Hydrogel capable of regulating the redox homeostasis of the microenvironment, preparation method and application thereof

Abstract

The present invention belongs to the field of compositions of biomedical materials and high molecular compounds, and provides a hydrogel capable of regulating the redox homeostasis of the microenvironment, its preparation method and application. The polymer network of the hydrogel capable of regulating the redox homeostasis of the microenvironment is formed by the hybridization cross-linking reaction of catechol-modified hyaluronic acid, carboxylated chitosan, oxidized dextran and iron ions; under physiological conditions, the catechol groups in the hydrogel will undergo autoxidation reaction to generate H<subgt;2< / subgt;O<subgt;2< / subgt>, and at the same time, the iron ions coordinated on the catechol groups in the hydrogel will catalyze the decomposition of H<subgt;2< / subgt;O<subgt;2< / subgt> into water and oxygen, thereby regulating the level of reactive oxygen species in the microenvironment. The hydrogel of the present invention can achieve bidirectional regulation of reactive oxygen species in the pathological microenvironment after myocardial infarction, thereby maintaining the redox homeostasis of the pathological microenvironment after myocardial infarction and improving the regenerative repair ability of the existing hydrogel materials with antioxidant ability to the myocardial tissue after myocardial infarction.

Description

Technical Field

[0001] The present invention belongs to the field of compositions of biomedical materials and high molecular compounds, and relates to a hydrogel capable of regulating the redox homeostasis of the microenvironment, and a preparation method and application thereof. Background Art

[0002] Myocardial infarction is a common disease that seriously threatens human health, causing more than 7 million deaths every year. Patients who survive are also prone to secondary complications, which affect the quality of life of patients and impose a heavy economic burden. The main cause of myocardial infarction is the obstruction of the coronary artery, resulting in a reduction in blood flow to cardiomyocytes, which in turn causes the death of a large number of cardiomyocytes and is accompanied by ischemia-reperfusion injury and inflammatory responses, leading to adverse adaptive remodeling of the ventricle and thus developing into ischemic heart failure. After myocardial infarction occurs, the body's self-repair mechanism will be quickly activated to prevent further aggravation of the injury. However, due to the limited regenerative ability of the adult heart, myocardial infarction often results in the death of cardiomyocytes or replacement by fibrotic tissue. The body's self-repair process is often accompanied by the formation of scar tissue and the degradation of the vascular network, which not only fails to restore cardiac function but may also lead to further deterioration of cardiac function.

[0003] Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), are considered to be mediators in the development of ischemia-reperfusion injury and inflammatory diseases. During ischemia-reperfusion and inflammation, ROS have been shown to be released from activated neutrophils and macrophages. The excessive generation and accumulation of free radicals or their oxidation products can lead to oxidative stress during heart failure or ischemia-reperfusion. Although appropriate amounts of ROS can promote the synthesis of cardiac collagen after myocardial infarction, oxidative stress in mitochondria and impaired mitochondrial oxidative capacity after myocardial infarction can lead to abnormal remodeling of the left ventricle, mainly manifested in that ROS can activate multiple hypertrophic signaling kinases and transcription factors and mediate apoptosis, and ROS can also stimulate the proliferation of cardiac fibroblasts and activate matrix metalloproteinases (MMPs), resulting in extracellular matrix remodeling. The excessive generation of ROS in cardiomyocytes can directly or indirectly damage cell ion channels and transporters. That is to say, while ROS play a biological signal function in the body, they are also a double-edged sword. Excessive ROS will mediate cellular oxidative stress, activate signal pathways such as inflammation and apoptosis, and lead to consequences such as apoptosis and tissue necrosis.

[0004] Given the toxic effects of ROS, existing research on related tissue engineering materials has mainly focused on promoting tissue regeneration by scavenging ROS in the microenvironment. However, due to the neglect of the necessity of ROS in regenerative signal transduction, simple ROS scavenging or antioxidant intervention may instead interfere with the initiation and maintenance of regenerative signals, having an adverse impact on tissue regeneration. In the case of myocardial infarction, after myocardial infarction occurs, the concentration level of ROS in the early microenvironment is relatively high, which can activate signaling pathways such as inflammation and apoptosis, while an appropriate concentration of ROS in the mid- and late-stage microenvironment can prolong the maintenance time of the repair phenotype of myocardial epithelial cells. Therefore, if the concentration level of ROS in the microenvironment can be reduced in the early stage after myocardial infarction through tissue engineering materials and maintained at a relatively low level in the mid- and late stages, it is beneficial for improving ischemic heart failure after myocardial infarction and promoting myocardial repair. Currently, although there are reports of injectable hydrogel materials with antioxidant properties, they are difficult to achieve precise adaptation to the redox state of the myocardial pathological microenvironment, and thus have relatively limited regenerative repair ability for the tissue after myocardial infarction. How to control the ROS concentration in the microenvironment after myocardial infarction within the level range where it plays a positive role through tissue engineering materials, so as to better promote the repair of the tissue after myocardial infarction, is a major challenge in this field. Summary of the Invention

[0005] Aiming at the problem that existing injectable hydrogel materials with antioxidant ability can scavenge ROS but are difficult to achieve precise adaptation to the redox state of the myocardial pathological microenvironment, the present invention provides a hydrogel capable of regulating the redox homeostasis of the microenvironment, its preparation method and application, to achieve bidirectional regulation of ROS in the pathological microenvironment after myocardial infarction, which can not only rapidly scavenge excessive H2O2 but also continuously generate a certain physiological concentration of H2O2 for a long time, thereby maintaining the redox homeostasis of the pathological microenvironment after myocardial infarction and improving the regenerative repair ability of the existing hydrogel materials with antioxidant ability for the myocardial tissue after myocardial infarction.

[0006] To achieve the above invention objectives, the technical solutions adopted by the present invention are as follows:

[0007] A hydrogel capable of regulating the redox homeostasis of the microenvironment. The polymer network of this hydrogel is formed by the hybrid cross-linking reaction of catechol-modified hyaluronic acid, carboxylated chitosan, oxidized dextran and iron ions. The hybrid cross-linking reaction includes the Schiff base reaction between the amino group of carboxylated chitosan and the aldehyde group of oxidized dextran, the oxidative self-cross-linking reaction of catechol-modified hyaluronic acid, the Michael addition reaction between the quinone group of catechol-modified hyaluronic acid after oxidative self-cross-linking and the amino group of carboxylated chitosan, and the dynamic coordination reaction between the catechol group of catechol-modified hyaluronic acid after oxidative self-cross-linking and iron ions. Under physiological conditions, the catechol groups in this hydrogel will undergo self-oxidation reaction to generate H2O2. At the same time, the iron ions coordinated on the catechol groups in this hydrogel will catalyze the decomposition of H2O2 into water and oxygen, thereby regulating the level of reactive oxygen species in the microenvironment.

[0008] In the technical solution of the above-mentioned hydrogel capable of regulating the redox homeostasis of the microenvironment, the hydrogel is formed by the hybrid cross-linking reaction of a gel precursor solution containing catechol-modified hyaluronic acid, carboxylated chitosan, oxidized dextran and water-soluble iron salt.

[0009] Further, in the technical solution of the above-mentioned hydrogel capable of regulating the redox homeostasis of the microenvironment, in the gel precursor solution, the concentration of catechol-modified hyaluronic acid is 5 - 50 mg / mL, the concentration of carboxylated chitosan is 20 - 100 mg / mL, the concentration of oxidized dextran is 10 - 80 mg / mL, and the concentration of iron ions is 0.01 - 0.1 mol / L. Even further, in the gel precursor solution, the concentration of catechol-modified hyaluronic acid is preferably 30 - 50 mg / mL, the concentration of carboxylated chitosan is preferably 50 - 80 mg / mL, the concentration of oxidized dextran is preferably 20 - 40 mg / mL, and the concentration of iron ions is preferably 0.01 - 0.05 mol / L.

[0010] In the technical solution of the above-mentioned hydrogel capable of regulating the redox homeostasis of the microenvironment, the water-soluble iron salt can be ferric chloride, ferric sulfate or ferric nitrate.

