Hydrogel capable of regulating and controlling redox steady state of microenvironment as well as preparation method and application of hydrogel

Abstract

The invention belongs to the field of compositions of biomedical materials and high-molecular compounds, and provides hydrogel capable of regulating and controlling the redox steady state of a microenvironment as well as a preparation method and application of the hydrogel. A macromolecular network of the hydrogel capable of regulating and controlling the redox steady state of the microenvironment is formed by catechol modified hyaluronic acid, carboxylated chitosan, oxidized dextran and iron ions through a hybridization cross-linking reaction; under physiological conditions, catechol groups in the hydrogel can be subjected to an autoxidation reaction to generate H2O2, and meanwhile, iron ions coordinated on the catechol groups in the hydrogel can catalytically decompose H2O2 into water and oxygen, so that the level of active oxygen in a microenvironment is adjusted. The hydrogel disclosed by the invention can realize bidirectional regulation and control on active oxygen in a pathological microenvironment after myocardial infarction, so that the redox steady state of the pathological microenvironment after myocardial infarction is maintained, and the regeneration and repair capacity of an existing hydrogel material with antioxidant capacity on myocardial tissues after myocardial infarction is improved.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials and compositions of polymer compounds, and relates to a hydrogel capable of regulating the redox homeostasis of a 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 each year. Survivors are also prone to secondary complications, which affect their quality of life and face a large economic burden. The main cause of myocardial infarction is the obstruction of the coronary arteries, which causes a decrease in blood flow to the myocardial cells, which in turn causes a large number of myocardial cell death accompanied by ischemia-reperfusion injury and inflammatory response, leading to maladaptive remodeling of the ventricle, and then developing into ischemic heart failure. After myocardial infarction, the body's self-repair mechanism will be rapidly activated to prevent further damage. However, due to the limited regenerative capacity of the adult heart, myocardial infarction often causes the death of myocardial cells or their 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 achieve the recovery of heart function, but may lead to further deterioration of heart function.

[0003] Including hydrogen peroxide (H 2 O 2 ) are considered to be substances that play a mediator role 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. Excessive production and accumulation of free radicals or their oxidation products can lead to heart failure or oxidative stress during ischemia-reperfusion. Although a moderate amount 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 ROS activating a variety of hypertrophic signaling kinases and transcription factors and mediating cell apoptosis. ROS also stimulates the proliferation of cardiac fibroblasts and activates matrix metalloproteinases (MMPs), leading to extracellular matrix remodeling. Excessive production of ROS in cardiomyocytes can directly or indirectly lead to damage to cellular ion channels and transporters. In other words, while ROS plays a biological signaling function in the body, it is also a double-edged sword. Excessive ROS can mediate cellular oxidative stress, activate signaling pathways such as inflammation and apoptosis, and lead to consequences such as cell apoptosis and tissue necrosis.

[0004] In view of the toxic effects of ROS, existing research on related tissue engineering materials is mainly focused on promoting tissue regeneration by removing ROS in the microenvironment. However, due to ignoring the necessity of ROS in regenerative signal transduction, simple ROS removal or antioxidant intervention may interfere with the initiation and maintenance of regenerative signals, and have an adverse effect on tissue regeneration. For myocardial infarction, after myocardial infarction, the concentration level of ROS in the early microenvironment is high, which will stimulate signal pathways such as inflammation and apoptosis, while the appropriate concentration of ROS in the mid-to-late microenvironment can prolong the maintenance time of the repair phenotype of myocardial epithelial cells. Therefore, if the ROS concentration level in the microenvironment can be reduced in the early stage after myocardial infarction through tissue engineering materials, and the ROS concentration in the microenvironment can be maintained at a low level in the mid-to-late stage, it will be beneficial to improve ischemic heart failure after myocardial infarction and promote myocardial repair. At present, 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 therefore have limited regenerative repair ability for tissues after myocardial infarction. How to control the ROS concentration in the microenvironment after myocardial infarction to a level where it can play a positive role through tissue engineering materials, so as to better promote the repair of tissues after myocardial infarction, is a major challenge facing this field. Summary of the invention

[0005] In view of the problem that the existing injectable hydrogel materials with antioxidant capacity can remove ROS but are difficult to achieve accurate 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 and a preparation method and application thereof, so as to achieve bidirectional regulation of ROS in the pathological microenvironment after myocardial infarction, which can not only quickly remove excess H 2 O 2 , and also plays a role in producing a certain physiological concentration of H 2 O 2 The effect of hydrogel materials with antioxidant capacity on the regeneration and repair of myocardial tissue after myocardial infarction is maintained, thereby maintaining the redox homeostasis of the pathological microenvironment after myocardial infarction and improving the ability of existing hydrogel materials with antioxidant capacity to repair myocardial tissue after myocardial infarction.

[0006] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

[0007] A hydrogel capable of regulating the redox homeostasis of a microenvironment, wherein a polymer network of the hydrogel is formed by a hybrid crosslinking reaction of catechol-modified hyaluronic acid, carboxylated chitosan, oxidized dextran and iron ions, wherein the hybrid crosslinking reaction comprises a Schiff base reaction between an amino group of carboxylated chitosan and an aldehyde group of oxidized dextran, an oxidative self-crosslinking reaction of the catechol-modified hyaluronic acid, a Michael addition reaction between a quinone group of the catechol-modified hyaluronic acid after oxidative self-crosslinking and an amino group of the carboxylated chitosan, and a dynamic coordination reaction between a catechol group of the catechol-modified hyaluronic acid after oxidative self-crosslinking and iron ions; under physiological conditions, the catechol groups in the hydrogel undergo a self-oxidation reaction to generate H 2 O 2 At the same time, the iron ions coordinated to the catechol groups in the hydrogel will convert H 2 O 2 It catalyzes decomposition into water and oxygen, thereby regulating the level of reactive oxygen species in the microenvironment.

[0008] In the technical solution of the hydrogel capable of regulating the redox homeostasis of 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.

[0009] Furthermore, in the technical solution of the 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. Furthermore, 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 above-mentioned technical solution of the hydrogel capable of regulating the redox homeostasis of the microenvironment, the water-soluble iron salt may be ferric chloride, ferric sulfate or ferric nitrate.

