A bioactive polypeptide hydrogel and its preparation method and application

Abstract

The present invention discloses a bioactive polypeptide hydrogel and a preparation method and application thereof, belonging to the field of biomedical technology. The present invention synthesizes an antioxidant peptide MMP12 for scavenging ROS in the MI area, thereby protecting myocardial cells; a pro-angiogenic peptide KRX is used to treat myocardial ischemia during myocardial infarction. Experimental results show that in addition to the antioxidant and pro-angiogenic functions of the bioactive polypeptide hydrogel prepared by the present invention, the combination of the two polypeptides also has significant anti-apoptosis, anti-inflammatory and myocardial fibrosis inhibition effects, which can significantly improve cardiac function, promote the repair of damaged myocardial tissue, and inhibit ventricular wall remodeling, providing a feasible treatment plan for myocardial infarction treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a bioactive polypeptide hydrogel and a preparation method and application thereof. Background Art

[0002] Myocardial Infarction (MI), hereinafter referred to as MI, is a serious heart disease. It is usually caused by blockage of the coronary arteries (the main blood vessels that supply blood to the myocardium) of the heart, resulting in a sharp reduction or interruption of blood supply to the myocardium, which in turn causes myocardial cell necrosis. This blockage is often due to thrombosis in the coronary arteries, which may be triggered by the rupture of unstable plaques of atherosclerosis. Symptoms of MI may include chest pain, difficulty breathing, nausea, sweating, etc., and in severe cases may lead to heart failure, shock, and even death. MI is an emergency medical condition that requires rapid diagnosis and treatment to restore blood supply to the myocardium, reduce myocardial damage, and prevent complications.

[0003] During myocardial infarction, the blockage of the coronary arteries leads to a sharp decrease in myocardial blood supply, causing ischemia and hypoxia of myocardial cells. This ischemic and hypoxic environment will lead to abnormal production of reactive oxygen species (ROS) in myocardial tissue, breaking the dynamic balance of ROS production and elimination. The abnormal accumulation of ROS will damage myocardial cells and aggravate the pathological process of myocardial infarction. At the same time, myocardial infarction is accompanied by a series of changes in blood vessels, such as rupture of atherosclerotic plaques, thrombosis, lumen obstruction, and insufficient blood supply, which further aggravate myocardial damage and dysfunction. Therefore, in the treatment of myocardial infarction, timely and effective elimination of excessive ROS and inhibition of the inflammatory microenvironment, as well as improvement of myocardial blood supply, are of great significance to promoting the repair and regeneration of myocardial tissue.

[0004] Currently, synthetic antioxidant nanozymes-related antioxidants are commonly used to remove ROS in food and biological systems, but increasing evidence shows that the negative effects of artificial antioxidants on health have severely limited their application. Therefore, it is urgent to develop a medical method that has good biosafety, is easily absorbed and metabolized by organisms, and can effectively promote the repair and regeneration of myocardial tissue. Summary of the invention

[0005] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a bioactive polypeptide hydrogel and a preparation method and application thereof, so as to provide a therapeutic means that can effectively promote the repair and regeneration of myocardial tissue and has good biosafety.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: providing a bioactive polypeptide hydrogel, including a hydrogel loaded with bioactive polypeptides; the bioactive polypeptides include an antioxidant peptide MMP12 and an angiogenic peptide KRX; wherein the amino acid sequence of the antioxidant peptide MMP12 is shown in SEQ ID NO.1; the amino acid sequence of the angiogenic peptide KRX is shown in SEQ ID NO.2.

[0007] Furthermore, the hydrogel is methacrylated gelatin.

[0008] The present invention provides a method for preparing the above-mentioned bioactive polypeptide hydrogel, comprising the following steps:

[0009] (1) Methacrylic gelatin is prepared by grafting methacrylic acid groups onto the molecular chains of gelatin;

[0010] (2) Synthesis of biologically active peptides by solid-phase peptide synthesis;

[0011] (3) Using a photoinitiator, the products obtained in step (1) and step (2) are subjected to ultraviolet cross-linking to obtain a crystalline polyol.

[0012] Furthermore, in step (1), methacrylic acid groups are grafted onto the molecular chains of gelatin to prepare methacrylated gelatin by dissolving gelatin in deionized water, heating and stirring, then adding methacrylic anhydride and NaOH solution and reacting for 45-50 hours, dialyzing the reaction product and centrifuging it, collecting the supernatant and freeze-drying it to obtain the obtained product.

[0013] Furthermore, the gelatin is pig skin-derived gelatin or fish skin-derived gelatin.

[0014] Furthermore, the synthesis of the biologically active polypeptide by the solid phase polypeptide synthesis method in step (2) comprises the following steps:

[0015] (1) Weigh the amino acid and dissolve it in N,N-dimethylformamide solution; then add the resin and condensation agent, heat it with microwave, take out the resin, and wash it with detergent;

[0016] (2) Deprotecting the 9-fluorenylmethoxycarbonyl protecting group on the amino acid using DMF containing piperidine, then removing the resin and washing it with a detergent;

[0017] (3) Repeat steps (1)-(2) according to the amino acid sequence of the polypeptide;

[0018] (4) using a lysis solution to lyse the product obtained in step (3) for 2-3 hours, and then blowing the lysis solution to obtain a crude polypeptide product after precipitation;

[0019] (5) dissolving the crude polypeptide product obtained in step (4), and then purifying and separating it;

[0020] (6) The product obtained in step (5) is subjected to rotary evaporation and freeze drying.

[0021] Furthermore, the condensation 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.

[0022] Furthermore, in step (3), the products obtained in step (1) and step (2) are subjected to ultraviolet crosslinking using a photoinitiator. Specifically, the methacryloyl gelatin obtained in step (1) and the bioactive polypeptide obtained in step (2) are dissolved in an aqueous solution, a photoinitiator is added, and then ultraviolet crosslinking is performed for 20-30 seconds to obtain the obtained product.

[0023] Furthermore, the photoinitiator is phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate, diacetophenone or phenylacetone.

[0024] The present invention also provides an application of the bioactive polypeptide hydrogel in preparing medicines for treating myocardial infarction, skin wounds, diabetes or tumors.

