Microenvironment response type injectable hydrogel as well as preparation method and application thereof

Abstract

The invention discloses microenvironment response type injectable hydrogel as well as a preparation method and application thereof. The hydrogel is of a dual-network structure formed by crosslinking phenylboronic acid modified oxidized hyaluronic acid and dopamine grafted gelatin through a dynamic Schiff base bond and a boric acid ester bond; wherein black phosphorus nanosheets and puerarin-loaded honeycomb-shaped manganese dioxide nanoparticles coated with polydopamine are dispersed in the nano-particles. The hydrogel can intelligently respond to acidic pH and a high-active oxygen microenvironment, and controllable degradation and on-demand release of contents are realized. Honeycomb-shaped manganese dioxide nanoparticles can catalyze decomposition of hydrogen peroxide to generate oxygen under an acidic condition, and meanwhile, active oxygen is removed; the black phosphorus nanosheets endow the hydrogel with excellent conductivity. The multi-layer function integrated design synchronously solves the problems of function maintenance and electrical conduction performance of the material in an anoxic environment, the adaptability and functionality of the material in a complex physiological environment are remarkably enhanced, and a new strategy is provided for development of multifunctional intelligent materials in biomedical engineering.

Description

Technical Field

[0001] This invention relates to the field of hydrogel technology, specifically to a microenvironment-responsive injectable hydrogel, its preparation method, and its applications. Background Technology

[0002] The pathological microenvironment of myocardial infarction has complex biochemical characteristics, including acidic pH (6.5-6.8), high levels of reactive oxygen species (ROS), hypoxia, and overexpression of specific enzymes. Against this backdrop, injectable hydrogels have become a hot topic in biomaterials research due to their minimally invasive implantation properties and ability to mimic the natural extracellular matrix.

[0003] However, existing hydrogel technologies face a key technical challenge: they cannot simultaneously and effectively address the dual functional challenges of hypoxic microenvironments and interrupted electrical signal conduction. Most current smart hydrogel systems are designed for single stimuli (such as pH or ROS) and generally lack the ability to actively generate oxygen under pathological conditions. Furthermore, the inherent electrical conductivity of the materials is insufficient, making it difficult to maintain effective electrical conduction performance in dynamic cardiac environments. These functional limitations severely restrict the application effectiveness of hydrogels in complex physiological environments. Summary of the Invention

[0004] This invention aims to provide a microenvironment-responsive injectable hydrogel, its preparation method, and its application. It solves the problems of maintaining the function and electrical conductivity of materials in hypoxic environments, and enhances the adaptability and functionality of materials in complex physiological environments through a dual-response mechanism, providing a new technical path for developing multifunctional smart materials in biomedical engineering.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a microenvironment-responsive injectable hydrogel, wherein the hydrogel is formed by cross-linking oxidized hyaluronic acid modified with phenylboronic acid and dopamine-grafted gelatin through dynamic Schiff base bonds and dynamic borate ester bonds to form a double cross-linked network structure, and the hydrogel contains dispersed black phosphorus nanosheets and honeycomb manganese dioxide nanoparticles loaded with puerarin and coated with polydopamine.

[0006] Preferably, the concentrations of black phosphorus nanosheets and polydopamine-coated puerarin-loaded honeycomb manganese dioxide nanoparticles in the hydrogel are both 200 μg / mL.

[0007] Preferably, the hydrogel has dual pH / ROS responsiveness, enabling it to break Schiff base bonds under acidic pH conditions in the myocardial infarction region and cleave borate ester bonds under high reactive oxygen species conditions, thereby achieving controlled degradation of the hydrogel and drug release.

