Hydrogel loaded with oxygen self-producing nanoparticles as well as preparation method and application of hydrogel

Abstract

The invention relates to hydrogel loaded with oxygen self-producing nanoparticles as well as a preparation method and application of the hydrogel. The preparation method comprises the following steps: 1, taking cross-linked methacrylamide silk fibroin and hyaluronic acid modified by arginine-glycine-aspartic acid peptide as base materials, and loading self-oxygen-producing nano particles CaO2 (at) PVP (at) Cu-EGCG, and the self-oxygen-producing nano particles CaO2 (at) PVP (at) Cu-EGCG are prepared by coating Cu-EGCG on calcium peroxide nano particles modified by PVP modified polyvinylpyrrolidone. The hydrogel can improve energy metabolism and regulate immune environment through dual mechanisms, realizes efficient promotion of myocardial repair, and provides a new strategy and thought for tissue regeneration and functional recovery after myocardial infarction.

Description

Technical Field

[0001] The present invention belongs to the technical field of hydrogels, and in particular relates to a hydrogel loaded with self-generated oxygen nanoparticles, a preparation method and an application thereof. Background Art

[0002] Stem cell therapy, gene therapy, hydrogel technology, tissue engineering and biomaterials are currently one of the important research directions for myocardial repair. Mesenchymal stem cells (MSCs) and others can differentiate into cardiomyocytes, endothelial cells and smooth muscle cells, thereby promoting the recovery of cardiac structure and function. In addition, stem cells can promote angiogenesis, reduce inflammation and inhibit cell apoptosis by secreting paracrine factors. However, stem cell therapy faces challenges such as low cell survival rate, immune rejection, ethical issues and limited cell sources. Gene therapy fundamentally solves the problem of myocardial cell dysfunction by repairing or replacing damaged genes. Although gene therapy has shown good efficacy in animal models, its clinical application still faces problems such as the safety of gene carriers, the accuracy of gene editing and long-term efficacy. Hydrogels can serve as cell carriers to protect cells from damage in the adverse microenvironment of ischemic myocardial tissue and help cells adhere, spread and form intercellular connections.

[0003] It can be seen that although certain progress has been made in existing technologies such as stem cell therapy, gene therapy, hydrogel technology and tissue engineering, they still face many challenges, such as the hypoxic environment in the myocardial infarction area limits the survival and function of cells and drugs, the loading and delivery efficiency of cells and drugs is low, the biocompatibility and operability of biomaterials are insufficient, as well as immune rejection, ethical issues and technical complexity. However, the repair effect of existing hydrogel technology in hypoxic environments is limited, and it lacks the ability to improve the hypoxic environment. In addition, tissue engineering faces problems such as biocompatibility of biomaterials, in vivo degradability, host immune rejection and biological toxicity. Existing technologies still face challenges such as low cell survival rate, immune rejection, technical complexity and hypoxic environment. The limitations of these technologies in clinical applications indicate that the development of a new technology that can effectively improve the hypoxic environment and promote myocardial repair is of great clinical significance. Summary of the Invention

[0004] In response to these problems, the present invention introduces self-generated oxygen nanoparticles, which can continuously release oxygen in hypoxic myocardial tissue, improve the local microenvironment, thereby improving the survival rate and function of cells and enhancing the myocardial repair effect. At the same time, the hydrogel system adopted by the present invention has good biocompatibility, injectability and mechanical stability, and can be directly applied to the damaged myocardial area through minimally invasive surgery, reducing surgical risks and patient trauma. The hydrogel system of the present invention also avoids immune rejection and ethical issues, simplifies the technical operation process, and is more suitable for clinical promotion. In summary, the present invention is expected to provide a new method for myocardial repair that is more efficient, safe and has clinical application potential by improving the local microenvironment of the myocardium, improving load and delivery efficiency, enhancing biocompatibility and operability, and realizing multifunctional repair.

[0005] The specific technical solutions of the present invention are as follows:

[0006] The first aspect of the present invention provides a hydrogel loaded with self-generated oxygen nanoparticles. The hydrogel is based on cross-linked methacrylamidated silk fibroin (SFMA) and arginine-glycine-aspartic acid peptide (RGD)-modified hyaluronic acid (HA-RGD), and is loaded with self-generated oxygen nanoparticles CaO2@PVP@Cu-EGCG (CPCE). The self-generated oxygen nanoparticles CaO2@PVP@Cu-EGCG are prepared by coating Cu-EGCG with calcium peroxide nanoparticles modified with PVP-modified polyvinylpyrrolidone.

[0007] In this hydrogel system, SFMA serves as a structural support material, providing good biocompatibility and biodegradability. At the same time, silk fibroin itself is rich in amino acids and can serve as a nutrient source for cells, promoting cell survival and proliferation. HA-RGD binds to the cell surface through its modified RGD peptide, significantly enhancing the adhesion, proliferation, and migration capabilities of cells, accelerating tissue regeneration, and playing an important role in myocardial repair. CPCE, as the core functional component, can continuously release oxygen and also has anti-inflammatory and antibacterial effects. By organically combining SFMA, HA-RGD, and CPCE, the hydrogel system fully utilizes the synergistic effects of each component to achieve dual regulation of energy metabolism and immune environment.