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

[0012] In the technical solution of the hydrogel capable of regulating the redox homeostasis of the microenvironment, the catechol-modified hyaluronic acid is formed by grafting a catechol group onto the carboxyl group of hyaluronic acid, and the grafting rate of the catechol group in the catechol-modified hyaluronic acid is 5% to 15%. Furthermore, the molecular weight of the sodium hyaluronate used as the modification basis is 8 to 2000 kDa, preferably 200 to 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 capable of regulating the redox homeostasis of the microenvironment, the catechol-modified hyaluronic acid can be prepared by referring to the prior art. A feasible method for preparing the catechol-modified hyaluronic acid is as follows:

[0015] Add N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide aqueous solution to the sodium hyaluronate aqueous solution, then add N-hydroxysuccinimide aqueous solution, stir and react at room temperature for 2-8 hours under nitrogen protection, then add dopamine hydrochloride aqueous solution dropwise to the obtained reaction solution under light-proof conditions, stir and react at room temperature for 12-48 hours under light-proof and nitrogen protection, and control the pH value to 5-6 during the two stirring reactions; remove 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 ratio of each reaction raw material will affect the grafting rate of the catechol group in the catechol-modified hyaluronic acid. In actual application, the ratio of each raw material can be determined according to the application requirements and with reference to the prior art. For example, the molar ratio of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, N-hydroxysuccinimide, dopamine hydrochloride and the carboxyl group on sodium hyaluronate can be controlled to be (3~6):(1~5):(1~5):1. Usually, the concentration of the sodium hyaluronate aqueous solution can be controlled to be 10~50 mg / mL.

[0017] In the technical solution of the hydrogel capable of regulating the redox homeostasis of the microenvironment, the oxidized dextran is obtained by oxidizing part of the hydroxyl groups in the dextran into aldehyde groups, the oxidation degree of the dextran is 15% to 40%, and the structure of the oxidized dextran is shown in formula (II):

[0018] (II).

[0019] In the technical scheme of the hydrogel capable of regulating the redox homeostasis of the microenvironment, the oxidized dextran can be prepared by referring to the prior art. A feasible method for preparing oxidized dextran is: adding sodium periodate aqueous solution to the dextran aqueous solution under light-proof conditions, stirring and reacting at room temperature for 2 to 5 hours under light-proof conditions after the addition is completed, then removing the unreacted raw materials, and freeze-drying to obtain. Generally, the molecular weight of the dextran used as the basis for oxidative modification is 100 to 1000 kDa.

[0020] In the technical solution of the hydrogel capable of regulating the redox homeostasis of the microenvironment, 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 having a pH value of 7.2 to 7.4 to obtain a carboxylated chitosan solution; dissolving oxidized dextran in an aqueous solution of an iron salt to obtain an oxidized dextran / iron salt mixed solution;

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

[0025] (3) The gel precursor solution is allowed to stand until it turns into a gel state, and the resulting hydrogel is repeatedly soaked in PBS buffer and water with a pH value of 7.2 to 7.4 to remove iron ions that are 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 preparation method, the reason for adopting the 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 obtained 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 reducing or avoiding as much as possible the reaction between the aldehyde groups of the oxidized dextran and 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 preparation method, when the obtained hydrogel is repeatedly soaked in PBS buffer with a pH value of 7.2 to 7.4 and water, the hydrogel is first soaked in PBS buffer 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 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, and the average pore size of the porous structure is about 71 μm and the porosity is about 92%. This porous structure is very beneficial to the transmission of nutrients and oxygen, cell migration, penetration and new tissue growth.

[0030] (2) The gelation time of the hydrogel of the present invention is short, not exceeding 2 min, and it has better mechanical properties after gelation, which is conducive to rapid gelation and exertion 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 to maintain the stability of its own structure in the myocardium with a complex mechanical environment, so as to exert its effect more lastingly.

[0031] (3) The hydrogel of the present invention has excellent H 2O 2 Bidirectional regulation capability. For example, COFH prepared in Example 5 can generate H 2 O 2 After 16 days, H 2 O 2 The concentration gradually increased from 0 to about 34 μmol / L; at the same time, COFH could remove H 2 O 2 After 4 days, H 2 O 2 The concentration dropped rapidly from 1340 μmol / L to about 525 μmol / L, and then slowly decreased. By the 16th day, H 2 O 2 The concentration was reduced to about 53 μmol / L. This is beneficial for clearing excess H in the microenvironment in the early stage of myocardial infarction. 2 O 2 , avoiding tissue damage caused by oxidative stress, and also helping to maintain a low and appropriate concentration of H in the late stage of myocardial infarction 2 O 2 , continuously providing reactive oxygen 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 vascular endothelial cells (HUVECs), and can meet the safety requirements of injectable hydrogels for minimally invasive intervention after myocardial infarction.

[0033] (5) The hydrogel of the present invention has a good ability to promote HUVECs angiogenesis. For example, COFH prepared in Example 5 can produce H at an appropriate concentration level in the microenvironment through the self-oxidation of its catechol group and the coordination reaction of trivalent iron ions. 2 O 2 To promote angiogenesis of HUVECs.

[0034] (6) The hydrogel of the present invention has a good ability to promote myocardial regeneration and repair after myocardial infarction. For example, the COFH gel precursor solution used to prepare the COFH gel in Example 5 was injected into the myocardium of rats with myocardial infarction to form COFH. After 28 days of myocardial repair in the myocardium of the rats with myocardial infarction, the results of the rats' echocardiogram, electrocardiogram and Masson trichrome staining of the rats' heart tissue showed 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 use of the hydrogel capable of regulating the redox homeostasis of the microenvironment in preparing myocardial tissue repair materials, especially the use of the hydrogel capable of regulating the redox homeostasis of the microenvironment in preparing myocardial tissue repair materials after myocardial infarction.