[0008] On the other hand, this invention proposes a method for preparing a microenvironment-responsive injectable hydrogel, comprising the following steps: KMnO4 was dissolved in water, and after vigorous stirring, oleic acid was added dropwise to form a stable emulsion. The mixture was then stirred continuously, and the precipitate was collected by centrifugation to obtain honeycomb-shaped manganese dioxide. Honeycomb-shaped manganese dioxide was dispersed in a mixed solvent containing puerarin, stirred, and then centrifuged to collect the precipitate, thus obtaining honeycomb-shaped manganese dioxide loaded with puerarin. Honeycomb manganese dioxide loaded with puerarin was resuspended in Tris-HCl buffer, dopamine hydrochloride was added, the reaction was stirred continuously, and the precipitate was collected by centrifugation to obtain polydopamine-coated honeycomb manganese dioxide nanoparticles loaded with puerarin. Bulk black phosphorus was dispersed in N-methyl-2-pyrrolidone at a concentration of 0.2 mg / mL, sonicated under ice bath conditions, and the unpeeled black phosphorus crystals were removed by centrifugation. The supernatant was collected and centrifuged again to obtain black phosphorus nanosheets. Hyaluronic acid was dissolved in water, sodium periodate aqueous solution was added, the reaction was stirred in the dark, ethylene glycol was added to deactivate unreacted sodium periodate, the mixture was dialyzed and then freeze-dried to obtain oxidized hyaluronic acid; oxidized hyaluronic acid was dissolved in water, 3-aminophenylboronic acid was added, the reaction was stirred at room temperature, the mixture was dialyzed and then freeze-dried to obtain phenylboronic acid-grafted oxidized hyaluronic acid. Gelatin was dissolved in PBS solution, EDC and NHS were added to activate the carboxyl groups, and then dopamine hydrochloride was added. The reaction was carried out under nitrogen protection, dialyzed, and then freeze-dried to obtain dopamine-grafted gelatin. A solution of phenylboronic acid grafted with oxidized hyaluronic acid, a solution of dopamine grafted with gelatin, honeycomb manganese dioxide nanoparticles loaded with puerarin and black phosphorus nanosheets were mixed to form the microenvironment-responsive injectable hydrogel.

[0009] Preferably, the mixed solvent is a mixed solution of methanol and water, with a volume ratio of methanol to water of 1:3.

[0010] Preferably, the ultrasonic treatment includes: ultrasonic treatment for 1 hour using an ultrasonic cell disruptor, with an ultrasonic cycle of 3 seconds on and 2 seconds off, followed by further ultrasonic treatment for 20 minutes in an ultrasonic cleaner, for a total of 9 cycles.

[0011] Preferably, the concentration of the sodium periodate aqueous solution is 0.5 mol / L, the reaction time is 8 hours, and a dialysis bag with a molecular weight cutoff of 7000 Da is used for dialysis.

[0012] Preferably, the pH of the PBS solution is 5.0, the ratio of EDC to NHS is 2.0g:1.2g, the reaction temperature is 37℃, the reaction time is 15 hours, and the dialysis uses a dialysis bag with a molecular weight cutoff of 8000-14000 Da.

[0013] Preferably, the concentrations of the phenylboronic acid-grafted hyaluronic acid solution and the dopamine-grafted gelatin solution are both 6% w / v.

[0014] On the other hand, this invention proposes the application of a microenvironment-responsive injectable hydrogel in the preparation of drugs for treating myocardial infarction. The hydrogel, in response to the acidic pH and highly reactive oxygen species microenvironment of the myocardial infarction area, triggers hydrogel degradation and releases black phosphorus nanosheets and polydopamine-coated honeycomb manganese dioxide nanoparticles loaded with puerarin. The black phosphorus nanosheets restore the electrical signal conduction in the myocardial infarction area, and the polydopamine-coated honeycomb manganese dioxide nanoparticles loaded with puerarin catalyze the decomposition of endogenous hydrogen peroxide to generate oxygen, thereby alleviating tissue hypoxia and scavenging reactive oxygen species. At the same time, the release of puerarin exerts anti-inflammatory, antioxidant, and angiogenesis-promoting effects.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The hydrogel proposed in this invention contains polydopamine-coated, puerarin-loaded, honeycomb-shaped manganese dioxide nanoparticles dispersed within it. These nanoparticles catalyze the decomposition of hydrogen peroxide to produce oxygen under acidic conditions, significantly enhancing the material's oxygen generation capacity in oxygen-deficient environments. Simultaneously, these nanoparticles exhibit excellent reactive oxygen species (ROS) scavenging properties. Uniformly dispersed black phosphorus nanosheets within the hydrogel impart superior electrical conductivity, significantly higher than that of traditional hydrogel materials. More importantly, the phenylboronic acid-modified oxidized hyaluronic acid and dopamine-grafted gelatin form a dual cross-linked network through dynamic Schiff base bonds and dynamic borate ester bonds. This network intelligently responds to acidic pH and highly reactive oxygen species microenvironments, enabling controlled degradation and on-demand release of the material's contents. This multi-level functional integration design strategy not only simultaneously addresses the issues of maintaining function and electrical conductivity in oxygen-deficient environments but also enhances the material's adaptability and functionality in complex physiological environments through a dual-response mechanism. Attached Figure Description

