Active oxygen responsive hydrogel for relieving myocardial ischemia-reperfusion injury as well as preparation method and application of active oxygen responsive hydrogel

Abstract

The invention discloses active oxygen responsive hydrogel for relieving myocardial ischemia-reperfusion injury and a preparation method and application thereof.The preparation method comprises the following steps that a hydrophobic anti-oxidation medicine solution is added into a water-phase solution of dendritic macromolecules, a product obtained after reaction is dialyzed, purified and freeze-dried, and a compound A is obtained; mixing the water-phase solution of the compound A with the water-phase solution of the copper death related gene to form a compound B solution, and then adding an oxidized polysaccharide water-phase solution to react to form gel, so as to obtain the active oxygen responsive hydrogel. According to the invention, the hydrophobic antioxidant drug and the copper death related gene are loaded on the dendrimer, and cross-linked with the oxidized polysaccharide, so that the myocardial ischemia reperfusion injury can be effectively relieved by utilizing the mutual synergistic effect of the three compounds, and long-acting and controllable treatment is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to an active oxygen responsive hydrogel for alleviating myocardial ischemia-reperfusion injury, and a preparation method and application thereof. Background Art

[0002] Myocardial ischemia-reperfusion injury (IRI) is a common and serious complication in the treatment of cardiovascular diseases, severely impacting patients' prognosis and quality of life. During IRI, reactive oxygen species (ROS) are generated in large quantities, triggering oxidative stress and leading to a series of pathological processes, including myocardial cell damage, apoptosis, and inflammation. Currently, treatment options for IRI are limited. Traditional drug therapies struggle to precisely target the site of injury, and due to their low bioavailability, they are unable to effectively address the complex pathological changes induced by ROS.

[0003] In the field of biomedical materials, hydrogels have attracted widespread attention due to their excellent biocompatibility and adjustable physicochemical properties. CN112168952A discloses the use of hydrogel-loaded cyclosporine A in the preparation of a drug for treating myocardial ischemia-reperfusion injury. This addresses the existing issue of excessive toxicity of cyclosporine A administered directly. By using hydrogel-loaded cyclosporine A for administration, not only can the efficacy of cyclosporine A be locally increased while reducing systemic toxicity, but the hydrogel A cardiac patch, with its local electrical conductivity and adhesive properties, can also improve myocardial damage after myocardial infarction.

[0004] CN119019856A discloses the preparation of a polypeptide hydrogel and its application in myocardial ischemia-reperfusion injury. The hydrogel is assembled from the polypeptide gelling factor YFF-TK-FFY (structural sequence: tyrosine-phenylalanine-phenylalanine-thioketal-phenylalanine-phenylalanine-tyrosine) and the adipocyte factor Apelin-13 to form a ROS-responsive polypeptide hydrogel. The polypeptide hydrogel Apelin-13@GelTK has good biocompatibility and biodegradability. The thioketal bonds in the gelling factor can respond to high levels of ROS in the core area of ​​myocardial ischemia-reperfusion injury and then break. Studies have shown that the polypeptide hydrogel Apelin-13@GelTK has a significant therapeutic effect in alleviating myocardial ischemia-reperfusion injury.

[0005] However, most existing hydrogel materials lack the ability to specifically respond to reactive oxygen species, making it difficult to achieve localized, controlled, and long-lasting release of drugs and genes in the pathological environment of myocardial ischemia-reperfusion injury. Therefore, developing a hydrogel material that can precisely respond to reactive oxygen species and achieve efficient drug and gene release at the site of myocardial ischemia-reperfusion injury is of great significance for improving treatment outcomes. Summary of the Invention

[0006] The present invention aims to develop a hydrogel material that can accurately respond to reactive oxygen species and achieve efficient and controllable release of drugs and genes at the site of myocardial ischemia-reperfusion injury, thereby breaking the existing treatment dilemma and significantly improving the treatment effect.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] A method for preparing an active oxygen responsive hydrogel for alleviating myocardial ischemia-reperfusion injury, characterized by comprising the steps of:

[0009] Step 1, adding a hydrophobic antioxidant drug solution to an aqueous solution of a dendrimer macromolecule, and the reaction product is dialyzed, purified, and freeze-dried to obtain a complex A;

[0010] Step 2: Mix the aqueous solution of complex A and the aqueous solution of copper death-related genes to form a complex B solution, and then add the aqueous solution of oxidized polysaccharide to react and form a gel to obtain the active oxygen responsive hydrogel.