[0036] Compared with the prior art, the technical solution provided by the present invention produces 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 a 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 undergo a self-oxidation reaction to generate H 2 O 2 At the same time, the iron ions coordinated to the catechol groups in the hydrogel will convert H 2 O 2 Compared with the existing hydrogel materials with antioxidant capacity, the hydrogel provided by the present invention can achieve two-way regulation of ROS in the pathological microenvironment after myocardial infarction, which can not only quickly remove the excessive H in the myocardial microenvironment in the early stage after myocardial infarction, but also effectively regulate the level of reactive oxygen in the microenvironment. 2 O 2 It also plays a role in maintaining a low concentration of H in the myocardial microenvironment in the middle and late stages of myocardial infarction. 2 O 2 It can solve the problem that the existing hydrogels with antioxidant capacity mainly play the role of removing ROS and are difficult to achieve precise adaptation to the redox state of the myocardial pathological microenvironment, and can enhance 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 described in the present invention forms a multiple cross-linked 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 the body, so as to better match the tissue repair process. In addition, the rich catechol functional groups in the hydrogel can give it good tissue adhesion properties, and after being injected into the tissue to form a gel, it can be fixed in the injection area, thereby playing a role in long-term regulation of the redox homeostasis of the pathological microenvironment. The above characteristics provide favorable conditions for the hydrogel described in the present invention to promote myocardial tissue repair after myocardial infarction.

[0039] 3. The present invention has been verified through experiments that the hydrogel of the present invention has good cell compatibility and can meet the safety requirements of injectable hydrogels for minimally invasive intervention; the hydrogel of the present invention can produce H at an appropriate concentration level in the microenvironment through the self-oxidation of its catechol groups and the coordination reaction of trivalent iron ions. 2 O 2To promote the angiogenesis of HUVECs; the gel precursor solution used to prepare the hydrogel of the present invention was injected into the myocardium of rats with myocardial infarction to form a hydrogel. After 28 days of repair in the myocardium of the rats with myocardial infarction, the results of the rats' echocardiogram, electrocardiogram and Masson trichrome staining of the rats' heart tissue showed 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 use of the hydrogel capable of regulating the redox homeostasis of the microenvironment in the preparation of myocardial tissue repair materials, so as to remove excess H in the microenvironment in the early stage after myocardial infarction. 2 O 2 This can reduce or avoid myocardial tissue damage caused by oxidative stress and reduce H in the microenvironment in the middle and late stages of myocardial infarction. 2 O 2 The concentration is maintained at a low level, thereby continuously providing active oxygen signals to stimulate angiogenesis. The present invention can solve the problem that the prior art repairs myocardium by simply removing ROS or intervening with antioxidants, but has poor regenerative repair performance on myocardial tissue. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

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

[0046] Figure 7 Figure A shows the changes in the storage modulus and loss modulus of the gel precursor solution used to prepare COFH, CO, COH and COF over time. Figure 7 Figure B shows the storage modulus and loss modulus of COFH, CO, COH and COF under different strain conditions.

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

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

[0049] Fig.10 Figure A shows the hydrogel bidirectionally regulating H 2 O 2 Schematic diagram of the concentration level principle, Fig.10 Figure B shows the H production in vitro by each experimental group. 2 O 2 How the ability of Fig.10 Figure C shows the results of H clearance in vitro by each experimental group. 2 O 2 How capabilities change over time.

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

[0051] Fig.12 The growth status and distribution of COFH, CO, COH and COF cells after co-culture with HUVECs for 1 day and 3 days.

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

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

[0054] Fig.15 These are photos of the surgical process, in which A to D are photos of the rat's chest opening, ligation of the left anterior descending coronary artery, injection of gel precursor solution, and before suturing.

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

[0056] Fig.17 This is the electrocardiogram of rats in each experimental group on the 28th day after surgery.

[0057] Fig.18 This is the result of Masson trichrome staining of cardiac tissues of rats in each experimental group on the 28th day after surgery. DETAILED DESCRIPTION

[0058] The following examples further illustrate the hydrogel with adjustable microenvironment redox homeostasis and its preparation method and application provided by the present invention. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. The technical personnel in the relevant field make some non-essential improvements and adjustments to the present invention according to the above invention content for specific implementation, which still falls within the scope of protection of the present invention.

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

[0060] (III).

[0061] Example 1

[0062] In this embodiment, catechol-modified hyaluronic acid (HAD) is prepared in the following steps:

[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 the round-bottom flask was evacuated, it was stirred at room temperature under nitrogen protection until HA was completely dissolved to obtain a 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) Add EDC solution to HA solution, then add NHS solution, stir and react at room temperature for 2 hours under nitrogen protection. After the reaction is completed, add DOPA solution dropwise to the resulting reaction solution under light-shielding conditions, and stir and react at room temperature for 12 hours under light-shielding and nitrogen protection. During the two stirring reactions, the pH value was controlled to be 5.5. In this step, the molar ratio of EDC, NHS, DOPA and the carboxyl group of HA was controlled to be 4:2:1:1.

[0065] (3) The reaction solution obtained in step (2) was centrifuged at 2000 rpm for 5 min, and the supernatant was placed in a dialysis bag (MWCO: 8-14 kDa), placed in a dialysate with a pH of 5.0 (obtained by adjusting the pH value of ultrapure water to 5.0 with hydrochloric acid), and dialyzed for 3 days at room temperature and a stirring condition of 400 rpm in the dark; during the dialysis period, the dialysate was replaced every 6 h; after the dialysis was completed, the liquid in the dialysis bag was freeze-dried to obtain HAD, which was sealed in a bag and stored in the dark for later use.

[0066] The raw material HA and the prepared HAD used in this example were tested by nuclear magnetic resonance hydrogen spectrum, and the results are as follows: Figure 1 As shown in FIG. 1 , the grafting rate of catechol groups in HAD was calculated by integrating the nuclear magnetic resonance hydrogen spectrum, and the result was 10%. The HA, DOPA and HAD prepared in this example were tested by infrared spectroscopy, and the results were as follows: Figure 3 As shown. Figure 1 and 3 It can be seen that the characteristic peak of the catechol group (δ = 6.8-7.2 ppm) appeared in the hydrogen spectrum of HAD, indicating that DOPA was successfully grafted onto the molecular chain of HA. -1 A strong absorption peak appeared near 1235 cm-1, which is related to the stretching vibration of the carboxyl group (C=O), indicating that HA contains a large number of carboxyl groups. -1 A strong absorption peak appeared at 900-1500 cm, which is related to the CN stretching vibration, which is a typical characteristic absorption peak of the amino group in DOPA. -1 region, and also benzene rings and amino groups (-NH 2 ) related absorption peaks. In the infrared spectrum of HAD, at 1720 cm -1 The absorption peak near 1235 cm is stronger than that of HA, which may be related to the introduction of DOPA to enhance the conjugation effect of carboxyl groups or the intermolecular hydrogen bonding effect. -1 The absorption peak at is related to the amino group of dopamine, which further indicates that DOPA is successfully grafted onto the molecular chain of HA through the amidation reaction. The structure of HAD is shown in formula (I):

[0067] (I).