[0016] Figure 1 Transmission electron microscopy images of the prepared HM and PHMP nanoparticles; Figure 2 Transmission electron microscope image of the prepared black phosphorus nanosheets; Figure 3 The proton NMR spectrum of the prepared phenylboronic acid grafted with oxidized hyaluronic acid; Figure 4 The hydrogen spectrum of the prepared dopamine-grafted gelatin; Figure 5 The result of oxygen generation in the prepared injectable hydrogel is shown in the figure. Figure 6 The image shows the results of fluorescence staining for cell viability and death in the prepared injectable hydrogel. Figure 7The graph shows the expression results of ROS in H9C2 cells treated with the prepared injectable hydrogel. Figure 8 Echocardiogram of mice treated with the prepared injectable hydrogel for myocardial infarction; Figure 9 Figure 1 shows the results of left ventricular ejection fraction and shortening fraction in mice treated with the injectable hydrogel. Figure 10 Electrocardiogram of mice with myocardial infarction prepared with injectable hydrogel. Detailed Implementation

[0017] The following description is intended to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art.

[0018] Example 1 This invention proposes a microenvironment-responsive injectable hydrogel. The hydrogel is formed by cross-linking phenylboronic acid-modified oxidized hyaluronic acid with dopamine-grafted gelatin through dynamic Schiff base bonds and dynamic borate ester bonds, creating a double cross-linked network structure. The hydrogel disperses black phosphorus nanosheets and polydopamine-coated, puerarin-loaded, honeycomb-shaped manganese dioxide nanoparticles. The concentrations of both black phosphorus nanosheets and polydopamine-coated, puerarin-loaded, honeycomb-shaped manganese dioxide nanoparticles in the hydrogel are 200 μg / mL. The hydrogel exhibits dual pH / ROS responsiveness, capable of cleaving Schiff base bonds under acidic pH conditions in the myocardial infarction region and cleaving borate ester bonds under high reactive oxygen species levels, achieving controlled degradation of the hydrogel and drug release.

[0019] This invention uses phenylboronic acid-modified oxidized hyaluronic acid (OHA-PBA) and dopamine-grafted gelatin (GelDA) as precursor solutions. The two are doubly cross-linked via dynamic Schiff base bonds and dynamic borate ester bonds, forming a reversible three-dimensional network. This dynamic network endows the hydrogel with excellent injectability (suitable for minimally invasive surgery) and self-healing ability (automatically repairing structural integrity after damage), providing a stable carrier for functional nanomaterials.

[0020] Black phosphorus nanosheets (BPNs) are dispersed in a hydrogel network. The excellent conductivity of BPNs enables the reconstruction of electrical signal transduction pathways in the infarcted region, promoting the electrical synchronization of cardiomyocytes, thereby improving cardiac pump function and reducing the risk of arrhythmias.

[0021] Honeycomb-shaped manganese dioxide nanoparticles (HM), utilizing their large specific surface area and mesoporous structure, are loaded with the hydrophobic therapeutic drug puerarin (PUE). To further enhance stability and biocompatibility, the drug-loaded HM is coated with polydopamine (PDA) to form the final PHMPNPs. As a catalytic nanozyme, PHMPNPs can efficiently catalyze the decomposition of endogenously overexpressed hydrogen peroxide (H2O2) into oxygen in the acidic microenvironment of myocardial infarction, thereby actively alleviating tissue hypoxia; simultaneously, this process can scavenge harmful reactive oxygen species (ROS) and reduce oxidative stress damage.

[0022] Hyaluronic acid (HA) is oxidized to its vicinal diol structure via sodium periodate to generate oxidized hyaluronic acid (OHA). Subsequently, the amino group of 3-aminophenylboronic acid undergoes a Schiff base reaction with the aldehyde group of oxidized hyaluronic acid to prepare phenylboronic acid-grafted oxidized hyaluronic acid (OHA-PBA). An amidation reaction between the carboxyl group of gelatin and the amino group of dopamine synthesizes dopamine-grafted gelatin (GelDA). Subsequently, the phenylboronic acid group of OHA-PBA and the catechol group of GelDA form a borate ester bond, ultimately forming a hydrogel. This hydrogel exhibits microenvironment-responsive properties, breaking chemical bonds under the acidic pH and high reactive oxygen species (ROS) levels in the myocardial infarction region.

