A polyphenol-coated Mn-Co3O4 metal polyphenol nanozyme, preparation method and application

Abstract

The invention discloses a polyphenol-coated Mn-Co3O4 metal polyphenol nanozyme, a preparation method and an application thereof, comprising the following steps: step 1: heating a Co(NO3)2·6H2O solution to a temperature T1, adding aqueous ammonia under vigorous stirring, and fully reacting; step 2: adding a Mn(NO3)2·6H2O solution to the solution obtained in step 1, and heating to a temperature T2; step 3: adding a plant polyphenol solution to the solution formed in step 2, and after sufficient reaction, separating to obtain the desired metal polyphenol nanozyme; the invention uses tannic acid as a shell to encapsulate MC particles to form a pomegranate-like structure; MC is formed by partially replacing Co3O4 with manganese atoms; since the introduction of manganese atoms greatly improves the activity of Co3O4, MC becomes a powerful ROS scavenger, which is used to reduce oxidative stress after myocardial infarction; the provided MCT hydrogel provides a potential treatment strategy for myocardial infarction, which can be extended to other diseases mediated by ROS, and also expands the application of biomimetic nanostructures in biomedicine.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a polyphenol-coated Mn-Co3O4 metal polyphenol nanozyme, a preparation method and an application thereof. Background Art

[0002] With the increasing global aging population, cardiovascular disease (CVD), a "fatal killer" threatening human health, has placed a tremendous burden on global health. Almost all cardiovascular diseases progress to heart failure in their late stages, with myocardial infarction (MI) caused by coronary artery disease, such as atherosclerosis, being particularly prevalent. The ischemic and hypoxic microenvironment in the early stages of MI triggers a shutdown of the mitochondrial electron respiratory chain, leading to proton accumulation. This impacts the synthesis of antioxidant molecules and oxidoreductases, further causing abnormal accumulation of reactive oxygen species (ROS) in mitochondria. This is the primary factor contributing to massive cardiomyocyte apoptosis and persistent myocardial fibrosis. Furthermore, high levels of ROS and massive apoptosis stimulate the polarization of immune-infiltrating macrophages toward the M1 phenotype, leading to the release of multiple chemokines and inflammatory factors, thereby inducing a negative immune cycle, ultimately causing a severe inflammatory response and exacerbating myocardial damage. Therefore, alleviating the ischemic and hypoxic environment, protecting myocardial tissue from ROS damage, and inhibiting the inflammatory response are promising approaches for the treatment of MI.

[0003] Oxidative stress is a characteristic of the pathological conditions of myocardial infarction, which has prompted the study of ROS scavengers as potential remedies. These scavengers are mainly divided into two categories: antioxidant molecules (such as resveratrol, vitamins) and natural antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). Despite the impressive ROS scavenging ability of natural enzymes, their wider application still faces major challenges. Extraction costs, inactivity and limited cell permeability hinder their practical application. The emergence of metallo-nanozymes provides an attractive avenue for innovative ROS scavenging solutions. These artificial enzymes have significant advantages such as low cost, high production efficiency and long stability. However, such nanozymes have potential toxic side effects and cellular endocytosis barriers. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides a polyphenol-coated Mn-Co3O4 metal polyphenol nanozyme, a preparation method and an application thereof.

[0005] The technical solution adopted in the present invention is:

[0006] A method for preparing polyphenol-coated Mn-Co3O4 metallopolyphenol nanozyme comprises the following steps:

[0007] Step 1: Heat the Co(NO3)2·6H2O solution to T1 temperature, add ammonia water under vigorous stirring conditions, and fully react; wherein the molar ratio of Co(NO3)2·6H2O to ammonia water is 1:10-65;

[0008] Step 2: Add Mn(NO3)2·6H2O solution to the solution obtained in step 1 and heat to temperature T2; the molar ratio of Mn(NO3)2·6H2O to Co(NO3)2·6H2O is 1:3-6;

[0009] Step 3: Add the plant polyphenol solution to the solution formed in step 2, and after sufficient reaction, separate to obtain the desired metal polyphenol nanozyme; the molar ratio of the plant polyphenol to Co(NO3)2·6H2O is 1:2-6.

[0010] Furthermore, the plant polyphenol is tannic acid.

[0011] Furthermore, T1 in step 1 is 80°C, and T2 in step 2 is 100°C.

[0012] Furthermore, the reaction time in step 1 is 15 min, and the reaction time in step 3 is 20 h.

[0013] A polyphenol-coated Mn-Co3O4 metal polyphenol nanozyme, wherein the metal polyphenol nanozyme uses plant polyphenol as a shell and encapsulates Mn-Co3O4 particles.

[0014] A method for preparing a metal polyphenol nanozyme hydrogel comprises the following steps:

[0015] dissolving collagen in an acetic acid buffer solution to obtain a collagen solution;

[0016] Adding chondroitin sulfate and metal polyphenol nanozyme solution to the collagen solution; mixing thoroughly to obtain the desired hydrogel;

[0017] The mass ratio of collagen, chondroitin sulfate and metal polyphenol nanozymes is 2:2:0.1.