[0011] In the present invention, hydrophobic antioxidant drugs and copper-related death genes are loaded onto dendrimers. Dendrimers have unique structures and properties. Their internal hydrophobic cavities can load hydrophobic drugs, and the abundant amino groups on their surfaces can load copper-related death genes through electrostatic interactions, providing a good foundation for the construction of multifunctional hydrogels. Oxidized polysaccharides can cross-link with dendrimers through Schiff base reactions to form a stable hydrogel network, which imparts reactive oxygen species responsiveness to the hydrogel. Hydrophobic drugs such as caffeic acid and its derivatives have antioxidant properties; copper-related death genes such as siFDX1 can inhibit cell death pathways associated with reactive oxygen species (such as copper death). The synergistic effect can effectively alleviate myocardial ischemia-reperfusion injury.

[0012] The dendrimers include one or more of polyamidoamine (PAMAM), polylysine (PLL), polypropyleneimine (PPI), polyethyleneimine (PEI), and polyarylether. The type of dendrimer directly impacts drug loading efficiency (determined by its internal cavity size and hydrophobicity), gene loading capacity, complex stability (influenced by surface charge density), and ultimately the hydrogel's mechanical strength, degradability, and biocompatibility / cytotoxicity. Selection should be based on a comprehensive consideration of drug loading capacity, gene loading capacity, gel-forming properties, and safety.

[0013] The hydrophobic antioxidant drug includes one or more of hydrophobic polyphenols, hydrophobic quinones, hydrophobic flavonoids, hydrophobic statins, and hydrophobic carotenoids. The type of hydrophobic antioxidant drug directly affects its loading capacity in the dendrimer, its release rate from the hydrogel, its antioxidant efficacy (including the targeted reactive oxygen species and their activity), and its ultimate biological effect. The appropriate drug should be selected based on the characteristics of the target oxidative stress in myocardial ischemia-reperfusion injury.

[0014] The copper death-related genes include siRNA or shRNA of one or more of FDX1, LIAS, LIPT1, DLAT, PDHA1, and PDHB; preferably, siRNA or shRNA of FDX1.

[0015] The oxidized polysaccharide includes one or more of oxidized dextran, oxidized sodium alginate, oxidized glucomannan, oxidized bletilla striata polysaccharide, oxidized fucoidan, oxidized hyaluronic acid, etc. The type of oxidized polysaccharide and its oxidation degree (aldehyde content) will affect the gelation speed and strength, the responsiveness to active oxygen species, and the release behavior of drugs / genes.

[0016] The molecular weight of the dendrimer is between 10,000 and 30,000. A molecular weight that is too low (<10,000) significantly reduces drug and gene loading capacity, and the resulting hydrogel has weak mechanical strength. A molecular weight that is too high (>30,000) increases the risk of cytotoxicity, increases solution viscosity, and can lead to an overly dense gel network, hindering drug / gene release and reactive oxygen species response, while also reducing cellular uptake efficiency.

[0017] The mass ratio of the dendrimer to the hydrophobic antioxidant drug is 1:(0.5-2). An appropriate drug loading amount can achieve effective therapeutic effects while avoiding drug waste, damage to the stability of the complex, and interference with subsequent steps.

[0018] The mass ratio of the dendrimer to the copper death-related gene is 1:(0.1-5). The appropriate gene ratio can reduce the risk of cytotoxicity and is not likely to cause degradation of free genes, resulting in unstable phenomena such as increase in complex particle size or precipitation, reducing transfection efficiency or possibly interfering with gel formation.

[0019] The mass ratio of the dendrimer to the oxidized polysaccharide is (0.1-10): 1. The gel obtained at this ratio has high mechanical strength, suitable solubility, suitable drug / gene release rate, low cytotoxicity risk, and good reactive oxygen species responsiveness.

[0020] The mass ratio of the hydrophobic antioxidant drug to the copper-related death gene is (0.1-20):1. A ratio that is too low may result in insufficient initial antioxidant protection, failing to create a favorable microenvironment for gene therapy; a ratio that is too high may weaken the effectiveness of targeted gene intervention in the corresponding pathway. Optimizing the ratio is crucial to achieve a synergistic effect between the drug's rapid scavenging of reactive oxygen species and the gene's targeted regulation of the death pathway.

[0021] The mass concentration of the oxidized polysaccharide aqueous solution is 2-10 wt %, the reaction temperature after adding the oxidized polysaccharide is 25-37° C., and the reaction time is 5 seconds to 10 minutes.

