Conductive hydrogel based on bioactive polypeptide as well as preparation method and application of conductive hydrogel

Abstract

The invention discloses conductive hydrogel based on bioactive polypeptide as well as a preparation method and application of the conductive hydrogel, and belongs to the technical field of biological medicines. The invention discloses conductive hydrogel based on bioactive polypeptide. The conductive hydrogel is beneficial to stable transmission of an electric signal, regulation of the level of active oxygen in a body and synchronous contraction of myocardial cells. And a stable platform is provided for continuous release of the angiogenic peptide. In-vitro research shows that the compound has excellent cell compatibility, can remarkably promote endothelial cell proliferation, and has efficient active oxygen scavenging capacity. In a rat myocardial infarction model, the heart function of a mouse is remarkably improved by the composite hydrogel. Histological and immunohistochemical tests further prove the therapeutic effect of the traditional Chinese medicine composition. Therefore, the prepared composite hydrogel can adjust the myocardial microenvironment and electrical activity at the same time, so that the recovery process of myocardial infarction is accelerated, and the composite hydrogel has wide application potential in the aspect of treating myocardial infarction.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a conductive hydrogel based on bioactive polypeptides, and a preparation method and application thereof. Background Art

[0002] Myocardial infarction (MI) remains a leading cause of morbidity and mortality worldwide and is characterized by the irreversible loss of cardiomyocytes and subsequent adverse cardiac remodeling. This condition is typically triggered by coronary artery occlusion, resulting in ischemia and massive cell death in the affected myocardial region. Despite significant advances in pharmacological therapy, percutaneous coronary intervention, and surgical revascularization, the limited regenerative capacity of adult cardiomyocytes often results in incomplete recovery of cardiac function. Following MI, the cardiac microenvironment is characterized by excessive oxidative stress and insufficient vascularization, creating a hostile environment that is not conducive to tissue repair. Over time, fibrotic scarring develops in the infarcted area, impairing the heart's contractile capacity and electrical conduction capacity, ultimately leading to heart failure. Therefore, developing strategies to modulate the microenvironment and restore the conductive microenvironment to promote myocardial repair, restore cardiac function, and prevent adverse remodeling has become a key focus of cardiovascular research.

[0003] After a myocardial infarction, the heart faces significant functional challenges, and promoting angiogenesis and establishing effective electrical conductivity are crucial for cardiac repair. Ischemic events disrupt the blood supply to the myocardial region, leading to damage and death of numerous cardiomyocytes due to lack of oxygen and nutrients. Angiogenesis plays a key role in rebuilding the microvascular network within the infarcted area, providing the oxygen and nutrients necessary for cardiomyocyte survival and functional recovery. At the same time, the rhythmic contraction of the heart depends on well-coordinated electrical signaling to synchronize the activity of cardiomyocytes. Myocardial infarction often disrupts the heart's conduction system, leading to arrhythmias and other electrical abnormalities. Restoring electrical conductivity ensures the stable and accurate propagation of electrical impulses, maintains the synchronized contraction of cardiomyocytes, and preserves normal cardiac pumping function. Disruption of electrical conduction can severely impair the heart's pumping efficiency, increase cardiac workload, and hinder recovery after infarction.

[0004] With the advancement of medical technology, it is clinically possible to restore blood supply to the infarcted area through intravascular stent implantation and thrombolytic and anticoagulant drug treatment, save surviving myocardial tissue, and promote the recovery of cardiac function. For some patients, including those with stent implantation failure, restenosis, ineffective drug treatment, and patency of the distal end of the stenosis, coronary artery bypass grafting can be performed, which has enabled a large number of myocardial infarction patients to survive. However, for patients who further deteriorate into heart failure, although heart transplantation is the gold standard for treating advanced heart failure, its clinical application is limited by the scarcity of donor sources and strong postoperative immune rejection reactions. Therefore, there is an urgent need to develop a new composite hydrogel that can promote angiogenesis to restore blood supply while maintaining normal cardiac electrical conduction. Summary of the Invention

[0005] In order to address the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a conductive hydrogel based on bioactive polypeptides, and a preparation method and application thereof, so as to remove reactive oxygen species in the infarct area, promote the propagation of electrical signals, and transmit pro-angiogenic signals to promote angiogenesis and tissue regeneration.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: 1. A conductive hydrogel based on bioactive polypeptides is provided, characterized in that it includes a hydrogel loaded with cationic angiogenic peptides and carbon nanotubes.

[0007] Furthermore, the mass ratio of the cationic angiogenic peptide to the carbon nanotubes is 1:50-100.

[0008] Furthermore, the cationic angiogenic peptides include KRX peptide, Peptide 12 peptide and Peptide-2 peptide; wherein the amino acid sequence of KRX peptide is shown in SEQ ID NO.1; the amino acid sequence of Peptide 12 peptide is shown in SEQ ID NO.2; and the amino acid sequence of Peptide-2 peptide is shown in SEQ ID NO.3.

[0009] Furthermore, the hydrogel is an oxidized hyaluronic acid-carboxymethyl chitosan hydrogel.

[0010] The present invention provides a method for preparing the above-mentioned conductive hydrogel based on bioactive polypeptides, comprising the following steps: (1) Synthesis of cationic angiogenic peptides by solid-phase peptide synthesis; (2) Synthesizing hyaluronic acid into oxidized hyaluronic acid; (3) The cationic angiogenic peptide prepared in step (1), the oxidized hyaluronic acid prepared in step (2), carboxymethyl chitosan and carbon nanotubes are dissolved in an aqueous solution and stirred evenly to obtain a product.

[0011] Furthermore, the synthesis of cationic angiogenic peptides by solid phase peptide synthesis in step (1) includes the following steps: (1) Dissolve the amino acid in N,N-dimethylformamide solution; then add the resin and condensing agent, microwave heat, remove the resin, and wash with detergent; (2) Deprotect the 9-fluorenylmethyloxycarbonyl protecting group on the amino acid using DMF containing piperidine, then remove the resin and wash with detergent; (3) Repeat steps (1)-(2) according to the amino acid sequence of the polypeptide; (4) Using a lysis buffer to lyse the product obtained in step (3) for 2-3 hours, then precipitating the lysis buffer and drying it to obtain a crude polypeptide product; (5) dissolving the crude polypeptide product obtained in step (4) and then purifying and separating it; (6) The product obtained in step (5) is subjected to rotary evaporation and freeze drying.

