Preparation method of sequential targeting mesoporous manganese oxide nano-enzyme and application of sequential targeting mesoporous manganese oxide nano-enzyme in ischemia reperfusion induced acute kidney injury model treatment

Abstract

The invention relates to the technical field of biomedicine, in particular to a preparation method of sequential targeting mesoporous manganese oxide nano-enzyme and application of the sequential targeting mesoporous manganese oxide nano-enzyme in treatment of ischemia reperfusion induced acute kidney injury. Adding a potassium permanganate solution into the silicon dioxide nanoparticle dispersion liquid under ultrasonic waves, and carrying out alkali etching to obtain mesoporous manganese oxide nanoparticles (HMN); and after surface amination modification, connecting with a mixed PEG spacer layer, and reacting with thiolated SS31 and azidoacyl hyaluronic acid to obtain the nano-enzyme. The preparation can effectively penetrate through a damaged glomerulus filtration barrier and is passively enriched in a damaged kidney, damaged renal tubular epithelial cells over-express CD44 receptor mediated hyaluronic acid modified nano-particle endocytosis is enhanced, SS31 modified nano-particles achieve lysosome escape mitochondrial targeting in cells, the level of active oxygen in mitochondria can be reduced, and the activity of the mitochondria can be improved. The mitochondrial function is recovered, the renal function and tissue pathological injury are improved, and accurate anti-oxidation treatment on the acute kidney injury is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, and particularly relates to a preparation method of a sequential target mesoporous manganese oxide nanoscale enzyme and application thereof in treatment of an acute kidney injury model induced by blood reperfusion. BACKGROUND

[0002] Ischemia-reperfusion (I / R) induced acute kidney injury (AKI) remains a formidable challenge in clinical treatment, which is characterized by sudden decline in renal function, accompanied by high incidence of serious complications, economic burden and mortality. In clinical practice, I / R is the main cause of AKI in kidney transplantation, major surgery, trauma and hemorrhagic shock. If not timely and effective intervention, the risk of progression to chronic kidney disease will increase, and transplant recipients may experience delayed graft function and even rejection. The kidney has a rich mitochondrial, second only to the brain, and I / R will damage mitochondrial energy metabolism and homeostasis, thereby impairing organ function.

[0003] Antioxidant strategies include small molecules, natural nanoscale enzymes and synthetic nanoscale enzyme materials. Compared with the former two types, synthetic nanoscale enzyme materials usually have excellent stability and multiple reactive oxygen species (ROS) scavenging efficiency, thereby highlighting their superiority in non-drug therapy. However, their intracellular effectiveness is often limited, and excessive hydrodynamic size will hinder endocytosis, lysosomal recognition and isolation may reduce catalytic efficiency, and the diffusion radius of mitochondrial reactive oxygen species is limited (about 20 nm), which requires scavenging near organelles to ensure effective protection and repair of mitochondria.

[0004] Although significant progress has been made in the development of mitochondrial-targeted delivery systems, achieving precise and effective treatment of acute kidney injury (AKI) remains a considerable challenge. Many mitochondrial ligands employ cationic or hydrophobic motifs, which can exhibit instability in vivo, and these ligands can reduce targeting efficiency through binding to circulating anionic proteins. At the same time, insufficient targeting of proximal tubular epithelial cells (PTECs), which are the main site of ischemia-reperfusion injury (IRI), can lead to excretion of nanomedicines from urine. SUMMARY

[0005] The purpose of the present application is to construct a mesoporous manganese oxide nanoscale enzyme platform (HMN@HA-SS31), which realizes multi-level targeted delivery of the kidney, proximal tubular epithelial cells and mitochondria by synergistically modifying hyaluronic acid (HA) and mitochondrial targeting peptide SS31, so as to accurately remove mitochondrial reactive oxygen species in acute kidney injury. The design aims to enhance the exposure of catalytic sites, improve the biodistribution of nanoscale enzymes, and realize organelle-level antioxidant protection by specifically binding to CD44 receptors and mitochondrial inner membrane cardiolipin, so as to inhibit mitochondrial dysfunction, apoptosis and inflammatory response, and improve ischemia-reperfusion (I / R) induced kidney injury.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The present application provides a preparation method of a sequential targeting mesoporous manganese oxide nanoscale enzyme, comprising the following steps: (1) Under continuous ultrasonic conditions, potassium permanganate solution is gradually added to a silica nanoparticle dispersion, continuous ultrasonic treatment is continued, the precipitate is centrifuged, washed, and then etched with an alkaline solution, the precipitate is centrifuged, washed, and HMN nanoparticles are obtained; (2) After the HMN nanoparticles are modified with amino groups, they are combined with a mixed spacer layer of Mal-PEG-NHS and DBCO-PEG-NHS, and then thiolated SS31 and azido-lactyl hyaluronic acid are added, and the product is purified by ultrafiltration to obtain a sequential targeting mesoporous manganese oxide nanoscale enzyme.

