Self-adaptive multifunctional hydrogel system for cardiac microenvironment regulation after myocardial infarction as well as preparation method and application of self-adaptive multifunctional hydrogel system

Abstract

The invention provides a self-adaptive multifunctional hydrogel system for cardiac microenvironment regulation after myocardial infarction as well as a preparation method and application of the self-adaptive multifunctional hydrogel system, and belongs to the technical field of biomedical materials. The hydrogel system is hydrogel prepared by taking a micromolecular cross-linking agent, a polymer matrix and drug-loaded microspheres as raw materials. The hydrogel can realize staged response drug release in oxidative stress, inflammation and fibrosis microenvironments after myocardial infarction, has good injectability and self-healing property, and has the functions of ROS removal, nitric oxide release and the like. The invention provides a new strategy for tissue repair after myocardial infarction with multi-channel collaboration and accurate pathological response control.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to a self-adaptive multifunctional hydrogel system for regulating the cardiac microenvironment after myocardial infarction, a preparation method and applications. BACKGROUND

[0002] Myocardial infarction (MI) is one of the cardiovascular diseases with the highest incidence and mortality worldwide, mainly caused by acute or persistent obstruction of the coronary artery, leading to large area ischemic necrosis of myocardial tissue. After MI, a series of adverse remodeling phenomena such as irreversible death of myocardial cells, thinning of ventricular wall, and dilation of ventricle will rapidly progress, eventually leading to heart failure, seriously endangering the life and health of patients.

[0003] Although modern clinical treatment methods (such as thrombolysis, coronary intervention or coronary bypass surgery) can restore blood perfusion to some extent, they cannot effectively reverse the myocardial damage that has already occurred. The pathological repair process after MI is complex and multi-stage, mainly including early intense oxidative stress response, inflammatory cascade, mid-term angiogenesis and granulation tissue formation, and late fibrosis remodeling. If precise intervention cannot be carried out at each stage, it will inevitably lead to scar tissue formation, deterioration of heart function, and even sudden death.

[0004] Injectable hydrogels, as a new type of biomaterials with good tissue compatibility and plasticity, show good application prospects in the treatment of myocardial infarction. This kind of material can simulate the extracellular matrix microenvironment, regulate cell behavior, carry and release therapeutic factors, and has mechanical properties that can adapt to the deformation of the beating heart. Compared with traditional systemic drug delivery, injectable hydrogels can achieve the advantages of strong targeting, stable sustained-release effect, and high local drug concentration. However, most existing hydrogels still face key problems such as poor structural stability, single drug release mode, limited response mechanism, and single treatment function, making it difficult to meet the treatment needs of continuous, phased, and multi-mechanism collaborative repair after myocardial infarction.

[0005] In practical applications, the myocardial infarction area has microenvironment characteristics such as high concentration of reactive oxygen species (ROS) and local acidity, and most hydrogels are difficult to exist stably and respond specifically in this pathological environment. At the same time, the myocardial repair process often requires multiple time-dependent treatment functions, including early antioxidant, promotion of angiogenesis, mid-term regulation of inflammation, and late inhibition of abnormal proliferation of fibroblasts and collagen deposition. Traditional single-function hydrogel systems usually cannot achieve precise regulation of drugs in space and time, making it difficult to simultaneously cover the above treatment needs. In addition, in the mechanical environment of the continuously beating heart, hydrogels without good mechanical strength or self-repairing ability are prone to rupture and failure, seriously affecting the continuity and stability of treatment.

[0006] Therefore, it is urgent to develop a new hydrogel system with comprehensive advantages in structural stability, biological responsiveness, drug carrier capacity and time sequence treatment efficiency. The system should not only have intelligent response ability to ROS and acidic environment after MI, but also be able to carry and programmed release a variety of drug factors, match multiple stages of myocardial repair, have synergistic treatment ability, improve the targeting and accuracy of treatment, and have good injection, self-healing and tissue compatibility, so as to more effectively realize the tissue function reconstruction after myocardial infarction. SUMMARY

[0007] The purpose of the present application is to provide a self-adaptive multifunctional hydrogel platform with novel structure, integrated function and accurate response, so as to solve the problems of single response, limited treatment path and uncoordinated drug release of the prior art, and improve the treatment effect of myocardial infarction.