[0011] In the technical solution of the above-mentioned hydrogel capable of regulating the redox homeostasis of the microenvironment, the hydrogel has a mutually interconnected porous structure after freeze-drying. The average pore diameter of the porous structure is 50 - 100 μm, and the porosity is 90% - 95%.

[0012] In the technical solution of the hydrogel that can regulate the redox homeostasis of the microenvironment, the catechol-modified hyaluronic acid is formed by grafting catechol groups onto the carboxyl groups of hyaluronic acid, and the grafting rate of catechol groups in the catechol-modified hyaluronic acid is 5% - 15%. Further, the molecular weight of sodium hyaluronate as the modification basis is 8 - 2000 kDa, preferably 200 - 1000 kDa. The structure of the catechol-modified hyaluronic acid is shown in formula (I):

[0013] (I).

[0014] In the technical solution of the hydrogel that can regulate the redox homeostasis of the microenvironment, the catechol-modified hyaluronic acid can be prepared with reference to the existing technology. A feasible preparation method of the catechol-modified hyaluronic acid is as follows:

[0015] Add an aqueous solution of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide to an aqueous solution of sodium hyaluronate, then add an aqueous solution of N-hydroxysuccinimide, stir and react at room temperature for 2 - 8 h under nitrogen protection. After that, dropwise add an aqueous solution of dopamine hydrochloride to the obtained reaction solution under light avoidance conditions, and stir and react at room temperature for 12 - 48 h under light avoidance and nitrogen protection. During both stirring reactions, control the pH value to be 5 - 6; remove the unreacted raw materials in the obtained reaction solution, and freeze-dry to obtain the product.

[0016] In the method for preparing catechol-modified hyaluronic acid, the proportional relationship of each reaction raw material will affect the grafting rate of catechol groups in the catechol-modified hyaluronic acid. In actual application, the proportional relationship of each raw material can be determined with reference to the existing technology according to the application requirements. For example, the molar ratio of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, N-hydroxysuccinimide, dopamine hydrochloride to the carboxyl groups on sodium hyaluronate can be controlled to be (3 - 6):(1 - 5):(1 - 5):1. Generally, the concentration of the aqueous solution of sodium hyaluronate can be controlled to be 10 - 50 mg / mL.

[0017] In the technical solution of the hydrogel that can regulate the redox homeostasis of the microenvironment, the oxidized dextran is obtained by oxidizing some hydroxyl groups in dextran into aldehyde groups. The oxidation degree of dextran is 15% - 40%. The structure of the oxidized dextran is shown in formula (II):

[0018] (II).

[0019] In the technical solution for the hydrogel capable of regulating the redox homeostasis of the microenvironment, the oxidized dextran can be prepared by referring to existing techniques. A feasible method for preparing oxidized dextran is to add an aqueous sodium periodate solution dropwise to an aqueous dextran solution in the dark. After the addition is complete, stir the mixture at room temperature in the dark for 2-5 hours. Unreacted material is then removed and freeze-dried to obtain the product. Typically, the molecular weight of the dextran used as the basis for oxidative modification is 100-1000 kDa.

[0020] In the above-mentioned technical solution for the hydrogel capable of regulating microenvironmental redox homeostasis, the carboxylated chitosan is formed by introducing carboxyl groups into the hydroxyl groups of chitosan through a carboxylation reaction, and the carboxylation degree of the carboxylated chitosan is at least 60%. Preferably, the carboxylation degree of the carboxylated chitosan is 60% to 90%, and the structure of the carboxylated chitosan is shown in Formula (III):

[0021] (III).

[0022] The present invention also provides a method for preparing the hydrogel capable of regulating the redox homeostasis of the microenvironment, comprising the following steps:

[0023] (1) Dissolving catechol-modified hyaluronic acid in water to obtain a catechol-modified hyaluronic acid solution; dissolving carboxylated chitosan in a PBS buffer solution with a pH value of 7.2 to 7.4 to obtain a carboxylated chitosan solution; dissolving oxidized dextran in an iron salt aqueous solution to obtain an oxidized dextran / iron salt mixed solution;

[0024] (2) fully mixing the carboxylated chitosan solution with the oxidized dextran / iron salt mixed solution, and then fully mixing the resulting mixed solution with the catechol-modified hyaluronic acid solution to obtain a gel precursor solution;

[0025] (3) The gel precursor solution was allowed to stand until it turned into a gel state, and the resulting hydrogel was repeatedly soaked in PBS buffer solution with a pH value of 7.2-7.4 and water to remove the iron ions not bound to the catechol groups, thereby obtaining a hydrogel that can regulate the redox homeostasis of the microenvironment.

[0026] In step (2) of the technical solution of the above-mentioned preparation method, the reason for adopting a two-step mixing method of first fully mixing the carboxylated chitosan solution with the oxidized dextran / iron salt mixed solution and then fully mixing the resulting mixed solution with the catechol-modified hyaluronic acid solution is mainly to allow the amino groups of the carboxylated chitosan to fully react with the aldehyde groups of the oxidized dextran first, thereby minimizing or avoiding the reaction of the aldehyde groups of the oxidized dextran with the phenolic hydroxyl groups of the catechol-modified hyaluronic acid to form a hemiacetal structure. In the gel precursor solution of step (2), the concentration of the catechol-modified hyaluronic acid is 5-50 mg / mL, the concentration of the carboxylated chitosan is 20-100 mg / mL, the concentration of the oxidized dextran is 10-80 mg / mL, and the concentration of the iron ion is 0.01-0.1 mol / L. Furthermore, in the gel precursor solution of step (2), the concentration of catechol-modified hyaluronic acid is preferably 30-50 mg / mL, the concentration of carboxylated chitosan is preferably 50-80 mg / mL, the concentration of oxidized dextran is preferably 20-40 mg / mL, and the concentration of iron ions is preferably 0.01-0.05 mol / L.

[0027] In step (3) of the technical solution of the above-mentioned preparation method, when the obtained hydrogel is repeatedly soaked in PBS buffer solution with a pH value of 7.2 to 7.4 and water, the hydrogel is first soaked in PBS buffer solution with a pH value of 7.2 to 7.4 for 12 to 24 hours, then transferred to water and soaked for 2 to 4 hours, and then the aforementioned soaking operation in PBS buffer solution with a pH value of 7.2 to 7.4 and water is repeated 2 to 3 times.

[0028] The present invention is confirmed by experiments:

[0029] (1) The hydrogel of the present invention has an interconnected porous structure after freeze-drying. For example, the COFH prepared in Example 5 has an interconnected porous structure after freeze-drying. The average pore size of the porous structure is about 71 μm and the porosity is about 92%. This porous structure is very beneficial for the transmission of nutrients and oxygen, cell migration, infiltration, and new tissue ingrowth.

[0030] (2) The hydrogel of the present invention has a short gelation time of no more than 2 minutes, and has better mechanical properties after gelation, which is conducive to rapid gelation and function in the target tissue in practical applications. For example, at a frequency of 2 to 10 Hz, the storage modulus (G') of the COFH prepared in Example 5 is 43 to 67 KPa, and its compressive strength reaches 38 ± 5 KPa. The excellent mechanical properties are conducive to the hydrogel of the present invention maintaining its own structural stability in the myocardium with a complex mechanical environment, thereby playing a more lasting role.

[0031] (3) The hydrogel of the present invention has excellent bidirectional regulation ability of H2O2. For example, the COFH prepared in Example 5 can generate H2O2. After 16 days, the concentration of H2O2 can be gradually increased from 0 to about 34 μmol / L. At the same time, COFH can scavenge H2O2. After 4 days, the concentration of H2O2 can be rapidly decreased from 1340 μmol / L to about 525 μmol / L, and then slowly decreased. By the 16th day, the concentration of H2O2 can be decreased to about 53 μmol / L. This is beneficial to removing excess H2O2 in the microenvironment in the early stage of myocardial infarction to avoid tissue damage caused by oxidative stress, and is also beneficial to maintaining a low and appropriate concentration level of H2O2 in the late stage of myocardial infarction to continuously provide reactive oxygen species signals to stimulate myocardial angiogenesis.