[0018] Furthermore, the mass concentration of the collagen solution is 1.5 wt.%, and the solution is mixed by oscillation.

[0019] The invention discloses an application of a polyphenol-coated Mn-Co3O4 metal polyphenol nanozyme, and an application of the metal polyphenol nanozyme in the preparation of a drug for treating myocardial infarction.

[0020] Furthermore, the metal polyphenol nanozyme hydrogel is used in the preparation of drugs for treating myocardial infarction.

[0021] The beneficial effects of the present invention are:

[0022] (1) The present invention uses tannic acid as a shell to encapsulate MC particles to form a pomegranate-like structure; MC is formed by partial substitution of Co3O4 by manganese atoms; the introduction of manganese atoms greatly increases the activity of Co3O4, making MC a powerful ROS scavenger for alleviating oxidative stress after myocardial infarction;

[0023] (2) The present invention uses collagen hydrogel as a carrier to achieve in situ delivery in the infarct area, which can solve the problem of heart failure caused by excessive ROS-mediated acute inflammatory response and myocardial cell apoptosis after myocardial infarction;

[0024] (3) The MCT hydrogel provided by the present invention provides a potential therapeutic strategy for myocardial infarction, which can be extended to other diseases mediated by ROS, and also expands the application of biomimetic nanostructures in biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 These are images of the metal polyphenol nanozyme MCT obtained in Example 1, a and b are TEM images, c is a combined imaging image of EDS and HAADF, d is a HAADF image, and e, f and g are EDS images.

[0026] Figure 2 This is a schematic diagram of the dynamic light scattering measurement results of the metal polyphenol nanozyme MCT obtained in Example 1.

[0027] Figure 3 Schematic diagram of the Zeta potential of the metal polyphenol nanozyme obtained in Example 1.

[0028] Figure 4 Schematic diagram of XPS of the metal polyphenol nanozyme obtained in Example 1, a is the XPS graph, b is the C1s peak, c is the Mn2p orbital fitting diagram, d is the Co2p orbital fitting diagram, and e is the O1s orbital fitting peak.

[0029] Figure 5 The antioxidant activity of the metal polyphenol nanozymes obtained in Example 1 under different concentration conditions, a is the ABTS+ test result, and b is the DPPH test result.

[0030] Figure 6 This is the absorption spectrum of the metal polyphenol nanozyme obtained in Example 1 at a wavelength of 200 to 800 nm.

[0031] Figure 7 The SOD activity of the metal polyphenol nanozyme obtained in Example 1 under different concentration conditions.

[0032] Figure 8 is the dissolved oxygen concentration in the H2O2 solution of the metal polyphenol nanozyme and other materials obtained in Example 1.

[0033] Figure 9 These are the mechanical property test results of the hydrogel prepared by the metal polyphenol nanozyme obtained in Example 1, a is the storage modulus and loss modulus test results, b is the frequency sweep test result, c is the strain sweep curve, and d is the self-healing ability of the gel under 100% strain.

[0034] Figure 10 These are the toxicity test results of the hydrogel prepared by the metal polyphenol nanozymes obtained in Example 1. A is the activity of the cells after co-culture with H9C2 cells at different concentrations of MC and MCT for 24 hours, B is the activity of the cells under conditions of different concentrations of H2O2, and C is the activity of the cells under conditions of different concentrations of MCT when the H2O2 concentration is 200 μM.

[0035] Figure 11 CLSM images of the metal polyphenol nanozyme hydrogel obtained in Example 1, A is a graph for evaluating mitochondrial targeting ability, B is a graph for evaluating the Pearson coefficient, C is a graph for evaluating the reactive oxygen species scavenging ability, and D is a quantitative statistical data of graph C.

[0036] Figure 12 This is the test result of the cell apoptosis rate of the metal polyphenol nanozyme hydrogel obtained in Example 1.

[0037] Figure 13 The hemolysis rate of the metal polyphenol nanozyme formed hydrogel under different concentration conditions obtained in Example 1.

[0038] Figure 14 These are the test results of the metal polyphenol nanozyme hydrogel obtained in Example 1 on the pathological changes of rat organs.

[0039] Figure 15 Schematic diagram of the therapeutic effect of the metal polyphenol nanozyme forming hydrogel obtained in Example 1, A is a flow chart, B is pictures at different times after surgery, C is an echocardiogram, D is the left ventricular diastolic ejection fraction EF result, E is the shortening fraction FS result, F is the left ventricular terminal volume EDV result, and G is the left ventricular end-systolic volume ESV result. DETAILED DESCRIPTION

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0041] A method for preparing polyphenol-coated Mn-Co3O4 metallopolyphenol nanozyme comprises the following steps:

[0042] Step 1: Heat the Co(NO3)2·6H2O solution to 80°C, add aqueous ammonia under vigorous stirring, and react for 15 minutes; wherein the molar ratio of Co(NO3)2·6H2O to aqueous ammonia is 1:10-65;

[0043] Step 2: Add Mn(NO3)2·6H2O solution to the solution obtained in step 1 and heat to 100°C; wherein the molar ratio of Mn(NO3)2·6H2O to Co(NO3)2·6H2O is 1:3-6;

[0044] Step 3: Add plant polyphenol (tannic acid) solution to the solution formed in step 2. After 20 hours, separate and obtain the desired metal polyphenol nanozyme; the molar ratio of the plant polyphenol to Co(NO3)2·6H2O is 1:2-6.