[0025] The present invention has the following beneficial effects:

[0026] (1) The present invention synthesizes an antioxidant peptide MMP12 from monkfish muscle protein hydrolysate for removing reactive oxygen species in the myocardial infarction area, thereby protecting myocardial cells. The G protein-coupled receptors of the endothelial differentiation gene (EDG) family have the ability to mediate angiogenic activity, promote endothelial cell proliferation, chemotaxis, and the development of vascular morphology. Therefore, the present invention synthesizes a 9-amino acid angiogenic peptide KRX derived from the second intracellular loop of S1P3 (EDG3) for the treatment of myocardial ischemia during myocardial infarction.

[0027] (2) Methacryloyl gelatin has good biocompatibility and degradability. It can coexist peacefully with biological tissues without causing rejection reactions. It can be gradually degraded into harmless small molecules in the body and absorbed or excreted by the organism. The introduction of methacrylic acid groups makes it photosensitive and can trigger polymerization reactions through ultraviolet light irradiation to form a three-dimensional network structure, making it a carrier for the delivery of bioactive peptides.

[0028] (3) The in vitro and in vivo experimental results of the present invention show that in addition to the antioxidant and angiogenic effects of the bioactive polypeptide hydrogel prepared by the present invention, the combination of the antioxidant peptide MMP12 and the angiogenic peptide KRX also has significant anti-apoptotic, anti-inflammatory and myocardial fibrosis inhibitory effects. These novel combined effects can significantly improve cardiac function and inhibit ventricular wall remodeling. The antioxidant, anti-apoptotic, anti-inflammatory, angiogenic and anti-fibrotic functions effectively improve cardiac function after MI and promote the repair of damaged myocardial tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : are mass spectra of MMP12 polypeptide and KRX polypeptide; wherein, Figure A is the mass spectrum of MMP12 polypeptide; Figure B is the mass spectrum of KRX polypeptide;

[0030] Figure 2 Graphs A and B are high performance liquid chromatograms of MMP12 and KRX polypeptides; wherein, Graph A is a high performance liquid chromatogram of MMP12 polypeptide; Graph B is a high performance liquid chromatogram of KRX polypeptide;

[0031] Figure 3 : are circular dichroism spectra of MMP12 polypeptide and KRX polypeptide; wherein, Figure A is the circular dichroism spectra of MMP12 polypeptide; Figure B is the circular dichroism spectra of KRX polypeptide;

[0032] Figure 4 The antioxidant effects of MMP12 polypeptide at concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL and 3 mg / mL; Figure A shows the DPPH active oxygen scavenging rate; Figure B shows the ABTS active oxygen scavenging rate; Figure C shows the scavenging effect;

[0033] Figure 5 The results are as follows: HUVECs cells were incubated with KRX polypeptide at 10 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL for 3 days;

[0034] Figure 6 The results are shown in Figure 1 after incubation of scratched HUVECs cells with KRX peptide at 50 μg / mL, 100 μg / mL and 200 μg / mL for 0 h, 12 h and 36 h; Figure A is the bright field shooting result; Figure B is the migration rate statistics of HUVECs cells;

[0035] Figure 7 The figure shows the effect of KRX peptide on blood vessels at 50 μg / mL, 100 μg / mL and 200 μg / mL; Figure A shows blood vessel imaging; Figure B shows blood vessel length and node number;

[0036] Figure 8The infrared spectrum and nuclear magnetic resonance hydrogen spectrum of methacryloyl gelatin are shown in Figure A; Figure B is the infrared spectrum;

[0037] Fig. 9 The diagrams are different states of the bioactive polypeptide hydrogel; wherein, Figure A is the injectable state of the bioactive polypeptide hydrogel; Figure B is the gelling state of the bioactive polypeptide hydrogel;

[0038] Fig.10 is a scanning electron microscope image of a hydrogel containing different polypeptides;

[0039] Fig.11 The antioxidant activity of each group of hydrogels; Figure A is the scavenging effect of DPPH and ABTS; Figure B is the active oxygen scavenging rate of DPPH and ABTS;

[0040] Fig.12 The survival rates of cells treated with hydrogels in each group after incubation with H2O2 for 2 h; Figure A is the cell activity staining image; Figure B is the cell survival rate;

[0041] Fig.13 Figure 3 shows the reactive oxygen species in cells treated with hydrogels in each group after incubation with H2O2 for 2 hours; Figure A shows the cell activity staining; Figure B shows the fluorescence intensity;

[0042] Fig.14 After scratching HUVECs cells, they were treated with various groups of hydrogels to show the migration of cells at 0h, 12h, and 36h; Figure A is the cell migration diagram; Figure B is the cell migration rate;

[0043] Fig.15 The effects of each group of hydrogels on blood vessels; Figure A is vascular imaging; Figure B is vascular length and node number;

[0044] Fig.16 Figure 1 is the cardiac ultrasound image of rats on the 14th and 28th day, as well as the statistical results of blood fraction, shortening fraction, end-diastolic volume and end-systolic volume; Figure A is the cardiac ultrasound image of rats; Figure B is the statistical results of ejection fraction, shortening fraction, end-diastolic volume and end-systolic volume;

[0045] Fig.17 HE staining results of liver, spleen, lung and kidney in each group treated with hydrogel on day 28;

[0046] Fig.18 The status and related indicators of the hearts treated with hydrogels in each group on the 28th day; Figure A is ROS staining; Figure B is TUNEL staining; Figure C is CD31 staining; Figure D is the relevant fluorescence intensity; Figure E is the positive cell rate; Figure F is the CD31 positive area;

[0047] Fig.19 The CD206 / CD86, CX43 / α-actinin and collagen I / collagen III staining and related fluorescence area statistics of the hydrogel-treated hearts in each group on the 28th day; Figure A is the immunofluorescence staining of CD86 and CD206; Figure B is the immunofluorescence staining of α-actinin and CX 43; Figure C is the immunofluorescence staining of collagen I and collagen III; Figure D is the fluorescence intensity of CD206; Figure E is the fluorescence intensity of CD86; Figure F is the positive cell efficiency of CX43; Figure G is the positive cell efficiency of α-actinin; Figure H is the ratio of collagen III / collagen I;

