Hydrogel and application thereof in ischemic heart disease

Abstract

The invention discloses hydrogel and application thereof in ischemic heart disease, and relates to the technical field of myocardial infarction repair. The invention provides the multifunctional hydrogel which has injectability and can realize in-situ injection. The hydrogel has the comprehensive characteristics of improving the conductivity of an infarction area, generating oxygen, removing ROS, promoting M2 type polarization of macrophages and promoting angiogenesis. The excellent performance of the compound is outstanding in the aspects of relieving MI / RI, improving the local microenvironment and enhancing the myocardial repair function, and a new direction is provided for heart injury treatment. Meanwhile, the CSA-delta hydrogel formed after the drug is loaded has the drug sustained release capability, so that the drug is continuously and stably released, and the drug action time is prolonged.

Description

Technical Field

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

[0002] Myocardial infarction (MI) is a major cause of disability and death worldwide. Its pathogenesis is mainly related to the rupture of coronary atherosclerotic plaques or endothelial erosion, especially in elderly patients. Although current treatment strategies focus 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 proliferation capacity of adult mammalian cardiomyocytes, existing treatments are difficult to effectively repair the large number of cardiomyocytes lost 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 aggravate myocardial cell loss, expand the area of ​​necrosis, 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, cardiac function decline, and eventually develop into chronic heart failure. In addition, reperfusion injury may also cause serious arrhythmias, further endangering the patient's life safety. These complications significantly affect the overall effect of reperfusion therapy and become an important obstacle to improving the long-term prognosis of patients.

[0004] Recent studies have shown that by targeting and inhibiting the key factor CBX7 through gene editing, cardiomyocytes can be induced to re-enter a proliferative state, thereby reducing the degree of injury in young mice during ischemia-reperfusion injury. However, MI should pay more attention to the biological characteristics of the elderly. In addition, gene therapy is limited by the long clinical transformation cycle and high technical barriers. In contrast, the development of small molecule inhibitors targeting CBX7 has significant advantages such as strong specificity and clear mechanism of action, and is easier to achieve clinical transformation.

[0005] However, traditional oral or injection administration methods have problems such as poor targeting and low utilization rate, and innovative drug delivery systems are urgently needed. 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 simulating human ECM. Studies have revealed that this biomaterial can not only provide the necessary mechanical support for myocardial repair, but also act as a transport medium for cells or drugs, playing a key role. With the in-depth understanding of the changes in the myocardial microenvironment after myocardial infarction, smart and functional hydrogels have been developed for cardiac repair. Zou et al. designed Gel@Exo conductive hydrogel to enhance the electrical coupling between myocardial cells. The application of this hydrogel effectively improved cardiac function, including improving ejection fraction and fractional shortening, and reducing the area of ​​fibrosis. Li et al. developed a microRNA-21-5p delivery system based on functionalized mesoporous silica nanoparticles, which can achieve controllable delivery through injection of hydrogels, promote angiogenesis, and significantly reduce the area of ​​infarction. However, there are still problems such as hypoxia, oxidative stress, insufficient angiogenesis, and insufficient electrical coupling, which cannot provide a good environment for myocardial regeneration.

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

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

[0008] The present invention is achieved in that: In a first aspect, the present invention provides a hydrogel, which comprises: a sodium alginate aqueous solution containing melanin nanoparticles, a reducing agent, and a calcium gluconate aqueous solution containing calcium peroxide nanoparticles, wherein 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 - 3.5:1, the final concentration of the melanin nanoparticles in the hydrogel is 0.8-1.5 mg / ml, the mass fraction of sodium alginate in the sodium alginate aqueous solution is 2.5%-3.5%, the concentration of calcium peroxide nanoparticles in the calcium gluconate aqueous solution is 0.8-1.5 mg / ml, and the mass fraction of calcium gluconate in the calcium gluconate aqueous solution is 2.5%-3.5%; the ratio of the sodium alginate aqueous 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.

[0009] 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.

[0010] In a third aspect, the present invention further provides a hydrogel containing a small molecule inhibitor, which comprises the above hydrogel or the hydrogel prepared by the above hydrogel preparation method and a small molecule inhibitor, wherein the small molecule inhibitor is δ-Amyrenone.

[0011] 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 solution of calcium gluconate containing calcium peroxide nanoparticles, mixing to obtain an aqueous solution of calcium gluconate containing δ-Amyrenone and calcium peroxide nanoparticles, and then mixing an aqueous solution of sodium alginate containing melanin nanoparticles and a reducing agent with the aqueous solution of calcium gluconate containing δ-Amyrenone and calcium peroxide nanoparticles.

[0012] In a fifth aspect, the present invention also provides the use of a hydrogel or a hydrogel containing a small molecule inhibitor in the preparation of a drug for preventing and / or treating ischemic heart disease.

[0013] The present invention has the following beneficial effects: The present invention provides a hydrogel (CSA), which is injectable and can be injected in situ. In addition, the CSA hydrogel exhibits a strong long-term oxygen production capacity and has significant potential to alleviate the hypoxic environment in the myocardial infarction area.

[0014] CSA hydrogel exhibits good electrical conductivity and can provide an electrical environment similar to that of myocardial tissue. By introducing melanin and calcium peroxide, the conductivity of 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.

[0015] CSA hydrogel showed good biocompatibility in cell experiments. Even at a concentration of 10%, no obvious cytotoxicity was observed, and cell viability was well maintained, providing solid biocompatibility support for its further application in animal experiments.

[0016] CSA hydrogel effectively removes ROS, reduces oxidative stress, and reduces damage to cardiomyocytes and human umbilical vein endothelial cells in a ROS environment. In addition, 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 the degree of cardiac fibrosis, increase the thickness of the ventricular wall, and thus have a positive effect on cardiac function.

[0017] At the same time, the CSA-δ hydrogel (CSA containing δ-Amyrenone) formed after loading the drug has the ability to release the drug slowly, which enables the drug to be released continuously and stably, thereby prolonging the drug action time; 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. The experimental results of the present invention indicate that 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

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 Preparation and characterization of CaO2NPs ((a) XRD spectrum of CaO2; (b) FTIR spectrum of CaO2; (c) SEM image of CaO2, scale bar = 500nm); Figure 2 The preparation and characterization results of MNPs are shown in Figure 2 ((a) FTIR image of MNPs; (b) SEM image of MNPs, scale bar = 500 nm); Figure 3 Verification of the fluidity and injectability of SA and CSA hydrogels (demonstration of the fluidity of the two hydrogels in an inverted container and syringe writing experimental results); Figure 4 Rheological and FT-IR characterization of hydrogels ((a) Rheological analysis of SA and CSA hydrogels; (b) IR spectra of calcium peroxide, ascorbic acid, melanin, SA, and CSA hydrogels); Figure 5 The conductivity 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. The data in the figure represent Mean±SD. Significant difference: **p<0.01); Figure 6 is the drug release curve of CSA-δ hydrogel; Figure 7 The results of the determination of various parameters of hydrogel degradation are shown in Figure 2 ((a) oxygen production curve of CSA hydrogel; (b) degradation curve of CSA hydrogel; (c) statistics of pH value of CSA hydrogel products); Figure 8 This is the result of the hydrogel biocompatibility test (the green line in the figure represents 80% cell viability); Fig. 9 The results of the hydrogel scavenging ROS level test are shown in Figure 2 ((a) SA and CSA hydrogels scavenging DPPH free radicals; (b) representative fluorescence images of total intracellular reactive oxygen species (DCFH-DA) activity after SA and CSA hydrogel treatment; scale bar = 100 um); Fig.10 The results of the hydrogel angiogenesis-promoting ability test are shown in Figure 2 ((a) CCK-8 test results of HUVEC after being treated with SA or CSA hydrogel for 3 days in ROS environment; (b) Western-blot test results of angiogenesis-related proteins (VWF, Vcam1 and CD31) of HUVEC after being treated with SA or CSA hydrogel for 3 days in ROS environment. The data in the figure represent Mean±SD. Significant difference: ****p<0.0001); Fig.11 The results of the hydrogel-induced macrophage polarization test are shown in Figure 1 (representative immunofluorescence images showing the expression of CD86 and CD206 in THP-1-derived macrophages under different treatments; scale bar = 100 um); Fig.12 Detection of the ROS scavenging ability of hydrogels in vivo (representative fluorescence images of superoxide anion radical activity (DHE, red) in heart slices 1 day after treatment with different conditions; scale bar = 100 um); Fig.13 The results of the hydrogel in vivo angiogenesis-promoting ability test are shown in Figure 2 (representative fluorescent images of α-smooth muscle actin (α-SMA, red) in heart slices 28 days after treatment with different conditions; scale bar = 100 um); Fig.14 The results of the hydrogel's ability to promote macrophage polarization in vivo are shown in Figure 2 ((a) Immunofluorescence images of CD86 and CD206 in myocardial tissue on the first day after treatment. (b) Immunofluorescence images of CD86 and CD206 in myocardial tissue on the third day after treatment; scale bar = 50 um); Fig.15After 28 days of treatment, the results of cardiac function indexes of rats were analyzed by echocardiography ((a) M-mode echocardiography images of rats in each group after 28 days of treatment, which directly reflect the structure and movement state of the heart; (b) Doppler echocardiography images, which further reveal the characteristics of cardiac hemodynamics; (ce) quantitative comparison of left ventricular function of rats in different groups after 28 days of treatment. The data in the figure represent Mean±SD. Significant differences: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001); Fig.16 The Masson staining results of the hearts of each group 28 days after MI / R ((a) The figure shows the Masson trichrome staining results of the heart regional sections of each group, the scale bar of the complete section = 1mm, the scale bar of the regional screenshot = 100um; (b) Based on these Masson trichrome staining images, we conducted a quantitative analysis of the thickness of the left ventricular anterior wall. The data in the figure represent Mean±SD. Significant difference: *p<0.05, ***p<0.001, ****p<0.0001); Fig.17 HE staining images of various organs of rats in each group 28 days after MI / R, scale bar = 200 μm. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0021] CSA-δ hydrogel: a hydrogel containing δ-Amyrenone.