[0008] The present application provides a kind of hydrogel, it is with small molecule crosslinking agent, polymer matrix, drug-loaded microspheres as raw material preparation hydrogel, wherein, the mass ratio of small molecule crosslinking agent, polymer matrix, drug-loaded microspheres is 1~3:3~10:1~2;The polymer matrix is nitric oxide donor modified polyvinyl alcohol;The drug-loaded microspheres are polymeric microspheres loaded with fibrosis inhibitors and anti-inflammatory drugs.

[0009] Further, the mass ratio of the small molecule crosslinking agent, the polymer matrix and the drug-loaded microspheres is 1:10:2.

[0010] Further, the small molecule crosslinking agent is the product obtained by reacting 3-aminobenzene boronic acid under the action of a condensing agent; The polymer matrix is the product obtained by reacting a nitric oxide donor and polyvinyl alcohol under the action of a condensing agent and a base; The drug-loaded microspheres are the product obtained by reacting a benzene boronic acid modified polymer, a fibrosis inhibitor and an anti-inflammatory drug under the action of a photoinitiator.

[0011] Further, the mass ratio of the nitric oxide donor, polyvinyl alcohol, condensing agent and base is 1~2:10:1~3:0.05~0.1; The nitric oxide donor is S-nitrosoglutathione; The mass ratio of the benzene boronic acid modified polymer and the photoinitiator is 1~3:1; The benzene boronic acid modified polymer is the product obtained by reacting hyaluronic acid with methacrylic anhydride, and then with a condensing agent and benzene boronic acid; The condensing agent is selected from one or both of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide; The base is an organic base; The photoinitiator is a lithium salt.

[0012] The base is 4-dimethylaminopyridine. The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

[0013] Further, the structure of the small molecule crosslinking agent is ; The structure of the polymer matrix is: , wherein n is 1700. In the drug-loaded microspheres, the fibrosis inhibitor is Galunisertib, and the anti-inflammatory drug is Danshensuan B, and the mass ratio of the fibrosis inhibitor and the anti-inflammatory drug is 0.1-0.3:0.2-0.4.

[0014] Further, in the drug-loaded microspheres, the mass ratio of the phenylboronic acid modified polymer, the fibrosis inhibitor and the photoinitiator is 1-3:0.5-1:0.5-1.

[0015] The application also provides a method for preparing the above hydrogel, which comprises the following steps: (1) adding the polymer matrix into water and mixing uniformly to obtain solution A; (2) adding the small molecule crosslinking agent into water and mixing uniformly to obtain solution B; (3) mixing solution A and solution B, and adding drug-loaded microspheres to form a hydrogel.

[0016] Further, in the solution A, the concentration of the polymer matrix is 1-10 mg / mL; in the solution B, the concentration of the small molecule crosslinking agent is 50-150 mg / mL; and the volume ratio of the solution A, the solution B and the drug-loaded microspheres is 8-12:0.5-2:0.5-2.

[0017] Further, in the solution A, the concentration of the polymer matrix is 30 mg / mL; in the solution B, the concentration of the small molecule crosslinking agent is 100 mg / mL; and the volume ratio of the solution A, the solution B and the drug-loaded microspheres is 10:1:1.

[0018] Further, the drug is a drug for promoting angiogenesis, improving ischemic environment, resisting oxidation, inhibiting fibroblast differentiation, inhibiting collagen deposition of heart tissue and / or inhibiting ventricular remodeling.

[0019] The application has the following beneficial effects: 1、The hydrogel system constructed in the application has a multi-stage pathological response mechanism, which can accurately adapt to the complex microenvironment after myocardial infarction. Through the dual response of reactive oxygen species (ROS) and acidic pH, the hydrogel can automatically change its structure under typical pathological conditions such as increased oxidative stress and decreased pH, realize on-demand drug release, and significantly improve the targeting and effectiveness of treatment.