[0032] (4) The hydrogel of the present invention has good cell compatibility. For example, the COFH prepared in Example 5 can promote the proliferation of human umbilical vein endothelial cells (HUVECs), which can meet the safety requirements of minimally invasive injectable hydrogels after myocardial infarction.

[0033] (5) The hydrogel of the present invention has good ability to promote angiogenesis of HUVECs. For example, the COFH prepared in Example 5 can generate an appropriate concentration level of H2O2 in the microenvironment through the autoxidation of its catechol groups and the coordination reaction of ferric ions to promote the angiogenesis of HUVECs.

[0034] (6) The hydrogel of the present invention has good ability to promote myocardial regeneration and repair after myocardial infarction. For example, injecting the COFH gel precursor solution prepared in Example 5 into the myocardium of myocardial infarction rats to form COFH, and repairing in the myocardium of myocardial infarction rats for 28 days. The results of echocardiogram, electrocardiogram of rats and Masson's trichrome staining of rat heart tissue show that COFH can effectively reduce myocardial fibrosis after myocardial infarction and promote the recovery of cardiac function after myocardial infarction.

[0035] Based on the above experimental results, the present invention also provides the application of the hydrogel capable of regulating the redox homeostasis of the microenvironment in the preparation of myocardial tissue repair materials. In particular, the application of the hydrogel capable of regulating the redox homeostasis of the microenvironment in the preparation of myocardial tissue repair materials after myocardial infarction.

[0036] Compared with the prior art, the technical solution provided by the present invention has the following beneficial technical effects:

[0037] 1. The present invention provides a hydrogel capable of regulating the redox homeostasis of the microenvironment. The polymer network of the hydrogel is formed by the hybrid cross-linking reaction of catechol-modified hyaluronic acid, carboxylated chitosan, oxidized dextran and iron ions. Under physiological conditions, the catechol groups in the hydrogel will undergo autoxidation reaction to generate H2O2. At the same time, the iron ions coordinated on the catechol groups in the hydrogel will catalyze the decomposition of H2O2 into water and oxygen, thereby regulating the level of reactive oxygen species in the microenvironment. Compared with the existing hydrogel materials with antioxidant ability, the hydrogel provided by the present invention can achieve bidirectional regulation of ROS in the pathological microenvironment after myocardial infarction, which not only rapidly scavenges excessive H2O2 in the myocardial microenvironment in the early stage after myocardial infarction, but also maintains a low concentration of H2O2 in the myocardial microenvironment in the middle and late stages of myocardial infarction. It can solve the problem that the existing hydrogel materials with antioxidant ability mainly play the role of scavenging ROS and are difficult to achieve precise adaptation to the redox state of the myocardial pathological microenvironment, and can improve the ability of hydrogel materials to regenerate and repair myocardial tissue after myocardial infarction.

[0038] 2. The hydrogel capable of regulating the redox homeostasis of the microenvironment according to the present invention forms a multiple cross-linking network through multiple reactions including Schiff base reaction, Michael addition reaction, oxidative self-crosslinking and dynamic coordination reaction, which can not only optimize the mechanical properties of the hydrogel, but also delay the degradation of the hydrogel in vivo, so as to better match the tissue repair process. In addition, the rich catechol functional groups in the hydrogel can endow it with good tissue adhesion characteristics, and it can be fixed in the injection area after being injected into a gel in the tissue, thereby playing a role in long-term regulating the redox homeostasis of the pathological microenvironment. The above characteristics provide favorable conditions for the hydrogel according to the present invention to promote myocardial tissue repair after myocardial infarction.

[0039] 3. The present invention has been experimentally verified that the hydrogel of the present invention has good cell compatibility and can meet the safety requirements of minimally invasive injectable hydrogels; through the autoxidation of its catechol groups and the coordination reaction of ferric ions, the hydrogel of the present invention can generate an appropriate concentration level of H2O2 in the microenvironment to promote the angiogenesis of HUVECs; injecting the gel precursor solution for preparing the hydrogel of the present invention into the myocardium of myocardial infarction rats to form a hydrogel, and repairing it in the myocardium of myocardial infarction rats for 28 days. The echocardiogram, electrocardiogram of the rats and the Masson trichrome staining results of the rat heart tissue show that the hydrogel of the present invention can effectively reduce myocardial fibrosis after myocardial infarction and promote the recovery of cardiac function after myocardial infarction. Based on this, the present invention also provides the application of the hydrogel capable of regulating the redox homeostasis of the microenvironment in the preparation of myocardial tissue repair materials, so as to reduce or avoid myocardial tissue damage caused by oxidative stress by scavenging excessive H2O2 in the microenvironment in the early stage after myocardial infarction, and maintain the H2O2 concentration in the microenvironment at a low level in the middle and late stages of myocardial infarction, thereby continuously providing reactive oxygen species signal to stimulate angiogenesis. The present invention can solve the problem that the existing technology has poor regenerative repair performance for myocardial tissue by simply scavenging ROS or antioxidant intervention for myocardial repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of the raw material HA and the prepared HAD used in Example 1.

[0041] Figure 2 is the nuclear magnetic resonance hydrogen spectrum of the raw material Dex and the prepared OD used in Example 3.

[0042] Figure 3 is the infrared spectrum of HA, DOPA and the prepared HAD used in Example 1.

[0043] Figure 4 is the infrared spectrum of Dex and the prepared OD used in Example 3.

[0044] Figure 5 are the SEM images of freeze-dried CO, COF, COH and COFH at different magnifications.

[0045] Figure 6 is the infrared spectrum of freeze-dried CO, COF, COH and COFH.

[0046] Figure 7 Figure A of is the change of the storage modulus and loss modulus of the gel precursor solution for preparing COFH, CO, COH and COF with time, Figure 7 Figure B of is the storage modulus and loss modulus of COFH, CO, COH and COF under different strain conditions.

[0047] Figure 8 are the storage modulus and loss modulus of CO, COF, COH, and COFH at different frequencies.

[0048] Figure 9 are the compressive stress-strain curves of CO, COF, COH, and COFH.

[0049] Figure 10 Figure A shows the schematic diagram of the principle of the hydrogel for bidirectionally regulating the H2O2 concentration level in the present invention. Figure 10 Figure B shows the change of the ability of each experimental group to generate H2O2 in vitro over time. Figure 10 Figure C shows the change of the ability of each experimental group to scavenge H2O2 in vitro over time.

[0050] Figure 11 are the test results of the cell proliferation of COFH, CO, COH, and COF after co-culturing with HUVECs for 1 day and 3 days.

[0051] Figure 12 are the growth states and distribution of the cells of COFH, CO, COH, and COF after co-culturing with HUVECs for 1 day and 3 days.

[0052] Figure 13 are the original tube formation images and the tube formation images processed by Image J software of each experimental group at 6 h of incubation.

[0053] Figure 14 are the number of cell grids, nodes, and intersection points of each experimental group at 6 h of incubation.

[0054] Figure 15 are the photos of the surgical process, and Figures A - D are the photos before thoracotomy, ligation of the left anterior descending coronary artery, injection of the gel precursor solution, and suturing of the rats, respectively.

[0055] Figure 16 are the echocardiograms of the rats in each experimental group on the 28th day after surgery.

[0056] Figure 17 are the electrocardiograms of the rats in each experimental group on the 28th day after surgery.

[0057] Figure 18 are the Masson trichrome staining results of the heart tissues of the rats in each experimental group on the 28th day after surgery. Specific implementation manners

[0058] The following examples are used to further illustrate the hydrogel capable of regulating the redox homeostasis of the microenvironment provided by the present invention, its preparation method and application. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as a limitation of the protection scope of the present invention. Those skilled in the art make some non-essential improvements and adjustments to the present invention according to the above-mentioned invention content for specific implementation, and still fall within the protection scope of the present invention.

[0059] In the following examples, the carboxylated chitosan (CCS, degree of carboxylation ≥ 80%, CAS: 9012-76-4) used was purchased from Yuanye Bio-Technology Co., Ltd., Shanghai, China. The structural formula of CCS is shown in formula (III):

[0060] (III).

