Application of injectable hydrogel in preparation of medicine for treating myocardial infarction

Abstract

The invention provides application of injectable hydrogel in preparation of a medicine for treating myocardial infarction. The injectable hydrogel is obtained by mixing aniline tetramer grafted gelatin, adipic dihydrazide modified hyaluronic acid and an aldehyde group-containing metal polyphenol complex. The injectable hydrogel has antioxidant activity, conductivity and angiogenesis promoting activity performance, and myocardial repair is promoted through synergistic regulation and control.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically to the application of an injectable hydrogel in the preparation of drugs for treating myocardial infarction. Background Technology

[0002] Myocardial infarction is the leading cause of death and disability worldwide, and it is showing a trend towards affecting younger people. After a heart attack, the infarcted area forms non-electrically active scar tissue that gradually expands, leading to arrhythmias, ventricular remodeling, and ultimately heart failure. Current clinical treatments can only alleviate symptoms and slow disease progression; heart transplantation faces challenges such as high costs, severe donor shortages, and immune rejection; stem cell transplantation can reduce inflammation in the infarcted area, promote angiogenesis, and improve cardiac function; however, clinical trials of stem cell therapy have been unsuccessful, with severe limitations including low cell retention and survival rates. Therefore, researching new treatment methods to promote myocardial repair is of great significance.

[0003] Tissue engineering based on biomaterials offers new hope for the treatment of myocardial infarction. Current research primarily focuses on constructing functional materials to regulate key pathological processes in myocardial infarction repair. After a myocardial infarction, a large number of cardiomyocytes die or undergo apoptosis due to ischemia and hypoxia. Insufficient blood and oxygen supply leads to blockage of the mitochondrial respiratory chain, resulting in a significant increase in reactive oxygen species (ROS) levels, causing cellular dysfunction, oxidative damage to proteins, lipids, and DNA, and ultimately irreversible cell damage and death. Using biomaterials to remove excess ROS and alleviate oxidative stress can reduce inflammation and improve cell survival. Scar tissue formed after a myocardial infarction lacks electrical activity, and surviving cardiomyocytes lose coupling in the non-conductive scar, leading to abnormal electrical signal propagation and ventricular dysfunction. Electroactive biomaterials can establish electrical conduction between non-infarcted myocardial cells, facilitating synchronized cardiac contraction, promoting cardiomyocyte proliferation, migration, and differentiation, and promoting myocardial tissue repair and regeneration. Furthermore, pro-angiogenic biomaterials can promote the rapid formation of a dense capillary network from the boundary region to the infarct center to meet the high metabolic demands of myocardial infarction repair, helping to promote waste removal, nutrient diffusion, and gas exchange, and limiting the continued death of cardiomyocytes in the infarct periphery. However, there is still a lack of novel functional materials with multiple biological activities that can simultaneously regulate oxidative stress, promote electrocoupling, and promote angiogenesis.

[0004] Furthermore, the construction of existing functional materials for myocardial infarction treatment still faces key bottlenecks: Biomaterials with antioxidant and angiogenesis-promoting properties are mainly produced by grafting drugs onto a polymer backbone or loading them into the material's cross-linked network through physical means / carriers. However, pre-grafting presents challenges such as altered drug bioactivity, limited loading capacity, and mismatched release behavior; physical mixing can lead to drug burst release. To achieve controlled drug release, drugs typically need to be pre-encapsulated in micelles, liposomes, or other nanomaterials, resulting in complex biomaterial systems. The preparation of biomaterials with conductive properties faces challenges such as the lack of biodegradability of conductive components and the potential to induce chronic inflammation. The rigid structure of conductive components makes them prone to aggregation, leading to uneven mechanical and electrical properties, thus limiting their clinical application. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides an application of an injectable hydrogel in the preparation of drugs for treating myocardial infarction. This injectable hydrogel possesses antioxidant activity, conductivity, and pro-angiogenic activity, synergistically promoting myocardial repair. An aldehyde-containing metal polyphenol complex is used as a crosslinking agent to crosslink aniline tetramer grafted with gelatin and modified with adipic acid dihydrazide, thereby constructing a hydrogel network. Furthermore, through physical mixing and dynamic Schiff base bonding, a carrier with pro-angiogenic activity is loaded into the hydrogel, achieving sustained release of the carrier at the site of myocardial infarction, improving the therapeutic effect of myocardial infarction repair, and overcoming the drawbacks of traditional drug delivery methods.

[0006] To achieve the above objectives, the present invention provides the following technical solution: an injectable hydrogel, which is obtained by mixing aniline tetramer grafted gelatin, adipic acid dihydrazide modified hyaluronic acid and an aldehyde-containing metal polyphenol complex.

[0007] Furthermore, the concentration of the aniline tetramer grafted gelatin solution was 2 wt%–6 wt%, the concentration of the adipic acid dihydrazide-modified hyaluronic acid solution was 1 wt%–3 wt%, and the concentration of the aldehyde-containing metal polyphenol complex was 1 × 10⁻⁶. -3 mol / L ~ 2.5 × 10 -3 mol / L; The volume ratio of aniline tetramer grafted gelatin solution, adipic acid dihydrazide modified hyaluronic acid solution, and aldehyde-containing metal polyphenol complex solution is 1:1:(0.02~0.05).

[0008] Furthermore, the hydrogel is also loaded with a carrier that promotes angiogenesis; after the carrier is mixed with aniline tetramer grafted gelatin, it is then mixed with adipic acid dihydrazide-modified hyaluronic acid and an aldehyde-containing metal polyphenol complex to obtain an injectable hydrogel.

[0009] Furthermore, the carrier with pro-angiogenic activity is L-arginine, and the mass concentration of L-arginine is 0.5 mg / mL to 1 mg / mL.