[0020] 2、The traditional phenylboronic acid-based hydrogel is prepared by randomly modifying phenylboronic acid on a high molecular polymer. However, this method makes it difficult to accurately control the molecular structure and dynamic crosslinking mechanism. In contrast, the application uses a small molecule phenylboronic acid-based crosslinking agent N(BA)4 with a four-arm topological structure. The crosslinking agent has a precise molecular structure, each molecule contains four phenylboronic acid groups, and the molecular weight is definite. This structure enables N(BA)4 to realize a rapid gelation process. The drug-loaded hydrogel system of the application provides a new choice for the application of self-adaptive multifunctional hydrogel system in myocardial infarction treatment.

[0021] 3、The self-adaptive multifunctional hydrogel system of the application integrates various biological treatment functions into one material platform, and achieves highly synergistic antioxidant, pro-vascular, anti-inflammatory, and anti-fibrosis treatment effects. NO release and Sal B synergistic effect to remove ROS, repair mitochondrial function; Sal B and Gal release are staggered in time, the former deals with acute oxidative stress, and the latter delays intervention in the chronic fibrosis process, building a dynamic, multi-path intervention system.

[0022] It is particularly noteworthy that experiments have found that the hydrogel system of the application has a significant treatment effect in a rat myocardial infarction model. After injecting PG / Ms@GS hydrogel, the ejection fraction of the animal significantly improves within 28 days, and the ventricular structure is obviously restored; HE and Masson staining shows that inflammatory cell infiltration is reduced, and collagen deposition is significantly decreased; immunohistochemical analysis further confirms that angiogenesis is enhanced and fibrosis markers are down-regulated, verifying the excellent in vivo treatment effect.

[0023] In summary, the self-adaptive multifunctional hydrogel system of the application has a broad application prospect in the preparation of drugs for preventing and / or treating myocardial infarction.

[0024] Obviously, according to the above content of the application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modifications, replacements or changes can be made without departing from the above basic technical idea of the application.

[0025] The above content of the application will be further described in detail through the specific embodiments below. However, this should not be understood as limiting the scope of the above subject matter of the application to the following examples. Any technology realized based on the above content of the application belongs to the scope of the application. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the synthesis pathway of HAMABA.

[0027] Figure 2 This is the NMR mass spectrometry of HAMABA.

[0028] Figure 3 The infrared spectrum of HAMABA.

[0029] Figure 4 The ultraviolet absorption spectrum of the microspheres Figure 5 This is a scanning electron microscope image of Ms@GS.

[0030] Figure 6 This is a schematic diagram of the synthesis path of N(BA)4.

[0031] Figure 7 This is the mass spectrum of N(BA)4.

[0032] Figure 8 The NMR spectrum of N(BA)4.

[0033] Figure 9 This is a schematic diagram of the synthesis pathway of PVA-GSNO.

[0034] Figure 10 This is the NMR mass spectrometry of PVA-GSNO.

[0035] Figure 11 This is a schematic diagram of hydrogel formation.

[0036] Figure 12 This is a scanning electron microscope (SEM) image of the hydrogel.

[0037] Figure 13 The rheological results for the hydrogels are as follows: (A) Frequency-dependent storage modulus (G′) and loss modulus (G″) of different hydrogels; (B) Strain-dependent G′ and G″ curves; (C) Strain scan curves of hydrogels under alternating low strain (1%) and high strain (500%) conditions; (D) Curves showing the change of G′ and G″ over time.

[0038] Figure 14 The results of in vitro drug release from hydrogel-loaded drugs under different conditions: (A) Cumulative release rate of Sal B.; (B) Cumulative release rate of Gal.

[0039] Figure 15 Figure showing the cell viability of cardiomyocytes after different hydrogel treatments at 24 and 72 hours. **** indicates p < 0.0001.