[0022] The present invention also provides an active oxygen responsive hydrogel for alleviating myocardial ischemia-reperfusion injury prepared by the preparation method.

[0023] The present invention also provides the use of the hydrogel in the preparation of drugs or medical materials for alleviating myocardial ischemia-reperfusion injury. The hydrogel can respond intelligently under the stimulation of high reactive oxygen species in the pathological site, release drugs and genes, and achieve long-term, controllable treatment. It also has good injectability and stability, can protect and deliver antioxidant drugs to the target tissue, so that the loaded antioxidant drugs can effectively remove local excess reactive oxygen species and significantly reduce the oxidative stress level at the site of myocardial damage. At the same time, copper death-related genes can efficiently enter myocardial cells, inhibit related cell death pathways (such as copper death), thereby effectively saving myocardial tissue.

[0024] The present invention also provides the use of siRNA or shRNA targeting copper-death-related genes in the preparation of drugs or medical materials for alleviating myocardial ischemia-reperfusion injury. The copper-death-related genes include FDX1. The inventors have first revealed the mechanism by which genes such as FDX1 exacerbate myocardial injury by activating the copper-death pathway. Silencing this gene can achieve:

[0025] (1) Inhibition of Cu 2+ →Cu + Transformation, blocking toxic oligomerization driven by copper ion-lipoylated protein binding;

[0026] (2) Inhibit the explosive generation of reactive oxygen species and interrupt the oxidative damage cascade;

[0027] (3) Significantly reduce myocardial cell death and improve cardiac function.

[0028] The relief of myocardial ischemia-reperfusion injury in the present invention is specifically manifested as follows:

[0029] (1) Blockade of the copper death pathway: downregulation of the key protein FDX1, reduced DLAT oligomerization, and restoration of the stability of the lipoylase LIAS;

[0030] (2) Oxidative stress inhibition: significantly reduces the content of reactive oxygen species in myocardial tissue and improves cell activity;

[0031] (3) Recovery of cardiac function: Improve left ventricular ejection fraction, etc., and improve cardiac function.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The hydrogel of the present invention can intelligently respond to the stimulation of highly reactive oxygen species at the pathological site, releasing drugs and genes to achieve long-term, controllable treatment. Furthermore, the hydrogel has good injectability and stability, enabling precise delivery to the site of myocardial injury, significantly improving the bioavailability of functional molecules at that site.

[0034] (2) Under the protection and delivery of the hydrogel, the loaded antioxidant drugs can effectively remove local excess reactive oxygen species and significantly reduce the level of oxidative stress in the myocardial injury site. At the same time, copper death-related genes can efficiently enter myocardial cells and inhibit related cell death pathways (such as copper death), thereby effectively saving myocardial tissue.

[0035] (3) The preparation method of the present invention is simple, mild, and has a wide range of raw materials. It has good reproducibility and large-scale production potential. The obtained hydrogel is biodegradable and has good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the design of active oxygen responsive hydrogel.

[0037] Figure 2 Transmission electron microscopy image of the dendrimer complex loaded with antioxidant drugs and copper death-related genes.

[0038] Figure 3 Particle size distribution of dendrimer complexes loaded with antioxidant drugs and copper-related death genes.

[0039] Figure 4 Particle size and potential of dendrimer complexes loaded with antioxidant drugs and copper-induced death-related genes.

[0040] Figure 5 Cytocompatibility of dendrimer complexes loaded with antioxidant drugs and copper-mediated death-related genes.

[0041] Figure 6 In vitro intracellular delivery of dendrimer complexes loaded with antioxidant drugs and copper-induced death-related genes.

[0042] Figure 7 The reactive oxygen free radical scavenging ability of dendrimer complexes loaded with antioxidant drugs and copper death-related genes.

[0043] Figure 8 The microscopic morphology of the active oxygen responsive hydrogel with a G4CA:si-FDX1 mass ratio of 2:1.

[0044] Figure 9 The active oxygen responsive drug release behavior of the active oxygen responsive hydrogel with a G4CA:si-FDX1 mass ratio of 2:1.

[0045] Figure 10 The in vivo gel degradation of the active oxygen responsive hydrogel with a G4CA:si-FDX1 mass ratio of 2:1.

[0046] Figure 11 The copper cell death inhibition effect of the active oxygen responsive hydrogel with a G4CA:si-FDX1 mass ratio of 2:1.

