 15 d of Figure [X]). The data showed that the CSA-δ group performed optimally in promoting the recovery of cardiac function. In summary, the CSA-δ hydrogel can significantly enhance the cardiac function of elderly rats after MI / RI.

[0221] 3.5 Verification of the repair effect of CSA-δ hydrogel by Masson staining.

[0222] Myocardial tissue sections were stained using Masson trichrome staining to quantify the myocardial repair effects in each group. The stained images ( Figure 16 a of Figure [X]) visually showed that the normal heart should have all red myocardial tissues in the Masson staining result. Due to the extensive necrosis caused by MI / R in the PBS group, the area of blue fibrous tissue was significantly increased compared with other groups. There was almost no red myocardial tissue in the infarct zone (IZ) and the border zone (BZ). The CSA-δ group showed the least blue fibrous tissue except for the Sham group, showing a more significant recovery of myocardial tissue, especially in the infarct area and the border area, where there was more red myocardial tissue. Further, the images were processed using Zenbio image analysis software to accurately measure the infarct wall thickness, and the results showed significant statistical differences among the groups ( Figure 16In b). Specifically, the left ventricular wall thickness of the CSA-δ group reached 1.762 ± 0.139 mm, which was much higher than that of the PBS group (0.3954 ± 0.0345 mm). These data strongly support that the CSA-δ hydrogel can not only promote the recovery of cardiac function in elderly rats with MI / R, but also effectively reduce the formation of scar tissue, thus revealing that CSA-δ has significant repair efficacy in the field of myocardial injury repair.

[0223] 3.6 Effects of CSA-δ hydrogel treatment on organ histopathology.

[0224] To deeply evaluate the safety performance of the CSA-δ hydrogel system in vivo, we performed HE staining analysis on the core organs (heart, liver, spleen, lung, and kidney) of all 28-day treatment group elderly rats, aiming to explore its potential organ toxicity at the histological level. As Figure 17 can be seen, HE staining of each experimental group showed that the cardiac tissue structure was normal, myocardial cells were arranged neatly, and there were no abnormal phenomena such as inflammation or necrosis. The liver tissue structure was normal, the hepatic lobule structure was intact, hepatocytes showed no fatty degeneration or necrosis, and there were no significant changes in blood vessels and bile ducts. In the spleen tissue, the white pulp and red pulp were clearly demarcated, and there were no significant cell infiltrations or injuries. The alveolar structure was intact, and there were no pathological changes such as pulmonary edema, inflammation, or fibrosis. The renal tubules and glomeruli were intact, and there were no toxic manifestations such as renal tubular necrosis or interstitial fibrosis. In summary, after 28 days of treatment, no significant abnormalities were found in the main organs of each group of SD elderly rats, which strongly proves that the CSA-δ hydrogel, as a drug delivery platform, has high in vivo safety and good biocompatibility, laying a solid foundation for its further application in the biomedical field.

[0225] The experimental results of this example show that SA, CSA hydrogels, and δ-Amyrenone all exhibit certain cardioprotective effects, specifically manifested as the ability to reduce the infarct area, alleviate the degree of cardiac fibrosis, and increase the ventricular wall thickness, thus having a positive impact on the cardiac function of rats. However, in comparison, the CSA-δ hydrogel not only significantly promotes the regeneration and repair of myocardial cells, but also further enhances its repair effect by constructing a microenvironment conducive to myocardial recovery. These series of experimental results indicate that the CSA-δ hydrogel has significant potential in the field of myocardial infarction repair, providing a new treatment strategy for the recovery of cardiac function after MI / R and possibly having important clinical application prospects.

[0226] In summary, the CSA hydrogel drug delivery system prepared by the present invention can improve the local microenvironment of myocardial infarction: the conductivity of the hydrogel is 0.115±0.006 S / m, which meets the requirements of cardiac electrical signal conduction, and can slowly release oxygen in the local hypoxic environment. It can also scavenge ROS in vivo and in vitro, promote angiogenesis and macrophage M2 polarization function. The drug sustained-release and biocompatibility experiments verified its sustained-release performance and safety, laying a foundation for its application in the treatment of myocardial infarction. The CSA-δ hydrogel significantly promotes myocardial repair: the CSA-δ hydrogel is superior to SA, CSA hydrogel or δ-Amyrenone alone in reducing the myocardial infarction area, reducing fibrosis and improving cardiac function. The results of HE staining of organs show that the CSA-δ hydrogel has good safety, providing favorable support for its application in the treatment of cardiac injury.

[0227] The foregoing are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A hydrogel containing a small molecule inhibitor, characterized in that, It includes a hydrogel and a small molecule inhibitor, and the small molecule inhibitor is δ-Amyrenone. The hydrogel includes: an aqueous sodium alginate solution containing melanin nanoparticles, a reducing agent, and an aqueous calcium gluconate solution containing calcium peroxide nanoparticles. The volume ratio of the aqueous sodium alginate solution containing melanin nanoparticles to the aqueous calcium gluconate solution containing calcium peroxide nanoparticles is 2.5:1 - 3.5:

1. The final concentration of melanin nanoparticles in the hydrogel is 0.8 - 1.5 mg / ml. The mass fraction of sodium alginate in the aqueous sodium alginate solution is 2.5%–3.5%. The concentration of calcium peroxide nanoparticles in the aqueous calcium gluconate solution is 0.8–1.5 mg / ml. The mass fraction of calcium gluconate in the aqueous calcium gluconate solution is 2.5%–3.5%. The ratio of the aqueous sodium alginate solution to the reducing agent is 1 mL: 0.4–0.6 mg, and the reducing agent is selected from ascorbic acid, glutathione or cysteine.

2. The hydrogel containing a small molecule inhibitor according to claim 1, wherein The melanin nanoparticles are natural melanin nanoparticles or modified melanin nanoparticles.

3. The hydrogel containing the small molecule inhibitor according to claim 2, wherein The natural melanin nanoparticles are prepared from the raw material of squid ink sac.

4. The hydrogel containing a small molecule inhibitor according to claim 1, wherein Each ml of the hydrogel contains 10 mg of the small molecule inhibitor.

5. The hydrogel containing a small molecule inhibitor according to claim 1, wherein The preparation method of the hydrogel includes the following steps: adding a reducing agent to the aqueous sodium alginate solution containing melanin nanoparticles, and then mixing with the aqueous calcium gluconate solution containing calcium peroxide nanoparticles to obtain a mixture.

6. The hydrogel containing a small molecule inhibitor according to claim 5, wherein, The preparation method further includes: centrifuging the mixture.

7. The preparation method of the hydrogel containing a small molecule inhibitor according to any one of claims 1-4, characterized in that, It includes: First, add δ-Amyrenone to the aqueous calcium gluconate solution containing calcium peroxide nanoparticles, and mix to obtain an aqueous calcium gluconate solution containing δ-Amyrenone and calcium peroxide nanoparticles. Then, mix the aqueous sodium alginate solution containing melanin nanoparticles, the reducing agent with the aqueous calcium gluconate solution containing δ-Amyrenone and calcium peroxide nanoparticles.

8. Use of the hydrogel containing a small molecule inhibitor as described in any one of claims 1-4 or the hydrogel containing a small molecule inhibitor prepared by the preparation method of the hydrogel containing a small molecule inhibitor as described in claim 7 in the preparation of a drug for preventing and / or treating ischemic heart disease, characterized in that The ischemic heart disease is myocardial infarction reperfusion injury.

9. The application according to claim 8, wherein The hydrogel containing the small molecule inhibitor is administered by injection.

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