Metal polyphenol-based nanoparticles, methods of making and use thereof in treating acute kidney injury

Abstract

The application discloses an antioxidative metal polyphenol-based nanoparticle, and belongs to the technical field of biomaterials, which is composed of a nanoenzyme component and a non-enzyme molecular component; the application further discloses a preparation method of the nanoparticle, that is, the antioxidative metal polyphenol-based nanoparticle with broad-spectrum free radical scavenging activity and pro-oxidant (ferric ion) chelating capacity is synthesized through the interaction between metal ions and polyphenols; the nanoparticle has good cell compatibility for renal tubular epithelial cells and good scavenging capacity for active oxygen and active nitrogen free radicals in the renal tubular epithelial cells; the nanoparticle can be applied to the prevention and treatment of acute kidney injury; the nanoparticle can realize rapid and effective aggregation at a kidney site, as an efficient antioxidant, can effectively scavenge active oxygen at the kidney site, reduce oxidative damage and inflammatory response, and prevent and treat glycerol-induced acute kidney injury, and simultaneously has excellent biocompatibility and biosafety.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, in particular to a metal polyphenol-based nanoparticle, a preparation method thereof and application thereof in treating acute kidney injury. BACKGROUND

[0002] Acute kidney injury is a critical illness with high morbidity and mortality, especially for patients in intensive care units. There are more than 13.3 million new cases of acute kidney injury every year, about 5,000 cases per million people, and the number of related deaths is 1.7 million. At present, the clinical treatment of acute kidney injury mainly relies on supportive treatment, including hemodialysis, kidney transplantation and fluid infusion. Although basic research and clinical treatment methods have been rapidly advanced in recent decades, so far there is no clinical method to cure acute kidney injury.

[0003] During the occurrence of acute kidney injury, a large number of toxic reactive oxygen species are produced by renal tubular epithelial cells, which react with biological molecules in the body to induce oxidative stress, thereby causing abnormal expression of inflammatory factors and pro-adhesion factors, activating the NF-κB inflammatory pathway, inducing mitochondrial apoptosis, and ultimately exacerbating kidney injury and dysfunction. Effective reactive oxygen species scavenging can improve the renal microenvironment, inhibit oxidative stress, and alleviate acute kidney injury. In recent years, antioxidant nanodrugs have been used to treat acute kidney injury and have achieved remarkable results. However, the therapeutic effect is still limited by many factors: (1) most of them exhibit peroxidase activity, which produces highly active hydroxyl radicals during treatment, causing secondary oxidative damage; (2) most nanomaterials cannot achieve targeting and effective aggregation in the kidney; and (3) the metabolic mechanism of many nanomaterials is not clear, and the biological safety cannot be guaranteed. Therefore, the treatment of acute kidney injury urgently needs to develop more effective and safer antioxidant materials.

[0004] Metal-phenolic networks (MPNs) are constructed by coordination bonds between natural polyphenols and metal ions (such as Fe 3+ , Cu 2+ , Zn 2+ , Ce 3+ ), which have the dual properties of natural polyphenols and metal ions and good biocompatibility, and have received widespread attention in the construction of biomedical materials in recent years. Existing technologies such as CN 116019929 A and CN 115317516 A have reported the use of metal-polyphenol materials for reactive oxygen species scavenging and for acute kidney injury, but due to the selection of raw materials, preparation methods, etc., their effects, safety, etc. need to be improved. SUMMARY

[0005] One of the purposes of the present application is to provide an antioxidant metal polyphenol-based nanoparticle to solve the problems in the treatment of acute kidney injury.

[0006] In order to solve the above technical problems, the technical scheme adopted is an antioxidant metal polyphenol-based nanoparticle, which is composed of two parts of nano-enzyme components and non-enzyme molecular components.

[0007] As a preferred technical scheme, the antioxidant metal polyphenol-based nanoparticle is prepared by an oxidation-reduction reaction of natural polyphenols and metal salt precursors under alkaline conditions.

[0008] As a preferred technical scheme, the nano-enzyme component is selected from one or more of copper oxide, cuprous oxide or elemental copper.