[0047] Figure 12 The cardiac function recovery of the active oxygen responsive hydrogel with a G4CA:si-FDX1 mass ratio of 2:1.

[0048] Figure 13 The active oxygen scavenging ability of the dendrimer complex lacking a certain component under the hypoxia-reoxygenation condition in Comparative Example 1.

[0049] Figure 14 The cell activities of the dendrimer complex loaded with antioxidant drugs and with or without copper death-related genes in Comparative Example 2 under normal or reoxygenation conditions are shown.

[0050] Figure 15 The in vivo drug retention of the active oxygen responsive hydrogel with a G4CA:si-FDX1 mass ratio of 2:1 in Comparative Example 3 and the dendrimer macromolecular complex simply loaded with antioxidant drugs and copper death-related genes.

[0051] Figure 16 This is the copper death-related gene inhibition status of the active oxygen responsive hydrogel with a G4CA:si-FDX1 mass ratio of 2:1 in Comparative Example 3 and the dendrimer macromolecular complex simply loaded with antioxidant drugs and copper death-related genes. DETAILED DESCRIPTION

[0052] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail with reference to the following examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Those skilled in the art can make modifications or equivalent substitutions based on their understanding of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, which should all be included in the protection scope of the present invention. Unless otherwise defined, the professional terms used below are consistent with the meanings understood by professionals in this field; unless otherwise specified, the raw materials and reagents involved in this article can be purchased from the market or prepared by known methods.

[0053] The raw materials used in the following specific embodiments are all purchased from the market.

[0054] Example 1

[0055] Step 1: Preparation of dendrimers loaded with antioxidant drugs. The preparation process diagram is as follows: Figure 1 As shown, the following steps are included:

[0056] 1) Prepare 10 mg / ml PAMAM G4 solution and 10 mg / ml ethyl caffeate (EA) solution for later use;

[0057] 2) Place a flask containing 50 ml of PAMAM G4 solution on a magnetic stirrer and add EA solution dropwise to the flask. The amount of EA is 1.2 times that of PAMAM G4. The reaction conditions are pH 7.4, reaction temperature 37°C, and stirring for 12 hours in the dark.

[0058] 3) The solution was placed in a dialysis bag with a cutoff of 3500, and the dialysis bag was submerged in a beaker filled with 2 L of deionized water. A magnetic stirrer was placed at the bottom of the beaker. The deionized water was replaced every 2-3 hours for the first 12 hours of dialysis and every 5-6 hours for the next 36 hours. The solution was lyophilized to obtain G4EA.

[0059] Step 2: Preparation of dendrimers loaded with antioxidant drugs and copper death-related genes, including the following steps:

[0060] 1) Re-disperse the dried G4EA in water to prepare a 4 mg / ml solution;

[0061] 2) Dissolve 2 mg of si-FDX1 in 1 ml of DEPC water to prepare a 2 mg / ml solution.

[0062] 3) Add 1 ml of si-FDX1 solution to 1 ml of G4EA solution at room temperature. Gently pipette to mix thoroughly. Incubate at 4°C for several minutes to allow si-FDX1 to be stably loaded onto the PAMAM G4 surface through electrostatic interaction, yielding a G4CAsi-FDX1 solution.

[0063] Step 3: Preparation of oxidized dextran (OD), comprising the following steps:

[0064] 1) Weigh 100 g of dextran and dissolve it completely in 150 ml of deionized water. Place the mixture in a magnetic stirrer equipped with a 37°C water bath.

[0065] 2) Weigh 6.4 g of NaIO4 and dissolve it completely in 50 ml of deionized water;

[0066] 3) NaIO4 solution was added dropwise to the dextran solution, and the mixture was stirred in the dark for 6 hours;

[0067] 4) Add 10 ml of ethylene glycol to the reaction solution and continue the reaction for 1 hour;

[0068] 5) After the reaction is terminated, the resulting product solution is transferred to a dialysis bag with a MWCO of 3500 Da. The dialysis bag is submerged in a beaker filled with 2 L of deionized water, and a magnetic stirrer is placed at the bottom of the beaker. The deionized water is replaced every 2-3 hours for the first 12 hours of dialysis and every 5-6 hours for the next 36 hours. The resulting product is frozen at -80°C, solidified, and then lyophilized to obtain the final product.