[0048] Fig. 20 The ridge plots of up-regulated and down-regulated gene enrichment channels in the myocardial infarction group and the dipeptide glue loading group on the 28th day. DETAILED DESCRIPTION

[0049] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0050] Example 1

[0051] A bioactive polypeptide hydrogel includes a hydrogel loaded with bioactive polypeptides; the bioactive polypeptides include an antioxidant peptide MMP12 and an angiogenic peptide KRX; wherein the amino acid sequence of the antioxidant peptide MMP12 is shown in SEQ ID NO.1; and the amino acid sequence of the angiogenic peptide KRX is shown in SEQ ID NO.2. The preparation method thereof includes the following steps:

[0052] (1) Preparation method of methacryloyl gelatin (GelMA): 15.25g of pig skin gelatin was placed in a 500mL round-bottom flask and dissolved in 250mL of deionized water. It was heated and stirred in a 50℃ water bath for 3h to fully dissolve. Then, 12mL of methacrylic anhydride was added dropwise to the fully dissolved gelatin solution under stirring, and then 3mol / L NaOH solution was added to make the pH = 8, so that the methacrylic anhydride reacted to the molecular chain of gelatin in an alkaline environment. After the pH no longer changed, the reaction was maintained for 48h. After the reaction was completed, the obtained product was transferred to a dialysis bag (MWCO: 10000) and dialyzed for 72h to remove the unreacted methacrylic anhydride in the solution and the alkali used to adjust the pH. After the dialysis was completed, high-speed centrifugation (8000r / min, 20min) was performed to remove the flocculent precipitate in the solution, and finally the supernatant was collected and freeze-dried for 3 days to obtain;

[0053] (2) Preparation of antioxidant peptide MMP12 and pro-angiogenic peptide KRX: In order to synthesize polypeptides of corresponding sequences, amino acids at a concentration of 0.2 mM (MMP12: alanine 0.38 g / 6 mL, aspartic acid 0.5 g / 6 mL, tryptophan 1.16 g / 11 mL, and tyrosine 0.56 g / 6 mL; KRX: alanine 0.5 g / 8 mL, aspartic acid 0.66 g / 8 mL, lysine 0.75 g / 8 mL, methionine 0.6 g / 8 mL, asparagine 0.96 g / 8 mL, proline 0.54 / 8 mL, arginine 4.16 g / 32 mL, and tyrosine 0.74 g / 8 mL) were weighed and completely dissolved in 0.2 mmol / L N,N-dimethylformamide (DMF) solution, and then the amino acid solution was added to the peptide synthesizer. The coupling reaction of the resin with amino acids, 500 mL 142.1 mg / mL 1-hydroxyphenyl-4,5-diphenylimidazole (Oxyma), and 200 mL 98% N,N'-diisopropylcarbodiimide (DIC) in the peptide synthesizer was heated by microwave and reacted at 90°C for 2 min. The reaction between each amino acid was washed 4 times with DMF. The 9-fluorenylmethoxycarbonyl protecting group (Fmoc) on the amino acid was deprotected at 90°C using DMF containing 20% ​​piperidine, and then the resin connected with the amino acid was taken out and washed 3 times with DMF and dichloromethane (DCM) (or methanol), respectively, and the above steps were repeated according to the amino acid sequence of the peptide. The resin was then placed in a cleavage solution consisting of 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIPS) and 2.5% deionized water for 3 h, and then the cleavage solution was added to ice anhydrous ether for precipitation, and the precipitate was blown dry to obtain a crude peptide product. After the crude polypeptide product is dissolved in water and acetonitrile, it is purified and separated by high performance liquid chromatography. The gradient elution method is used in the eluent for 30 minutes. The initial proportion of water in the eluent is 95%, and the initial proportion of acetonitrile is 5%. During the elution process, the proportion of water is linearly reduced from 95% to 30%, and the proportion of acetonitrile is linearly increased from 5% to 70%. In this process, the pure product will peak when the proportion of water is 55%. The solution of this peak is collected to obtain the purified polypeptide. The purified polypeptide solution is stored in a -20°C refrigerator after rotary evaporation and freeze drying;

[0054] (3) Preparation method of bioactive peptide hydrogel: 80 mg of GelMA, 2 mg of antioxidant peptide and 0.1 mg of angiogenic peptide were dissolved in 1 mL of aqueous solution, 0.5 mg of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) was used as a photoinitiator, and then UV cross-linked for 20 seconds to obtain the hydrogel.

[0055] Test Example 1 Detection of Antioxidant Performance

[0056] The antioxidant capacity of each group of hydrogels was tested using DPPH and ABTS. For DPPH, the DPPH free radical has a single electron and has a strong absorption at 519nm, and its alcohol solution is purple. When an antioxidant is present, the DPPH free radical is removed, the color of the solution will fade, and the degree of fading is related to the degree of removal. Take 1 mg of DPPH solid and dissolve it in 24 mL of anhydrous ethanol, protect from light and ultrasonic for 3 minutes, and shake it thoroughly to make the upper and lower parts uniform. Take 1 mL of the DPPH solution prepared in the above steps for detection, so that its absorbance is between 0.6-1.0. If the absorbance is too large, continue to add solvent; if the absorbance is too small, add DPPH solid or original solution. Add 0.5 mL of freeze-dried hydrogels of each group to 2.5 mL of DPPH solution and react at 37°C in the dark for 30 minutes. Then transfer it to a cuvette and use a UV-visible spectrophotometer to detect at 500nm-600nm, and record the absorbance at 519nm. The calculation formula of clearance rate is: Clearance rate % = (A0-A / A0) × 100% (A0 is the absorbance without adding sample, A is the absorbance after adding sample).