[0009] The reasons for selecting copper ions are as follows: 1) Copper ions, as essential trace elements for the human body, have good biocompatibility, and a small amount of intake is harmless to the human body, so safety is the primary consideration for selecting copper ions, while Ce ions and their compounds have strong biological toxicity and are not considered in material design; 2) Copper ions have multiple valence states that can be regulated and are important components of antioxidant enzymes in the human body. In this study, after the oxidation-reduction reaction of copper ions and polyphenols to form metal polyphenol nanoparticles, the nanoparticles exhibit good superoxide dismutase activity and catalase activity, while metal polyphenol nanoparticles formed with metals such as Zn do not have good superoxide dismutase activity and catalase activity; 3) Although Fe and other metals have multiple valence states that can be regulated, Fe ions are the main components of peroxidase in the human body, so nanoparticles formed with polyphenols exhibit weak superoxide dismutase activity and catalase activity. Therefore, copper ions are selected for reaction with polyphenols to obtain nanoparticles for further study.

[0010] As a preferred technical scheme, the non-enzyme molecular component is selected from one or more of tannic acid, ellagic acid, gallic acid, tea polyphenols and dopamine.

[0011] As a preferred technical scheme, the alkaline condition is achieved by adding one or more of sodium hydroxide solution, ammonia, and potassium hydroxide solution.

[0012] The present application is mainly through the reducibility of the phenolic hydroxyl group of the polyphenol and the oxidizability of the metal ion to occur redox reaction, the polyphenol is partially oxidized to quinone by copper ion, and the polyphenol oligomer is formed through covalent bond, in this process, the copper ion is also partially reduced to monovalent cuprous ion or copper element, and gradually matures into polyvalent metal oxide under alkaline conditions, so that the metal polyphenol-based nanoparticles are obtained, in which the polyphenol oligomer serves as the core skeleton, and the polyvalent metal oxide is uniformly dispersed on the polyphenol oligomer core skeleton. Such combination brings superoxide dismutase activity and catalase activity to the polyphenol, and in situ removes the hydroxyl radicals generated in the acidic environment, thereby avoiding the peroxidase activity of the nanoscale enzyme components and the generation of highly active hydroxyl radicals.

[0013] The polyphenol material alone cannot effectively remove hydrogen peroxide, and the metal oxide alone cannot effectively chelate pro-oxidants such as ferrous ions, and also exhibits peroxidase activity that can generate highly active radicals. Compared with traditional polyphenols and nanoscale enzyme materials, the metal polyphenol-based nanoparticles for treating acute kidney injury in the present application are composed of nanoscale enzyme components and non-enzyme molecular components, and can simultaneously realize the effects of antioxidant enzymes and antioxidant non-enzyme molecules. The nanoscale enzyme components can effectively remove superoxide anion radicals and hydrogen peroxide, and the non-enzyme molecular components can effectively remove hydroxyl radicals and pro-oxidants (Fe 2+ ), and can also in situ remove the hydroxyl radicals generated by peroxidase activity, thereby realizing the removal of a broad spectrum of reactive oxygen species. More importantly, the surface of the nanoparticles is negatively charged, so it is repelled by the negatively charged glomerular filtration barrier, achieving passive targeting of the kidney, and the size of the nanoparticles is greater than 10 nm, so they will not be quickly excreted into the urine from the kidney, thus achieving long-term aggregation and accumulation in the kidney, thereby achieving the targeting effect and normal metabolism after playing a role, which makes it possible to treat acute kidney injury.

[0014] The second object of the present application is to provide a preparation method of the above-mentioned antioxidant metal polyphenol-based nanoparticles, and the specific steps are as follows:

[0015] The copper salt solution is added to the mixed solution of phenylalanine and polyvinylpyrrolidone, and stirred to obtain a uniform solution. Under vigorous stirring, the polyphenol solution is added and heated to a certain temperature. After the redox reaction is fully carried out under the protection of nitrogen atmosphere for a certain period of time, the pH of the solution is adjusted to alkaline by sodium hydroxide, and the reaction is continued for a period of time. The product is naturally cooled to room temperature, centrifuged and collected, washed with deionized water and ethanol for several times to remove unreacted reactants and impurities, thereby obtaining the metal polyphenol-based nanoparticles.

[0016] Preferably, the copper salt is one of copper chloride, copper sulfate, copper nitrate, and the like, but is not limited thereto.