[0008] The SFMA in the hydrogel of the present invention can be modified by modifying the silk fibroin with glycidyl methacrylate (GMA), introducing methacryloyl groups on the SF molecular chain, and making it have the ability to photocuring. In a preferred embodiment, the methacrylamide silk fibroin of the present invention is obtained by dissolving the degummed silk fibroin in a lithium bromide solution, dispersing it at 60°C until it is completely dissolved, adding glycidyl methacrylate, and stirring the reaction at 60°C. After the reaction is completed, the solution is filtered through a polyester rayon filter cloth, and then dialyzed in pure water for 3 days to remove salts, and the product with a molecular weight cutoff of 12-14kDa is obtained. Finally, the solution is frozen at -80°C and freeze-dried. Preferably, the degummed silk fibroin: lithium bromide: glycidyl methacrylate = 10g: 75-85g: 6mL.

[0009] The HA-RGD in the hydrogel of the present invention is chemically modified, retaining the biocompatibility and moisture retention properties of hyaluronic acid while also enhancing compatibility, stability, and targeting through the RGD peptide. Hyaluronic acid is a high-molecular-weight polysaccharide with excellent biocompatibility and moisture retention. The RGD peptide is a short peptide that specifically binds to integrin receptors on the cell surface. Modification is achieved by forming an amide bond between the carboxyl groups of hyaluronic acid and the amino groups of the RGD peptide.

[0010] In a preferred embodiment, the HA-RGD is obtained by activating HA with EDC and NHS, adding the HA dropwise to an RGD solution, reacting at room temperature, and dialyzing the HA in pure water for 3 days using a dialysis bag with a molecular weight cutoff of 2000 Da. The solution is then frozen at -80°C and lyophilized.

[0011] Preferably, the mass ratio is hyaluronic acid: EDC: NHS: arginine-glycine-aspartic acid peptide = 1:0.8:1.2:0.5.

[0012] The self-generated oxygen nanoparticles CaO2@PVP@Cu-EGCG in the hydrogel of the present invention are obtained by coating and modification. They serve as an oxygen source, continuously releasing oxygen and improving the local myocardial microenvironment.

[0013] In a preferred embodiment, the preparation method of the self-generated oxygen nanoparticles CaO2@PVP@Cu-EGCG comprises the following steps:

[0014] 1) Preparation of polyvinylpyrrolidone-modified calcium peroxide nanoparticles CaO2@PVP;

[0015] 2) Preparation of Cu-EGCG complex; Cu 2+ The solution was added dropwise to the EGCG solution, and after mixing, the pH was adjusted to 7, and the mixture was stirred at a constant temperature of 40°C to fully complex the solution. The precipitate was then washed and freeze-dried in vacuum to obtain the Cu-EGCG complex.

[0016] 3) Coating Cu-EGCG; mixing the CaO2@PVP obtained in step 1) and the Cu-EGCG complex obtained in step 2), then adding MOPS buffer, and finally washing to obtain CaO2@PVP particles coated with Cu-EGCG.

[0017] In an embodiment of the present invention, the CaO2@PVP can be prepared according to a conventional method. In one embodiment, in the step 1), the CaO2@PVP is prepared by dissolving CaCl2 and PVP in anhydrous methanol, and then adding a mixed solution of H2O2 and ammonia water; the obtained mixed solution is stirred and reacted at 0°C, the obtained precipitate is collected by centrifugation, and washed with methanol. Preferably, the mass ratio of the CaCl2 and PVP is 1:1.

[0018] In the embodiment of the present invention, the Cu-EGCG complex is prepared by 2+ Salt and EGCG are mixed and complexed in the solution, wherein Cu 2+ The salt can be copper sulfate, copper chloride, etc. Preferably, according to the molar ratio, Cu 2+ : EGCG = 1:1-2. Preferably, the molar concentration of EGCG in the EGCG solution is 0.01-0.02 mol / L, Cu 2+ Cu in solution 2+ The molar concentration is 0.1~0.2mol / L.

[0019] In an embodiment of the present invention, when CaO2@PVP nanoparticles are coated with Cu-EGCG complex, preferably, the mass ratio is CaO2@PVP:Cu-EGCG=1:1; multiple coatings can be performed. For example, in one embodiment of the present invention, the coating is repeated twice to obtain CaO2@PVP particles coated with three layers of Cu-EGCG.

[0020] The second aspect of the present invention further provides a method for preparing the hydrogel, comprising:

[0021] Dissolving methacrylamide silk fibroin in an aqueous solution containing a photoinitiator to obtain a methacrylamide silk fibroin solution, which is referred to as solution A;

[0022] A hyaluronic acid solution modified with an arginine-glycine-aspartic acid peptide was prepared, and CaO2@PVP@Cu-EGCG was dispersed in HA-RGD and vortexed to completely and evenly disperse the solution therein to obtain solution B.

[0023] Then, solution A and solution B are mixed, and cross-linked by ultraviolet irradiation to obtain the hydrogel.

[0024] The gel is prepared by UV cross-linking, and the process is simple.

[0025] In a preferred embodiment, solution A and solution B are mixed in a volume ratio of 1:1, and in solution A, the concentration of the methacrylamide silk fibroin solution is 7.5%-12% by mass volume ratio, the concentration of the arginine-glycine-aspartic acid peptide-modified hyaluronic acid in solution B is 1-2%, and the amount of CaO2@PVP@Cu-EGCG is 10-200 μg / mL.