[0010] Furthermore, 1) the preparation of aniline tetramer grafted gelatin is as follows: Under a protective atmosphere, a solution of N-hydroxysuccinimide-terminated aniline tetramer was added dropwise to a gelatin solution, the reaction was carried out, and the precipitate was collected. The precipitate was washed, dried, reconstituted, and filtered to obtain the final product. Hydrochloric acid was added to the filtrate to adjust the pH to 2-3, followed by alcohol precipitation. The precipitate was collected to obtain HCl-doped aniline tetramer grafted gelatin. 2) The preparation of adipic acid dihydrazide-modified hyaluronic acid solution is as follows: Hyaluronic acid solution and adipic acid dihydrazide were mixed to obtain a mixture. A condensing agent was added dropwise to the mixture to adjust the pH to 6.8. The reaction product was purified to obtain adipic acid dihydrazide-modified hyaluronic acid. 3) The preparation of aldehyde-containing metal polyphenol complexes is as follows: Iron salt solution was added dropwise to 2,3,4-trihydroxybenzaldehyde solution, the pH was adjusted to 10, and the reaction was stirred at room temperature to obtain 2,3,4-trihydroxybenzaldehyde complex with iron ions, which is a metal polyphenol complex containing aldehyde group.

[0011] The present invention also provides the application of injectable hydrogel in the preparation of drugs for regulating the oxidative stress environment after myocardial infarction. An effective dose of injectable hydrogel is taken and injected into the myocardial infarction and the edge area of ​​myocardial infarction at five points.

[0012] Furthermore, the effective dose is 100 μL.

[0013] The present invention also provides the application of an injectable hydrogel in the preparation of angiogenic active pharmaceutical ingredients.

[0014] The present invention also provides the application of injectable hydrogel in the preparation of myocardial repair drugs after myocardial infarction, wherein an effective dose of injectable hydrogel is taken and injected into the myocardial infarction and myocardial infarction edge areas at five points.

[0015] Furthermore, the effective dose is 100 μL.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: This invention provides an injectable hydrogel. By using an aldehyde-containing metal polyphenol complex as a crosslinking agent in the hydrogel system, the crosslinking network of the hydrogel can be easily constructed. Simultaneously, the catechol and adjacent phenolic hydroxyl groups of this complex endow the hydrogel with excellent antioxidant properties. By grafting a conductive oligomer—aniline tetramer—on gelatin, the hydrogel is endowed with uniform conductivity, avoiding the introduction of rigid conductive components such as metal nanomaterials and conductive polymers. The aniline tetramer and iron ions give the hydrogel electronic and ionic conductivity, reducing the contact impedance of the hydrogel and facilitating the reconstruction of electrical conduction between non-infarcted areas. L-arginine is loaded onto the hydrogel through physical action and Schiff base bonds, avoiding burst release of small molecule drugs and achieving long-term controlled release, promoting angiogenesis after myocardial infarction. Furthermore, the hydrogel is crosslinked by Schiff base bonds and metal ion ligand coordination bonds, exhibiting dynamic and reversible crosslinking characteristics, endowing the hydrogel with good mechanical stability and self-healing properties, and the dynamic crosslinking network is conducive to cell migration and infiltration. Catechins and adjacent phenolic hydroxyl groups endow the hydrogel with excellent tissue adhesion properties, facilitating the formation of a fixed, seamless contact between the hydrogel and surrounding myocardial tissue, promoting cell infiltration and electrical signal transduction. Therefore, this hydrogel shows great potential in the treatment of myocardial infarction. Attached Figure Description

[0017] Figure 1 These are representative SEM images of the lyophilized hydrogel.

[0018] Figure 2 For the injectability of hydrogels, Figure 2 A represents the viscosity-shear rate curve of the hydrogel. Figure 2 B is a hydrogel injected into deionized water using a syringe.

[0019] Figure 3 For the mechanical properties of hydrogels, Figure 3 A is the compression curve. Figure 3 B represents the energy storage modulus and the loss modulus.

[0020] Figure 4 The curves show the changes in storage modulus and loss modulus of the hydrogel with respect to angular frequency.

[0021] Figure 5 The self-healing properties of hydrogels, Figure 5 A represents the hydrogel strain amplitude scanning curve. Figure 5 B represents the cyclic alternating strain scan curve of the hydrogel. Figure 5 C represents the self-healing properties of the hydrogel.

[0022] Figure 6 The antioxidant activity of the hydrogel, Figure 6 A, Figure 6 C Figure 6 E, Figure 6G is DPPH • ABTS •+ O2 •- and • The mechanism of OH removal process; Figure 6 B Figure 6 D、 Figure 6 F, Figure 6 H represents the hydrogel's effect on DPPH. • ABTS •+ O2 •- and • Statistical results of OH removal efficiency.

[0023] Figure 7 The results show the statistical results of RAW 264.7 cell viability under oxidative stress. The Blank group served as a blank control group and was not treated with hydrogel.

[0024] Figure 8 These are representative images of RAW 264.7 cells stained with DCFH-DA fluorescence. The Blank group served as the blank control group, without any hydrogel treatment.

[0025] Figure 9 This refers to the electrical conductivity of the hydrogel. Figure 9 A represents the conductivity of the hydrogel; Figure 9 B is the frequency impedance curve of the hydrogel; Figure 9 C represents the statistical results of the interfacial impedance of the hydrogel at 20 Hz.

[0026] Figure 10 The curve shows the release of L-arginine in PBS buffer.

[0027] Figure 11 This refers to the tissue adhesion properties of hydrogels. Figure 11 A is a schematic diagram of the overlap shear test, using pig skin and pig myocardium to test the tissue adhesion strength of the hydrogel; Figure 11 B represents the statistical results of the adhesion strength of the hydrogel to pigskin; Figure 11 C represents the statistical results of the adhesion strength of the hydrogel to porcine myocardium; Figure 11 Photograph D shows the adhesion of hydrogel to porcine myocardium.