[0045] Metal polyphenol nanozymes use plant polyphenols as the shell and encapsulate Mn-Co3O4 particles to form a pomegranate-like structure, with plant polyphenols as the pomegranate shell and Mn-Co3O4 particles as pomegranate seeds.

[0046] A method for preparing a metal polyphenol nanozyme hydrogel comprises the following steps:

[0047] Collagen was dissolved in an acetic acid buffer solution to obtain a collagen solution; the mass concentration of the collagen solution was 1.5 wt.%, and the solution was mixed by oscillation.

[0048] Chondroitin sulfate and metal polyphenol nanozyme solution were added to the collagen solution; the desired hydrogel was obtained by thorough mixing; the mass ratio of collagen, chondroitin sulfate and metal polyphenol nanozyme was 2:2:0.1.

[0049] Application of polyphenol-coated Mn-Co3O4 metallopolyphenol nanozyme, application of metallopolyphenol nanozyme in the preparation of drugs for treating myocardial infarction, and application of metallopolyphenol nanozyme hydrogel in the preparation of drugs for treating myocardial infarction.

[0050] Example 1

[0051] A method for preparing polyphenol-coated Mn-Co3O4 metallopolyphenol nanozyme comprises the following steps:

[0052] Step 1: Dissolve 0.375 mmol of Co(NO₃)₂·6H₂O in 15 mL of double-deionized (ddH₂O) / deuterium-depleted (DDW) water. Heat to 80°C, add 1 mL of aqueous ammonia, and react for 15 minutes while stirring vigorously.

[0053] Step 2: Add 10 mL of 12.5 μmol / mL Mn(NO3)2·6H2O solution to the solution obtained in step 1, and then heat to 100°C.

[0054] Step 3: Add 5 mL of a mixed solution containing 0.1 mmol of tannic acid (TA) to the solution in Step 2, wherein the solvent is a mixture of acetonitrile and water in a volume ratio of 3:2. Continue the reaction for 20 hours. After the reaction is completed, the reaction solution is cooled to room temperature, filtered through a 1 μm filter, centrifuged at 10,000 rpm for 15 minutes, and washed three times with ultrapure water to obtain the metallopolyphenol nanozyme MCT.

[0055] Example 2

[0056] A method for preparing polyphenol-coated Mn-Co3O4 metallopolyphenol nanozyme comprises the following steps:

[0057] Step 1: Dissolve 0.75 mmol of Co(NO₃)₂·6H₂O in 15 mL of double-deionized (ddH₂O) / deuterium-depleted (DDW) water. Heat to 80°C, add 1 mL of aqueous ammonia, and react for 15 minutes while stirring vigorously.

[0058] Step 2: Add 10 mL of 12.5 μmol / mL Mn(NO3)2·6H2O solution to the solution obtained in step 1, and then heat to 100°C.

[0059] Step 3: Add 5 mL of a mixed solution containing 0.125 mmol of tannic acid (TA) to the solution in Step 2, wherein the solvent is a mixture of acetonitrile and water in a volume ratio of 3:2. Continue the reaction for 20 hours. After the reaction is completed, the reaction solution is cooled to room temperature, filtered through a 1 μm filter, centrifuged at 10,000 rpm for 15 minutes, and washed three times with ultrapure water to obtain the metallopolyphenol nanozyme MCT.

[0060] Example 3

[0061] A method for preparing polyphenol-coated Mn-Co3O4 metallopolyphenol nanozyme comprises the following steps:

[0062] Step 1: Dissolve 0.625 mmol of Co(NO₃)₂·6H₂O in 15 mL of double-deionized (ddH₂O) / deuterium-depleted (DDW) water. Heat to 80°C, add 1 mL of aqueous ammonia, and react for 15 minutes while stirring vigorously.

[0063] Step 2: Add 10 mL of 12.5 μmol / mL Mn(NO3)2·6H2O solution to the solution obtained in step 1, and then heat to 100°C.

[0064] Step 3: Add 5 mL of a mixed solution containing 0.3125 mmol of tannic acid (TA) to the solution in step 2, wherein the solvent is a mixture of acetonitrile and water in a volume ratio of 3:2. Continue the reaction for 20 hours. After the reaction is completed, the reaction solution is cooled to room temperature, filtered through a 1 μm filter, centrifuged at 10,000 rpm for 15 minutes, and washed three times with ultrapure water to obtain the metallopolyphenol nanozyme MCT.