[0026] The third aspect of the present invention further provides the use of the hydrogel or the hydrogel prepared by the preparation method in the preparation of myocardial repair therapeutic drugs.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The hydrogel of the present invention, on the one hand, the self-oxygenating complex CPCE significantly improves the local myocardial hypoxia state by continuously releasing oxygen, enhancing the energy metabolism and repair capacity of myocardial cells; on the other hand, the anti-inflammatory effect of EGCG regulates the immune microenvironment, reduces the damage of inflammatory response to myocardial cells, and promotes tissue regeneration. At the same time, the cell support effect of SFMA and HA-RGD further enhances the survival rate and function of cells in the hydrogel, improving the overall effect of myocardial repair. Therefore, the multifunctional novel hydrogel system provided by the present invention can achieve efficient promotion of myocardial repair through a dual mechanism - improving energy metabolism and regulating the immune environment, providing new strategies and ideas for tissue regeneration and functional recovery after myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a SEM image of CPCE nanoparticles of Example 3-1 of the present invention;

[0030] Figure 2 is a TEM image of CPCE nanoparticles of Example 3-1 of the present invention;

[0031] Figure 3 is the XRD spectra of CaO2, CaO2@PVP, and CaO2@PVP@Cu-EGCG nanoparticles;

[0032] Figure 4 is a cross-sectional SEM image of a hydrogel according to an embodiment of the present invention;

[0033] Figure 5 Immunohistochemical staining images of IL-6 and IFN-γ in rat myocardial tissue after 2 and 4 weeks of treatment with the hydrogel according to the embodiment of the present invention. DETAILED DESCRIPTION

[0034] The present invention provides a hydrogel material that can be used for myocardial repair. The hydrogel material adopts methacrylamidated silk fibroin (SFMA) and arginine-glycine-aspartic acid peptide (RGD)-modified hyaluronic acid (HA-RGD) as a substrate, prepares a hydrogel carrier by chemical cross-linking, and loads self-generated oxygen nanoparticles CaO2@PVP@Cu-EGCG (CPCE). The self-generated oxygen nanoparticles CaO2@PVP@Cu-EGCG are prepared by coating Cu-EGCG with calcium peroxide nanoparticles modified with PVP-modified polyvinylpyrrolidone.

[0035] In this hydrogel system, the self-oxygenating complex CPCE significantly improves the local hypoxia of the myocardium by continuously releasing oxygen. In addition, the anti-inflammatory effect of CPCE can regulate the immune microenvironment, reduce the damage of inflammatory response to myocardial cells, and promote tissue regeneration. SFMA, as a structural support material, provides good biocompatibility and biodegradability. At the same time, silk fibroin itself is rich in amino acids and can serve as a nutrient source for cells, promoting cell survival and proliferation. HA-RGD binds to the cell surface through its modified RGD peptide, significantly enhancing the adhesion, proliferation and migration ability of cells, accelerating tissue regeneration, and playing an important role in the myocardial repair process, especially. By organically combining SFMA, HA-RGD and CPCE, the multifunctional new hydrogel system of the present invention can achieve efficient promotion of myocardial repair through a dual mechanism - improving energy metabolism and regulating the immune environment, providing new strategies and ideas for tissue regeneration and functional recovery after myocardial infarction.

[0036] Since SFMA has a photocuring effect after modification, the hydrogel of the present invention can be prepared by photocuring. Specifically,

[0037] Dissolving methacrylamide silk fibroin in an aqueous solution containing a photoinitiator to obtain a methacrylamide silk fibroin solution, which is referred to as solution A;

[0038] A hyaluronic acid solution modified with an arginine-glycine-aspartic acid peptide was prepared, and CaO2@PVP@Cu-EGCG was dispersed in HA-RGD and vortexed to completely and evenly disperse the solution therein to obtain solution B.

[0039] Then, solution A and solution B are mixed, and cross-linked by ultraviolet irradiation to obtain the hydrogel.

[0040] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and specific embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Unless otherwise specified, the reagents, methods and equipment used in the practice of the present invention are conventional reagents, methods and equipment in this technical field.

[0042] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0043] Example 1 Preparation of Methacrylamide Silk Fibroin (SFMA)

[0044] 40g of silkworm cocoons, after removing the pupae, were weighed and cut into four pieces. The cocoons were boiled in 1L of 0.05M Na2CO3 solution at 100°C for 30 minutes and washed several times with distilled water. The degummed silk fibroin (SF) was then oven-dried for 36 hours. 10g of degummed SF was dissolved in 100mL of 9.3M lithium bromide (LiBr) solution and stirred at 60°C for 1 hour. Once the degummed SF was completely dissolved, 6mL of glycidyl methacrylate was added and stirred at 60°C at 1000rpm for 6 hours. After the reaction was complete, the solution was filtered through a polyester rayon filter cloth (miracloth) and placed into a dialysis bag (molecular weight cutoff: 12-14 kDa). The solution was dialyzed against pure water for 3 days to remove salts. The solution was frozen at -80°C for 12 hours and then freeze-dried to obtain SFMA.

[0045] Example 2 Preparation of RGD-modified hyaluronic acid (HA-RGD)

[0046] Weigh 1g of hyaluronic acid and add 100mL of pure water. Once dissolved, add 0.8g of EDC to activate the carboxyl groups for 15 minutes. Then, add 1.2g of NHS. After 15 minutes, slowly add the activated hyaluronic acid solution dropwise to a 10mL, 0.05g / mL RGD solution. The mixture is then allowed to react at room temperature for 12 hours. After the reaction is complete, the solution is placed in a dialysis bag (molecular weight cutoff: 2000Da) and dialyzed in pure water for 3 days. The solution is frozen at -80°C for 12 hours and then lyophilized to obtain HA-RGD.

[0047] Example 3 Synthesis and Screening of Self-generated Oxygen Nanoparticles

[0048] 3-1 A self-generated oxygen nanocomposite particle, and a preparation method thereof, comprising the following steps:

[0049] 1) Preparation of polyvinylpyrrolidone-modified calcium peroxide nanoparticles CaO2@PVP.