[0022] MI / RI: myocardial infarction-reperfusion injury.

[0023] CSA: Injectable composite sodium alginate hydrogel.

[0024] SA: injectable sodium alginate hydrogel.

[0025] MNPs: melanin nanoparticles.

[0026] In a first aspect, the present invention provides a hydrogel, which comprises: a sodium alginate aqueous solution containing melanin nanoparticles, a reducing agent, and a calcium gluconate aqueous solution containing calcium peroxide nanoparticles, wherein 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 - 3.5:1, the final concentration of the melanin nanoparticles in the hydrogel is 0.8-1.5 mg / ml, the mass fraction of sodium alginate in the sodium alginate aqueous solution is 2.5%-3.5%, the concentration of calcium peroxide nanoparticles in the calcium gluconate aqueous solution is 0.8-1.5 mg / ml, and the mass fraction of calcium gluconate in the calcium gluconate aqueous solution is 2.5%-3.5%; the ratio of the sodium alginate aqueous 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.

[0027] CaO2 (calcium peroxide) is a widely used solid oxygen release agent. Compared with H2O2, it exhibits a more gradual and sustained oxygen release characteristic, which can effectively prevent the rapid accumulation of local reactive oxygen species and maintain redox homeostasis. However, conventional-sized CaO2 particles are large in size and tend to agglomerate when exposed to water, which significantly slows down the oxygen release rate. In contrast, nanoscale CaO2 particles can not only greatly accelerate the reaction kinetics, but also effectively overcome the aggregation phenomenon between particles and optimize their performance. The preparation method of calcium peroxide nanoparticles refers to the method reported in the prior art, such as (HUANG Y, FU Z, WANGH, 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 replenish oxygen in the infarct site.

[0028] Ascorbic acid can regulate the acid-base balance and prevent the alkaline environment produced by calcium peroxide from having adverse effects on tissues.

[0029] The natural MNPs contained in squid ink are regarded as an important bioactive substance with excellent protective functions, which can effectively shield ultraviolet radiation, resist reactive oxygen attack, neutralize toxins and chelate metal ions. These naturally occurring melanins not only have good biocompatibility and biodegradability, but also show excellent redox activity and semiconductive properties, thus showing great potential in biomedical applications. The present invention adds melanin nanoparticles to the hydrogel, which has the effect of removing excess ROS and can promote the polarization of macrophages to M2 type, which further helps to inhibit inflammatory response.

[0030] By introducing melanin and calcium peroxide, the conductivity of CSA hydrogel was significantly improved, which is consistent with the electrical signal conduction level of natural myocardial tissue, showing great application potential in cardiac tissue engineering repair.

[0031] In order to achieve in situ drug release and optimize the microenvironment after myocardial infarction reperfusion, it is particularly appropriate to use a fluid hydrogel that can be directly injected into the myocardium as a drug delivery carrier. The present invention prepares a fluid injectable hydrogel by adjusting the ratio of sodium alginate and calcium ions. Furthermore, using this hydrogel as a base, ascorbic acid, melanin nanoparticles and calcium peroxide nanoparticles are incorporated into the preparation process to successfully synthesize CSA hydrogel.

[0032] CSA hydrogel also exhibits strong long-term oxygen production capacity and has significant potential to alleviate the hypoxic environment in the myocardial infarction area.

[0033] CSA hydrogel showed good biocompatibility in cell experiments. Even at a concentration of 10%, no obvious cytotoxicity was observed, and cell viability was well maintained, providing solid biocompatibility support for its further application in animal experiments.

[0034] In addition, CSA hydrogel can effectively remove ROS, reduce oxidative stress, and reduce the damage of cardiomyocytes and human umbilical vein endothelial cells in the ROS environment. In addition, 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 reduce the degree of cardiac fibrosis, increase the thickness of the ventricular wall, and thus have a positive effect on cardiac function.

[0035] In an optional 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 the 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, and 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.

[0036] In a preferred embodiment of the present invention, the melanin nanoparticles are natural melanin nanoparticles or modified melanin nanoparticles.

[0037] In a preferred embodiment of the present invention, natural melanin nanoparticles are prepared from raw material squid ink sacs. In one embodiment, squid ink sacs are mixed with water, impurities are removed, and the precipitate is freeze-dried to prepare natural melanin nanoparticles. In addition, the powder in the squid ink sac can be directly ultrasonically washed in deionized water, and the melanin nanoparticles are separated by centrifugation, washed and dried.

[0038] The modification is carried out, for example, by using an organic solvent, such as APTES (triaminopropyltriethoxysilane), followed by drying and ultrasonic dispersion. The modification is performed to improve mechanical properties, UV shielding efficiency, etc.

[0039] In a second aspect, the present invention also provides a method for preparing a hydrogel, which comprises the following steps: adding ascorbic acid 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.

[0040] In a preferred embodiment of the present invention, the preparation method further comprises: centrifuging the mixed solution.

[0041] In a third aspect, the present invention further provides a hydrogel containing a small molecule inhibitor, which comprises the above hydrogel or the hydrogel prepared by the above hydrogel preparation method and a small molecule inhibitor, wherein the small molecule inhibitor is δ-Amyrenone.

[0042] δ-Amyrenone is an effective small molecule inhibitor of CBX7 identified by the inventors through screening. It can regulate the cell cycle, inhibit apoptosis, and promote cardiomyocyte proliferation. When loaded into a hydrogel, it exhibits a certain cardioprotective effect, which is specifically manifested in the ability to reduce the infarct area and reduce the degree of cardiac fibrosis, increase the thickness of the ventricular wall, and thus have a positive effect on cardiac function. CSA-δ hydrogel not only significantly promotes the regeneration and repair of cardiomyocytes, but also further enhances its repair effect by constructing a microenvironment that is conducive to myocardial recovery. The experimental results of the present invention indicate that CSA-δ hydrogel has significant potential in the field of myocardial infarction repair, provides a new therapeutic strategy for the recovery of cardiac function after MI / R, and may have important clinical application prospects.