[0012] Furthermore, the condensing agent is at least one of 1-hydroxyphenyl-4,5-diphenylimidazole, N,N'-diisopropylcarbodiimide, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbopolamide, O-benzotriazole-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine; the detergent is at least one of N,N-dimethylformamide, dichloromethane and methanol; and the cleavage solution includes 95wt% trifluoroacetic acid, 2.5wt% triisopropylsilane and 2.5wt% deionized water.

[0013] Furthermore, in step (2), synthesizing hyaluronic acid into oxidized hyaluronic acid includes the following steps: dispersing hyaluronic acid in deionized water, then adding sodium periodate solution dropwise to the hyaluronic acid solution, and reacting at room temperature under light-proof conditions; after the reaction is completed, purifying the obtained product to obtain.

[0014] Furthermore, before purifying the obtained product, ethylene glycol needs to be added to quench the excess sodium periodate.

[0015] Furthermore, the obtained product was purified using a dialysis membrane.

[0016] The present invention also provides a use of the above-mentioned conductive hydrogel based on bioactive polypeptide in the preparation of a drug for treating myocardial infarction.

[0017] The present invention has the following beneficial effects: (1) The present invention integrates carbon nanotubes and a cationic angiogenic peptide into an oxidized hyaluronic acid-carboxymethyl chitosan hydrogel by electrostatic adsorption to develop a multifunctional composite hydrogel. This innovative design not only enhances the biocompatibility and electrical conductivity of the hydrogel, but also promotes angiogenesis and scavenges reactive oxygen species. In vitro experiments show that the hydrogel has excellent biocompatibility, significant ROS scavenging ability, and can promote the proliferation and migration of endothelial cells. In a rat myocardial infarction model, the hydrogel effectively improved cardiac function, reduced infarct size, promoted angiogenesis, and alleviated oxidative stress. These findings demonstrate the potential of Gel@CK hydrogel as a promising strategy for the treatment of myocardial infarction and other cardiovascular diseases, providing a comprehensive solution for cardiac repair and functional recovery.

[0018] (2) The present invention combines cationic angiogenic peptides with carbon nanotubes, which have both excellent electrical conductivity and angiogenic effects. This design not only gives the hydrogel excellent electrical conductivity, which helps to stabilize the propagation of electrical signals and the synchronous contraction of cardiomyocytes, but also provides a stable platform for the sustained release of angiogenic peptides, preventing their rapid diffusion. By precisely combining electrical properties with biological activity, a synergistic microenvironment is created that can promote angiogenesis to restore blood supply while maintaining normal cardiac electrical conduction. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of CNT and KRX adsorption; Figure 2 The mass spectra of KRX, the conductivity of CNTs at different concentrations, and the effects of different concentrations of KRX on HUVE cell activity on the first and third days; Figure 3 Graph showing the effects of different concentrations of CNT and KRX peptide on cell activity; Figure 4 TEM images and Zeta potential images of CNT before and after KRX adsorption, IR spectra and H-NMR spectra of HA and OHA; Figure 5 Figure 1 is an injectable diagram of the hydrogel; Figure 6 SEM images and rheological images of each group of hydrogels; Figure 7 Graph showing the antioxidant capacity of each group of hydrogels; Figure 8 EIS images and CV images of Gel and Gel@CK hydrogels, and the effect of each group of hydrogels on H9C2 cell activity on day 1; Figure 9 The LED experiment diagram of Gel@CK hydrogel and the conductive pen experiment diagram are shown; Figure 10 Figure 1 shows the protective effect of each group of hydrogels on H9C2 cells and the ROS scavenging ability of each group of hydrogels on H9C2 cells treated with hydrogen peroxide on day 1; Figure 11 This is the effect of each group of hydrogels on the proliferation of HUVE cells on the 3rd day; Figure 12 The effect of each group of hydrogels on the migration of HUVE cells; Figure 13 The diagram shows the tube-forming effect of each group of hydrogels on HUVE cells; Figure 14 The cardiac function test diagrams of each group of hydrogels; Figure 15 The degradation diagram of the hydrogel in vivo and the heart diagram of the experimental mice on the 28th day; Figure 16 The staining sections of each group of hydrogels on ROS, TUNEL and WGA in the heart; Figure 17 Figure 2 shows the composite hydrogels in each group promoting angiogenesis; Figure 18 The images of heart thickness, infarct size and electrocardiogram processing of each group of hydrogels on day 28. DETAILED DESCRIPTION

[0020] The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, the experiments were performed under conventional conditions or those recommended by the manufacturer. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0021] Example: A conductive hydrogel based on bioactive polypeptides, including a hydrogel loaded with cationic angiogenic peptides and carbon nanotubes, wherein the preparation method comprises the following steps: (1) Synthesis of cationic angiogenic peptide KRX: KRX peptide was synthesized by solid-phase peptide synthesis. Alanine (0.5 g / 8 mL), d-aspartic acid (0.66 g / 8 mL), lysine (0.75 g / 8 mL), methionine (0.6 g / 8 mL), d-asparagine (0.96 g / 8 mL), proline (0.54 g / 8 mL), arginine (4.16 g / 32 mL), and tyrosine (0.74 g / 8 mL) were accurately weighed at a concentration of 0.2 mM, dissolved in DMF (N,N-dimethylformamide), and then placed in a synthesizer. The coupling reaction was carried out by mixing the resin with an amino acid solution, Oxyma (1 mM), and N,N-diisopropylcarbodiimide (DIC, 0.5 mM), followed by microwave heating at 90 °C for 2 min. The resin was washed four times with DMF between each deprotection and condensation step. The fluorenylmethyloxycarbonyl (Fmoc) protecting group was removed using 20% piperidine in DMF at 90°C. The resin was then washed three times with DMF and three times with dichloromethane (DCM). The peptide was cleaved from the resin using a cleavage solution consisting of 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIPS), and 2.5% deionized water for 3 hours. The resulting peptide was precipitated with cold anhydrous ether and then dried to obtain a crude product. The crude peptide was dissolved in a water-acetonitrile mixture (4:1 by volume) and purified by high-performance liquid chromatography (HPLC). The purified peptide solution was concentrated by rotary evaporation, freeze-dried, and stored at -20°C.