[0050] CaCl2 (0.6 g) and PVP (0.6 g) were dissolved in 30 mL of anhydrous methanol and stirred. Subsequently, a mixture of 480 μL of H2O2 and 1 mL of aqueous ammonia was added. The mixture was stirred at 0°C for 1 hour. The resulting precipitate was collected by centrifugation and washed three times with methanol. The resulting sample was named CaO2@PVP.

[0051] 2) Preparation of Cu-EGCG complex.

[0052] 0.5 mmol of EGCG was weighed and dissolved in 30 mL of distilled water. 1 mmol of copper sulfate was prepared into 10 mL of solution and added dropwise. After mixing, the pH was adjusted to 7 with 1 mol / L sodium bicarbonate. The mixture was stirred at 40 °C with a constant temperature magnetic stirrer for 1 h to fully complex it. The precipitate was then washed several times with a 1:1 ethanol-water solution and freeze-dried in vacuum to obtain Cu-EGCG.

[0053] 3) Coating with Cu-EGCG.

[0054] Weigh 0.5 g of CaO2@PVP obtained in step 1) and 0.5 g of Cu-EGCG obtained in step 2); add 5 mL of MOPS buffer (100 mM, pH = 8.0) and wash to obtain CaO2@PVP particles coated with a layer of Cu-EGCG. Then, repeat the above coating process twice to obtain CaO2@PVP particles coated with three layers of Cu-EGCG, CaO2@PVP@Cu-EGCG, abbreviated as CPCE.

[0055] 3-2 A self-generated oxygen nanocomposite particle, the preparation method thereof comprises the following steps:

[0056] 1) Preparation of polyvinylpyrrolidone-modified calcium peroxide nanoparticles CaO2@PVP.

[0057] CaCl2 (0.6 g) and PVP (0.6 g) were dissolved in 30 mL of anhydrous methanol and stirred. Subsequently, a mixture of 480 μL of H2O2 and 1 mL of aqueous ammonia was added. The mixture was stirred at 0°C for 1 hour. The resulting precipitate was collected by centrifugation and washed three times with methanol. The resulting sample was designated CaO2@PVP.

[0058] 2) Preparation of Cu-EGCG complex.

[0059] Weigh 0.5 mmol of EGCG and dissolve it in 30 mL of distilled water. Then, prepare 5 mL of solution with 0.5 mmol of CuCl2 and add it dropwise. After mixing, adjust the pH to 7 with 1 mol / L sodium bicarbonate. Stir the mixture with a constant temperature magnetic stirrer at 40°C for 1 h to fully complex it. Then, wash the precipitate several times with a 1:1 ethanol-water solution and freeze-dry it in vacuum to obtain Cu-EGCG.

[0060] 3) Coating with Cu-EGCG.

[0061] Weigh 0.5 g of CaO2@PVP obtained in step 1) and 0.5 g of Cu-EGCG obtained in step 2); add 5 mL of MOPS buffer (100 mM, pH = 8.0) and wash to obtain CaO2@PVP particles coated with a layer of Cu-EGCG; then, repeat the above coating process twice to obtain CaO2@PVP particles coated with three layers of Cu-EGCG.

[0062] 3-3 A self-generated oxygen nanocomposite particle, which differs from 3-1 in that step 3) does not repeat the coating, and a layer of Cu-EGCG-coated CaO2@PVP particles is obtained.

[0063] 3-4 A self-generated oxygen nanocomposite particle, which differs from 3-2 in that step 3) does not repeat the coating, and a layer of Cu-EGCG-coated CaO2@PVP particles is obtained.

[0064] 3-5 3-1 Step 1) Prepare polyvinyl pyrrolidone-modified calcium peroxide nanoparticles CaO2@PVP.

[0065] 3-6 3-1 Step 2) Prepared Cu-EGCG complex.

[0066] The samples obtained in the above examples and comparative examples were respectively prepared into solutions of different concentrations using PBS (pH=7.4), and relevant tests were carried out to evaluate their performance. The results are as follows:

[0067] (1) Analysis of the antioxidant effect of nanoparticles in vitro (DPPH)

[0068] Table 1 DPPH free radical scavenging rate (%)

[0069]

[0070] Test Method: Samples were prepared according to the instructions of a commercially available DPPH free radical scavenging ability test kit. The extracts from the corresponding examples and comparative examples were added to the DPPH solution. The mixture was incubated in the dark for 30 minutes. The absorbance at 515 nm was measured using a UV-visible spectrophotometer, and the free radical scavenging rate was calculated. The test results are shown in Table 1.

[0071] The results indicate that the Cu-EGCG complex possesses strong antioxidant capacity, scavenging free radicals in the body and reducing cellular damage caused by oxidative stress. CaO2@PVP alone exhibits poor free radical scavenging ability, but the synergistic effect of the CPCE particles is enhanced, with the synergistic effect becoming more pronounced with increasing dosage. The greater the number of coating layers, the better the free radical scavenging results due to the increased incorporation of Cu-EGCG.

[0072] (2) Analysis of the antibacterial properties of nanoparticles.

[0073] Table 2 Antibacterial rate (%)

[0074]

[0075]

[0076] Test method: Escherichia coli and Staphylococcus aureus were revived and cultured to the logarithmic growth phase, centrifuged, and the bacteria were collected and resuspended in physiological saline. The final bacterial solution concentration was 1×10 8 CFU / mL. Add 500 μL of the solution of the corresponding concentration to a 24-well plate. Add 500 μL of normal saline to the control group. Then, add 100 μL of the diluted bacterial suspension and 500 μL of sterile saline to each well. Incubate at 37°C for 24 hours. After serial dilution, spread the suspension onto LB agar plates and count the cells.