[0023] Black phosphorus nanosheets have excellent electrical conductivity, enabling them to build "electronic bridges" between myocardial cells and restore electrical signal conduction in the infarcted area.

[0024] Honeycomb manganese dioxide (HM) alleviates oxidative damage and improves tissue hypoxia. In acidic environments, it acts as a catalase, catalyzing the decomposition of endogenous harmful hydrogen peroxide into water and oxygen. This directly provides valuable oxygen to hypoxic tissues. It also scavenges ROS: the reaction process itself consumes hydrogen peroxide, a major ROS, thereby mitigating oxidative stress damage. The mesoporous structure of HM also endows it with nanocarrier function, enabling the synergistic delivery of oxygen and therapeutic drugs. Introducing puerarin-loaded honeycomb manganese dioxide nanozymes into hydrogels endows them with multiple bioactivities, including oxygen production, anti-inflammation, anti-oxidation, and angiogenesis. Embedding them into the hydrogel matrix significantly improves their stability and allows for localized, controlled release in infarcted areas. Its mesoporous structure makes it a natural "drug reservoir" for loading hydrophobic drugs. Puerarin is a multi-functional natural drug, but its water solubility is extremely poor. Loading puerarin molecules into the mesopores of manganese dioxide and encapsulating them with polydopamine to form PHMPNPs improves stability and biocompatibility. When the hydrogel matrix degrades in the microenvironment, PHMPNPs are released; subsequently, puerarin is slowly and continuously released from PHMPNPs, exerting its anti-inflammatory, antioxidant, and angiogenesis-promoting effects in situ.

[0025] Example 2 This invention proposes a method for preparing a microenvironment-responsive injectable hydrogel, comprising the following steps: Synthesis and characterization of PHMP nanoparticles (PHMPNPs): 50 mg KMnO4 was dissolved in 25 mL of water and stirred vigorously for 30 min. 1 mL of oleic acid was slowly added dropwise using a syringe, and after forming a stable emulsion, stirring was continued for 12 h. The mixture was centrifuged at 15000 rpm for 20 min, and HM nanoparticles were collected and washed three times with deionized water. 10 mL of HM solution (1 mg / mL) was dispersed in a mixed solvent containing 2 mg puerarin (V:V water = 1:3), and stirred at room temperature for 2 h. The mixture was centrifuged at 12000 rpm for 10 min and washed three times with deionized water. The precipitate was resuspended in 10 mL of Tris-HCl buffer (10 mM, pH 8.5), and 10 mg of dopamine hydrochloride was added. The reaction was stirred continuously for 6 h. PHMP nanoparticles were collected by centrifugation and washed twice with deionized water. The nanoparticles were characterized by transmission electron microscopy (TEM).

[0026] Synthesis and characterization of black phosphorus nanosheets (BPNs): BPNs were synthesized via liquid-phase exfoliation. Bulk black phosphorus was dispersed in N-methyl-2-pyrrolidone (NMP) at a concentration of 0.2 mg / mL. The solution was sonicated for 1 h using an ultrasonic cell disruptor (BILON92-IID) under ice bath conditions, with a sonication cycle of 3 s on and 2 s off. It was then further sonicated for 20 min in an ultrasonic cleaner (KQ-500DE) for a total of 9 cycles. The BP suspension was centrifuged at 4000 rpm to remove unexfoliated BP crystals, and the supernatant was then centrifuged at 13000 rpm. The precipitate was collected and washed three times with anhydrous ethanol. The BP was characterized by TEM.

[0027] Synthesis and characterization of phenylboronic acid-grafted oxidized hyaluronic acid (OHA-PBA): 2.0 g of hyaluronic acid (HA) was dissolved in 200 mL of water and stirred continuously until completely dissolved. 10 mL of 0.5 mol / L NaIO4 aqueous solution was added dropwise to the HA solution, and the reaction was carried out at 250 rpm for 8 h under dark conditions at room temperature. 2 mL of ethylene glycol was added to the solution, and stirring was continued for 1 h to ensure the inactivation of unreacted NaIO4. The reaction solution was dialyzed against a dialysis bag (MWCO: 7000 Da) for 3 days, and then freeze-dried to obtain a white sponge-like solid (OHA). Oxidized hyaluronic acid was dissolved in water, and 0.68 g of 3-aminophenylboronic acid was added. The reaction was stirred at room temperature for 24 h. After dialyzing against ultrapure water for 3 days, the solution was freeze-dried to obtain a brown sponge-like solid (OHA-PBA), which was stored at -20°C. The chemical structure of OHA-PBA was determined by ¹H NMR spectroscopy.