[0068] Example 2

[0069] In this embodiment, catechol-modified hyaluronic acid (HAD) is prepared in the following steps:

[0070] (1) Add HA (Mw = 340 kDa) into a round-bottom flask containing 0.01 mol / L PBS buffer at a pH of 5.5. Evacuate the round-bottom flask and stir at room temperature under nitrogen until HA is completely dissolved to obtain a 16.7 mg / mL HA solution. Dissolve EDC in 0.01 mol / L PBS buffer at a pH of 5.5 to obtain a 300 mg / mL EDC solution. Dissolve NHS in 0.01 mol / L PBS buffer at a pH of 5.5 to obtain a 125 mg / mL NHS solution. Dissolve DOPA in 0.01 mol / L PBS buffer at a pH of 5.5 to obtain a 250 mg / mL DOPA solution.

[0071] (2) Add EDC solution to HA solution, then add NHS solution, stir and react at room temperature for 2 hours under nitrogen protection. After the reaction is completed, add DOPA solution dropwise to the resulting reaction solution under light-proof conditions, and stir and react at room temperature for 12 hours under light-proof and nitrogen protection. During the two stirring reactions, the pH value was controlled to be 5.5. In this step, the molar ratio of EDC, NHS, DOPA and the carboxyl group of HA was controlled to be 3:1:1:1.

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

[0073] The HAD prepared in this example was tested by hydrogen nuclear magnetic resonance spectrum and the grafting rate of catechol groups in HAD was calculated by hydrogen nuclear magnetic resonance spectrum integration, and the result was 6%.

[0074] Example 3

[0075] Dextran (Dex) has a large number of hydroxyl groups on its molecular chain, which are easily oxidized to aldehyde groups with higher reaction activity under the action of a strong oxidant. In this embodiment, Dex is oxidized to prepare oxidized dextran (OD), and the steps are as follows:

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

[0077] (2) Slowly add a 30 mg / mL sodium periodate aqueous solution to the Dex solution under light-proof conditions. After the addition is complete, stir the mixture at room temperature under light-proof conditions for 2 h. The amount of sodium periodate used is controlled to be 16.6% of the mass of Dex.

[0078] (3) The reaction solution obtained in step (2) was placed in a dialysis bag (MWCO: 3.5 kDa) and dialyzed in ultrapure water for 3 days; during the dialysis period, the dialysate was replaced every 6 hours; after the dialysis was completed, the liquid in the dialysis bag was freeze-dried to obtain the OD, which was then sealed in a bag and stored away from light for later use.

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

[0080] (II).

[0081] Example 4

[0082] In this example, oxidized dextran (OD) was prepared in the following steps:

[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 a 50 mg / mL sodium periodate aqueous solution to the Dex solution under light-proof conditions. After the addition is complete, stir the mixture at room temperature under light-proof conditions for 2 h. The amount of sodium periodate used is controlled to be 28.2% of the mass of Dex.

[0085] (3) The reaction solution obtained in step (2) was placed in a dialysis bag (MWCO: 3.5 kDa) and dialyzed in ultrapure water for 3 days; during the dialysis period, the dialysate was replaced every 6 hours; after the dialysis was completed, the liquid in the dialysis bag was freeze-dried to obtain the OD, which was then sealed in a bag and stored away from light for later use.

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

[0087] Example 5

[0088] In this embodiment, a hydrogel capable of regulating the redox homeostasis of a microenvironment is prepared by the following steps:

[0089] (1) Dissolve the HAD prepared in Example 1 in ultrapure water to obtain a HAD solution with a concentration of 80 mg / mL. Dissolve carboxylated chitosan (CCS) in 0.01 mol / L PBS buffer with a pH of 7.4 to obtain a CCS solution with a concentration of 240 mg / mL. Dissolve the OD prepared in Example 3 in FeCl 3 In aqueous solution, the OD concentration was 120 mg / mL, FeCl 3 OD / FeCl concentration of 0.04 mol / L 3 Mix the solution.

[0090] (2) Mix CCS solution with OD / FeCl 3 The mixed solutions were mixed in equal volumes to obtain a mixed solution A, in which the concentration of CCS was 120 mg / mL, the concentration of OD was 60 mg / mL, and the concentration of FeCl 3 The concentration is 0.02 mol / L.

[0091] Then, 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 FeCl 3 The concentration is 0.01mol / L.

[0092] (3) The gel precursor solution was quickly transferred to the mold and allowed to react at room temperature for 10 min. The gel precursor solution was then converted into a hydrogel state. The resulting hydrogel was placed in a 0.01 mol / L PBS buffer solution with a pH of 7.4 and soaked at room temperature for 24 h. It was then transferred to ultrapure water and soaked at room temperature for 2 h. The aforementioned operation of soaking the hydrogel in PBS buffer and ultrapure water was repeated three times to remove Fe2+ that was not bound to the catechol group. 3+The obtained hydrogel was transferred to a PBS buffer with a concentration of 0.01 mol / L and a pH value of 7.4 and immersed in it at room temperature for 2 h to obtain a hydrogel with adjustable microenvironment redox homeostasis, which was recorded as COFH.