[0069] Step 4: preparing a drug-loaded reactive oxygen species-responsive hydrogel, comprising the following steps:

[0070] 2.5 mg of OD was weighed and dissolved in 1 ml of deionized water; 2 ml of G4EAsi-FDX1 solution was thoroughly mixed with the OD solution, and after standing at room temperature for 5 minutes, a Schiff base reaction was used to form a gel to prepare a drug-loaded active oxygen responsive hydrogel.

[0071] The performance of the nanoparticles prepared in step 1 was tested using the following test method:

[0072] The morphology of the nanoparticles was observed using a transmission electron microscope (TEM). Figure 2 As shown, the nanoparticles are spherical and have a relatively uniform particle size distribution between 10-100 nm.

[0073] The particle size and potential were measured by dynamic light scattering (DLS). Figure 3 As shown in Figure 2, the average particle size of G4EAsi-FDX1 is about 58.7±2.45nm. Figure 4As shown, the potential is +30-40 mV, indicating that the nanoparticles have good stability and dispersibility.

[0074] The biocompatibility of PAMAM G4 loaded with EA and si-FDX1 at different concentrations (G4EAsi-FDX1) was tested. Primary neonatal rat cardiomyocytes were cultured in 96-well plates and G4EAsi-FDX1 at concentrations of 1μg / ml, 2μg / ml, 4μg / ml, 6μg / ml, 8μg / ml, 10μg / ml, 20μg / ml, 50μg / ml, 75μg / ml, and 100μg / ml were added and incubated for 24 hours. According to the test instructions, 10μl CCK8 solution was added to each well and the cells were placed in an incubator for another 4 hours. The culture was then terminated, the supernatant was aspirated, and the absorbance of each well was measured at OD450nm on a microplate reader. Figure 5 As shown, after the cells were treated with 20 μg / ml G4CAsi-FDX1 for 24 hours, the cell viability was still above 80%.

[0075] Example 2

[0076] Step 1: Preparation of dendrimers loaded with antioxidant drugs, comprising the following steps:

[0077] 1) Prepare 10 mg / ml PAMAM G4 solution and 10 mg / ml CA solution for later use:

[0078] Weigh 20 mg of PAMAM G4 and dissolve it in 20 mL of deionized water. Stir at room temperature for 30 minutes to allow it to fully dissolve, to obtain a PAMAM G4 solution with a concentration of 1 mg / mL.

[0079] Weigh 30 mg of caffeic acid CA and dissolve it in 30 ml of methanol to prepare a solution with a concentration of 1 mg / ml;

[0080] 2) Slowly add the prepared CA solution dropwise to the PAMAM G4 solution. The reaction conditions are pH 8.0, reaction temperature 26°C, and stirring for 10 hours in the dark.

[0081] 3) The solution was poured into a dialysis bag with a cutoff of 3500, and the dialysis bag was submerged in a beaker filled with 2 L of deionized water. A magnetic stirrer was placed at the bottom of the beaker. The deionized water was replaced every 2-3 hours for the first 12 hours of dialysis and every 5-6 hours for the next 36 hours. The obtained product was frozen at -80°C, solidified, and then lyophilized to obtain a CA-loaded PAMAM G4 complex.

[0082] Step 2: Preparation of dendrimers loaded with antioxidant drugs and copper death-related genes, including the following steps:

[0083] 1) Re-disperse the dried G4CA in water to prepare a 1 mg / ml solution;

[0084] 2) Dissolve 80 μg of si-FDX1 in 80 μl of G4CA solution, incubate at room temperature for 10 minutes, and gently shake the EP tube to better load si-FDX1 onto the PAMAM G4 surface to obtain G4CAsi-FDX1.

[0085] Step 3 is the same as in Example 1.

[0086] Step 4: preparing a drug-loaded reactive oxygen species-responsive hydrogel, comprising the following steps:

[0087] Weigh 2.5 mg of OD and dissolve it in 2.5 ml of deionized water; thoroughly mix 80 μl of G4CAsi-FDX1 solution with 80 μl of OD solution. After standing, allow OD to react with PAMAM G4 to undergo a Schiff base reaction to form an active oxygen responsive hydrogel.