[0043] In a preferred embodiment of the present invention, each ml of hydrogel contains 10 mg of small molecule inhibitor.

[0044] In a fourth aspect, the present invention also provides a method for preparing a hydrogel containing a small molecule inhibitor, which comprises: firstly mixing δ-Amyrenone with a calcium gluconate aqueous solution containing calcium peroxide nanoparticles to obtain a calcium gluconate aqueous solution containing δ-Amyrenone and calcium peroxide nanoparticles, and then mixing a sodium alginate aqueous solution containing melanin nanoparticles and ascorbic acid with the calcium gluconate aqueous solution containing δ-Amyrenone and calcium peroxide nanoparticles.

[0045] In a fifth aspect, the present invention also provides the use of a hydrogel or a hydrogel containing a small molecule inhibitor in the preparation of a drug for preventing and / or treating ischemic heart disease.

[0046] In a preferred embodiment 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.

[0047] This embodiment develops a multifunctional hydrogel drug delivery system, which aims to achieve the precise in situ release of δ-Amyrenone in the damaged part of the heart and the improvement of the microenvironment. This hydrogel drug delivery system consists of a skeleton composed of sodium alginate and calcium gluconate, which can be injected in situ into the area of ​​myocardial infarction and has a conductive property similar to that of myocardial tissue. In addition, the hydrogel is filled with calcium peroxide nanoparticles, which can slowly release oxygen to help replenish oxygen in the infarcted area, and ascorbic acid is added to adjust the acid-base balance to prevent the alkaline environment produced by calcium peroxide from having adverse effects on the tissue and removing ROS at the same time. At the same time, melanin nanoparticles are added to the hydrogel, which has the effect of removing excess ROS, and can promote the polarization of macrophages to M2 type, which further contributes to the inhibition of inflammatory response. In other words, the core of the present invention focuses on building a stable carrier platform to ensure that δ-Amyrenone can be efficiently released in the specific environment of the heart, and simultaneously create a microenvironment that is conducive to the repair of cardiac tissue, opening up a novel and effective strategic approach for the treatment of ischemic heart disease.

[0048] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0049] Example 1 Screening of compound δ-Amyrenone.

[0050] The inventors obtained the information of CBX7 protein from the Uniport database, obtained the small molecule compound library from Taoshu Biology, and screened out the compounds with the most potential specific binding ability through Discovery Studio software. Based on 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 with docking scores, and the binding mode and energy stability of the candidate compound δ-Amyrenone were verified by 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 that the binding is very stable.

[0051] Table 1 The top 10 small molecule compounds with the best docking scores after high-throughput screening

[0052] Example 2 This embodiment develops a multifunctional hydrogel drug delivery system, aiming to achieve the precise in situ release of δ-Amyrenone in the damaged part of the heart and the improvement of the microenvironment. This embodiment provides a preparation method of an injectable composite hydrogel drug delivery system (CSA hydrogel).

[0053] 1. Experimental Materials 1.1 Experimental cell lines Human cardiomyocyte cell line AC16, human monocytic cell line THP-1 and human umbilical vein endothelial cells HUVEC were all from ATCC.

[0054] 1.2 Experimental Reagents Table 2 Main experimental materials and reagents

[0055] 1.3 Reagent preparation Cell culture medium: add 5.6 ml of double antibody and 56 ml of FBS to every 500 ml of basal culture medium (DMEM / F-12 for AC16, RPMI 1640 for THP-1, and DMEM for HUVEC).

[0056] 2. Experimental Methods 2.1 Preparation of calcium peroxide nanoparticles (CaO2NPs) (1) Preparation of reaction solution: First, completely dissolve 3 g of calcium chloride in 30 ml of distilled water, then add 15 ml of 1 mol / L ammonia solution and 120 ml of polyethylene glycol 200 (PEG200), and continue stirring at room temperature until the mixture is well mixed.

[0057] (2) Slow addition of hydrogen peroxide: Slowly add 30 ml of 30% concentration hydrogen peroxide solution to the above stirring mixture at a rate not exceeding 20-30 seconds / drop.

[0058] (3) Adjust the pH value and observe the precipitation: After the addition of hydrogen peroxide is complete, continue to add ammonia solution to adjust the pH value of the mixed solution to 10. During this process, a white or light yellow precipitate will gradually form in the solution. Continue stirring for 2 hours to ensure that the reaction is fully carried out.

[0059] (4) Extraction and purification of the product: The mixture was centrifuged at a centrifugal force of 12,000 g for 5 minutes to separate the precipitate. After discarding the supernatant, the precipitate was washed three times with a sodium hydroxide solution with a pH value of 13, and then washed twice with distilled water. Finally, the washed precipitate was placed in an oven at 80 degrees Celsius and dried for 2 hours to obtain the final product.

[0060] 2.2 Preparation of natural melanin nanoparticles (MNPs) (1) Sample preparation The squid ink sacs were purchased from the Taobao store “Qingdao Xinjie Aquatic Products” and taken out from a -20°C refrigerator.

[0061] (2) Dissolution and filtration Take a certain amount of ink sac and add distilled water. Crush and stir until dissolved. Stir with a stirrer at a constant speed for 12 hours to dissolve lipids and other impurities. After precipitation, discard the supernatant and add distilled water again. Repeat this step 6 times.

[0062] (3) Freezing and freeze-drying The final precipitate was frozen at -80°C and then freeze-dried.

[0063] 2.3 Scanning electron microscopy characterization of MNPs and CaO2NPs To preserve the original morphology of MNPs and CaO2 NPs, we took out samples from the freeze-dried state. Then, scanning electron microscopy was used to characterize the microstructure of the freeze-dried samples.

[0064] 2.4 X-ray diffraction characterization of CaO2NPs The freeze-dried CaO2NPs were taken for X-ray diffraction (XRD) analysis. First, the sample was ground and evenly spread to ensure that its surface was flat, and then scanned in an X-ray diffractometer with a scanning range set to 2θ 20° to 60°, using Cu Kα radiation (wavelength 1.5406 Å), and tested at a scanning rate of 0.5° / min.

[0065] 2.5 Preparation of injectable composite hydrogel drug delivery system (CSA hydrogel).

[0066] Preparation of CSA hydrogel: First, prepare 3 ml of melanin nanoparticle aqueous solution with a concentration of 1.34 mg / ml (the final concentration after gelation is 1.34 mg / ml), and prepare a 3% mass fraction sodium alginate aqueous solution containing melanin nanoparticles based on this, and add 1.5 mg of ascorbic acid during stirring. Next, prepare a 3% mass fraction calcium gluconate aqueous solution (the reaction temperature needs to be appropriately increased), add 1 mg of calcium peroxide nanoparticles, and stir well to make it evenly dispersed. Subsequently, the sodium alginate aqueous solution and the calcium gluconate aqueous solution are mixed in a volume ratio of 3:1 (in this embodiment, 3 ml of melanin nanoparticle aqueous solution and 1 ml of calcium gluconate aqueous solution), ensuring that the solution does not clump and evenly merges during the process. After completion, continue stirring for 10 minutes to ensure sufficient mixing. Finally, remove bubbles by centrifugation to obtain an injectable composite hydrogel.

[0067] Example 3 In this example, CSA-δ (CSA-δ-Amyrenone) hydrogel was prepared.

[0068] On the basis of CSA hydrogel, 40 mg of small molecule inhibitor δ-Amyrenone was added to the system, and calcium gluconate aqueous solution was added with calcium peroxide, and then δ-Amyrenone was added, and finally CSA-δ hydrogel was synthesized with sodium alginate solution with melanin.

[0069] Comparative Example 1 Preparation of injectable sodium alginate hydrogel (SA hydrogel) First, prepare a 3% (mass fraction) sodium alginate aqueous solution and a 3% (mass fraction) calcium gluconate aqueous solution. It is worth noting that the operating temperature needs to be moderately increased when preparing the calcium gluconate solution. While continuously stirring 3 ml of the sodium alginate aqueous solution, add 1 ml of the calcium gluconate aqueous solution dropwise to ensure that the solution does not clump and is evenly mixed during the addition process. After the addition is completed, continue stirring for 10 minutes to fully mix. Afterwards, remove the bubbles in the solution by high-speed centrifugation, and finally obtain an injectable sodium alginate hydrogel.