[0007] Preferably, the continuous ultrasonic conditions in step (1) are 80-120 W for 8-12 min.

[0008] Preferably, the volume ratio of the potassium permanganate solution to the silica nanoparticle dispersion in step (1) is 25-35:4, and the silica nanoparticle dispersion is prepared by the Stöber method.

[0009] Preferably, the alkaline solution in step (1) is a sodium carbonate solution.

[0010] Preferably, the molar ratio of Mal-PEG-NHS (MV 2000) to DBCO-PEG-NHS (MV 3400) in step (2) is 5-7:4.

[0011] Preferably, the ratio of thiolated SS31 to HMN nanoparticles in step (2) is 0.2-1.0 µmol:10 mg, and the mass ratio of azido-lactyl hyaluronic acid to HMN nanoparticles is 1-1.4:1.

[0012] Preferably, the reaction time in step (2) is 4-6 h, and the molecular weight cut-off of the ultrafiltration is 90-110 kDa.

[0013] The application provides the sequential targeting mesoporous manganese oxide nanoscale enzyme prepared by the preparation method.

[0014] The application provides application of the sequential targeting mesoporous manganese oxide nanoscale enzyme in treatment of an ischemia-reperfusion-induced acute kidney injury model.

[0015] The technical solution of the application is to construct a sequential targeting mesoporous manganese oxide nanoscale enzyme composite system (HMN@HA-SS31), and the specific technical implementation path is as follows: 1. Nanoscale enzyme core construction and catalytic function design: taking mesoporous manganese nanoparticles with a large specific surface area and an ordered pore structure as a catalytic core. The manganese-based active center simulates the function of natural manganese superoxide dismutase (Mn-SOD), efficiently catalyzes the dismutation reaction of superoxide anion (O2 ) to generate H2O2 and O2. The mesoporous structure greatly increases the exposure of active sites and significantly improves the efficiency of enzymatic reaction, and the SOD-like activity is better than that of Fe3O4, CeO2 and other nanoscale enzymes.

[0016] 2. Surface functionalization and kidney / cell level targeting design: a layer of hyaluronic acid (HA) is coated on the surface of the HMN to form an "HA corona". The modification on one hand gives the nanoparticles a strong negative surface charge, effectively reduces the adsorption of plasma proteins and the capture of the reticuloendothelial system through the electric repulsion effect, prolongs the blood circulation time, and promotes the passive enrichment in the kidney tissue by using the nanometer size effect. On the other hand, HA can be used as a specific ligand to actively target the CD44 receptor highly expressed on the surface of ischemia-reperfusion injury renal tubular epithelial cells. This receptor-ligand combination achieves active aggregation of the nanodrug in the injured kidney and efficient endocytosis of the renal tubular epithelial cells.

[0017] 3. Organelle level targeting and positioning control design: a mitochondrion-targeting peptide SS31 (D-Arg-dimethylTyr-Lys-Phe-NH2) is further covalently coupled on the nanometer platform. The peptide segment has a unique targeting mechanism: it can specifically recognize and bind to cardiolipin on the inner membrane of mitochondria, and the binding process does not depend on the mitochondrial membrane potential. After the nanodrug is endocytosed by PTECs, the SS31 peptide guides the entire nanocomposite to cross the cytoplasm and finally precisely anchors to the mitochondria, especially the key microdomains of mtROS generation.

[0018] 4. Cascade catalysis and synergistic therapeutic effect: after the nanoscale enzyme is positioned to the mitochondria, the HMN core can not only catalyze the clearance of O2 Further, the H2O2 generated by the reaction can be decomposed into water and oxygen by peroxidase-like or CAT-like enzyme activity, realizing the cascade catalytic reaction of "superoxide dismutation-hydrogen peroxide scavenging", and effectively eliminating mtROS from the root. This in-situ catalysis in the lesion organelle maximally protects the mitochondrial membrane potential and morphological integrity, thereby inhibiting mitochondrial pathway apoptosis and reducing the sterile inflammation mediated by mtROS overflow and the cGAS / STING pathway.