[0088] Performance Testing

[0089] The in vitro intracellular delivery ability of PAMAMG4 (G4CAsi-FDX1) loaded with caffeic acid (CA) and copper death-related gene (si-FDX1) at different ratios and concentrations was tested. Primary neonatal rat cardiomyocytes were cultured in 24-well plates and G4CA:si-FDX1 were added respectively. FITC The ratios were 0.5:1, 1:1, 2:1, and 5:1, and the concentrations were 0 μg / ml, 1 μg / ml, 2 μg / ml, 4 μg / ml, 8 μg / ml, and 10 μg / ml of G4CAsi-FDX1 linked to fluorescein isothiocyanate (G4CAsi-FDX1 FITC ), incubated for 4 hours, and after the incubation, the cell nuclei were stained with DAPI, the stained cells were observed and images were collected using a fluorescence microscope, and the fluorescence intensity in the cells was quantitatively analyzed using a flow cytometer. The results are shown in Figure 2. Figure 6 As shown, uptake of G4CAsi-FDX1 FITC The cells showed obvious green fluorescence signals, indicating that G4CAsi-FDX1 FITC It has good intracellular delivery ability in vitro. When the mass ratio of G4CA:si-FDX1 was (2-4):1 and the concentration was 2-10 μg / mL, flow cytometry showed that 92% of cardiomyocytes took up FITC signals (MFI>50%), which was significantly higher than that of other ratio groups.

[0090] PAMAM G4 (G4CAsi-FDX1) loaded with caffeic acid (CA) and copper death-related gene (si-FDX1) can effectively scavenge free radicals and significantly reduce the level of oxidative stress in the area of ​​myocardial ischemia-reperfusion injury. The G4CAsi-FDX1 extract with a mass ratio of G4CA:si-FDX1 of 2:1 was prepared into sample solutions of 0μg / m, 0.1μg / m, 0.2μg / m, 0.5μg / m, 1μg / m, 1.5μg / m, 2μg / m, and 3μg / ml for testing. The sample solution / positive control, distilled water, and working solution were added to a 96-well plate, mixed thoroughly, and allowed to stand at room temperature in the dark for 6 minutes. The absorbance at 405nm was measured. ABTS free radical scavenging rate D% = [A blank - (A determination - A control)] ÷ A blank × 100%. As Figure 7 As shown, when 1 μg / ml of the complex was added, the ABTS clearance rate was about 60%. When the concentration continued to increase to 2 μg / ml, the clearance rate reached more than 80%, proving that the complex has potential application value in antioxidant properties.

[0091] The hydrogel obtained in this example (OD:G4CA:si-FDX1 mass ratio is 1:1) was observed by scanning electron microscopy. Figure 8 As shown ( Figure 8 a is the cross section, Figure 8 b is the longitudinal section), the hydrogel has good uniformity.

[0092] The hydrogel was placed in 0.1mM and 1mM H2O2 solutions to simulate physiological environment, and the release of caffeic acid (CA) in the hydrogel was detected at regular intervals. The results are as follows: Figure 9 As shown, the gel exhibits excellent responsiveness to reactive oxygen species. At a concentration of 0.1 mM, caffeic acid (CA) release is slow; at a concentration of 1 mM, the cumulative release of caffeic acid (CA) reaches 70-80% within 720 minutes. This demonstrates excellent reactive oxygen species-responsive drug release properties, enabling on-demand drug release at sites of myocardial ischemia-reperfusion injury (a high reactive oxygen species environment), enhancing therapeutic efficacy.

[0093] To detect the in vivo degradation of the reactive oxygen species-responsive hydrogel, it was mixed with indocyanine green (ICG) dye and injected into the myocardial injury site of mice under direct vision after thoracotomy. The results were detected using IVIS at different time points. Figure 10 As shown, 0.25-1.5 hours after injection, the total fluorescence flux decreased slowly and the hydrogel slowly degraded; 1.5-24 hours, the downward trend gradually became apparent; 24-96 hours, it continued to decline steadily; 96-192 hours, it dropped to a lower level, indicating that the hydrogel has the characteristics of responsive release under high reactive oxygen levels in pathological sites, and can achieve long-term and controllable release of drugs and genes.

[0094] Under the protection and delivery of hydrogels, copper-related death genes can efficiently enter myocardial cells, inhibit related cell death pathways (such as copper death), and save the myocardium. Figure 11 As shown in the figure, in the animal experiment, Sham was the sham operation group, IR+PBS was the control group 1, IR+OD@G4CA:si-NC was the control group 2, and IR+OD@G4CA:si-FDX1 was the experimental group. After myocardial ischemia-reperfusion injury surgery, the corresponding reagents were injected into the damaged myocardial area. By detecting the expression levels of related proteins in myocardial tissue, it was found that the expression of copper death-related proteins in the myocardial tissue of the experimental group mice was significantly downregulated compared with the control group. At the same time, cardiac function was effectively improved, as shown in Figure 2. Figure 12 As shown in the results, the ejection fraction increased by about 25% after 7 days of treatment, basically returning to normal levels. In addition, the shortening fraction, cardiac output and stroke volume also improved.