[0022] (2) Synthesis of oxidized hyaluronic acid (OHA): 10 g of hyaluronic acid (HA) was dispersed in 300 mL of deionized water. Under continuous stirring, 20 mL of sodium periodate solution (150 mg / mL) was added dropwise to the completely dissolved HA solution. The reaction was carried out at room temperature in the dark for 6 h to ensure that the hydroxyl groups on the HA backbone were completely oxidized to form aldehyde groups. After the reaction was completed, 20 mL of ethylene glycol was added to quench the excess sodium periodate. The solution was then dialyzed using a dialysis membrane with a molecular weight cutoff of 3.5 kDa to remove unreacted reagents and by-products. The dialysis process lasted for 3 days. After dialysis, the purified OHA solution was freeze-dried to obtain a white porous solid.

[0023] (3) Preparation of conductive hydrogel based on bioactive peptides: OHA (30 mg), carboxymethyl chitosan (CMCS, 60 mg), carbon nanotubes (CNT, 15 mg) from step (2) and cationic angiogenic peptide KRX (0.2 mg) from step (1) were dissolved in 1 mL of aqueous solution. The mixture was then vigorously stirred for 1 min to ensure complete dissolution and homogeneity, thereby forming a composite hydrogel named Gel@CK.

[0024] Comparative Example 1: A method for preparing a hydrogel (1) Prepare OHA by referring to the method of step (2) of the example.

[0025] (2) OHA (30 mg) and carboxymethyl chitosan (CMCS, 60 mg) from step (1) were dissolved in 1 mL of aqueous solution. The mixture was then vigorously stirred for 1 min to ensure complete dissolution and homogeneity, thereby forming a hydrogel, named Gel.

[0026] Comparative Example 2: Preparation method of a composite hydrogel (1) Prepare KRX peptide according to the method of step (1) of Example; (2) Prepare OHA by referring to the method of step (2) of the embodiment; (3) OHA (30 mg), carboxymethyl chitosan (CMCS, 60 mg) from step (2), and cationic angiogenic peptide KRX (0.2 mg) from step (1) were dissolved in 1 mL of aqueous solution. The mixture was then vigorously stirred for 1 min to ensure complete dissolution and homogeneity, thereby forming a composite hydrogel, named Gel@KRX.

[0027] Comparative Example 3: Preparation method of a composite hydrogel (1) Prepare OHA by referring to the method of step (2) of the embodiment; (2) OHA (30 mg), carboxymethyl chitosan (CMCS, 60 mg), and carbon nanotubes (CNT, 15 mg) from step (1) were dissolved in 1 mL of aqueous solution. The mixture was then vigorously stirred for 1 min to ensure complete dissolution and homogeneity, thereby forming a composite hydrogel, named Gel@CNT.

[0028] Experimental Example 1: Mechanical Properties of Conductive Hydrogels Based on Bioactive Peptides The rheological and self-healing properties of the hydrogels were determined using a rheometer (MCR 302, Anton Paar GmbH, Austria). Frequency sweep tests were performed at 37°C with a strain setting of 1% and a frequency range of 1 to 100 rad / s. Strain sweep tests were performed at a frequency of 1 Hz and a strain range of 1% to 2000%.

[0029] Experimental Example 2: Antioxidant Properties of Conductive Hydrogels Based on Bioactive Peptides The antioxidant capacity of each composite hydrogel was tested using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 3-ethylbenzothiazoline-6-sulfonic acid (ABTS). The DPPH free radical has a single electron and strongly absorbs at 519 nm, resulting in a purple ethanolic solution. In the presence of an antioxidant, the DPPH free radical is scavenged, causing the solution to lighten, with the degree of fading correlated with the extent of scavenging. 1 mg of solid DPPH was dissolved in 24 mL of anhydrous ethanol, sonicated in the dark for 3 minutes, and shaken thoroughly to ensure uniform distribution. 1 mL of the DPPH solution prepared in the above steps was taken to an absorbance between 0.6 and 1.0. If the absorbance was too high, additional solvent was added. If the absorbance was too low, additional DPPH solid or the original solution was added. 0.5 mL of the lyophilized hydrogel was added to 2.5 mL of the DPPH solution and incubated at 37°C in the dark for 30 minutes. The sample was then transferred to a cuvette using a UV-visible spectrophotometer (UV-2401PC, Shimadzu Corporation), and the absorbance at 519 nm was measured and recorded. The clearance rate was calculated as follows: Clearance rate (%) = (A0 - A0) / A0 × 100% (A0 is the absorbance before sample addition, and A is the absorbance after sample addition).

[0030] ABTS reacts with potassium persulfate (K2S2O8) to form a green ABTS radical. This radical has a maximum absorption at 734 nm, so its concentration can be determined by measuring the absorbance at 734 nm. Mix 0.2 mL of a 7.4 mM ABTS diammonium salt stock solution with 0.2 mL of a 2.6 mM K2S2O8 stock solution and incubate at room temperature in the dark for 12 hours. Then, dilute the mixture with pH 7.4 phosphate buffer until the absorbance reaches approximately 0.8. This solution serves as the ABTS radical working solution. Add 0.5 mL of the lyophilized hydrogel to 2.5 mL of the ABTS solution and allow the reaction to proceed in the dark for 6 minutes. The solution is then transferred to a cuvette using a UV-visible spectrophotometer (UV-2401PC, Shimadzu Corporation), and the absorbance at 734 nm is measured and recorded. The formula for calculating the clearance rate is: Clearance rate (%) = (B1-B) / B1×100% (B1 is the absorbance before adding the sample, and B is the absorbance after adding the sample).