[0017] Preferably, the molar ratio of the copper salt to sodium hydroxide is 1:4.

[0018] Preferably, the stabilizer is polyvinylpyrrolidone and phenylalanine.

[0019] Preferably, the reaction time of the polyphenol and the metal salt is 4 hours, and the reaction time after adding sodium hydroxide is 2 hours.

[0020] Preferably, the reaction temperature is 90°C.

[0021] Preferably, the metal polyphenol-based nanoparticles have broad-spectrum antioxidant activity. Specifically, the metal polyphenol-based nanoparticles have one or more of superoxide dismutase activity, catalase activity, hydroxyl radical scavenging ability, and ferrous ion chelating ability.

[0022] Preferably, the metal polyphenol-based nanoparticles can remove intracellular reactive oxygen species and inhibit apoptosis. Acute kidney injury is one of the serious renal dysfunction syndromes, and its pathological mechanism is derived from the excessive production of endogenous reactive oxygen species, which in turn induces oxidative stress and apoptosis of renal tubular epithelial cells, and damages the structure and function of the kidney. Therefore, inhibiting oxidative stress and apoptosis of renal tubular epithelial cells is effective in treating and / or preventing acute kidney injury.

[0023] According to a specific embodiment of the present application, the metal polyphenol-based nanoparticles have superoxide dismutase activity, catalase activity, hydroxyl radical scavenging ability, and ferrous ion chelating ability, as well as intracellular reactive oxygen species scavenging ability and inflammation regulation ability, and have good aqueous dispersion and biocompatibility. As an antioxidant nanomaterial, it can be applied to the treatment of acute kidney injury and other diseases related to reactive oxygen species.

[0024] The third object of the present application is to provide a use of the metal polyphenol-based nanoparticles as described above in the preparation of a medicament for treating acute kidney injury.

[0025] The metal polyphenol-based nanoparticles of the present application are nanoparticles obtained by redox reaction of polyphenolic compounds and metal ions, and have broad-spectrum antioxidant enzyme and non-enzyme activity, including superoxide dismutase, catalase, hydroxyl radical scavenging ability, and ferrous ion chelating ability.

[0026] The metal polyphenol-based nano drug of the application can effectively enrich in the kidney of a mouse, clear a large amount of active oxygen in the renal tubule, and relieve cell apoptosis.

[0027] The metal polyphenol-based nanoparticle of the application can effectively inhibit the activation of the NF-κB inflammatory pathway and reduce the release of inflammatory factors, so as to relieve and treat acute kidney injury.

[0028] The metal polyphenol-based nanoparticle of the application has good treatment effect on acute kidney injury and has excellent biocompatibility and biosafety.

[0029] The biggest feature of the application is that the metal polyphenol-based nanoparticles with good antioxidant activity are synthesized by using metal polyphenol interaction as an antioxidant defense system mimetic, the metal polyphenol-based nanoparticles have superoxide dismutase activity, catalase activity, hydroxyl radical scavenging capacity, ferrous ion chelation capacity and intracellular active oxygen scavenging capacity, can effectively accumulate in the kidney, and can realize prevention and treatment of acute kidney injury.

[0030] Compared with the prior art, the application has the following advantages:

[0031] 1. The metal polyphenol-based nanoparticle has good broad-spectrum free radical scavenging performance and excellent blood compatibility.

[0032] 2. The metal polyphenol-based nanoparticle can be used for relieving oxidative stress, reducing cell apoptosis, reducing kidney injury and restoring kidney function.

[0033] 3. The presence of phenolic hydroxyl groups of polyphenols in the metal polyphenol-based nanoparticle improves the blood compatibility and antioxidant activity of the nanoparticle.

[0034] 4. The nanenzyme component of the metal polyphenol-based nanoparticle prepared by the application has a wide selection range.

[0035] 5. The metal polyphenol-based nanoparticle prepared by the application can quickly reduce the free radical level of the kidney and protect biological macromolecules from oxidative damage.

[0036] 6. The raw materials used in the metal polyphenol-based nanoparticle prepared by the application are common chemical raw materials, which can be prepared in large quantities through chemical industry, and are rich in resources and low in cost, which is conducive to industrialization. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The synthesis route diagram of the metal polyphenol-based nanoparticle in the specific embodiment 1 of the application is shown in the figure.