[0095] Comparative Example 1: No hydrophobic antioxidant drug added

[0096] Step 1: Prepare 4 mg / ml PAMAM G4 solution for later use.

[0097] Step 2: Preparation of dendrimers loaded with copper death-related genes, including the following steps:

[0098] 1) Dissolve 2 mg of si-FDX1 in 1 ml of DEPC water to prepare a 2 mg / ml solution.

[0099] 2) Add 1 ml of si-FDX1 solution to 1 ml of PAMAM G4 solution at room temperature and let it stand for several minutes to stably load si-FDX1.

[0100] Step 3: Same as step 3 in Example 1.

[0101] Step 4: preparing a drug-loaded reactive oxygen species-responsive hydrogel, comprising the following steps:

[0102] Weigh 2.5 mg of OD and dissolve it in 1 ml of deionized water; mix 2 ml of G4si-FDX1 solution with the OD solution and allow it to stand for a Schiff base reaction to form a hydrogel.

[0103] Performance Testing

[0104] The inhibitory ability of four groups of samples (HR, HR+G4siFDX1, HR+G4CA, HR+G4CAsiFDX1) on reactive oxygen species in myocardial ischemia-reperfusion (HR) model was tested by reactive oxygen species probe. Figure 13As shown, the absence of the CA component significantly reduced the nanomedicine's ability to inhibit reactive oxygen species compared to the group retaining the CA component. Specifically, measurements of the mean fluorescence intensity, cumulative density, and raw cumulative density of reactive oxygen species in the four groups revealed that the CA-deficient group had higher levels of these indicators than the group retaining CA. This result suggests that CA plays a crucial antioxidant role in this system, and that its absence significantly reduced the nanomedicine's ability to inhibit reactive oxygen species, thereby impacting the overall therapeutic efficacy.

[0105] Comparative Example 2: Death-related genes without copper addition

[0106] Step 1: Same as step 1 in Example 2

[0107] Step 2: Preparation of dendrimers loaded with non-functional genes, including the following steps:

[0108] 1) Prepare 4 mg / ml PAMAM G4 solution for later use;

[0109] 2) Dissolve 2 mg of si-NC in 1 ml of DEPC water to prepare a 2 mg / ml solution;

[0110] 3) Add 1 ml of si-NC solution to 1 ml of PAMAM G4 solution at room temperature and let it stand for several minutes.

[0111] Step 3: Same as step 3 in Example 1

[0112] Step 4: preparing a drug-loaded reactive oxygen species-responsive hydrogel, comprising the following steps:

[0113] 2.5 mg of OD was weighed and dissolved in 2.5 ml of deionized water; 80 μl of G4CAsi-NC solution was thoroughly mixed with 80 μl of OD solution. After standing, OD reacted with PAMAM G4 to form an active oxygen responsive hydrogel.

[0114] Performance Testing

[0115] Biocompatibility testing was performed on four groups: NC+G4CAsi-NC, NC+G4CAsi-NC, HR+G4CAsi-NC, and HR+G4CAsiFDX1. Primary neonatal rat cardiomyocytes were cultured in 96-well plates with or without HR treatment. G4CAsi-NC or G4CAsi-NC was added, and CCK8 assay was performed after incubation for 0, 12, 24, and 48 hours. Figure 14As shown in the figure, in the HR model, the cell viability of the G4CAsi-FDX1-treated group was significantly higher than that of the G4CAsi-NC-treated group. This result indicates that G4CAsi-FDX1 has a positive effect on cell growth in the HR model, while the empty vector G4CAsi-NC group did not show the same effect, indicating that the loading of si-FDX1 exerts an effective therapeutic effect.

[0116] Comparative Example 3: No oxidized polysaccharide added

[0117] Step 1: preparing dendrimers loaded with antioxidant drugs, similar to step 1 of Example 2;

[0118] Step 2: Prepare dendrimers loaded with antioxidant drugs and copper death-related genes, similar to step 2 of Example 2, to obtain G4CAsi-FDX1.