[0031] Test Example 3: Cell Viability The cell viability of human umbilical vein endothelial cells (HUVECs) and rat cardiac myoblasts (H9C2 cells) was assessed using the CCK-8 assay. Cell viability was assessed using a co-culture method, with 50 μL of each composite hydrogel added to a 96-well plate. For fluorescein diacetate (FDA) / propidium iodide (PI) staining, H9C2 cells and HUVECs were uniformly seeded in 96-well plates (6 × 10 cells / well). After treatment with 50 μL of the different hydrogel samples for 24 and 72 hours, the cultured cells were stained with a mixed solution of FDA (40 μg / mL) and PI (20 μg / mL) in PBS at 37°C for 1 minute in the dark. Cells were then imaged using an inverted fluorescence microscope (Leica DMI 4000, Germany) with excitation and emission wavelengths of 488 nm and 530 nm for FDA and 535 nm and 615 nm for PI, respectively.

[0032] To simulate in vivo reactive oxygen species (ROS) damage to cardiomyocytes and evaluate the protective effect of the hydrogel on damaged cardiomyocytes, H9C2 cells were treated with 300 μM hydrogen peroxide (H2O2) for 2 hours. The H2O2 was then removed and the hydrogel (50 μL) was added for co-culture for 48 and 72 hours. The cells were then stained for viability and death using the aforementioned protocol with FDA / PI and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). According to the manufacturer's instructions, DCFH-DA was diluted 1:1000 in serum-free medium to a final concentration of 10 μM. The cell culture medium was removed, and an appropriate volume of the diluted DCFH-DA solution was added to completely cover the cells. The cells were incubated at 37°C for 20 minutes. The cells were washed three times with serum-free medium and observed under an inverted fluorescence microscope (Leica DMI 4000, Germany) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0033] Experimental Example 4: HUVEC cell migration assay HUVEC cells were seeded in a 6-well plate and cultured for 24 hours. After HUVEC adhered, they were scratched using a 10μL pipette tip. The cells were then washed twice with PBS buffer. Subsequently, HUVEC were co-cultured with different hydrogel samples. At fixed time points (0 hours, 12 hours, and 36 hours), the scratched cells were photographed under bright field background. Cell migration ability was calculated by comparing the area of the enclosed area to the initial scratch area.

[0034] Experimental Example 5: HUVEC tube formation experiment 20 μL / well of Matrigel (BD Biosciences) was added to a 24-well plate and allowed to spread overnight in a 4°C refrigerator. The plate was incubated in a cell culture incubator for 30 minutes. 1 mL of standard culture medium containing HUVECs was then seeded onto the Matrigel surface (1 × 10 cells per well). 6 After 12 h, FDA-stained HUVECs were added and the cells were observed using a fluorescence microscope. Imaging software was used to calculate the head length of the tubes and the number of tubes.

[0035] Experimental Example 6: Rat Myocardial Infarction (MI) Model The animal part of this experiment was reviewed and approved by the Medical Ethics Committee of Sichuan University (approval number K2023010). Male Sprague-Dawley myocardial infarction model rats (Chengdu Dashuo Experimental Animal Co., Ltd., 120 g) were used. The rats were anesthetized with 3% isoflurane-oxygen mixture via a ventilator (Nanjing Calvin Biotechnology Co., Ltd.). Subsequently, the left anterior descending artery (LAD) was permanently ligated at the fourth intercostal space through left thoracotomy as described in previous studies. During this procedure, electrocardiogram monitoring showed ST-T segment elevation, which confirmed that the rat myocardial infarction model was successfully established. The infarcted area was pale in the anterior part of the heart. Then, 30 μL of hydrogel was injected into two sites of the infarcted area, and the treated animals were divided into 5 groups (n = 8 per group) as follows: (1) myocardial infarction group (MI group), (2) Gel hydrogel group, (3) Gel@KRX hydrogel group, (4) Gel@CNT hydrogel group, and (5) Gel@CK hydrogel group. The sham-operated control group underwent the same procedure, but without LAD ligation. After surgery, the cannula was removed and the rats were observed for approximately 30 minutes until they regained activity. The rats were then sacrificed at the desired time point for sampling.

[0036] Test Example 7: Cardiac Function Assessment To examine cardiac function in each hydrogel-treated rat group, echocardiography was performed 14 and 28 days after myocardial infarction surgery, after the rats were anesthetized with a 3% isoflurane-oxygen mixture. Echocardiography was performed using a Philips CX 50 ultrasound system equipped with a Philips L15-7io high-frequency probe. To assess each parameter, measurements from three different imaging periods were averaged. Ejection fraction (EF), fractional shortening (FS), left ventricular end-diastolic volume (EDV), and end-systolic volume (ESV) were measured. All measurements were averaged over three consecutive periods.

[0037] Experimental Example 8: Characterization of hydrogel degradation in vivo Hydrogel degradation was assessed by injecting Gel-Cy 7 into rats undergoing myocardial infarction. The Gel-Cy7 conjugate was synthesized by reacting CMCS with Cy 7 (cyanine 7 dye). The final product, in a total volume of 1 mL, contained 6% (w / v) CMCS and 0.01% (w / v) Cy 7-N-hydroxysuccinimide (NHS). The in vivo degradation of the hydrogel was monitored at predetermined time points (1, 3, 5, 7, 14, and 28 days) using a small animal 3D in vivo optical imaging system (IVIS Spectrum).