[0038] Figure 2 The scanning electron microscope image of the metal polyphenol-based nanoparticle in the specific embodiment 1 of the application is shown in the figure.

[0039] Figure 3 X-ray diffraction pattern of metal polyphenol-based nanoparticles in specific embodiment 1 of the present invention;

[0040] Figure 4 X-ray photoelectron spectroscopy pattern of metal polyphenol-based nanoparticles in specific embodiment 1 of the present invention;

[0041] Figure 5 Superoxide dismutase activity pattern of metal polyphenol-based nanoparticles in specific embodiment 1 of the present invention;

[0042] Figure 6 Catalase activity pattern of metal polyphenol-based nanoparticles in specific embodiment 1 of the present invention;

[0043] Figure 7 Hydroxyl radical scavenging rate pattern of metal polyphenol-based nanoparticles in specific embodiment 1 of the present invention;

[0044] Figure 8 Ferrous ion chelating ability pattern of metal polyphenol-based nanoparticles in specific embodiment 1 of the present invention;

[0045] Figure 9 Protective effect of metal polyphenol-based nanoparticles in specific embodiment 1 of the present invention on the morphology of renal tubular epithelial cells (staining of cytoskeleton and nucleus by phalloidin and 4,6-diamidino-2 phenylindole (DAPI), respectively, indicates that CuxO@MPNs can protect the nucleus and cytoskeleton from oxidative stress damage);

[0046] Figure 10 Fluorescence pattern of reactive oxygen species in renal tubular epithelial cells under hydrogen peroxide stimulation by metal polyphenol-based nanoparticles in specific embodiment 1 of the present invention (the stronger the fluorescence signal in the FITC channel, the higher the content of reactive oxygen species in the cell);

[0047] Figure 11 Apoptosis pattern of renal tubular epithelial cells under oxidative stress by metal polyphenol-based nanoparticles in specific embodiment 1 of the present invention (upper left quadrant (UL) is dead cells; lower left quadrant (LL) is normal cells; upper right quadrant (UR) is late apoptotic cells; lower right quadrant (LR) is early apoptotic cells);

[0048] Figure 12 Distribution pattern of metal polyphenol-based nanoparticles in the major organs of rats at different times in specific embodiment 1 of the present invention (the stronger the fluorescence signal, the higher the content of fluorescence in the organ);

[0049] Figure 13 Blood urea nitrogen content pattern in serum of rats in different treatment groups by metal polyphenol-based nanoparticles in specific embodiment 1 of the present invention;

[0050] Figure 14 Figure 1 is a graph showing the serum creatinine levels in rats of different treatment groups after injection of metal polyphenol-based nanoparticles according to a specific embodiment of the present application;

[0051] Figure 15 Figure 2 is a graph showing the body weight of rats over time after injection of metal polyphenol-based nanoparticles and phosphate buffer (control) according to a specific embodiment of the present application;

[0052] Figure 16 Figure 3 is a graph showing the hydrogen peroxide levels in the kidneys of rats of different treatment groups after injection of metal polyphenol-based nanoparticles according to a specific embodiment of the present application;

[0053] Figure 17 Figure 4 is a graph showing the malondialdehyde levels in the kidneys of rats of different treatment groups after injection of metal polyphenol-based nanoparticles according to a specific embodiment of the present application;

[0054] Figure 18 Figure 5 is a graph showing the superoxide dismutase levels in the kidneys of rats of different treatment groups after injection of metal polyphenol-based nanoparticles according to a specific embodiment of the present application;

[0055] Figure 19 Figure 6 is a graph showing the TNF-α levels in the serum of rats of different treatment groups after injection of metal polyphenol-based nanoparticles according to a specific embodiment of the present application;

[0056] Figure 20 Figure 7 is a graph showing the IL-6 levels in the serum of rats of different treatment groups after injection of metal polyphenol-based nanoparticles according to a specific embodiment of the present application. Embodiment

[0057] The technical solutions of the present application will be further described below through specific embodiments.