[0065] The same method as in Example 1 was used, except that polyethyleneimine was replaced with tannic acid, to obtain non-targeted nanoparticles MC.

[0066] For subsequent studies, MC and MCT were reacted with Cy5.5NHS for 8 hours to obtain Cy5.5-labeled MC and MCT.

[0067] X-ray photoelectron spectroscopy (XPS) spectra were obtained on a KAlpha+X-ray photoelectron spectrometer system. Figure 1 Dynamic light scattering (DLS) was performed using a Malvern Zetasizer Nano ZS to record the particle size and zeta potential of the nanoparticles. The morphology of the nanoparticles was observed using a Hitachi H-600 transmission electron microscope (TEM) at an accelerating voltage of 100K.

[0068] from Figure 1 In images a and b, it can be seen that MC is a regular sphere with uniform size. By improving the image resolution, it can be observed that MC has clear lattice fringes with a lattice spacing of 0.245-0.255nm, indicating that the lattice plane is A(103).

[0069] The uniform distribution of manganese atoms in MC was observed using a combined EDS and HAADF imaging method. Figure 1 As can be seen in c and d, the manganese atoms in the partially substituted Co3O4 nanozyme were successfully synthesized.

[0070] Figure 2 This is a schematic diagram of the dynamic light scattering measurement results of the metal polyphenol nanozyme MCT obtained in Example 1. It can be seen from the figure that the particle size of MCT in the aqueous solution is 239.3nm, PDI=0.243,

[0071] Figure 3 Schematic diagram of the Zeta potential of the metal polyphenol nanozyme obtained in Example 1. The obtained metal polyphenol nanozyme was dissolved in RO, PBS and DMEM, respectively, and the Zeta potentials were measured to be -25.3 mV, -20.4 mV and -9.59 mV, respectively.

[0072] The morphology and elemental distribution of MCT were observed using a combined TEM and EDS method, revealing a distinct core-shell structure. The carbon element is primarily concentrated in the shell region, indicating a "pomegranate peel" formed by TA. The MC nanozyme exhibits a distinct Co signal in the inner layer, with a certain amount of Mn evenly scattered.

[0073] XPS was used to characterize the elemental composition and valence distribution of MCT. Figure 4 As shown in the figure, the C1s peak can be fitted to three characteristic peaks corresponding to CC, CO, and C=O, further confirming the presence of a TA coating in the MCT. Peak fitting of the Mn2p and Co2p orbitals also revealed that they exist in multivalent forms, capable of exerting a variety of enzyme-like activities. Calculation of the elemental ratios revealed a manganese to cobalt ratio of 12:5, with a trivalent to divalent ratio of the primary cobalt element of 6:5.

[0074] The total antioxidant capacity of MCT nanozymes was verified by using DPPH and ABTS free radical scavenging ability test kits. The ability of MCT to scavenge DPPH and ABTS+ at different concentrations was calculated. Figure 5 As shown. The SOD-like activity of MCT was determined using a superoxide dismutase (SOD) activity detection kit. The SOD-like activity of MCT was expressed by the inhibition rate of different concentrations of nanozymes to eliminate superoxide on the water-soluble tetrazolium reaction (WST). The results are shown in Figure 7 As shown. The CAT-like activity of MCT was identified by measuring its catalytic oxygen production activity. At room temperature, 1M H2O2 was mixed with 200μL of MC, MCT, MT (the preparation process was the same as in Example 1, except that Co(NO3)2·6H2O was not added in step 1, and tannic acid was not added in step 3, as a control group), CT (the preparation process was the same as in Example 1, except that Mn(NO3)2·6H2O was not included in step 2, and tannic acid was not added in step 3, as a control group) and TA solution in 20mL PBS. The oxygen production activity of different materials was then measured using a dissolved oxygen meter (INESA, JPSJ-605F) (measured every 10 seconds until 600 seconds). Finally, TMB solution was used as a substrate to detect the POD-like activity of MCT. Specifically, different amounts of TMB were dissolved in 0.1M acetic acid / sodium acetate buffer (pH=4.5), and then 50μg of MCT solution and 100μL of 30% H2O2 were added. The results are shown in Figure 2. Figure 8 The absorption spectra of MCT at different concentrations at wavelengths of 200-800 nm were measured using UV-visible spectroscopy. The results are shown in Figure 2. Figure 6 shown.

[0075] from Figure 5It can be seen that when the solution contains 1 mM DPPH· and ABTS·+, MCT can achieve free radical scavenging rates of 95.01% and 77.66% respectively at a concentration of 0.25 mg / mL, showing excellent antioxidant capacity.

[0076] The process of ROS clearance in vivo is primarily achieved through a cascade of enzymatic reactions involving superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). The SOD enzymatic reaction initiates the ROS clearance process, converting superoxide anions (O·-) into O2 and hydrogen peroxide (H2O2). CAT then catalyzes the production of O2 from H2O2. Using the WST-8 assay, MCTs have been shown to exhibit concentration-dependent SOD-like activity, inhibiting the formation of thyroxine dye by scavenging O·-.