[0038] Experimental Example 9: Cardiac Morphometry and Immunohistochemistry (IHC) On days 3, 7, 14, and 28, rats were anesthetized and sacrificed. Hearts were collected, stained for reactive oxygen species (ROS) in liquid nitrogen, frozen, and embedded. The remaining sections were stained with 4% paraformaldehyde. Cardiac morphology and infarct size were assessed using hematoxylin and eosin (HE) and Masson's trichrome staining. Immunohistochemical staining was performed as previously described. ROS levels during myocardial infarction were assessed using ROS staining. Cell apoptosis within the infarcted area was assessed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Neovascularization was assessed using α-smooth muscle actin (α-SMA) and CD31. The cross-sectional area of cardiomyocytes (CMs) within the infarcted area was determined using wheat germ agglutinin (WGA). Connexin 43 (CX43) and α-actin staining were used to further assess cardiac functional recovery after hydrogel treatment. All images were analyzed and measured using ImageJ software.

[0039] Experimental Example 10: Quantitative Analysis of Gene Expression Using Real-Time Polymerase Chain Reaction (qPCR) Myocardial infarction tissues were collected from each experimental group, and the relative mRNA expression levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were quantitatively evaluated using the methods described in detail in the Supporting Information section.

[0040] Data are presented as mean ± standard deviation (n = 3). Statistical analysis was performed using two-way analysis of variance (ANOVA) with a Student's t-test. Differences were considered statistically significant when ****p < 0.001, ***p < 0.005, **p < 0.01, and *p < 0.05 were considered statistically significant; ns indicates no significant difference.

[0041] Results and Analysis (1) Characterization and optimal concentration of carbon nanotube / angiogenic peptide complex (CNT / KRX) The mammalian endothelial differentiation gene (EDG) promotes endothelial cell proliferation, chemotaxis, and vascular morphogenesis. A cationic nine-amino acid peptide (KRX) derived from EDG3 was synthesized to allow electrostatic interaction with negatively charged carboxylated carbon nanotubes (CNTs). The peptide was introduced to improve the biocompatibility, stability, and dispersibility of the carboxylated carbon nanotubes, thereby enhancing their therapeutic potential for treating myocardial ischemia in the setting of myocardial infarction (MI). Figure 1 As shown, carbon nanotubes are electrostatically adsorbed on KRX peptides through the negative charge on their surfaces and the positive charge on the surface of KRX peptides, forming a CNT / KRX complex.

[0042] The theoretical relative molecular mass of KRX peptide is M=1149.9 Da. The mass spectrum of KRX peptide is shown in Figure 2 As shown in A, M+H + =1150.3Da and M+Na + =1173.2Da, which is consistent with the theoretical relative molecular mass, indicating that the peptide has been successfully synthesized. Subsequently, the optimal concentration of KRX peptide to promote angiogenesis was screened. The cell viability of human umbilical vein endothelial cells (HUVECs) was quantitatively detected using a CCK-8 detection kit at different concentrations of 50μg / mL, 100μg / mL, 200μg / mL, and 400μg / mL. The results are shown in Figure 2. Figure 2 As shown in Figures CD, HUVEC cell proliferation was measured on days 1 and 3. The results showed that cell survival significantly increased with increasing peptide concentration. At 200 μg / mL, the relative cell survival rate reached approximately 122.08%, which was not significantly different from the survival rate at 400 μg / mL.

[0043] The optimal concentration of carbon nanotubes was determined by evaluating the conductivity and cytotoxicity of carbon nanotubes on rat cardiac myoblasts (H9C2 cells). The conductivity of carbon nanotubes at different concentrations was measured using a TruEbox 01RC system. Figure 2 As shown in Figure 2, the conductivity increased with increasing concentration, reaching 0.59 mS / cm at 15 mg / mL, and no significant difference was observed at 20 mg / mL. To evaluate cytotoxicity, H9C2 cells were treated with carbon nanotubes at concentrations of 10, 15, 20, and 30 mg / mL, and cell viability was quantitatively analyzed using a CCK-8 assay kit. Figure 3As shown in Figures AB, cell viability was measured 24 and 72 hours after treatment. The results showed that cells treated with 15 mg / mL of carbon nanotubes maintained viability comparable to that of the untreated control. Based on the standard range of cardiac conductivity of 0.5-0.9 mS / cm and the cytotoxicity profile, 15 mg / mL was determined to be the optimal concentration of carbon nanotubes.

[0044] In the electrostatically adsorbed CNT / KRX complex, according to the concentration range determined above, KRX peptides at concentrations of 200 μg / mL, 300 μg / mL, and 400 μg / mL were further selected and adsorbed with 15 mg / mL of carbon nanotubes, respectively, and then used to screen the survival rate of HUVECs. Figure 3 As shown in Figure C, in the CNT / KRX system, the 300 μg / mL KRX peptide concentration significantly increased HUVEC proliferation on day 3, with no significant difference compared to the 400 μg / mL KRX peptide concentration. This result further confirms that 300 μg / mL is the optimal working concentration of KRX peptide in CNT / KRX. Therefore, this concentration was ultimately selected for further characterization and subsequent hydrogel preparation.

[0045] In order to confirm the electrostatic adsorption between carbon nanotubes and KRX peptide, the morphological characteristics of the original carbon nanotubes and the carbon nanotubes mixed with KRX peptide were characterized by transmission electron microscopy (TEM). Figure 4 As shown in Figure A, a clear peptide layer was observed between the carbon nanotubes, which provided direct evidence for the adsorption of KRX peptide on the surface of carbon nanotubes. In addition, Zeta potential analysis showed that the surface charge of carbon nanotubes significantly shifted from -30.1 mV to -12.5 mV after the addition of KRX peptide (see Figure 4). Figure 4 B), further confirming the electrostatic interaction between KRX peptide and carbon nanotubes, and also showing that most of the KRX peptide has been adsorbed on the carbon nanotubes.