[0057] ABTS reacts with potassium persulfate to generate green ABTS•+ free radicals. This free radical has a maximum absorption at 734nm, so its concentration can be determined by detecting the absorbance at 734nm. Take 0.2mL (7.4mM) ABTS diammonium salt stock solution and 0.2mL 2.6mM potassium persulfate stock solution and mix them. Place them at room temperature in a dark environment for 12h. Dilute the mixture 10-20 times with pH7.4 phosphate buffer (PBS) until the absorbance is 0.8. This solution is the ABTS•+ free radical working solution. Then add 0.5mL of each group of freeze-dried hydrogels to 2.5mL of ABTS•+ solution and react for 6min in the dark. Then transfer it to a cuvette and use a UV-visible spectrophotometer (UV-2401PC, Shimadzu) to detect at 600nm to 750nm and record the absorbance at 734nm. The calculation formula of clearance rate is: Clearance rate % = (B1-B) / B1×100% (B1 is the absorbance without adding sample, B is the absorbance after adding sample).

[0058] Experimental Example 2 Detection of cell viability

[0059] The CCK-8 assay was used to detect the cell viability of each group of hydrogel samples on human umbilical vein endothelial cells (HUVECs) and rat cardiac myoblasts (H9C2 cells). Using the co-culture method, 50 μL of each group of hydrogels were added to a 96-well plate to detect cell viability. For PDA / PI staining, H9C2 cells and HUVECs cells were uniformly seeded in 96-well plates (6×103 cells / well). After being treated with different hydrogel samples in a volume of 50 μL for 24 h and 72 h, the cultured cells were stained with a mixed PBS solution containing 40 μg / mL fluorescein diacetate (FDA) and 20 μg / mL propidium iodide (PI) at 37 °C in the dark for 1 min. Then, the cells were imaged using an inverted fluorescence microscope (Leica DMI 4000, Germany), with the excitation and emission wavelengths of FDA at 488 nm and 530 nm, respectively, and PI at 535 nm and 615 nm, respectively.

[0060] In order to simulate the damage of ROS to cardiomyocytes in vivo and evaluate the protective effect of hydrogel on damaged cardiomyocytes. H9C2 cells were treated with 300μM H2O2 for 2h, then H2O2 was removed and hydrogel (50μL) was added for co-culture treatment for 48h and 72h. Then, the cells were stained for live and dead by FDA / PI and 2,7-dichlorofluorescein diacetate (DCFH-DA) dyes using the above experimental procedures. According to the manufacturer's instructions, DCFH-DA was diluted 1:1000 with serum-free medium to a final concentration of 10μM. The cell culture medium was removed, and the diluted DCFH-DA was added to fully cover the cells. The cells were incubated in a 37℃ cell culture incubator for 20min, and then the cells were washed three times with serum-free medium. The cells were then observed under an inverted fluorescence microscope with an excitation wavelength of 488nm and an emission wavelength of 525nm.

[0061] Test Example 3 Cell migration test

[0062] HUVECs cells were inoculated in 6-well plates and cultured for 24 hours to adhere to the wall. The cells were scratched with a 10μL pipette tip, and then washed twice with PBS buffer solution. HUVECs cells were then co-cultured with hydrogel samples of different combinations. The scratches on the cells were photographed under a bright field background at fixed time points (0h, 12h, and 36h), and the cell migration rate was calculated by comparing the ratio of the area of ​​the closed area to the initial scratch area.

[0063] Test Example 4 Tube Forming Experiment

[0064] 20uL / well of Matrigel (BD Bioscience) was added to a 24-well plate and placed in a 4°C refrigerator overnight. The plate was then placed in a cell culture incubator for 30 minutes, and then 1mL of standard culture medium containing HUVECs cells was inoculated on the surface of the Matrigel (1×106 cells per well). After 12 hours, FDA-stained HUVECs cells were added, and cell images were observed using a fluorescence microscope. The total tube segment length and the number of tubes were calculated using ImaginJ software.

[0065] Experimental Example 5 Construction of rat myocardial infarction model

[0066] Male Sprague-Dawley MI model rats (Chengdu Dashuo Experimental Animal Co, Ltd., 120 g) were used with a 3% isoflurane-oxygen mixture via a ventilator (Nanjing Calvin Biotechnology Co., Ltd.). Subsequently, the left anterior descending coronary artery (LAD) was permanently ligated at the 4th intercostal space through a left thoracotomy as described in previous studies, with the animal body temperature maintained at 37°C. During this process, the electrocardiogram was monitored, and the test showed ST-T segment elevation, which proved that the rat MI model was successfully established. The color of the infarct area turned pale in the anterior part of the heart. Then, 30 μL of hydrogel was injected into two sites of the infarct area, and the treated animals were divided into 5 groups (n=8): myocardial infarction group (MI), blank gel group (Gel), MMP12-loaded gel group (Gel@MMP12), KRX-loaded gel group (Gel@KRX), and dual-peptide gel group (MMP12 peptide and KRX peptide, Gel@MK). The control group mice underwent the same surgery, but without ligating the LAD. After the operation, the intubation tube was removed and the rats were observed for about 30 minutes until they regained consciousness and were then killed at the time when sampling was required.

[0067] Test Example 6 Cardiac Function Assessment

[0068] To examine the cardiac function of the rats treated with hydrogels in each group, echocardiography of the rats was performed on days 14 and 28 after MI surgery after the rats were anesthetized with a 3% isoflurane-oxygen mixture. Echocardiography was performed using a Philips CX 50 ultrasound system and a Philips L15-7io high-frequency probe configured with it. To evaluate each parameter, the average of three measurements was obtained from three different cycles of the image to determine the end-diastolic volume (EDV), left ventricular end-systolic volume (ESV), left ventricular ejection fraction (EF), and fractional shortening (FS). All measurements were averaged over three consecutive cycles.

[0069] Experimental Example 7 Cardiac morphology measurement and immunohistochemistry (IHC)

[0070] On days 3, 14, and 28, the rats were anesthetized and sacrificed. The hearts of the rats were collected, frozen in liquid nitrogen for ROS staining, and fixed with 4% paraformaldehyde solution for the rest of the staining. ROS staining was used to evaluate the level of reactive oxygen species during myocardial infarction. CD31 was used to evaluate the formation of new blood vessels, TUNEL was used to evaluate cell apoptosis, and CD206 / CD86 was used to evaluate inflammation in vivo. CX43 / α-actinin was used to evaluate cardiac function. The ratio of collagen I / collagen III was used to evaluate the degree of fibrosis. All the images of staining were characterized and measured by ImageJ software.