[0028] Figure 12 This relates to the biocompatibility of hydrogels. Figure 12 A represents the statistical results of H9c2 cell viability after co-culturing with hydrogel extracts of different concentrations; Figure 12 B represents the statistical results of cell proliferation rate of H9c2 cells after 1 day and 2 days of co-culture with hydrogel; Figure 12 C represents a representative live / dead staining image of H9c2 cells after 1 day of co-culture with hydrogel; the TCP group is a blank control group without hydrogel.

[0029] Figure 13 The hydrogel exhibits pro-angiogenic activity. Figure 13 A is a schematic diagram of the Transwell cell migration experiment; Figure 13 B shows representative live / dead staining images of HUVECs observed on the Z-axis, in three dimensions, and on the X-axis after 1 day of Transwell migration. Figure 13 C represents the quantitative statistical results of HUVECs cell migration depth; Figure 13 D represents the quantitative statistical results of cell migration observed from the Z-axis; Figure 13 E represents the quantitative statistical results of the fluorescence area of ​​HUVECs migrating in each region after being averaged along the Z-axis (each region is 8 μm high).

[0030] Figure 14 28 days after hydrogel treatment for myocardial infarction Figure 14 A represents heart tissue H&E and Figure 14 B is a representative photograph of Masson staining.

[0031] Figure 15 Hydrogels can improve heart structure. Figure 15 A represents the thickness of the left ventricular wall. Figure 15 B represents the statistical results of myocardial infarction area and... Figure 15 C represents the statistical results of collagen content.

[0032] Figure 16 Hydrogels can improve heart function. Figure 16 A represents the M-mode ultrasound images of rats in each group at specific time points after surgery; Figure 16 B represents the ejection fraction EF and Figure 16 C represents the statistical result of the shortened fraction FS.

[0033] Figure 17 This is a hydrogen nuclear magnetic resonance spectrum. Figure 17 A is an aniline tetramer; Figure 17 B is the 1H NMR spectrum of aniline tetramer grafted gelatin.

[0034] Figure 18 This is the UV-Vis absorption standard curve of aniline tetramer.

[0035] Figure 19 The 1H NMR spectrum of hyaluronic acid modified with adipic acid dihydrazide.

[0036] Figure 20 The UV-Vis absorption spectra of aniline tetramer, aniline tetramer-grafted gelatin, and hydrochloric acid-doped aniline tetramer-grafted gelatin are shown.

[0037] Figure 21 Fourier transform infrared spectra of three precursor materials and the prepared injectable hydrogel. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0039] This invention provides a multifunctional injectable hydrogel for the treatment of myocardial infarction, which modulates the post-infarction oxidative stress environment and promotes electrical conduction and angiogenesis. This system avoids the problems of traditional functionalized hydrogel drug delivery systems, such as complexity, tissue inflammation caused by non-degradation of conductive components, and inhomogeneous electromechanical properties due to the easy aggregation of conductive components. In preparing the multifunctional injectable hydrogel of this invention, aniline tetramer is grafted onto gelatin (AT). -g A mixed solution of α-GA and L-arginine, adipic acid dihydrazide-modified hyaluronic acid (AHA), and a solution of 2,3,4-trihydroxybenzaldehyde and iron ion complex (THA@Fe) were mixed evenly and reacted to obtain the above-mentioned multifunctional injectable hydrogel. Among them, aniline tetramer grafted gelatin (AT) -g A hydrogel network was constructed using α-GA, adipic acid dihydrazide-modified hyaluronic acid (AHA), and 2,3,4-trihydroxybenzaldehyde with an iron complex (THA@Fe). This hydrogel network loaded with a pro-angiogenic carrier. The hydrogel was formed through a Schiff base reaction between the aldehyde groups on the complex and the amino groups on gelatin and hyaluronic acid. Aniline tetramers and iron ions endowed the hydrogel with electrical conductivity, while the crosslinking agent 2,3,4-trihydroxybenzaldehyde with the iron complex endowed the hydrogel with antioxidant properties. The aldehyde groups, catechols, and ortho-phenolic hydroxyl groups endowed the hydrogel with tissue adhesion. The pro-angiogenic carrier was loaded into the hydrogel through physical mixing and the formation of Schiff base bonds with the crosslinking agent.

[0040] Injecting the multifunctional injectable hydrogel provided by this invention into the myocardial infarction area can effectively scavenge reactive oxygen species (ROS) and regulate oxidative stress in the infarct area. This hydrogel possesses conductivity similar to natural myocardial tissue, allowing for controlled release of carriers with pro-angiogenic activity, thus promoting angiogenesis. This hydrogel system, by intervening in key pathological processes after myocardial infarction, multidimensionally regulates the myocardial infarction microenvironment, providing a new strategy for the treatment of myocardial infarction.

[0041] The specific preparation steps of the multifunctional injectable hydrogel provided by this invention are as follows: (1) Preparation of hydrogel precursor 1.1) Preparation of aniline tetramer grafted gelatin Synthesis of N-hydroxysuccinimide-terminated aniline tetramer (AT-NHS): Under nitrogen protection, aniline tetramer (AT, 5 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (15 mmol), and N-hydroxysuccinimide (NHS, 15 mmol) were dissolved in anhydrous DMF, and the solution was stirred at room temperature for 24 h. The reaction mixture was then poured into pre-cooled diethyl ether, the precipitate was collected by filtration, washed three times with diethyl ether, and dried at room temperature in a vacuum oven.