[0046] (2) Characterization of oxidized hyaluronic acid (OHA) polymers and conductive hydrogels based on bioactive peptides To treat myocardial infarction, CNT / KRX was incorporated into an injectable natural polysaccharide hydrogel with good biosafety to achieve minimally invasive delivery and enhance its retention in the infarcted area, thereby making it a long-lasting conductive matrix. The OHA polymer was fully characterized by Fourier transform infrared spectroscopy (FT-IR) and hydrogen nuclear magnetic resonance (¹H NMR), confirming its successful synthesis. Figure 4 As shown in C, compared with pure hyaluronic acid (HA), the FT-IR spectrum of OHA at 1720 cm -1 There is an obvious C=O stretching vibration peak at 2720cm-1 and 2820cm -1 The CH stretching vibration peak at α is enhanced. These spectral changes confirm that the aldehyde group is successfully introduced into the HA molecular chain after oxidation. Figure 4 As shown in Figure D, the ¹H NMR spectrum of OHA shows three distinct peaks at 5.01 ppm, 5.10 ppm, and 5.20 ppm, confirming the formation of aldehyde groups in the OHA structure. Subsequently, a hydrogel was synthesized via a reaction between carboxymethyl chitosan (CMCS) and OHA, driven by Schiff base crosslinking between the amino groups of CMCS and the aldehyde groups of OHA. By optimizing the composition, a hydrogel with optimal performance was prepared using a formulation containing 3% OHA and 6% CMCS, which completely gelled within 10 minutes. The resulting hydrogel exhibited excellent injectability, as shown in Figure 3. Figure 5 As shown, it can be squeezed out smoothly through a 1 mL syringe needle and accurately write the word "MI".

[0047] (3) Characterization of the morphology and properties of conductive hydrogels based on bioactive peptides The cross-sectional morphology of the freeze-dried hydrogels was examined using field emission scanning electron microscopy (FE-SEM). Figure 6 As shown in Figure A, the hydrogel samples exhibited a relatively uniform pore structure. These results indicate that the hydrogels have well-defined and structurally similar networks, and their porous structures provide sufficient space for cell growth, adhesion, and proliferation. In addition, rheological tests were performed to evaluate the mechanical properties of the hydrogel samples. Figure 6 As shown in B, the storage modulus (G') of all hydrogel samples is greater than the loss modulus (G''), confirming that they have solid-like properties, with an average G' of approximately 100 Pa. When the strain exceeds 1000%, G' is lower than G'', indicating that the hydrogel has broken; when the strain exceeds 1000%, the G' of all hydrogel samples drops rapidly, but when the strain returns to 1%, G' quickly returns to its original level. Rheological experiments show that the hydrogel has excellent self-healing ability. The reactive oxygen species (ROS) scavenging ability of the hydrogel was systematically evaluated using DPPH and ABTS experiments. The results are shown in Figure 7 As shown in the figure, compared with the groups without carbon nanotubes (Gel group and Gel@KRX group), the solution color of the carbon nanotube-wrapped hydrogel groups (Gel@CNT group and Gel@CK group, among which the Gel@CK group is a hydrogel wrapped with CNT / KRX complex) is significantly lighter, indicating that its ROS scavenging activity is enhanced; quantitative analysis shows that the ROS scavenging efficiency of the carbon nanotube-wrapped hydrogel is about 60%, which is significantly better than that of other groups.

[0048] To further investigate the electrochemical properties of the conductive hydrogel, an electrochemical workstation was used to perform tests using a three-electrode system. Electrochemical impedance spectroscopy (EIS) was used to evaluate the resistance of the conductive hydrogel, where the charge transfer resistance (Rct) was represented by the diameter of the semicircle in the Nyquist plot. Figure 8 As shown in A, Gel@CK hydrogel exhibits lower Rct (1400Ω) and better conductivity (0.59mS / cm) compared with Gel hydrogel. Cyclic voltammetry (CV) curve ( Figure 8 B) It further shows that Gel@CK hydrogel has excellent charge storage capacity and electrochemical activity, which is consistent with the EIS results. To intuitively characterize its conductive properties, Gel@CK hydrogel was applied to multi-color light-emitting diode (LED) circuit experiments and conductive pen lighting experiments. The results show that the hydrogel can stably power the LED and conductive pen and emit bright light, indicating that it has excellent electrical signal transmission capabilities (see Figure 9 In summary, these analyses confirmed that the Gel@CK hydrogel exhibited excellent electrical conductivity, effectively mimicking the conductive microenvironment of native cardiac tissue. This laid a solid foundation for improving electrical integration in the infarcted area and ultimately achieving cardiac regeneration.

[0049] (4) Evaluation of cytotoxicity and oxidative stress protection of conductive hydrogels based on bioactive peptides on H9C2 cells Initially, the cytotoxicity of the composite hydrogel was evaluated using CCK-8 assay and live / dead staining. Figure 8 As shown in Figures CD, on day 1, there was no significant difference in cell viability between the carbon nanotube-encapsulated hydrogel group and the control group. This confirms that carbon nanotube encapsulation does not induce significant cytotoxicity in H9C2 cells. These results collectively demonstrate that the composite hydrogel exhibits excellent biocompatibility and is suitable for further biological applications.

[0050] In order to evaluate the protective effect of composite hydrogels on cardiomyocytes, an oxidative stress model was established using H9C2 cells treated with hydrogen peroxide (H2O2). First, the protective effect of various hydrogels on H9C2 cell oxidative damage induced by H2O2 was evaluated using the live cell staining method. Figure 10 As shown in Figures A and 10C, after 1 day of treatment, a large number of dead cells were observed in the H2O2 (300 μM) group, while the cell viability of the hydrogel-treated groups increased to varying degrees; the cell viability of the Gel@CNT group and the Gel@CK group reached 69.48% and 73.26%, respectively, which were significantly higher than that of the H2O2 group (51.16%). Subsequently, DCFH-DA staining was used to detect the intracellular ROS level to evaluate the ROS scavenging ability of the hydrogel. The results are shown in Figure 10C. Figure 10As shown in Figure 2, the ROS level in the control group was extremely low, while the ROS accumulation in the H2O2 group increased significantly. In contrast, the ROS levels (green fluorescence) in the Gel@CNT and Gel@CK groups containing carbon nanotubes were significantly lower than those in the H2O2 group. Quantitative analysis of ROS fluorescence intensity showed that, relative to the H2O2 group set as 100%, the ROS levels in the Gel@CNT and Gel@CK groups decreased to 45.14% and 37.37%, respectively. In contrast, the ROS level in the Gel@KRX group without carbon nanotubes was higher, at 82.21% (see Figure 2). Figure 10 D). These results indicate that carbon nanotubes play a key role in the effective scavenging of ROS.