[0070] Experimental Example 1 This experimental example experiments the hydrogels prepared in Examples 2-3 and Comparative Example 1 respectively.

[0071] 1. Experimental Methods 1. Fourier transform infrared spectroscopy (FT-IR) characterization.

[0072] In order to improve the stability of the samples, CaO2, MNPs, ascorbic acid, and two hydrogels, SA (Comparative Example 1) and CSA (Example 2), were first freeze-dried. Subsequently, these samples were placed in the sample slot of the Fourier transform infrared spectrometer, and the spectral scanning range was set. By executing the spectral scanning program, the FT-IR spectrum of each sample was obtained.

[0073] 2. Conductive characterization of hydrogels prepared by SA (Comparative Example 1) and CSA (Example 2).

[0074] A constant voltage circuit system is designed, which uses a light-emitting diode (LED) as an indicator element. In this circuit, hydrogel and components such as light-emitting diodes are connected in sequence. By directly observing the changes in the brightness of the light-emitting diodes, we can indirectly evaluate the conductive performance of the hydrogel. In order to further quantitatively evaluate the conductivity of the hydrogel, a resistivity tester is used to conduct a systematic experiment. First, a hydrogel sample is prepared. Then, the electrodes of the resistivity tester are precisely placed on both sides of the hydrogel sample to ensure that the electrodes are in full contact with the sample, so as to accurately measure the resistivity.

[0075] 3. The oxygen production capacity of the CSA hydrogel prepared in Example 2 was tested.

[0076] A volume of 20 ml of CSA was prepared and added to 40 ml of water. After evacuation to exclude air, the system was connected to a gas chromatograph to monitor and determine the amount of oxygen generated regularly (once every two days).

[0077] 4. Test the pH change during the degradation process of the CSA hydrogel prepared in Example 2.

[0078] Prepare 2 ml of composite hydrogel CSA, place the hydrogel in several times the volume of neutral water, observe its degradation time, detect the pH value of the degraded liquid every day, and weigh the undegraded part after 1, 2, 4, 7, and 15 days respectively.

[0079] 5. Rheological properties of the hydrogels prepared by SA (Comparative Example 1) and CSA (Example 2) were tested.

[0080] In order to verify the rheological properties of the two hydrogels, a systematic experiment was conducted using a rheometer. First, the 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 (0.1 Hz to 100 Hz).

[0081] 6. Determination of drug release curve of CSA-δ hydrogel prepared in Example 3.

[0082] A hydrogel containing 1 mg of the small molecule inhibitor δ-Amyrenone was synthesized and added to a brown bottle containing 2 mL of PBS buffer at pH 7.4. The bottle was placed in a 37°C constant temperature shaking incubator. In the initial stage of the experiment (0-2 days), we took out 200 microliters of samples from the bottle every 12 hours and supplemented with an equal amount of PBS to keep the total volume constant; in the next five days (2-7 days), the sampling was changed to every 24 hours; and in the next eight days (7-15 days), the sampling was adjusted to every 48 hours. The absorbance of each sample was measured at a wavelength of 230 nanometers using a microplate reader equipped with a UV spectrophotometer function, and the obtained data was recorded in detail. By converting these absorbance values ​​to the corresponding drug concentration, we further drew the drug release curve to evaluate the slow release drug performance of CSA-δ hydrogel.

[0083] 7. The cell biocompatibility of the CSA hydrogel prepared in Example 2 was tested.

[0084] Human cardiomyocytes were seeded on a 96-well plate at different densities until the cells were stable. The hydrogel was prepared according to the scheme in Example 2, and the aqueous solution in the production process was replaced with a cell culture medium. The prepared hydrogel was mixed with a complete culture medium in different proportions (0%, 1%, 5%, 8%, 10%, 20%). The cell culture medium was replaced with the previously prepared hydrogel culture medium mixture, 200ul was added to each well, and the culture was continued for 24h, 72h, and 168h (the pre-prepared mixed solution was replaced in the middle of the longer time group). After the culture cycle, the culture solution in each well was removed, and then the CCK-8 mixed solution was added to each well. The 96-well plate containing the CCK-8 mixed solution was placed in a 37°C incubator for 2 hours. Afterwards, the 96-well plate was transferred to a microplate reader to measure the absorbance value at 450 nanometers. These absorbance data were used to analyze cell survival rates to evaluate the biocompatibility of the prepared hydrogels for human cardiomyocytes.

[0085] 8. The hydrogels prepared by SA (Comparative Example 1) and CSA (Example 2) were tested for their DPPH scavenging performance.

[0086] Weigh an appropriate amount of DPPH powder, dissolve it in anhydrous ethanol, mix and vortex thoroughly to prepare a DPPH mother solution with a concentration of 1 mM. Dilute the mother solution to 0.1 mM with anhydrous ethanol to obtain a DPPH sub-liquid. Prepare 1 ml of SA hydrogel and CSA hydrogel according to the schemes of Comparative Example 1 and Example 2, respectively. Take 300 ul of PBS (as a control group), 300 ul of SA hydrogel and CSA hydrogel (as an experimental group) and add them to 800 ul of DPPH sub-liquid, incubate in the dark for 6 hours, and then measure the absorbance of the three groups of mixed liquids at 517 nm with an ELISA reader. Calculate the clearance rate using Formula 1. Set up 3 parallel samples for each group.

[0087] (Formula 1) 9. The hydrogels prepared by SA (Comparative Example 1) and CSA (Example 2) were tested for their ROS performance.

[0088] The levels of multiple reactive oxygen species (ROS) in cells were evaluated by staining with 2',7'-dichlorodifluorofluorescein diacetate (DCFH-DA). Cardiomyocytes were cultured in a normal environment or in a ROS microenvironment induced by 200 μM H2O2, with or without the addition of a certain concentration of SA or CSA hydrogel. After 72 hours, 10 μM DCFH-DA was used for incubation in the dark for 15-25 minutes. After washing with PBS, 4',6-diamidino-2-phenylindole (DAPI) was used for staining. Samples were observed and recorded under a fluorescence microscope.

[0089] 10. The hydrogels prepared by SA (Comparative Example 1) and CSA (Example 2) were tested for their angiogenesis-promoting properties.

[0090] HUVEC cells were cultured in a special culture medium, seeded in a 96-well plate at a certain density, and placed in an incubator for 8-12 hours. Subsequently, HUVEC cells were divided into two groups: a normal culture environment group and a 200 μM H2O2-induced ROS microenvironment group. In the two environments, SA and CSA hydrogels were added or not added at a certain concentration, and a total of four experimental conditions were set. After 72 hours of culture, the culture medium was removed and CCK-8 working solution (prepared according to the reagent instructions) was added to each well. The 96-well plate containing CCK-8 working solution was placed in an incubator and incubated for 2 hours to allow the reaction to fully occur. After the incubation, the 96-well plate was transferred to a microplate reader to measure the absorbance at 450 nanometers. The absorbance value was analyzed and the cell survival rate was calculated to determine the promoting effect of the hydrogel on the proliferation of HUVEC cells under ROS microenvironment conditions.

[0091] At the same time, HUVEC cells were cultured under the above four conditions. After the culture, cell samples were collected, protein was extracted, and the expression of angiogenesis-related indicators in HUVEC cells was verified by Western blot experiments to further evaluate the functional effects of the hydrogel.

[0092] 11. The hydrogels prepared by SA (Comparative Example 1) and CSA (Example 2) were tested for their ability to promote macrophage polarization.

[0093] (1) Cell culture: THP-1 cells were cultured in culture medium and induced with PMA for 24 hours to adhere to the wall. Subsequently, the cells were seeded into 6-well plates at a certain density and divided into two groups after the cells stabilized: a normal culture environment group and a 200 μM H2O2-induced ROS microenvironment group. In these two environments, SA and CSA hydrogels were added or not at a certain concentration, and four experimental conditions were set up for 24 hours of culture.