[0058] The performance test method of the metal polyphenol-based nanoparticle medicine prepared by the present application is as follows:

[0059] For the blood compatibility test of metal polyphenol nanoparticles, the hemolysis rate test is taken as an example. CuxO@MPNs solution (10-50 μg / mL) was pretreated by soaking in phosphate buffer for 12 hours and incubated at 37 °C for 1 hour. Phosphate buffer was added to whole blood at a volume ratio of 1:1, and the mixture was centrifuged for 15 minutes at 2000 rpm to obtain red blood cells. This red blood cell separation process was repeated 5 times. 0.2 mL of red blood cells and 0.8 mL of phosphate buffer were added to the pretreated microsphere sample, and the mixture was shaken in a 37 °C incubator for 2 hours. The suspension was obtained by centrifugation at 8000 rpm for 5 minutes. The absorbance of the suspension was measured using a UV-Vis spectrometer. Deionized water and phosphate buffer were set as positive and negative controls, respectively. The hemolysis rate was calculated using the following formula:

[0060] Hemolysis rate (%) = (Absorbance of suspension - Absorbance of negative control) / (Absorbance of positive control – Absorbance of negative control) × 100%. Example

[0061] This embodiment aims to provide a relatively ideal antioxidant metal polyphenol nanoparticle, and its preparation method is as follows:

[0062] like Figure 1 As shown, 10 mL of copper sulfate solution (1 mmol) was added to a mixed solution of 10 mL of phenylalanine (1 mmol) and polyvinylpyrrolidone (1 mmol) and stirred until a homogeneous solution was obtained. Under vigorous stirring, 10 mL of glucose (1 mmol), 20 mL of dopamine solution (1 mmol), and 30 mL of tannic acid solution (0.1 mmol) were added and heated to 90 °C. After reacting for 4 h under a nitrogen atmosphere, the pH of the solution was adjusted to alkaline by adding 4 mL of 1 M sodium hydroxide (4 mmol) and the reaction was continued for 2 h. The product was collected by centrifugation and washed repeatedly with deionized water and ethanol to remove unreacted reactants and impurities, thus obtaining metal polyphenol nanoparticles, abbreviated as CuxO@MPNs.

[0063] Figure 1 Cu 2+ Representing copper sulfate pentahydrate, the polyvinylpyrrolidone surface ligand in the nanoparticles effectively stabilizes the nanoparticles. Figure 2 This is a SEM image of the nanoparticles synthesized in this embodiment, showing that the nanoparticles have a spherical morphology and a suitable size; Figure 3 This is the XRD pattern of the nanoparticles synthesized in this embodiment, characterizing the successful synthesis of copper oxide nanoparticles; Figure 4 This is an XPS image of the nanoparticles synthesized in this embodiment, which characterizes the multivalent states of copper in the nanoparticles.

[0064] Test Example 1: This test example is intended to demonstrate the radical scavenging effect of metal polyphenol-based nanoparticles.

[0065] (1) Superoxide radical scavenging ability: In a 1.5 mL "mixed solution containing riboflavin (20 μM), methionine (12.5 mM), and nitro blue tetrazolium chloride (75 μM)", CuxO@MPNs prepared in Example 1 were added to make the concentration of CuxO@MPNs in the mixed solution 0-10 μg / mL. The mixed solution was irradiated with constant intensity of ultraviolet light for 5 minutes, and then the absorbance of the solution was measured at 560 nm. By measuring the inhibition rate of nitro blue tetrazolium chloride photoreduction, the superoxide radical (O2 •− ) scavenging efficiency of CuxO@MPNs was obtained.

[0066] The results are shown in Table 1, and it can be seen from Table 1 that the metal polyphenol-based nanoparticles prepared in Example 1 can effectively mimic superoxide dismutase, scavenge superoxide anion radicals, and have concentration-dependent properties. Figure 5 Figure 5

[0067] (2) Hydrogen peroxide scavenging ability: In general, 500 μL of CuxO@MPNs was added to 500 μL of 2.5 mM H2O2 solution to make the concentration of CuxO@MPNs in the mixed solution 10-50 μg / mL. The mixed solution was incubated overnight (12 h), and then 50 μL of the mixed solution was added to 100 μL of the above Ti(SO4)2 solution. The absorbance of the mixed solution was measured at 405 nm every 30 minutes.