[0040] Figure 16Figure showing the 24-hour ROS scavenging rate of cardiomyocytes treated with different hydrogels. **** indicates p<0.0001.

[0041] Figure 17 Figure showing the tube formation of endothelial cells treated with different hydrogels. ** indicates p<0.01, **** indicates p<0.0001.

[0042] Figure 18 Figure showing the cell proliferation rate of cardiac fibroblasts treated with different hydrogels at 5 days. ** indicates p<0.01.

[0043] Figure 19 Figure showing the treatment effect evaluation in a rat myocardial infarction model treated with different hydrogels: (A) Hematoxylin-eosin (H&E) staining of left ventricular tissue at 28 days after myocardial infarction in different treatment groups; (B) Masson trichrome staining. DETAILED DESCRIPTION

[0044] The raw materials and equipment used in the present application are known products, which can be obtained by purchasing commercially available products.

[0045] In the present application, unless otherwise specified, the solution refers to an aqueous solution; unless otherwise specified, the operation is carried out at room temperature (25±5°C).

[0046] Example 1, synthesis of adaptive multifunctional hydrogel system PG / Ms@GS 1. Synthesis of four-arm phenylboronic acid crosslinker N(BA)4 Dissolve 21.92 g of 3-aminophenylboronic acid in 500 mL of anhydrous DMSO, and then add 15.36 g of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and 9.2 g of NHS (N-hydroxysuccinimide) successively for activation reaction, and stir at room temperature for 48 hours. After the reaction is completed, evaporate the solvent under reduced pressure. Add an excess of tetrahydrofuran (THF) to the concentrated product in an ice bath to precipitate the reaction product, repeat the precipitation and washing 3 times, and dry to obtain a white powder crosslinker N(BA)4. The structure is confirmed by mass spectrometry (MS) and 1H NMR, and the main peak is consistent with the theoretical molecular weight.

[0047] 2. Preparation of S-nitrosoglutathione (GSNO) modified polyvinyl alcohol (PVA-GSNO) PVA-GSNO was prepared by the following method. 10 g of polyvinyl alcohol (PVA) was dissolved in 200 mL of DMSO, and 1.6 g of S-nitrosoglutathione (GSNO), 1.9 g of EDC and 80 mg of DMAP (4-dimethylaminopyridine) were added. The reaction was stirred at room temperature for 48 hours. After the reaction was completed, the product was transferred to a dialysis bag (MWCO = 3500 Da) and dialyzed in deionized water for 3 days (water change every 6 hours). After freeze-drying, a powdery PVA-GSNO was obtained. The success of GSNO grafting was verified by 1H NMR and UV absorption spectrum.

[0048] 3. Preparation of HAMABA microspheres and drug loading 1 g of hyaluronic acid (HA) was dissolved in 300 mL of deionized water, 3 mL of methacrylic anhydride (MA) was slowly added, the pH was adjusted to 8-10, and the solution was stirred in the dark for 24 h. After the reaction solution was dialyzed for 3 days, it was freeze-dried to obtain HAMA. Further, HAMA (1 g) was dissolved in 200 mL of deionized water, 12 g of EDC, 8 g of NHS and 10 g of phenylboronic acid (PBA) were added, and the reaction was carried out for 24 h. After dialysis and freeze-drying, HAMA modified with phenylboronic acid (HAMABA) was obtained. 0.02 g of HAMABA was dissolved in 1 mL of water, 0.01 g of LAP (photoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphinate) and 10 mg of Galunisertib (Gal) were added, and a water droplet structure was prepared using a microfluidic chip device. The solution was irradiated under a UV lamp (365 nm) for 30 seconds to prepare Gal-loaded microspheres (Ms@G). The obtained Ms@G microspheres were suspended in a PBS solution containing Salvianolic acid B (Sal B) and incubated for 2 hours. Sal B was combined with the phenylboronic acid groups on the surface of the microspheres through the formation of a borate ester bond to obtain double-drug-loaded microspheres Ms@GS.