[0094] (2) Cell fixation: Gently rinse the cells with pre-cooled PBS to remove the culture medium. Add 4% paraformaldehyde and fix the cells for 10-15 minutes at room temperature. Wash the cells 3 times with PBS, 5 minutes each time, and remove the excess fixative.

[0095] (3) Cell permeabilization: Use permeabilization solution 0.1% Triton X-100 for 5-10 minutes to help the antibody penetrate into the cells. Wash the cells 3 times with PBS, 5 minutes each time.

[0096] (4) Blocking: Use 5% goat serum as the blocking solution and perform the blocking treatment at room temperature for 30 to 60 minutes to prevent the occurrence of nonspecific binding reactions.

[0097] (5) Antibody incubation: Add primary antibody (such as primary antibody against target protein), dilute according to manufacturer's instructions, and incubate at 4°C for 10-12 hours. Then wash with PBS three times, 5 minutes each time.

[0098] (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 with PBS three times, 5 minutes each time.

[0099] (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.

[0100] (8) Microscope observation: Use a fluorescence microscope to observe and record the fluorescence signal.

[0101] 12. Statistical analysis methods The data obtained from the experiment were expressed as 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, where * represents p<0.05, ** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001, all of which are used to indicate statistically significant differences.

[0102] 2. Experimental Results 1. Preparation and characterization of CaO2NPs.

[0103] CaO2, as a widely used solid oxygen release agent, exhibits a more gradual and sustained oxygen release characteristic compared to H2O2, which can effectively prevent the rapid accumulation of local reactive oxygen species and thus maintain redox homeostasis. However, conventional-sized CaO2 particles are large in size and tend to agglomerate when exposed to water, significantly slowing down the oxygen release rate. In contrast, nanoscale CaO2 particles can not only significantly accelerate the reaction kinetics, but also effectively overcome the aggregation phenomenon between particles and optimize their performance.

[0104] We prepared calcium peroxide nanoparticles using a previously reported method. The X-ray diffraction (XRD) pattern of the obtained sample is shown in Figure 1As shown in Figure 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 crystal phase purity of the sample. At the same time, we conducted FTIR testing on the prepared samples, and the results are shown in Figure 5. Figure 1 As shown in b. In the infrared spectrum, the positions of the characteristic peaks are consistent with those reported in the literature, which further proves the chemical composition and structure of the sample. In addition, we used scanning electron microscopy (SEM) to observe the morphology of the sample. The results are shown in Figure 1 As shown in Figure c. The SEM image clearly shows that most of the prepared calcium peroxide particles are distributed at the nanometer level, further verifying the successful preparation of nanoparticles. In summary, through the comprehensive characterization of XRD, FTIR and SEM, we have successfully prepared calcium peroxide nanoparticles with pure crystal phase, clear chemical structure and uniform particle size, laying a solid foundation for its further research and application of oxygen release performance.

[0105] 2. Preparation and characterization of MNPs.

[0106] The natural MNPs contained in squid ink are regarded as an important bioactive substance with excellent protective functions, which can effectively shield ultraviolet radiation, resist reactive oxygen attack, neutralize toxins and chelate metal ions. These naturally occurring melanins not only have good biocompatibility and biodegradability, but also show excellent redox activity and semiconducting properties, thus showing great potential in biomedical applications.

[0107] We successfully extracted melanin nanoparticles using a simple centrifugation and stirring method. According to the scanning electron microscopy (SEM) results ( Figure 2 (b) shows that the obtained MNPs are spherical, with uniform size distribution and particle size less than 500 nm. In addition, we conducted Fourier transform infrared spectroscopy (FTIR) test on the prepared samples, and the results are shown in Figure 2 As shown in a. The characteristic peaks of the FTIR spectrum are completely consistent with those reported in the literature, further confirming the accuracy of the chemical composition and structure of the sample.

[0108] 3. Characterization of SA hydrogel and CSA hydrogel.

[0109] In order to achieve in situ drug release and optimize the microenvironment after myocardial infarction and reperfusion, it is particularly appropriate to use a fluid hydrogel that can be directly injected into the myocardium as a drug delivery carrier. In this experimental section, we prepared a fluid injectable hydrogel by adjusting the ratio of sodium alginate and calcium ions. Furthermore, using this hydrogel as the base, ascorbic acid, MNPs and CaO2NPs were incorporated into the preparation process to successfully synthesize CSA hydrogel. Figure 3As shown in the figure, both hydrogels exhibit moderate fluidity, which is not as good as pure water, but slight flow can be observed when the container is inverted. By loading the syringe and performing writing tests, we verified that these hydrogels have excellent injectable properties.

[0110] Figure 4 Figure a shows that the storage modulus (G') of SA and CSA hydrogels is higher than the loss modulus (G''), which reveals that the prepared materials have the typical characteristics of hydrogels. However, G' is relatively low, suggesting that the hydrogel is in a weak gel state. In order to confirm that CaO2, ascorbic acid and MNPs were successfully loaded into the composite hydrogel, FT-IR analysis was used to detect each component and CSA. Figure 4 As shown in (b), the correspondence between the characteristic peaks of CSA and the components not only confirms the existence of CaO2 and MNPs, but also effectively verifies the loading of ascorbic acid.

[0111] 4. Characterization of the conductive properties of injectable hydrogels and injectable composite hydrogels.

[0112] To quantify the conductivity properties of SA and CSA, we constructed a circuit system with integrated light-emitting diodes to visualize their electrical performance. Figure 5 As shown in Figure a, although SA hydrogel is able to drive light-emitting diodes to emit light, its brightness is relatively low. In contrast, 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 improving the conductive properties. Comprehensive analysis shows that CSA hydrogel shows good application potential in regulating cardiac tissue electrophysiology.

[0113] The conductivity of natural myocardium ranges from 10 -4 ~10 S / cm, which provides an important reference for designing conductive hydrogels for repair after myocardial infarction. To further verify the conductive properties of the two hydrogels, we used a resistivity tester to quantify their conductivity. Figure 5 As shown in middle b, the conductivity of SA hydrogel is 0.084±0.007 S / m, showing relatively low conductivity; while the conductivity of CSA hydrogel is increased to 0.115±0.006 S / m, which is consistent with the electrical signal conduction level of natural myocardial tissue.

[0114] 5. Determination of drug release curve of CSA-δ hydrogel.

[0115] In the in vitro drug release test, we used CSA-δ hydrogel loaded with 1 mg of δ-Amyrenone. During the experiment, samples were continuously collected and analyzed to draw a drug release curve (see Figure 6 ). The experimental results showed that CSA exhibited obvious sustained-release properties under neutral conditions: on the second day, the drug release rate exceeded 50%, and on the 15th day, the release rate exceeded 85%. These data show that the hydrogel has excellent drug sustained-release properties, providing strong support for its application in long-term drug delivery.

[0116] 6. Detection of oxygen production capacity, degradation and pH change of CSA hydrogel When CaO2 comes into contact with water, it decomposes into H2O2 and Ca(OH)2, and H2O2 then continues to decompose into oxygen and water. In order to control the amount of H2O2 accumulation, we introduced ascorbic acid 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 neutralize the local alkaline environment caused by Ca(OH)2. Not only that, the antioxidant properties of ascorbic acid can also prevent calcium overload caused by damaged cellular calcium channels, thereby maintaining the normal function of cells.

[0117] like Figure 7 As shown in Figure a, CSA exhibits stable oxygen release performance, with a sustained release time of about 8 to 10 days, and then at about the 10th day, oxygen generation slows down significantly to almost stop. In order to monitor the degradation process of the hydrogel, we took out the hydrogel samples on the 1st, 2nd, 4th, 7th and 15th days of the experiment and weighed them, and then drew the degradation curve (see Figure 7 b). The results showed that the hydrogel achieved almost complete degradation in about 15 days. In addition, the pH value of the degradation product solution was found to be always maintained in the range of 7.1 to 7.4, which is close to the physiological pH (reference Figure 7 This finding indicates that the composite hydrogel can effectively maintain the physiological stability of the environment during the degradation process, laying a solid foundation for its potential application in the biomedical field.