[0068] To measure the oxygen production activity of CuxO@MPNs, H2O2 and CuxO@MPNs were dissolved in 20 mL of deionized water, the final concentration of H2O2 was 2 M, and the final concentration of CuxO@MPNs was 10-50 μg / mL, and then mixed at room temperature, and then the O2 concentration was measured every 1 minute using a dissolved oxygen meter.

[0069] The results are shown in Table 2, and "Control" in Table 2 refers to the aforementioned "H2O2 (2 M)", and it can be seen from Table 2 that the antioxidant metal polyphenol-based nanoparticles prepared in Example 1 can mimic catalase, effectively scavenge hydrogen peroxide, and produce oxygen. Figure 6 Figure 6 Figure 6

[0070] (3) Hydroxyl radical scavenging ability: Fe 2+ ​​​​​Fenton reaction of / H2O2 system generates •OH, which can be detected by the reaction of specific probe salicylic acid (SA) to generate 2-hydroxy salicylic acid. SA and Fe 2+ After mixing / H2O2 system, there is a clear absorption peak at 510 nm. Prepare FeSO4 (2 mM), H2O2 (5 mM), SA (1.5 mM) and CuxO@MPNs solution with different concentrations in phosphate buffer solution, incubate at 37 °C for 30 min, and measure the absorbance of the mixed solution at 510 nm.

[0071] The results are shown in Figure 7 From Figure 7 it can be seen that the metal polyphenol-based nanoparticles prepared in Example 1 can effectively scavenge hydroxyl radicals and exhibit concentration-dependent characteristics.

[0072] (4) Ferrous ion chelating ability: different concentrations of CuxO@MPNs were added to FeCl2 solution. Add phenothiazine to initiate the reaction, adjust the total volume of the mixed solution to 10 mL (final CuxO@MPNs concentration: 0-500 μg / mL, FeCl2: 0.2 mM, phenothiazine: 1 mM). Then shake the mixed solution well and stand at room temperature for 10 min. The absorbance of the mixed solution at 562 nm was measured by spectrophotometry. The lower the absorbance of the reaction mixture, the stronger the Fe 2+ chelating ability of CuxO@MPNs.

[0073] The results are shown in Figure 8 From Figure 8 it can be seen that the metal polyphenol-based nanoparticles prepared in Example 1 can effectively chelate ferrous ions and exhibit concentration-dependent characteristics.

[0074] Test Example 2: Evaluate the effect of nanoparticles on the survival rate of human renal tubular epithelial cells using fluorescence imaging and flow cytometry.

[0075] Renal tubular epithelial cells were seeded in a 6-well plate at a density of 10 5 cells per well and incubated at 37 °C, 5% CO2 for 24 h. Then, the old culture medium in the 6-well plate was aspirated and different nanoparticle-containing culture medium solutions were added. Continue to incubate for 4 h, then aspirate the old culture medium in the 6-well plate and replace it with fresh culture medium containing Rosup (i.e. reactive oxygen species positive control reagent, Biotium, S0033S) (containing 100 μM H2O2) reagent to induce intracellular oxidative stress for 1 h. Subsequently, add reactive oxygen probe (DCFH-DA, Biotium, S0033S) to the renal tubular epithelial cells and incubate at 37 °C in the dark for 0.5 h. HK-2 cells treated with Rosup reagent serve as positive controls (i.e.Figure 11 "Rosup" in the "Rosup" column), the renal tubular epithelial cells without any treatment were used as the negative control (i.e. Figure 11 "Control" in the "Control" column). The renal tubular epithelial cells co-treated with Rosup and the nanoparticles were used as the experimental group. The fluorescence imaging analysis of the cells and the reactive oxygen species was performed using a fluorescence microscope (Zeiss, OBSERVER D1 / AX10 cam HRC, Germany). The apoptosis of the renal tubular epithelial cells was detected by a flow cytometer (Beckman, cytoflex) using an Annexin V-FITC / PI apoptosis detection kit (Bi Yun Tian, C1062S) to evaluate the anti-apoptosis effect of the nanoparticles.

[0076] As shown in FIG. 1, the metal polyphenol-based nanoparticles prepared in Example 1 can protect the cell nucleus and the cytoskeleton from oxidative stress and exhibit normal morphology of the renal tubular epithelial cells. Figure 9 As shown in FIG. 2, compared with the cells stimulated by hydrogen peroxide, the fluorescence in the renal tubular epithelial cells pretreated with the metal polyphenol-based nanoparticles is significantly weakened, which is close to that of the control group. This indicates that the metal polyphenol-based nanoparticles can effectively scavenge the reactive oxygen species in the cells and thus protect the cells.