[0061] Example 1

[0062] In this example, the preparation of catechol-modified hyaluronic acid (HAD) was carried out as follows:

[0063] (1) Sodium hyaluronate (HA, Mw = 340 kDa) was added to a round-bottom flask containing PBS buffer with a concentration of 0.01 mol / L and a pH value of 5.5. After evacuating the round-bottom flask, it was stirred at room temperature under nitrogen protection until HA was completely dissolved to obtain an HA solution with a concentration of 16.7 mg / mL. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) was dissolved in PBS buffer with a concentration of 0.01 mol / L and a pH value of 5.5 to obtain an EDC solution with a concentration of 450 mg / mL. N-hydroxysuccinimide (NHS) was dissolved in PBS buffer with a concentration of 0.01 mol / L and a pH value of 5.5 to obtain an NHS solution with a concentration of 175 mg / mL. Dopamine hydrochloride (DOPA) was dissolved in PBS buffer with a concentration of 0.01 mol / L and a pH value of 5.5 to obtain a DOPA solution with a concentration of 450 mg / mL.

[0064] (2) The EDC solution was added to the HA solution, and then the NHS solution was added. The mixture was stirred at room temperature under nitrogen protection for 2 h. After the reaction was completed, the DOPA solution was added dropwise to the obtained reaction solution under light-shielded conditions, and the mixture was stirred at room temperature under light-shielded and nitrogen protection for 12 h. During both stirring reactions, the pH value was controlled at 5.5. In this step, the molar ratio of EDC, NHS, DOPA to the carboxyl group of HA was controlled at 4:2:1:1.

[0065] (3) Centrifuge the reaction solution obtained in step (2) at a speed of 2000 rpm for 5 min, take the supernatant and put it into a dialysis bag (MWCO: 8 - 14 kDa), and place it in a dialysis solution with pH = 5.0 (obtained by adjusting the pH of ultrapure water to 5.0 with hydrochloric acid). Under the stirring condition of 400 rpm at room temperature and in the dark, dialyze for 3 days; during dialysis, change the dialysis solution every 6 h; after dialysis is completed, lyophilize the liquid in the dialysis bag to obtain HAD, bag it and seal it, and store it in the dark for later use.

[0066] Perform nuclear magnetic resonance hydrogen spectrum tests on the raw material HA and the prepared HAD used in this example. The results are as Figure 1 shown. The grafting rate of catechol groups in HAD is calculated by nuclear magnetic resonance hydrogen spectrum integration, and the result is 10%. Perform infrared spectrum tests on the HA, DOPA, and the prepared HAD used in this example. The results are as Figure 3 shown. From Figure 1 and 3 it can be seen that characteristic peaks of catechol groups (δ = 6.8 - 7.2 ppm) appear in the hydrogen spectrum of HAD, indicating that DOPA has been successfully grafted onto the molecular chain of HA. A strong absorption peak appears near 1720 cm -1 in the infrared spectrum of HA, which is related to the stretching vibration of the carboxyl group (C=O), indicating that HA contains a large number of carboxyl groups. A relatively strong absorption peak appears at 1235 cm -1 in the infrared spectrum of DOPA, which is related to the C-N stretching vibration. This is a typical characteristic absorption peak of the amino group in DOPA. In the range of 900 - 1500 cm -1 regions, absorption peaks related to the benzene ring and amino group (-NH2) can also be seen. In the infrared spectrum of HAD, the absorption peak near 1720 cm -1 is stronger than that of HA, which may be related to the enhanced conjugation effect or intermolecular hydrogen bond interaction of the carboxyl group due to the introduction of DOPA. The absorption peak at 1235 cm -1 is related to the amino group of dopamine, further indicating that DOPA has been successfully grafted onto the molecular chain of HA through an amidation reaction. The structure of HAD is as shown in formula (I):

[0067] (I).

[0068] Example 2

[0069] In this example, to prepare catechol-modified hyaluronic acid (HAD), the steps are as follows:

[0070] (1) HA (Mw = 340 kDa) was added to a round-bottom flask containing PBS buffer with a concentration of 0.01 mol / L and a pH value of 5.5. After evacuating the round-bottom flask, it was stirred at room temperature under nitrogen protection until HA was completely dissolved, obtaining an HA solution with a concentration of 16.7 mg / mL. EDC was dissolved in PBS buffer with a concentration of 0.01 mol / L and a pH value of 5.5, obtaining an EDC solution with a concentration of 300 mg / mL. NHS was dissolved in PBS buffer with a concentration of 0.01 mol / L and a pH value of 5.5, obtaining an NHS solution with a concentration of 125 mg / mL. DOPA was dissolved in PBS buffer with a concentration of 0.01 mol / L and a pH value of 5.5, obtaining a DOPA solution with a concentration of 250 mg / mL.

[0071] (2) The EDC solution was added to the HA solution, and then the NHS solution was added. The mixture was stirred and reacted at room temperature under nitrogen protection for 2 h. After the reaction was completed, the DOPA solution was added dropwise to the obtained reaction solution under light-shielded conditions, and the mixture was stirred and reacted at room temperature under light-shielded and nitrogen protection for 12 h. During the two stirring reactions, the pH value was controlled at 5.5. In this step, the molar ratio of EDC, NHS, DOPA to the carboxyl group of HA was controlled at 3:1:1:1.

[0072] (3) The reaction solution obtained in step (2) was centrifuged at a speed of 2000 rpm for 5 min. The supernatant was taken and aliquoted into a dialysis bag (MWCO: 8 - 14 kDa), and placed in a dialysis solution with a pH of 5.0 (obtained by adjusting the pH value of ultrapure water to 5.0 with hydrochloric acid). Under the stirring condition at room temperature and a speed of 400 rpm, it was dialyzed for 3 days in the dark. During dialysis, the dialysis solution was changed every 6 h. After dialysis was completed, the liquid in the dialysis bag was freeze-dried to obtain HAD, which was bagged and sealed, and stored for later use in the dark.

[0073] The 1H NMR test was carried out on the HAD prepared in this example, and the grafting rate of catechol groups in HAD was calculated by 1H NMR integration. The result was 6%. [[ID=IQ]]

[0074] Example 3

[0075] A large number of hydroxyl groups exist on the molecular chain of dextran (Dex). These hydroxyl groups are easily oxidized to aldehyde groups with higher reactivity under the action of strong oxidants. In this example, Dex was oxidized to prepare oxidized dextran (OD), and the steps were as follows:

[0076] (1) Dex (Mw = 100 kDa) was dissolved in ultrapure water to obtain a Dex solution with a concentration of 10 mg / mL.

[0077] (2) Slowly add an aqueous sodium periodate solution with a concentration of 30 mg / mL to the Dex solution under light - shielding conditions. After the addition is complete, stir and react at room temperature for 2 h under light - shielding conditions, and control the amount of sodium periodate used to be 16.6% of the mass of Dex.

[0078] (3) Load the reaction solution obtained in step (2) into a dialysis bag (MWCO: 3.5 kDa), and dialyze it in ultrapure water for 3 days; during dialysis, change the dialysis fluid every 6 h; after dialysis is completed, lyophilize the liquid in the dialysis bag to obtain OD, pack it in a bag and seal it, and store it in the dark for later use.

[0079] Perform nuclear magnetic resonance hydrogen spectrum tests on the raw material Dex and the prepared OD used in this example. The results are as Figure 2 shown. The 1 H of the Dex sugar ring appears at broad peaks at δ = 3.2 - 4.0 ppm and δ = 4.8 ppm, and a new broad peak appears at δ = 5.0 - 5.8 ppm for OD. Perform infrared spectrum tests on the Dex and the prepared OD used in this example. The results are as Figure 4 shown. The main characteristic absorption peak of the aldehyde is located at 1750 - 1700 cm -1 - 1. In the infrared spectrum of OD, the absorption peak near 1710 cm -1 - 1 is stronger than that of Dex, which may be related to the introduction of the aldehyde group enhancing the stretching vibration of C = O. The above characterization data confirm the successful preparation of OD. The oxidation degree of OD is determined by the hydroxylamine hydrochloride titration method, and the result is 17.8%. The structure of OD is shown in formula (II):

[0080] (II).

[0081] Example 4

[0082] In this example, the preparation of oxidized dextran (OD) is as follows:

[0083] (1) Dissolve Dex (Mw = 200 kDa) in ultrapure water to obtain a Dex solution with a concentration of 10 mg / mL.