[0051] (5) Effects of bioactive peptide-based conductive hydrogels on HUVECs proliferation, migration, and tube formation The effect of hydrogel on the proliferation of HUVECs was evaluated by co-culture experiments. Figure 11 As shown, the relative cell viabilities of the KRX peptide-encapsulated Gel@KRX and Gel@CK groups at 72 h were 124.56% and 125.68%, respectively, indicating that they had a significant proliferative effect on HUVECs. A major challenge in the repair of myocardial infarction is the lack of vascularization. Therefore, promoting angiogenesis is a promising therapeutic strategy for the treatment of myocardial infarction. First, the proliferation and migration of pro-angiogenic HUVECs were evaluated. A scratch assay was performed to evaluate the effect of hydrogel samples on the proliferation / migration of HUVECs in vitro. At 12 h, significant cell migration was observed in the KRX peptide-encapsulated hydrogel groups (Gel@KRX and Gel@CK) compared with the other groups without KRX peptide; by 36 h, the scratches in the Gel@KRX and Gel@CK groups were almost completely healed, while the other groups still had unhealed areas. These results indicate that encapsulation of KRX peptide significantly promoted the migration of HUVECs. Quantitative analysis of HUVECs migration further supported these findings (see Figure 12 ).

[0052] The above results indicate that KRX peptide enhances the migration ability of HUVECs. Therefore, HUVEC tube formation experiments were performed to verify the angiogenic potential of the hydrogel. Quantitative analysis of tube formation, including the number of junctions and total tube length, showed that the KRX peptide-encapsulated hydrogel group significantly promoted HUVEC tube formation compared with the control group and the group without KRX peptide (see Figure 13 These findings highlight the potential of KRX peptide-encapsulated hydrogels to promote vascularization for the treatment of myocardial infarction.

[0053] (6) Evaluation of the therapeutic effect of conductive hydrogels based on bioactive peptides in vivo To evaluate the therapeutic efficacy of the composite hydrogel on myocardial infarction in vivo, an acute myocardial infarction model was established in rats by permanent ligation of the left anterior descending (LAD) coronary artery. The rats were randomly divided into six groups (n = 8 per group): a sham-operated group and five myocardial infarction injury groups, each receiving different interventions, including injection of phosphate-buffered saline (PBS) (MI group), CMC / OHA hydrogel (Gel group), Gel@CNT hydrogel, Gel@KRX hydrogel, and Gel@CK hydrogel. Cardiac function parameters, including ejection fraction (EF), fractional shortening (FS), end-diastolic volume (EDV), and end-systolic volume, were measured by echocardiography on days 14 and 28. Compared with the sham-operated group, EF and FS were significantly reduced in all rats with myocardial infarction injury. Fourteen days after treatment, echocardiography revealed that the Gel@CK group had the highest EF and FS among all myocardial infarction injury groups. By day 28, the myocardial infarction condition in the Gel@CK group was further improved, with an average EF of 82.86% and FS of 46.26% (see Figure 14 In order to evaluate the retention of the hydrogel in vivo, in vivo fluorescence imaging was performed by binding Cyanine 7 dye to the hydrogel molecular chains. Figure 15 As shown in A, the hydrogel gradually degraded, and even after 28 days, a portion of it remained, effectively retaining the carbon nanotubes to maintain the conductivity. Figure 15 B) further supports these findings and is consistent with the above conclusions. These results indicate that the composite hydrogel has the potential for long-term therapeutic application in the treatment of myocardial infarction. On day 28, macroscopic evaluation of cardiac morphology showed that the Gel@CK group exhibited better therapeutic effects compared with other myocardial infarction injury groups, and the retention of carbon nanotubes was observed in the infarct area (see Figure 15 C).

[0054] (7) Evaluation of the antioxidant, anti-apoptotic and anti-cardiomyocyte hypertrophy effects of conductive hydrogels based on bioactive peptides in vivo After myocardial infarction, a large surge of ROS can be observed. To clarify the therapeutic mechanism of hydrogel in the treatment of myocardial infarction, its efficacy in alleviating the increase of ROS after myocardial infarction in vivo was first studied. Figure 16As shown in AB, after 3 days of treatment, the ROS fluorescence signal and relative fluorescence intensity of the Gel@CK group were significantly reduced compared with other myocardial infarction injury groups, indicating that it has a strong ROS scavenging ability at the infarct site. If the excessive accumulation of ROS cannot be properly neutralized, it will induce cardiomyocyte apoptosis. Therefore, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining method was used to evaluate the apoptosis of cardiomyocytes. Compared with other myocardial infarction injury groups, the Gel@CK group showed the most significant reduction in cardiomyocyte apoptosis by 25.68% ( Figure 16 CD). This therapeutic effect is likely attributed to the synergistic effect between the CNTs and the proangiogenic peptide KRX in the hydrogel, which together exert antioxidant effects and promote cell proliferation. In addition, on the 7th day after treatment, the cross-sectional area of cardiomyocytes in the peri-infarct region was quantitatively assessed using wheat germ agglutinin (WGA) staining. Morphometric analysis showed that Gel@CK treatment significantly reduced pathological cardiomyocyte hypertrophy compared to the untreated myocardial infarction injury group, with an average cross-sectional area of 234.4 μm. 2 (See Figure 16 EF). These histological results provide strong evidence that Gel@CK effectively inhibits maladaptive cardiac hypertrophy in the border zone of infarcted myocardium, suggesting its potential therapeutic role in prevention.