[0077] Figure 10 Meanwhile, as shown in FIG. 3, after the cells receiving the hydrogen peroxide stimulation are treated with the nanoparticles at different concentrations for 6 h, the survival rate is greatly improved, which indicates that the metal polyphenol-based nanoparticles can protect the renal cells from the damage of the reactive oxygen species.

[0078] Test Example 3: Accumulation of the Metal Polyphenol-Based Nanoparticles Prepared in Example 1 in the Kidney and Treatment Figure 11 All the experimental operations are in accordance with the animal use and care system approved by the Clinical Center for Animal Care and Use Committee, and all the animal experiments are approved by the Animal Ethics and Welfare Committee of the West China Hospital of Sichuan University, Chengdu, China, with the approval number: 20211087A.

[0079] Before the model of acute kidney injury is started, all the female Sprague Dawley (S.D.) rats (250 ~ 300 g) are given normal food and no water for 15 h; after the water restriction is over, the S.D. rats are intramuscularly injected with 50% glycerol (8 mL / kg) in the hind legs; then, all the rats are given free water and food, so as to establish the model of acute kidney injury for further research. Before the model of acute kidney injury is established, the nanoparticles are injected to test the effect of the nanoparticles in preventing acute kidney injury. 2 h after the model of acute kidney injury is established, the nanoparticles are injected to test the effect of the nanoparticles in treating acute kidney injury.

[0080] Before the model of acute kidney injury is started, all the female Sprague Dawley (S.D.) rats (250 ~ 300 g) are given normal food and no water for 15 h; after the water restriction is over, the S.D. rats are intramuscularly injected with 50% glycerol (8 mL / kg) in the hind legs; then, all the rats are given free water and food, so as to establish the model of acute kidney injury for further research. Before the model of acute kidney injury is established, the nanoparticles are injected to test the effect of the nanoparticles in preventing acute kidney injury. 2 h after the model of acute kidney injury is established, the nanoparticles are injected to test the effect of the nanoparticles in treating acute kidney injury.

[0081] ​The main organs of the mice were taken out at different time points, and the distribution of nanoparticles in the organs of the mice was detected using live imaging. As shown in FIG. 8, the nanoparticles were mainly distributed in the kidneys and livers of the mice, with the highest content at 12 hours, indicating that the nanoparticles prepared in Example 1 can quickly reach the kidneys of the mice and achieve effective kidney accumulation. Figure 12

[0082] Test Example 4: Evaluation of the treatment of acute kidney injury and biological safety of the metal polyphenol-based nanoparticles prepared in Example 1.

[0083] All experimental operations for the treatment of acute kidney injury by the nanoparticles were in accordance with the animal use and care system approved by the clinical center for animal care and use committee, and all animal experiments were approved by the animal ethics and welfare committee of West China Hospital of Sichuan University in Chengdu, China, and the research approval number was 20211087A.

[0084] Before the start of the model of acute kidney injury, all female S.D. rats were given normal food and no water for 15 h; after the end of water restriction, the S.D. rats were intramuscularly injected with 50% glycerol (8 mL / kg); then, all the rats were given free water and food, thereby establishing the model of acute kidney injury for further study. Before the establishment of the model of acute kidney injury, the nanoparticles were injected to test the effect of the nanoparticles in preventing acute kidney injury. Two hours after the establishment of the model of acute kidney injury, the nanoparticles were injected to test the effect of the nanoparticles in treating acute kidney injury.

[0085] The rats were randomly divided into 5 groups: (1) healthy S.D. rats injected with phosphate buffer; (2) healthy S.D. rats injected with metal polyphenol-based nanodrugs; (3) glycerol-induced acute kidney injury S.D. rats injected with phosphate buffer; (4) glycerol-induced acute kidney injury S.D. rats, injected with metal polyphenol-based nanodrugs before the establishment of the model; (5) glycerol-induced acute kidney injury S.D. rats, injected with metal polyphenol-based nanodrugs 2 h after the establishment of the model. The healthy rats and the glycerol-induced acute kidney injury S.D. rats were euthanized after 24 hours, the blood of the rats was centrifuged to obtain serum, and the contents of creatinine and blood urea nitrogen were measured, and various biochemical indicators were detected. The injection volume of phosphate buffer was 1000 μL, and the injection amount of nanoparticles was 200 μg.