[0049] 4. Construction of PG / Ms@GS hydrogel Two aqueous solutions were prepared as follows: Solution A: PVA-GSNO mass fraction of 5% (w / v); Solution B: N(BA)4 mass fraction of 5% (w / v). Solution A and solution B were mixed at a volume ratio of 10:1 and stirred uniformly, and a transparent injectable hydrogel (PG) was formed within 5 seconds. 1 mg / mL of Ms@GS microspheres (solution A, solution B and Ms@GS microspheres at a volume ratio of 10:1:1) were added, and the solution A, solution B and Ms@GS microspheres were stirred uniformly at a volume ratio of 10:1:1 to form a composite hydrogel PG / Ms@GS.

[0050] The following is a control example.

[0051] Control Example 1: Preparation of PG blank hydrogel PVA-GSNO was dissolved in deionized water (5% (w / v), 50 mg / mL) and adjusted to neutral pH to obtain solution A. Small molecule crosslinker N(BA)4 was dissolved in deionized water to prepare a solution B with a concentration of 5% W / V (50 mg / mL). Solution A and solution B were mixed in a ratio of 10:1 by volume, and stirred immediately to form a PG blank hydrogel without drug loading.

[0052] Example 2, preparation of PG / Ms hydrogel PVA-GSNO was dissolved in deionized water (5% (w / v), 50 mg / mL) and adjusted to neutral pH to obtain solution A. Small molecule crosslinker N(BA)4 was dissolved in deionized water to prepare a solution B with a concentration of 5% W / V (50 mg / mL). Solution A and solution B were mixed in a ratio of 10:1 by volume, and stirred immediately to form a PG / Ms hydrogel loaded with blank microspheres.

[0053] Example 3, preparation of PG / Ms@G hydrogel PVA-GSNO was dissolved in deionized water (5% (w / v), 50 mg / mL) and adjusted to neutral pH to obtain solution A. Small molecule crosslinker N(BA)4 was dissolved in deionized water to prepare a solution B with a concentration of 5% W / V (50 mg / mL). Solution A and solution B were mixed in a ratio of 10:1 by volume, and stirred immediately to form a PG / Ms@G hydrogel loaded with blank microspheres.

[0054] The beneficial effects of the present application are demonstrated by the following experimental examples.

[0055] Experimental Example 1, preparation and characterization of active drug carrier microspheres 1. Experimental method Dual-drug loaded microspheres (Ms@GS) were prepared according to the method described in Example 1.

[0056] 2. Experimental results Figure 1 The synthesis of HAMABA is shown in the schematic diagram.

[0057] Figure 2 The synthesis of HAMABA is shown in the schematic diagram. 1 HNMR spectrum, the results confirmed the successful functionalization of HAMABA. Characteristic proton signals related to the methacrylate (MA) and boric acid (BA) molecules were observed in the ranges of 5.5-6.5 ppm and 7.5-8.0 ppm, respectively, which did not exist in the unmodified HA, indicating that the chemical modification was effective.

[0058] Figure 3 FT-IR further confirmed the synthesis of HAMABA, 1715 cm - - New absorption bands appeared at 1715 cm

[0059] Figure 4 UV-Vis was recorded to verify the dual drug loading capacity of the system, with Sal B and Gal loading of 31.4% and 22.8%, respectively.

[0060] Figure 5 SEM observation showed that the microspheres had uniform size (about 100-130 μm) and porous surface, which was beneficial to drug release and reactive degradation.

[0061] Example 2, Synthesis and characterization of four-arm small molecule crosslinker N(BA)4 and PVA-GSNO 1. Experimental methods The small molecule crosslinker N(BA)4 and PVA-GSNO polymer were prepared according to the method described in Example 1.

[0062] 2. Experimental results Figure 6 The synthesis route of N(BA)4 is shown in the figure.