[0118] 7. CSA hydrogel cell biocompatibility testing.

[0119] Biocompatibility is a key indicator for measuring the applicability of hydrogels as biomedical materials. In order to comprehensively evaluate the potential toxic effects of hydrogels on cells, we used CCK-8 detection technology to conduct relevant experiments. The results are as follows: Figure 8As shown. For the four groups with CSA hydrogel accounting for 1% to 10%, compared with the control group, the cell viability in short-term culture (24 hours) and long-term culture (72 hours and 168 hours) exceeded 80%, that is, no obvious cytotoxicity was shown. However, the CSA hydrogel in the 20% group may have a certain toxic or inhibitory effect on cells, especially in long-term culture, this effect may gradually appear. The results of the study show that CSA hydrogel has good biocompatibility with cells within a certain concentration range, proving its potential for application in the biomedical field. This finding also provides a reference concentration for subsequent studies.

[0120] 8. Detection of ROS removal levels by CSA hydrogel.

[0121] 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 scavenge reactive oxygen species in high oxidative stress myocardial infarction tissue, we evaluated the antioxidant potential of SA and CSA hydrogels. Fig. 9 As shown in Figure a, the DPPH scavenging ability of CSA hydrogel is significantly higher than that of 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 key factors in scavenging ROS. The excellent antioxidant activity exhibited by CSA hydrogel helps to effectively remove harmful ROS in the myocardial infarction area, thereby improving and reshaping the infarction microenvironment.

[0122] To further verify the ability of CSA hydrogel to scavenge ROS, we used DCFH-DA staining to evaluate the level of ROS in cells. The results showed that after 3 days of culture, Fig. 9 (b) The green intensity of DCFH-DA was almost the same as that of the hydrogen peroxide culture group, indicating a high level of ROS. However, the fluorescence intensity of DCFH-DA in the CSA hydrogel group was significantly reduced and close to the level of normal cultured cells.

[0123] In summary, CSA hydrogel exhibits excellent free radical scavenging ability through the synergistic effect of MNPs and ascorbic acid. This performance not only effectively alleviates the oxidative damage caused by ROS, but also provides strong support for the optimization of the myocardial infarction microenvironment and functional recovery, highlighting the potential application value of CSA hydrogel in cardiac tissue engineering.

[0124] 9. Detection of the angiogenesis-promoting ability of CSA hydrogel.

[0125] The survival ability of human umbilical vein endothelial cells (HUVECs) in the ROS microenvironment was evaluated by CCK-8 assay. After 3 days of continuous culture, oxidative stress damage caused a significant decrease in the survival ability of HUVECs, while the application of CSA hydrogel enhanced the survival rate of these endothelial cells to a certain extent ( Fig.10 Subsequently, the expression of angiogenesis-related proteins, including von Willebrand factor (VWF), vascular cell adhesion molecule 1 (Vcam1), and CD31, was detected by Westren-blot assay. Fig.10 As shown in (b), CSA hydrogel significantly upregulated the expression of angiogenesis-related genes compared with SA hydrogel. In other words, CSA hydrogel improved the survival ability of HUVECs by scavenging ROS, thus having the potential to promote angiogenesis in the MI area.

[0126] 10. Detection of the ability of CSA hydrogel to promote macrophage polarization.

[0127] Immunofluorescence experiments were performed to investigate the effects of SA or CSA hydrogels on THP-1-derived macrophage polarization in a ROS microenvironment. After 1 day of continuous culture, immunofluorescence staining was performed to demonstrate the expression of CD86 (M1 macrophage surface marker) and CD206 (M2 macrophage surface marker) in THP-1-derived macrophages after different treatments. Fig.11 ). After the addition of hydrogen peroxide, the number of CD86-positive THP-1-derived macrophages increased significantly, indicating their polarization to the M1 phenotype. At the same time, the number of CD206-positive cells did not change significantly. Obviously, CSA hydrogel reduced the signal of CD86 and increased the signal of CD206, suggesting that CSA hydrogel induced macrophage polarization from M1 to M2.

[0128] The experimental results of this embodiment show that: (1) CSA hydrogel has excellent injectability and can be injected in situ. At the same time, the CSA-δ hydrogel formed after loading drugs has the ability to release drugs continuously and stably, thereby prolonging the drug action time. In addition, CSA hydrogel exhibits strong long-term oxygen production capacity and has significant potential to alleviate the hypoxic environment in the myocardial infarction area.

[0129] (2) CSA hydrogel exhibits good electrical conductivity and can provide an electrical environment similar to that of myocardial tissue. By introducing melanin and calcium peroxide, the conductivity of CSA hydrogel was significantly improved, with an electrical conductivity of 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.

[0130] (3) CSA hydrogel showed good biocompatibility in cell experiments. Even at a concentration of 10%, no obvious cytotoxicity was observed, and cell viability was well maintained, providing solid biocompatibility support for its further application in animal experiments.

[0131] (4) CSA hydrogel effectively removes ROS, reduces oxidative stress, and reduces damage to cardiomyocytes and human umbilical vein endothelial cells in a ROS environment. In addition, CSA hydrogel can promote the proliferation of vascular endothelial cells and the M2 polarization of macrophages, which is expected to promote angiogenesis and tissue repair in the myocardial infarction area.

[0132] Experimental Example 2 The therapeutic effect of CSA-δ hydrogel on MI / R aged rats was evaluated.

[0133] In this experiment, we established an MI / R model in aged rats and set up a sham operation (Sham) group 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, and then evaluate the therapeutic effect of the hydrogel. We will use a variety of evaluation methods including DHE staining, immunofluorescence analysis, echocardiography, cardiac function parameter determination, Masson trichrome staining and HE staining to comprehensively and deeply explore the performance of CSA-δ hydrogel in therapeutic applications.

[0134] 1. Experimental Materials 1.1 Experimental animals SPF SD rats provided by Chengdu Dashuo were used as experimental subjects, regardless of gender, and continued to be cultured until the age of 2. To ensure the sanitary conditions of the animals, the experimental rats were kept in an environment with a 12-hour light-dark cycle control and free access to clean drinking water and high-quality feed. All animal experiments were carried out in strict accordance with the approval and guidelines of the Experimental Animal Ethics Review Committee of Sichuan Provincial People's Hospital.

[0135] 2.2 Experimental Materials and Reagents Table 3 Main experimental materials and reagents

[0136] 2. Experimental Methods 2.1 SA hydrogel, CSA hydrogel and CSA-δ hydrogel were prepared according to the methods of Examples 2-3 and Comparative Example 1.

[0137] 2.2 MI / R model construction and drug administration The MI / R model was constructed for aged SD rats: First, the pleura was exposed and the thoracotomy was performed at the second and third intercostal spaces on the left side of the chest. After that, the thoracotomy was performed at the second and third intercostal spaces on the left side to expose the heart. A slipknot was tied at the origin of the left anterior descending coronary artery using 7-0 silk suture and the left anterior descending coronary artery was closed. It was ensured that there was no obvious bleeding in the heart cavity during the operation, while keeping the operation area clean. Finally, the surgical area was sutured layer by layer to ensure that the tissue layers were fully adhered. After 40 minutes of ischemic treatment, the coronary artery ligature was removed. At the end of the experimental period, the animals were euthanized using an overdose of isoflurane, and major organ tissues were removed for further analysis.

[0138] Afterwards, SA, CSA hydrogel and CSA-δ hydrogel were injected into the myocardium at multiple points around the ischemic area of ​​the heart to avoid overflow. The sham operation group only underwent thoracotomy without ligation of the left anterior descending artery, and the PBS group and the drug-treated group were injected with PBS and δ-Amyrenone through the tail vein, respectively.

[0139] 2.3 DHE staining of frozen sections.

[0140] (1) Tissue collection: The rat hearts were collected on the first day after infarction and immediately stored in a -80°C refrigerator to ensure tissue activity and integrity.

[0141] (2) Cryoembedding and sectioning: Place the preserved heart tissue in a cryosection embedding medium. After the tissue is completely frozen, use a cryostat to cut the tissue into 5 μm thick slices. Transfer the slices to a glass slide for subsequent experiments.