[0071] Experimental Example 8 mRNA Sequencing Analysis of Myocardial Infarction Site

[0072] The hearts of the dipeptide gel loading group and the myocardial infarction group were quickly collected and stored in dry ice. Total RNA was extracted according to conventional methods. After passing the quality inspection, the double-stranded cDNA was further purified and reverse transcribed, and then the product was purified and PCR enriched. For samples with biological replicates, DESeq2 software was used to perform differential expression analysis between sample groups to obtain the differentially expressed gene set between the two biological conditions. For samples without biological replicates, edgeR software was used for analysis. It is required to input the unstandardized read count data of the gene, rather than the standardized data such as RPKM and FPKM. The read count of the gene was implemented using featureCounts software. After the differential analysis, the Benjamini-Hochberg method was also used to correct the hypothesis test probability (Pvalue) for multiple hypothesis testing to obtain the false discovery rate (FDR). The screening conditions for differential genes were |log2Fold Change|>=1 and pvalue<0.05.

[0073] Data statistics and analysis

[0074] Data in the experiments are shown as mean ± SD (at least n = 3). Statistical analysis was performed by two-way analysis of variance (ANOVA) according to Tukey's test. **** means p < 0.001, *** means p < 0.005, ** means p < 0.01, and * means p < 0.05. Differences were considered statistically significant, and ns means no significant difference.

[0075] Test results

[0076] 1. Characterization of Bioactive Peptides

[0077] Mass spectrometry, high performance liquid chromatography, and circular dichroism spectroscopy were used to determine the purity and structure of the peptides.

[0078] Figure 1 is the MMP12 polypeptide ( Figure 1 Middle A) and KRX peptide ( Figure 1 The mass spectrum of Figure 1 It can be seen that the relative molecular masses of MMP12 polypeptide and KRX polypeptide are 738.33Da and 1149.9Da, respectively, which are consistent with their theoretical relative molecular masses, indicating that MMP12 polypeptide and KRX polypeptide were successfully synthesized.

[0079] Figure 2 is the MMP12 polypeptide ( Figure 2 Middle A) and KRX peptide ( Figure 2 Figure B shows a high performance liquid chromatogram (HPLC). Through HPLC analysis, the purity of the MMP12 polypeptide and KRX polypeptide prepared by the present invention can reach 99%.

[0080] Figure 3 is the MMP12 polypeptide ( Figure 3 Middle A) and KRX peptide ( Figure 3 Circular dichroism spectrum (CD) of Figure 3 As shown in Figure A, in the CD map of the MMP12 polypeptide, there is a positive peak at about 185nm and 225nm, and a negative peak at about 220nm and 215nm. Analysis of the peaks shows that the secondary structure of the MMP12 polypeptide contains 6.5% α-helix and 93.5% β-fold. Figure 3 As shown in Figure B, there is a negative peak near 200nm in the CD map of the KRX polypeptide. Analysis of the peak shows that the secondary structure of the KRX polypeptide contains 50% α-helix and 50% β-fold.

[0081] 2. Optimal concentrations of MMP12 peptide and KRX peptide

[0082] (1) Screening of the optimal antioxidant concentration of MMP12 polypeptide: Using DPPH and ABTS free radicals as indicators, the ROS scavenging activity of MMP12 polypeptide at concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL and 3 mg / mL was evaluated. Figure 4 As shown. Figure 4 As shown in Figure AB, as the concentration of MMP12 peptide increases, its clearance rate of ROS gradually increases, reaching 50%, 69% and 91% respectively, while the clearance rates of 3mg / mL and 2mg / mL reach more than 90% with no significant difference. This indicates that MMP12 peptide can achieve the best ROS clearance effect at a concentration of 2mg / mL, so 2mg / mL is selected as the optimal concentration of MMP12 peptide. Figure 4 Figure C is the color map of the corresponding solution.

[0083] (2) Screening for the optimal angiogenesis-promoting concentration of KRX peptide:

[0084] ① The CCK-8 detection kit was used to quantitatively determine the cell viability of human umbilical vein endothelial cells (HUVECs) at different concentrations of 10μg / mL, 25μg / mL, 50μg / mL, 100μg / mL and 200μg / mL. The cell proliferation of HUVECs cells was determined on the third day. The results are as follows: Figure 5 As shown. Figure 5 It can be seen that as the concentration of KRX polypeptide increases, the cell survival rate is significantly improved. At a concentration of 100 μg / mL, the cell survival rate reaches 140%, and there is no significant difference with that at 200 μg / mL.

[0085] ② The HUVECs cells were tested for cell migration at concentrations of 50 μg / mL, 100 μg / mL and 200 μg / mL. The results are as follows: Figure 6 As shown. Figure 6 As shown in Figure A, at 36h, the distance between the cells on both sides of the 100μg / mL and 200μg / mL groups was smaller than that of the control group and 50μg / mL, indicating that the migration rate of the 100μg / mL and 200μg / mL groups was greater than that of the control group and 50μg / mL groups. Quantitative testing showed that at 12h and 36h, the migration rate of cells at the concentrations of 100μg / mL and 200μg / mL reached about 40% and 60% with no significant difference, which was significantly greater than that of the control group and 50μg / mL, which is consistent with the results in the previous part (see Figure 6 (Figure B).

[0086] ③ The above concentrations were used to test HUVECs tube formation to verify the angiogenic ability of the peptide. The results are as follows Figure 7 As shown. Figure 7 As shown in Figure A, angiogenesis was observed in the group containing KRX peptide after 12 hours of incubation with Matrigel, and vascularization was more obvious in the 100μg / mL and 200μg / mL groups compared with the control group and the 50μg / mL group. The number of vascular nodes and the length of blood vessels were quantified, and the results showed that the 100μg / mL group and the 200μg / mL group significantly promoted tube formation in HUVECs compared with the control group and the 50μg / mL group, while there was no statistically significant difference between the 100μg / mL group and the 200μg / mL group (see Figure 7 Therefore, 100 μg / mL of KRX peptide was selected as the optimal concentration in this experiment.