[0084] (2) Slowly add an aqueous sodium periodate solution with a concentration of 50 mg / mL to the Dex solution under light - shielding conditions. After the addition is complete, stir and react at room temperature for 2 h under light - shielding conditions, and control the amount of sodium periodate used to be 28.2% of the mass of Dex.

[0085] (3) The reaction solution obtained in step (2) was loaded into a dialysis bag (MWCO: 3.5 kDa) and dialyzed in ultrapure water for 3 days; during dialysis, the dialysis solution was changed every 6 h; after dialysis was completed, the liquid in the dialysis bag was lyophilized to obtain OD, which was bagged and sealed and stored in the dark for later use.

[0086] The oxidation degree of OD prepared in this example was determined by the hydroxylamine hydrochloride titration method, and the result was 35%.

[0087] Example 5

[0088] In this example, a hydrogel capable of regulating the redox homeostasis of the microenvironment was prepared as follows:

[0089] (1) The HAD prepared in Example 1 was dissolved in ultrapure water to obtain a HAD solution with a concentration of 80 mg / mL. The carboxylated chitosan (CCS) was dissolved in a PBS buffer solution with a concentration of 0.01 mol / L and a pH value of 7.4 to obtain a CCS solution with a concentration of 240 mg / mL. The OD prepared in Example 3 was dissolved in an aqueous FeCl3 solution to obtain an OD / FeCl3 mixed solution with an OD concentration of 120 mg / mL and an FeCl3 concentration of 0.04 mol / L.

[0090] (2) The CCS solution and the OD / FeCl3 mixed solution were mixed thoroughly in equal volumes to obtain a mixed solution A. In the mixed solution A, the concentration of CCS was 120 mg / mL, the concentration of OD was 60 mg / mL, and the concentration of FeCl3 was 0.02 mol / L.

[0091] Subsequently, the HAD solution was added to the mixed solution A and mixed thoroughly to obtain a gel precursor solution. In the gel precursor solution, the concentration of HAD was 40 mg / mL, the concentration of CCS was 60 mg / mL, the concentration of OD was 30 mg / mL, and the concentration of FeCl3 was 0.01 mol / L.

[0092] (3) The gel precursor solution was quickly transferred to a mold and allowed to stand at room temperature for 10 min. The gel precursor solution immediately turned into a hydrogel state. The obtained hydrogel was soaked in a PBS buffer solution with a concentration of 0.01 mol / L and a pH of 7.4 at room temperature for 24 h, and then transferred to ultrapure water and soaked at room temperature for 2 h. The operation of soaking the hydrogel in the PBS buffer solution and ultrapure water was repeated 3 times to remove the Fe that was not bound to the catechol group 3+ , and the obtained hydrogel was transferred to a PBS buffer solution with a concentration of 0.01 mol / L and a pH value of 7.4 and soaked at room temperature for 2 h to obtain a hydrogel capable of regulating the redox homeostasis of the microenvironment, denoted as COFH.

[0093] Comparative Example 1

[0094] In this comparative example, an injectable single-network hydrogel was prepared as follows:

[0095] (1) CCS was dissolved in a PBS buffer solution with a concentration of 0.01 mol / L and a pH value of 7.4 to obtain a CCS solution with a concentration of 120 mg / mL. The OD prepared in Example 3 was dissolved in ultrapure water to obtain an OD solution with a concentration of 60 mg / mL.

[0096] (2) The CCS solution and the OD solution were fully mixed to obtain a mixed solution B. In the mixed solution B, the concentration of CCS was 60 mg / mL and the concentration of OD was 30 mg / mL.

[0097] (3) The mixed solution B was quickly transferred to a mold and allowed to stand at room temperature for reaction for 10 min. The gel precursor solution immediately changed into a hydrogel state. The obtained hydrogel was immersed in a PBS buffer solution with a concentration of 0.01 mol / L and a pH value of 7.4 at room temperature for 24 h, and then transferred to ultrapure water and immersed at room temperature for 2 h. The operation of immersing the hydrogel in the PBS buffer solution and ultrapure water was repeated 3 times. The obtained hydrogel was transferred to a PBS buffer solution with a concentration of 0.01 mol / L and a pH value of 7.4 and immersed at room temperature for 2 h to obtain an injectable single-network hydrogel, denoted as CO.

[0098] Comparative Example 2

[0099] In this comparative example, an injectable double-network hydrogel was prepared as follows:

[0100] (1) The HAD prepared in Example 1 was dissolved in ultrapure water to obtain a HAD solution with a concentration of 120 mg / mL. CCS was dissolved in a PBS buffer solution with a concentration of 0.01 mol / L and a pH value of 7.4 to obtain a CCS solution with a concentration of 180 mg / mL. The OD prepared in Example 3 was dissolved in ultrapure water to obtain an OD solution with a concentration of 30 mg / mL.

[0101] (2) The HAD solution, the CCS solution and the OD solution were fully mixed to obtain a mixed solution C. In the mixed solution C, the concentration of HAD was 40 mg / mL, the concentration of CCS was 60 mg / mL, and the concentration of OD was 30 mg / mL.

[0102] (3) Rapidly transfer the mixed solution C to a mold and let it stand at room temperature for 10 min. The gel precursor solution will turn into a hydrogel state. Immerse the obtained hydrogel in a PBS buffer solution with a concentration of 0.01 mol / L and a pH value of 7.4 at room temperature for 24 h, then transfer it to ultrapure water and soak it at room temperature for 2 h. Repeat the operation of soaking the hydrogel in the PBS buffer solution and ultrapure water mentioned above 3 times. Transfer the obtained hydrogel to a PBS buffer solution with a concentration of 0.01 mol / L and a pH value of 7.4 and soak it at room temperature for 2 h to obtain an injectable double-network hydrogel, denoted as COH.

[0103] Comparative Example 3

[0104] In this comparative example, an injectable single-network ferric ion composite hydrogel was prepared as follows:

[0105] (1) Dissolve CCS in a PBS buffer solution with a concentration of 0.01 mol / L and a pH value of 7.4 to obtain a CCS solution with a concentration of 120 mg / mL. Dissolve OD prepared in Example 3 in an FeCl3 solution to obtain an OD / FeCl3 mixed solution with an OD concentration of 60 mg / mL and an FeCl3 concentration of 0.02 mol / L.

[0106] (2) Thoroughly mix the CCS solution and the OD / FeCl3 mixed solution to obtain a mixed solution D. In the mixed solution D, the concentration of CCS is 60 mg / mL, the concentration of OD is 30 mg / mL, and the concentration of FeCl3 is 0.1 mol / L.

[0107] (3) Rapidly transfer the mixed solution D to a mold and let it stand at room temperature for 10 min. The gel precursor solution will turn into a hydrogel state. Immerse the obtained hydrogel in a PBS buffer solution with a concentration of 0.01 mol / L and a pH value of 7.4 at room temperature for 24 h, then transfer it to ultrapure water and soak it at room temperature for 2 h. Repeat the operation of soaking the hydrogel in the PBS buffer solution and ultrapure water mentioned above 3 times. Transfer the obtained hydrogel to a PBS buffer solution with a concentration of 0.01 mol / L and a pH value of 7.4 and soak it at room temperature for 2 h to obtain an injectable single-network ferric ion composite hydrogel, denoted as COF.

[0108] Example 6

[0109] In this example, COFH, CO, COH, and COF prepared in Example 5 and Comparative Examples 1 - 3 were subjected to scanning electron microscopy (SEM) tests.

[0110] Freeze-dry COFH, CO, COH, and COF and conduct SEM tests. Figure 5SEM images of CO, COF, COH, and COFH after lyophilization at different magnifications. It can be seen from Figure 5 that CO, COF, COH, and COFH after lyophilization all have interconnected porous structures, which are beneficial for the delivery of cytokines and hydrogen peroxide. Among them, the average pore size of CO after lyophilization is about 178 μm, and the porosity is about 83%; the average pore size of COF after lyophilization is about 165 μm, and the porosity is about 86%; the average pore size of COH after lyophilization is about 122 μm, and the porosity is about 89%; the average pore size of COFH after lyophilization is about 71 μm, and the porosity is about 92%. Relatively speaking, COFH after lyophilization has a denser pore structure and a higher porosity, which may be due to the Schiff base reaction between the amino group of CCS and the aldehyde group of OD, the oxidative self-crosslinking reaction of HAD, and the Michael addition reaction between the quinone group of the oxidized self-crosslinked HAD and the residual amino group of CCS, and the coordination reaction with Fe 3+ occurred, forming a multiple cross-linked network.