[0042] Synthesis of aniline tetramer-grafted gelatin: Gelatin (GT, 3 wt%) was dissolved in a mixed solution of deionized water and dimethyl sulfoxide (DMSO) (H₂O:DMSO (v / v) = 2:1) at 50 °C. An N,N-dimethylformamide solution of N-hydroxysuccinimide-terminated aniline tetramer was added dropwise to the gelatin solution. The reaction was carried out at 50 °C under nitrogen protection for 24 h. After cooling the solution to room temperature, it was poured into ethanol pre-cooled to -80 °C, and the precipitate was collected. After washing with ethanol and diethyl ether, the precipitate was dried at room temperature in a vacuum chamber. The dried solid was dissolved in deionized water to remove unreacted AT-NHS. Hydrochloric acid (HCl, 1 M) was added dropwise to the filtrate until the pH of the solution reached 2-3. The solution was then poured into a large volume of ethanol, and the precipitate was collected to obtain HCl-doped aniline tetramer-grafted gelatin, which was dried at room temperature in a vacuum chamber.

[0043] 1.2) Hyaluronic acid modified with adipic acid dihydrazide 0.5 g of hyaluronic acid was dissolved in 100 mL of deionized water to obtain a 0.5 wt% hyaluronic acid solution. Then, 10 g of adipic acid dihydrazide was added to this solution. Next, 0.8 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.7 g of 1-hydroxybenzotriazole were dissolved in DMSO / H₂O (1:1 v / v, 5 mL each) and added to the above reaction mixture. The pH of the solution was adjusted to 6.8 with 1 M HCl. The mixture was stirred at room temperature for 12 hours, and the product was dialyzed against deionized water for 3 days. Then, 5 wt% NaCl was added, dissolved, and precipitated in pre-cooled ice-cold ethanol. The precipitate was redissolved in deionized water and dialyzed for 3 days. Finally, the product was freeze-dried to obtain adipic acid dihydrazide-modified hyaluronic acid (AHA), with an average amination grafting rate of approximately 30%.

[0044] 1.3) Complexes of 2,3,4-trihydroxybenzaldehyde with iron ions 693.54 mg of 2,3,4-trihydroxybenzaldehyde was added to 8 mL of distilled water and dissolved under nitrogen atmosphere at 80 °C. 243.3 mg of anhydrous ferric chloride was dissolved in 5 mL of distilled water and added dropwise to the 2,3,4-trihydroxybenzaldehyde solution. The pH of the solution was adjusted to 10 using 10 M sodium hydroxide solution. After stirring at room temperature for 3 hours, distilled water was added to maintain a total solution volume of 15 mL, yielding the 2,3,4-trihydroxybenzaldehyde-ferric complex THA@Fe. The structure of the complex was characterized using a UV-Vis spectrophotometer.

[0045] (2) Preparation of injectable hydrogel 2.1) The concentration of the aniline tetramer grafted gelatin solution was 2 wt%~6 wt%, the concentration of the adipic acid dihydrazide-modified hyaluronic acid solution was 1 wt%~3 wt%, and the concentration of the 2,3,4-trihydroxybenzaldehyde complex with iron ions was 1×10⁻⁶. -3 mol / L ~ 2.5 × 10 -3 The volume ratio of the three precursor solutions is 1:1:(0.02~0.05), and the injectable hydrogel is also loaded with a carrier with pro-angiogenic activity, with a mass concentration of 0.5 mg / mL~1 mg / mL. Hydrogels containing carriers with pro-angiogenic activity were prepared by first dissolving the carriers in aniline tetramer-grafted gelatin to obtain a mixed solution. Subsequently, drug-loaded hydrogels were prepared using the same method.

[0046] Carriers with pro-angiogenic activity can be small molecule drugs containing amino groups, such as L-arginine, spermidine, spermine, nitric oxide precursor drugs, etc. 2.2) Preferably, the concentration of the aniline tetramer grafted gelatin solution is 6 wt%, the concentration of the adipic acid dihydrazide-modified hyaluronic acid solution is 2%, the concentration of the 2,3,4-trihydroxybenzaldehyde-iron ion complex is 0.1 M, the volume of the aniline tetramer grafted gelatin solution is 100 μL, the volume of the adipic acid dihydrazide-modified hyaluronic acid solution is 100 μL, and the volumes of the 2,3,4-trihydroxybenzaldehyde-iron ion complex are 2 μL, 3 μL, 4 μL, and 5 μL, respectively, to obtain a hydrogel named AT- g- GT / AHA / THA@FeX, where X is the volume of the complex solution X μL under the above preparation conditions; Preferably, the carrier with pro-angiogenic activity is L-arginine, with a mass concentration of 1 mg / mL, and is transported using AT- g -GA / AHA / THA@Fe4 loaded with L-arginine yields AT-g -GA / AHA / THA@Fe4 / Arg.

[0047] The structure and properties of the hydrogel prepared using the above (2.2) were verified. The structures of the raw materials, aniline tetramer grafted gelatin, adipic acid dihydrazide modified hyaluronic acid, and the prepared hydrogel were characterized by proton nuclear magnetic resonance spectroscopy, ultraviolet-visible spectroscopy, and infrared spectroscopy. Figures 17-21 Place: The peaks between 7.0 and 7.4 ppm in the NMR spectrum of aniline tetramer-grafted gelatin represent hydrogen atoms on the benzene ring of the aniline tetramer. The peak at 1504 cm⁻¹ in the infrared spectrum of aniline tetramer-grafted gelatin is also relevant. -1 The characteristic peak at [location] is attributed to the vibrational peak of the benzene ring skeleton in the aniline tetramer, while the amide I band of gelatin is located between 1700 and 1600 cm⁻¹. -1 Within this range, other characteristic peaks of aniline tetramer are covered.