[0063] Figure 7 The molecular weight of N(BA)4 was verified by mass spectrometry analysis, which revealed a characteristic molecular ion peak consistent with its theoretical value. The sharp and obvious peak indicated the success of the synthesis, laying a solid structural foundation for the subsequent formation of hydrogel through dynamic borate-diol interaction.

[0064] Figure 8 The 1H nuclear magnetic resonance (NMR) spectrum confirmed that N(BA)4 exhibited multiple symmetrical and strong signals in the range of 7.8-8.2 ppm, which was attributed to the aromatic protons (Ar-H) of the para-benzene ring, verifying the successful functionalization of boronic acid.

[0065] Figure 9 The synthesis route of PVA-GSNO is shown in the figure.

[0066] Figure 10 The results showed that the 1H NMR spectrum showed peaks corresponding to the GSNO moiety, confirming the successful coupling of the NO donor GSNO with the PVA backbone. These chemical modifications formed dynamic borate ester bonds between the boronic acid groups on the crosslinker and the diols on the PVA chain.

[0067] ​Experimental Example 3, Preparation and characterization of multifunctional responsive hydrogel 1. Experimental method PG hydrogel was prepared according to the method described in Example 1. Different microspheres were added to the PG hydrogel to prepare PG / Ms (empty microspheres), PG / Ms@G (Gal-loaded microspheres) and PG / Ms@GS (Gal+Sal B-loaded microspheres).

[0068] 2. Experimental results As shown in Figure 11 Photographic observation of each component and the obtained hydrogel further proved the successful gelation of the PG hydrogel.

[0069] Figure 12 For the SEM results of the freeze-dried hydrogel, the SEM imaging of the freeze-dried hydrogel showed a three-dimensional porous microstructure.

[0070] Figure 13 It was proved that the hydrogel had good rheological properties, could gel quickly, and had good self-healing characteristics.

[0071] Experimental Example 4, Drug release performance test 1. Experimental method Different hydrogels were placed in PBS (pH 7.4) and simulated pathological environment (pH 6.5, 200 μM H2O2), and the Sal B and Gal release curves (UV method) were detected.

[0072] 2. Experimental results Figure 14 The drug release results of the hydrogel under different experimental conditions are shown, and both drugs show an initial rapid release, followed by a stable trend. It is worth noting that the cumulative release of both drugs under acidic and high ROS conditions (pH=6.5, 200 μM H2O2) is higher than that at pH 7.4, indicating that the hydrogel has good pH responsiveness and acid-responsive release characteristics.

[0073] Experimental Example 5, Functional evaluation at the cellular level 1. Experimental method First, H9C2 cells were seeded in 96-well plates at a density of 1*10 6 / Wells were seeded at a density of 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum to 80-90% confluence. Then, different concentrations of hydrogels were added into the cell culture medium and incubated for 2-4 h. Next, oxidative stress was induced with 200 μM H2O2 and cells were treated for 6 hours. After that, 10 μM DCFH-DA probe solution was added and incubated for 30-60 min to allow DCFH-DA to penetrate the cells and convert to fluorescent product DCF under oxidative stress. After treatment, cells were washed with PBS and the fluorescence intensity of DCF was measured with a fluorescence microscope, reflecting the level of oxidative stress in cells. Lower fluorescence intensity indicates that the hydrogel has antioxidant protection and can effectively reduce oxidative damage.

[0074] 2. Experimental results As shown in Figure 15 , the cell viability of the H2O2 group decreased to about 55%, and the viability after PG / Ms@GS hydrogel treatment recovered to ~85%. This demonstrates the good cell protection function of the hydrogel.

[0075] Figure 16 The results demonstrate the superior antioxidant properties of PG / Ms@GS hydrogel. This composite hydrogel significantly attenuates hypoxia-induced oxidative stress, thereby effectively reducing myocardial cell damage.