[0087] 3. Characterization of Bioactive Peptide Hydrogels

[0088] (1) Using Fourier transform infrared spectroscopy (FT-IR) and 1 H NMR spectroscopy ( 1H NMR) was used to detect the synthesized GelMA to determine whether the polymer was successfully prepared. The results are shown in Figure 8 As shown. Figure 8 As shown in Figure A, the infrared spectrum of GelMA is at 1634 cm-1 in the amide I band. -1 The band caused by C=O stretching vibration and amide II is 1540 cm -1 The peaks caused by CN band stretching vibration and NH bending vibration are stronger than those of pure gelatin, indicating that methacrylamide groups have been introduced into the gelatin molecular chain. Figure 8 As shown in Figure B, GelMA 1 In the H NMR spectrum, proton peaks of methacrylamide appeared at 5.44 and 5.68 ppm, which also proved that the methacrylamide groups were grafted onto the gelatin molecular chain.

[0089] (2) Characterization of bioactive peptide hydrogels:

[0090] ① By adjusting the content of GelMA and photoinitiator, 8% GelMA and 0.05% LAP were finally used. After irradiation with UV-visible light at a wavelength of 405 nm for 40 seconds, the obtained bioactive polypeptide hydrogel showed excellent injectability (see Fig. 9 The hydrogel is squeezed through a 1 mL needle to write the word "MI" and can also be injected into the heart (see Figure 1). Fig. 9 (Figure B).

[0091] ② The cross-sectional morphology of each group of hydrogels after freeze-drying was observed using a field emission scanning electron microscope. Fig.10 It can be seen that the pore structure of each group of hydrogel samples is relatively uniform, indicating that each group of hydrogels has a similar, well-structured network, and the porous structure can provide sufficient space for cell growth, attachment and proliferation.

[0092] ③ The ROS scavenging rate of each group of hydrogels was evaluated using DPPH and ABTS free radicals as indicators. Fig.11 It can be seen that the color of the solution in the hydrogel group loaded with MMP12 polypeptide is significantly lighter than that in the group without loading, indicating that the MMP12-loaded gel and the dipeptide-loaded gel have the ability to remove ROS. At the same time, quantitative detection shows that the removal rate of the hydrogel loaded with MMP12 polypeptide is about 60%, which is significantly higher than that of other groups, indicating that the dipeptide-loaded hydrogel has an antioxidant function.

[0093] 4. Protective effect of bioactive peptide hydrogel on H9C2 cells under oxidative stimulation

[0094] (1) The protective effect of the loaded dipeptide hydrogel on cardiomyocytes was evaluated by establishing a H2O2-induced H9C2 cell oxidative stress model. Live / dead staining was used to evaluate the protective effect of each group of hydrogels on H2O2-induced H9C2 cells. The results are shown in Fig.12 As shown. Fig.12 As shown in Figure A, after 3 days of H2O2 treatment, almost no dead cells (red) were detected in the control group (no H2O2 treatment), while some dead cells were observed in the H2O2 (300 μM) group. Importantly, the bioactive peptide hydrogel group increased the proportion of live cells to varying degrees. Fig.12 As shown in Figure B, the cell viability of the group loaded with MMP12 gel (Gel@MMP12) and the group loaded with MMP12 and KRX dipeptide gel (Gel@MK) is about 79%, while that of the H2O2 group is about 65%, indicating that the group loaded with MMP12 gel and the group loaded with dipeptide gel have significantly higher cell activity than other groups.

[0095] (2) DCFH-DA was used to stain cells for reactive oxygen species to detect the scavenging effect of the hydrogel on intracellular reactive oxygen species. Fig.13 As shown in Figure A, in the control group, the intracellular ROS content was very low, while a large amount of ROS was observed in the H2O2 group. The hydrogel group showed a decrease compared to the H2O2 group, especially the Gel@MMP12 and Gel@MK groups showed lower ROS (green). The fluorescence intensity was quantitatively detected. Taking the fluorescence intensity of the H2O2 group as 100%, the Gel@MMP12 and Gel@MK groups were 46% and 37% respectively, and the hydrogel group without MMP12 peptide was about 90% (see Fig.13 (Figure B).

[0096] The above results indicate that the bioactive peptide hydrogel loaded with MMP12 polypeptide and KRX polypeptide of the present invention has good biocompatibility and has a protective effect on cardiomyocytes under oxidative stress.

[0097] 5. Effects of Bioactive Peptide Hydrogels on Migration and Tube Formation of HUVECs

[0098] (1) Another difficulty that hinders the effective repair of MI is the lack of vascularization. Therefore, accelerating angiogenesis is an effective way to treat myocardial infarction. The scratch test is a simple and low-cost in vitro cell proliferation / migration assay. The scratch test was used to evaluate the effect of hydrogel samples on in vitro HUVECs proliferation / migration, which is a key step in angiogenesis. The results are shown in Fig.14 .Depend on Fig.14As shown in Figure A, at 12h, compared with the control group, obvious cell migration can be observed in the hydrogel groups loaded with KRX polypeptide (Gel@KRX and Gel@MK groups); the hydrogel groups not loaded with KRX polypeptide (blank gel group (Gel) and Gel@MMP12 group) did not show obvious cell migration. In addition, at 36h, it was observed that the scratches in the Gel@KRX and Gel@MK groups were almost completely migrated, while there were still certain unhealed areas in the control group, Gel group and Gel@MMP12 group. These results indicate that hydrogel-loaded KRX polypeptide can significantly increase cell migration rate. At the same time, Fig.14 The quantitative results of cell migration rate in Figure B are also consistent with the above results.

[0099] (2) Endothelial cell vascularization is a very important step in the angiogenesis process. The above results have proved that KRX peptide can promote cell migration. Therefore, the HUVECs tube formation test was used to verify the angiogenesis ability of the hydrogel. The results are as follows Fig.15 As shown. Fig.15 As shown in Figure A, angiogenesis was observed in the Gel@KRX group after 12 h of incubation. The number of connections and total length of the tubes were obtained by quantifying the tube formation. Fig.15 As shown in Figure B, compared with the control group, Gel group and Gel@MMP12 group, the Gel@KRX group and Gel@MK group significantly promoted the tube formation in HUVECs.