[0077] Results: The test results in Table 2 show that the CPCE complex exhibited significant antibacterial activity against both Escherichia coli and Staphylococcus aureus, compared to the Cu-EGCG complex and CaO2@PVP particles alone. This is attributed to the synergistic effects of oxygen released by CaO2, the antibacterial effects of copper, and EGCG, which together enhanced the antibacterial efficacy of the complex. Copper and EGCG disrupted bacterial cell structure through different mechanisms, inhibiting their growth. Simultaneously, the continuous release of oxygen improved the local environment, further inhibiting bacterial growth.

[0078] (3) Nanoparticle biocompatibility assessment

[0079] Table 3 Cell survival rate (%)

[0080]

[0081] Test method: The nanoparticles were sterilized by irradiating them with ultraviolet light for 24 hours in advance. Nanozyme solutions of different concentrations were then prepared with culture medium for cell culture in subsequent experiments. H9C2 / 293T cells were digested with 0.25% trypsin, resuspended in DMEM, and seeded in 48-well plates at a density of 10,000 cells per well. They were cultured in a 37°C constant temperature carbon dioxide incubator (containing 5% CO2) for 24 hours until the cells adhered to the wall. The culture medium was discarded, washed with PBS, and the corresponding concentration of nanozyme solution was added and cultured for another 24 hours. After the culture was completed, the culture medium was discarded, washed 3 times with PBS, 500 μL of CCK-8 working solution (DMEM basal culture medium containing 10% CCK-8) was added to each well, and the cells were incubated in a 37°C constant temperature carbon dioxide incubator (containing 5% CO2) for 30 minutes. The absorbance (OD) was measured at a wavelength of 450 nm using a microplate reader, and the cell survival rate was calculated according to the formula: Cell survival rate (%) = (OD 实验组 -OD Blank ) / (OD 对照组 -OD Blank )×100%

[0082] Results: The above results show that the cell survival rate in all treatment groups was above 85%, indicating that the cells treated with nanoparticles did not suffer significant toxicity or damage and were able to maintain good growth and proliferation. This suggests that the prepared composite self-generating oxygen nanoparticle CPCE has good cell biocompatibility.

[0083] (4) Evaluation of the oxygen release ability of nanoparticles

[0084] Table 4 Dissolved oxygen content (ppm)

[0085]

[0086] Test method: 1 mL of 30% H2O2 is added to 9 mL of pure water to obtain 3% H2O2. The electrode of a portable dissolved oxygen meter is calibrated with 5% sodium sulfite. Then, 1 mL of H2O2 is added to 100 mL of H2O, followed by the nanomaterial at the desired final concentration. The dissolved oxygen concentration is then measured after incubation for two days.

[0087] The results show that the hydrolysis reaction of CaO2 is: CaO2 + 2H2O → Ca(OH)2 + O2. In this reaction, water decomposes calcium peroxide to produce oxygen, while Ca(OH)2 is a byproduct. In the composite self-generating oxygen nanoparticle CPCE of the present invention, PVP acts as a stabilizer to help disperse CaO2, control its hydrolysis rate, avoid excessive oxygen release, and maintain the stability of the material. Cu-EGCG may further regulate the reaction rate or promote oxygen release through catalysis. Furthermore, CaO2@PVP@Cu-EGCG can stably release oxygen over a long period of time, showing potential as an oxygen source material.

[0088] The above test results show that the combination of CaO2 / PVP and Cu-EGCG synergistically improves the antioxidant and antibacterial properties of the resulting nanoparticles, as well as their oxygen-releasing capacity. Furthermore, the resulting composite particles exhibit excellent biocompatibility and can be used as oxygen sources. Furthermore, as the concentration increases, the relevant properties also improve, demonstrating a significant synergistic effect. The most effective effect is achieved at a concentration of 50-200 μg / mL.

[0089] Furthermore, the CPCE particles obtained in 3-1 were characterized, and the results were as follows: Figure 1-3 shown. Figure 1 SEM image of CPCE nanoparticles 3-1, Figure 2 The TEM image of CPCE nanoparticles in Figure 3-1 shows a granular structure. The use of PVP effectively improves particle dispersion and reduces agglomeration, thereby enhancing the overall performance of the material.

[0090] Figure 3 The XRD spectra of CaO2, CaO2@PVP in 3-1, and CaO2@PVP@Cu-EGCG nanoparticles obtained from 3-1 are shown. In the spectrum, CaO2 shows significant peaks at about 20° and 32°, corresponding to the main crystal planes (such as (002), (110), and (202), respectively. These diffraction peaks are consistent with the known diffraction pattern of CaO2 (refer to PDF 03-0865), proving its crystal structure. Under the modification of PVP, the core structure of CaO2 is basically retained. The diffraction peaks of CaO2@PVP@Cu-EGCG show that they are broader and weaker than those of CaO2 and CaO2@PVP, which may be due to the further influence of the addition of Cu-EGCG on the structure of CaO2, resulting in further degradation of the crystal structure. The degradation of the crystal structure will increase the reaction activity of CaO2, which is beneficial to the improvement of antioxidant properties and dissolved oxygen capacity, which to a certain extent also supports the previous performance test results.