[0076] Example 6: Hydrogel promotes tube formation of endothelial cells 1. Experimental method 50 μL of Matrigel per well was added to a 96-well plate and incubated in a cell culture incubator for 40 minutes. Then, 50 μL of standard medium containing HUVECs was seeded on the surface of Matrigel (5 x 10 4 cells) and HUVECs were pretreated with various hydrogel samples for 6 h. Images were obtained by fluorescence microscopy.

[0077] 2. Experimental results To further evaluate the angiogenic ability, a Matrigel-based tube formation assay was performed Figure 17 . The results show that H2O2 treatment significantly impaired the formation of capillary-like networks of endothelial cells, and a significant decrease in the number of connections demonstrated this. In contrast, the PG / Ms@G and PG / Ms@GS treatment groups had significantly more capillary-like structures formed by cells, and a significant increase in the number of connections, indicating that the hydrogel can promote angiogenesis even under oxidative stress conditions through antioxidant and sustained release of NO.

[0078] Example 7: Hydrogel inhibits the proliferation of cardiac fibroblasts 1. Experimental method To perform the proliferation evaluation, cardiac fibroblasts were seeded at a density of 1 x 10 5Cells were seeded at a density of 1x105 cells / well in 24-well plates and allowed to attach overnight. Then the cells were co-cultured with hydrogels for 24 hours. To assess proliferation, EDU probe was added to the culture medium for the last 4 hours of culture. Cells were then fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.5% Triton X-100 for 10 minutes, and stained according to the manufacturer's instructions using the Click-iT™ EdU Imaging Kit. Nuclei were stained with DAPI.

[0079] 2. Experimental results As shown in Figure 18 , the PG and PG / Ms groups had little inhibitory effect on CF proliferation. In contrast, the Galunisertib-loaded PG / Ms@G and PG / Ms@GS groups significantly reduced the proportion of EDU-positive cells, indicating that the addition of Galunisertib effectively inhibited the abnormal proliferation of CF. Notably, the PG / Ms@GS group had the lowest proliferative activity, indicating that Galunisertib had a synergistic inhibitory effect when used in combination with SalB, providing a solid foundation for anti-fibrotic intervention.

[0080] Experimental Example 8. Detection of the in vivo heart repair effect of hydrogels 1. Experimental methods To study the effect of hydrogels on in vivo heart repair, a rat myocardial infarction (MI) disease model was established. The method for establishing the MI model was as follows: All procedures involving animals in this study were strictly in accordance with the guidelines and ethical standards established by the Experimental Animal Ethics Committee of Sichuan University. Forty-eight male SD rats were randomly divided into six groups. Sham, MI, PDB hydrogel, PDB@NPs hydrogel, PDB@Ms hydrogel, and PDB@NPs&Ms hydrogel constructs were used in the MI rat model. The experiment was performed under sterile conditions, and all animals were anesthetized using isoflurane via a respirator. Continuous electrocardiogram monitoring was performed. To simulate the pathological changes of myocardial infarction, the experimenter ligated the left anterior descending branch from the left fourth intercostal space. This resulted in coronary artery occlusion, and the downstream blood supply area suffered acute ischemia and myocardial necrosis. The successful construction of the rat myocardial infarction model was evidenced by the elevation of the ST-T segment or the formation of Q waves on the electrocardiogram. Then, 25 μL of PBS or different hydrogels were injected into the infarcted area and the surrounding two areas using a 27 G syringe. The rat chest was sutured and iodophor was applied for disinfection. The Sham group underwent thoracotomy without ligation of the left anterior descending branch.

[0081] On day 28, hematoxylin-eosin (H&E) staining and Masson staining were performed on the heart tissue of rats in each group.

[0082] 2. Experimental results As shown in Figure 19The ventricular wall thickness of the heart tissue of the PG / Ms@GS group was the highest, and the collagen deposition area was the smallest. It was proved that the PG / Ms@GS hydrogel treatment group could effectively reduce the collagen deposition at the myocardial infarction site, and played an excellent heart repair function.