[0086] As shown in FIG. 9, the metal polyphenol-based nanodrugs prepared in Example 1 can effectively prevent acute kidney injury, and can also effectively treat acute kidney injury. Figures 13-14 ​As shown, the creatinine and blood urea nitrogen levels of healthy S.D. rats injected with metal polyphenol-based nanodrugs did not change significantly. The creatinine and blood urea nitrogen levels of S.D. rats with acute kidney injury injected with metal polyphenol-based nanoparticles were significantly lower than those of S.D. rats injected with only phosphate buffer and close to the levels of healthy S.D. rats. In addition, S.D. rats injected with phosphate buffer and metal polyphenol-based nanoparticles were used to record the body weight changes of S.D. rats with healthy and glycerol-induced acute kidney injury. As shown in Figure 15 As shown, there was no significant difference in the body weight of mice injected with nanoparticles compared with the control group. At the same time, biochemical analysis showed that, as shown in Figures 16-20 The levels of H2O2, malondialdehyde, superoxide dismutase, TNF-α and IL-6 of rats treated with metal polyphenol-based nanoparticles returned to normal levels. These all indicate that metal polyphenol-based nanoparticles can effectively alleviate and treat acute kidney injury.

[0087] In summary, the metal polyphenol-based nanoparticles of the present application can be prepared by a simple synthesis method to realize the antioxidant function of nanoparticles, which can effectively scavenge various active oxygen and chelate pro-oxidant ferrous ions. Metal polyphenol-based nanoparticles can protect cells from oxidative stress by scavenging excess active oxygen in renal tubular epithelial cells. With the special properties of metal polyphenol-based nanoparticles, effective accumulation of metal polyphenol-based nanoparticles in the kidneys of mice can be observed by in vivo imaging. In addition, metal polyphenol-based nanoparticles show good prevention and treatment effect in S.D. rats with glycerol-induced acute kidney injury. More importantly, metal polyphenol-based nanoparticles have good biocompatibility and biosafety.

[0088] Example 2-5

[0089] Example 2-5 aims to illustrate the influence of different nanenzyme components, different polyphenol components, different stabilizer components and reaction time on the antioxidant metal polyphenol-based nanoparticles, as shown in Table 1.

[0090] Table 1: Scheme and corresponding effect of Example 2-5

[0091]

Claims

1. A method of preparing antioxidant metal polyphenol-based nanoparticles, characterized by, The method comprises the following steps: adding the copper salt solution into the mixed solution containing the stabilizer, stirring to obtain a uniform solution; adding the polyphenol compound solution under stirring, heating to the reaction temperature, and performing a redox reaction under the protection of nitrogen atmosphere, then adjusting the pH of the solution to alkaline by a basic solution, continuing the reaction for a period of time, naturally cooling the product to room temperature, centrifuging and collecting, washing with deionized water and ethanol for several times, removing the unreacted reactants and impurities, and obtaining the antioxidant metal polyphenol-based nanoparticles; the time of the redox reaction is 4-6 hours, and the reaction time after adjusting to the alkaline environment is 2-6 hours; the base in the basic solution is selected from one of sodium hydroxide, ammonia, and potassium hydroxide, and the molar ratio of the copper salt to the base is 0.25-1; the reaction temperature is 60-90℃; the copper salt is selected from one of copper sulfate, copper chloride, and copper nitrate; the solute in the mixed solution containing the stabilizer is selected from a mixture of polyvinylpyrrolidone and phenylalanine; the polyphenol compound is a mixture of tannic acid and dopamine.

2. The method of claim 1, wherein: the concentration of the mixed solution containing the stabilizer is 0.1-0.3 M.

3. The antioxidant metal polyphenol-based nanoparticles prepared by the preparation method in any one of claims 1-2.

4. The use of the metal polyphenol-based nanoparticles prepared by the preparation method in any one of claims 1-2 in the preparation of a drug for treating acute kidney injury.

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