[0111] CO, COF, COH, and COFH were lyophilized and subjected to infrared spectroscopy tests. The results are as Figure 6 shown. Compared with CO and COF, due to the oxidation of HAD in COH and COFH, the peak intensities at 1033 cm -1 and 1082 cm -1 showed obvious changes, which may be related to the enhanced stretching vibrations of the C-OH bond and the C-O bond due to the introduction of dopamine, which confirmed that HAD played a "bridge" role in the multiple cross-linked network of COFH.

[0112] Example 7

[0113] In this example, the rheological mechanical properties of COFH, CO, COH, and COF prepared in Example 5 and Comparative Examples 1-3 were tested.

[0114] Prepare the gel precursor solution, mixture B, mixture C, and mixture D according to the operations in Example 5 and Comparative Examples 1-3, that is, prepare the gel precursor solution for preparing COFH, CO, COH, and COF, and test the changes of their storage modulus (G') and loss modulus (G'') with time. The results are as Figure 7 shown in Figure A. Test the storage modulus (G') and loss modulus (G'') of COFH, CO, COH, and COF prepared in Example 5 and Comparative Examples 1-3 under different strain conditions. The results are as Figure 7 shown in Figure B.

[0115] The gelation time is an important parameter of injectable hydrogels. It can be seen from Figure 7As can be seen from Figure A, for COFH, CO, COH, and COF, the storage modulus and loss modulus of their gel precursor solutions showed slight fluctuations at the initial stage of the test, then increased rapidly, and finally reached an equilibrium state. Their initial storage modulus was lower than the loss modulus, and then the storage modulus increased relatively rapidly and exceeded the loss modulus. The formation of the intersection point of the storage modulus and the loss modulus marked the gelation of the hydrogel, and the time of the intersection point was the gelation time. From Figure 7 It can be seen that the storage modulus of COFH and COH is higher than that of CO and COF, indicating that the catechol-mediated multiple cross-linking mechanism enhances the mechanical properties of the hydrogel, which helps to improve the structural stability of the hydrogel in the myocardium with a complex mechanical environment.

[0116] Example 8

[0117] In this example, according to the operations of Example 5 and Comparative Examples 1 to 3, COFH, CO, COH, and COF hydrogels were prepared using a cylindrical mold with a diameter of 0.5 cm and a depth of 0.15 cm, and the rheological mechanical properties of each hydrogel were tested.

[0118] A dynamic mechanical tester was used to perform compression stress-strain tests on each hydrogel sample. Four parallel samples were set for each hydrogel sample. The displacement rate used during the test was 1 mm / min until the hydrogel sample was damaged, and the storage modulus (G') and loss modulus (G'') of each hydrogel at different frequencies and the compression stress-strain curves of each hydrogel were obtained, as shown in Figure 8 、 9 respectively. As can be seen from Figure 8 it, the storage modulus and loss modulus of each hydrogel increased with the increase in frequency, indicating that each hydrogel showed a stronger elastic and viscous response under high-frequency conditions. Among them, the storage modulus of COFH was the highest. As the frequency increased from 2 Hz to 10 Hz, the storage modulus of COFH increased from about 43 KPa to about 67 KPa, showing the strongest rigidity and elasticity. As can be seen from Figure 9 it, the maximum compressive strength of COFH was the highest, reaching 38 ± 5 KPa, which was mainly due to the multiple cross-linking network of COFH enhancing its mechanical properties.

[0119] Example 9

[0120] In this example, the bidirectional regulation performance of COFH, CO, COH, and COF prepared in Example 5 and Comparative Examples 1 to 3 on H2O2 in vitro was tested. The following experimental groups were set:

[0121] COFH group and COFH + H2O2 group: To test the ability of COFH to generate H2O2 in vitro, COFH with a volume of 70 μL was prepared using a cylindrical mold according to the method of Example 5, freeze-dried, and the freeze-dried COFH was placed in 300 μL of PBS buffer (pH = 7.4). It was placed in an incubator at a temperature of 37 °C, a relative humidity of 95%, and 5% CO2 for 16 days. During this period, samples were taken at regular intervals to measure the H2O2 concentration using the Amplex Red method. To test the ability of COFH to scavenge H2O2 in vitro, COFH with a volume of 70 μL was prepared using a cylindrical mold according to the method of Example 5, freeze-dried, and the freeze-dried COFH was placed in 300 μL of PBS buffer containing H2O2 (pH = 7.4, H2O2 concentration of 1340 μmol / L). It was placed in an incubator at a temperature of 37 °C, a relative humidity of 95%, and 5% CO2 for 16 days. During this period, samples were taken at regular intervals to measure the H2O2 concentration using the Amplex Red method.

[0122] CO group and CO + H2O2 group: The operations were basically the same as those of the COFH group and the COFH + H2O2 group, except that COFH was replaced with CO prepared according to the method of Comparative Example 1 with the same shape and volume.

[0123] COH group and COH + H2O2 group: The operations were basically the same as those of the COFH + H2O2 group and the COFH group, except that COFH was replaced with COH prepared according to the method of Comparative Example 2 with the same shape and volume.

[0124] COF group or COF + H2O2 group: The operations were basically the same as those of the COFH group and the COFH + H2O2 group, except that COFH was replaced with COF prepared according to the method of Comparative Example 3 with the same shape and volume.

[0125] Control group or Control + H2O2 group: The operations were basically the same as those of the COFH group and the COFH + H2O2 group, except that no hydrogel was placed in the PBS buffer and the PBS buffer containing H2O2 (pH = 7.4, H2O2 concentration of 1340 μmol / L).

[0126] Figure 10 Figure B shows the change in the ability of each experimental group to generate H2O2 in vitro over time. Figure 10Figure C shows the change in the ability of each experimental group to scavenge H2O2 in vitro over time. For the ability to generate H2O2 in vitro, after 16 days, the H2O2 concentration in the COFH group gradually increased from 0 to approximately 34 μmol / L. For the ability to scavenge H2O2 in vitro, after 4 days, the H2O2 concentration in the COFH + H2O2 group rapidly decreased from 1340 μmol / L to approximately 525 μmol / L, and then decreased slowly. By the 16th day, the H2O2 concentration in the COFH + H2O2 group decreased to approximately 53 μmol / L. Compared with the situation where COH maintained a relatively high H2O2 release level, the H2O2 generated by COFH was rapidly catalytically decomposed into water and oxygen by Fe 3+ coordinated on the catechol group of COFH, which was beneficial for COFH to scavenge excess H2O2 in the microenvironment in the early stage of myocardial infarction and avoid tissue damage caused by oxidative stress. Compared with the situation where CO and COF could only maintain a very low H2O2 release level, the catechol group of COFH would undergo auto-oxidation under physiological conditions to generate H2O2, which could maintain a relatively low and appropriate level of H2O2 in the late stage of myocardial infarction and continuously provide reactive oxygen species signals to stimulate myocardial angiogenesis. This indicated that COFH had a better ability to bidirectionally regulate the H2O2 concentration level compared with CO, COH, and COF. In addition, the multiple cross-linked network of COFH endowed it with higher mechanical strength and structural stability, which was beneficial for COFH covering the myocardial infarction interface area to sustainably release a certain concentration of H2O2, thereby promoting the repair effect of myocardial tissue and function.