[0093] Comparative Example 1

[0094] In this comparative example, the injectable single network hydrogel was prepared by the following steps:

[0095] (1) CCS was dissolved in 0.01 mol / L PBS buffer with a pH 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 thoroughly 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 the mold and allowed to react at room temperature for 10 min. The gel precursor solution was then converted into a hydrogel state. The resulting hydrogel was placed in a 0.01 mol / L PBS buffer solution with a pH value of 7.4 and soaked at room temperature for 24 h. The mixture was then transferred to ultrapure water and soaked at room temperature for 2 h. The aforementioned operation of soaking the hydrogel in PBS buffer and ultrapure water was repeated three times. The resulting hydrogel was transferred to a 0.01 mol / L PBS buffer solution with a pH value of 7.4 and soaked at room temperature for 2 h to obtain an injectable single network hydrogel, which was recorded as CO.

[0098] Comparative Example 2

[0099] In this comparative example, the injectable double network hydrogel was prepared in the following steps:

[0100] (1) 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 0.01 mol / L PBS buffer with a pH value of 7.4 to obtain a CCS solution with a concentration of 180 mg / mL. 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, CCS solution and 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) The mixed liquid C was quickly transferred to the mold and allowed to react at room temperature for 10 min. The gel precursor was then converted into a hydrogel state. The resulting hydrogel was placed in a 0.01 mol / L PBS buffer solution with a pH value of 7.4 and soaked at room temperature for 24 h. It was then transferred to ultrapure water and soaked at room temperature for 2 h. The aforementioned operation of soaking the hydrogel in PBS buffer and ultrapure water was repeated three times. The resulting hydrogel was transferred to a 0.01 mol / L PBS buffer solution with a pH value of 7.4 and soaked at room temperature for 2 h to obtain an injectable double network hydrogel, which was recorded as COH.

[0103] Comparative Example 3

[0104] In this comparative example, an injectable single-network trivalent iron ion composite hydrogel was prepared in the following steps:

[0105] (1) CCS was dissolved in 0.01 mol / L PBS buffer with a pH of 7.4 to obtain a CCS solution with a concentration of 120 mg / mL. 3 The solution was obtained with an OD concentration of 60 mg / mL and FeCl 3 OD / FeCl at a concentration of 0.02 mol / L 3 Mix the solution.

[0106] (2) Mix CCS solution with OD / FeCl 3 The mixed solutions were thoroughly mixed to obtain mixed solution D, in which the concentration of CCS was 60 mg / mL, the concentration of OD was 30 mg / mL, and the concentration of FeCl 3 The concentration is 0.1 mol / L.

[0107] (3) The mixed solution D was quickly transferred to the mold and allowed to react at room temperature for 10 min. The gel precursor solution was converted into a hydrogel state. The obtained hydrogel was placed in 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 24 h. Then, it was transferred to ultrapure water and soaked at room temperature for 2 h. The above operation of soaking the hydrogel in PBS buffer and ultrapure water was repeated three 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 soaked at room temperature for 2 h to obtain an injectable single-network trivalent iron ion composite hydrogel, recorded as COF.

[0108] Example 6

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

[0110] COFH, CO, COH and COF were freeze-dried and subjected to SEM analysis. Figure 5 The following are SEM images of freeze-dried CO, COF, COH and COFH at different magnifications. Figure 5 It can be seen that freeze-dried CO, COF, COH and COFH all have interconnected porous structures, which are beneficial to the delivery of cytokines and hydrogen peroxide. Among them, the average pore size of freeze-dried CO is about 178 μm and the porosity is about 83%, the average pore size of freeze-dried COF is about 165 μm and the porosity is about 86%, the average pore size of freeze-dried COH is about 122 μm and the porosity is about 89%, and the average pore size of freeze-dried COFH is about 71 μm and the porosity is about 92%. Relatively speaking, freeze-dried COFH has a denser pore structure and 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, the Michael addition reaction between the quinone group of oxidative self-crosslinking HAD and the residual amino group of CCS and Fe 3+ This is caused by a coordination reaction that forms a multiple cross-linked network.

[0111] CO, COF, COH and COFH were freeze-dried and tested by infrared spectroscopy. Figure 6 As shown in the figure, compared with CO and COF, COH and COFH have a higher concentration of 1033 cm due to the oxidation of HAD. -1 and 1082 cm -1 The peak intensity at showed an obvious change, which may be related to the introduction of dopamine enhancing the stretching vibration of C-OH bond and CO bond, which confirms that HAD plays a "bridge" role in the multi-cross-linked network of COFH.

[0112] Example 7

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

[0114] According to the operations in Example 5 and Comparative Examples 1 to 3, gel precursor solutions, mixed solutions B, mixed solutions C and mixed solutions D were prepared, i.e., gel precursor solutions for preparing COFH, CO, COH and COF were prepared, and their storage modulus (G') and loss modulus (G'') were tested over time. The results are shown in FIG. Figure 7 The storage modulus (G') and loss modulus (G'') of COFH, CO, COH and COF prepared in Example 5 and Comparative Examples 1 to 3 were tested under different strain conditions. The results are shown in FIG. Figure 7 As shown in Figure B.

[0115] The gelation time is an important parameter of injectable hydrogels. Figure 7 As shown in Figure A, for COFH, CO, COH and COF, the storage modulus and loss modulus of their gel precursors fluctuated slightly at the beginning of the test, then increased rapidly and finally reached a balance state. The initial storage modulus was lower than the loss modulus, and then the storage modulus increased rapidly and exceeded the loss modulus. The formation of the intersection of the storage modulus and the loss modulus marked the gelation of the hydrogel, and the time of the intersection was the gelation time. 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 embodiment, according to the operations of Example 5 and Comparative Examples 1 to 3, four hydrogels of COFH, CO, COH and COF 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 compressive stress-strain tests on each hydrogel sample. Four parallel samples were set for each hydrogel sample. The displacement rate used in the test was 1 mm / min until the hydrogel sample was destroyed. The storage modulus (G') and loss modulus (G'') of each hydrogel at different frequencies and the compressive stress-strain curve of each hydrogel were obtained, as shown in the figure below. Figure 8 , 9 As shown. Figure 8 It can be seen that the storage modulus and loss modulus of each hydrogel increase with the increase of frequency, indicating that each hydrogel exhibits stronger elastic and viscous response under high-frequency conditions. Among them, COFH has the highest storage modulus. As the frequency increases from 2 Hz to 10 Hz, the storage modulus of COFH increases from about 43 KPa to about 67 KPa, showing the strongest rigidity and elasticity. Fig. 9 It can be seen that COFH has the highest maximum compressive strength, reaching 38 ± 5 KPa, which is mainly due to the multiple cross-linked network of COFH enhancing its mechanical properties.