[0100] 6. In vivo evaluation of the therapeutic efficacy of bioactive peptide hydrogels

[0101] (1) In order to evaluate the therapeutic effect of loaded dipeptide hydrogel on myocardial infarction in vivo, an acute myocardial infarction model was established by permanent ligation of the left anterior descending artery (LAD) of rats. All rats were randomly divided into 6 groups (n=8 / group): control group, MI group, Gel group, Gel@MMP12 group, Gel@KRX group and Gel@MK group. Rats with MI injury were treated by injection of PBS, Gel, Gel@MMP12, Gel@KRX and Gel@MK. On the 14th and 28th days, the cardiac function parameters of the rats, including ejection fraction (EF), shortening fraction (FS), end-diastolic volume (EDV) and end-systolic volume (ESV), were detected by echocardiography. Fig.16 As shown in Figure B, all MI-injured rats had similar decreases in EF and FS levels compared with the control group, due to increased EDV and ESV measurements. These results indicate that all groups of rats had inflicted the same degree of myocardial infarction damage, resulting in weakened left ventricular myocardial contractility. After 14 days of treatment, echocardiography showed (see Fig.16(Figure A in the middle), the EF and FS of rats in the Gel, Gel@MMP12, Gel@KRX, and Gel@MK groups were higher than those in the MI group. At the same time, compared with the Gel group, the EF and FS of the Gel@MK group were improved, proving that the MMP12 peptide and the KRX peptide have a clear therapeutic effect in the repair of myocardial infarction tissue. On the 28th day, the cardiac echocardiogram and related parameters of the myocardial infarction rats in the Gel@MK group were further normalized, indicating that the therapeutic effect of the hydrogel loaded with the MMP12 peptide and the KRX peptide is getting better and better.

[0102] (2) Myocardial infarction often leads to severe ventricular wall thinning and abnormal collagen deposition. On day 28 after treatment, hematoxylin-eosin (H&E) staining was used to evaluate the toxicity of the hydrogel. Masson trichrome staining showed (see Fig.17 ), the injected hydrogel had no obvious toxic effects on the liver, spleen, lungs, and kidneys of rats.

[0103] 7. In vivo evaluation of bioactive peptide hydrogels

[0104] (1) After myocardial infarction occurs, a large amount of ROS will be generated in a short period of time, which cannot be completely eliminated by the body. In order to reveal the potential mechanism of hydrogel in treating myocardial infarction, we first evaluated whether bioactive peptide hydrogel can effectively reduce the high level of reactive oxygen species after myocardial infarction in vivo. The results are as follows: Fig.18 As shown. Fig.18 As shown in Figures A and D, the ROS fluorescence signal and relative fluorescence intensity of MI-injured rats were higher than those of the control group, indicating that the ROS level increased after myocardial infarction. At the same time, weak fluorescence signals and lower relative fluorescence intensities were detected in the MMP12-loaded gel and dipeptide-loaded gel groups, indicating that the MMP12 polypeptide can effectively reduce the ROS level in the myocardial infarction site, which is mainly due to the antioxidant effect of the MMP12 polypeptide.

[0105] (2) Next, the mechanism of hydrogel repairing damaged hearts was studied, and the TUNEL method was used to study the apoptosis of cells in myocardial tissue in the MI area. Fig.18 As shown in Figures B and E, 3 days after surgery, significant levels of apoptosis were observed in the cardiac tissue of the MI group, and injection of the loaded dipeptide hydrogel significantly reduced apoptosis, indicating that the loaded dipeptide hydrogel can effectively reduce apoptosis in the MI area. In addition, we evaluated the inflammatory response after hydrogel treatment by immunofluorescence staining of CD86 and CD206 (CD86 is a typical pro-inflammatory factor, and CD206 is a typical anti-inflammatory factor). Fig.19As shown in Figure E, the expression level of CD86 in the MI group was significantly higher than that in the control group, confirming the inflammatory response after MI. After treatment with dipeptide glue, the expression level of CD86 was significantly reduced and CD206 was significantly increased due to the combined effects of MMP12 peptide and KRX peptide. Quantitative data confirmed these trends. At 7 days after MI, the percentage of CD86 cells in the MI group was significantly higher than that in the control group, and the percentage of CD86 cells after dipeptide glue treatment was significantly lower than that in the MI group. These results indicate that the hydrogel effectively reduced apoptosis and inflammation levels in the ROS microenvironment after MI.

[0106] (3) The above results have demonstrated that KRX peptide has the effect of promoting microtubule formation in vitro. Therefore, whether the bioactive peptide hydrogel accelerates the formation of new blood vessels in the infarcted heart was evaluated. Fig.18 Figure EF in the middle shows the immunohistochemical results of CD31 expression in the myocardial infarction site. Fig.18 As shown in Figures C and F, on day 28, the vascular density of the dipeptide glue group was higher than that of the MI group and other hydrogel treatment groups. The quantitative data of vascular density was consistent with the fluorescence image. This indicates that the dipeptide glue can show a good effect in treating myocardial infarction in vivo by promoting cell proliferation and angiogenesis around the infarct.

[0107] (4) The synchronous contraction of cardiomyocytes mainly depends on the expression of α-actinin, which is closely related to the maturation of the myocardium and the regulation of muscle contraction. Connexin 43 (CX 43) is a myocardial protein that is mainly involved in the coupling of myocardial electrical contraction. α-actinin and CX 43 were used as cardiac-related factors to detect the cardiac function of rats after hydrogel treatment. Fig.19 As shown in Figures B and G, the least expression of α-actinin was observed in the MI groups at 14 and 28 days compared with the other treatment groups. At the same time, the expression of α-actinin in the dipeptide gel-loaded group was more obvious than that in the other hydrogel-treated groups, indicating that the cardiac function was greatly improved. Fig.19 As shown in Figure F, CX43 was hardly expressed in the MI group, but in the hydrogel-treated groups, the expression of CX43 was enhanced, especially in the dipeptide-loaded group, indicating better electrical contraction 14 and 28 days after treatment. Most importantly, the expression of α-actinin and CX43 in the dipeptide-loaded group was higher than that in the MMP12-loaded group or the KRX-loaded group, demonstrating the enhanced therapeutic effect of the combined therapy.