[0091] Example 4 Synthesis of hydrogel

[0092] 4-1 to 4-6 A hydrogel, the preparation method of which is as follows:

[0093] Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was dissolved in PBS buffer or normal saline to form a 0.2% aqueous solution. SFMA synthesized in Example 1 was then dissolved in the 0.2% LAP aqueous solution and completely dissolved by sonication to obtain Solution A. The specific mass fraction of SFMA in Solution A is shown in Table 5.

[0094] After dissolving the corresponding amount of HA-RGD prepared in Example 2 in PBS buffer, the CaO2@PVP@Cu-EGCG synthesized in Example 3-1 was dispersed in the HA-RGD at the desired concentration and vortexed for 10 minutes to ensure complete and uniform dispersion, yielding Solution B. The mass fraction of HA-RGD and the CPCE content in Solution B are shown in Table 5.

[0095] Then, solution A and solution B were mixed at a volume ratio of 1:1 and poured into a dressing mold. Bubbles were removed under a vacuum of -1 bar and the dressing was irradiated with 365 nm ultraviolet light for 60 seconds to complete cross-linking of the hydrogel dressing. The hydrogel dressing was then demolded to obtain the dressing.

[0096] Table 5 Experimental formulas in Example 4

[0097] Example SFMA HA-RGD LAP CPCE 4-1 7.5％ 1％ 0.2％ 50 μg / mL 4-2 7.5％ 1％ 0.2％ 100 μg / mL 4-3 7.5％ 1％ 0.2％ 200 μg / mL 4-4 10％ 1.5％ 0.2％ 200 μg / mL 4-5 10％ 2％ 0.2％ 200 μg / mL 4-6 12％ 2％ 0.2％ 200 μg / mL 4-7 12％ 2％ 0.2％ / 4-8 12％ / 0.2％ 200 μg / mL 4-9 12％ 2％ / 200 μg / mL 4-10 12％ / 0.2％ /

[0098] The difference between Example 4-7 and Example 4-6 is that CaO2@PVP@Cu-EGCG is not added. The specific steps are as follows:

[0099] LAP was dissolved in PBS buffer or saline to form a 0.2% aqueous solution. The 0.2% LAP aqueous solution was then used to dissolve the SFMA synthesized in Example 1, and sonicated to completely dissolve it, to obtain Solution A. The specific mass fraction of SFMA in Solution A is shown in Table 5.

[0100] A corresponding amount of HA-RGD prepared in Example 2 was dissolved in PBS buffer to obtain solution B'.

[0101] Then, the A and B' solutions were mixed at a volume ratio of 1:1 and poured into a dressing mold. Bubbles were removed under a vacuum of -1 bar and the hydrogel dressing was irradiated with 365 nm ultraviolet light for 60 seconds to complete cross-linking. The hydrogel dressing was then demolded to obtain the dressing.

[0102] The difference between Example 4-8 and Example 4-6 is that HA-RGD is not added. The specific steps are as follows:

[0103] Dissolve LAP in PBS buffer or saline to form a 0.2% aqueous solution. Then, dissolve the SFMA synthesized in Example 1 in the 0.2% LAP aqueous solution and completely dissolve it by sonication. Disperse the CaO2@PVP@Cu-EGCG synthesized in Example 3-1 into the SFMA solution at the desired concentration and vortex for 10 minutes to ensure complete and uniform dispersion.

[0104] The above solution was then poured into a dressing mold, and bubbles were removed under a vacuum of -1 bar. The hydrogel dressing was irradiated with 365 nm wavelength ultraviolet light for 60 seconds to complete cross-linking. The hydrogel dressing was then demoulded to obtain the hydrogel dressing.

[0105] The difference between Example 4-9 and Example 4-6 is that the photoinitiator LAP is not added. The specific steps are as follows:

[0106] The SFMA synthesized in Example 1 was dissolved in PBS buffer and completely dissolved by ultrasound;

[0107] The HA-RGD synthesized in Example 2 was dissolved in PBS buffer and completely dissolved by ultrasound. Then, the CaO2@PVP@Cu-EGCG synthesized in Example 3-1 was dispersed in the HA-RGD solution at a set concentration and vortexed for 10 minutes to completely and evenly disperse it therein.

[0108] Then the above solutions were mixed at a volume ratio of 1:1 to maintain the solution state.

[0109] The difference between Example 4-10 and Example 4-6 is that HA-RGD and CaO2@PVP@Cu-EGCG are not added. The specific steps are as follows:

[0110] Dissolve LAP in PBS buffer to form a 0.2% aqueous solution. The 0.2% LAP aqueous solution was then used to dissolve the SFMA synthesized in Example 1, and sonication was performed to completely dissolve the solution. The solution was then poured into a dressing mold, and air bubbles were removed under a vacuum of -1 bar. The hydrogel dressing was then irradiated with 365 nm UV light for 60 seconds to complete crosslinking. The mold was then removed to obtain the hydrogel dressing.

[0111] Analysis of hydrogel effects:

[0112] (1) Analysis of storage modulus of hydrogel scaffolds

[0113] Table 6 Hydrogel storage modulus

[0114] Example 4-1 4-2 4-3 4-4 4-5 4-6 4-7 4-8 4-9 4-10 Storage modulus (Pa) 85 86 88 456 480 1015 999 385 / 859

[0115] Storage modulus testing method: A cylindrical hydrogel sample containing 400 μL of liquid was demolded and placed on a 25 mm diameter stainless steel parallel plate rotor for rheological measurement using a rotational rheometer. The test temperature was 37°C and the sweep strain was 1%. G' represents the elastic modulus of the sample.