[0119] Example 9

[0120] In this example, the COFH, CO, COH and COF prepared in Example 5 and Comparative Examples 1 to 3 were tested for their in vitro 2 O 2 The following experimental groups are set up:

[0121] COFH group and COFH+H2 O 2 Group: To test the ability of COFH to generate H in vitro 2 O 2 According to the method of Example 5, a cylindrical mold was used to prepare 70 μL of COFH, freeze-dried, and the freeze-dried COFH was placed in 300 μL PBS buffer (pH = 7.4) at a temperature of 37 ° C, a relative humidity of 95% and 5% CO 2 The samples were placed in an incubator for 16 days, and the Amplex Red method was used to measure H 2 O 2 To test the ability of COFH to scavenge H 2 O 2 According to the method of Example 5, a cylindrical mold was used to prepare 70 μL of COFH, freeze-dried, and the freeze-dried COFH was placed in 300 μL of H 2 O 2 PBS buffer (pH = 7.4, H 2 O 2 concentration of 1340 μmol / L) at 37°C, 95% relative humidity and 5% CO 2 The samples were placed in an incubator for 16 days, and the Amplex Red method was used to measure H 2 O 2 concentration.

[0122] CO group and CO+H 2 O 2 Group: Operation and COFH group and COFH+H 2 O 2 The groups are basically the same, the only difference is that COFH is replaced by CO prepared according to the method of Comparative Example 1 with the same shape and volume.

[0123] COH group and COH+H 2 O 2 Group: Operation and COFH+H 2 O 2 The operation of the COFH group is basically the same, except that COFH is replaced by COH of the same shape and volume prepared according to the method of Comparative Example 2.

[0124] COF group or COF+H 2 O 2 Group: Operation with COFH group and COFH+H 2 O 2 The operation of the groups is basically the same, the only difference is that COFH is replaced by COF prepared according to the method of Comparative Example 3 with the same shape and volume.

[0125] Control group or Control+H 2 O 2 Group: Operation with COFH group and COFH+H 2 O 2 The operation of the two groups was basically the same, except that no PBS buffer or H 2 O 2 PBS buffer (pH = 7.4, H 2 O 2 concentration of 1340 μmol / L).

[0126] Fig.10 Figure B shows the H production in vitro by each experimental group. 2 O 2 How the ability of Fig.10 Figure C shows the results of H clearance in vitro by each experimental group. 2 O 2 The ability to generate H in vitro changes over time. 2 O 2 In terms of the ability to 2 O 2 The concentration gradually increased from 0 to about 34 μmol / L; for in vitro clearance of H 2 O 2 In terms of capacity, after 4 days, COFH+H 2 O 2 Group H 2 O 2 The concentration dropped rapidly from 1340 μmol / L to about 525 μmol / L, and then slowly decreased. On the 16th day, COFH+H 2 O 2 Group H 2 O 2 The concentration dropped to about 53 μmol / L. Compared with COH, which maintains a higher H 2 O 2 In terms of the release level, the H produced by COFH 2 O 2 The Fe2+ coordinated to the catechol group of COFH 3+ Rapid catalytic decomposition into water and oxygen is beneficial for COFH to remove excess H in the microenvironment in the early stage of myocardial infarction. 2 O 2 , avoiding tissue damage caused by oxidative stress. Compared with CO and COF, which can only maintain very low H 2 O 2 In terms of the release level, the catechol group of COFH undergoes self-oxidation under physiological conditions to produce H2 O 2 , which can maintain a low and appropriate level of H in the late stage of myocardial infarction 2 O 2 , continuously providing reactive oxygen signals to stimulate myocardial angiogenesis. This shows that compared with CO, COH and COF, COFH has a better bidirectional regulation of H 2 O 2 In addition, the multiple cross-linked network of COFH gives it higher mechanical strength and structural stability, which is conducive to the long-term sustained release of a certain concentration of H by COFH covering the interface area of ​​MI. 2 O 2 , thereby promoting the repair of myocardial tissue and function.

[0127] Fig.10 Figure A shows the hydrogel bidirectionally regulating H 2 O 2 Schematic diagram of the principle of concentration level, as shown in the figure, the catechol groups in the hydrogel (such as COFH) of the present invention undergo a self-oxidation reaction to convert the catechol groups into quinone or semiquinone structures to generate H 2 O 2 At the same time, H 2 O 2 The ferrous ions coordinated to the catechol groups in the hydrogel will be catalytically decomposed into water and oxygen, and the quinone or semiquinone structure will be converted into a catechol structure, that is, H 2 O 2 The generation and H 2 O 2 decomposition process, forming a dynamic balance and regulating H 2 O 2 The concentration level can regulate the ROS level. 2 O 2 The changes in concentration levels play different roles at different stages after myocardial infarction. In the early stage of myocardial infarction, it plays a role in clearing excess H in the microenvironment. 2 O 2 , to avoid tissue damage caused by oxidative stress, and to maintain a low and appropriate level of H in the late stage of myocardial infarction 2 O 2 , which plays a role in continuously providing reactive oxygen signals to stimulate myocardial angiogenesis.

[0128] Example 10

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

[0130] According to the methods of Example 5 and Comparative Examples 1 to 3, COFH, CO, COH and COF were prepared using molds with a diameter of 8 mm and a height of 3 mm, and then a hydrogel-HUVECs two-dimensional co-culture model was constructed. Specifically, each hydrogel was soaked in a 75% ethanol aqueous solution for 24 h, and then transferred to a sterile PBS buffer (0.01 mol / L, pH = 7.4) and soaked for 24 h. 10 μL of a cell suspension containing 50,000 HUVECs was added dropwise to the upper surface of each hydrogel treated as described above, and the mixture was incubated in an incubator (5% CO 2 , 20%O 2 ) for 2 h, then 10 μL of cell suspension containing 50,000 HUVECs was added to the lower surface of each hydrogel and incubated for another 2 h. The culture medium was supplemented with high-glucose DMEM medium containing 10% serum and 1% dual antibody and then incubated in the incubator for 1 day and 3 days, respectively. The high-glucose DMEM medium containing 10% serum and 1% dual antibody was replaced every 24 h during the incubation period.