[0077] The CAT-like activity was evaluated by measuring the dissolved oxygen concentration in H2O2 solutions containing different materials. Figure 8 As shown in the figure, at the same metal concentration, MC and MCT exhibited greater increases in CAT activity than CT and MT. Compared to the rapid initial oxygen production efficiency of MC, the oxygen production rate of MCT was more stable. This is due to the tannic acid shell of MCT's pomegranate-like structure initially performing a certain H2O2 scavenging function, slowing the direct reaction between H2O2 and the MC core. Due to the limited antioxidant capacity of TA, pomegranate seeds (MC) remain the primary scavenger of hydrogen peroxide. Therefore, after 5 minutes of reaction, the oxygen production of MCT remained close to that of the MC group.

[0078] In the process of scavenging ROS, hydroxyl radicals (OH - ) is inevitable, which will aggravate the oxidative stress in cells. Similarly, metal ions (such as Mn 2+ and CO 3+ ) will also trigger a Fenton-like reaction, thus becoming OH - The additional source of OH after MC and MCT reacted in H2O2 solution was determined by TMB method. - Content, to characterize the effect of MCT on OH - The inhibitory effect of Figure 7 As shown in the figure, it can be seen that as the MCT concentration increases, its catalytic activity also increases.

[0079] The MCT prepared in Example 1 was used to prepare MCT hydrogel.

[0080] Dissolve 15 mg of collagen in 1 mL of PBS containing 0.5 M acetic acid to obtain a 1.5% collagen solution, which was then stored at 4°C. Subsequently, add 0.5 mL of PBS containing 3% chondroitin sulfate and 0.15% MCT to the collagen solution and immediately shake vigorously to mix. The final ratio of the different components in the composite hydrogel was 2:2:0.1 for collagen:chondroitin sulfate.

[0081] The same method was used to replace MCT to obtain MC hydrogel and TA hydrogel.

[0082] The rheological properties of the hydrogels, including the elastic modulus (G'), loss modulus (G") and self-healing properties, were determined using a rheofluid spectrometer (MCR302). First, a frequency sweep was performed on each set of hydrogel samples, using a constant strain value of 1%. The angular frequency was increased logarithmically from 0.1 to 100 rad / s. Subsequently, a dynamic strain sweep test was performed with a fixed frequency of 1 Hz and a strain range of 0.01% to 1000%. In a subsequent step, a self-healing examination was performed. For this purpose, alternating strain cycles from 1% to 100% were applied. The tests were performed at a consistent frequency of 1 Hz. All evaluations were performed at a temperature of 37°C, and the results are shown in Figure 2. Figure 9 shown.

[0083] The storage modulus (G') of the hydrogel measured at 37 °C, 1% shear strain and 1 Hz frequency was between 230 and 460 Pa, which was much larger than the loss modulus (G"), indicating that it was in a gel state. Figure 9 As shown in Figure a. TA and MCT have higher mechanical strength than MC, which is due to the stronger hydrogen bonding between the polyphenolic structure of tannic acid and the hydrogel matrix. Frequency sweep tests show that the G' and G" of the three hydrogels increase slightly with increasing angular frequency, indicating that the crosslinking within the gel is mainly achieved through physical bonds (ionic bonds and hydrogen bonds), as shown in Figure 3. Figure 9 As shown in b.

[0084] It can be seen from the strain scanning curve that under 100% strain conditions, the structures of TA, MC and MCT hydrogels are destroyed, which is manifested by the decrease of G' and G", and G' is smaller than G". Figure 9 The self-healing ability of the hydrogel at 100% strain was measured. The results showed that although the structure of the MCT hydrogel was destroyed under high strain, the initial modulus was immediately restored when the strain was reduced. This shows that the hydrogel has good self-healing ability and can immediately return to the gel state after injection needle shearing to exert subsequent therapeutic functions.

[0085] In the following tests, the cells used were mouse mononuclear macrophages (RAW264.7) and rat cardiac myoblasts (H9C2 cells), cultured at 37°C in a 5% CO2 environment, and the DMEM culture medium used was supplemented with 10% (volume fraction) fetal bovine serum (FBS) and 1% (volume fraction) penicillin-streptomycin.

[0086] The biocompatibility of the hydrogel was evaluated using the CCK-8 assay to evaluate the potential cytotoxic effects of the hydrogel extract on H9C2 cells and RAW 264.7 cells. 1.5 mL of the hydrogel sample was immersed in 15 mL of DMEM medium; the medium was supplemented with 10% (volume fraction) fetal bovine serum (FBS) and 1% (volume fraction) penicillin-streptomycin. After culturing at 37°C for 48 hours, H9C2 and RAW 264.7 cells were evenly seeded into 96-well plates and co-cultured for 24 hours. After co-culture, cell viability was evaluated by CCK-8 assay. The cytotoxicity of different concentrations of MC and MCT on H9C2 cells was also determined using the CCK-8 assay; the results are shown in Figure 3. Figure 10 shown.