[0048] Adipic acid dihydrazide was used to modify hyaluronic acid methyl hydroxyl groups (HH groups) at 2.26 and 1.66 ppm, representing the methylene hydrogen atoms on the hydrazide. By comparing the number of HH groups on the methyl hydroxyl group of hyaluronic acid at 2.02 ppm with those on the methylene hydroxyl group of the hydrazide, the grafting degree of the hydrazide was calculated to be 30%. The 1715 cm⁻¹ infrared spectrum was observed. -1 C=O stretching vibration peak (amide I) at 1600 cm⁻¹ -1 The bending vibration peak (amide II) in the NH plane at 3290 cm⁻¹, and the peak at 3290 cm⁻¹. -1 The nearby NH stretching broadband indicates the successful preparation of AHA.

[0049] The grafting amount of aniline tetramer was measured using a UV-Vis spectrophotometer according to the aniline tetramer standard curve, and the results are shown in the figure. The calculated grafting amount of aniline tetramer was 3.6 wt%. Furthermore, the UV absorption spectrum of the aniline tetramer showed two characteristic peaks: 315 nm, originating from the π-π* electronic transition of the benzene ring, and 588 nm, originating from the exciton transition (πb-πq) from the benzene ring to the quinone ring. However, on the aniline tetramer-grafted gelatin, the absorption peak of the π-π* electronic transition showed a blue shift of approximately 7 nm. This is because the polymer chain leads to significant steric hindrance, affecting the conjugated structure of the aniline tetramer. After doping the conductive polymer with hydrochloric acid, the polymer chain segments generate charge-carrying polarons, which is the fundamental reason for the conductivity of the conductive polymer. Therefore, hydrochloric acid-doped aniline tetramer-grafted gelatin was used for the subsequent preparation of hydrogels. After HCl doping, a new absorption peak appeared near 433 nm in its UV-Vis absorption spectrum. This peak originated from the polaron band to the π* transition, indicating that the doping of aniline tetramer grafted gelatin with hydrochloric acid was successfully achieved, thereby generating polarons.

[0050] Under alkaline conditions, THA reacts with Fe. 3+ The coordination compound THA@Fe was formed at a molar ratio of 3:1. The absorption peak at approximately 425 nm in the UV-Vis spectrum confirmed the formation of this compound.

[0051] In the infrared spectrum of the hydrogel, it is located at 1638 cm⁻¹. -1 The sharp peak at that point belongs to the aldehyde group of THA@Fe and AT- g- Schiff base bonds are formed between the amino groups of GA and AHA.

[0052] Morphology verification of hydrogels After the hydrogel was freeze-dried, its surface was sputtered with gold and SEM was performed to observe its morphology. Figure 1 Representative SEM images of the lyophilized hydrogel, such as... Figure 1 As shown, the hydrogel possesses a connected porous structure, which facilitates substance exchange and cell infiltration. With increasing cross-linking agent content, the pore size of the hydrogel gradually decreases, indicating the formation of a denser cross-linked network. The addition of L-arginine slightly increases the pore size of the hydrogel; this is because L-arginine undergoes a Schiff base reaction with some of the cross-linking agents, leading to a decrease in the cross-linking density of the hydrogel.

[0053] Injectability verification Figure 2 The results of the hydrogel's injectability verification, such as... Figure 2 As shown in Figure A, the hydrogel exhibits shear-thinning properties, with the viscosity gradually decreasing as the shear rate increases. Figure 2 Photo B shows that the hydrogel can be injected using a syringe and maintain a specific shape after injection.

[0054] Verification of compressive and rheological properties A compression test was performed on a cylindrical hydrogel sample (8 mm diameter, 6 mm height) using a rheometer. The temperature was set at 25 °C, and the sample was compressed at a rate of 6 mm / min until 60% strain was achieved. Figure 3 As shown in Figure A, the hydrogel exhibits good compressibility, maintaining its shape intact even at a compressive strain of 60%. Adjusting the crosslinking agent content significantly alters the hydrogel's strength, which gradually increases with increasing crosslinking agent content. Figure 3 B recorded the rheological properties of the hydrogels. The results showed that the storage modulus (G′) of the hydrogels was greater than the loss modulus (G′′), indicating that the hydrogels possess characteristic viscoelasticity, which is beneficial for maintaining their structural integrity by dissipating energy in dynamic environments. The content of the crosslinking agent also affected the rheological properties of the hydrogels; as the content of the crosslinking agent increased, the storage modulus of the hydrogels gradually increased.

[0055] Stability verification Using a rheometer, a 310 μL hydrogel sample was placed between platforms with a diameter of 20 mm. The distance between the test platforms was set to 1000 μm and the strain to be constant at 1%. The hydrogel was scanned at angular frequencies ranging from 0.1 to 100 rad / s. Figure 4 As shown, the storage modulus (G′) of the hydrogel is consistently higher than its loss modulus (G′′) in the angular frequency range of 0.1 to 100 rad / s, indicating that the hydrogel can maintain its gel state under the dynamic environment of the heart.

[0056] Self-healing performance verification Figure 5 Data to validate the self-healing properties of the hydrogel, such as Figure 5 As shown in Figure A, the critical point of the hydrogel is determined to be 497% by the crossover point in the strain amplitude scan. Figure 5 Results B show that in alternating strain scanning tests, the hydrogel network collapses when the strain exceeds a critical point of 500%, but immediately undergoes self-healing when the strain is reduced to 1%. (Macroscopic illustration) Figure 5 In Figure C, a hydrogel disk was cut in half. After gently placing the two halves together and allowing them to stand at room temperature for 30 seconds, they healed into a single unit and could be stretched without any breakage. These results strongly demonstrate the good stability, viscoelasticity, and self-healing properties of hydrogels. This indicates that hydrogels are not only stable but also mechanically adaptable in dynamic environments.