[0019] 5. System advantages and technical problems avoided: The HMN@HA-SS31 system integrates efficient manganese-based catalysis, overexpression of CD44 receptors in damaged renal tubular epithelial cells, and membrane potential-independent mitochondrial targeting technology, successfully avoiding the technical bottlenecks of poor stability, high systemic toxicity, and uncontrollable in vivo distribution of traditional cationic mitochondrial targeting carriers, providing a reliable technical path for high-resolution and precise antioxidant therapy of acute kidney injury.

[0020] Compared with the prior art, the present application has the following beneficial effects: The HMN@HA-SS31 NPs developed in this study as a layered targeted nanozyme platform exhibit significant technical effects in various aspects under experimental conditions: The composite nanosystem successfully realizes sequential cascade delivery of kidney→proximal tubular epithelial cells→mitochondria through the synergistic action of HA / CD44-mediated cell targeting and SS-31 mitochondrial localization, and exhibits concentrated multi-antioxidant activity in the target mitochondrial region. In vitro, it not only exhibits comprehensive reactive oxygen species scavenging capacity (including SOD-like, CAT-like activity and direct scavenging of ·OH and O2 Functions), effectively managing mitochondrial reactive oxygen species (mtROS), and also showing good blood compatibility.

[0021] At the mechanism level, HMN@HA-SS31 NPs can stabilize mitochondrial membrane potential, restore mitochondrial network morphology and ATP levels, balance mitochondrial division / fusion dynamics, and significantly inhibit the intrinsic apoptosis pathway (manifested as reduced cytochrome c release and decreased caspase-3 activation).

[0022] In the ischemia-reperfusion (I / R) injury model, the preparation successfully penetrates the damaged glomerular filtration barrier, enriches in the damaged kidney, significantly reduces the ROS level in the kidney, improves kidney function and histopathological injury, and reduces tubular cell apoptosis. Its mitochondrial protection effect is further manifested as reduced mtDNA leakage, inhibited cGAS-STING pathway activation, and promoted macrophage polarization to the M2 phenotype, thereby collectively reducing inflammation and inhibiting kidney fibrosis remodeling under multiple dosing regimens. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0024] Figure 1 (A) High-resolution transmission electron microscopy (HR-TEM) shows the hollow structure of the nanoparticle HMN@HA-SS31, which is characterized by a rough porous shell. Scale bar = 100 nm. (B) Dynamic light scattering (DLS) measurement shows that the average hydrodynamic diameter of the HMN@HA-SS31 nanoszyme is 105 ± 2.26 nm. (C) Changes in the Zeta potential of HMN, HMN@HA, and HMN@HA-SS31 nanoszymes.

[0025] Figure 2 (B) In the ABTS assay, the ROS scavenging activity of the HMN@HA-SS31 nanoszyme increased from 25% to 90% in the range of 5 to 40 μg mL -1 (A). Similarly, the DPPH radical scavenging efficiency also showed a concentration-dependent decrease after contacting the nanoscale preparation, and reached about 80% at a concentration of 40 μg mL -1 (B). The TMB method was used to determine the hydroxyl radical scavenging capacity, and the results showed that the scavenging rate increased linearly with the nanoscale dose. More than 50% of the hydroxyl radicals were removed at a concentration of 10 μg mL -1 (C). The superoxide anion neutralization capacity was evaluated by WTS-1 detection, and the HMN@HA-SS31 nanoszyme showed a significant concentration-dependent decay, consistent with the behavior of superoxide dismutase mimics (D). The HMN@HA-SS31 nanoszyme showed a concentration-dependent SOD-like activity, and the activity increased from 25% to nearly 100% as the concentration increased from 5 to 30 μg mL -1 (E). At a concentration of 40 μg mL-1, the nanoszyme decomposed about 70% of H2O2, indicating that it had CAT-like activity (F).

[0026] Figure 3 Fluorescence microscopy observation of time-dependent cellular uptake of Cy5.5-labeled HMN@HA-SS31 nanoszyme (red) by untreated and H2O2-stimulated HK-2 cells. Scale bar = 100 μm.