[0087] like Figure 10 As shown in Figure A, high concentrations of MC (0.2 mg / ml) have significant cytotoxicity. After 24 hours of co-culture with H9C2 cells, the cell survival rate is only 60.02%. In contrast, the cytotoxicity of MCT is almost negligible, which also verifies that the introduction of TA reduces the original toxic side effects of MC.

[0088] The degree of damage to cells caused by different concentrations of H2O2, such as Figure 10 As shown in B. 200 μM was selected as the fixed H2O2 concentration for subsequent experiments. Figure 10 Middle C shows the addition of different concentrations of MCT in a 200 μM H2O2 environment. It was observed that 100 μg / mL MCT could effectively protect cells from oxidative stress damage.

[0089] Figure 11The confocal laser scanning microscopy (CLSM) images of the metallopolyphenol nanozyme hydrogels obtained in Example 1 are shown. A is a graph showing the evaluation of mitochondrial targeting ability, B is a graph showing the Pearson coefficient, C is a graph showing the evaluation of the reactive oxygen species scavenging ability, and D is a quantitative statistical data for graph C. The upper left corner of A shows Cy5.5-labeled nanoparticles (MC, MCT), the upper right corner shows mitochondrial staining (mitotracker green), and the bottom is a superposition of the two graphs above. B is a graph showing the Pearson coefficients of cy5.5 and mitotracker green. The higher the Pearson coefficient, the better the mitochondrial targeting. C is a graph showing the evaluation of the reactive oxygen species scavenging ability. The stronger the fluorescence signal in the large graph below, the higher the reactive oxygen species content, and relatively speaking, the weaker the reactive oxygen species scavenging ability. D is a quantitative statistical data for graph C.

[0090] After adding TA to the nanozyme, the mitochondrial targeting ability of MCT nanopomegranate was enhanced. In a typical procedure, H9C2 cells were exposed to a solution containing Cy5.5-labeled MC and MC (1 mg / ml). Subsequently, after 4 hours of incubation, the cell mitochondria were stained using Mito-Tracker Green. The samples were observed using CLSM (Nikon Air MP+) and the fluorescence signals of Cy5.5 and Mito-Tracker Green were recorded; as shown in Figure 2. Figure 11 As shown in A. In addition, the mean fluorescence intensity (MFI) and Pearson correlation coefficient of the confocal images were quantified. To verify that the targeting efficacy of MCT is independent of the mitochondrial membrane potential theory, we used H9C2 cells to injure them with H2O2 for 12 hours; the results are shown in Figure 11 D and B in the middle.

[0091] RAW264.7 and H9C2 cells were cultured at 1 × 10 4 The cells were seeded in glass dishes at a density of 100 μg / mL for 24 hours. After incubation with physiological saline and hydrogel extracts of TA, MC, and MCT for 3 hours, LPS (500 ng mL-1) was added for activation for 3 hours. No LPS was added to the control group. Afterwards, the cells were stained with MitoSOX Red and DCFH-DA for 30 minutes, respectively, and then observed under CLSM. The MFI of the confocal images was quantified by Image-Pro Plus 6.0 software; as shown in Figure 5. Figure 11 As shown in C.

[0092] from Figure 11As can be seen in Figure A, cell damage caused by oxidative stress is often accompanied by a loss of mitochondrial membrane potential, which weakens the targeting ability of traditional mitochondrial targeting molecules (such as TPP). It is worth noting that the TA coating of MCT has a targeting mechanism independent of the traditional mitochondrial membrane potential theory and can achieve targeting function through high affinity with mitochondrial outer membrane specific proteins (such as voltage-dependent anion selective channel (VDAC) and outer membrane translocase (TOM).

[0093] from Figure 11 As can be seen in Figure B, the Pearson coefficient increased from 0.81 to 0.90 (p = 0.0094) and was not affected by mitochondrial damage mediated by oxidative stress. The outstanding mitochondrial targeting ability of MCT paves the way for its multiple therapeutic functions.

[0094] DCFH-DA and Mito-SOX were used to stain the cytoplasmic broad spectrum ROS (cROS) and mitochondrial ROS (mROS), respectively. Figure 11 As shown in Figure C, significant cROS and mROS fluorescence signals were detected in macrophages (RAW264.7) after LPS injury and cardiomyocytes (H9C2) after high-level H2O2 injury. After co-culture with hydrogel extracts, the ROS levels in RAW264.7 and H9C2 were reduced to varying degrees. Although MC has an excellent ability to scavenge ROS, its targeted scavenging ability for superoxide in mitochondria is poor. In contrast, relying on the efficient mitochondrial targeting function of TA, MCT exhibits unparalleled ability in scavenging cROS and mROS. The introduction of TA shell can effectively enhance the mitochondrial targeting ability. During the recovery process after myocardial infarction, an inflammatory period will be experienced, the main feature of which is oxidative stress caused by the overexpression of reactive oxygen species in mitochondria. MCT has good mitochondrial targeting function.