[0028] Synthesis and characterization of dopamine-grafted gelatin (GelDA): 4.0 g of gelatin was dissolved in 100 ml of 0.1 M PBS solution (pH=5.0) and stirred in a 50 °C water bath until completely dissolved. 2.0 g of EDC and 1.2 g of NHS were added to activate the carboxyl groups, and the mixture was stirred for 20 min. 4.0 g of dopamine hydrochloride was added, and the reaction was carried out at 37 °C under nitrogen protection for 15 h. The reaction solution was dialyzed for 2 days using a dialysis bag with a molecular weight cutoff of 8000-14000 Da, then freeze-dried and stored at -20 °C. The chemical structure of GelDA was determined by HNMR spectroscopy.

[0029] Preparation and characterization of hydrogels: The OHA-PBA solution (6%, w / v) and GelDA solution (6%, w / v) were mixed at room temperature, and PHMP and BPNs were added to form a BP / PHMP@OP-GD hydrogel. The final concentration of PHMP and BPNs in the hydrogel was 200 μg / mL.

[0030] Injectable properties of hydrogels: The hydrogel is loaded into a 1mL syringe and injected to form a specific letter shape.

[0031] pH / ROS response characteristics of hydrogels: The hydrogel was placed in a centrifuge tube, and 200 μL of PBS solution (pH 5.0) was added to assess its pH response characteristics. 200 μL of H₂O₂ solution (100 μM) was added to the system to assess its ROS response characteristics. The hydrogel morphology was photographed after 24 h.

[0032] Oxygen generation properties of hydrogels: 1 mL of hydrogel was placed in a simulated physiological environment (pH 7.4) and a pathological microenvironment (pH 5.0, containing 100 µM H₂O₂). Oxygen production was monitored using a portable dissolved oxygen meter (JPBJ-607A) and recorded continuously for 20 min.

[0033] Safety assessment: Cell compatibility: Human umbilical vein endothelial cells (HUVECs) and rat embryonic cardiomyocytes (H9C2) were seeded at a density of 5000 cells per well in 96-well plates and pre-cultured at 37°C for 24 h. After adding hydrogel extraction buffer, the cells were cultured for 24 h and 48 h, respectively. Then, 10 μL of LCK-8 solution was added to each well, and the cells were incubated at 37°C for 1 h. The absorbance at 450 nm was measured using a microplate reader. The relative cell viability was calculated using the following formula: Cellviability (%) = (A2 - A0) / (A1 - A0) × 100%; A0, A1, and A2 represent the absorbance of the solvent control group, the normal culture control group, and the experimental group, respectively.

[0034] Evaluation of intracellular antioxidant activity: The ability of the hydrogel to scavenge reactive oxygen species (ROS) in H9C2 cells was assessed using the DCFH-DA staining method. H9C2 cells were inoculated at a concentration of 1×10⁻⁶ cells / cells. 5 Cells were seeded at a density of [number] cells / well in 24-well plates and co-cultured with different hydrogels for 24 hours. After removing the medium, fresh medium containing 200 µM H₂O₂ was added to stimulate the cells for 1 hour. Subsequently, the H₂O₂ medium was discarded, and the cells were incubated with 10 µM MDCFH-DA at 37°C in the dark for 30 minutes. Finally, the cells were observed and images were acquired under a fluorescence microscope to assess intracellular ROS levels.

[0035] Establishment of a mouse model of myocardial infarction and in vivo injection of hydrogel: A myocardial infarction model was established using 8-week-old male C57BL / 6 mice. After piloerection, anesthesia, and mechanical ventilation, the heart was exposed via left thoracotomy. The left anterior descending coronary artery was ligated with 6-0 sutures to induce myocardial infarction; whitening of the cardiac tissue after ligation was considered a successful model establishment indicator. Mice were then randomly divided into PBS, OP-GD, BP@OP-GD, BP / PHMP@OP-GD, and sham-operated groups. 20 μL of the corresponding PBS or hydrogel preparation was injected into the infarct area and surrounding tissue. The sham-operated group underwent only thoracotomy and suturing, without coronary artery ligation or injection treatment.