[0142] (3) DHE staining: Wash the tissue sections three times with PBS for 3 minutes each time to completely remove the residual embedding agent. According to the instructions of the DHE kit, dilute the stock solution to 10 uM and stain at 37°C for 30 minutes in the dark. Then, wash the sections three times with PBS again, and stain the nuclei with DAPI for 10 minutes. Wash three times with PBS and seal the sections. Finally, observe the sections under a fluorescence microscope and record the results.

[0143] 2.4 Organ tissue collection, pathological HE staining and Masson staining of heart tissue.

[0144] (1) Tissue sample collection: Rats were divided into three groups according to different experimental purposes. The heart tissue was collected on the first day after administration; the heart tissue was collected on the third day after administration; and the liver, spleen, lung, and kidney organs were collected in the 28-day administration group in addition to the heart. The heart of the first day group was frozen at -80°C for future use, and the organs of the other groups were fixed with 4% paraformaldehyde for more than 24 hours for future use.

[0145] (2) Embedding and sectioning of tissue: Dehydrate the sample in (1) in a dehydrator, and then paraffin embed in a paraffin embedding machine. Slice the embedded and frozen tissue to a thickness of 5 microns. Pick it up with a slide in warm water for subsequent processing.

[0146] (3) HE staining process: Place the sample in the fully automatic staining machine and select the program dewaxing + HE staining. After staining, cover the slide with neutral resin. Observe and record the image under a microscope.

[0147] (4) Masson trichrome staining: Perform staining according to Section 2.3.8.

[0148] (5) Immunofluorescence staining analysis: After the heart tissue samples were dewaxed and pre-treated by an automatic staining machine, immunofluorescence staining was performed according to the following steps: a: Process of obtaining, fixing and dehydrating heart samples: The MI / R model of aged mice was constructed according to the steps shown in 2.2. After 24 hours, the mice were anesthetized and humanely killed, and then the hearts were removed for experiments. The heart tissue was immersed in a 4% paraformaldehyde solution for 24 hours to ensure that the integrity of the tissue structure was maintained. Next, the heart tissue was dehydrated using a fully automatic tissue dehydration device.

[0149] b: Paraffin embedding and thin section preparation of cardiac tissue: The dehydrated cardiac tissue needs to be further embedded in paraffin for subsequent sectioning. When sectioning, it is necessary to ensure that the thickness of the cardiac tissue slice is 5 microns to meet the specific needs of the experiment.

[0150] c: Dewaxing and hydration: Place the paraffin sections into the fully automatic staining machine, bake and dewax them, and complete the hydration.

[0151] d: Antigen repair: Place the sections in a steam pressure cooker containing sodium citrate buffer (pH 6.0) for high temperature and high pressure repair for 5-10 minutes. After cooling naturally to room temperature, rinse the sections with PBS 3 times, 5 minutes each time.

[0152] e: Penetration and blocking: The slices were treated with 0.5% Triton X-100 solution to enhance the penetration of antibodies in tissues. Subsequently, the slices were blocked with goat serum for 30 minutes at room temperature to reduce the interference of nonspecific binding and ensure the accuracy of the experimental results.

[0153] f: Primary antibody incubation: Add primary antibody according to the ratio in the instruction manual, cover the sliced ​​tissue, and incubate overnight in a humidified box at 4°C. Then wash the slices 3 times with PBS, 5 minutes each time.

[0154] g: Secondary antibody incubation: Then add fluorescently labeled secondary antibody (select the corresponding secondary antibody according to the primary antibody host, and the dilution ratio is shown in the instructions), incubate at 37°C for 1 hour, and wash again with PBS 3 times, 5 minutes each time.

[0155] h: DAPI staining and sealing: At 37 °C, stain with DAPI for 20 minutes to mark the cell nucleus. Wash with PBS 3 times, 5 minutes each time, to remove excess stain. Add appropriate amount of sealing solution, cover the slice with a cover glass, and gently flatten to prevent bubbles.

[0156] i: Microscopic observation and photography: Observe the slices using a fluorescence microscope, use appropriate excitation wavelengths to capture the target fluorescence signal and nuclear staining signal in sequence, and save the images for subsequent analysis.

[0157] 2.5 Cardiac function index detection In order to evaluate the effects of different treatments on cardiac function, echocardiography was used to detect relevant indicators. After the successful construction of the MI / R model and the implementation of different treatments, after 28 days, the mice in each group were anesthetized with isoflurane, and the cardiac function was tested using the Fujifilm brand small animal ultrasound imaging system. Doppler ultrasound in C mode and M-mode ultrasound technology in B mode were mainly used to accurately measure mitral valve blood flow and comprehensively evaluate cardiac function indicators, respectively.

[0158] 3. Experimental results: 3.1 ROS scavenging effect of CSA-δ hydrogel in vivo.

[0159] MI / R can induce a lot of oxidative stress. In order to verify the antioxidant effect of CSA-δ hydrogel, we used DHE staining technology to visually display the distribution of superoxide anions (a type of ROS) in cells. Specifically, Fig.12 As shown in the figure, on the first day after PBS injection in the myocardial infarction area, we clearly observed a strong DHE signal, which directly indicated the large accumulation of ROS in the area. Furthermore, compared with the SA hydrogel and δ-Amyrenone treatment groups, when we injected CSA and CSA-δ hydrogel on the first day, the DHE fluorescence was significantly weakened, indicating that CSA hydrogel can effectively reduce the accumulation of ROS, and the addition of δ-Amyrenone will not affect its ability to scavenge ROS. The above results verify the antioxidant properties of CSA and CSA-δ hydrogel, indicating that it can protect myocardial cells from oxidative damage by scavenging ROS in the early stage of myocardial infarction reperfusion.

[0160] 3.2 The angiogenic effect of CSA-δ hydrogel in vivo.

[0161] After MI / R, angiogenesis plays a vital role in the effective repair of the myocardium, which can promote the recovery of oxygen supply and save dying myocardial cells. In view of this, we deeply explored the effect of CSA-δ hydrogel on the vascular density in the myocardial infarction area. Fig.13 As shown. In the study, we used immunofluorescence staining to detect α-smooth muscle actin (α-SMA), a marker protein of mature new blood vessels, as a basis for evaluating vascular density. Compared with the PBS group, more new blood vessels were 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 intuitive observation, it can be found that the number of blood vessels in the CSA and CSA-δ hydrogel groups is significantly higher than that in other groups. These results indicate that CSA-δ hydrogel has a strong ability to promote angiogenesis in vivo, that is, CSA-δ hydrogel may achieve a therapeutic effect on left ventricular reconstruction by increasing the vascular density in the infarct area.

[0162] 3.3 The effect of CSA-δ hydrogel on promoting macrophage polarization in vivo.

[0163] To evaluate the polarization of macrophages, we performed immunofluorescence staining analysis on cardiac tissues at day 1 and day 3 after myocardial infarction to detect surface markers and analyze the pro-inflammatory and anti-inflammatory phases. Fig.14 In the immunofluorescence image shown in a, it can be observed that on the first day of treatment, CD86, a marker of M1 macrophages, 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 in the CSA and CSA-δ hydrogel groups were relatively reduced. In contrast, almost no CD206-positive M2 macrophages were observed in each group on day 1. These results indicate that in the early stages of myocardial infarction (day 1), the introduction of CSA and CSA-δ hydrogels can downregulate the number of M1 macrophages, while no significant changes were observed in M2 macrophages. It is worth noting that on the third day after treatment, the expression of CD206 showed significant differences.

[0164] Fig.14The immunofluorescence image in middle b shows that compared with the PBS, δ-Amyrenone and SA hydrogel groups, the expression of CD206 in the CSA and CSA-δ hydrogel groups was significantly upregulated, indicating that 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 CSA and CSA-δ hydrogels downregulated M1 macrophages and inhibited the inflammatory response of early myocardial infarction. In summary, CSA-δ hydrogel treatment effectively reduced the number of pro-inflammatory M1 macrophages 1 to 3 days after treatment, and promoted the polarization of macrophages to the repair-promoting M2 type, which was beneficial to the recovery process of myocardial tissue.