[0057] In vitro antioxidant performance verification The free radical scavenging ability of hydrogels is primarily mediated by DPPH. • and ABTS •+ It was evaluated as a nitrogen-based free radical model. Figure 6 As shown in B and D, all four types of AT- g -GA / AHA / THA@Fe hydrogels all exhibited significant free radical scavenging activity. The hydrogels also showed resistance to DPPH. • The removal efficiency increased from 63.64% to 91.45%, while for ABTS... •+ The removal rate increased from 72.81% to 100%, both showing a concentration-dependent increase with increasing THA@Fe crosslinking agent content. The O2 removal rate of these hydrogels was studied using the nitroblue tetrazolium method. •- Cleaning efficiency Figure 6 C shows the O2 of the hydrogel. •- The clearance rate also showed a similar upward trend, ranging from 74.80% to 88.08%. Subsequently, Fe was established. 2+ The / H2O2 / salicylic acid reaction system was used to evaluate the hydrogel. •OH removal performance Figure 6 The results showed that the hydrogel exhibited effective •OH scavenging activity, with a scavenging rate ranging from 58.70% to 69.16%.

[0058] Cellular antioxidant performance verification The protective effect of hydrogel on cells under oxidative stress and its ability to scavenge ROS were investigated using RAW 264.7 macrophages. RAW 264.7 cells were incubated with 400 μM H2O2 and lyophilized hydrogel for 24 hours. Cell viability was measured using AlamarBlue®. Figure 7 The results showed that both unloaded and drug-loaded hydrogels exhibited significant protective effects against RAW 264.7 cells, with cell viability values ​​approximately 12 times higher in both hydrogel groups compared to the control group.

[0059] Intracellular ROS levels were characterized using the fluorescent probe DCFH-DA. Figure 8 The results showed that the blank group cells exhibited strong fluorescence after H2O2 treatment, indicating a significant increase in intracellular ROS. In contrast, almost no green fluorescence was detected in either of the hydrogel-treated groups, further confirming the hydrogel's effective ROS scavenging ability.

[0060] Conductivity verification The electrical conductivity of the hydrogel was measured using a digital multimeter, and the results are as follows: Figure 9 A showed that with increasing THA@Fe content, the conductivity of the hydrogel significantly increased by an order of magnitude, from 8.4 × 10⁻⁵ S / cm to 17.9 × 10⁻⁵ S / cm, comparable to the conductivity of healthy native cardiac muscle (5 × 10⁻⁵ to 1.6 × 10⁻³ S / cm). Electrical impedance is an important indicator of charge transfer resistance in conductive hydrogels. The low resistance of the hydrogel will facilitate intercellular communication via bioelectrical signals. To investigate the impedance of the hydrogel, frequency-scanning impedance testing was performed to measure the contact impedance of these hydrogels with human skin. Figure 9 B and 9C show that at high frequencies, the four hydrogels exhibit low impedance, which is attributed to the large capacitive current. At a low frequency of 20 Hz, the interfacial impedance of the hydrogels decreases from 30.81 kΩ to 25.37 kΩ with increasing THA@Fe content, which is a result of enhanced conductivity of the hydrogels and synergistic conduction by ions and electrons.

[0061] Verification of arginine release behavior Add 1 mL of AT- gThe GA / AHA / THA@Fe4 / Arg hydrogel was placed in a 50 mL centrifuge tube containing 10 mL of PBS (pH 7.4) and incubated in a shaker at 37°C and 100 rpm. At predetermined time points, 1 mL of solution was drawn off each time, and 1 mL of fresh PBS was added to maintain a constant volume. The arginine content detection kit from Beijing Box Biotechnology Co., Ltd. was used to quantitatively determine the Arg released from the hydrogel; all operations were strictly performed according to the kit instructions. The release behavior of L-arginine from the hydrogel was tested in PBS pH 7.4 buffer solution. Figure 10 The results showed that the hydrogel released approximately 48% of its volume within the first 24 hours, followed by a slow release over the next 10 days.

[0062] tissue adhesion performance verification The test was conducted by overlapping and shearing experiments on pig skin and pig myocardium. Figure 11 A) The tissue adhesion properties of the hydrogel were studied. For example... Figure 11 As shown in B and C, with the increase of cross-linking agent content, the tissue adhesion strength of the hydrogel to pig skin and pig myocardium gradually increased, with the adhesion strength to pig skin ranging from 4.06 ± 1.28 kPa to 11.56 ± 0.79 kPa, and the adhesion strength to pig myocardium ranging from 3.43 ± 0.30 kPa to 7.60 ± 0.51 kPa. Figure 11 D visually demonstrates that the hydrogel can maintain its adhesion to the myocardium even under torsion, bending, and immersion conditions.

[0063] Biocompatibility verification The cell compatibility of the hydrogel was assessed using leaching and direct contact tests, with untreated rat cardiomyocytes (H9c2 cells) serving as the control group (TCP group). Sterilized lyophilized hydrogels were pre-incubated in serum-free cell culture medium for 24 hours to prepare the leaching solution. A concentration gradient of 1–5 mg / mL was established for culturing H9c2 cells. Figure 12 As shown in Figure A, all hydrogel groups exhibited significant cell compatibility, with cell viability exceeding 90%. To further evaluate cell compatibility, 100 μL of lyophilized hydrogel discs were immersed in culture medium for direct contact experiments. Figure 12 As shown in Figure B, all hydrogel groups exhibited good cell compatibility, indicating that co-culturing with hydrogels did not adversely affect the proliferation of H9c2 cells. These quantitative data are consistent with the cell live / dead staining results, such as... Figure 12 The results showed a large number of live H9c2 cells, exhibiting green fluorescence and a spindle-shaped morphology, with only a small number of dead cells due to cell metabolism and apoptosis (marked with red fluorescence).