[0036] Echocardiographic and electrocardiographic assessment: On postoperative days 14 and 28, mice with myocardial infarction were anesthetized with isoflurane, fixed to the operating table, and kept under anesthesia. Their limbs were secured with tape, and ultrasound coupling gel was applied to their chests for imaging. Subsequently, M-mode echocardiograms of the heart were acquired using a small animal ultrasound imaging system, and cardiac function parameters such as ejection fraction (EF) and fractional shortening (FS) were measured. All data were averaged over three consecutive cardiac cycles. Electrocardiograms of the mice in each group were recorded at week 4 postoperatively.

[0037] Experimental results: Synthesis of PHMPNPs and BPNs: TEM images confirmed that both the original and drug-loaded nanoparticles maintained a spherical morphology and a uniform size distribution. Figure 1 Meanwhile, TEM characterization of BPNs revealed a typical hierarchical two-dimensional morphology. Figure 2 ).

[0038] Preparation and characterization of phenylboronic acid-grafted oxidized hyaluronic acid (OHA-PBA) and dopamine-grafted gelatin (GelDA): ¹H NMR spectroscopy confirmed the successful synthesis. OHA showed characteristic aldehyde hydrogen peaks at 4.83–5.15 ppm, and OHA-PBA showed proton signals on the benzene ring at 6.52–7.62 ppm, corresponding to the grafted phenylboronic acid moiety. Figure 3 The HNMR spectrum of GelDA showed new peaks corresponding to the aromatic protons (6.56–6.76 ppm) and methylene protons (2.73 ppm) of conjugated dopamine, confirming successful functionalization. Figure 4 ).

[0039] Preparation and characterization of hydrogels: At room temperature, OP-GD hydrogels are formed by simply mixing aqueous solutions of OHA-PBA and GelDA through dynamic crosslinking of Schiff bases and borate ester bonds. Simultaneous addition of BPNs and PHMPNPs during mixing forms BP / PHMP@OP-GD hydrogels.

[0040] pH / ROS response characteristics of hydrogels: The BP / PHMP@OP-GD hydrogel is constructed from a dual cross-linked network based on dynamic Schiff bases and borate ester bonds, achieving a dual response by selectively cleaving imine bonds at acidic pH and selectively cleaving borate ester bonds at elevated ROS levels. The hydrogel gradually decomposes upon exposure to simulated pathological microenvironments with low pH or high ROS.

[0041] Oxygen generation properties of hydrogels: The oxygen release capacity of the hydrogel was evaluated under different pH and ROS conditions to simulate the microenvironment of ischemic myocardium. Figure 5 As shown, dissolved oxygen levels continuously increased over time, due to the catalytic decomposition of H₂O₂ into O₂ by PHMPNPs. The fastest oxygen release was observed under synergistic conditions of pH 5.0 and 100 μM H₂O₂. This sustained oxygen generation capacity helps alleviate myocardial hypoxia and reduce oxidative stress, thereby supporting the repair process.

[0042] Safety evaluation, cell compatibility: Cell compatibility was further assessed using CCK-8 assay. The results showed that the cell viability of H9C2 cardiomyocytes and HUVECs remained above 90% after 24 and 48 hours of culture with hydrogel. Figure 6 This confirms that the cytotoxicity is negligible.

[0043] Evaluation of intracellular antioxidant activity: Excessive accumulation of reactive oxygen species (ROS) after myocardial infarction can lead to persistent inflammation and hinder tissue regeneration. Hydrogels with free radical scavenging capabilities can mitigate these effects by neutralizing oxidants. Intracellular ROS levels in H9C2 cardiomyocytes were measured using a DCFH-DA fluorescent probe under oxidative stress conditions (200 μM H₂O₂). Figure 7 As shown, the green fluorescence intensity of cells cultured with BP / PHMP@OP-GD hydrogel was significantly lower than that of the control group, indicating that intracellular oxidative stress was effectively reduced.