[0119] Performance Testing

[0120] Cy5-labeled si-FDX1 was used to inject OD@G4CAsi-FDX1 into the myocardium of mice with myocardial ischemia-reperfusion (IR) model. Cy5 or G4CAsi-FDX1 Cy5 The retention and distribution of drugs in the body were observed by IVIS technology. Figure 15 As shown, OD@G4CAsi-FDX1 Cy5 The drug of group A can be continuously released in the target organ and maintain a high concentration within 72 hours, while G4CAsi-FDX1 Cy5 The drugs in the hydrogel group were rapidly metabolized within 12 hours, indicating that hydrogel drugs can better exert long-term controlled release effects.

[0121] The mRNA levels of Fdx1, Dlat and Lias in the myocardial tissues of the five groups of mice (Sham, IR, IR+OD@G4CAsi-NC, IR+OD@G4CAsi-FDX1, IR+G4CAsi-FDX1) were detected by qRT-PCR analysis. Figure 16 As shown, the IR+G4CAsi-FDX1 group showed excessive inhibition of genes regulating enzymes related to the tricarboxylic acid cycle, which may lead to cellular metabolic disorders and pose potential risks. This suggests that the hydrogel may reduce the negative effects of si-FDX1 when overregulating metabolic pathways, thereby maintaining normal cellular metabolic activity.

Claims

1. A method for preparing an active oxygen responsive hydrogel for alleviating myocardial ischemia-reperfusion injury, characterized in that: The following steps are involved: Step 1, adding a hydrophobic antioxidant drug solution to an aqueous solution of a dendrimer macromolecule, and the reaction product is dialyzed, purified, and freeze-dried to obtain a complex A; Step 2: Mix the aqueous solution of complex A and the aqueous solution of copper death-related genes to form a complex B solution, and then add the aqueous solution of oxidized polysaccharide to react into a gel to obtain the active oxygen responsive hydrogel.

2. The method for preparing the reactive oxygen species responsive hydrogel for alleviating myocardial ischemia-reperfusion injury according to claim 1, wherein: The dendrimers include one or more of polyamidoamine, polylysine, polypropyleneimine, polyethyleneimine, and polyarylether; The hydrophobic antioxidant drug includes one or more of hydrophobic polyphenol compounds, hydrophobic quinone compounds, hydrophobic flavonoid compounds, hydrophobic statins, and hydrophobic carotenoids.

3. The method for preparing the reactive oxygen species responsive hydrogel for alleviating myocardial ischemia-reperfusion injury according to claim 1, characterized in that: The copper death-related genes include siRNA or shRNA of one or more of FDX1, LIAS, LIPT1, DLAT, PDHA1, and PDHB; The oxidized polysaccharide includes one or more of oxidized dextran, oxidized sodium alginate, oxidized glucomannan, oxidized bletilla striata polysaccharide, oxidized fucoidan, oxidized hyaluronic acid, and the like.

4. The method for preparing the reactive oxygen species responsive hydrogel for alleviating myocardial ischemia-reperfusion injury according to claim 1, wherein: The molecular weight of the dendrimer is 10,000 to 30,000.

5. The method for preparing the reactive oxygen species responsive hydrogel for alleviating myocardial ischemia-reperfusion injury according to claim 1, wherein: The mass ratio of the dendrimer to the hydrophobic antioxidant drug is 1:(0.5-2); The mass ratio of the dendrimer to the copper death-related gene is 1:(0.1-5); The mass ratio of the dendrimer to the oxidized polysaccharide is (0.1-10):

1.

6. The method for preparing the reactive oxygen species responsive hydrogel for alleviating myocardial ischemia-reperfusion injury according to claim 1, characterized in that: The mass ratio of the hydrophobic antioxidant drug to the copper death-related gene is (0.1-20):

1.

7. The method for preparing the reactive oxygen species responsive hydrogel for alleviating myocardial ischemia-reperfusion injury according to claim 1, characterized in that: The mass concentration of the oxidized polysaccharide aqueous solution is 2-10 wt %, the reaction temperature after adding the oxidized polysaccharide is 25-37° C., and the reaction time is 5 seconds to 10 minutes.

8. An active oxygen responsive hydrogel for alleviating myocardial ischemia-reperfusion injury obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the hydrogel according to claim 8 in preparing drugs or medical materials for alleviating myocardial ischemia-reperfusion injury.

10. Use of siRNA or shRNA targeting copper death-related genes in the preparation of drugs or medical materials for alleviating myocardial ischemia-reperfusion injury.

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