[0095] While paying attention to local inflammation, cell apoptosis is also an important indicator of myocardial remodeling after infarction. The apoptosis cycle was deeply tested using cell flow cytometry. Figure 12 The test method is as follows: H9C2 cells are plated at 5×10 5 The cells were seeded at a density of 100 μg / ml into 6-well plates. After 12 hours of culture, the cells were exposed to different hydrogel extracts and H2O2 (1 mM) for 12 hours. After treatment, the cells were harvested and stained with the Annexin V-FITC / PI cell apoptosis detection kit. Finally, the proportion of cell apoptosis was assessed using flow cytometry (Attune, ThermoFisher). The above steps were repeated, and the protein expression of mitochondrial-related apoptosis pathways (Bax, Bcl-2, and cleaved caspase-3) in the cells was detected by Western Blot.

[0096] As can be seen from the figure, compared with cardiomyocytes treated with H2O2 alone, the late apoptosis rate in the TA, MC, and MCT groups was significantly reduced. Although the early apoptosis rate in the MCT group was slightly higher than that in the MC and TA groups, the overall apoptosis level was lower, indicating that MCT can delay the apoptosis process and reduce the apoptosis rate of cells under oxidative stress.

[0097] After centrifugation of rat whole blood, red blood cells were collected to test the hemolysis rate of the hydrogel. The in vivo toxicity and degradation characteristics of the MCT hydrogel were evaluated. The test methods are as follows:

[0098] The hemolysis rate of MCT hydrogel was evaluated using rat red blood cells. Typically, fresh mouse blood was obtained from SD rats. The blood was centrifuged at 1500 rpm for 10 minutes to obtain RBCs. Then, the RBCs were diluted with PBS to a 5% RBC solution. The diluted RBCs (500 μL) were mixed with 100 μL of MCT hydrogels of different concentrations for 2 hours. All samples were centrifuged at 1200 rpm for 6 minutes, and the absorbance of the supernatant was measured at a wavelength of 541 nm to calculate the hemolysis rate; the results are shown in Figure 2. Figure 13 shown.

[0099] from Figure 13 It can be seen that the hemolysis rate of hydrogels containing different concentrations of MCT is less than 2%, showing good blood compatibility.

[0100] Subsequently, a rat myocardial infarction model was established by ligating the left anterior descending artery (LAD), and the hydrogel was injected 30 minutes after ligation to treat myocardial infarction. Ultrasound diagnosis and multiple immunohistological analyses were performed on the rats after a specific time to verify the efficacy of the hydrogel on the infarcted myocardium. MCT was fluorescently labeled with Cy 5.5 to observe the retention of MCT in the rat heart after delivery through the hydrogel; the results are shown in Figure 2. Figure 14 Seven days after treatment, H&E staining was used to observe the pathological changes in the rats' major organs (heart, liver, spleen, lungs, kidneys, and brain). No significant differences in H&E staining were found between the hydrogel-injected group and the disease model group, indicating that organ damage caused by metabolic toxicity was not significant. MCT's excellent biosafety and in vivo retention ensure its effectiveness in subsequent therapeutic actions.

[0101] After myocardial infarction occurs, the myocardium will first enter the inflammatory phase. The main pathological characteristics of the inflammatory phase are massive necrosis of myocardial cells and infiltration of immune cells. The management of apoptosis and macrophage polarization mediated by sudden ROS generation is the key to controlling inflammation. Echocardiography is one of the most commonly used diagnostic tools for heart failure in clinical practice. Diagnostic ultrasound imaging technology was first used to directly observe myocardial infarction in rats after treatment with different hydrogel groups. We collected echocardiograms of rats in different treatment groups on days 7, 14, and 28 after surgery, and analyzed the cardiac pumping function in detail by measuring the left ventricular diastolic ejection fraction (EF) and shortening fraction (FS), left ventricular terminal volume (EDV), and left ventricular end-systolic volume (ESV); the results are as follows: Figure 15 shown.

[0102] The specific method for constructing the myocardial ischemia animal model is as follows:

[0103] All animal experiments were approved by the Medical Ethics Committee of Sichuan University. Sprague-Dawley (SD) rats (male) were purchased from Beijing Huolihe Experimental Animal Technology Co., Ltd. (China). (SD rats (male) were purchased from Beijing Huolihe Experimental Animal Technology Co., Ltd. (China) and housed in an SPF-grade animal facility with a temperature controlled at 20–22°C, a relative humidity of 50–60%, and a light–dark cycle of 12 h. The in vivo animal model was established according to previously reported methods. Briefly, 150 ± 20 g male SD rats were anesthetized with 3% isoflurane-oxygen mixed gas through a ventilator (Nanjing Kevin Biotechnology Co., Ltd.), and an acute myocardial infarction model was established by left thoracotomy and LAD ligation at the fourth intercostal space. All surgical operations were performed under sterile conditions. Rat ECG The ST-T segment elevation in the figure demonstrates successful establishment of the acute myocardial infarction model. Thirty minutes later, 50 μL of hydrogel or PBS was injected twice around the acute myocardial infarction site. All rats were randomly divided into the following five treatment groups (n = 8 per group): 1) Sham: chest closure alone without ligation or injection of any medication; 2) MI (control group, in which rats underwent LAD ligation without any other treatment): PBS injection after MI; 3) TA: TA hydrogel injection after MI; 4) MC: MC hydrogel injection after MI; and 5) MCT: MCT hydrogel injection after MI. All rats were euthanized on the 7th or 28th day after model establishment.