[0027] Figure 4For confocal microscopy, HMN@HA-SS31 nanoszyme showed weak colocalization with lysosomes, while the highest degree of colocalization was observed with mitochondria (correlation coefficient R = 0.811) (left panel). Line scan profile showed overlapping peaks in the mitochondrial channel, indicating preferential accumulation (right panel).

[0028] Figure 5 For evaluation of renal cortical morphology using H&E staining, ischemia reperfusion resulted in significant tubular injury in the IRI group. The injury was characterized by significant tubular dilation and distortion, disarrayed arrangement between tubules, cast formation in the lumen, loss of brush border in proximal tubules, swelling, necrosis and detachment of epithelial cells. Administration of HMN@HA-SS31 nanoszyme at a dose of 5 mg kg -1 The pathological condition was significantly improved by administration of HMN@HA-SS31 nanoszyme at a dose of 5 mg kg DETAILED DESCRIPTION

[0029] The technical solutions provided by the present application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0030] Example 1

[0031] The sequential targeted manganese nanoszyme described in the present application is synthesized by the following method: (1) Synthesis of silica nanoparticles: silica nanoparticles (SiO2 NPs) were synthesized by the Stöber method; 1 mL of NH3-H2O was mixed with 40 mL of ethanol (95 wt%) and stirred at 40°C for 30 minutes. Then, tetraethyl orthosilicate (TEOS) (0.5 mL) was added dropwise under magnetic stirring, and the reaction was carried out for 24 hours to obtain SiO2 NPs. The product was purified by repeated washing with deionized water and ethanol and stored in deionized water to obtain a SiO2 NP dispersion, until further use.

[0032] (2) Synthesis process of hollow mesoporous manganese nanoparticles (HMN NPs; hMnOx): 30 mL of KMnO4 solution (10 mg / mL) was gradually added to 4 mL of SiO2 NP dispersion (10 mg / mL) under continuous ultrasound at 100 W for 10 min. After 6 hours of ultrasound (100 W, 10 min), the Si@MnOx composite material was separated by centrifugation and repeatedly washed with anhydrous ethanol and deionized water.

[0033] The prepared Si@MnOx was then etched in a Na2CO3 (2 M) aqueous solution to remove the template by stirring at 60 °C for 12 h. The obtained hollow manganese oxide nanoparticles (HMN NPs) were collected by centrifugation and carefully washed with deionized water (DIW) and ethanol to remove any residual reagents. These materials were used as such in the subsequent experiments.

[0034] (3) Sequentially targeted mesoporous manganese oxide nanoszyme preparation: After the initial preparation, the HMN NPs were modified with amines (3-aminopropyltriethoxysilane APTES 150 pL, added to 15 mL of anhydrous ethanol solution, 10% v / v, 65 C, reaction 3 h) and then bound to a mixed spacer layer of Mal-PEG-NHS (MV 2000) and DBCO-PEG-NHS (MV 3400) (molar ratio 6:4, reaction at room temperature for 2 h). Simultaneously, thiolated SS31 (0.2-1.0 pmol per 10 mg of mesoporous manganese oxide) and azido hyaluronan (N3-HA: mesoporous manganese oxide = 1.2:1, w / w) were added to the same reaction system (HEPES, 10 mM, pH 7.2) and reacted for 5 h, enabling a strain-promoted azide-alkyne cycloaddition reaction (SPAAC) with maleimide thiol and copper-free, without consuming the carboxylate of HA.

[0035] The product was purified by ultrafiltration (100 kDa) to obtain HMN@HA-SS31 nanoparticles (HMN@HA-SS31 NPs). As shown in Figure 1 , the obtained nanoszyme had a hydrated particle size of 105 ± 2.26 nm. High-resolution transmission electron microscopy (HRTEM) revealed the hollow structure of the HMN@HA-SS31, characterized by a rough, porous shell.

[0036] Example 2

[0037] 1. Evaluation of the multiple antioxidant enzyme activities of the HMN@HA-SS31 nanoparticles prepared in Example 1 at different concentrations.