[0165] 3.4 Rat heart function test verified the repair effect of CSA-δ hydrogel.

[0166] In order to explore the effect of CSA-δ hydrogel on the repair of cardiac function in aged rats after MI / R, cardiac ultrasound was detected in each group after 28 days of treatment. The contraction and relaxation capacity of the heart in each group was evaluated by M-mode ultrasound in B mode. Fig.15 As shown in a, among the 6 groups, the Sham group had the largest range of contraction activity, and the CSA-δ treatment group showed a similar contraction amplitude. The anterior wall of the ventricle in the PBS group and the SA group was almost horizontal, showing the characteristics of fibrosis. The CSA group and the δ-Amyrenone group had a certain improvement compared with the PBS group, and the contraction amplitude was larger. In addition, Doppler ultrasound in C mode was used to further evaluate cardiac function. The results showed that the δ-Amyrenone group, the SA group, and the CSA group were able to improve the E / A peak ratio, but the E / A peak ratio of the CSA-δ group was higher, demonstrating a more prominent therapeutic effect (see Fig.15 Middle b and Fig.15 At the same time, we also conducted in-depth analysis of key echocardiographic parameters such as ejection fraction (EF) and shortening rate (FS) ( Fig.15 Middle c, Fig.15 (d) The data showed that the CSA-δ group performed best in promoting cardiac function recovery. In summary, CSA-δ hydrogel can significantly enhance the cardiac function of elderly rats after MI / RI.

[0167] 3.5 Masson staining verified the repair effect of CSA-δ hydrogel.

[0168] Masson trichrome staining was used to stain myocardial tissue sections to quantify the myocardial repair effect of each group. Fig.16Middle a) Visually shows that the Masson staining results of a normal heart should all be red myocardial tissue, but the PBS group had extensive necrosis caused by MI / R, resulting in a significant increase in the area of ​​blue fibrous tissue compared with other groups. There was almost no red myocardial tissue in the infarct zone (IZ) and the marginal zone (BZ), while the CSA-δ group showed the least blue fibrous tissue except for the sham operation group. It showed more significant myocardial tissue recovery, especially in the infarct area and marginal zone, where red myocardial tissue was more abundant. Zenbio image analysis software was further used to process the images and accurately measure the thickness of the infarct wall. The results showed statistically significant differences between the groups ( Fig.16 b). Specifically, the thickness of the left ventricular wall in the CSA-δ group reached 1.762±0.139 mm, which was much higher than that in the PBS group (0.3954±0.0345 mm). These data strongly support that CSA-δ hydrogel can not only promote the recovery of cardiac function in MI / R aged rats, 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.

[0169] 3.6 Effect of CSA-δ hydrogel treatment on organ tissue pathology.

[0170] In order to further evaluate the safety performance of the CSA-δ hydrogel system in vivo, we performed HE staining analysis on the core organs (heart, liver, spleen, lungs and kidneys) of all 28-day treated elderly rats, aiming to explore its potential organ toxicity at the histological level. Fig.17 It can be seen that HE staining of each experimental group showed that the cardiac tissue structure was normal, the myocardial cells were neatly arranged, and there was no abnormal phenomenon such as inflammation or necrosis. There was no abnormality in the liver tissue structure, the liver lobule structure was intact, the hepatocytes did not show 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 divided, and no significant cell infiltration or damage was observed. The alveolar structure was intact, and no pathological changes such as pulmonary edema, inflammation or fibrosis were observed. The renal tubules and glomeruli were intact, and there were no toxic manifestations such as tubular necrosis or interstitial fibrosis. In summary, after 28 days of treatment, no significant abnormalities were found in the main organs of SD elderly rats in each group, which strongly proved that CSA-δ hydrogel, as a drug delivery platform, has a high degree of in vivo safety and good biocompatibility, laying a solid foundation for its further application in the biomedical field.

[0171] The experimental results of this experiment showed that SA, CSA hydrogel and δ-Amyrenone all showed certain cardioprotective effects, which were specifically manifested in reducing the infarct area and reducing the degree of cardiac fibrosis, increasing the thickness of the ventricular wall, and thus having a positive effect on the cardiac function of rats. However, in comparison, CSA-δ hydrogel not only significantly promoted the regeneration and repair of myocardial cells, but also further enhanced its repair effect by constructing a microenvironment conducive to myocardial recovery. This series of experimental results indicate that CSA-δ hydrogel has significant potential in the field of myocardial infarction repair, provides a new therapeutic strategy for the recovery of cardiac function after MI / R, and may have important clinical application prospects.

[0172] 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 a local hypoxic environment. It can also remove ROS in vivo and in vitro, promote angiogenesis and macrophage M2 polarization function. Drug sustained release and biocompatibility experiments verified its sustained release performance and safety, laying the foundation for its application in the treatment of myocardial infarction. CSA-δ hydrogel significantly promotes myocardial repair: CSA-δ hydrogel is superior to the use of SA, CSA hydrogel or δ-Amyrenone alone in reducing the area of ​​myocardial infarction, reducing fibrosis and improving cardiac function. The results of organ HE staining showed that CSA-δ hydrogel has good safety, which provides favorable support for its application in the treatment of cardiac injury.

[0173] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A hydrogel, characterized in that: The hydrogel comprises: a sodium alginate aqueous solution containing melanin nanoparticles, a reducing agent, and a calcium gluconate aqueous solution containing calcium peroxide nanoparticles, wherein the volume ratio of the sodium alginate aqueous solution containing melanin nanoparticles to the calcium gluconate aqueous solution containing calcium peroxide nanoparticles is 2.5:1-3.5:1, the final concentration of the melanin nanoparticles in the hydrogel is 0.8-1.5 mg / ml, the mass fraction of sodium alginate in the sodium alginate aqueous solution is 2.5%-3.5%, the concentration of calcium peroxide nanoparticles in the calcium gluconate aqueous solution is 0.8-1.5 mg / ml, and the mass fraction of calcium gluconate in the calcium gluconate aqueous solution is 2.5%-3.5%; the ratio of the sodium alginate aqueous 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 according to claim 1, characterized in that The melanin nanoparticles are natural melanin nanoparticles or modified melanin nanoparticles.

3. The hydrogel according to claim 2, characterized in that The natural melanin nanoparticles are prepared from raw material squid ink sac.

4. The method for preparing the hydrogel according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: adding a reducing agent into a sodium alginate aqueous solution containing melanin nanoparticles, and then mixing the solution with a calcium gluconate aqueous solution containing calcium peroxide nanoparticles to obtain a mixed solution.

5. The method for preparing the hydrogel according to claim 4, characterized in that: The preparation method further comprises: centrifuging the mixed solution.

6. A hydrogel containing a small molecule inhibitor, characterized in that: The invention comprises the hydrogel according to any one of claims 1 to 3 or the hydrogel prepared by the method for preparing the hydrogel according to claims 4 to 5 and a small molecule inhibitor, wherein the small molecule inhibitor is δ-Amyrenone.

7. The hydrogel containing small molecule inhibitors according to claim 6, characterized in that: Each ml of the hydrogel contains 10 mg of the small molecule inhibitor.

8. The method for preparing a hydrogel containing a small molecule inhibitor according to any one of claims 6 to 7, characterized in that: It includes: First, δ-Amyrenone is added to a calcium gluconate aqueous solution containing calcium peroxide nanoparticles, and mixed to obtain a calcium gluconate aqueous solution containing δ-Amyrenone and calcium peroxide nanoparticles, and then a sodium alginate aqueous solution containing melanin nanoparticles and a reducing agent are mixed with the calcium gluconate aqueous solution containing δ-Amyrenone and calcium peroxide nanoparticles.

9. Use of the hydrogel according to any one of claims 1 to 3, the hydrogel prepared by the method for preparing the hydrogel according to claims 4 to 5, or the hydrogel containing a small molecule inhibitor according to any one of claims 6 to 7 in the preparation of a medicament for preventing and / or treating ischemic heart disease.

10. The use according to claim 9, characterized in that: The 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.

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