[0131] After the incubation time reached the required time, each hydrogel was taken out and the proliferation of HUVECs in each hydrogel was tested using the CCK-8 method. During the test, the absorbance was measured at a wavelength of 450 nm. The results were as follows: Fig.11 After the incubation time reached the required level, each hydrogel was removed, and the cells were washed twice with PBS buffer. The washed cells were stained with 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 confocal laser scanning microscopy (CLSM). The results are shown in Fig.12 shown.

[0132] HUVECs are important cells involved in the whole process of myocardial angiogenesis. Fig.11 It can be seen that the absorbance value of COFH after co-culture with HUVECs for 3 days was the highest, and it was significantly different from CO, COF and COH. Fig.12 It can be seen that HUVECs grew well after co-culture with each hydrogel for 1 day and 3 days. Compared with CO, COF and COH, COFH had better cell proliferation and higher live cell ratio after co-culture with HUVECs for 3 days. The above experimental results show that COFH has higher cell compatibility with HUVECs and better ability to promote HUVECs proliferation, which can meet the safety requirements of injectable hydrogels for minimally invasive intervention after myocardial infarction.

[0133] Embodiment 11

[0134] In this example, the ability of COFH, CO, COH and COF prepared in Example 5 and Comparative Examples 1 to 3 to promote HUVECs angiogenesis was evaluated.

[0135] HUVECs at the fifth generation were selected for tube formation experiments. The matrix gel was taken out of the -20 ℃ refrigerator and placed in a 4 ℃ refrigerator until it was completely thawed. Use a pre-cooled pipette tip to mix the matrix gel evenly. Place the 96-well plate on ice and add 50 μL of completely thawed matrix gel to each well to avoid bubbles. Then, place the 96-well plate in a cell culture incubator (37 ℃, 5% CO 2 ) for 30 min to ensure that the matrix gel forms a gel base. The fifth-generation HUVECs were digested with trypsin, centrifuged, and the cell suspension concentration was adjusted to 600,000 / mL using serum-free 1640 medium containing 1% double antibody.

[0136] Prepare COFH, CO, COH and COF extracts and store them at 4 °C for future use. The preparation of COFH extract is used as an example. Place COFH hydrogel in a 6-well plate and add 4 mL of serum-free 1640 medium containing 1% double antibody to each well. Place the plate at 37 °C and 5% CO 2 The cells were cultured in an incubator for 3 days. Then, the culture medium extract was extracted and the H 2 O 2 concentration, and diluted to H with serum-free 1640 medium containing 1% double antibody 2 O 2 The concentration was 10 μmol / L, and the diluted COFH extract was obtained, and the dilution multiple was recorded.

[0137] Set up the following experimental groups:

[0138] COFH group: The diluted COFH extract was added into a 96-well plate, and then the HUVECs cell suspension was added.

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

[0140] COH group: COH extract was diluted with serum-free 1640 medium containing 1% double antibody according to the dilution multiple when preparing the diluted COFH extract. The obtained diluted COH extract was added to a 96-well plate, and then the HUVECs cell suspension was added.

[0141] COFH+CAT group: According to the dilution multiple when preparing the diluted COFH extract, the COFH extract was diluted with serum-free 1640 culture medium containing 1% double antibody, and the diluted COFH extract was added to a 96-well plate, and then catalase (CAT) was added at a rate of 1000 U / well, and then the HUVECs cell suspension was added.

[0142] H 2 O 2 Group: Serum-free 1640 medium containing 1% double antibody was added to the 96-well plate, and then H 2 O 2 To 96-well plate 2 O 2 The concentration was 10 μmol / L, and then HUVECs cell suspension was added.

[0143] Ctrl group: Serum-free 1640 medium containing 1% double antibody was added to the 96-well plate, and then HUVECs cell suspension was added.

[0144] In the above experimental groups, the amount of HUVECs cell suspension added to each well of the 96-well plate was the same. The 96-well plate was placed in a real-time living cell workstation and incubated at 37°C and 5% CO. 2 The cells were incubated under the conditions of 4% paraformaldehyde and observed dynamically for 24 h. During this period, photos were taken every 1 h to observe the formation status of the vascular network and the cell status. The images taken at 6 h of incubation were quantitatively analyzed using the AngiogenesisAnalyzer plug-in of Image J software, and the angiogenesis-related data of HUVECs in each experimental group were calculated, including the number of cell grids, nodes, and intersections. The results are shown in Figure 2. Figures 13 and 14 shown.

[0145] Depend on Fig.14 It can be seen that compared with the CO group, COH group and COF group, the COFH group has the highest number of grids, nodes and intersections, which is consistent with H 2 O 2 The results of the test in this example showed that the tube formation effect of the COFH+CAT group was similar to that of the Ctrl group. 2 O 2 The concentration of COFH has a significant effect on the angiogenesis of HUVECs. COFH can produce H with appropriate concentration in the microenvironment through the self-oxidation of its catechol group and the coordination reaction of trivalent iron ions. 2 O 2 to promote angiogenesis. It also shows that compared with H 2 O 2 Insufficient or H 2 O 2 Excessive amount of H in the microenvironment 2 O 2 The concentration is stable within a certain range (H 2 O 2 homeostasis), which is beneficial to better promote angiogenesis.

[0146] Example 12

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

[0148] COFH group: After the rats were anesthetized by breathing, the left thoracotomy was performed under a ventilator, and the left anterior descending coronary artery was ligated to simulate myocardial infarction. Then, the gel precursor solution used to prepare COFH in Example 5 was injected into the myocardium, and then sutured. The photos of the operation process are shown in Fig.15 As shown, Fig.15 Figures A to D are photos of rats before thoracotomy, ligation of the left anterior descending coronary artery, injection of gel precursor solution, and suturing. The recovery of cardiac function was evaluated by cardiac ultrasound and electrocardiogram on the 28th day after surgery. The heart tissue of the rats was removed and sliced ​​after the experiment ended on the 28th day after surgery, and the area of ​​cardiac fibrosis was observed by Masson trichrome staining to evaluate the pathological remodeling of the infarct area.