[0127] Figure 10 Figure A shows the schematic diagram of the principle of the hydrogel of the present invention for bidirectionally regulating the H_{2}O_{2} concentration level. As shown in this figure, the catechol group in the hydrogel (such as COFH) of the present invention will undergo an auto-oxidation reaction to transform the catechol group into a quinone or semiquinone structure to generate H_{2}O_{2}. At the same time, H_{2}O_{2} will be catalytically decomposed into water and oxygen by the iron ions coordinated on the catechol group in the hydrogel, and the quinone or semiquinone structure will be transformed into a catechol structure, that is, there are both the generation and decomposition processes of H_{2}O_{2} in the system, forming a dynamic equilibrium to regulate the H_{2}O_{2} concentration level, and further regulating the ROS level. This enables the hydrogel of the present invention to play different roles at different stages after myocardial infarction with the change of the H_{2}O_{2} concentration level in the myocardial infarction microenvironment. In the early stage of myocardial infarction, it plays a role in scavenging excess H_{2}O_{2} in the microenvironment and avoiding tissue damage caused by oxidative stress. In the late stage of myocardial infarction, it maintains a relatively low and appropriate level of H_{2}O_{2}, playing a role in continuously providing reactive oxygen species signals to stimulate myocardial angiogenesis.

[0128] Example 10

[0129] In this example, the COFH, CO, COH, and COF prepared in Example 5 and Comparative Examples 1-3 were co-cultured with human umbilical vein endothelial cells (HUVECs) to investigate the biocompatibility of each hydrogel.

[0130] COFH, CO, COH, and COF were prepared using a mold with a diameter of 8 mm and a height of 3 mm according to the methods of Example 5 and Comparative Examples 1-3, respectively. Subsequently, a hydrogel-HUVECs two-dimensional co-culture model was constructed. Specifically: each hydrogel was immersed in an ethanol aqueous solution with a volume fraction of 75% for 24 h, and then transferred to a sterile PBS buffer (0.01 mol / L, pH = 7.4) and immersed for 24 h. 10 μL of a cell suspension containing 50,000 HUVECs was dropped onto the upper surface of each hydrogel treated as described above. After incubating in an incubator (5% CO2, 20% O2) for 2 h, 10 μL of a cell suspension containing 50,000 HUVECs was dropped onto the lower surface of each hydrogel. After continuing to incubate for 2 h, high-glucose DMEM medium containing 10% serum and 1% double antibody was supplemented, and the samples were incubated in the incubator for 1 day and 3 days respectively. During the incubation period, the high-glucose DMEM medium containing 10% serum and 1% double antibody was replaced every 24 h.

[0131] After the incubation time reached the requirement, each hydrogel was taken out, and the CCK-8 method was used to test the proliferation of HUVECs in each hydrogel. When testing, the absorbance was measured at a wavelength of 450 nm, and the results are as Figure 11 shown. After the incubation time reached the requirement, each hydrogel was taken out, and the cells were washed twice with PBS buffer. The washed cells were stained with a PBS buffer containing fluorescein diacetate (FDA) and propidium iodide (PI) for 1 min, and the growth state and distribution of the cells were observed by a confocal laser scanning microscope (CLSM). The results are as Figure 12 shown.

[0132] HUVECs are important cells involved in the entire process of myocardial vascular regeneration. As Figure 11 can be seen, the absorbance value of COFH co-cultured with HUVECs after 3 days was the highest, and there were significant differences from CO, COF, and COH. As Figure 12 can be seen, HUVECs grew well after co-culturing with each hydrogel for 1 day and 3 days. Compared with CO, COF, and COH, COFH had better cell proliferation and a higher proportion of live cells after co-culturing with HUVECs for 3 days. The above experimental results indicate that COFH has higher cell compatibility with HUVECs and better ability to promote the proliferation of HUVECs, and can meet the safety requirements of minimally invasive injectable hydrogels after myocardial infarction.

[0133] Example 11

[0134] In this example, the promoting abilities of COFH, CO, COH, and COF prepared in Example 5 and Comparative Examples 1-3 on the angiogenesis of HUVECs were evaluated.

[0135] HUVECs in the fifth generation were selected for the tube formation experiment. The Matrigel was taken out of the -20 °C refrigerator and placed in the 4 °C refrigerator until completely thawed. The Matrigel was mixed evenly using pre-cooled pipette tips. The 96-well plate was placed on ice, and 50 μL of the completely thawed Matrigel was added to each well, avoiding the generation of bubbles. Subsequently, the 96-well plate was placed in a cell culture incubator (37 °C, 5% CO2) and incubated for 30 min to ensure the formation of a gel substrate by the Matrigel. The fifth-generation HUVECs were digested with trypsin, and after centrifugation, the cell suspension concentration was adjusted to 600,000 / mL using serum-free 1640 medium containing 1% double antibodies.

[0136] The extraction solutions of COFH, CO, COH, and COF were prepared and stored at 4 °C for standby use. Taking the preparation of the COFH extraction solution as an example for illustration. The COFH hydrogel was placed in a 6-well plate, and 4 mL of serum-free 1640 medium containing 1% double antibodies was added to each well. The plate was placed in a 37 °C, 5% CO2 incubator and cultured for 3 days. Subsequently, the culture medium extraction solution was extracted, the H2O2 concentration was measured using the Amplex Red method, and it was diluted to an H2O2 concentration of 10 μmol / L with serum-free 1640 medium containing 1% double antibodies to obtain the diluted COFH extraction solution, and the dilution factor was recorded.

[0137] The following experimental groups were set up:

[0138] COFH group: The diluted COFH extraction solution was added to the 96-well plate, and then the HUVECs cell suspension was added.

[0139] CO group: According to the dilution factor when preparing the diluted COFH extraction solution, the CO extraction solution was diluted with serum-free 1640 medium containing 1% double antibodies. The obtained diluted CO extraction solution was added to the 96-well plate, and then the HUVECs cell suspension was added.

[0140] COH group: According to the dilution factor when preparing the diluted COFH extraction solution, the COH extraction solution was diluted with serum-free 1640 medium containing 1% double antibodies. The obtained diluted COH extraction solution was added to the 96-well plate, and then the HUVECs cell suspension was added.

[0141] COFH + CAT group: According to the dilution factor when preparing the diluted COFH extraction solution, the COFH extraction solution was diluted with serum-free 1640 medium containing 1% double antibodies. The obtained diluted COFH extraction solution was added to the 96-well plate, and then catalase (CAT) was added at a dose of 1000 U / well, and then the HUVECs cell suspension was added.

[0142] H2O2 group: Add serum-free 1640 medium containing 1% double antibiotics to a 96-well plate, then add H2O2 to the 96-well plate until the H2O2 concentration is 10 μmol / L, and then add the HUVECs cell suspension.

[0143] Ctrl group: Add serum-free 1640 medium containing 1% double antibiotics to a 96-well plate, and then add the HUVECs cell suspension.

[0144] In the above experimental groups, the addition amount of the HUVECs cell suspension in each well of the 96-well plate is the same. Place the 96-well plate in a real-time live cell workstation, incubate it under the conditions of 37 °C and 5% CO2, and dynamically observe for 24 h. Take pictures and record every 1 h during this period to observe the formation state of the vascular network and the cell state. Use the Angiogenesis Analyzer plug-in of Image J software to quantitatively analyze the images taken at 6 h of incubation, and calculate the angiogenesis-related data of HUVECs in each experimental group, including the number of cell grids, nodes, and intersection points. The results are as Figure 13 - 14 shown.

[0145] As Figure 14 can be seen, compared with the CO group, COH group, and COF group, the COFH group has the highest number of grids, nodes, and intersection points, which is comparable to the effect of the H2O2 group, while the tube formation effect of the COFH + CAT group is close to that of the Ctrl group. The test results of this example show that the H2O2 concentration has a significant effect on the angiogenesis of HUVECs. Through the autoxidation of its catechol group and the coordination reaction of ferric ions, COFH can generate H2O2 with an appropriate concentration in the microenvironment to promote angiogenesis. At the same time, it also shows that compared with insufficient or excessive H2O2, stabilizing the H2O2 concentration in a certain range (H2O2 homeostasis) in the microenvironment is beneficial to better promoting angiogenesis.