[0038] As shown in Figure 2 , the antioxidant capacity of the HMN@HA-SS31 nanoszyme at different concentrations was evaluated by ABTS, DPPH, TMB, and WTS probes. In the ABTS experiment, potassium persulfate (K2S2O8) oxidized ABTS to generate blue-green cation radical ABTS +, the degree of reduction of which reflected the antioxidant activity of the sample. After adding the HMN@HA-SS31 nanoszyme, the color of the solution gradually changed from green to colorless with increasing dose, and the scavenging activity in the range of 5~40 pg mL -1 increased from 25% to 90% (Figure 2 A). Similarly, DPPH radicals have a characteristic absorption peak at 517 nm. Upon encountering this nano-formulation, the absorption signal shows a concentration-dependent decrease, decreasing at 40 μg / mL. -1 The free radical scavenging rate is approximately 80% ( Figure 2 B). These results confirm that the HMN@HA-SS31 nanozyme is a highly efficient free radical scavenger.

[0039] Using the TMB method Quantitative analysis of OH scavenging ability showed a monotonically increasing relationship between the amount of nanoparticles used and the scavenging efficiency. At a concentration of 10 μg / m³, the OH scavenging efficiency was significantly improved. ¹At that time, it can remove more than 50% OH ( Figure 2 C). Superoxide anion scavenging was assessed using the WTS-1 method, which reflected the change in absorbance of the reduction product (formazan) at 560 nm. - The neutralizing effect of HMN@HA-SS31 nanozyme showed a significant concentration-dependent decreasing trend, which is similar to the behavior of SOD mimicry. Figure 2 D). These results confirm that the HMN@HA-SS31 nanozyme exhibits broad-spectrum multi-antioxidant and multi-enzyme activities against various RONS in vitro.

[0040] Furthermore, the HMN@HA-SS31 nanozyme exhibited concentration-dependent SOD-like activity, which increased with increasing concentration from 5 to 30 μg / mL. -1 The activity increased from 25% to nearly 100% (E). At 40 μg / mL -1 At a concentration of [specific concentration not specified], the nanozyme decomposed approximately 70% of H2O2, indicating that it possesses CAT-like activity. Figure 2 F). The overall results indicate that HMN@HA-SS31 NPs achieve broad-spectrum reactive oxygen species (RONS) scavenging function through a multi-enzyme synergistic mechanism.

[0041] 2. Validation of targeted uptake by damaged renal tubular epithelial cells: Using Cy5.5-labeled HMN@HA-SS31 nanozymes, uptake experiments showed that in HK-2 cells, cellular uptake gradually increased with prolonged co-culture time. Figure 3 Compared with unstimulated cells, H2O2-stimulated HK-2 cells showed significant intracellular accumulation, with extensive intracytoplasmic distribution at each time point (1, 2, 4, 6 and 8 hours after stimulation).

[0042] 3. Mitochondrial targeting validation: To verify the subcellular localization, key organelles were fluorescently labeled and the distribution of Cy5.5-labeled HMN@SS-HA31 NPs was visualized using the Pearson coefficient (R: -1~ +1) for co-localization analysis. Confocal microscopy showed that the consistency of the nucleus and mitochondria was the largest (R = 0.811); the line scan curve showed that there was a coincidence peak in the mitochondria channel, indicating preferential accumulation. It was confirmed that SS31-mediated organelle-specific delivery and emphasized the potential of mitochondrial therapy. Mechanistically, HMN@SS-HA31 NPs present HA and SS31 on their surface; particles are mainly absorbed by HK-2 cells through CD44-mediated endocytosis. After lysosomal escape, SS31 binds to cardiolipin on the inner membrane of mitochondria, thereby promoting mitochondrial entry and accumulation in the inner membrane. Figure 4

[0043] 4. Nanoprotease efficacy evaluation on ischemia-reperfusion injury model: By different nano-drug intervention, the therapeutic effect of nanoprotease on the ischemia-reperfusion-induced acute kidney injury model was evaluated. Specifically, C57 mice (6-7 weeks old, 20-22 g, male) were fasted for 12 hours and placed on a thermostatic plate (37±2℃) to maintain body temperature after anesthesia. The kidneys were exposed through a midline abdominal incision, and the renal artery blood vessels were isolated and clamped with a non-traumatic clamp to block the blood vessels. Successful blood flow blockage was confirmed by the gradual uniformity of the kidney turning into a dull gray color, and after 20 minutes of ischemia, the artery clamp was removed to restore blood flow, whereby the kidney blood flow gradually returned to the initial healthy light reddish brown color in a short period of time (5 minutes), indicating successful reperfusion.