[0108] (5) Myocardial fibrosis is often accompanied by excessive accumulation of collagen I. We evaluated fibrosis in the MI area by immunofluorescence staining of collagen I and collagen III at 28 days. Fig.19As can be seen from Figures C and H, the collagen III / I ratio in the dipeptide-loaded hydrogel was significantly higher than that in the MI group, indicating that the dipeptide-loaded hydrogel also has the function of regulating and changing the deposition of collagen I and collagen III, which is helpful for MI tissue repair.

[0109] 8. Evaluation of mRNA Sequencing in Myocardial Infarction Site Tissue

[0110] Depend on Fig. 20 It can be seen that the downregulation of genes enriched in the focal adhesion pathway can inhibit the abnormal proliferation and migration of cells in the fibrosis process, thereby reducing the degree of fibrosis by downregulating the expression of focal adhesion kinase (FAK). Downregulation of genes enriched in the tumor necrosis factor (TNF) signaling pathway can reduce the production and release of TNF, thereby reducing the level of inflammatory response and helping to reduce tissue damage in the fibrosis process. Downregulation of genes enriched in the lipid and atherosclerosis pathway can balance the production and clearance of the extracellular matrix and reduce the excessive deposition of the extracellular matrix in the fibrosis process by downregulating genes related to the synthesis and degradation of the extracellular matrix.

[0111] In summary, the present invention prepares an injectable hydrogel loaded with bioactive polypeptides, which, in addition to anti-oxidation and angiogenesis, also has anti-apoptosis, anti-inflammatory and anti-fibrosis mechanisms, realizing the whole process repair of the damaged heart, achieving the effect of 1+1>2. In the rat MI model, we injected different hydrogel samples into the damaged myocardium and observed unexpected therapeutic effects. In the inflammatory stage, MMP12 and KRX released by the hydrogel can simultaneously reduce apoptosis and inflammatory response; in the hyperplastic stage, the sustained release of KRX promotes angiogenesis and is significantly stronger than the single KRX group; in the remodeling stage, what is relatively novel is that the simultaneous slow release of the two polypeptides inhibits myocardial fibrosis in vivo, reduces ventricular wall remodeling and improves cardiac function after MI. These results show that the hydrogel with dual polypeptides can effectively accelerate the repair of MI tissues and provide a promising solution for MI treatment.

[0112] The amino acid sequences of the antioxidant peptide MMP12 and the angiogenic peptide KRX in the present invention are YWDAW (SEQ ID NO. 1) and MRPYDANKR (SEQ ID NO. 2), respectively.

[0113] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A bioactive polypeptide hydrogel, characterized in that: It comprises a hydrogel loaded with bioactive polypeptides; the bioactive polypeptides comprise an antioxidant peptide MMP12 and an angiogenic peptide KRX; wherein the amino acid sequence of the antioxidant peptide MMP12 is as shown in SEQ ID NO.1; and the amino acid sequence of the angiogenic peptide KRX is as shown in SEQ ID NO.

2.

2. The bioactive polypeptide hydrogel according to claim 1, characterized in that: The hydrogel is methacrylated gelatin.

3. The method for preparing the bioactive polypeptide hydrogel according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) Methacrylic gelatin is prepared by grafting methacrylic acid groups onto the molecular chains of gelatin; (2) Synthesis of biologically active peptides by solid-phase peptide synthesis; (3) Using a photoinitiator, the products obtained in step (1) and step (2) are subjected to ultraviolet cross-linking to obtain a crystalline polyol.

4. The method for preparing the bioactive polypeptide hydrogel according to claim 3, characterized in that: The method of preparing methacrylated gelatin by grafting methacrylic acid groups onto the molecular chains of gelatin in step (1) is as follows: gelatin is dissolved in deionized water and heated and stirred, and then methacrylic anhydride and NaOH solution are added and reacted for 45-50 hours, the reaction product is dialyzed and then centrifuged, and the supernatant is collected and freeze-dried to obtain the obtained product.

5. The method for preparing the bioactive polypeptide hydrogel according to claim 4, characterized in that: The gelatin is pig skin-derived gelatin or fish skin-derived gelatin.

6. The method for preparing the bioactive polypeptide hydrogel according to claim 3, characterized in that: The step (2) of synthesizing the biologically active polypeptide by solid phase polypeptide synthesis method comprises the following steps: (1) Weigh the amino acid and dissolve it in N,N-dimethylformamide solution; then add the resin and condensation agent, heat it with microwave, take out the resin, and wash it with detergent; (2) Deprotecting the 9-fluorenylmethoxycarbonyl protecting group on the amino acid using DMF containing piperidine, then removing the resin and washing it with a detergent; (3) Repeat steps (1)-(2) according to the amino acid sequence of the polypeptide; (4) using a lysis solution to lyse the product obtained in step (3) for 2-3 hours, and then blowing the lysis solution to obtain a crude polypeptide product after precipitation; (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.

7. The method for preparing the bioactive polypeptide hydrogel according to claim 6, 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 and O-benzotriazole-tetramethyluronium hexafluorophosphate; the detergent is at least one of N,N-dimethylformamide, dichloromethane and methanol; the lysate includes 95wt% trifluoroacetic acid, 2.5wt% triisopropylsilane and 2.5wt% deionized water.

8. The method for preparing the bioactive polypeptide hydrogel according to claim 3, characterized in that: The method of using a photoinitiator to UV-crosslink the products obtained in step (1) and step (2) in step (3) is specifically as follows: dissolving the methacryloyl gelatin obtained in step (1) and the bioactive polypeptide obtained in step (2) in an aqueous solution, adding a photoinitiator, and then UV-crosslinking for 20-30 seconds to obtain the obtained product.

9. The method for preparing the bioactive polypeptide hydrogel according to claim 8, characterized in that: The photoinitiator is phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate, diacetophenone or phenylacetone.

10. Use of the bioactive polypeptide hydrogel according to any one of claims 1 to 2 in the preparation of a drug for treating myocardial infarction.

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