[0064] Validation of angiogenesis-promoting activity The pro-angiogenic activity of the hydrogel was evaluated by observing the migration behavior of human umbilical vein endothelial cells (HUVECs) in a Transwell migration assay. Figure 13 A). Due to the hydrogel AT- g -GA / AHA / THA@Fe4 exhibits superior biocompatibility compared to other hydrogels, and also demonstrates excellent bioactivity in scavenging reactive oxygen species (ROS). Therefore, AT was chosen. -g -GA / AHA / THA@Fe4 and AT- g -GA / AHA / THA@Fe4 / Arg was used in studies to promote endothelial cell migration. For example... Figure 13 As shown in Figure B, it can be observed from the Z-axis and 3D projection that both groups of hydrogels exhibit enhanced migration ability of HUVECs compared to the blank group. Figure 13 The quantitative analysis results shown in C and 13D reveal that AT- g The migration rate of HUVECs in the GA / AHA / THA@Fe4 / Arg hydrogel group was significantly higher than that in the blank control group and AT- g -GA / AHA / THA@Fe4 hydrogel assembly, including migration depth (P<0.05) and number of migrating cells (P<0.01). Subsequently, the migration depth along the Z-axis was divided into 11 consecutive 8-micrometer segments, and the distribution of migrating cells was analyzed based on the maximum fluorescent area within each segment. Figure 13 As shown in E, AT- g The migrating cells in the GA / AHA / THA@Fe4 / Arg hydrogel group were mainly concentrated in segments 3 to 8, while the migrating cells in the control group were mainly distributed in segments 2 to 7. g The migrating cells in the -GA / AHA / THA@Fe4 hydrogel group were mainly concentrated in segments 3 to 7. In addition, AT- g The L-arginine (Arg) group showed a significantly higher number of migrating cells in segments 3 through 9 compared to the other two groups. In conclusion, the addition of L-arginine (Arg) endowed the hydrogel with AT- g -GA / AHA / THA@Fe4 / Arg exhibits significant pro-angiogenic activity.

[0065] Verification of the efficacy of hydrogel in repairing myocardial infarction Histological analysis using hematoxylin-eosin (H&E) and Masson staining was used to assess the structural changes in the hearts of different groups four weeks post-surgery. Figure 14 As shown, Sham: sham surgery group, only open-chest surgery was performed; MI: myocardial infarction group, no treatment was received; H&E staining results showed as follows: Figure 14A. Compared with the MI group showing severe inflammatory cell infiltration, both hydrogel groups exhibited significant moderate inflammatory cell infiltration in the infarct area, and the left ventricular (LV) wall thickness was greater in both hydrogel groups than in the control group. Masson staining results showed... Figure 14 Compared to the extensive infarct area and widespread collagen deposition in the MI group, both hydrogel groups showed significantly reduced infarct area and collagen content.

[0066] Furthermore, quantitative analysis was performed on cardiac-related myocardial infarction repair indicators in each group, such as... Figure 15 As shown in Figure A, compared to the Sham group, the left ventricular wall thickness in the MI group was significantly reduced to 1.12 ± 0.12 mm, while the AT- g The LV wall thickness of the GA / AHA / THA@Fe4 group increased to 1.75 ± 0.12 mm (P<0.01). Furthermore, AT- g The LV wall thickness was highest in the GA / AHA / THA@Fe4 / Arg group, reaching 2.15 ± 0.08 mm, showing similarity to AT- g The -GA / AHA / THA@Fe4 group (P<0.05) showed a significantly improved ability to improve cardiac structure compared to the MI group (P<0.01). Figure 15 B and 15C indicate that the hydrogel AT- g -GA / AHA / THA@Fe4 / Arg showed the best therapeutic effect, with the smallest infarct area (11.58 ± 2.12%, P<0.01) and the lowest level of collagen deposition (4.42 ± 1.28%, P<0.01). The therapeutic effect of the hydrogel mainly stems from synergistic therapeutic effects, including mechanical support, scavenging of reactive oxygen species (ROS), and enhanced electrical signal transduction. AT- g The -GA / AHA / THA@Fe4 / Arg hydrogel exhibited the most significant improvements in improving cardiac pumping function and reducing cardiac structural collapse, primarily attributed to the pro-angiogenic effect of the Arg loading. Overall, the injectable hydrogel of this invention demonstrates a significant reversal effect on cardiac function and adverse remodeling following acute myocardial infarction.

[0067] Hydrogels improve heart function. Echocardiography performed at 2 and 4 weeks after surgical treatment for myocardial infarction (MI) assessed the left ventricular function in each group of rats, evaluating the effect of hydrogel on improving cardiac function. Figure 16As shown, compared with the sham group, rats in the MI group exhibited severe impaired pumping function, with a significant and gradual decrease in ejection fraction (EF) (56.19 ± 3.70% after 2 weeks, P<0.01; 53.18 ± 5.58% after 4 weeks, P<0.01) and a decrease in fractional shortening (FS) (25.90 ± 2.43% after 2 weeks, P<0.01; 24.43 ± 3.56% after 4 weeks, P<0.01). Conversely, treatment with the hydrogel injection group (100 μL of injectable hydrogel injected at five points into the myocardial infarction and periphery regions) significantly improved cardiac pumping function. Compared with the MI group, the hydrogel AT- loaded with the pro-angiogenic active drug significantly improved cardiac pumping function. g The GA / AHA / THA@Fe4 / Arg group rats showed the most significant improvement in cardiac function, with EF reaching 79.27 ± 7.50% (P<0.05) and FS reaching 56.58 ± 3.71% (P<0.05) after 4 weeks of hydrogel treatment.