[0055] (8) Evaluation of the effects of bioactive peptide-based conductive hydrogels on angiogenesis and cardiac function in vivo To explore the mechanism by which the composite hydrogel promotes angiogenesis, the key factors involved in this process, namely hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF), were evaluated. Figure 17 As shown in Figures CD, on day 28, the expression levels of HIF-1α and VEGF were the highest in the Gel@CK group. These results indicate that Gel@CK enhances the expression of pro-angiogenic factors. To further explore whether the hydrogel can accelerate the formation of new blood vessels in the infarcted area, immunofluorescence staining and quantitative analysis of CD31 and α-smooth muscle actin (α-SMA) were performed. Figure 17 As shown in Figures A and 17E, on day 28, the positive cell rates of these two markers in the Gel@CK group were significantly higher than those in the other myocardial infarction injury groups. These findings suggest that Gel@CK promotes cell proliferation and angiogenesis in the peri-infarct area in vivo.

[0056] CX 43 is a key protein located in the gap junctions of myocardial cells. It plays a key role in regulating intercellular electrical signal conduction and mechanical coupling, thereby maintaining normal cardiac electrophysiological function. At the same time, α-actin, as an important component of the myocardial cell skeleton, not only contributes to the stability of cell structure, but also plays a key role in myocardial contraction. In this study, CX 43 and α-actin were used as biomarkers to evaluate the therapeutic effect of hydrogel treatment on the recovery of cardiac function in a rat myocardial infarction model. The results are as follows Figure 17 As shown in Figures B and 17F, the expression levels of CX43 and α-actin in the Gel@CK-treated group were significantly increased compared to the other myocardial infarction injury groups, reaching 53.94% and 51.78%, respectively. These findings indicate that Gel@CK effectively promotes the structural repair and functional recovery of cardiac tissue after myocardial infarction, further confirming its potential application in cardiac regenerative therapy.

[0057] (9) Evaluation of the in vivo effects of conductive hydrogels based on bioactive peptides on cardiac repair and electrical integration Myocardial infarction usually leads to significant thinning of the ventricular wall and abnormal collagen deposition. To evaluate the therapeutic effect, HE staining and Masson trichrome staining were performed on the 28th day after treatment ( Figure 18 A). Quantitative analysis showed that among all myocardial infarction injury groups, the Gel@CK group had the thickest myocardial wall and the smallest infarct area (see Figure 18 CD).

[0058] In vitro experiments confirmed that Gel@CK hydrogel has suitable electrical conductivity. Therefore, the effect of Gel@CK hydrogel on the electrical integration of the myocardial infarction area was further evaluated in vivo. The QRS complex duration ( Figure 18 B). Previous studies have shown that scar tissue prolongs the QRS interval. Figure 18 As shown in Figure E, the QRS duration of the Gel@CK group was significantly shorter than that of the myocardial infarction group (MI group) and the Gel group. These results indicate that Gel@CK hydrogel has the potential to improve electrical integration and has application value in myocardial repair.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A conductive hydrogel based on bioactive polypeptides, characterized in that: The invention comprises a hydrogel loaded with cationic angiogenic peptides and carbon nanotubes.

2. The conductive hydrogel based on bioactive polypeptide according to claim 1, characterized in that The cationic angiogenic peptides include KRX peptide, Peptide 12 peptide and Peptide-2 peptide; wherein the amino acid sequence of the KRX peptide is shown as SEQ ID NO.1; the amino acid sequence of the Peptide 12 peptide is shown as SEQ ID NO.2; and the amino acid sequence of the Peptide-2 peptide is shown as SEQ ID NO.

3.

3. The conductive hydrogel based on bioactive polypeptide according to claim 1, characterized in that The hydrogel is an oxidized hyaluronic acid-carboxymethyl chitosan hydrogel.

4. The method for preparing a conductive hydrogel based on a bioactive polypeptide according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Synthesis of cationic angiogenic peptides by solid-phase peptide synthesis; (2) Synthesizing hyaluronic acid into oxidized hyaluronic acid; (3) The cationic angiogenic peptide prepared in step (1), the oxidized hyaluronic acid prepared in step (2), carboxymethyl chitosan and carbon nanotubes are dissolved in an aqueous solution and stirred evenly to obtain a product.

5. The preparation method according to claim 4, characterized in that The synthesis of cationic angiogenic peptides by solid phase peptide synthesis in step (1) comprises the following steps: (1) Dissolve the amino acid in N,N-dimethylformamide solution; then add the resin and condensing agent, heat in a microwave, remove the resin, and wash with detergent; (2) Deprotect the 9-fluorenylmethyloxycarbonyl protecting group on the amino acid using DMF containing piperidine, then remove the resin and wash with detergent; (3) Repeat steps (1)-(2) according to the amino acid sequence of the polypeptide; (4) Using a lysis buffer to lyse the product obtained in step (3) for 2-3 hours, then precipitating the lysis buffer and drying it to obtain a crude polypeptide product; (5) dissolving the crude polypeptide product obtained in step (4) and then purifying and separating it; (6) The product obtained in step (5) is subjected to rotary evaporation and freeze drying.

6. The preparation method according to claim 5, characterized in that The condensing agent is at least one of 1-hydroxyphenyl-4,5-diphenylimidazole, N,N'-diisopropylcarbodiimide, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbopolamide, O-benzotriazole-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine; the detergent is at least one of N,N-dimethylformamide, dichloromethane and methanol; and the lysis solution includes 95wt% trifluoroacetic acid, 2.5wt% triisopropylsilane and 2.5wt% deionized water.

7. The preparation method according to claim 4, characterized in that The synthesis of hyaluronic acid into oxidized hyaluronic acid in step (2) includes the following steps: dispersing hyaluronic acid in deionized water, then adding sodium periodate solution dropwise to the hyaluronic acid solution, and reacting at room temperature under light-proof conditions; after the reaction is completed, purifying the obtained product to obtain.

8. The preparation method according to claim 7, characterized in that Before purifying the obtained product, ethylene glycol needs to be added to quench the excess sodium periodate.

9. The preparation method according to claim 7, characterized in that The purification is to purify the obtained product using a dialysis membrane.

10. Use of the conductive hydrogel based on the bioactive polypeptide according to any one of claims 1 to 3 in the preparation of a drug for treating myocardial infarction.

Images