[0146] Example 12

[0147] In this example, the myocardial regeneration and repair abilities of COFH, CO, COH, and COF prepared in Example 5 and Comparative Examples 1 to 3 were investigated. Set the following experimental groups:

[0148] COFH group: After anesthetizing the rats by respiration and opening the chest in the left lateral position under a ventilator, ligate the left anterior descending coronary artery to simulate myocardial infarction, and then perform intramyocardial injection of the gel precursor solution used to prepare COFH in Example 5, and then suture. The photos of the surgical process are as Figure 15 shown, Figure 15Figures A - D are the photos of the rat before thoracotomy, ligation of the left anterior descending coronary artery, injection of the gel precursor solution, and suture. On the 28th day after surgery, the cardiac function recovery was evaluated by echocardiogram and electrocardiogram. After the experiment ended on the 28th day after surgery, the rat heart tissue was removed and sectioned, and the cardiac fibrosis area was observed by Masson trichrome staining to evaluate the pathological remodeling of the infarcted area.

[0149] CO group: The operation was basically the same as that of the COFH group, except that the injected gel precursor liquid was replaced with the mixture B in Comparative Example 1.

[0150] COH group: The operation was basically the same as that of the COFH group, except that the injected gel precursor liquid was replaced with the mixture C in Comparative Example 2.

[0151] COF group: The operation was basically the same as that of the COFH group, except that the injected gel precursor liquid was replaced with the mixture D in Comparative Example 3.

[0152] Sham group: The rat was anesthetized by respiration and then thoracotomized on the left side under the ventilator and sutured (only thoracotomy and suture, without ligation or injection). On the 28th day after surgery, the cardiac function recovery was evaluated by echocardiogram and electrocardiogram. After the experiment ended on the 28th day after surgery, the rat heart tissue was removed and sectioned, and the cardiac fibrosis area was observed by Masson trichrome staining.

[0153] Saline group: The operation was basically the same as that of the COFH group, except that the injected COFH was replaced with an equal volume of normal saline.

[0154] The echocardiograms of the rats in each experimental group on the 28th day after surgery are as Figure 16 shown. The ejection fraction EF value of the COFH group was significantly higher than that of the CO group, COH group, COF group, and Saline group, indicating that the COFH group had a better effect on promoting cardiac function recovery after myocardial infarction. The electrocardiograms of the rats in each experimental group on the 28th day after surgery are as Figure 17 shown. Compared with the Saline group, CO group, COF group, and COH group, the electrocardiograms of the COFH group and Sham group showed no pathological Q waves, ST - segment changes, or other manifestations of myocardial injury or myocardial ischemia, and had the best cardiac function.

[0155] The results of Masson trichrome staining of the rat heart tissue in each experimental group on the 28th day after surgery are as Figure 18 shown. Compared with the CO group, COH group, COF group, and Saline group, the COFH group had the smallest myocardial fibrosis area and the lightest degree of fibrosis, indicating that COFH could reduce myocardial fibrosis after myocardial infarction and thus promote cardiac function recovery.

[0156] Example 13

[0157] In this example, the hydrogel for regulating the redox homeostasis of the microenvironment was prepared. The operation was basically the same as that in Example 5, except that in steps (1) and (2), HAD prepared in Example 2, OD prepared in Example 4, CCS, and Fe2(SO4)3 were used as raw materials, and the gel precursor solution was prepared according to the method in Example 5. In the gel precursor solution, the concentration of HAD was 30 mg / mL, the concentration of CCS was 50 mg / mL, the concentration of OD was 20 mg / mL, and the concentration of Fe2(SO4)3 was 0.01 mol / L.

[0158] Example 14

[0159] In this example, the hydrogel for regulating the redox homeostasis of the microenvironment was prepared. The operation was basically the same as that in Example 5, except that in steps (1) and (2), HAD prepared in Example 1, OD prepared in Example 4, CCS, and Fe(NO3)3 were used as raw materials, and the gel precursor solution was prepared according to the method in Example 5. In the gel precursor solution, the concentration of HAD was 50 mg / mL, the concentration of CCS was 80 mg / mL, the concentration of OD was 40 mg / mL, and the concentration of Fe(NO3)3 was 0.05 mol / L.

[0160] Example 15

[0161] In this example, the hydrogel for regulating the redox homeostasis of the microenvironment was prepared. The operation was basically the same as that in Example 5, except that HAD with a grafting rate of catechol groups of 15% was prepared by referring to the method in Example 1, OD with an oxidation degree of 40% was prepared by referring to the method in Example 4, and HAD and OD prepared in this example, CCS, and FeCl3 were used as raw materials, and the gel precursor solution was prepared according to the method in Example 5. In the gel precursor solution, the concentration of HAD was 40 mg / mL, the concentration of CCS was 70 mg / mL, the concentration of OD was 25 mg / mL, and the concentration of Fe(NO3)3 was 0.1 mol / L.

Claims

1. A hydrogel capable of regulating the redox homeostasis of the microenvironment, characterized in that, The polymer network of the hydrogel is formed by a hybrid cross-linking reaction of catechol-modified hyaluronic acid, carboxylated chitosan, oxidized dextran, and iron ions. The hybrid cross-linking reaction includes a Schiff base reaction between the amino groups of the carboxylated chitosan and the aldehyde groups of the oxidized dextran, an oxidative self-cross-linking reaction of the catechol-modified hyaluronic acid, a Michael addition reaction between the quinone groups of the oxidative self-cross-linked catechol-modified hyaluronic acid and the amino groups of the carboxylated chitosan, and a dynamic coordination reaction between the catechol groups of the oxidative self-cross-linked catechol-modified hyaluronic acid and the iron ions. Under physiological conditions, the catechol groups in the hydrogel undergo a self-oxidation reaction to generate H2O2. At the same time, the iron ions coordinated to the catechol groups in the hydrogel catalyze the decomposition of H2O2 into water and oxygen, thereby regulating the level of reactive oxygen species in the microenvironment. The hydrogel is formed by a hybrid cross-linking reaction of a gel precursor solution containing catechol-modified hyaluronic acid, carboxylated chitosan, oxidized dextran and a water-soluble iron salt. The catechol-modified hyaluronic acid is formed by grafting catechol groups onto the carboxyl groups of hyaluronic acid, and the grafting rate of catechol groups in the catechol-modified hyaluronic acid is 5% to 15%. The oxidized dextran is obtained by oxidizing some hydroxyl groups in dextran to aldehyde groups, and the degree of oxidation of the dextran is 15% to 40%. The preparation method of the hydrogel comprises the following steps: (1) Dissolving catechol-modified hyaluronic acid in water to obtain a catechol-modified hyaluronic acid solution; dissolving carboxylated chitosan in a PBS buffer solution with a pH value of 7.2 to 7.4 to obtain a carboxylated chitosan solution; dissolving oxidized dextran in an iron salt aqueous solution to obtain an oxidized dextran / iron salt mixed solution; (2) fully mixing the carboxylated chitosan solution with the oxidized dextran / iron salt mixed solution, and then fully mixing the resulting mixed solution with the catechol-modified hyaluronic acid solution to obtain a gel precursor solution; in the gel precursor solution, the concentration of the catechol-modified hyaluronic acid is 30-50 mg / mL, the concentration of the carboxylated chitosan is 50-80 mg / mL, the concentration of the oxidized dextran is 20-40 mg / mL, and the concentration of the iron ion is 0.01-0.05 mol / L; (3) The gel precursor solution was allowed to stand until it turned into a gel state, and the resulting hydrogel was repeatedly soaked in PBS buffer solution with a pH value of 7.2-7.4 and water to remove the iron ions not bound to the catechol groups, thereby obtaining a hydrogel that can regulate the redox homeostasis of the microenvironment.

2. The hydrogel capable of regulating the redox homeostasis of the microenvironment according to claim 1, wherein After freeze-drying, the hydrogel has an interconnected porous structure, wherein the average pore size of the porous structure is 50-100 μm and the porosity is 90%-95%.

3. The hydrogel capable of regulating the redox homeostasis of the microenvironment according to claim 1 or 2, characterized in that The carboxylated chitosan is formed by introducing carboxyl groups into the hydroxyl groups of chitosan through a carboxylation reaction, and the carboxylation degree of the carboxylated chitosan is at least 60%.

4. Use of the hydrogel capable of regulating microenvironment redox homeostasis according to claim 1 or 2 in the preparation of myocardial tissue repair materials.

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