[0104] from Figure 15 As can be seen in the results, compared with the MI group, the EF and FS of the hydrogel-treated rats increased to varying degrees over time, while the EDV and ESV approached those of the sham group. The MCT group was significantly superior to the other treatment groups at all three time points, suggesting that targeted clearance of highly expressed ROS in mitochondria can optimize cardiac function recovery and improve long-term prognosis in rats with myocardial infarction.

[0105] The present invention provides a new type of metal polyphenol nanozyme with mitochondrial targeting function, and uses collagen hydrogel as a carrier to achieve in situ delivery in the infarct area, thereby solving the problem of heart failure caused by excessive ROS-mediated acute inflammatory response and myocardial cell apoptosis after myocardial infarction. Inspired by the core-shell structure of pomegranate, a nanozyme (MC) in which manganese atoms partially replaced Co3O4 was synthesized and used as pomegranate seeds. Subsequently, a large number of MC small particles were encapsulated into metal polyphenol nanopomegranate (MCT) using TA as pomegranate peel. The introduction of Mn atoms greatly improves the enzyme-like activity of Co3O4, making MC a powerful ROS scavenger for alleviating oxidative stress after myocardial infarction. Utilizing TA's specific affinity for TOM / VDAC and inherent ROS scavenging ability, MCT has a mitochondrial targeting function independent of membrane potential theory, and by chelating Mn 2+ / Co 3+ Inhibit the occurrence of Fenton-type reactions and inhibit POD-type activity. In addition, type III collagen and chondroitin sulfate are used as hydrogel carriers to deliver MCT to the myocardial infarction area, thereby improving the infarct microenvironment and alleviating the adverse effects of ventricular remodeling.

[0106] In vitro experiments have confirmed that MCT hydrogel can effectively eliminate mitochondrial reactive oxygen species (mROS) in damaged cells by inhibiting the Caspase apoptosis pathway, thereby protecting cardiomyocytes from ROS invasion. Studies have also found that MCT hydrogel can convert ROS in cells into O2 through enzyme-like activity, upregulate the expression of HIF-1α in LPS-induced macrophages, stimulate macrophage polarization to the M2 phenotype, and thus alleviate post-infarction inflammation. In a rat model of myocardial infarction, injection of MCT hydrogel into the infarct site can reduce collagen deposition, restore mitochondrial function, effectively reduce the degree of cell apoptosis and inflammatory response, and ultimately improve cardiac function. In summary, MCT hydrogel provides a potential therapeutic strategy for myocardial infarction and can be extended to other diseases mediated by ROS. It also expands the application of biomimetic nanostructures in biomedicine.

Claims

1. A method for preparing a metal polyphenol nanozyme hydrogel, characterized in that: The following steps are included: dissolving collagen in an acetic acid buffer solution to obtain a collagen solution; Adding chondroitin sulfate and metal polyphenol nanozyme solution to the collagen solution; mixing thoroughly to obtain the desired hydrogel; The mass ratio of collagen, chondroitin sulfate, and metal polyphenol nanozymes is 2:2:0.

1. The metal polyphenol nanozymes are composed of plant polyphenols as shells, encapsulating Mn-Co3O4 particles; the plant polyphenols are tannic acid. The preparation method of metal polyphenol nanozymes comprises the following steps: Step 1: Heat the Co(NO3)2·6H2O solution to T1, add aqueous ammonia under vigorous stirring, and allow to react fully; wherein the molar ratio of Co(NO3)2·6H2O to aqueous ammonia is 1:10-65; T1 is 80°C; Step 2: Add Mn(NO3)2·6H2O solution to the solution obtained in step 1 and heat to temperature T2; wherein the molar ratio of Mn(NO3)2·6H2O to Co(NO3)2·6H2O is 1:3-6; T2 is 100°C; Step 3: Add the plant polyphenol solution to the solution formed in step 2, and after sufficient reaction, separate to obtain the desired metal polyphenol nanozyme; the molar ratio of the plant polyphenol to Co(NO3)2·6H2O is 1:2-6.

2. The method for preparing a metal polyphenol nanozyme hydrogel according to claim 1, wherein: The reaction time in step 1 is 15 min, and the reaction time in step 3 is 20 h.

3. The method for preparing a metal polyphenol nanozyme hydrogel according to claim 1, wherein: The mass concentration of the collagen solution is 1.5 wt.%, and the solution is mixed by oscillation.

4. The metal polyphenol nanozyme hydrogel obtained by the preparation method according to any one of claims 1 to 3.

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