[0068] In summary, the hydrogel provided by this invention possesses the following properties: It exhibits shear-thinning properties, allowing for injection into the myocardial infarction area via a syringe. It possesses excellent mechanical properties and self-healing capabilities, maintaining structural stability and integrity under dynamic cardiac conditions. It has a three-dimensional interconnected porous network structure, facilitating gas and substance exchange. It exhibits excellent antioxidant properties, efficiently scavenging reactive oxygen species and reducing oxidative stress. Under oxidative stress, it enhances macrophage activity and reduces intracellular ROS levels. It is conductive, with conductivity similar to that of natural myocardial tissue; increasing the crosslinking agent content leads to an increasing conductivity and decreasing contact resistance. It displays sustained arginine release, effectively prolonging the duration of arginine action. It significantly promotes vascular endothelial cell migration. It exhibits good blood and cell compatibility, meeting the biosafety requirements for in vivo application of biomaterials. It promotes myocardial repair after myocardial infarction. Tissue staining, echocardiography, and immunofluorescence analysis showed that the hydrogel could effectively reduce the infarct area, reduce fibrosis deposition, and improve the recovery of cardiac function after myocardial infarction.

[0069] Therefore, this invention provides an injectable hydrogel prepared by a simple method that combines ideal properties such as anti-oxidation, conductivity, and angiogenesis. When used for the repair of myocardial infarction (MI), the THA@Fe coordination compound acts as a cross-linking agent, endowing the hydrogel with excellent injectability, self-healing properties, stability, and mechanical adaptability, allowing it to adapt to the dynamic environment of the heart. It also endows the hydrogel with good reactive oxygen species (ROS) scavenging ability. Furthermore, THA@Fe promotes the loading of angiogenic amino acids (L-arginine) through dynamic Schiff base reactions and physical mixing, achieving its controllable long-term release. In addition, THA@Fe provides tissue adhesion to the hydrogel, which is beneficial for substance exchange and signal transduction. Grafting the conductive oligomer aniline onto gelatin imparts good conductivity to the hydrogel, effectively solving the challenges faced by traditional conductive hydrogels, such as limited biodegradability, poor solubility, and the inherent mechanical inconsistency between rigid conductive components and the soft hydrogel matrix. Furthermore, the ionic and electronic conductivity of the hydrogel work together to reduce the interfacial impedance of the hydrogel. In a rat MI model, injectable multifunctional hydrogels were applied to the infarct area, showing significant therapeutic effects and effectively improving cardiac function and structure.

Claims

1. An injectable hydrogel, characterized in that, An injectable hydrogel was prepared by mixing aniline tetramer grafted gelatin, adipic acid dihydrazide-modified hyaluronic acid, and an aldehyde-containing metal polyphenol complex.

2. The injectable hydrogel according to claim 1, characterized in that, The concentration of the aniline tetramer grafted gelatin solution was 2 wt%–6 wt%, the concentration of the adipic acid dihydrazide-modified hyaluronic acid solution was 1 wt%–3 wt%, and the concentration of the aldehyde-containing metal polyphenol complex was 1 × 10⁻⁶. -3 mol / L ~ 2.5 × 10 -3 mol / L; The volume ratio of aniline tetramer grafted gelatin solution, adipic acid dihydrazide modified hyaluronic acid solution, and aldehyde-containing metal polyphenol complex solution is 1:1:(0.02~0.05).

3. The injectable hydrogel according to claim 1, characterized in that, The injectable hydrogel is also loaded with a carrier that promotes angiogenesis; the carrier is mixed with aniline tetramer grafted gelatin, and then mixed with adipic acid dihydrazide-modified hyaluronic acid and an aldehyde-containing metal polyphenol complex to obtain the injectable hydrogel.

4. The injectable hydrogel according to claim 3, characterized in that, The carrier with pro-angiogenic activity is L-arginine, and the mass concentration of L-arginine is 0.5 mg / mL to 1 mg / mL.

5. The injectable hydrogel according to claim 1, characterized in that, 1) The specific preparation of aniline tetramer grafted gelatin is as follows: Under a protective atmosphere, a solution of N-hydroxysuccinimide-terminated aniline tetramer was added dropwise to a gelatin solution, the reaction was carried out, and the precipitate was collected. The precipitate was washed, dried, reconstituted, and filtered to obtain the final product. Hydrochloric acid was added to the filtrate to adjust the pH to 2-3, followed by alcohol precipitation. The precipitate was collected to obtain HCl-doped aniline tetramer grafted gelatin. 2) The preparation of adipic acid dihydrazide-modified hyaluronic acid solution is as follows: Hyaluronic acid solution and adipic acid dihydrazide were mixed to obtain a mixture. A condensing agent was added dropwise to the mixture to adjust the pH to 6.

8. The reaction product was purified to obtain adipic acid dihydrazide-modified hyaluronic acid. 3) The preparation of aldehyde-containing metal polyphenol complexes is as follows: Iron salt solution was added dropwise to 2,3,4-trihydroxybenzaldehyde solution, the pH was adjusted to 10, and the reaction was stirred to obtain 2,3,4-trihydroxybenzaldehyde complex with iron ions, which is a metal polyphenol complex containing aldehyde group.

6. The use of any one of the injectable hydrogels according to claims 1 to 5 in the preparation of pharmaceuticals for regulating post-myocardial infarction oxidative stress environment.

7. The application according to claim 6, characterized in that, Take an effective dose of injectable hydrogel and inject it into five points in the myocardial infarction and periphery areas.

8. The use of any one of the injectable hydrogels according to claims 1 to 5 in the preparation of angiogenic active pharmaceutical ingredients.

9. The use of any one of the injectable hydrogels according to claims 1 to 5 in the preparation of myocardial repair drugs after myocardial infarction.

10. The application according to claim 9, characterized in that, Take an effective dose of injectable hydrogel and inject it into five points in the myocardial infarction and periphery areas.

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