[0044] After the construction of the bilateral renal ischemia-reperfusion model of C57 mice, the mice were grouped for treatment evaluation: the first group: sham operation group (Sham); the second group: ischemia-reperfusion injury model (IRI); the third group: IRI+acetylcysteine (NAC); the fourth group: IRI+mesoporous manganese oxide nanoparticles (HMN); the fifth group: IRI+simple hyaluronic acid modified nanoparticles (HMN@HA); the sixth group: IRI+double-targeted modified nanoparticles (HMN@HA-SS31). Hematoxylin and eosin (H&E) staining was used to evaluate the morphology of the renal cortex. As shown in FIG. 6, the renal cortex of the sham group was normal, and the renal cortex of the IRI group was damaged, with a large number of inflammatory cells and a large number of tubular epithelial cells with pyknosis and necrosis. The NAC group showed a certain degree of improvement, but the damage was still serious. The HMN group showed a certain degree of improvement, but the damage was still serious. The HMN@HA group showed a certain degree of improvement, but the damage was still serious. The HMN@HA-SS31 group showed a certain degree of improvement, but the damage was still serious. Figure 5 ​As shown, ischemia / reperfusion (I / R) resulted in severe damage to the tubules in the ischemia reperfusion injury (IRI) group. This damage was characterized by significant dilation and distortion of the tubules, disorganized arrangement between tubules, presence of casts within the lumen, loss of brush border in proximal tubules, swelling, necrosis and sloughing of the epithelium, and a damage score of 6.8. Administration of HMN@HA-SS31 NPs at a concentration of 5 mg / kg significantly improved the pathological condition and helped restore the tubular structure to near normal. In contrast, there was a persistent presence of partial lesions in the acetylcysteine NAC, HMN NPs, and HMN@HA NPs cohorts, with kidney damage scores of 4.8, 3.8, and 3.0, respectively. This result was primarily due to reduced targeting and accumulation of these treatments within the proximal tubular epithelial cells. This demonstrates that in vivo, HMN@HA-SS31 NPs successfully permeated the damaged glomerular filtration barrier in I / R, concentrated in the injured kidney and AQP1 -positive tubules, reduced kidney ROS, enhanced kidney function, and reduced tubular apoptosis.

[0045] The above only is the preferred embodiment of the present application, it should be pointed out that, for those skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for preparing a sequential targeted mesoporous manganese oxide nanoszyme, characterized in that, The method comprises the following steps: (1) under continuous ultrasonic condition, potassium permanganate solution is gradually added into silica nanoparticle dispersion, continuous ultrasonic is continued, precipitate is obtained by centrifugation, cleaning is performed, after etching by alkali solution, precipitate is obtained by centrifugation, cleaning is performed, and HMN nanoparticles are obtained; (2) after amino modification of the HMN nanoparticles, mixed spacer layer of Mal-PEG-NHS and DBCO-PEG-NHS is combined, then thiolated SS31 and azido hyaluronate are added, the product is purified by ultrafiltration, and sequential targeting mesoporous manganese oxide nanoscale enzyme is obtained.

2. The production method according to claim 1, characterized by, The continuous ultrasonic condition in step (1) is 80-120 W, 8-12 min.

3. The preparation method according to claim 1, characterized in that, The volume ratio of the potassium permanganate solution to the silica nanoparticle dispersion in step (1) is 25-35:4; the silica nanoparticle dispersion is prepared by Stöber method.

4. The production method according to claim 1, characterized by, The alkali solution in step (1) is sodium carbonate solution.

5. The preparation method according to claim 1, characterized in that, The molar ratio of Mal-PEG-NHS to DBCO-PEG-NHS in step (2) is 5-7:

4.

6. The method of claim 1, wherein, The ratio of thiolated SS31 to HMN nanoparticles in step (2) is 0.2-1.0 µmol:10 mg; the mass ratio of azido hyaluronate to HMN nanoparticles is 1-1.4:

1.

7. The preparation method according to claim 1, characterized in that, The reaction time in step (2) is 4-6 h; the molecular weight cut-off of ultrafiltration is 90-110 kDa.

8. Sequential targeting mesoporous manganese oxide nanoscale enzyme prepared by the preparation method in any one of claims 1-7.

9. Application of the sequential targeting mesoporous manganese oxide nanoscale enzyme in claim 8 in treatment of ischemia-reperfusion-induced acute kidney injury model.

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