[0044] Establishment of a mouse model of myocardial infarction and evaluation of cardiac function: Based on the favorable properties and biocompatibility of hydrogels in vitro, this study further evaluated the therapeutic potential of a drug-loaded injectable conductive hydrogel system in a mouse model of myocardial infarction. A myocardial infarction model was successfully established by ligating the left anterior descending coronary artery. Changes in cardiac function were assessed by echocardiography at weeks 2 and 4 post-treatment. Figure 8 As shown, the M-mode ultrasound pattern of mice in the PBS group tended to be flatter, with significant left ventricular dilation. This is consistent with the typical pathological manifestations of ventricular wall thinning, cardiac dysfunction, and left ventricular remodeling after myocardial infarction, further validating the successful establishment of the model. Compared with the PBS group, the hydrogel treatment group showed more significant dynamic changes in left ventricular diameter waveform, suggesting partial improvement in cardiac systolic function. Two weeks after treatment, echocardiography showed that EF and FS were significantly reduced in the PBS group, while the left ventricular end-systolic diameter (LVIDs) and end-diastolic diameter (LVIDd) were significantly increased, indicating severe infarct dilation and left ventricular remodeling. Figure 9 All hydrogel treatment groups showed higher EF and FS than the PBS group. The OP-GD hydrogel group also showed some improvement in cardiac function, possibly due to the physical support provided by the hydrogel and its relatively weak ROS scavenging ability. Meanwhile, the EF and FS of the BP@OP-GD and BP / PHMP@OP-GD hydrogel groups were further superior to the OP-GD group, indicating that BP and PHMP nanoparticles have a clear promoting effect on myocardial tissue repair. Notably, the BP / PHMP@OP-GD hydrogel group exhibited the best therapeutic effect, suggesting a synergistic protective effect between BP and PHMP nanoparticles. At week 4, cardiac function in the PBS group further deteriorated, while all hydrogel treatment groups showed sustained improvement, demonstrating the stable and enhanced therapeutic effect of hydrogels in myocardial repair.

[0045] In addition to mechanical improvement, restoring electrical conduction in the infarcted area is crucial for functional recovery. Conductive biomaterials can enhance the electrical properties of scar tissue, bridging healthy and infarcted areas to improve signal propagation. Given the previously established conductivity of the BP / PHMP@OP-GD hydrogel, this study investigated the hydrogel's ability to modulate post-infarction electrophysiology. Electrocardiographic analysis revealed significant pathological Q waves in the PBS group, indicating extensive myocardial necrosis and fibrosis. Figure 10 In contrast, neither the BP@OP-GD nor the BP / PHMP@OP-GD hydrogel groups exhibited this pathological Q wave, confirming that the incorporation of conductive BPNs effectively restored electrical continuity and improved signal transmission in the infarcted myocardium. Furthermore, the BP@OP-GD hydrogel group showed T-wave inversion on the electrocardiogram, indicating myocardial ischemia. This phenomenon was alleviated in the BP / PHMP@OP-GD hydrogel group, possibly due to the combined effect of the continuous oxygen release from PHMPNPs and the angiogenic effect of PUE in improving the ischemic state.

[0046] Example 3 This invention proposes the application of a microenvironment-responsive injectable hydrogel in the preparation of drugs for treating myocardial infarction. The hydrogel, responding to the acidic pH and highly reactive oxygen species microenvironment of the infarcted area, triggers hydrogel degradation and releases black phosphorus nanosheets and polydopamine-coated puerarin-loaded honeycomb manganese dioxide nanoparticles. The black phosphorus nanosheets restore electrical signal conduction in the infarcted area, while the polydopamine-coated puerarin-loaded honeycomb manganese dioxide nanoparticles catalyze the decomposition of endogenous hydrogen peroxide to generate oxygen, alleviating tissue hypoxia and scavenging reactive oxygen species. Simultaneously, the released puerarin exerts anti-inflammatory, antioxidant, and angiogenesis-promoting effects.

[0047] This system overcomes the limitations of traditional single-function therapies by simultaneously addressing key pathological aspects such as hypoxia, oxidative stress, and electrical conduction disruption through a single integrated system. Its pH / ROS dual-response mechanism intelligently and precisely controls drug release, while its catalytic endogenous hydrogen peroxide oxygen production technology actively reverses the vicious cycle of tissue hypoxia. This system integrates efficient hydrophobic drug delivery, injectability, and electrophysiological reconstructive capabilities, achieving perfect implantation through minimally invasive techniques while maintaining long-term structural integrity and functional continuity in a dynamic cardiac environment. It improves EF and FS in mice with myocardial infarction, restores synchronous electrical signal conduction in the infarct area, and improves cardiac function to some extent, providing a novel therapeutic paradigm for functional cardiac regeneration.