[0083] In conclusion, the application provides a self-adaptive multifunctional hydrogel system for regulating the cardiac microenvironment after myocardial infarction, a preparation method and an application. The hydrogel system is prepared from a small molecule crosslinking agent, a polymer matrix and drug-loaded microspheres. The hydrogel can achieve staged drug release in the oxidative stress, inflammation and fibrosis microenvironment after myocardial infarction, and has good injection and self-healing properties, and functions such as ROS removal and nitric oxide release. The application provides a new strategy for myocardial infarction tissue repair with multi-channel synergy and precise control of pathological response.

Claims

1. A hydrogel, characterized by, It is a hydrogel prepared from small molecule crosslinking agent, polymer matrix and drug-loaded microspheres, wherein the mass ratio of the small molecule crosslinking agent, the polymer matrix and the drug-loaded microspheres is 1-3:3-10:1-2; the polymer matrix is a polyvinyl alcohol modified by a nitric oxide donor; and the drug-loaded microspheres are polymer microspheres loaded with a fibrosis inhibitor and an anti-inflammatory drug.

2. The hydrogel of claim 1, wherein, The mass ratio of the small molecule crosslinking agent, the polymer matrix and the drug-loaded microspheres is 1:10:

2.

3. The hydrogel according to claim 1 or 2, characterized in that, The small molecule crosslinking agent is a product obtained by reacting 3-aminobenzene boronic acid under the action of a condensing agent; The polymer matrix is a product obtained by reacting a nitric oxide donor and polyvinyl alcohol under the action of a condensing agent and a base; The drug-loaded microspheres are a product obtained by reacting a benzene boronic acid modified polymer, a fibrosis inhibitor and an anti-inflammatory drug under the action of a photoinitiator.

4. The hydrogel of claim 3, wherein, The mass ratio of the nitric oxide donor, polyvinyl alcohol, condensing agent and base is 1-2:10:1-3:0.05-0.1; The nitric oxide donor is S-nitrosoglutathione; The mass ratio of the benzene boronic acid modified polymer and the photoinitiator is 1-3:1; The benzene boronic acid modified polymer is a product obtained by reacting hyaluronic acid with methacrylic anhydride, and then with a condensing agent and benzene boronic acid; The condensing agent is selected from one or both of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide; The base is an organic base; The photoinitiator is a lithium salt.

5. The hydrogel according to any one of claims 1 to 4, wherein The structure of the small molecule crosslinking agent is ; The structure of the polymer matrix is: wherein n is 1700; In the drug-loaded microspheres, the fibrosis inhibitor is Galunisertib, the anti-inflammatory drug is Danshensuan B, and the mass ratio of the fibrosis inhibitor and the anti-inflammatory drug is 0.1-0.3:0.2-0.

4.

6. A method of preparing the hydrogel according to any one of claims 1 to 5, characterized in that, The method comprises the following steps: (1) adding the polymer matrix into water and mixing uniformly to obtain solution A; (2) adding the small molecule crosslinking agent into water and mixing uniformly to obtain solution B; (3) mixing solution A and solution B, and adding drug-loaded microspheres to form a hydrogel.

7. The method of claim 6, wherein: In the solution A, the concentration of the polymer matrix is 1-10 mg / mL; in the solution B, the concentration of the small molecule crosslinking agent is 50-150 mg / mL; and the volume ratio of the solution A, the solution B and the drug-loaded microspheres is 8-12:0.5-2:0.5-2.

8. The method of claim 7, wherein: In the solution A, the concentration of the polymer matrix is 30 mg / mL; in the solution B, the concentration of the small molecule crosslinking agent is 100 mg / mL; and the volume ratio of the solution A, the solution B and the drug-loaded microspheres is 10:1:

1.

9. Use of the hydrogel of any one of claims 1-5 in the preparation of a drug for preventing and / or treating myocardial infarction.

10. Use according to claim 9, characterized in that, The drug is a drug for promoting angiogenesis, improving ischemic environment, resisting oxidation, inhibiting fibroblast differentiation, inhibiting collagen deposition in cardiac tissue and / or inhibiting ventricular remodeling.

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