[0116] Explanation of the results: Referring to Table 6, the storage modulus of the hydrogel is mainly affected by the concentration of the hydrogel substrate SFMA and HA-RGD. The higher the concentration of SFMA and HA-RGD, the higher the storage modulus. The addition of CPCE will also have a certain effect on its storage modulus. Among them, in Examples 4-1 to 4-3, due to the low concentration of SFMA, the hydrogel gelation effect is not good, the gel is too soft, and the storage modulus is low. The gelation effect of Examples 4-4 to 4-6 is better. If Example 4-9 does not add LAP, it cannot be photocured to form a hydrogel and the storage modulus test cannot be performed.

[0117] (2) Evaluation of blood compatibility of hydrogel scaffolds

[0118] Table 7 Hydrogel hemolysis rate (%)

[0119]

[0120] Examples 4-9 were liquids and were not tested.

[0121] Hemolysis rate test method: First, fresh whole blood is centrifuged at 2000rpm for 15min to remove serum. Repeat washing with PBS 5 times to ensure that pure red blood cells are obtained. The pure red blood cells are diluted with PBS solution to obtain a red blood cell suspension with a concentration of 2% (v / v). Then 1ml of red blood cell suspension is added to the sample to be tested (100μL gel sample) and incubated at 37°C for 1h. After this mixture is centrifuged at 1600rpm for 10min, the absorbance value of the supernatant is measured at a wavelength of 524nm. In addition, distilled water is used as a positive control and PBS is used as a negative control. The calculation formula for the sponge hemolysis rate (H) is as follows: H(%) = (OD sample -OD negative ) / (OD positive -OD negative )×100, where H is the hemolysis rate, in %; OD sample -Absorbance value of sample group; OD negative -Absorbance value of negative control group; OD positive -The absorbance value of the positive control group;.

[0122] Results: Referring to Table 7, the hemolysis rate of the hydrogel scaffolds in each example was less than 5%, meeting the requirements of biomedical materials. Although the addition of CPCE had a slight effect on red blood cells, it was within a controllable range.

[0123] (3) Evaluation of the anti-inflammatory effect of hydrogel scaffolds

[0124] Table 8 Relative expression of inflammatory factors

[0125] Example 4-1 4-2 4-3 4-4 4-5 4-6 4-7 4-8 4-9 4-10 iNOS 1.25 0.59 0.32 0.29 0.31 0.25 1.51 0.36 0.22 1.63 TNF-α 0.75 0.25 0.09 0.09 0.14 0.05 0.95 0.39 0.11 0.99 IL-1β 1.16 0.84 0.16 0.12 0.11 0.11 1.46 0.51 0.15 1.51 IL-10 1.29 2.38 3.15 3.10 3.18 3.2 1.42 2.86 3.09 1.49 TGF-β1 0.56 0.89 1.21 1.25 1.32 1.26 0.71 1.15 1.23 0.82 Arg-1 1.11 2.57 3.89 3.88 3.91 3.95 1.36 3.52 3.89 1.45

[0126] Testing method: H9C2 cells were induced with LPS and incubated with extracts from different hydrogel scaffold groups for 24 hours. The cells were then washed with PBS and placed on ice. Trizol was then added for lysis and RNA extraction. After concentration determination, the RNA was added to a gDNA adsorption column and centrifuged to collect the DNA-free RNA. The RNA was denatured at 65°C, cooled on ice, and reverse transcribed to generate cDNA. A PCR reaction was set up, and the cDNA and reaction solution were combined and analyzed on the PCR instrument to generate melting and amplification curves. The data were then exported and analyzed.

[0127] Results: As shown in Table 8, increasing CPCE content significantly reduced the expression of pro-inflammatory factors (iNOS, TNF-α, IL-1β) and increased the expression of anti-inflammatory factors (IL-10, TGF-β1, Arg-1). Furthermore, the dosage of SFMA and HA-RGD also had a certain impact on the anti-inflammatory effect.

[0128] (4) Analysis of the in vitro antioxidant properties of hydrogel scaffolds

[0129] Table 9 DPPH free radical scavenging rate (%)

[0130]

[0131] Test method: Prepare the sample according to the instructions of the commercially available DPPH free radical scavenging ability test kit. Add the extracts of the corresponding examples and comparative examples to the DPPH solution, incubate in the dark for 30 minutes, measure the absorbance at 515 nm using a UV-visible spectrophotometer, and calculate the free radical scavenging rate.

[0132] Results: Table 9 shows that CPCE plays a major role in the in vitro antioxidant effect of the gel. With increasing CPCE concentration, the free radical scavenging rate significantly increased. CPCE complexes, through their metal ion properties, may directly participate in free radical scavenging reactions and also regulate intracellular redox status. Cu may activate antioxidant-related genes by regulating intracellular ROS levels, while EGCG's antioxidant effects may be exerted by directly neutralizing free radicals and inhibiting inflammatory signaling pathways.

[0133] (5) Hydrogel structure characterization

[0134] Figure 4These are SEM images of some hydrogel samples in Example 4. It can be seen that the cross section of the formed hydrogel has a good pore structure and a certain degree of roughness.

[0135] (6) Animal experiments

[0136] A mouse myocardial injury model was established, and the hydrogel samples of Examples 4-6, 4-7, and 4-8 were selected for treatment, respectively. The immunohistochemical staining results of IL-6 and IFN-γ in myocardial tissue after 2 and 4 weeks of treatment were as follows: Figure 5 shown.

[0137] It can be seen that Examples 4-6 have the best effect, and compared with other experimental groups, the inflammatory response is weakened, which can play a role in immune regulation.