[0048] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. A microenvironment-responsive injectable hydrogel, characterized in that, The hydrogel is formed by cross-linking phenylboronic acid-modified oxidized hyaluronic acid and dopamine-grafted gelatin through dynamic Schiff base bonds and dynamic borate ester bonds to form a double cross-linked network structure. The hydrogel contains black phosphorus nanosheets and polydopamine-coated honeycomb manganese dioxide nanoparticles loaded with puerarin.

2. The microenvironment-responsive injectable hydrogel according to claim 1, characterized in that, The concentrations of black phosphorus nanosheets and polydopamine-coated puerarin-loaded honeycomb manganese dioxide nanoparticles in the hydrogel were both 200 μg / mL.

3. The microenvironment-responsive injectable hydrogel according to claim 1, characterized in that, The hydrogel exhibits dual pH / ROS responsiveness, enabling it to break Schiff base bonds under acidic pH conditions in the myocardial infarction region and cleave borate ester bonds under high reactive oxygen species conditions, thereby achieving controlled degradation of the hydrogel and drug release.

4. A method for preparing a microenvironment-responsive injectable hydrogel as described in any one of claims 1-3, characterized in that, Includes the following steps: KMnO4 was dissolved in water, and after vigorous stirring, oleic acid was added dropwise to form a stable emulsion. The mixture was then stirred continuously, and the precipitate was collected by centrifugation to obtain honeycomb-shaped manganese dioxide. Honeycomb-shaped manganese dioxide was dispersed in a mixed solvent containing puerarin, stirred, and then centrifuged to collect the precipitate, thus obtaining honeycomb-shaped manganese dioxide loaded with puerarin. Honeycomb manganese dioxide loaded with puerarin was resuspended in Tris-HCl buffer, dopamine hydrochloride was added, the reaction was stirred continuously, and the precipitate was collected by centrifugation to obtain polydopamine-coated honeycomb manganese dioxide nanoparticles loaded with puerarin. Bulk black phosphorus was dispersed in N-methyl-2-pyrrolidone at a concentration of 0.2 mg / mL, sonicated under ice bath conditions, and the unpeeled black phosphorus crystals were removed by centrifugation. The supernatant was collected and centrifuged again to obtain black phosphorus nanosheets. Hyaluronic acid was dissolved in water, sodium periodate aqueous solution was added, the reaction was stirred in the dark, ethylene glycol was added to deactivate unreacted sodium periodate, the mixture was dialyzed and then freeze-dried to obtain oxidized hyaluronic acid; oxidized hyaluronic acid was dissolved in water, 3-aminophenylboronic acid was added, the reaction was stirred at room temperature, the mixture was dialyzed and then freeze-dried to obtain phenylboronic acid-grafted oxidized hyaluronic acid. Gelatin was dissolved in PBS solution, EDC and NHS were added to activate the carboxyl groups, and then dopamine hydrochloride was added. The reaction was carried out under nitrogen protection, dialyzed, and then freeze-dried to obtain dopamine-grafted gelatin. A solution of phenylboronic acid grafted with oxidized hyaluronic acid, a solution of dopamine grafted with gelatin, honeycomb manganese dioxide nanoparticles loaded with puerarin and black phosphorus nanosheets were mixed to form the microenvironment-responsive injectable hydrogel.

5. The method according to claim 4, characterized in that, The mixed solvent is a mixture of methanol and water, with a volume ratio of methanol to water of 1:

3.

6. The method according to claim 4, characterized in that, The ultrasonic treatment includes: ultrasonic treatment for 1 hour using an ultrasonic cell disruptor, with an ultrasonic cycle of 3 seconds on and 2 seconds off, followed by further ultrasonic treatment for 20 minutes in an ultrasonic cleaner, for a total of 9 cycles.

7. The method according to claim 4, characterized in that, The concentration of the sodium periodate aqueous solution was 0.5 mol / L, the reaction time was 8 hours, and a dialysis bag with a molecular weight cutoff of 7000 Da was used for dialysis.

8. The method according to claim 4, characterized in that, The pH of the PBS solution was 5.0, the ratio of EDC to NHS was 2.0g:1.2g, the reaction temperature was 37℃, the reaction time was 15 hours, and dialysis was performed using dialysis bags with a molecular weight cutoff of 8000-14000 Da.

9. The method according to claim 4, characterized in that, The concentrations of the phenylboronic acid-grafted hyaluronic acid solution and the dopamine-grafted gelatin solution are both 6% w / v.

10. The use of a microenvironment-responsive injectable hydrogel as described in any one of claims 1-3 in the preparation of a drug for treating myocardial infarction.

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