[0149] CO group: The operation was basically the same as that of COFH group, except that the injected gel precursor liquid was replaced by the mixed liquid 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 by the mixed liquid 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 by the mixed liquid D in Comparative Example 3.

[0152] Sham group: The rats were anesthetized by breathing, and the left thoracotomy was performed under a ventilator and then sutured (only thoracotomy and suture, without ligation or injection). The recovery of cardiac function was evaluated by cardiac ultrasound and electrocardiogram on the 28th day after surgery. At the end of the experiment on the 28th day after surgery, the rat heart tissues were removed and sliced, and the area of ​​cardiac fibrosis was observed by Masson's trichrome staining.

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

[0154] Echocardiograms of rats in each experimental group on the 28th day after surgery Fig.16 As shown in the figure, 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 has a better effect in promoting the recovery of cardiac function after myocardial infarction. Fig.17As shown, compared with the Saline group, CO group, COF group and COH group, the electrocardiograms of the COFH group and the Sham group showed no pathological Q waves, ST segment changes and other manifestations of myocardial damage or myocardial ischemia, and had the best cardiac function.

[0155] The results of Masson trichrome staining of the heart tissues of rats in each experimental group on the 28th day after surgery are as follows: Fig.18 As shown in the figure, compared with the CO group, COH group, COF group and Saline group, the COFH group had the smallest myocardial fibrosis area and the mildest degree of fibrosis, indicating that COFH can reduce myocardial fibrosis after myocardial infarction and thus promote the recovery of cardiac function.

[0156] Example 13

[0157] In this example, a hydrogel capable of regulating the redox homeostasis of a microenvironment was prepared. The operation was basically the same as that of Example 5, except that: in steps (1) and (2), HAD prepared in Example 2, OD prepared in Example 4, and CCS, Fe 2 (SO 4 ) 3 As raw materials, a 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 Fe 2 (SO 4 ) 3 The concentration is 0.01 mol / L.

[0158] Embodiment 14

[0159] In this example, a hydrogel capable of regulating the redox homeostasis of a microenvironment was prepared. The operation was basically the same as that of Example 5, except that: in steps (1) and (2), HAD prepared in Example 1, OD prepared in Example 4, and CCS, Fe(NO 3 ) 3 As raw materials, a 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(NO 3 ) 3 The concentration is 0.05 mol / L.

[0160] Embodiment 15

[0161] In this example, a hydrogel capable of 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 15% of catechol groups was prepared according to the method in Example 1, and OD with an oxidation degree of 40% was prepared according to the method in Example 4. The HAD and OD prepared in this example, as well as CCS, FeCl 3 As raw materials, a 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(NO 3 ) 3 The concentration is 0.1 mol / L.

Claims

1. A hydrogel capable of regulating the redox homeostasis of a 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, wherein the hybrid cross-linking reaction includes a Schiff base reaction between the amino group of the carboxylated chitosan and the aldehyde group of the oxidized dextran, an oxidative self-cross-linking reaction of the catechol-modified hyaluronic acid, a Michael addition reaction between the quinone group of the catechol-modified hyaluronic acid after the oxidative self-cross-linking and the amino group of the carboxylated chitosan, and a dynamic coordination reaction between the catechol group of the catechol-modified hyaluronic acid after the oxidative self-cross-linking and the iron ions; under physiological conditions, the catechol groups in the hydrogel will undergo a self-oxidation reaction to generate H2O2, and at the same time, the iron ions coordinated to the catechol groups in the hydrogel will catalyze the decomposition of H2O2 into water and oxygen, thereby regulating the level of active oxygen in the microenvironment.

2. The hydrogel capable of regulating the redox homeostasis of the microenvironment according to claim 1, characterized in that: The hydrogel is formed by a gel precursor solution containing catechol-modified hyaluronic acid, carboxylated chitosan, oxidized dextran and water-soluble iron salt through a hybrid cross-linking reaction.

3. The hydrogel capable of regulating the redox homeostasis of the microenvironment according to claim 2, characterized in that: 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.

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

5. The hydrogel capable of regulating the redox homeostasis of the microenvironment according to any one of claims 1 to 4, characterized in that: 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%.

6. The hydrogel capable of regulating the redox homeostasis of the microenvironment according to any one of claims 1 to 4, characterized in that: The oxidized dextran is obtained by oxidizing part of the hydroxyl groups in the dextran into aldehyde groups, and the oxidation degree of the dextran is 15% to 40%.

7. The hydrogel capable of regulating the redox homeostasis of a microenvironment according to any one of claims 1 to 4, 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%.

8. The method for preparing the hydrogel capable of regulating the redox homeostasis of the microenvironment according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) dissolving catechol-modified hyaluronic acid in water to obtain a catechol-modified hyaluronic acid solution; dissolving carboxylated chitosan in a PBS buffer solution having a pH value of 7.2 to 7.4 to obtain a carboxylated chitosan solution; dissolving oxidized dextran in an aqueous solution of an iron salt to obtain an oxidized dextran / iron salt mixed solution; (2) fully mixing the carboxylated chitosan solution and the oxidized dextran / iron salt mixed solution, and then fully mixing the obtained mixed solution with the catechol-modified hyaluronic acid solution to obtain a gel precursor solution; (3) The gel precursor solution is allowed to stand until it turns into a gel state, and the resulting hydrogel is repeatedly soaked in PBS buffer and water with a pH value of 7.2 to 7.4 to remove iron ions that are not bound to the catechol groups, thereby obtaining a hydrogel that can regulate the redox homeostasis of the microenvironment.

9. The method for preparing a hydrogel capable of regulating microenvironment redox homeostasis according to claim 8, characterized in that: In the gel precursor solution of step (2), 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.

10. Use of the hydrogel capable of regulating the redox homeostasis of the microenvironment according to any one of claims 1 to 7 in the preparation of myocardial tissue repair materials.

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