[0138] In summary, the embodiments of the present invention synthesize and screen self-generated oxygen nanoparticles, and then load them into a hyaluronic acid cross-linking system modified with methacrylamide silk fibroin and arginine-glycine-aspartic acid peptide to obtain a hydrogel containing self-generated oxygen nanoparticles. The hydrogel has good strength, anti-inflammatory and antioxidant effects, and can be used for myocardial repair.

[0139] The technical features of the above-described embodiments can be combined in any combination. To simplify the description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent of this invention shall be based on the attached claims.

Claims

1. A hydrogel loaded with self-generated oxygen nanoparticles, characterized by: The hydrogel is based on cross-linked methacrylamidated silk protein and arginine-glycine-aspartic acid peptide-modified hyaluronic acid, and is loaded with self-generated oxygen nanoparticles CaO2@PVP@Cu-EGCG. The self-generated oxygen nanoparticles CaO2@PVP@Cu-EGCG are prepared by coating Cu-EGCG with calcium peroxide nanoparticles modified with PVP-modified polyvinyl pyrrolidone.

2. The hydrogel according to claim 1, wherein The methacrylamide silk fibroin is prepared by dissolving degummed silk fibroin in a lithium bromide solution, dispersing the solution at 60°C until completely dissolved, adding glycidyl methacrylate, and reacting the solution at 60°C with stirring. After the reaction is complete, the solution is filtered through a polyester rayon filter cloth, and then dialyzed in pure water for 3 days to remove salt, obtaining a product with a molecular weight cutoff of 12-14 kDa. Finally, the solution is frozen at -80°C and then freeze-dried. Preferably, the degummed silk fibroin: lithium bromide: glycidyl methacrylate = 10 g: 75-85 g: 6 mL.

3. The hydrogel according to claim 1, wherein The arginine-glycine-aspartic acid peptide-modified hyaluronic acid is obtained by activating hyaluronic acid with EDC and NHS, adding the hyaluronic acid dropwise to an arginine-glycine-aspartic acid peptide solution, reacting at room temperature, and dialyzing the hyaluronic acid in pure water for 3 days using a dialysis bag with a molecular weight cutoff of 2000 Da. The solution is then frozen at -80°C and lyophilized. Preferably, the mass ratio is hyaluronic acid: EDC: NHS: arginine-glycine-aspartic acid peptide = 1:0.8:1.2:0.

5.

4. The hydrogel according to claim 1, wherein: The preparation method of the self-generated oxygen nanoparticles CaO2@PVP@Cu-EGCG comprises the following steps: 1) Preparation of polyvinylpyrrolidone-modified calcium peroxide nanoparticles CaO2@PVP; 2) Preparation of Cu-EGCG complex; Cu 2+ The solution was added dropwise to the EGCG solution, and after mixing, the pH was adjusted to 7, and the mixture was stirred at a constant temperature of 40°C to fully complex the solution. The precipitate was then washed and freeze-dried in vacuum to obtain the Cu-EGCG complex. 3) Coating Cu-EGCG; mixing the CaO2@PVP obtained in step 1) and the Cu-EGCG complex obtained in step 2), then adding MOPS buffer, and finally washing to obtain CaO2@PVP particles coated with Cu-EGCG.

5. The hydrogel according to claim 2, characterized in that In the step 1), CaO2@PVP is prepared by dissolving CaCl2 and PVP in anhydrous methanol, and then adding a mixed solution of H2O2 and ammonia; the obtained mixed solution is stirred at 0°C for reaction, and the obtained precipitate is collected by centrifugation and washed with methanol; Preferably, the mass ratio of CaCl2 to PVP is 1:1; Preferably, the molar concentration of EGCG in the EGCG solution is 0.01-0.02 mol / L, and the Cu 2+ Cu in solution 2+ The molar concentration is 0.05~0.1mol / L.

6. The hydrogel according to claim 2, characterized in that In the step 2), according to the molar ratio, Cu 2+ :EGCG=1:1-2.

7. The hydrogel according to claim 2, characterized in that In the step 3), the mass ratio of CaO2@PVP:Cu-EGCG is 1:

1. Preferably, step 3) can be repeated at least once to obtain CaO2@PVP particles coated with multiple layers of Cu-EGCG.

8. The method for preparing the hydrogel according to any one of claims 1 to 7, characterized in that: include: Dissolving methacrylamide silk fibroin in an aqueous solution containing a photoinitiator to obtain a methacrylamide silk fibroin solution, which is referred to as solution A; A hyaluronic acid solution modified with an arginine-glycine-aspartic acid peptide was prepared, and CaO2@PVP@Cu-EGCG was dispersed in HA-RGD and vortexed to completely and evenly disperse the solution therein to obtain solution B. Then, solution A and solution B are mixed, and cross-linked by ultraviolet irradiation to obtain the hydrogel.

9. The method for preparing the hydrogel according to claim 8, wherein: Solution A and solution B were mixed in a volume ratio of 1:

1. In addition, according to the mass-to-volume ratio, the concentration of the methacrylamide silk fibroin solution in solution A was 7.5%-12%, the concentration of the arginine-glycine-aspartic acid peptide-modified hyaluronic acid in solution B was 1-2%, and the dosage of CaO2@PVP@Cu-EGCG was 10-200 μg / mL.

10. Use of the hydrogel according to any one of claims 1 to 7 or the hydrogel prepared by the preparation method according to any one of claims 8 to 9 in preparing a drug for myocardial repair therapy.

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